UNIVERSIDADE FEDERAL DA BAHIA FACULDADE DE MEDICINA

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UNIVERSIDADE FEDERAL DA BAHIA FACULDADE DE MEDICINA FUNDAÇÃO OSWALDO CRUZ INSTITUTO GONÇALO MONIZ UFBA Curso de Pós-Graduação Em Patologia Humana

TESE DE DOUTORADO

AVALIAÇÃO DE BIOMARCADORES DE GRAVIDADE EM CÃES NATURALMENTE INFECTADOS POR LEISHMANIA INFANTUM CLASSIFICADOS CLINICAMENTE E ESTRATIFICADOS QUANTO À CARGA PARASITÁRIA EM ESTUDOS DE CORTE TRANSVERSAL

MANUELA DA SILVA SOLCÀ

Salvador – Bahia 2017

UNIVERSIDADE FEDERAL DA BAHIA FACULDADE DE MEDICINA FUNDAÇÃO OSWALDO CRUZ INSTITUTO GONÇALO MONIZ

Curso de Pós-Graduação em Patologia Humana

AVALIAÇÃO DE BIOMARCADORES DE GRAVIDADE EM CÃES NATURALMENTE INFECTADOS POR LEISHMANIA INFANTUM CLASSIFICADOS CLINICAMENTE E ESTRATIFICADOS QUANTO À CARGA PARASITÁRIA EM ESTUDOS DE CORTE TRANSVERSAL

MANUELA DA SILVA SOLCÀ

Orientadora: Dra. Patrícia Sampaio Tavares Veras Co-orientadora: Dra. Deborah Bittencourt Mothé Fraga

Tese apresentada ao Curso de Pós-Graduação em Patologia Humana, para obtenção do grau de Doutora.

Salvador – Bahia 2017

Ficha Catalográfica elaborada pela Biblioteca do Instituto Gonçalo Moniz / FIOCRUZ - Salvador - Bahia.

S684a

Solcà, Manuela da Silva. Avaliação de biomarcadores de gravidade em cães naturalmente infectados por Leishmania infantum classificados clinicamente e estratificados quanto à carga parasitária em estudos de corte transversal. / Manuela da Silva. Solcà. 2016. 151 f. : il. ; 30 cm. Orientador: Profª. Drª. Patrícia Sampaio Tavares Verass, Laboratório de Patologia e Biologia Molecular. Tese (Doutorado em Patologia Humana) – Fundação Oswaldo Cruz, Instituto Gonçalo Moniz, 2016. 1. Leishmaniose Visceral Canina. 2. Biomarcadores. 3. PCR. 4. Cão. Título. CDU 616.993.161-08

da Tese: "AVALIAÇÃO DE BIOMARCADORES DE GRAVIDADE EM CÃES NATURALMENTE INFECTADOS POR LEISHMANIA INFANTUM" AVALIAÇÃO GRAVIDADE EMDE CÃES NATURALMENTE GRAVIDADE Título da DE Tese:BIOMARCADORES "AVALIAÇÃO DE DE BIOMARCADORES EM CÃES INFECTADOS POR INFECTADOS LEISHMANIA CLINICAMENTE E NATURALMENTE PORINFANTUM LEISHMANIA CLASSIFICADOS INFANTUM" Título

ESTRATIFICADOS QUANTO À CARGA PARASITÁRIA EM ESTUDOS DE CORTE TRANSVERSAL

MANUELA DA SILVA SOLCÁ MANUELA DA SILVA SOLCÁ FOLHA DE APROVAÇÃO FOLHA DE APROVAÇÃO Salvador, 09 de fevereiro de 2017 Salvador, 09 de fevereiro de 2017

COMISSÃO EXAMINADORA COMISSÃO EXAMINADORA

Professora Adjunta UFBA Professora Adjunta UFBA

Dr. FedericoCosta Professor Dr. Federico UFBA Costa Professor UFBA

;ggo-lo- 0&.«Ka{ Ü Dra. FlavlaneAlves Pinho ;ggo-lo0&.«K Professoraa{ Ü

Dra. Flavlane Alves Pinho UFBA Professora UFBA

Pesquisadora IGM/Fiocruz Pesquisadora IGM/Fiocruz

FONTES DE FINANCIAMENTO: CNPq – Bolsa de Doutorado (161750/2012-5) FAPESB – Projetos PPSUS (n° SUS0036/2013) e Projeto Doenças Negligenciadas (n° PET0024/2013) INCT – DT - Instituto Nacional de Ciência e Tecnologia de Doenças Tropicais

AGRADECIMENTOS Gostaria de agradecer a todos aqueles que participaram de forma direta ou indireta na realização deste trabalho: A meus pais e toda minha família pelo afeto, apoio e constante incentivo em todas as fases desse projeto e aos meus amigos que me ajudaram a seguir em frente sempre. Às minhas orientadoras Deborah e Dra. Patrícia, pela paciência, pelo tempo dedicado me acompanhando durante esse projeto e pelos conselhos valiosos durante cada momento desse doutorado. Gostaria de agradecer também Dra. Claudia, que mesmo não sendo orientadora oficial, contribui muito para meu crescimento pessoal e acadêmico. A toda a equipe do LPBI 1: Juliana, Carlos, José Geraldo, Antônio, Marcos, Isadora, Lairton, Kercia, Rosidete e aos ICs pela amizade e apoio durante a realização do trabalho. Obrigada especial a minhas amigas Luana, Beatriz e Niara, pelo apoio durante os momentos mais críticos desse doutorado, me motivando sempre. Agradeço com carinho a todo o “Grupo Cão” - membros atuais e passados: Bruna, Tiago, Miriam, Kelsilandia e Antonio Mendes. Gostaria de agradecer também a todos os ICs, sem os quais esse trabalho não seria possível: Liliane, Maiara, Lívia, Yuri, Junior Guedes, Rafael, Daniela, Amanda, Laís, Maria Helena, Luciano, Enice, Alice, Marcos David, Maria Carolina, Johnatan (espero não ter esquecido ninguém). A Dr. Geraldo, Dr. Freitas e Dr. Washington pela amizade e valiosos conselhos ao longo do projeto. A todos os colaboradores, cada um de forma distinta enriqueceu muito as análises realizadas: •

Dra. Rita, Dr. Alexandre e Dr. Marco Krieger (IBMP-FIOCRUZ) por toda ajuda na parte de biologia molecular, aprendi muito com essa parceria;



Dra. Valéria, Dr. Bruno, Dr. Ricardo e Dra. Melissa (LIMI-IGM FIOCRUZ) obrigada pela colaboração nas análises dos marcadores e pelas ideias excelentes;



Dra. Leila e Pétala (Instituo de Matemática - UFBA) pela ajuda com a análise da classe latente que tornou o trabalho muito mais interessante;



Profa. Daniela, Profa. Stella, Prof. Jairo e Rafaela (LIVE – ESCMEVZ – UFBA) pela colaboração nas coletas a campo e pela amizade;



Dr. Jesus Valenzuela e Dra. Shaden Kamhawi (Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, NIAIH, NIH) pelo fornecimento das proteínas recombinantes da saliva do vetor e especialmente a Dra. Shaden pela amizade e valiosos ensinamentos ao longo de todo o projeto;



Dra.

Patrícia

Torres

Bozza

(Laboratório

de

Imunofarmacologia,

Biomanguinhos FIOCRUZ) pela colaboração na análise dos biomarcadores por LUMINEX; •

Dra. Clarissa Teixeira (Fiocruz Piauí́) pela amizade e ajuda na parte das análises do ELISA da saliva do vetor;

Aos técnicos Lívia e Sergio pela disponibilidade; Às administradoras do LPBI pela ajuda e dedicação; A prefeitura de Camaçari, principalmente aos membros do Centro de Controle de Zoonoses, os veterinários Gilmar, Sandra e Adriana e aos agentes de saúde: Ana Maria, Antonio e Isa por todo apoio e colaboração no decorrer do trabalho; À Coordenação do curso PGPAT Fiocruz / UFBA; Aos funcionários da Biblioteca do IGM por toda a ajuda prestada nas pesquisas efetuadas na referida biblioteca; Aos membros da banca de qualificação e defesa de tese, pelas críticas construtivas que ajudaram a melhorar o trabalho; Ao CNPq pela bolsa de doutorado que ajudou na realização deste projeto; À Fiocruz e ao IGM, pela infraestrutura física e pessoal que possibilitaram a execução dos experimentos desta tese.

“Chi nega la ragion delle cose, pubblica la sua ignoranza”. LEONARDO DA VINCI

SOLCÀ, Manuela da Silva. Avaliação de biomarcadores de gravidade em cães naturalmente infectados por Leishmania infantum classificados clinicamente e estratificados quanto à carga parasitária em estudos de corte transversal. 151 f. il. Tese (Doutorado em Patologia Humana) – Fundação Oswaldo Cruz, Instituto Gonçalo Moniz, Salvador, 2017. RESUMO INTRODUÇÃO: A leishmaniose visceral (LV) é, principalmente, causada pelo protozoário Leishmania infantum nas Américas, podendo acometer o Homem e animais. Dentre estes, o cão é considerado o principal reservatório doméstico do parasito. O curso da LV canina (LVC) varia entre os animais, podendo alguns se mostrar resistentes à infecção, se mantendo subclínicos, e outros susceptíveis, que irão desenvolver sinais da doença. O estado de resistência ou susceptibilidade à LVC reflete na gravidade da infecção do animal, e não pode ser definido pelo quadro clínico apresentado ou por qualquer parâmetro isolado de resposta imune. OBJETIVO: Avaliar a carga parasitária como biomarcador parasitológico, as proteínas LJM11/LJM17 como biomarcadores de exposição à saliva do vetor, e identificar biomarcadores inflamatórios de gravidade da infecção por L. infantum em cães. Primeiramente, foi realizada a padronização de uma ferramenta diagnóstica de PCR quantitativa (qPCR), utilizando diferentes amostras biológicas (aspirado esplênico, linfonodos, pele, sangue, medula óssea e swab conjuntival) de cães sintomáticos provenientes da área endêmica de Jequié-BA. A avaliação da carga parasitária de L. infantum teve seu desempenho comparado com outras técnicas diagnósticas (cultura de aspirado esplênico, teste rápido e ELISA para LVC) empregando a análise de classe latente (ACL). Para essa análise, foi construída uma variável latente a ser empregada como padrão ouro para avaliação da acurácia desses métodos. Na avaliação inicia dos cães sintomáticos, a qPCR detectou DNA do parasita em 100% dos animais em pelo menos uma das amostras biológicas, sendo que todos foram positivos empregando-se o aspirado esplênico e 70% empregando-se o sangue. Utilizando a variável latente como padrão-ouro, a sensibilidade para a qPCR de aspirado esplênico (95,8%) foi maior do que as obtidas pela qPCR utilizando outras amostras biológicas e por outros testes diagnósticos. Desta forma, em uma segunda etapa, a acurácia da qPCR empregando-se aspirados esplênicos foi avaliada em uma amostra canina obtida de forma randômica (n = 800), coletada durante um estudo de corte transversal na área endêmica de Camaçari-BA. Utilizando a variável latente como padrão-ouro, novamente a sensibilidade para a qPCR de aspirado esplênico foi elevada (95%). Neste estudo de corte transversal foi possível também correlacionar a carga parasitária com a gravidade da infecção, existindo associação positiva significativa entre a intensidade da carga parasitária no baço e o número de sinais clínicos presentes nos cães. Nessa primeira parte do estudo, foi possível demonstrar que i) a utilização de aspirado esplênico na qPCR apresentou maior sensibilidade na detecção de DNA de L. infantum do que outras amostras biológicas; ii) a ACL pode ser usada para gerar um padrão ouro adequado para avaliação de técnicas diagnósticas, uma vez que esta técnica oferece uma avaliação mais completa dos resultados obtidos por diferentes métodos diagnósticos para LVC e iii) a carga parasitária se mostrou um bom biomarcador de gravidade clínica nos animais

infectados por L. infantum. Posteriormente, após a reação de qPCR ter sido padronizada e validada empregando-se aspirados esplênicos, esta foi aprimorada para utilização em estudos epidemiológicos, sendo padronizada em formato duplex tanto na forma líquida como no formato em gel (ready-to-use), adicionando-se à reação primers específicos para a detecção simultânea de um gene constitutivo canino. Esse protocolo permitiu a detecção de até 0,1 parasitas por amostra. Adicionalmente, a detecção de DNA do hospedeiro na mesma reação simultaneamente fortaleceu o diagnóstico da LVC, agindo como controle interno da reação, reduzindo tempo de execução e diminuindo custos da reação. Após a padronização da qPCR duplex, foi realizado um estudo exploratório de uma amostra de cães coletada durante outro estudo de corte transversal, em Camaçari-BA, no qual se objetivou avaliar biomarcadores de exposição à saliva do vetor (proteínas LJM11/LJM17) e identificar biomarcadores inflamatórios, correlacionando-os à gravidade da LVC e à carga parasitária. A análise identificou uma bioassinatura distinta em cães com diferentes manifestações clínicas, caracterizada por uma diminuição dos níveis de LTB4 e de PGE2 e um aumento de CXCL1 e CCL2, de acordo com o agravamento da doença. Além disso, utilizando uma combinação de 3 parâmetros distintos (LTB4, PGE2 e CXCL1) como um marcador, este permitiu a discriminação entre escores clínicos diferentes utilizando uma curva ROC. Foi detectado também, que cães com escores clínicos elevados apresentaram-se, mais frequentemente, positividade para anticorpos anti-saliva e elevadas cargas parasitárias. Este estudo permitiu a avaliação e identificação de vários biomarcadores em cães, que podem ser importantes para auxiliar na avaliação do curso da doença e prognóstico por médicos veterinários, além de futuramente poder ajudar na distinção entre cães resistentes ou susceptíveis direcionando estratégias de controle da LVC em áreas endêmicas. CONCLUSÃO: Existem biomarcadores parasitológicos como a carga parasitária, biomarcadores imunológicos e inflamatórios como CXCL1, CCL2, LTB-4, PGE-2, além da produção de anticorpos específicos contra as proteínas LJM 11 e LJM 17 da saliva do vetor, que são capazes de diferenciar animais infectados por L. infantum de acordo com seus diferentes quadros clínicos. Palavras-chave: Leishmaniose Visceral Canina, Biomarcadores, qPCR, Cão

SOLCÀ, Manuela da Silva. Severity biomarkers assessment in Leishmania infantum naturally infected dogs clinically classified and stratified by parasite load in cross sectional studies. 151 f. il. Thesis (PhD em Patologia Humana) – Fundação Oswaldo Cruz, Instituto Gonçalo Moniz, Salvador, 2017. ABSTRACT INTRODUCTION: In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, which can affect humans and animals. Among these, dog is considered the main domestic reservoir of this parasite. Canine VL (CVL) clinical outcome varies among animals, some of which may be resistant to infection remaining subclinical, and others may be susceptible showing signs of the disease. The state of resistance or susceptibility to CVL reflects on the severity of infection in the animal and cannot be defined solely by the clinical condition presented or by any isolated parameter of the immune response. OBJECTIVE: Assess parasite load as parasitological biomarkers, LJM11/LJM17 proteins as sandfly saliva exposure biomarkers, and identify inflammatory biomarkers that indicates L. infantum infection severity in dogs. Firstly, we performed the standardization of a quantitative PCR diagnostic tool (qPCR) using different biological samples (splenic aspirate, lymph nodes, skin, blood, bone marrow and conjunctival swab) of symptomatic dogs from the endemic area of Jequié-BA. The evaluation of the parasitic load of L. infantum had its performance compared to other diagnostic techniques (splenic aspirate culture, rapid test and CVL ELISA) using latent class analysis (LCA). In this analysis, a latent variable was constructed to be used as a gold standard to evaluate the accuracy of these methods. In the initial evaluation of the symptomatic dogs, qPCR detected DNA from the parasite in 100% of the animals in at least one of the biological samples, all of which were positive using the splenic aspirate and 70% using the blood. Employing the latent variable as a gold standard, splenic aspirate qPCR sensitivity (95.8%) was greater than that obtained by qPCR using other biological samples and other diagnostic tests. Thus, in a second stage, the accuracy of qPCR using splenic aspirates was evaluated in a randomly obtained canine sample (n = 800), collected during a cross-sectional study in the endemic area of Camaçari-BA. Using the latent variable as a gold standard, qPCR sensitivity of splenic aspirate was again high (95%). In this cross-sectional study, it was also possible to correlate the parasite load with the severity of the infection, and there was a significant positive association between the intensity of the parasite load in the spleen and the number of clinical signs present in dogs. In this first part of the study, it was possible to demonstrate that i) the use of splenic aspirate in the qPCR presented greater sensitivity in the detection of L. infantum DNA than other biological samples; Ii) LCA can be used to generate a suitable gold standard diagnostic techniques assessment, since this technique offers a more complete evaluation of the results obtained by different diagnostic methods for LVC and iii) the parasite load proved to be a good biomarker of clinical severity in animals infected with L. infantum. Next, after the qPCR reaction was standardized and validated using splenic aspirates, the same was improved for use in epidemiological studies, being standardized in both liquid and ready-to-use gel format, adding specific primers for the simultaneous detection of a canine constitutive gene. This protocol allowed the detection of up to 0.1 parasites per sample. In addition, the detection of host DNA in

the same reaction simultaneously strengthened the LVC diagnosis, acting as internal reaction control, reducing execution time and reducing reaction costs. After the standardization of the duplex qPCR, we performed an exploratory study of a sample of dogs collected during another cross-sectional study carried out in Camaçari-BA. We aimed to evaluate the biomarkers of vector saliva exposure (LJM11/ LJM17 proteins) and to identify inflammatory biomarkers, correlating them with CVL severity and parasite load. Assessment of protein expression of profile biomarkers identified a distinct biosignature that could cluster separately animal groups with different clinical scores. Increasing severity scores were associated with a gradual decrease of LTB-4 and PGE-2, and a gradual increase in CXCL1 and CCL2. Discriminant ROC analyses revealed that combined assessment of LTB-4, PGE-2 and CXCL1 was able to distinguish dogs with different clinical scores. Dogs with the highest clinical score values also exhibited high parasite loads and positivity for anti-saliva antibodies. This study allowed the evaluation and identification of several biomarkers in dogs, which may be important to assist in the evaluation of the disease course and prognosis by veterinarians, and in the future to be able to help distinguish between resistant or susceptible dogs directing CVL control strategies in endemic areas. CONCLUSION: There are parasitological biomarkers such as parasite load, immunological and inflammatory biomarkers such as CXCL1, CCL2, LTB-4, PGE-2, and the production of specific antibodies against LJM 11 and LJM 17 sandfly salivary proteins, which are able to differentiate animals infected by L. infantum according to their different clinical condition.

Key-Words: Canine Visceral Leishmaniasis, Biomarkers, qPCR, Dog

LISTA DE ABREVIATURAS

ALT

Alanina aminotransferase

AST

Aspartato aminotransferase

cPCR

Reação em cadeia da polimerase convencional

CCL2 CXCL1 CXL10 DPP LVC DNA DTH

Proteína quimioatratora de monócitos - do inglês: chemokine (C-C motif) ligand 2 Proteína ativadora de neutrófilos - do inglês: chemokine (C-X-C motif) ligand 1 Citocina Induzida por Interferon- do inglês: chemokine (C-X-C motif) ligand 10 Teste rápido para leishmaniose visceral canina do inglês Dual Path Platform Ácido desoxirribonucléico Hipersensibilidade do tipo tardio ao teste cutâneo a antígenos de Leishmania – do inglês: delayed type hypersensitivity

EIE LVC

Ensaio imunoenzimático para leishmaniose visceral canina

ELISA

Ensaio imunoenzimático

FA

Fosfatase alcalina

GGT

Gamaglutamiltransferase

IC

Intervalo de confiança

IgG

Imunoglobulina G

IgG1

Imunoglobulina G1

IgG2

Imunoglobulina G2

IL

Interleucina

IFN-γ

Interferon gama

kDNA

DNA do cinetoplasto

LJM11

Proteína salivar de Lutzomyia longipalpis LJM11

LJM17

Proteína salivar de Lutzomyia longipalpis LJM11

LV

Leishmaniose visceral

LVC

Leishmaniose visceral canina

LTB-4

Leucotrieno B4

MS

Ministério da Saúde

OMS

Organização Mundial da Saúde

PCR

Reação em cadeia da polimerase

PGE-2

Prostaglandina E2

qPCR

Reação em cadeia da polimerase quantitativa

RIFI

Reação de imunofluorescência indireta

RNA

Ácido Ribonucléico

rRNA

RNA ribossomal

SOD

Enzima superóxido desmutase

TH1

Linfócitos T auxiliares 1

TH2

Linfócitos T auxiliares 2

TCL

Teste cutâneo a antígenos de Leishmania

TGF-β TNF-α

Fator de transformação do crescimento beta - do inglês: transforming growth factor beta Fator de necrose tumoral alfa – do inglês: tumor necrosis fator alpha

SUMÁRIO

1

INTRODUÇÃO ................................................................................................ 15

1.1

DISTRIBUIÇÃO DA LEISHMANIOSE VISCERAL ........................................... 15

1.2

AGENTE ETIOLÓGICO E CICLO BIOLÓGICO DA LV ................................... 16

1.3

PAPEL DO CÃO NO CICLO DA LV ................................................................. 19

1.4

LEISHMANIOSE VISCERAL CANINA ............................................................. 19

1.4.1 Manifestações clínicas .................................................................................. 21 1.4.2 Diagnóstico ..................................................................................................... 22 1.4.3 Resposta imune .............................................................................................. 28 1.4.4 Resposta a saliva do vetor ............................................................................ 30 1.4.5 Biomarcadores de gravidade ........................................................................ 32 2

JUSTIFICATIVA .............................................................................................. 34

3

HIPÓTESE.......................................................................................................36

4

OBJETIVOS .................................................................................................... 36

4.1

OBJETIVO GERAL ......................................................................................... 36

4.2

OBJETIVOS ESPECÍFICOS ........................................................................... 36

CAPÍTULO 1 – Padronização de uma Ferramenta Diagnóstica de qPCR para avaliação da carga parasitária de L. infantum ........................................... 37 1.1

OBJETIVO GERAL ......................................................................................... 39

1.2

OBJETIVOS ESPECÍFICOS .......................................................................... 39

1.3

Artigo científico publicado na revista Plos One .............................................. 40

1.4

Artigo científico submetido à revista Diagnostic Microbiology and Infectious Disease ........................................................................................................... 54

CAPÍTULO 2 – Estudo exploratório para identificação de biomarcadores de gravidade de uma amostra de cães coletada durante um estudo de corte transversal em Camaçari-BA ....................................................................... 91 2.1

OBJETIVO GERAL ....................................................................................... 92

2.2

OBJETIVOS ESPECÍFICOS ......................................................................... 92

2.3

Artigo científico publicado na revista Scientific Reports ................................. 93

DISCUSSÃO GERAL.............................................................................................. 106 REFERÊNCIAS ....................................................................................................... 114 Anexo 1 – Colaboração em artigo científico publicado ................................... 132 Anexo 2 – Colaboração em artigo científico publicado ................................... 132

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1

INTRODUÇÃO

1.1 DISTRIBUIÇÃO DA LEISHMANIOSE VISCERAL Segundo a Organização Mundial da Saúde (OMS), a leishmaniose é classificada como uma doença negligenciada tropical, presente em cinco continentes, endêmica em 98 países, com mais de 350 milhões de pessoas em risco. Existem três manifestações clínicas diferentes de leishmaniose: cutânea, acometendo a pele; mucosa que acomete mucosas; e visceral, sendo esta a forma mais grave, que afeta os órgãos internos (BRASIL, 2014; WHO, 2017). A leishmaniose visceral (LV) acomete cerca de 300 mil pessoas mundialmente a cada ano, assim encontra-se entre as seis endemias consideradas prioritárias no mundo pela OMS, com registro de ocorrência em 88 países (DESJEUX, 2004; ALVAR et al., 2012; WHO, 2017). Anualmente, uma média de 4.000 casos de LV são diagnosticados, com uma taxa de mortalidade de 7%. Em 2014, mais de 90% dos novos casos notificados à OMS de LV ocorreram em seis países: Brasil, Etiópia, Índia, Somália, Sudão do Sul e Sudão (WHO, 2017). Já foi determinado que a presença de LV está diretamente ligada à pobreza, mas outros fatores sociais, assim como alguns ambientais e climatológicos também influenciam diretamente na distribuição da doença (FRANKE et al., 2002; QUINNELL e COURTENAY, 2009; WHO, 2017). Alterações ambientais e demográficas, além de uma intensa tendência migratória para centros urbanos, acarretaram que a partir da década de 70 a ocorrência da LV tenha sido mais frequente em áreas periurbanas e urbanas, com graves consequências para a saúde pública (BEVILACQUA et al., 2001). Na Região das Américas, foram registrados casos de leishmaniose do sul dos Estados Unidos até o norte da Argentina, com exceção das ilhas do Caribe, Chile e Uruguai. Desta forma, esta doença já́ foi descrita em pelo menos 12 países, sendo que Brasil, Argentina e Paraguai concentram 78% dos casos (LAINSON e RANGEL, 2005; WHO, 2017). No Brasil, casos de LV já foram notificados em 21 estados, atingindo as cinco regiões do país (MAIA-ELKHOURY et al., 2008; SINAN/SVS/MS, 2017), na região Nordeste localizam-se as principais áreas endêmicas, tendo em 2015, 1.806 casos de LV notificados (SINAN/SVS/MS, 2017).

16

Na Bahia, em 2015 foram registrados 320 casos de LV, sendo considerada como endêmica em 52% dos municípios, Sua ocorrência na Bahia é elevada, estando abaixo apenas de Maranhão, Ceará e Minas Gerais (SINAN/SVS/MS, 2017). A região central do estado da Bahia historicamente concentrava as taxas de prevalência mais elevadas da enfermidade, porem, há uma década, observou-se uma dispersão geográfica para o litoral e o sul do estado, que eram áreas tradicionalmente indenes, indicando mudanças no perfil ecoepidemiológico da doença (OLIVEIRA et al., 2010). A mudança no perfil epidemiológico com aumento do número de casos em áreas periurbanas e urbanas (BEVILACQUA et al., 2001) é um fenômeno que pode ser observado em diferentes capitais brasileiras, assim como na Região Metropolitana de Salvador, onde na última década tem se registrado um elevado número de casos humanos e caninos de LV (FRANKE et al., 2002; JULIÃO, 2004). Camaçari, uma das áreas de estudo do presente trabalho, é um município localizado na região metropolitana de Salvador, que foi identificado por estudos anteriores como uma área endêmica para LV e leishmaniose visceral canina (LVC) (CUNHA et al., 1995; JULIÃO et al., 2007.).

1.2 AGENTE ETIOLÓGICO E CICLO BIOLÓGICO DA LV A LV é causada por protozoários da ordem Kinetoplastida, família Trypanosomatidae, gênero Leishmania (MAURICIO et al., 1999), que acomete humanos, animais domésticos e silvestres (LAINSON e SHAW, 1978; KUHLS et al., 2011). Nas Américas, a LV é, principalmente, causada por Leishmania infantum, contudo, a espécie Leishmania amazonensis já foi reportada como responsável por causar casos raros de LV no Brasil (BARRAL et al., 1991; ALEIXO et al., 2006). Estudos de caracterização molecular do DNA do parasito mostraram que L. infantum e Leishmania chagasi são organismos indistinguíveis geneticamente, podendo uma espécie ser citada como sinônimo da outra (MAURICIO, STOTHARD e MILES, 2000; KUHLS et al., 2011). L. infantum é um protozoário bifásico, que se apresenta em duas formas distintas durante seu ciclo de vida (ALEXANDER e RUSSELL, 1992). No interior do trato digestório dos vetores flebotomíneos, o parasito encontra-se na forma de

17

promastigota (KILLICK KENDRICK e RIOUX, 1991). Enquanto que, no hospedeiro vertebrado prevalece a forma intracelular obrigatória, amastigota, encontrada no fagolisossomo de células do sistema fagocítico mononuclear, principalmente, macrófagos (ANTOINE et al., 1990; ALEXANDER, SATOSKAR e RUSSELL, 1999; KAYE e SCOTT, 2011). Estes protozoários necessitam no seu ciclo biológico de um inseto vetor, sendo transmitidos aos hospedeiros vertebrados pela picada de fêmeas de insetos hematófagos pertencentes à família Psychodidae, chamados flebotomíneos (NEVES, 2006; ROMICH, 2008). No Brasil, a espécie Lutzomyia longipalpis é considerada o principal vetor biológico da LV (SHERLOCK, 1996; LAINSON e RANGEL, 2005; MONTEIRO et al., 2005). O ciclo biológico da LV se inicia quando as fêmeas do inseto vetor alimentamse de algum hospedeiro vertebrado infectado e tornam-se infectadas (NEVES, 2006; ROMICH, 2008). Na pele do hospedeiro vertebrado, encontram-se células do sistema fagocítico mononuclear albergando formas amastigotas do parasito que são adquiridas durante o repasto sanguíneo pelo inseto vetor (KILLICK-KENDRICK, 1999). No interior do vetor, o sangue infectado ingurgitado passa para o intestino médio, uma vez que L. infantum é um parasito cujo desenvolvimento é restrito ao intestino médio do inseto (KAMHAWI, 2006). Posteriormente, os parasitos diferenciam-se em vários estádios de desenvolvimento distintos à medida que migram do intestino médio posterior para a válvula estomodeal, que forma uma junção com o intestino anterior (DESCOTEAUX e TURCO, 1999; KAMHAWI, 2006; BATES,

2007).

Após

cerca

de

6-9

dias,

os

parasitos

completam

seu

desenvolvimento no tubo digestório dos insetos, transformando-se da forma amastigota em forma promastigota, passando a expressar em sua superfície uma camada de moléculas como lipofosfoglicanos e glicoconjugados, capazes de proteger o parasito da ação das enzimas hidrolíticas presentes no intestino dos flebotomíneos (RUSSELL e WRIGHT, 1988; TALAMAS-ROHANA et al., 1990; ALEXANDER e RUSSELL, 1992). Essas formas infectantes chamadas de metacíclicas encontradas na porção posterior da válvula estomodeal são inoculadas na derme do hospedeiro vertebrado durante o repasto sanguíneo (KILLICKKENDRICK, 1999; KAMHAWI, 2006). Durante o repasto sanguíneo, o flebótomo danifica a pele do hospedeiro com sua probóscide, rasgando tecidos, lacerando capilares e criando lagos hemorrágicos

18

durante sua alimentação. Estes eventos provocam uma resposta imediata do sistema hemostático do hospedeiro, visando evitar a perda de sangue. Contudo, o inseto apresenta componentes farmacologicamente ativos na sua saliva que podem modular o sistema hemostático do hospedeiro (CHARLAB et al., 1999; RIBEIRO e FRANCISCHETTI, 2003; ABDELADHIM, KAMHAWI e VALENZUELA, 2014), como, por

exemplo,

maxadilan,

que

apresenta

propriedades

vasodilatadoras,

antiplaquetárias provocando hemorragia e lise tecidual (SAMUELSON et al., 1991; GUILPIN et al., 2002; ANDRADE et al., 2007; DE OLIVEIRA et al., 2009). Adicionalmente, outras substancias presentes na saliva do vetor, como por exemplo, a proteína amarela LJM11, que age como aglutinante de aminas biogênicas pró-inflamatórias como serotonina, histamina e catecolaminas, são responsáveis por influenciar o desenvolvimento e as funções de leucócitos, como neutrófilos e macrófagos (PRATES et al., 2011; XU et al., 2011; PRATES et al., 2012). Como consequência, estes eventos facilitam a transmissão das formas infectantes de Leishmania, uma vez que os vasodilatadores, anticoagulantes e moléculas imuno-moduladoras presentes na saliva promovem a interação inicial do parasito com as células imunes do hospedeiro vertebrado (PRATES et al., 2012). Estas substâncias liberadas pelos flebotomíneos provocam vasodilatação que facilita o contato dos parasitos com as células do hospedeiro, e a quimioatração que atrai os leucócitos para o local do repasto sanguíneo, favorecendo o estabelecimento da infecção no animal picado pelo inseto infectado (ABDELADHIM, KAMHAWI e VALENZUELA, 2014). Os parasitos inoculados na derme são fagocitados por macrófagos e no interior destas células, as formas promastigotas internalizam seu flagelo, sendo capazes de se adaptarem às condições do vacúolo parasitóforo (KAYE e SCOTT, 2011). No interior desses compartimentos fagolisossomais ácidos e ricos em enzimas

hidrolíticas,

algumas

promastigotas

se

transformam

nas

formas

amastigotas (NEVES, 2006; ROMICH, 2008), que se multiplicam intensamente por divisão binária simples. O resultante número elevado de parasitos pode levar à lise do macrófago, e uma vez liberadas, as amastigotas poderão ser novamente fagocitadas por outras células do sistema retículo endotelial, podendo se disseminar para outros órgão do sistema fagocítico mononuclear do hospedeiro (NEVES, 2006; REY, 2008).

19

1.3 PAPEL DO CÃO NO CICLO DA LV A LV pode acometer humanos, animais silvestres e domésticos (LAINSON e SHAW, 1978), dentre estes, o cão é considerado o principal reservatório doméstico do parasito, além de ser a principal fonte de infecção para o vetor no meio urbano (MOLINA et al., 1994; OLIVEIRA et al., 2001; ALVAR et al., 2004; MARGONARI et al., 2006; MADEIRA et al., 2009; BRASIL, 2014). As evidências que indicam o papel do cão como principal reservatório da LV nas áreas urbanas consistem em: I) presença da LV em 98 países e cerca de 50 desses países possuírem casos de LVC (ALVAR et al., 2012), II) elevado parasitismo na pele de cães infectados que pode funcionar como fonte de infecção para o vetor (MOLINA et al., 1994; MADEIRA et al., 2009); III) ocorrência da enzootia canina, precedendo tanto temporalmente quanto espacialmente a ocorrência da infecção humana (BEVILACQUA et al., 2001; OLIVEIRA et al., 2001; ALVAR et al., 2004; MARGONARI et al., 2006; BRASIL, 2014); IV) observação de elevada prevalência de LVC nas áreas endêmicas para LV, sendo esta superior à prevalência de LV humana (SHERLOCK, 1996; CAMARGO-NEVES et al., 2001; SILVA et al., 2001). Ademais, apesar de uma parte da população canina ser altamente susceptível à infecção por L. infantum e manifestar sinais característicos e clássicos da doença quando infectados (CIARAMELLA et al., 1997; KOUTINAS et al., 1999), a presença de cães sem manifestação clínica da doença que não são facilmente diagnosticados, mas que albergam o parasito na derme, também é frequente em áreas endêmicas, podendo estes animais desempenhar um papel na introdução, dispersão e manutenção da doença em áreas urbanas (MOLINA et al., 1994; GUARGA et al., 2000). Diante desses fatos, uma das principais medidas de controle da LV no Brasil preconizada pelo Ministério da Saúde (MS) consiste em identificar e eutanasiar os animais sorologicamente positivos (BRASIL, 2014).

1.4 LEISHMANIOSE VISCERAL CANINA Após a transmissão do parasito pelo inseto vetor, o surgimento dos primeiros sinais clínicos em cães pode ocorrer após semanas ou até alguns anos. Além disso,

20

uma parcela dos animais infectados pode nunca manifestar sinais característicos de LVC, se mantendo num estado subclínico (CIARAMELLA et al., 1997; KOUTINAS et al., 1999; ALVAR et al., 2004). Até o presente momento, não há uma definição clara de parâmetros que possam distinguir esses animais subclínicos daqueles prepatentes, ou seja aqueles que irão manifestar sinais clínicos decorrentes da infecção por L. Infantum, assim como não há uma distinção entre estes cães e aqueles infectados que nunca apresentarão manifestação clínica da doença, os resistentes. Adicionalmente, não existem evidências claras a respeito de quais desses animais (os que desenvolvem a doença e os que não desenvolvem) são importantes no ciclo de transmissão da doença, mas alguns autores consideram os cães que desenvolvem quadros de doença grave como susceptíveis, enquanto que animais subclínicos são considerados como resistentes à instalação da doença (PINELLI et al., 1994; PINELLI et al., 1995; PINELLI et al., 1999; CORREA et al., 2007; LAGE et al., 2007; REIS et al., 2009; MENEZES-SOUZA et al., 2011). Após a infecção, a evolução para a doença nos animais depende de diversos fatores como: a virulência do parasito (capacidade de inativar ou resistir ao efeito microbicida dos macrófagos ativados) e a suscetibilidade genética do hospedeiro, dentre outros, que são responsáveis pelo desencadeamento dos mecanismos imunológicos em resposta à infecção (ALVAR et al., 2004; LIPOLDOVA e DEMANT, 2006; BANETH et al., 2008) (GRIMALDI e TESH, 1993). Uma vez que as formas promastigotas são inoculadas na derme do cão, a infecção pode ser: limitada, sendo controlada localmente pelos macrófagos e outras células de defesa; não disseminada, quando os parasitos conseguem instalar-se apenas na pele e nos linfonodos e no baço, caracterizando um quadro de infecção subclínica; ou ainda disseminada, em caso de falha na resposta imune do hospedeiro, onde os parasitos espalham-se por todo o organismo podendo levar ao aparecimento da doença clínica (BANETH et al., 2008; SARIDOMICHELAKIS, 2009; SOLANO-GALLEGO et al., 2011). Após a picada do inseto vetor, L. infantum se distribui rapidamente nos linfonodos e baço, sucessivamente alcançando os rins e o fígado (MOREIRA et al., 2007). Finalmente o parasito se dissemina para os órgãos reprodutivos, pele, bexiga, trato digestório e respiratório do hospedeiro (MOLYNEUX e ASHFORD, 1983). Nos animais susceptíveis a distribuição sistêmica dos parasitos

21

é

extensa,

enquanto

que

nos

resistentes

ela

não

é

tão

disseminada

(SARIDOMICHELAKIS, 2009). Diversos estudos apontam que a população canina em áreas endêmicas é composta por três grupos de animais infectados, sendo estes: i) cães em fase de latência, que não apresentam sinais clínicos e que podem parecer cães resistentes, porém irão desenvolver a doença com o passar do tempo; ii) cães resistentes, que são os animais sem sinais clínicos e que nunca desenvolverão LVC; e iii) cães sintomáticos, que desenvolvem a doença após a infecção ou emergem da população de animais com infecção latente. (DYE, 1996; BURATTINI et al., 1998; NUNES et al., 2008; GRIMALDI et al., 2012b). Desta forma, a resistência ou a susceptibilidade dos cães à infecção por L. infantum não pode ser definida apenas por presença ou ausência de sinais clínicos, sendo indicada a análise conjunta destes com a carga parasitária e titulação de anticorpos anti-Leishmania e características relacionadas a diferentes parâmetros da resposta imune humoral ou celular. 1.4.1 Manifestações clínicas Em cães, a LV pode manifestar-se sob diferentes formas podendo produzir diversas manifestações clínicas, enquanto que no homem o quadro clínico é mais restrito (ALVAR et al., 2004). Os cães podem apresentar sinais que não são específicos, variando desde manifestações cutâneas até manifestações viscerais e sistêmicas (FOGLIA MANZILLO et al., 2013). Os sinais clínicos iniciais nos cães compreendem apatia, perda de peso, linfadenomegalia, dermatite periocular e nasal, além de alopecia localizada, onicogrifose e edema dos coxins plantares (NICOLE e COMTE, 1908; FERRER, 1999; MENDONCA et al., 1999; GREENE, 2006). Febre, diarreia, epistaxe, hemorragia

intestinal,

inapetência,

hepatoesplenomegalia,

hiperqueratose,

ulcerações cutâneas e ceratoconjuntivite também são achados clínicos nos animais acometidos (CIARAMELLA et al., 1997; KOUTINAS et al., 1999). Quadros severos se caracterizam por sinais típicos como ulcerações cutâneas disseminadas pelo corpo, cegueira, anorexia, onicogrifose, dermatite, alopecia generalizada, levando o animal à morte (REIS et al., 2009).

22

Além de manifestar sinais clínicos, cães infectados por L. infantum podem apresentar alteração de parâmetros hematológicos e bioquímicos (CIARAMELLA et al., 1997; KOUTINAS et al., 1999), que podem servir como marcadores de gravidade da doença, sendo úteis para avaliação do seu prognóstico (REIS et al., 2006a; DA COSTA-VAL et al., 2007; GEISWEID et al., 2012). O hemograma fornece subsídios importantes quanto à̀ resposta do cão infectado, pois já foi demostrado que anemia, hiperproteinemia e monocitose estão relacionadas com quadro de LVC disseminada, também chamada de LVC ativa (FEITOSA et al., 2003). Um estudo com animais tratados demonstrou que outros parâmetros hematológicos como a hipoalbuminemia e linfopenia também podem ser úteis para avaliar o prognóstico e prever a sobrevida dos animais (GEISWEID et al., 2012). A gravidade do quadro clínico da LVC também pode ser avaliada pela medida de alanina aminotransferase (ALT), aspartato aminotransferase (AST), fosfatase alcalina (FA) e gamaglutamiltransferase (GGT), enzimas que quando alteradas se relacionam com a gravidade de lesões hepáticas (BENDERITTER et al., 1988; IKEDA-GARCIA et al., 2007), tendo em vista que em quadros de LVC ativa já foi relatado o aumento sérico das enzimas hepáticas (CIARAMELLA et al., 1997). As dosagens séricas de ureia e creatinina também são importantes na LVC, pois são marcadores da redução da taxa de filtração glomerular, podendo seu aumento indicar início de insuficiência renal no animal, que é a principal causa de óbito do cão com LV (BENDERITTER et al., 1988; LOPEZ et al., 1996). 1.4.2 Diagnóstico Determinar o diagnóstico clínico de LVC é uma tarefa difícil e complexa, uma vez que a maior parte dos sinais associados a esta doença são variáveis e inespecíficos, podendo estar presentes em outras enfermidades que afetam os cães (GRAMICCIA, 2011). Para tentar aumentar a especificidade do diagnóstico clínico, alguns autores empregam técnicas padronizadas de registro clínico, como o cálculo de escore clínico baseado na intensidade e frequência de determinados sinais característicos de LVC apresentados pelos animais (SILVA et al., 2017). Assim, apesar do diagnóstico da LVC ser muitas vezes presumido pela identificação de manifestações clínicas características, este deve ser confirmado por testes

23

imunológicos

ou

pela

demonstração

do

parasito

utilizando-se

técnicas

parasitológicas (GRIMALDI e TESH, 1993). Testes sorológicos permitem a detecção de imunoglobulinas no soro, podendo ser utilizados para o diagnóstico de indivíduos infectados, assim como em estudos de prevalência (GOMES et al., 2008). A depender do tipo de antígeno empregado, esses testes podem apresentar sensibilidade e especificidade variáveis, sendo sujeitos ainda a reações cruzadas (KAR, 1995; SUNDAR e RAI, 2002; FERREIRA et al., 2007; TRONCARELLI et al., 2009; SILVA et al., 2011). As técnicas sorológicas, como o ensaio imunoenzimático (ELISA) e kits imunogromatográficos são os métodos mais empregados para o diagnóstico da LVC nos soroinquéritos promovidos pela saúde pública (ALVES e BEVILACQUA, 2004). Até o ano de 2011, segundo instrução do MS, os inquéritos caninos para triagem e identificação dos animais soropositivos para LVC eram realizados utilizando a técnica de ELISA (EIELVC), seguida pela reação de imunofluorescência indireta (RIFI) para confirmação da infecção (BRASIL, 2014). No entanto, a partir de 2011, uma nova instrução normativa foi emitida pelo MS, indicando o uso do teste rápido dual path platform (DPP-LVC) para triagem dos animais, e o EIE-LVC como exame confirmatório, eliminando a RIFI da rotina diagnóstica (BRASIL, 2011). A implantação do teste rápido DPP-LVC foi realizada gradativamente em todo o território nacional, abrangendo todos os estados brasileiros até o final de 2012 (BRASIL, 2011). Um estudo de Fraga et al., (2016) comparando os diferentes protocolos do MS mostrou que a adoção do novo protocolo (DPP LVC + EIE LVC) melhorou a especificidade no diagnóstico dos animais. Fato bastante relevante e que justifica a troca do método diagnóstico de triagem, uma vez que a eutanásia é a medida empregada para controle da população de reservatórios caninos sendo imprescindível um diagnóstico confirmatório específico do animal suspeito, para que animais sem LVC ou aqueles com outras patologias não sejam eutanasiados erroneamente. Os métodos parasitológicos como o exame direto de lâminas e o isolamento por cultura de tecidos, permitem a detecção do parasito, e podem ser empregados como métodos diagnósticos confirmatórios para LVC (BARROUIN-MELO et al., 2004). A observação de formas amastigotas ou promastigotas em material obtido de punção de medula óssea, baço ou de outros tecidos consiste no diagnóstico laboratorial definitivo da LV em humanos e caninos (BARROUIN-MELO et al., 2006; REIS et al., 2006a). Assim, as técnicas que permitem essa confirmação são

24

definidas como padrão-ouro (do inglês Gold standard) (DOURADO et al., 2007; GOMES et al., 2008). Seu uso é limitado para avaliação de cães em área endêmica, pois apesar da especificidade destes métodos parasitológicos ser muito elevada, a sensibilidade destas técnicas é baixa, principalmente na detecção de cães pouco parasitados, recém infectados ou subclínicos (PINELLI et al., 1994; SOLANOGALLEGO et al., 2001). De tal modo, o resultado de testes sorológicos e parasitológicos deve ser sempre avaliado com cuidado, procurando sempre associar este ao histórico epidemiológico e ao quadro clínico apresentado pelo animal e, quando possível, ao resultado de outro teste diagnóstico mais específico (DA SILVA et al., 2006). Nas últimas décadas, as técnicas moleculares como a reação em cadeia da polimerase (PCR), foram introduzidas para o diagnóstico da LVC, devido a sua elevada sensibilidade e especificidade (MIRÓ et al., 2008). Estas técnicas detectam o material genético do parasito, podendo ser empregadas como métodos confirmatórios em caso de animais recém infectados ou assintomáticos que, na maioria dos casos, não apresentam soroconversão e apresentam baixa carga parasitária, o que dificulta seu diagnóstico utilizando as técnicas sorológicas e parasitológicas, além de permitir a determinação da carga parasitária (SOLANOGALLEGO et al., 2001; ALVES e BEVILACQUA, 2004). 1.3.2.1 Diagnóstico Molecular Técnicas biomoleculares como a reação em cadeia da polimerase convencional (cPCR) e a PCR em tempo real (qPCR), são utilizadas para o diagnóstico da LVC, pois proporcionam elevada sensibilidade e especificidade, superando resultados obtidos pelas técnicas sorológicas e parasitológicas na detecção de animais infectados por Leishmania sp. (REALE et al., 1999; SOLANOGALLEGO et al., 2001; MIRÓ et al., 2008; SOLCÀ et al., 2012). O método da PCR consiste inicialmente na detecção de uma região específica do DNA do parasito, denominada alvo, por oligonucleotídeos iniciadores (primers) que, em condições ideais de temperatura e pH, hibridizam-se especificamente na região escolhida, levando à amplificação exponencial deste fragmento (SUNDAR e RAI, 2002; PAIVA-CAVALCANTI, REGIS-DA-SILVA e GOMES, 2010). A cPCR é uma técnica qualitativa, que apresenta a necessidade de eletroforese em gel de agarose ou poliacrilamida, para visualização do fragmento

25

amplificado, além de não permitir a quantificação da carga parasitária do animal, que consistem em desvantagens do método diagnóstico (REITHINGER e DUJARDIN, 2007). A qPCR por sua vez, é uma técnica quantitativa, com capacidade de monitorar e quantificar a amplificação do fragmento de DNA de interesse, utilizando corantes ou sondas fluorescentes, que são consumidas durante a amplificação do produto da qPCR, emitindo uma fluorescência específica ao passo que a reação progride, logo não há a necessidade de visualização de seu produto em gel (PAIVACAVALCANTI, REGIS-DA-SILVA e GOMES, 2010). As duas sondas utilizadas são: o SYBR Green (Applied Biosystems, CA, USA), um corante amplamente utilizado porém que tem como desvantagem sua ligação não específica nas sequências de DNA de fita dupla, produzidas durante a amplificação (REITHINGER e DUJARDIN, 2007); e o sistema TaqMan (Applied Biosystems, CA, USA), que consiste em uma sonda que se liga especificamente a sequencia alvo, aumentando a especificidade da qPCR (REITHINGER e DUJARDIN, 2007). A detecção de DNA de Leishmania sp. pelas técnicas de PCR pode ser realizada em uma ampla variedade de amostras clínicas (FISA et al., 2001; MANNA et al., 2004; FRANCINO et al., 2006; MAIA et al., 2009; GALLETTI et al., 2011). Manna et al. (2004), avaliaram através da técnica da cPCR diferentes tecidos de cães naturalmente infectados, obtendo elevada especificidade e sensibilidade: em 99% das amostras de linfonodos, 95% das biópsias de pele e 94% das amostras de sangue. Os tecidos linfoides estão incluídos dentre os órgãos onde é mais frequente a detecção de Leishmania sp. (MARZOCHI et al., 1985), dentre estes, a medula óssea é considerada o maior órgão hematopoiético e um dos principais tecidos linfoides, sendo considerada um local importante de armazenamento do parasito em cães infectados (TROPIA DE ABREU et al., 2011), assim como os linfonodos. Um inquérito realizado por Maia et al. (2009) em cachorros com diferentes quadros clínicos, demonstrou que o uso de aspirado de linfonodos pela técnica de cPCR é ideal para o diagnóstico precoce da LVC em cachorros sintomáticos. Todavia, em caso de ausência de linfadenomegalia, é indicado o emprego de aspirado de medula óssea, pois esta apresenta uma maior carga parasitária em relação aos linfonodos (QUARESMA et al., 2009). O baço é outro tecido linfático muito relevante para o diagnóstico e

26

patogênese da LVC (SOLCÀ et al., 2012). Reis et al. (2006) em um estudo citológico, demonstraram que o baço é um dos principais órgãos onde a densidade parasitária encontra-se elevada durante todo o curso da LVC, tanto em cães assintomáticos como sintomáticos. Saldarriaga et al. (2006) demonstraram em um estudo de infecção experimental intradérmica com promastigotas de Leishmania sp. em cães, que após 96 horas do inóculo, foi possível identificar o parasito nos linfonodos e baço dos animais. Outro estudo revelou graus variáveis de esplenomegalia na maioria dos cães naturalmente infectados por L. infantum sp. (BARROUIN-MELO et al., 2006). Estes achados fazem do baço um tecido importante e interessante para o diagnóstico da LVC (MAIA et al., 2009; CAVALCANTI et al., 2015). Assim como a coleta das amostras de medula óssea e linfonodo (CARVALHO et al., 2009), a de baço é considerada um procedimento invasivo, sobretudo pelo risco de causar lesões internas e, em consequência, hemorragias (LÉVEILLÉ et al., 1993). No entanto, existem descrições de que a coleta de amostras esplênicas é bem tolerada pelos animais (LÉVEILLÉ et al., 1993). Barrouin-Melo et al. (2006) descreveram que em um estudo com realização de mais de 257 punções esplênicas, somente em dois animais foi descrito o pinçamento de alça intestinal e hemorragia no local da punção. Estes riscos podem ser minimizados utilizando-se um aparelho de ultrassom que permite a visualização do órgão, favorecendo a realização de uma coleta de material esplênico guiada e segura (WATSON et al., 2011). Existe uma recomendação para utilização de amostras de sangue e esfregaços conjuntivais no diagnóstico da LVC, por estas serem obtidas de forma menos invasiva, mais fácil e mais rápida, além de terem baixo custo e por sua coleta ser bem aceita pelos proprietários dos animais (AOUN et al., 2009; MAIA et al., 2009; DE ALMEIDA FERREIRA et al., 2012; LOMBARDO et al., 2012). Apesar destas vantagens, alguns estudos evidenciaram que a detecção do parasito em sangue periférico é menos sensível, quando comparado com amostras de outros tecidos como baço, medula óssea, pele e linfonodos (REALE et al., 1999; FISA et al., 2001). No entanto, Francino et al. (2006), demonstraram que devido à elevada capacidade da qPCR em identificar DNA do parasito, mesmo que o sangue apresente cargas parasitárias mais baixas, este órgão pode vir a ser empregado para o diagnóstico de infecção por Leishmania sp. Amostras de coleta menos invasiva, como esfregaços conjuntivais podem ser

27

interessantes para serem empregadas em inquéritos epidemiológicos, uma vez que quase não apresenta risco para os animais, além de não necessitar muito treinamento na realização da técnica de coleta, exemplo dessas amostras podem ser esfregaços conjuntivais (STRAUSS-AYALI et al., 2004; LEITE et al., 2010; DE ALMEIDA FERREIRA et al., 2012; LOMBARDO et al., 2012). Utilizando-se esfregaços conjuntivais, Strauss-Ayali et al. (2004) conseguiram detectar pela técnica da cPCR 92% dos animais sintomáticos avaliados no estudo. Lombardo et al. (2012) pela técnica de qPCR em esfregaços conjuntivais, obtiveram resultados similares àqueles obtidos com amostras mais invasivas como aspirados de linfonodo. Leite et al. (2010) também conseguiram detectar o DNA do parasito a partir de esfregaços conjuntivais de animais assintomáticos pela técnica da cPCR seguida por hibridização. Dentre outras amostras menos invasivas investigadas, outros tecidos vem sendo avaliados como amostras de urina investigadas por Solano-Gallego et al. (2007) utilizando a técnica de qPCR, quando os resultados descritos mostraram positividade somente em cães com lesão renal severa; e Naranjo et al. (2011) que demonstraram a presença do DNA de Leishmania sp. pela qPCR em glândulas lacrimais de cães infectados, obtendo correlação entre positividade e presença de lesão ocular. Atualmente, o custo da qPCR, quanto aos reagentes e equipamentos utilizados ainda constitui um entrave para seu uso rotineiro. No entanto, esse método apresenta vantagens objetivas sobre a cPCR (MOREIRA et al., 2007; MAIA et al., 2009). Particularmente devido à possibilidade de quantificação do DNA alvo, permitindo monitorar a carga parasitária do animal no decorrer de infecção experimental, ou após tratamento, em países onde este é permitido (PENNISI et al., 2005; MAIA e CAMPINO, 2008; MANNA et al., 2008a; MARTINEZ et al., 2011). Também na qPCR há redução da probabilidade de contaminação e rapidez da reação, uma vez que não é realizada eletroforese para visualização dos resultados (ROLÃO et al., 2004; PAIVA-CAVALCANTI, REGIS-DA-SILVA e GOMES, 2010). A sensibilidade das técnicas de PCR para detecção de Leishmania sp. depende de vários fatores: as condições físico-químicas da reação, a concentração e a natureza do DNA da amostra e os iniciadores e sondas empregados para detecção da região alvo (BASTIEN, PROCOP e REISCHL, 2008). As regiões alvo mais frequentemente empregadas para detecção do DNA de Leishmania sp. tendem a ser altamente conservadas e repetitivas como o gene da subunidade do RNA

28

ribossomal (rRNA) ou o DNA do minicírculo do cinetoplasto (kDNA) (SUNDAR e RAI, 2002; ANTINORI et al., 2007; BASTIEN, PROCOP e REISCHL, 2008; MIRÓ et al., 2008). Nos últimos anos, diversos métodos de qPCR foram desenvolvidos para a detecção do parasito e o diagnóstico da LVC. No entanto, as metodologias empregadas variaram consideravelmente entre si, comprometendo a validade dos resultados obtidos, principalmente em relação à sensibilidade da técnica. Essas variações compreendem o emprego de diferentes alvos na reação, o uso de diferentes amostras biológicas, diferentes metodologias para o cálculo da carga parasitária dos tecidos de animais infectados (BASTIEN, PROCOP e REISCHL, 2008), além de muitas vezes empregar apenas animais sintomáticos para a análise do desempenho da técnica, superestimando assim sua performance. Adicionalmente, para evitar resultados falso-negativos é imprescindível a avaliação da qualidade do DNA extraído e usado nas reações de qPCR. A abordagem principal para resolver esta questão consiste em realizar uma PCR com o material empregado para detecção do DNA do parasito, para confirmar a integridade dos genes constitutivos no genoma do hospedeiro, assim como garantir a ausência de inibidores de amplificação. O melhor formato para tal análise é a realização da amplificação do DNA da Leishmania sp. e do gene constitutivo dentro da mesma reação (isto é, num formato multiplex). Vários genes domésticos, tais como GAPDH, RNase P e β-actina, já foram utilizados como controles internos para garantir a integridade do molde de DNA e prevenção de resultados falso-negativos melhorando assim a qualidade do resultado (ESPY et al., 2006; PIRON et al., 2007; PELEG et al., 2010; GONCALVES-DE-ALBUQUERQUE SDA et al., 2014). 1.4.3 Resposta imune Conforme comentado anteriormente, após a infecção, a evolução para a doença nos animais depende de alguns fatores, sendo, os mecanismos imunológicos desencadeados pelo hospedeiro após a infecção, um dos mais importantes (ALVAR et al., 2004; LIPOLDOVA e DEMANT, 2006; BANETH et al., 2008). O curso da infecção por L. infantum em cães está ligado à resposta imune do hospedeiro e à persistência e multiplicação do parasito. Os componentes da

29

imunidade inata e adaptativa envolvem uma gama de interações que é notavelmente diversificada e complexa (REIS et al., 2010). A resposta imune inata tem um papel relevante na proteção contra o parasito além de ter a capacidade de direcionar a resposta adaptativa, como mostrado por análise da caracterização das células mononucleares do sangue periférico por citometria de fluxo em animais experimentalmente infectados, capazes de controlar a infecção por Leishmania sp. sem desenvolver uma imunidade adaptativa específica (GUARGA et al., 2002; MORENO e ALVAR, 2002). Dados sugerem, que da mesma forma que ocorre na infecção humana, cães resistentes desenvolvem e controlam a LV por meio de mecanismos dependentes da resposta imune celular efetora, como foi explorado em diferentes estudos in vitro e in vivo (PINELLI et al., 1994; VOULDOUKIS et al., 1996; ALVAR et al., 2004); enquanto que, cães susceptíveis apresentam uma resposta imune humoral exacerbada, que é ineficaz no controle da infecção (REIS et al., 2006a). Essa ausência de resposta adequada das células T para controlar o parasito leva ao aparecimento de sinais clínicos, níveis elevados de anticorpos específicos anti-Leishmania e alta carga parasitária na pele, medula óssea, baço, fígado e linfonodos (REIS et al., 2006a; GIUNCHETTI et al., 2008; REIS et al., 2010). Estudos das subclasses de linfócitos presentes no sangue periférico de cães infectados mostraram uma significante redução de populações de células T CD4+ Leishmania-específicas e, consequentemente, da imunidade mediada por células nestes animais quando comparados com cães não infectados (MORENO et al., 1999). Além disso, a infectividade a flebótomos por xenodiagnóstico direto aumenta ao passo que ocorre uma redução dos níveis de células T CD4+, confirmando, deste modo, a importância destas células também para diminuição de parasitos circulantes na pele e, como consequência, na transmissibilidade da doença (GUARGA et al., 2000). Por outro lado, as células T CD8+, desempenham um importante papel no desenvolvimento

de

uma

resposta

imune

efetiva

contra

Leishmania

sp.,

possivelmente, devido a mecanismos citotóxicos durante a LVC. Observa-se que, números elevados de células T CD8+ no sangue periférico de cães assintomáticos estão associados a um baixo parasitismo na medula óssea desses animais (REIS et al., 2006a). Cães resistentes ao desenvolvimento da forma ativa de LVC, geralmente desenvolvem uma resposta imune celular do tipo helper 1 (TH1), com predominância

30

de produção de citocinas como IL-12, IFN-γ, IL-2 e fator de necrose tumoral (TNF-α) (PINELLI et al., 1994; PINELLI et al., 1995; PINELLI et al., 1999; CORREA et al., 2007; LAGE et al., 2007; REIS et al., 2009; MENEZES-SOUZA et al., 2011). Estas citocinas aumentam a eficiência das células fagocíticas e linfócitos citotóxicos, ativando uma resposta imune protetora, que leva a uma maior capacidade de eliminação do parasito (MANNA et al., 2006; CAVALCANTI et al., 2015). Já foi demonstrado que na transição do estado subclínico para aquele em que ocorre aparecimento de sinais, observa-se supressão da resposta proliferativa de linfócitos (MORENO et al., 1999; RHALEM et al., 1999). Desta forma, na ausência de resposta imune celular, pode ocorrer progressão para o desenvolvimento da doença (RHALEM et al., 1999; LEANDRO et al., 2001). Cães susceptíveis tendem a desenvolver uma resposta imune celular do tipo helper 2 (TH2), com predominância de citocinas como IL-4, IL-5, IL-10, IL-13 e TGF-β (PINELLI et al., 1994; PINELLI et al., 1995; PINELLI et al., 1999; CORREA et al., 2007; LAGE et al., 2007; REIS et al., 2009; MENEZES-SOUZA et al., 2011). Além disso, em animais infectados, o aumento do nível de anticorpos da classe IgG é observado e correlaciona-se com a presença de sintomatologia (KEENAN et al., 1984; RAFATI et al., 2005; REIS et al., 2006b). Há controvérsias na literatura relacionadas à̀ classe de anticorpos associada à suscetibilidade e resistência à doença. Alguns relatos apontam IgG1 como associada à suscetibilidade, enquanto outros observam um aumento de IgG2, no decorrer da infecção, independente do quadro clínico do animal (RAFATI et al., 2005; TRAVI et al., 2009). Até a presente data, existem poucos dados que correlacionem o desenvolvimento da infecção por L. infantum ou sua gravidade com a presença de quimiocinas específicas como CXCL1, CCL2, CXL10 em cães (MENEZES-SOUZA et al., 2011; MENEZES-SOUZA et al., 2012) (NASCIMENTO et al., 2013), desta forma são necessárias análises mais amplas, de múltiplos marcadores, visando identificar um perfil de resposta e correlaciona-lo com resistência ou susceptibilidade à infecção por L. infantum. 1.4.4 Resposta a saliva do vetor Fatores associados ao vetor podem ter reflexo sobre o curso da infecção nos hospedeiros, dentre estes, a exposição contínua à saliva do inseto pelas repetidas picadas pode influenciar o tipo de resposta do sistema imune (COURTENAY et al.,

31

2002; MORENO e ALVAR, 2002). Os aspectos envolvidos na resposta imune resultando em resistência ou suscetibilidade do hospedeiro dependem amplamente da primeira tentativa de resposta inata do hospedeiro para conter a infecção, que pode influenciar na predominância do tipo de padrão de resposta imune contra Leishmania sp. (PRATES et al., 2012). A constante exposição às picadas do vetor, resultando na produção de anticorpos anti-saliva foi demonstrada em diversas espécies de hospedeiro vertebrado (GOMES et al., 2002; COLLIN et al., 2009; AQUINO et al., 2010). Além disso, a exposição de hospedeiros mamíferos às proteínas salivares do flebotomíneos pode induzir imunidade celular (ANDRADE et al., 2007; VINHAS et al., 2007). Proteínas da saliva do vetor submetidas à sonicação e co-inoculadas em camundongos juntamente com promastigotas metacíclicas do parasito conferiram diminuição da gravidade do quadro clínico de LV (BELKAID et al., 1998). Outros estudos demonstraram o caráter imunogênico da saliva do vetor com estímulo da resposta Th1 e reação de hipersensibilidade tardia em ratos (GOMES et al., 2012) e camundongos (KAMHAWI, 2000). A exposição a proteínas da saliva do flebotomíneo Lu. longipalpis demonstrou um caráter imunogênico e conferiu proteção contra o desenvolvimento de quadro clínico da LV também em humanos. Este achado foi descrito em indivíduos residentes em área endêmica para LV, onde foi demonstrada uma correlação entre os níveis de anticorpos anti-saliva e o desenvolvimento de uma resposta de hipersensibilidade tardia contra o parasito (DTH+), sugerindo um perfil de resistência à infecção (GOMES et al., 2002). Estudos recentes identificaram duas proteínas presentes na saliva do Lu. longipalpis, LJM11 e LJM17, como marcadores específicos à exposição à saliva de Lu. longipalpis (SOUZA et al., 2010; TEIXEIRA et al., 2010). Em estudos experimentais com cães, demonstrou-se que as proteínas LJM11 e LJM17 da saliva do vetor também conferem um maior estímulo para resposta imune nestes animais (SOUZA et al., 2010; TEIXEIRA et al., 2010). Até a presente data, não existem dados que correlacionem o desenvolvimento da resposta imune anti-saliva do vetor com os achados clínicos e a gravidade da doença em cães.

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1.4.5 Biomarcadores de gravidade Um biomarcador é uma característica biológica objetivamente medida e avaliada como um indicador de processos biológicos ou patológicos normais, ou uma resposta a uma intervenção terapêutica (BIOMARKERS DEFINITIONS WORKING, 2001). Os biomarcadores podem ser usados para vários propósitos, dependendo da finalidade, do estudo e classificados como: de exposição, de efeito e de suscetibilidade, sendo ainda definidos como instrumentos que possibilitam identificar uma condição adversa antes que sejam evidenciados danos à saúde (AMORIM, 2003). A identificação de biomarcadores sensíveis e confiáveis de gravidade de doenças é importante para melhorar a qualidade do atendimento ao paciente, para permitir a estimativa mais correta do prognóstico e direcionar e otimizar medidas de tratamento e controle (ANDRADE et al., 2010). Na LVC existe a necessidade da avaliação de um conjunto de marcadores biológicos em estudos integrados para melhorar a compreensão do papel desses marcadores na classificação e predição do desfecho clínico dos cães. Embora não seja imperativo, um bom marcador deve ter uma estreita relação causal com a patogênese da doença (ANDRADE et al., 2010). O estado de resistência ou susceptibilidade à LVC reflete na gravidade da doença do animal, e não pode ser definido por qualquer parâmetro isolado da resposta imune celular ou humoral, sendo necessária uma análise conjunta. Evidências mostram que padrões de suscetibilidade e resistência (tipo de resposta imune apresentada) à infecção por L. infantum podem estar correlacionados ao quadro clínico e a carga parasitária do animal (CAVALCANTI et al., 2015). Adicionalmente, em cães naturalmente infectados com a presença de infecção ativa, definida pelo isolamento de parasitos por aspirado esplênico, existe associação entre altos níveis de anticorpos séricos contra o parasito e maior frequência de emagrecimento (DOS-SANTOS et al., 2008). Esta associação entre parasitismo esplênico e apresentações graves da LVC é intensificada pela ausência de resposta no teste cutâneo a antígenos de Leishmania sp. (TCL ou DTH) e pela desestruturação do tecido linfoide esplênico (SANTANA et al., 2008). Adicionalmente, existem evidências de correlação positiva entre sintomatologia manifestada por cães naturalmente infectados, níveis de citocinas produzidas e carga parasitária (MANNA et al., 2006). Não foi encontrada diferença

33

na produção de citocinas Th1 (como IFN-γ, IL-12 e TNF) no baço de cães com ou sem sinais clínicos (CORREA et al., 2007; LAGE et al., 2007), apesar de haver aumento dos níveis dessas citocinas durante o curso da infecção (LAGE et al., 2007). A predominância da produção de citocinas Th2 reguladoras (como IL-4, IL-10 e TGF-β1) determina o aumento da carga parasitária e a persistência do parasito, contudo sem associação com grupos clínicos de doença (leve, moderada ou grave) (LAGE et al., 2007; STRAUSS-AYALI, BANETH e JAFFE, 2007). No entanto, Correa et al. (2007) correlacionaram o aumento de citocinas Th2 reguladoras com a progressão da doença. Cavalcanti et al., (2015) descreveram uma diminuição na expressão de citocinas pro inflamatórias como IFN-γ, IL-12, TNF e IL-6 em cães infectados com carga parasitária elevada associada a uma modificação estrutural na micro-arquitetura do tecido linfoide esplênico. Mais recentemente, tem sido descrita a importância dos eicosanoides na regulação da resposta imune do hospedeiro na infecção por Leishmania sp. (DAUGSCHIES e JOACHIM, 2000; FRANCA-COSTA et al., 2015). Porém, existem poucos estudos em cães que avaliaram o comportamento desses marcadores inflamatórios no desenvolvimento e curso da LVC (VENTURIN et al., 2016). Até então, esse comportamento vem sendo analisado em seres humanos em resposta à infecção por Leishmania sp. nas formas cutâneas (ARAUJO-SANTOS et al., 2014; KHOURI et al., 2014). A avaliação da exposição à saliva do vetor pela detecção de anticorpos anti-proteínas salivares pode ser também empregada como um biomarcador em áreas endêmicas (TEIXEIRA et al., 2010). A detecção de anticorpos anti-saliva, além de determinar o grau de exposição ao vetor, parece também influenciar a resposta imune contra o parasito no hospedeiro (PRATES et al., 2012). O grau de exposição ao vetor em cães residentes em áreas endêmicas pode ser determinado através da detecção e determinação da cinética do desenvolvimento de anticorpos específicos anti-saliva do vetor. Contudo, no cão, a correlação entre o grau de exposição ao vetor e o desenvolvimento de resistência ou formas subclínica ou clínica da LV ainda não foi estudada.

34

2. JUSTIFICATIVA

Embora as medidas preventivas, como o uso de repelentes, possam vir a reduzir a prevalência de LVC, esta doença provavelmente continuará a causar preocupação a profissionais de saúde pública devido ao seu potencial zoonótico, assim como para os veterinários clínicos, principalmente pela falta de uma abordagem

diagnóstica

direta

e

de

um

tratamento

eficaz

(NOLI

e

SARIDOMICHELAKIS, 2014). As graves consequências do diagnóstico tardio ou da terapia ineficaz (nos países onde esta é permitida), implicam na manutenção do ciclo de transmissão do parasita, assim como na morte de animais doentes. A utilização de biomarcadores, para mensuração de processos biológicos e avaliação de sua relação com o desfecho clínico é importante para melhorar a abordagem do tratamento, além de promover a expansão do nosso arsenal terapêutico para diferentes doenças, e o aprofundamento de nossa compreensão quanto à fisiologia e patogênese destas enfermidades (STRIMBU e TAVEL, 2010). Assim, a busca e identificação de marcadores de gravidade pode não apenas ajudar a melhorar a compreensão da imunopatogênese da LV no hospedeiro mamífero, assim como direcionar medidas de diagnóstico, prevenção e controle nos animais. Citocinas circulantes, assim como outros mediadores inflamatórios podem ser empregados como biomarcadores para um diagnóstico precoce, gerador de prognóstico ou ainda como indicadores de falha terapêutica em doenças parasitárias (ANDRADE e BARRAL-NETTO, 2011). A produção de citocinas e quimiocinas que regulam o balanço entre uma resposta pro e anti-inflamatória apresentada pelos animais pode regular o espectro clínico da infecção por L. infantum. O espectro clínico pode ser correlacionado também com marcadores parasitológico como a intensidade da carga parasitária em cães infectados. Assim, a determinação da carga parasitária nos hospedeiros possibilita quantificar a intensidade da infecção, que pode ser relacionada com o grau de infectividade destes animais (BORJA et al., 2016), além de servir como uma importante ferramenta para monitorar a eficácia de tratamento, nos países onde este procedimento é permitido (ROURA et al., 2013). Nossa hipótese é que existem biomarcadores parasitológicos, imunológicos e inflamatórios capazes de predizer a gravidade da infecção por L. infantum em cães. Para explorar essa hipótese, objetivamos realizar uma análise exploratória para

35

avaliar e associar os achados clínicos, parasitológicos e imunológicos em cães naturalmente infectados para identificar dentre esses achados, biomarcadores preditivos quanto à gravidade da infecção por L. infantum. Para alcançar os objetivos propostos, o presente estudo foi realizado em duas etapas. Na primeira etapa, foi desenvolvida e validada uma ferramenta diagnóstica sensível e específica para quantificação da carga parasitária dos animais infectados (Capítulo 01). Na segunda etapa, foi realizado um estudo exploratório de uma amostra de cães coletada durante um estudo de corte transversal de uma área endêmica para LVC, visando avaliar diferentes possíveis biomarcadores e correlacioná-los com a gravidade da infecção por L. infantum apresentada pelos animais (Capítulo 02).

36

3. HIPÓTESE •

Existem biomarcadores parasitológicos, imunológicos e inflamatórios capazes de predizer a gravidade da infecção por L. infantum em cães.

4. OBJETIVOS 4.1 OBJETIVO GERAL •

Avaliar a carga parasitária como biomarcador parasitológico, as proteínas LJM11/LJM17 como biomarcadores de exposição à saliva do vetor, e identificar biomarcadores inflamatórios de gravidade da infecção por L. infantum em cães

4.2 OBJETIVOS ESPECÍFICOS •

Padronizar uma ferramenta de PCR quantitativa (qPCR) para detecção e quantificação da carga parasitária de L. infantum;



Correlacionar o biomarcador parasitológico (carga parasitária), determinada pela técnica de qPCR padronizada com o quadro clínico dos animais;



Correlacionar os biomarcadores de exposição à saliva do vetor e biomarcadores inflamatórios com o quadro clínico dos animais e a carga parasitária em um estudo de corte transversal.

37

CAPÍTULO 1 – Padronização de uma Ferramenta Diagnóstica de qPCR para avaliação da carga parasitária de L. infantum

A análise de carga parasitária dos animais pode ser considerada um biomarcador de gravidade, além de funcionar como um parâmetro para acompanhar o curso da infecção e a resposta a tratamento. Desta forma, nesta primeira etapa, um protocolo de qPCR para quantificar a carga parasitária foi desenvolvido e validado tendo se mostrado uma ferramenta diagnóstica para LVC, mais específica e acurada

quando

comparada

com

métodos

parasitológicos

e

sorológicos,

contribuindo para o alcance dos objetivos propostos na presente tese. A maioria dos cães expostos em áreas endêmicas, se torna infectada sem demonstrar sinais clínicos específicos e, frequentemente, com pouca ou nenhuma evidência sorológica, dificultando seu diagnóstico pelos testes sorológicos (CAMPINO et al., 2000; BANETH et al., 2008). Os testes sorológicos utilizados atualmente para identificação da infecção na população canina, podem apresentar baixa sensibilidade e especificidade especialmente em cães assintomáticos, recéminfectados, ou ainda em cães que apresentam um quadro clínico inespecífico (OLIVA et al., 2006; COURA-VITAL et al., 2011). Em área endêmica, é comum também à presença de cães com baixa carga parasitária, cujo diagnóstico resulta em resultados falso-negativos, principalmente quando são empregados testes parasitológicos para o diagnóstico (ALVAR et al., 2004; GOMES et al., 2008). Em conjunto, as limitações dos testes sorológicos e parasitológicos, apontam para a necessidade do emprego de técnicas mais sensíveis e específicas como as moleculares para a confirmação da infecção por Leishmania sp. em cães (GRIMALDI e TESH, 1993; REALE et al., 1999; SOLANO-GALLEGO et al., 2001; MIRÓ et al., 2008). A qPCR possibilita a identificação do DNA alvo do parasito, e sua quantificação, permitindo elaborar um diagnóstico de infecção assim como monitorar a carga parasitaria no animal durante o decorrer da infecção natural ou experimental ou, ainda, após o tratamento, em países onde este é permitido (PENNISI et al., 2005; MAIA e CAMPINO, 2008; MANNA et al., 2008b; MARTINEZ et al., 2011). A detecção de DNA de Leishmania sp. pode ser realizada em uma ampla variedade de

38

amostras clinicas (FISA et al., 2001; MANNA et al., 2004; FRANCINO et al., 2006; MAIA et al., 2009; GALLETTI et al., 2011). As amostras biológicas mais amplamente utilizadas para o diagnóstico molecular da infecção por Leishmania sp. são baço, medula óssea, linfonodos e pele (MIRÓ et al., 2008; MAIA et al., 2009; SOLCÀ et al., 2012). Todavia a literatura apresenta resultados divergentes e às vezes conflitantes em relação à sensibilidade na detecção do DNA de Leishmania sp. pela técnica de qPCR empregando-se diferentes tecidos. Desta forma, o primeiro capítulo da tese teve como objetivo padronizar uma técnica de qPCR para detecção e quantificação da infecção por L. infantum nos cães. Esse trabalho é uma continuidade do projeto desenvolvido anteriormente, no qual foi padronizada uma reação de qPCR e foi avaliado o desempenho diagnóstico de alguns tecidos (baço, medula óssea, sangue e linfonodo poplíteo) empregandose a cultura esplênica como padrão ouro (SOLCÀ, 2012). Contudo, os dados de acurácia obtidos foram subestimados uma vez que a técnica parasitológica empregada

como

padrão

ouro,

embora

altamente

específica,

carece

de

sensibilidade, prejudicando a análise de acurácia quando empregada como técnica padrão-ouro para avaliação do desempenho de outros métodos diagnósticos. Assim, no presente trabalho, a qPCR padronizada teve seu desempenho avaliado empregando a análise de classe latente. Várias técnicas diagnósticas foram empregadas para construção de uma variável latente que foi utilizada como padrãoouro na avaliação da acurácia da qPCR, comparando-a com outros testes diagnósticos e ao desempenho da qPCR. Adicionalmente, com o intuito de definir qual a melhor amostra para a detecção do DNA do parasito, foram empregados seis diferentes tecidos caninos para tal análise. Posteriormente, após a reação de qPCR ter sido validada, esta foi aprimorada para utilização em estudos epidemiológicos, sendo padronizada em formato duplex tanto na forma líquida como no formato em gel (ready-to-use), adicionando-se iniciadores específicos para a detecção de um gene constitutivo canino à reação padronizada anteriormente.

39

1.1. •

OBJETIVO GERAL Padronizar uma ferramenta de qPCR para detecção e quantificação da carga parasitária de L. infantum.

1.2. •

OBJETIVOS ESPECÍFICOS Avaliar a acurácia da qPCR padronizada para o diagnóstico da LVC empregando análise de classe latente;



Definir qual a amostra de tecido canino que fornece resultados mais acurados na detecção do DNA do parasito;



Adaptar a reação de qPCR padronizada para o formato duplex visando à detecção simultânea de um gene constitutivo canino e da carga parasitária de L. infantum;



Tornar o protocolo de qPCR duplex padronizado para o uso como um produto point of care na rotina diagnóstica de LVC.

40

1.3.

Artigo científico publicado na revista Plos One

Manuela da Silva Solcà, Leila Andrade Bastos, Carlos Eduardo Sampaio Guedes, Marcelo Bordoni, Lairton Souza Borja, Daniela Farias Larangeira, Pétala Gardênia da Silva Estrela Tuy, Leila Denise Alves Ferreira Amorim, Eliane Gomes Nascimento, Geraldo Gileno de Sá Oliveira, Washington Luis Conrado dos-Santos, Deborah Bittencourt Mothé Fraga, Patrícia Sampaio Tavares Veras. Evaluating the Accuracy of Molecular Diagnostic Testing for Canine Visceral Leishmaniasis Using Latent Class Analysis. 2014.

Este estudo foi realizado para avaliar a acurácia da técnica de qPCR e determinar qual o tipo de tecido fornece a maior taxa de detecção do DNA do parasito. A análise de classe latente foi realizada para avaliar a precisão de diferentes métodos diagnósticos e amostras biológicas avaliadas por qPCR em 51 cães de uma área endêmica. Usando a variável latente como padrão ouro, a qPCR alcançou uma sensibilidade de 95,8% (IC 90,4-100) empregando-se amostras de aspirado esplênico; 79,2% (IC 68-90,3) nos linfonodos; 77,3% (IC 64,5-90,1) na pele e 75% (IC 63,1-86,9) no sangue. A precisão da qPCR utilizando aspirados esplênicos no diagnóstico da LVC foi ainda avaliada utilizando uma amostra aleatória de 800 cães de um estudo de prevalência. A especificidade da qPCR empregando-se os 800 aspirados esplênicos foi de 76,7% (IC 73,7-79,6) e a sensibilidade foi de 95,0% (IC 93,5-96,5). A sensibilidade para a qPCR de aspirado esplênico foi maior do que as obtidas para os outros testes diagnósticos e foi semelhante à observada no estudo inicial com 51 animais. Estes dados reforçam que a qPCR é a técnica mais sensível para o diagnóstico de LVC, e o aspirado esplênico é a amostra biológica mais sensível para detectar a infecção por L. infantum. Adicionalmente, foi demonstrado que a análise de classe latente pode ser utilizada para gerar uma variável que funciona como padrão-ouro adequado para avaliação de técnicas diagnósticas de LVC.

Evaluating the Accuracy of Molecular Diagnostic Testing for Canine Visceral Leishmaniasis Using Latent Class Analysis Manuela da Silva Solca`1, Leila Andrade Bastos1, Carlos Eduardo Sampaio Guedes1, Marcelo Bordoni1, Lairton Souza Borja1, Daniela Farias Larangeira1,2, Pe´tala Gardeˆnia da Silva Estrela Tuy3, Leila Denise Alves Ferreira Amorim3, Eliane Gomes Nascimento4, Geraldo Gileno de Sa´ Oliveira1,5, Washington Luis Conrado dos-Santos1, Deborah Bittencourt Mothe´ Fraga1,2,5, Patrı´cia Sampaio Tavares Veras1,5* 1 Laborato´rio de Patologia e Biointervenc¸a˜o, Centro de Pesquisa Gonc¸alo Moniz–Fundac¸a˜o Oswaldo Cruz, Salvador, Bahia, Brazil, 2 Escola de Medicina Veterina´ria, Universidade Federal da Bahia, Salvador, Bahia, Brazil, 3 Instituto de Matema´tica –Departamento de Estatı´stica, Universidade Federal da Bahia, Salvador, Bahia, Brazil, 4 Centro de Refereˆncia em Doenc¸as Endeˆmicas Piraja´ da Silva (PIEJ), Jequie´, Bahia, Brazil, 5 Instituto Nacional de Cieˆncia e Tecnologia em Doenc¸as Tropicais (INCT - DT), Salvador, Bahia, Brazil

Abstract Host tissues affected by Leishmania infantum have differing degrees of parasitism. Previously, the use of different biological tissues to detect L. infantum DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4–100) in splenic aspirate; 79.2% (CI 68–90.3) in lymph nodes; 77.3% (CI 64.5–90.1) in skin; 75% (CI 63.1–86.9) in blood; 50% (CI 30–70) in bone marrow; 37.5% (CI 24.2– 50.8) in left-eye; and 29.2% (CI 16.7–41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7–79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5–96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting L. infantum infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing. Citation: Solca` MdS, Bastos LA, Guedes CES, Bordoni M, Borja LS, et al. (2014) Evaluating the Accuracy of Molecular Diagnostic Testing for Canine Visceral Leishmaniasis Using Latent Class Analysis. PLoS ONE 9(7): e103635. doi:10.1371/journal.pone.0103635 Editor: Yara M. Traub-Cseko¨, Instituto Oswaldo Cruz, Fiocruz, Brazil Received February 4, 2014; Accepted June 27, 2014; Published July 30, 2014 Copyright: ! 2014 Solca` et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants and fellowships from INCT (Instituto Nacional de Cieˆncia e Tecnologia em Doenc¸as Tropicais - http://inctdt.cebio. org - Grant number: 576269/2008-5) and PPUS - FAPESB (Programa de Pesquisa para o Sistema U´nico de Sau´de - Fundac¸a˜o de Amparo a Pesquisa no Estado da Bahia - http://www.fapesb.ba.gov.br - Grant number: SUS0011/2010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected]

Introduction

onychogryphosis, footpad swelling, localized or generalized alopecia, skin ulcers, and nasal or periocular dermatitis. They can also present with pathological alterations such as anemia or hepatic and renal failure [10,11]. Canine visceral leishmaniasis (CVL) can be diagnosed using parasitological, serological, or molecular methods in conjunction with clinical and epidemiological parameters [12]. Serological tests to diagnose CVL are the most common procedures used worldwide [13], however they lack sensitivity and specificity, which makes diagnosing the disease difficult when animals present with low antibody titers or there is cross-reactivity [14–17]. Hence, additional tests could be advantageous for confirming the diagnosis of inconclusive cases. For use as a confirmatory test,

Visceral leishmaniasis (VL) is a disease with both medical and veterinary importance that is endemic in Brazil, and in many other countries throughout Latin America, Asia, and Europe [1]. One of the etiological agents of VL is Leishmania infantum (syn. Leishmania chagasi), which is transmitted to vertebrate hosts through the bites of female sand flies [2–5]. Dogs are considered the main domestic reservoir for this parasite because of their high rates of infection and the high frequency of parasites found in their skin [6–9]. Once infected with L. infantum, dogs have clinical manifestations that range from asymptomatic to systemic, including weight loss or cachexia; hypertrophy of the lymph nodes; and changes to the skin such as

PLOS ONE | www.plosone.org

1

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Latent Class Analysis for Accuracy Evaluation of Leishmania qPCR

the molecular detection of Leishmania spp. provides greater sensitivity and specificity than other diagnostic techniques [8,18]. Numerous studies have described highly sensitive detection of low parasitic loads using quantitative real-time PCR (qPCR) [19– 21]. qPCR has also been used to monitor the tissue parasitic load in dogs following anti-Leishmania treatment in countries where this procedure is unrestricted [22,23]. Several invasive, and non-invasive, techniques have been used to obtain biological tissue samples to diagnose Leishmania infection using conventional PCR and qPCR. The biological samples most widely used for molecular diagnosis of Leishmania spp. infection in dogs are the spleen, bone marrow, lymph node, and skin [12,18,24]. However, molecular diagnostic tests in studies using these tissue types have produced variable, and sometimes conflicting results, for identifying Leishmania-infected dogs [19,25,26]. This might be because culturing the parasite, which has been used as the gold standard assay [27,28], has a low sensitivity threshold for detecting dogs with a low parasite burden [29,30], which compromises the accuracy evaluation of diagnostic testing. Therefore, the authors hypothesized that the lack of a reliable gold standard assay could account for the varying accuracy of the molecular diagnostic tests for Leishmania infection in different tissues. Latent class analysis (LCA) appraises tests with imperfect reference standards [31–33] using a statistical model to construct the latent class variable. Recently, LCA has been used to accurately evaluate the results of serological tests for diagnosing CVL [34]. The aim of the present study was to determine which type of canine tissue sample in an area with endemic VL provided the highest rate of Leishmania DNA detection by qPCR. In addition, qPCR results were compared to parasitological and serological diagnostic tests to determine which test provided the most accurate diagnosis of L. infantum infection.

3. Tissue Sampling Tissue samples were obtained during necropsies as previously described by Lima etal. (2014). Briefly, the dogs were anesthetized and then euthanized by intracardiac injection of a supersaturated solution of potassium chloride (2 mL/kg). Immediately before the lethal injection, 50 mL of blood were collected by intracardiac puncture. Blood samples were preserved in EDTA-2Na tubes (Greiner bio-one, Kremsmu¨nster, Austria) and in blood collection tubes (BD Vacutainer; Becton, Dickinson and Co). During the necropsy, splenic aspirate samples were collected by puncturing the central region of the spleen and bone marrow samples were obtained by puncturing the wing of the ilium, approaching from the dorsal crest. Conjunctival swabs of the right and left eyes were taken by rubbing the swab multiple times against the surface of the lower eyelid. A small fragment of the popliteal lymph node was cut from the whole organ and a skin fragment was collected using a sterile 5 mm punch (Kolplast, Brazil) from the medial portion of the pinna. Tissue samples were collected using sterile needles, swabs, and blades and all of the samples were stored in DNAaseand RNAase-free tubes at 270uC until DNA extraction.

4. Hematological and Biochemical Parameters Hematological and biochemical parameters were evaluated on the day of the necropsy. Total red blood cell and white blood cell counts were determined using an automated cell counter (Pentra 80 counter, ABX Diagnostics, Montpellier, France). Microhematocrit tubes containing blood samples were centrifuged at 12,000 rpm for 5 min, and then the hematocrit levels were estimated. Serum was collected by centrifuging the Vacutainer tubes, and was used for the biochemical tests including total protein, globulin, albumin, blood urea nitrogen, and creatinine, using an enzymatic colorimetric method with an A15 autoanalyzer (BioSystems, Barcelona, Spain).

5. Serological and Parasitological Tests Materials and Methods

The following serological tests were performed to detect antiLeishmania antibodies: the DPP CVL rapid test which detects rk28-specific antibodies and the EIE CVL with crude L. major antigen diagnostic test provided by FIOCRUZ (Bio-Manguinhos Unit, Rio de Janeiro, Brazil). These serum tests were performed in accordance with manufacturer instructions. An in-house ELISA, with crude L. infantum antigen was also performed as previously described [39,40]. Parasitological evaluation was performed by culturing part of the splenic aspirate collected during necropsy in Novy–MacNeal–Nicolle (NNN) biphasic medium supplemented with 20% Fetal Bovine Serum (FBS – Gibco BRL, New York, USA) and 100 mg/mL gentamicin to avoid contamination (Sigma Chemical Co., St. Louis, MO) for four weeks at 24uC [41]. Parasites were detected using microscopy performed at weekly intervals for no less than four weeks. Each splenic culture was prepared in duplicate. All of the culture labels were doublechecked to avoid misidentification. Parasite isolates were randomly selected from five dogs and sent to the national reference laboratory for Leishmania typing at the Oswaldo Cruz Institute (CLIOC, Rio de Janeiro, RJ, Brazil). The isolates were typed using monoclonal antibodies and enzyme electrophoresis analysis in order to determine the Leishmania species.

1. Ethics Statement Experimental procedures involving dogs were performed in accordance with Brazilian Federal Law on Animal Experimentation (Law no. 11794), the guidelines for animal research established by the Oswaldo Cruz Foundation [35], and the Brazilian Ministry of Health Manual for the Surveillance and Control of VL [36]. The CPqGM - FIOCRUZ Institutional Review Board for Animal Experimentation approved protocols for both animal euthanasia and sample collection procedures (Permit Number: 015/2009; Permit Number 017/2010).

2. Dogs As previously described by Lima etal. (2014), over a one week period in July 2010, 51 stray dogs were taken from the streets of Jequie´, a municipality located in the State of Bahia, Brazil, which is an area endemic for CVL. These dogs were selected as part of a surveillance and control program for VL that our group conducted in collaboration with the Endemic Diseases Surveillance Program of the State Health Service [37]. A CVL diagnosis was established based on the presence or absence of the following clinical signs: emaciation, alopecia, anemia, conjunctivitis, dehydration, dermatitis, erosion, ulcerations, lymphadenopathy, and onychogryphosis as previously detailed by Lima etal. (2014). Dogs from Jequie´ were clinically classified as having mild (stage I), moderate (stage II), and severe CVL (stage III) according to Solano-Gallego etal. (2009) [38]. PLOS ONE | www.plosone.org

6. Control Samples Splenic aspirate samples from 20 dogs that had previously been identified as Leishmania-positive from an endemic area [18] were used as positive controls. Splenic aspirates of 20 healthy dogs from 2

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the municipality of Pelotas, Rio Grande do Sul, Brazil, an area without endemic CVL, were used as negative controls. All of the healthy dogs had no clinical signs of CVL, and tested negative for infection using the in-house ELISA, parasite culturing, and qPCR techniques.

of Universal Mastermix (Life Technology Corporation, Carlsbad, CA-USA), the forward primer 59-AACTTTTCTGGTCCTCCGGGTAG-39 (LEISH-1) and the reverse primer 59-ACCCCCAGTTTCCCGCC-39 (LEISH-2) both at a final concentration of 900 nM, and a fluorogenic probe 59-AAAAATGGGTGCAGAAAT-39 with a FAM reporter molecule attached to the 59 end and an MGB-NFQ quencher (200 nM final concentration) linked to the 39-end (Life Technology Corporation). In order to overcome limitations caused by endogenous PCR inhibitors in the blood, skin fragment, and conjunctival swab samples, all of the steps leading up to DNA amplification were performed in the presence of bovine serum albumin (5 mg/each reaction) (Sigma Chemical) [44]. 9.3 Quantification of Leishmania kDNA. Quantification of Leishmania kDNA was performed using an absolute method based on comparing the cycle threshold (Ct) values from the samples to a standard curve, which was constructed using serial 10-fold dilutions from 105 to 1021 parasites performed in triplicate. Reactions were performed using the Applied Biosystems 7500 Fast Real-Time PCR System (Life Technology Corporation). The reaction was carried out under the following conditions: 1 cycle at 50uC for 2 min, 1 cycle at 95uC for 10 min, and 40 two-step cycles, first at 95uC for 15 s and then at 60uC for 1 min. In order to minimize variability between plates, the values from each plate were normalized using a common fluorescence detection baseline. Each sample’s Ct value was calculated by determining the point at which its fluorescence signal was above the established detection baseline. The Ct cut-off value was determined using a ReceiverOperator Characteristic (ROC) curve. The optimal Ct cut-off value for the parasite kDNA qPCR assay was determined by calculating sensitivity and specificity for different Ct cut-off points and the ROC curve derived from the amplification values of Leishmania-negative samples and Leishmania-positive samples (see item 6). Tissue samples were considered positive when the Ct values were equal to or less than the Ct cut-off point determined using the ROC curve analysis. If the standard deviation between triplicates was .0.38, the sample set was reanalyzed by qPCR [45]. The efficiency of the qPCR protocol was evaluated by calculating the slope value of the standard curve for the parasite kDNA. This value, 23.657 (SD = 0.148), was obtained from the mean slope values of nine independent experiments with a correlation coefficient (R2) of 0.998.

7. Sample Handling and Decontamination Procedures Due to the high degree of sensitivity inherent in qPCR, exceptional care was taken to avoid cross-contamination during not only the sample collection procedures, but also during DNA extraction and qPCR testing. As previously described [18], all procedures were carried out in an environment that was suitable for sample collection and qPCR procedures. All of the disposable surgical materials were used for a single animal, and the laminar flow hood was decontaminated by UV radiation before each procedure. Filter tips were routinely used throughout all DNA extraction steps and when performing the qPCR [42].

8. DNA Extraction

DNA was obtained from 200 mL of splenic and bone marrow aspirate, 200 mL of blood, 20 mg of lymph node, and 20 mg of a skin fragment using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocols. DNA samples from the conjunctival swabs were purified using a phenol–chloroform method as previously described [42]. The DNA pellets were suspended in 30 mL of Tris–EDTA buffer (10 mmol/L Tris and 1 mmol/L EDTA, pH 8.0). Once extracted, the quality and concentration of each DNA sample were evaluated using a digital spectrophotometer (NanoDrop ND-1000, Thermo Scientific, Wilmington, USA) [43]. All of the DNA samples were adjusted to a final concentration of 30 ng/mL, aliquoted, and kept at 220uC until the qPCR assays were performed. Parasite DNA was extracted from L. infantum (MHOM/ BR2000/MERIVALDO), Leishmania amazonensis (MHOM/ Br88/Ba-125), Leishmania braziliensis (MHOM/BR/94/ H3456), and Leishmania major (MHOM/RI//WR-173) promastigotes cultivated at 24uC. For the DNA extraction, the parasites were counted and centrifuged. DNA was extracted from pellets corresponding to a known number of parasites in accordance with the Qiagen protocols.

9.4 Assessment of qPCR Analytical Sensitivity and Specificity. Analytical sensitivity was evaluated by determining

9. Quantitative PCR (qPCR) 9.1 Inclusion and exclusion criteria.

To assess positivity, DNA samples were only included in the analysis if they met the minimum quality criteria: i) the DNA sample concentration was above 30ng/ml; ii) DNA samples amplified with the same efficiency as the DNA curve; and iii) amplification of the 18s rRNA housekeeping gene was successful. Any samples that did not fulfill one or more of the above inclusion criteria were excluded, only 10 out of 51 for skin fragments and 26 out of 51 for bone marrow aspirate. To compare parasitic load in different tissue types, DNA samples were only included in the analysis if they met the minimum quality criteria for all tissue types (samples from 20 dogs out of 51). 9.2 Quantitative PCR Assay. qPCR was used to determine the amount of parasite DNA in canine tissue samples. qPCR assays were performed following an amplification protocol previously described by Francino etal. (2006). The qPCR technique targeted a conserved region of L. infantum kDNA to obtain a 120-bp amplicon. All of the reactions were performed in triplicate. The reaction was in a final volume of 25 mL containing: 5 mL (150 ng) of each DNA sample diluted in deionized water and 20 mL of the PCR mixture. The PCR mixture contained: 12.5 mL PLOS ONE | www.plosone.org

whether the presence of host tissue interferes with the amplification profiles when using qPCR to detect L. infantum DNA in infected dogs. First, a standard curve was constructed using tenfold dilutions from reference strain L. infantum DNA (see item 9.3). Next, a ten-fold dilutions of reference strain L. infantum DNA was mixed with the splenic aspirate DNA from negative control animals (see item 6) and another standard curve was constructed from these dilutions. Finally, the amplification profiles of the two curves were compared. The analytical specificity of the qPCR analysis was assessed by comparing the amplification profiles of DNA samples from the L. infantum reference strain to profiles from several other Leishmania species, including the New World L. amazonensis and L. braziliensis, and the Old World L. major. As described in item 9.3, standard curves for each species were constructed from ten-fold serial dilutions ranging from 105 to 1021 parasites performed in triplicate. Analytical specificity was further assessed by evaluating the amplification profiles of DNA obtained from other canine pathogens, such as Ehrlichia canis and Babesia canis. Briefly, 150 ng of DNA from each pathogen was amplified and compared to the L. infantum amplification profile. 3

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ranges: 0 (no clinical signs), 1–3, 4–6, and .6 clinical signs. Fisher’s exact test was used to evaluate the association between the number of clinical signs and the splenic parasitic load ranges. LCA was performed using a statistical model to define a latent variable that could be used as a gold standard. To define a latent variable that could accurately identify L. infantum infection, three indicators representing serologic (DPP CVL), parasitological (culture from splenic samples), and molecular (splenic aspirate qPCR) diagnostic techniques were included. Animals were grouped into two categories, ‘infected dogs’, and ‘not-infected dogs’. The latent classes were estimated and characterized using two parameters: (a) item-response probabilities and (b) class prevalence, which is the probability of belonging to a latent class according to the response pattern. The estimate was performed using the maximum likelihood with expectation-maximization (EM) algorithm. The goodness of fit of the statistical model was evaluated using entropy, which varied between 0 and 1, with the value 1 indicating that the individuals are perfectly classified into the latent classes. Average probabilities for each latent class, which expresses the uncertainty of global classification, were also assessed a posteriori, considering a higher a posteriori probability to be a better goodness of fit for the statistical model. The Vuong-LoMendell-Rubin likelihood ratio test was used to choose the number of classes in LCA [48]. The Akaike information criterion (AIC) and Bayes information criterion (BIC) were also evaluated for each model. LCA was performed using the software Mplus 5.2, the syntax for fitting LCA in MPlus program is reported in Appendix S1 [49]. Additionally, the conditional independence was checked by evaluation of significant bivariate residuals [50,51]. The sensitivity and 95% confidence interval (CI) were calculated for each diagnostic technique and each tissue type analyzed, using the LCA latent variable as gold standard. The accuracy (sensitivity and specificity) of the qPCR technique using splenic aspirates was further evaluated with the LCA in a random sample of 800 dogs. Sensitivity of each test was measured as the proportion of positive results, only among those identified as such by the gold standard, while specificity was measured as the proportion of negative results, which were correctly identified as such by the gold standard.

9.5 Quantification of 18S rRNA Gene Expression. The expression of the canine housekeeping gene 18S rRNA was measured in order to normalize the concentration of input DNA for each sample and to obtain a reference amplification value to ensure the use of high-quality DNA samples [46]. TaqMan PreDeveloped Assay Reagents (Life Technology Corporation) were used to detect and quantify 18S rRNA gene expression. All of the reactions were performed at a final volume of 25 mL containing: 5 mL of DNA canine tissue sample diluted in deionized water and 20 mL of PCR mixture. The PCR mixture contained: 12.5 mL of Universal Mastermix (Life Technology Corporation), 1.25 mL of 18S GeneEx Assay primer and probe sets (Life Technology Corporation) at a concentration of 20x, and deionized water to obtain the final volume. The positive and negative controls for the housekeeping genes were plated in triplicate and the samples were plated in duplicate. Reactions were performed on an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technology Corporation) using the following protocol: 1 cycle at 50uC for 2 min; 1 cycle at 95uC for 10 min; and 40 two-step cycles, first at 95uC for 15 s and then 50uC for 1 min. A seven point standard curve was constructed for the housekeeping gene ranging from 450–18.75 ng. The slope of the standard curve for the 18s rRNA gene was 23.399 (SD = 0.296), which represents the mean slope value of 11 independent experiments with the corresponding coefficient of determination (R2) of 0.990. 9.6 Parasitic Load in DNA Samples. Samples from 20 of the 51 dogs were used to determine which tissue type harbored the highest parasitic load by comparing the splenic and bone marrow aspirates, blood, conjunctival swab of right and left eyes, lymph node and skin fragments. The parasitic load was expressed as the number of parasites normalized to the established reference amplification value for the 18S rRNA gene in 150 ng of DNA from each tissue sample [47]. Then the value obtained was calculated per 100 mg of host tissue DNA.

10. Evaluation of qPCR accuracy using splenic aspirate samples from a prevalence study The accuracy of the qPCR assay was evaluated using splenic samples obtained from 800 dogs during a random prevalence study performed in Camac¸ari, BA, an endemic area for CVL in Brazil. All 800 dogs were clinically evaluated and classified as described in item 2. They were also tested using the following CVL diagnostic methods: DPP CVL rapid test, EIE CVL, our inhouse ELISA, and parasite cultures from splenic aspirates as described in item 5. qPCR analysis of splenic aspirate samples was performed as described in item 9.

Results 1. Sample description All 51 dogs from the endemic area of Jequie´ were mixed-breed, their estimated ages varied from 1–10 years old, the animals weighed 5–30 kg, 45% (23/51) were males, and 55% (28/51) were females. All of the dogs exhibited clinical signs that could be related to CVL including splenomegaly (33/51), emaciation (17/ 51), hypertrophy of the lymph nodes (46/51), alopecia (21/51), cutaneous alterations (41/51), onychogryphosis (29/51), and ocular alterations (10/51). With respect to clinical pathology, 73% of the dogs presented with anemia (35/48), 98% with hypergammaglobulinemia (49/50), and 98% with hypoalbuminemia (49/50). Using the scale published by Solano-Gallego etal. (2009), all of the dogs were classified as having moderate CVL (stage II), except one animal that also exhibited a creatinine value greater than 1.4 mg/dL and was considered to have severe CVL (stage III).

11. Statistical Analysis In order to prevent bias, serological, parasitological and molecular techniques were performed and their results were judged without knowledge of the outcome of the other tests. The ROC curve data analysis described in item 9.3 was performed using GraphPad Prism software v.5.0 (GraphPad Prism Inc., San Diego, CA). Differences in the parasitic load between each type of biological sample were assessed using the Friedman test followed by the Dunn’s multiple comparison test. The relationship between parasitic load in the spleen and qPCR positivity in each infected tissue was assessed with the Spearman correlation test using log transformed values for the parasitic load (p,0.05). For the 800 dogs evaluated in the cross sectional study, the intensity of the parasitic load in the spleen (item 9.6) was categorized into three ranges: ,104, 104–106, and .106. The number of clinical signs in the dogs (item 2) was stratified into four PLOS ONE | www.plosone.org

2. Standardization of the qPCR Protocol The Ct cut-off value for parasite DNA detection was performed using a ROC analysis. This analysis showed an area under the curve of 1.0, indicating a high probability (p,0.001) that a randomly chosen positive sample would be correctly classified. 4

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24.1

100.0

75.9 6.7 65.8 0.0 2.0 Negative

4.2

93.3

12.2 86.8

34.2 100.0

81.5 45.8

95.8 98.0

64.7 Negative

Positive Splenic Aspirate qPCR

94.5

0.0 87.8 13.2 18.5 54.2 35.3 Positive Splenic Aspirate Culturing

5.5 82.9

17.1 83.4

16.6 0.0

Result Frequency (%) Not Infected n = 27 (52.9%)

100.0 0.0 52.9 Negative

Infected n = 120 (14.5%) Infected n = 24 (47.1%)

100.0 47.1 Positive

Latent Classes Dogs from Camac¸ari n = 800 Latent Classes

Conditional Probabilities (%)

DPP CVL

Latent class was used to provide a reliable estimate of sensitivity and specificity in order to select the tissue that provided the greatest accuracy for qPCR DNA detection. Serological, parasitological, and molecular techniques were used to determine prevalence of the latent classes and conditional probabilities in the LCA model for L. infantum infection in dogs. The probability that a dog from Jequie´ would be classified as infected using the LCA model was 47.1%. Among the animals considered infected by the LCA, the probability that a dog would test positive using qPCR of the splenic aspirate was 95.8%. The probability that a dog tested positive using either DPP CVL or by parasite culture from splenic aspirates was 100.0% or 54.2%, respectively (Table 1). Entropy was then calculated to assess how well the animals were classified a posteriori by the model. The entropy of the Jequie´ samples was 1.0; indicating accuracy in the classification of dogs using LCA. Moreover, a posteriori average probabilities that animals were properly classified into the latent classes ‘‘Infected’’ and ‘‘Not Infected’’ were 100% in both cases in the Jequie´ animals. The Lo-Mendel-Rubin test indicated that the model with 2 classes was a better fit for the data obtained from the Jequie´ dogs (p,0.01) when compared with the model with only 1 class (data not shown). These results are supported by the analysis of the AIC and BIC (data not shown). The sensitivity of the tests employed in Jequie´ to diagnose L. infantum infection was assessed employing the latent variable obtained by LCA as the gold standard (Figure 1). Splenic aspirates

Result Frequency (%)

4. Accuracy of the diagnostic tests

Result

Using qPCR, 100% of the dogs from Jequie´ (51/51) tested positive for parasite DNA in at least one of the tissue types analyzed. Among these, 98% (50/51) tested positive in the splenic aspirate samples; 80.4% (41/51) in blood samples; 68.3% (28/41) in skin fragments; 54.9% (28/51) in lymph node fragments; 35% (7/20) in bone marrow aspirate; 37.3% (19/51) in left eye conjunctival swabs, and 33.3% (17/51) in right eye conjunctival swabs. Parasites were observed in 35.3% (18/51) of the parasite cultures from splenic aspirate and anti-Leishmania antibodies were detected in 43.8% (21/48), 47.1% (24/51), and 66.7% (34/51) of the canine serum samples using the EIE CVL, DPP CVL rapid test, and in-house ELISA, respectively.

Technique

3. Positivity of diagnostic techniques

Dogs from Jequie´ n = 51

Table 1. Prevalence of latent classes and conditional probabilities to the LCA model for L. infantum infection detection in dogs.

Not Infected n = 680 (85.5%)

The Ct cut-off value of 37.0 had prediction rates of 100% sensitivity (CI 83.16–100) and 95% specificity (CI 75.13–99.87) with a likelihood ratio of 20. The analytical sensitivity was then determined. We found that the amplification profile of the reference strain L. infantum DNA was similar to that of the reference strain mixed with splenic aspirate DNA from negative control animals. The lower limit of detection was then determined and corresponded to 0.016 parasites per reaction. In terms of the analytical specificity, the Old World L. major parasite DNA samples were remarkably similar to those of L. infantum at all of the concentrations tested. In contrast, DNA from L. amazonensis and L. braziliensis could only be successfully amplified at concentrations of 104 and 105 parasites per reaction. This corresponded to the same number of cycles needed to amplify DNA from 0.02 parasites per reaction of the L. infantum reference strain (Figure S1). E. canis and B. canis DNA did not amplify using this qPCR protocol (data not shown). With respect to the housekeeping gene, attempts to amplify18S rRNA from DNA samples of Leishmania spp. resulted in no detectable qPCR amplification using the same primer set that successfully amplified the gene in canine DNA samples (data not shown).

Conditional Probabilities (%)

Latent Class Analysis for Accuracy Evaluation of Leishmania qPCR

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5. Parasitic load in different tissue types

provided the highest sensitivity of the available tissues sampled achieving 95.8% (95%CI 90.4–100) of sensitivity. The sensitivity attained in other tissues ranged from 80% to 30% as follows: lymph node fragments 79.2% (95%CI 68–90.3), skin fragments 77.3% (95%CI 64.5–90.1), blood 75% (95%CI 63.1–86.9), bone marrow aspirates 50% (95%CI 30–70), left eye swab 37.5% (95%CI 24.2–50.8), and right eye swab 29.2% (95%CI 16.7–41.6). It was not possible to calculate splenic qPCR specificity since only one sample tested negative in this method. Specificity of the other tissues achieved 66.7% for lymph node fragments (95%CI 53.7– 79.6) as well as for bone marrow aspirates (95%CI 47.8–85.6), 63% (95%CI 49.7–76.2) for right and left eye swabs, 42.1% (95%CI 27–57.2) for skin fragments and 14.8% (95%CI 5.1–24.6) for blood. Considering the other diagnostic tests, the sensitivity of the serological tests was 100% for the DPP CVL, followed by 79.2% (95%CI 68–90.3) for the in-house ELISA, 65.2% (95%CI 51.7–78.7) for EIE CVL, while sensitivity for the splenic aspirate culturing was 54.2% (95%CI 40.5–67.8). The specificity was highest for DPP CVL 100%, followed by splenic parasite cultures 81.5% (95%CI 70.8–92.1), EIE CVL 76% (95%CI 63.9–88.1), inhouse ELISA 44.4% (95%CI 30.8–58.1).

To further characterize tissue performance for the molecular diagnostic assay, parasitic loads were determined in the different tissues analyzed. As shown in Table 2 a considerable degree of variation was observed among the samples with values ranging from 120 parasites in a splenic aspirate sample up to 186 million parasites found in a bone marrow aspirate sample. However, the median parasitic load was higher in splenic aspirate samples than in the conjunctival swabs from either eye (p,0.05) or bone marrow aspirate (p,0.05). No statistically significant differences were observed when comparing parasitic loads in the splenic aspirate to the blood or skin tissue samples.

6. Distribution of parasitic load according to number of clinical signs The distribution of parasitic load according to the number of clinical signs is displayed in Table 3. We observed a significant positive association between the intensity of parasitic load in the spleen and the number of clinical signs present in the dogs. Animals with no clinical signs (p,0.01) or those exhibiting 1–3 clinical signs (p,0.001) had lower parasitic loads in splenic tissue

Figure 1. Sensitivity of the different diagnostic techniques employed in the biological samples obtained from Jequie´ animals (n = 51). Vertical bars represent the 95% confidence intervals. Sensitivity values were obtained using the latent variable as the gold standard. doi:10.1371/journal.pone.0103635.g001

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Table 2. Parasitic loads detected in different canine tissue types from a total of 20 dogs from the endemic area of Jequie´.

Tissue type

Positivity

Parasitic loadsa Minimum

25% Percentile

Median

75% Percentile

Maximum

Splenic Aspirate

100% (20/20)

120

1,088

4,365

14,325

74,000,000

Blood

70% (14/20)

0

0

7,960

19,800

228,000

Skin Fragment

60% (12/20)

0

0

1,870

21,500

32,400,000

Lymph node Fragment

60% (12/20)

0

0

830.5

9,288

7,800,000

Bone Marrow Aspirate

35% (07/20)

0

0

0.0*

28,275

186,000,000

Left Eye Swab

50% (10/20)

0

0

645.0*

2,073

240,000

Right Eye Swab

35% (07/20)

0

0

0.0*

3,141

147,000

a number of parasites normalized by the established reference amplification value for the housekeeping gene 18S rRNA in 100 mg of host tissue DNA. *p,0.05 Friedman’s together with Dunn’s multiple comparisons test of splenic aspirates and swab of right or left eye and splenic aspirates and bone marrow. doi:10.1371/journal.pone.0103635.t002

(,104). In contrast, animals with .6 clinical signs (p,0.01) showed relatively higher loads (.106). The dogs presenting with 4–6 clinical signs were homogeneously distributed throughout the three ranges.

The entropy of the Camac¸ari samples was 0.934, and the a posteriori average probabilities of being correctly classified as ‘‘Infected’’ and ‘‘Not Infected’’ were, respectively, 92.4% and 99.3%. Similar to the analysis performed with samples from Jequie´, using random samples, the Lo-Mendel-Rubin test indicated that the model with 2 classes was optimal and was supported by the analysis of the AIC and BIC (data not shown). Using LCA, the sensitivity of the splenic aspirate qPCR (95%; 95%CI 93.5–96.5) was higher than for the other diagnostic tests: DPP CVL (86.4%; 95%CI 84.1–88.8), splenic parasite cultures (83.5%; 95%CI 80.8–86.2), the in-house ELISA (78.3%; 95%CI 75.5–81.2), and EIE CVL (72.5%; 95% CI 69.4–75.6) (Figure 2A). However, the specificity was highest for splenic parasite cultures (100%), followed by DPP CVL (95.6%; 95%CI 94.2–97), the inhouse ELISA (90.6%; 95%CI 88.6–92.6), EIE CVL (84.1%; 95%CI 81.6–86.6), and splenic aspirate qPCR (76.7%; 95%CI 73.7–79.6) (Figure 2B).

7. Accuracy of qPCR using splenic aspirate samples from a prevalence study Splenic aspirate samples collected from a random study conducted in the endemic area of Camac¸ari were used to evaluate the high sensitivity observed for the qPCR technique developed using convenience sampling from Jequie´. Positive diagnoses in the samples from Camac¸ari varied according to diagnostic test. In this sample, 34.2% were positive using qPCR, 24.4% using EIE CVL, 19.8% using the in-house ELISA, and 16.6% using DPP CVL. Similar to the samples from Jequie´, LCA was used to analyze the results from the Camac¸ari samples. Reliability of the LCA model was evaluated and the probability of an animal being infected with L. infantum was calculated. The response patterns obtained from the latent class model that were used are listed in Table 4. Animals from Camac¸ari that had at least two positive test results were classified by the LCA model as ‘Infected’. However, the presence of a positive result from the splenic aspirate parasite culture implied a 100% probability of being infected with L. infantum, regardless of the DPP CVL and splenic aspirate qPCR results. When dogs from this endemic area tested negative by all three diagnostic techniques, the probability that the animal was infected with L. infantum was 0%. Furthermore, the probability of animals being infected was still very low when only splenic aspirate qPCR (2.7%) or DPP CVL (1.4%) tested positive according to this LCA model.

Discussion The present study found that a qPCR protocol targeting Leishmania kDNA provided the highest diagnostic sensitivity in dogs from Jequie´ when compared to standard serological and parasitological methods. In this endemic area, the DPP CVL rapid test and EIE CVL were able to detect infection in 47.1% and 43.8%, respectively, of a population of symptomatic dogs. Interestingly, 100% of these dogs tested positive with respect to at least one of the tissue types analyzed using qPCR. Similar results have been obtained by other studies, in which high sensitivity was achieved using molecular techniques [14,16,52]. Together these results reinforce the notion that the number of

Table 3. Distribution of parasitic load according to number of clinical signs in dogs from the prevalence study.

Number of Clinical Signs

Splenic Parasitic Load Ranges

Fisher Exact Test

,104

104–106

.106

0

8 (57.1%)

5 (35.7%)

1 (7.1%)

p,0.01

1–3

55 (42%)

49 (37.4%)

27 (20.6%)

p,0.001

4–6

37 (39.4%)

27 (28.7%)

30 (31.9%)

p = 0.11

.6

5 (16.1%)

9 (29.0%)

17 (54.8%)

p,0.01

Total

105

90

75

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Table 4. Response patternsa of Camac¸ari dogs for LCA model with 2 latent classes for diagnosis of CVL.

Response pattern DPP CVL

Splenic Aspirate Culturing

Splenic Aspirate qPCR

Frequency Observed % (n)

CVL Probability a posteriori (%)

Result Based on LCA

N

N

N

60.1 (429)

0.0

Not infected

N

N

P

20.5 (146)

1.4

Not infected

P

N

N

3.6 (26)

2.7

Not infected

N

P

N

0.1 (01)

100.0*

Infected

P

N

P

2.7 (19)

54.7

Infected

N

P

P

2.1 (15)

100.0

Infected

P

P

N

0.7 (05)

100.0

Infected

P

P

P

10.2 (73)

100.0

Infected

a Response patterns of all samples tested using the three techniques. *Estimation based on only one animal sample presenting this pattern. N: Negative; P: Positive. doi:10.1371/journal.pone.0103635.t004

parasite multiplication in naturally infected dogs [24,56]. Interestingly, following splenic aspirate samples, 80.4% of blood samples tested positive using qPCR. In addition, we found that the parasitic loads achieved were similar in the blood and splenic aspirate samples. These are promising results given that drawing blood is a much less invasive sampling technique to detect Leishmania infection in dogs than obtaining splenic aspirates. In contrast, several other studies have found that bone marrow and lymph node tissues offered a higher number of positive results than

infected dogs detected by serological surveys in endemic areas is severely underestimated [53,54]. Several methods have been recently developed for the molecular detection of Leishmania spp. [20,21,55], that provide divergent results when used in a variety of clinical canine samples [54]. Among the tissues analyzed, the authors observed that splenic aspirate samples provided the highest detection rate, successfully identifying 98% of the samples that tested positive. This result is supported by the fact that the spleen is a key site for

Figure 2. Sensitivity and specificity of the different diagnostic techniques employed in the biological samples obtained from Camac¸ari animals (n = 800). Vertical bars represent the 95% confidence intervals. A) Sensitivity and B) Specificity values obtained using the latent variable as the gold standard. doi:10.1371/journal.pone.0103635.g002

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were 95.8% and 100% respectively, in a population of symptomatic dogs in Jequie´. However, these results were limited since it was a small sample size. To address this, the results of the qPCR testing were evaluated using a larger random sampling of dogs that consisted of a population of positive and negative dogs, which are representative of the population of an endemic area for VL. In this random population survey using 800 dogs, the high sensitivity of splenic aspirate qPCR was confirmed achieving 95% of sensitivity, while the DPP CVL sensitivity was corrected to 83.5%. Despite the high sensitivity of the splenic aspirate qPCR, the specificity was relatively low (76.7%). This could be due to the large number of dogs from the randomly sampled population that tested positive only by splenic aspirate qPCR (20.5%) and were considered as ‘Not infected’ by the LCA. These animals were likely misclassified by LCA as false negatives, since the splenic aspirate qPCR is known to be the most sensitive diagnostic technique for CVL, most likely more sensitive than the variables used to define the variable latent class. Several studies have demonstrated a positive correlation between clinical manifestations of CVL and parasitic load in the spleen, lymph nodes and skin using several techniques [20,41,56,71]. Using qPCR of splenic aspirate in dogs, we also found a positive association between parasitic load and clinical manifestations of CVL, reinforcing the notion that can be used not only for detection of infection but also to monitor disease severity in dogs. Although splenic aspirate collection is considered an invasive procedure by many dog owners [27,72], Barrouin-Melo etal. (2006) noted that minor complications were observed in only three out of 257 dogs that underwent splenic aspiration. Complications can be further minimized by visualizing the spleen using an ultrasound device to guide splenic aspiration [72,73]. In our experience, during the prevalence study in the municipality of Camac¸ari, the splenic aspirate procedure assisted by ultrasonography was well tolerated in all 800 dogs without any reported complication. In conclusion, the authors found that, the splenic aspirates and blood, provided the greatest sensitivity for detecting Leishmania DNA using qPCR. In addition, the results indicated that LCA could be used to create a suitable gold standard for diagnosis, since this technique offers a more comprehensive evaluation of the results obtained using different diagnostic testing methods for CVL.

blood [46,55,57,58]. Francino etal. (2006) suggested that using qPCR to detect Leishmania parasites in blood samples might be sufficient to diagnose infection given the technique’s ability to quantify extremely low parasitemia. However, other authors consider the blood to be a poor source of Leishmania DNA [59], mostly because blood samples do not have satisfactory detection rates using conventional PCR. The underlying cause of these poor results may be the high frequency of PCR inhibitors found in blood, in addition to low parasitic loads, which could lead to false negatives especially in asymptomatic dogs [52]. Serum albumin can be added to avoid any potential inhibiting effects in qPCR reaction [44]. In the present study we added serum albumin to blood, skin, and conjunctival swab samples. Our results demonstrate that splenic aspirates or blood can be effectively used to detect parasite DNA using qPCR [18,19]. The analytical specificity of the qPCR technique was also evaluated in the present study by comparing the amplification profiles of L. infantum DNA to other Old and New World Leishmania species. The amplification profile of the Old World species L. major was remarkably similar to that of L. infantum (Figure S1). This corroborates other studies that have shown a great deal of similarity between the genomes of these species [60]. To the best of our knowledge, L. major is not known to be a causative agent of CVL, nor have any cases linked to this parasite been reported in Latin America [61]. kDNA from New World parasites, such as L. amazonensis and L. braziliensis, was successfully amplified using this protocol, but only at high concentrations of 104 and 105 parasites per reaction (Figure S1). Protocols capable of distinguishing between Leishmania species are preferable in endemics areas for both cutaneous and visceral forms of the disease [62]. In this study, five Leishmania species isolated from the dogs were identified by multilocus enzyme electrophoresis as L. infantum. Nonetheless, the use of splenic aspirate samples can avoid misleading diagnostic results since visceralization of L. braziliensis has not been reported and visceralization of L. amazonensis is a relatively rare event both in humans or dogs [62–64]. Regrettably, an ideal gold standard is still lacking for CVL diagnosis [65]. Historically, parasite culturing and immunofluorescence antibody test (IFAT) have been abundantly used. However, culturing is shown to have low sensitivity, while IFAT low specificity [65]. An alternative to using a single technique as the gold standard is to utilize LCA, once this method defines a latent variable to be used as gold standard, considering all diagnostic tests impartially. Indeed, LCA has been proved to successfully estimate the sensitivities and specificities of different diagnostic tests for several diseases [34,66–69]. LCA has been an useful tool for validating serological diagnostic methods for VL, since this analysis provides more realistic estimates of diagnostic test performance [34,67]. In the scientific community still exist concerns regarding the high sensitivity of qPCR results, especially when this technique is able to detect very low parasitic loads. In addition, some authors state that is impossible for qPCR to differentiate between the DNA of a living parasite and a dead one. Otherwise, Prina etal. (2007) [70] were the only ones that proved that as soon as 1 h after exposure to a substance able to kill the parasites, only less than 1% of the initial Leishmania DNA could be detected by qPCR. No other group demonstrated these results, especially using invivo experiments. Thus, in the present study, we have decided not to consider all the dogs as infected, even if they displayed parasite in at least one tissue by the qPCR, and perform the qPCR accuracy evaluation using the latent variable. Employing the latent class variable as the gold standard, we found that the sensitivity for splenic aspirate qPCR and DPP CVL PLOS ONE | www.plosone.org

Supporting Information Figure S1 Amplification profiles of DNA samples from Leishmania spp. A) L. infantum; B) L. major; C) L. amazonensis; D) L. braziliensis. DNA samples derived from the L. infantum reference strain, and several other Leishmania species, including New World L. amazonensis and L. braziliensis, and Old World L. major. Standard curves were constructed using amplification patterns from ten-fold serial dilutions performed in triplicate ranging from 105 to 1021 parasites per reaction. (TIF) Appendix S1 Syntax for fitting LCA in MPlus program.

(DOCX)

Acknowledgments The authors would like to thank Dr. Fla´via W. Cruz McBride for support to obtain negative control samples in Pelotas, Dr. Virgı´nia Maria G. da Silva and Joselli S. Silva for the help in the endemic area. Additionally, the authors acknowledge Kyoshi Fukutani for help in the qPCR analysis. Finally, we are grateful to Andris K. Walter for providing English revision

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and consulting services and manuscript edition by native English-speaking experts from BioMed Proofreading LLC.

MSS CESG PGSET LDAFA WLCS DBMF PSTV. Contributed reagents/materials/analysis tools: DFL GGSO EGN PGSET LDAFA. Wrote the paper: MSS CESG LDAFA DBMF PSTV.

Author Contributions Conceived and designed the experiments: MSS CESG DBMF PSTV. Performed the experiments: MSS LAB MB LSB DFL. Analyzed the data:

References 26. Lombardo G, Pennisi MG, Lupo T, Migliazzo A, Capri A, et al. (2012) Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques. Vet Parasitol 184: 10–17. 27. Carvalho D, Oliveira TMFS, Baldani CD, Machado RZ (2009) An enzymelinked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs. Pesquisa Veterina´ria Brasileira 29: 120–124. 28. Sundar S, Rai M (2002) Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol 9: 951–958. 29. Moreira MA, Luvizotto MC, Garcia JF, Corbett CE, Laurenti MD (2007) Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs. Vet Parasitol 145: 245–252. 30. Ndao M (2009) Diagnosis of parasitic diseases: old and new approaches. Interdiscip Perspect Infect Dis 2009: 278246. 31. Baughman AL, Bisgard KM, Cortese MM, Thompson WW, Sanden GN, et al. (2008) Utility of composite reference standards and latent class analysis in evaluating the clinical accuracy of diagnostic tests for pertussis. Clin Vaccine Immunol 15: 106–114. 32. Butler JC, Bosshardt SC, Phelan M, Moroney SM, Tondella ML, et al. (2003) Classical and latent class analysis evaluation of sputum polymerase chain reaction and urine antigen testing for diagnosis of pneumococcal pneumonia in adults. J Infect Dis 187: 1416–1423. 33. Nascimento MC, de Souza VA, Sumita LM, Freire W, Munoz F, et al. (2007) Comparative study of Kaposi’s sarcoma-associated herpesvirus serological assays using clinically and serologically defined reference standards and latent class analysis. J Clin Microbiol 45: 715–720. 34. Machado de Assis TS, Rabello A, Werneck GL (2012) Latent class analysis of diagnostic tests for visceral leishmaniasis in Brazil. Trop Med Int Health 17: 1202–1207. 35. Machado CJ, Filipecki AT, Teixeira MD, Klein HE (2010) Regulation of the use of animals in Brazil in the twentieth century and the process of forming the current regime applied to biomedical research. Histo´ria, Cieˆncias, Sau´deManguinhos 17: 87–105. 36. Brasil MdSd (2006) Manual de vigilancia e controle da leishmaniose visceral: Ministerio da Saude - Secretaria de Vigilancia em Saude. 37. Lima IS, Silva JS, Almeida VA, Junior FG, Souza PA, et al. (2014) Severe clinical presentation of visceral leishmaniasis in naturally infected dogs with disruption of the splenic white pulp. PLoS One 9: e87742. 38. Solano-Gallego L, Koutinas A, Miro G, Cardoso L, Pennisi MG, et al. (2009) Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol 165: 1–18. 39. Baleeiro CO, Paranhos-Silva M, dos Santos JC, Oliveira GG, Nascimento EG, et al. (2006) Montenegro’s skin reactions and antibodies against different Leishmania species in dogs from a visceral leishmaniosis endemic area. Vet Parasitol 139: 21–28. 40. Paranhos-Silva M, Freitas LA, Santos WC, Grimaldi GJ, Pontes-de-Carvalho LC, et al. (1996) A cross-sectional serodiagnostic survey of canine leishmaniasis due to Leishmania chagasi. Am J Trop Med Hyg 55: 39–44. 41. Barrouin-Melo SM, Larangeira DF, Trigo J, Aguiar PH, dos-Santos WL, et al. (2004) Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs. Mem Inst Oswaldo Cruz 99: 195–197. 42. Batista LF, Segatto M, Guedes CE, Sousa RS, Rodrigues CA, et al. (2012) An assessment of the genetic diversity of Leishmania infantum isolates from infected dogs in Brazil. Am J Trop Med Hyg 86: 799–806. 43. dos Santos Marques LH, Gomes LI, da Rocha IC, da Silva TA, Oliveira E, et al. (2012) Low parasite load estimated by qPCR in a cohort of children living in urban area endemic for visceral leishmaniasis in Brazil. PLoS Negl Trop Dis 6: e1955. 44. Giambernardi TA, Rodeck U, Klebe RJ (1998) Bovine serum albumin reverses inhibition of RT-PCR by melanin. Biotechniques 25: 564–566. 45. Naranjo C, Fondevila D, Altet L, Francino O, Rios J, et al. (2012) Evaluation of the presence of Leishmania spp. by real-time PCR in the lacrimal glands of dogs with leishmaniosis. Vet J 193: 168–173. 46. Solano-Gallego L, Rodriguez-Cortes A, Trotta M, Zampieron C, Razia L, et al. (2007) Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis. Vet Parasitol 147: 315–319. 47. Manna L, Reale S, Viola E, Vitale F, Foglia Manzillo V, et al. (2006) Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Vet Parasitol 142: 271–280. 48. Muthen B, Asparouhov T (2012) Bayesian structural equation modeling: a more flexible representation of substantive theory. Psychol Methods 17: 313–335.

1. Desjeux P (2004) Leishmaniasis. Nat Rev Microbiol 2: 692. 2. Killick-Kendrick R (1999) The biology and control of phlebotomine sand flies. Clin Dermatol 17: 279–289. 3. Kuhls K, Alam MZ, Cupolillo E, Ferreira GE, Mauricio IL, et al. (2011) Comparative microsatellite typing of new world Leishmania infantum reveals low heterogeneity among populations and its recent old world origin. PLoS Negl Trop Dis 5: e1155. 4. Lainson R, Shaw JJ (1978) Epidemiology and ecology of leishmaniasis in LatinAmerica. Nature 273: 595–600. 5. Mauricio IL, Stothard JR, Miles MA (2000) The strange case of Leishmania chagasi. Parasitol Today 16: 188–189. 6. Deane LM, Deane MP, Alencar JE (1955) [Control of Phlebotomus longipalpis by DDT house spraying endemic foci of kala-azar in Ceara]. Rev Bras Malariol Doencas Trop 7: 131–141. 7. Dye C (1996) The logic of visceral leishmaniasis control. Am J Trop Med Hyg 55: 125–130. 8. Gramiccia M, Gradoni L (2005) The current status of zoonotic leishmaniases and approaches to disease control. Int J Parasitol 35: 1169–1180. 9. Molina R, Amela C, Nieto J, San-Andres M, Gonzalez F, et al. (1994) Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus. Trans R Soc Trop Med Hyg 88: 491–493. 10. Ciaramella P, Oliva G, Luna RD, Gradoni L, Ambrosio R, et al. (1997) A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum. Vet Rec 141: 539–543. 11. Koutinas AF, Polizopoulou ZS, Saridomichelakis MN, Argyriadis D, Fytianou A, et al. (1999) Clinical considerations on canine visceral leishmaniasis in Greece: a retrospective study of 158 cases (1989–1996). J Am Anim Hosp Assoc 35: 376–383. 12. Miro G, Cardoso L, Pennisi MG, Oliva G, Baneth G (2008) Canine leishmaniosis–new concepts and insights on an expanding zoonosis: part two. Trends Parasitol 24: 371–377. 13. Gomes YM, Paiva Cavalcanti M, Lira RA, Abath FG, Alves LC (2008) Diagnosis of canine visceral leishmaniasis: biotechnological advances. Vet J 175: 45–52. 14. Coura-Vital W, Marques MJ, Veloso VM, Roatt BM, Aguiar-Soares RD, et al. (2011) Prevalence and factors associated with Leishmania infantum infection of dogs from an urban area of Brazil as identified by molecular methods. PLoS Negl Trop Dis 5: e1291. 15. Ferreira Ede C, de Lana M, Carneiro M, Reis AB, Paes DV, et al. (2007) Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations. Vet Parasitol 146: 235–241. 16. Solano-Gallego L, Morell P, Arboix M, Alberola J, Ferrer L (2001) Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology. J Clin Microbiol 39: 560– 563. 17. Troncarelli MZ, Camargo JB, Machado JG, Lucheis SB, Langoni H (2009) Leishmania spp. and/or Trypanosoma cruzi diagnosis in dogs from endemic and nonendemic areas for canine visceral leishmaniasis. Vet Parasitol 164: 118–123. 18. Solca Mda S, Guedes CE, Nascimento EG, Oliveira GG, dos Santos WL, et al. (2012) Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs. Vet Parasitol 184: 133–140. 19. Francino O, Altet L, Sanchez-Robert E, Rodriguez A, Solano-Gallego L, et al. (2006) Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis. Vet Parasitol 137: 214–221. 20. Manna L, Reale S, Vitale F, Gravino AE (2009) Evidence for a relationship between Leishmania load and clinical manifestations. Res Vet Sci 87: 76–78. 21. Mary C, Faraut F, Lascombe L, Dumon H (2004) Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol 42: 5249–5255. 22. Maia C, Campino L (2008) Methods for diagnosis of canine leishmaniasis and immune response to infection. Vet Parasitol 158: 274–287. 23. Martinez V, Quilez J, Sanchez A, Roura X, Francino O, et al. (2011) Canine leishmaniasis: the key points for qPCR result interpretation. Parasit Vectors 4: 57. 24. Maia C, Ramada J, Cristovao JM, Goncalves L, Campino L (2009) Diagnosis of canine leishmaniasis: conventional and molecular techniques using different tissues. Vet J 179: 142–144. 25. Ferreira Sde A, Ituassu LT, de Melo MN, de Andrade AS (2008) Evaluation of the conjunctival swab for canine visceral leishmaniasis diagnosis by PCRhybridization in Minas Gerais State, Brazil. Vet Parasitol 152: 257–263.

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Latent Class Analysis for Accuracy Evaluation of Leishmania qPCR

49. Muthen LK, Muthen BO (2007) Mplus - Statistical analysis with latent variable. Version 6. 50. Garrett ES, Zeger SL (2000) Latent class model diagnosis. Biometrics 56: 1055– 1067. 51. Uebersax J (2009) A Practical Guide to Conditional Dependence in Latent Class Models. John Uebersax Enterprises LLC. 52. Lachaud L, Chabbert E, Dubessay P, Dereure J, Lamothe J, et al. (2002) Value of two PCR methods for the diagnosis of canine visceral leishmaniasis and the detection of asymptomatic carriers. Parasitology 125: 197–207. 53. Alvar J, Canavate C, Molina R, Moreno J, Nieto J (2004) Canine leishmaniasis. Adv Parasitol 57: 1–88. 54. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L (2008) Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one. Trends Parasitol 24: 324–330. 55. Maia C, Nunes M, Cristovao J, Campino L (2010) Experimental canine leishmaniasis: clinical, parasitological and serological follow-up. Acta Trop 116: 193–199. 56. Reis AB, Martins-Filho OA, Teixeira-Carvalho A, Carvalho MG, Mayrink W, et al. (2006) Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis. Res Vet Sci 81: 68–75. 57. de Almeida Ferreira S, Leite RS, Ituassu LT, Almeida GG, Souza DM, et al. (2012) Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil. PLoS Negl Trop Dis 6: e1596. 58. Manna L, Reale S, Vitale F, Picillo E, Pavone LM, et al. (2008) Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol. Vet J 177: 279–282. 59. Reale S, Maxia L, Vitale F, Glorioso NS, Caracappa S, et al. (1999) Detection of Leishmania infantum in dogs by PCR with lymph node aspirates and blood. J Clin Microbiol 37: 2931–2935. 60. Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, et al. (2007) Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet 39: 839–847. 61. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, et al. (2012) Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7: e35671. 62. Madeira MF, Schubach A, Schubach TM, Pacheco RS, Oliveira FS, et al. (2006) Mixed infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil. Trans R Soc Trop Med Hyg 100: 442–445.

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63. Barral A, Pedral-Sampaio D, Grimaldi Junior G, Momen H, McMahon-Pratt D, et al. (1991) Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. Am J Trop Med Hyg 44: 536–546. 64. Tolezano JE, Uliana SR, Taniguchi HH, Araujo MF, Barbosa JA, et al. (2007) The first records of Leishmania (Leishmania) amazonensis in dogs (Canis familiaris) diagnosed clinically as having canine visceral leishmaniasis from Aracatuba County, Sao Paulo State, Brazil. Vet Parasitol 149: 280–284. 65. Rodriguez-Cortes A, Ojeda A, Francino O, Lopez-Fuertes L, Timon M, et al. (2010) Leishmania infection: laboratory diagnosing in the absence of a ‘‘gold standard’’. Am J Trop Med Hyg 82: 251–256. 66. Hartnack S, Budke CM, Craig PS, Jiamin Q, Boufana B, et al. (2013) Latentclass methods to evaluate diagnostics tests for Echinococcus infections in dogs. PLoS Negl Trop Dis 7: e2068. 67. Boelaert M, Rijal S, Regmi S, Singh R, Karki B, et al. (2004) A comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis. Am J Trop Med Hyg 70: 72–77. 68. Pan-ngum W, Blacksell SD, Lubell Y, Pukrittayakamee S, Bailey MS, et al. (2013) Estimating the true accuracy of diagnostic tests for dengue infection using bayesian latent class models. PLoS One 8: e50765. 69. Wu X, Berkow K, Frank DN, Li E, Gulati AS, et al. (2013) Comparative analysis of microbiome measurement platforms using latent variable structural equation modeling. BMC Bioinformatics 14: 79. 70. Prina E, Roux E, Mattei D, Milon G (2007) Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR. Microbes Infect 9: 1307–1315. 71. Sanchez MA, Diaz NL, Zerpa O, Negron E, Convit J, et al. (2004) Organspecific immunity in canine visceral leishmaniasis: analysis of symptomatic and asymptomatic dogs naturally infected with Leishmania chagasi. Am J Trop Med Hyg 70: 618–624. 72. Watson AT, Penninck D, Knoll JS, Keating JH, Sutherland-Smith J (2011) Safety and correlation of test results of combined ultrasound-guided fine-needle aspiration and needle core biopsy of the canine spleen. Vet Radiol Ultrasound 52: 317–322. 73. Barrouin-Melo SM, Larangeira DF, de Andrade Filho FA, Trigo J, Juliao FS, et al. (2006) Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniosis? A study on assymptomatic and polysymptomatic animals. Vet J 171: 331–339.

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Supporting Information Solcà et al., 2014

Figure S1. Amplification profiles of DNA samples from Leishmania spp. A) L. infantum; B) L. major; C) L. amazonensis; D) L. braziliensis. DNA samples derived from the L. infantum reference strain, and several other Leishmania species, including New World L. amazonensis and L. braziliensis, and Old World L. major. Standard curves were constructed using amplification patterns from ten-fold serial dilutions performed in triplicate ranging from 105 to 10−1 parasites per reaction. doi:10.1371/journal.pone.0103635.s001

Appendix S1. Syntax for fitting LCA in MPlus program. doi:10.1371/journal.pone.0103635.s002

APPENDIX

Mplus Commands for LCA TITLE: LCA with binary latent class indicators DATA: FILE IS "dogs.ascii"; VARIABLE: NAMES ARE id DPPCVL EIECVL ELISA culture spleen blood limph righte lefte skin bonemar; USEVARIABLES = DPPCVL culture spleen; CATEGORICAL= DPPCVL culture spleen; CLASSES = c (2); MISSING = .; AUXILIARY = id; ANALYSIS: TYPE = MIXTURE; STARTS = 100 10; STITERATIONS = 20; OUTPUT: TECH11 TECH14 TECH10; PLOT: TYPE IS plot3; SERIES IS DPPCVL (1) culture (2) spleen (3); SAVEDATA: FILE IS LCA_dogs.txt; SAVE IS cprob; FORMAT IS free;

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1.4.

Artigo científico submetido à revista Diagnostic Microbiology and Infectious Disease

Rita de Cássia Pontello Rampazzo*, Manuela da Silva Solcà*, Liliane Celestino Sales Santos, Lais de Novaes Pereira, José Carlos Oliveira Guedes Junior, Deborah Bittencourt Mothé Fraga, Patrícia Sampaio Tavares Veras, Marco Aurélio Krieger, Alexandre Dias Tavares Costa. Improvements on the detection of Leishmania spp. in naturally infected dogs by real time PCR: a duplex and ready-to-use reaction format. 2017.

O presente estudo teve como objetivo padronizar uma reação de qPCR duplex gelificada para a identificação de animais infectados por L. infantum. Este protocolo foi desenvolvido para detectar e quantificar simultaneamente uma sequência conservada do kDNA de L. infantum bem como uma sequência conservada do gene do rRNA canino. A detecção simultânea do gene 18S canino reforça a validade dos resultados da qPCR, principalmente, os resultados negativos por confirmar a presença do DNA genômico nas amostras caninas, excluindo a possibilidade de resultados falso negativos. Assim, quando ocorrer à amplificação do DNA do hospedeiro, associado à inexistência de amplificação do DNA de Leishmania pode-se concluir que há ausência de DNA do parasito. Ademais, a realização da qPCR em formato duplex, reduz o tempo de execução da técnica, combinando duas reações em uma, além de diminuir gastos com reagentes. O formato em gel (ready-to-use) permite que o usuário adicione apenas água e o DNA extraído das amostras, reduzindo ainda mais o tempo de execução, tornando-a mais fácil e evitando erros durante a manipulação de reagentes. Estas duas características são altamente importantes em um laboratório de diagnóstico de rotina podendo facilitar e acelerar o diagnóstico da LVC. A reação de qPCR em formato duplex foi avaliada em diferentes tecidos de cães com diferentes manifestações clínicas de LVC, provenientes de uma área endêmica. A sensibilidade da reação de qPCR em formato duplex foi testada em relação à sensibilidade da reação de qPCR singleplex, assim como, foi comparada à performance de detecção do DNA de L.

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infantum no formato líquido e gelificado da reação duplex. A padronização da qPCR em formato duplex gelificado garante especificidade e diminuição dos gastos com reagentes, sem perdas significativas de sensibilidade, que comprometam o diagnóstico de LVC.

Manuscript3 Click here to view linked References

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Click here to download Manuscript V2_artigo qPCR duplex_Set15.docx

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Improvements on the detection of Leishmania spp in naturally infected dogs by

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real time PCR: a duplex and ready-to-use reaction format

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Rita de Cássia Pontello Rampazzo1,2#, Manuela da Silva Solcà3#, Liliane

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Celestino Sales Santos4, Lais de Novaes Pereira4, José Carlos Oliveira Guedes

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Junior4, Patrícia Sampaio Tavares Veras3,4, Deborah Bittencourt Mothé

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Fraga3,4,5, Marco Aurélio Krieger1,2, Alexandre Dias Tavares Costa1,2*

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1 Instituto

de Biologia Molecular do Paraná (IBMP), Curitiba, Brazil

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2

Instituto Carlos Chagas (ICC), Fundação Oswaldo Cruz, Curitiba, Brazil

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Laboratório de Patologia e Biointervenção, Centro de Pesquisas Gonçalo

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Moniz, FIOCRUZ, Salvador, Bahia, Brazil

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4

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Brazil

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5

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de Medicina Veterinária e Zootecnia, Universidade Federal da Bahia, Salvador,

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Bahia, Brazil

Instituto de Ciência e Tecnologia de Doenças Tropicais, INCT-DT, Bahia,

Departamento de Medicina Veterinária Preventiva e Produção Animal, Escola

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#

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Email addresses: RCPR [email protected]; MSS

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[email protected]; LCSS [email protected]; LNP

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[email protected]; JCOGJ [email protected]; PSTV

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[email protected]; DBMF [email protected]; MAK

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[email protected]; ADTC [email protected]

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CORRESPONDING AUTHOR [email protected], [email protected]

these authors contributed equally to this work.

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Abstract

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Background. Canine visceral leishmaniasis is usually diagnosticated by

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serological tests, but these methods present limitations in accuracy. Thus,

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improvement in assessing Leishmania infection in dogs is needed. Quantitative

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PCR (qPCR) is a sensitive and specific techinique which presents limitations that

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hinder its implementation in diagnostic routines. Absolute requirement for freezer

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storage of qPCR reagents is one of them. Furthermore, internal controls used in

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the qPCR reaction for Leishmania detection have also not been developed, their

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use concomitantly would avoid false-negative results. The aim of this study was

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to develop a gelified duplex qPCR for the identification of infected animals. We

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combined a new reaction for detection of the 18S canine gene into a duplex qPCR

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that concomitantly amplifies a conserved sequence of L. infantum DNA in canine

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blood, spleen or skin tissue samples. The duplex reaction was compared to the

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traditional singleplex format. A gelified, freezer-free format was also produced

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and compared to the regular, liquid/frozen format. For comparison, samples were

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characterized by standard parasitological and serological protocols.

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Results. The new duplex qPCR exhibited the same detection limit of 0.1

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parasites/reaction as the singleplex reaction format. Such limit was observed for

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samples from all three tissues studied, using L. infantum DNA as target. We used

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samples of 82 asymptomatic and symptomatic dogs from an endemic area to

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compare our duplex qPCR to other diagnostic methods, and observed that spleen

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aspirates samples provided higher rate of positivity (92.9%), followed by skin

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(50%) and blood samples (35.7%). The gelified format showed a limit of detection

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of 1 parasite/reaction without affecting the reaction efficiency.

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Conclusions. Our results show that amplification of Leishmania and canine DNA

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in a single reaction exhibits the same sensitivity and specificity as amplification

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of Leishmania DNA alone. Detection of host genes strengthen qPCR results by

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confirming DNA presence in the samples and the absence of polymerase

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inhibitors. We also present a qPCR format that allows reagents to be stored on

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plate, which shows similar clinically-relevant performance as the regular format.

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Thus, the new format reduces the hand-on-time to set up a test while reducing

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the chance of operator’s mistakes.

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(350/350 words)

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Keywords: Canine visceral leishmaniasis; diagnosis; duplex; qPCR

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Background

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Leishmania infantum is the causative agent of visceral leishmaniasis (VL),

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a neglected tropical disease found throughout Europe and Latin America. VL, or

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kalazar, is considered the most serious presentation among the various clinical

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forms of leishmaniasis [1]. In Brazil, natural transmission occurs via the bite of

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infected female phlebotomine sandflies [2]. Dogs are considered the main urban

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reservoir of VL, mostly due to the high rate of canine infection in endemic areas

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and the intense parasitism in canine skin [3,4]. Since these animals live in close

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contact with humans and insect vectors, they are believed to be important to the

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maintenance of the L. infantum transmission cycle [2,5–7].

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In South America, Brazil has several endemic areas for VL, as well as a large and widespread canine population [8]. One of the strategies for the

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surveillance and control of VL recommended by the Brazilian Ministry of Health

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is to monitor canine visceral leishmaniasis (CVL) by detection and removal of

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seropositive dogs [9]. Currently, CVL diagnostic protocol established by

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Brazilian Ministry of Health employs the Dual Path Platform (DPP® CVL) as a

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screening method and the enzyme-linked immunosorbent assay (EIE® CVL) as

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a confirmatory test [9]. These serological tests rely on antibody detection and

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carry important limitations, such as: i) cross-reactions with antibodies produced

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against other canine pathogens, ii) fluctuations in antibody titers during

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Leishmania infection that may affect the sensitivity of these diagnostic methods,

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and iii) no or low production of antibodies by some infected animals that

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hampers accurate detection by serological testing [10–12]. Both conventional

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(cPCR) and quantitative (qPCR) PCR have proven to be capable of

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complementing the clinical diagnosis of leishmaniasis [13,14]. Therefore, VL

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diagnosing would greatly be improved by the inclusion of PCR as a confirmatory

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test, since it offers higher specificity and sensitivity than EIE® CVL. Additionally,

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qPCR has the advantage of parasite load quantification, allowing therapeutic

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monitoring of infected humans and dogs [13,15–17].

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Different biological tissues have been used to carry out molecular tests to

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detect L. infantum DNA, such as splenic, bone marrow or lymph node aspirates,

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skin fragments, blood, and conjunctival swabs [10]. At the time of tissue

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selection, factors such as the invasiveness of the protocol, the biology of L.

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infantum, and the animal’s clinical condition must be considered. Symptomatic

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animals tend to present higher parasite burden throughout their organism when

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compared to asymptomatic ones [18]. Less invasive collection protocols are

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preferable to epidemiological studies, such as blood, conjunctival swab or skins

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fragments [10,19]. However, in order to confirm Leishmania infeccion, the

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spleen is considered a key site for the accumulation and multiplication of L.

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infantum even in the early infection, since the parasite exhibits a known tropism

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to lymphoid tissues [19,20]. Thus, even though splenic aspiration is an invasive

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procedure, it is a safe sampling technique for the parasitological detection of

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Leishmania infantum [21], being a suitable sample to be used by veterinary

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practitioners, since splenic parasite density is high throughout the course of

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CVL in both symptomatic and asymptomatic dogs [19,20].

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Despite the accuracy of cPCR and qPCR in Leishmania DNA detection,

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some questions regarding the validity of test results still linger, mainly arising

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from the quality of the extracted DNA used in reactions with false-negative

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results. The main approach to address these issues is to perform a parallel

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PCR to confirm the integrity of constitutive genes in the host genome, preferably

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within the same reaction (i.e., in a multiplex format). Several housekeeping

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genes, such as GAPDH, RNase P, and -actin, have already been used as

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internal controls to ensure the DNA template’s integrity and prevention of false-

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negative results [22–26], improving the quality of the result.

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However, a major drawback of the PCR technique is the requirement for a

118

controlled, stable temperature of -20 oC during transport and storage of

119

reagents (a.k.a. “cold chain”), which steeply increases the costs and in extreme

120

cases precludes the adoption of qPCR as a diagnostic tool. In the last years,

121

several technologies have emerged trying to solve this issue. Among them,

122

gelification is particularly useful for its easiness of use at the laboratory bench

123

as well as introduction into a production line. The gelification process stabilizes

124

the qPCR reagents allowing the complete, ready-to-use reaction to be 5

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125

transported at room temperature and stored at 4 oC for extended periods [27–

126

29].

127

In order to improve the diagnostic protocols used to assess Leishmania

128

infection in dogs, the present study aimed to develop a gelified duplex qPCR for

129

the identification of infected animals. The gelified duplex qPCR was designed to

130

simultaneously identify both a conserved sequence of the host, as well as a

131

conserved repetitive sequence of L. infantum parasites in three clinically-

132

important canine tissues (blood, spleen and skin). We also show that the duplex

133

qPCR can be pre-loaded onto the 96-well plates in a ready-to-use format. This

134

format allows the user to add only water and the extracted DNA, preventing

135

mistakes during reagents manipulation as well as decreasing the hands-on time

136

needed for launching a reaction. These two features are highly praised in a

137

routine diagnostic laboratory and can expedite the sample-to-answer process,

138

while also increasing the robustness of the test. Furthermore, since the ready-

139

to-use format can be transported and stored at above-freezing temperatures,

140

users can expect a reduction in transportation/storage associated costs.

141 142 143

Methods 1. Serological and Parasitological Tests

144

We employed the serodiagnostic protocol recommended by the Brazilian

145

Ministry of Health to detect anti-Leishmania antibodies, consisting of a rapid test

146

(DPP® CVL, Bio-Manguinhos Unit, Rio de Janeiro, Brazil) followed by a

147

confirmatory test (EIE® CVL, Bio-Manguinhos Unit, Rio de Janeiro, Brazil).

148

Serological tests were performed in accordance with manufacturer instructions.

149

DPP® CVL consist in a chromatographic immunoassay for antibodies detection, 6

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150

based on Dual-Path Platform technology in which rK28 Leishmania antigens are

151

impregnated on a nitrocellulose membrane strip [30]. EIE CVL is an ELISA

152

immunoassay to detect anti-Leishmania antibodies which uses crude Leishmania

153

major extracts as antigen (as described in the test´s manual).

154

Parasitological evaluation of splenic aspirate was performed as previously

155

described [31,21]. Briefly, part of the splenic aspirate collected was cultured in

156

Novy–MacNeal–Nicolle biphasic medium supplemented with 20% Fetal Bovine

157

Serum (Gibco BRL, New York, USA) and 100 mg/mL gentamicin to prevent

158

contamination (Sigma Chemical Co., St. Louis, MO) during four weeks at 24°C .

159

Parasites presence was assessed using microscopy during four weeks. Each

160

splenic culture was cultured in duplicate. All of the culture labels were double-

161

checked to avoid misidentification.

162 163 164

2. Canine Tissue Sampling A random sample of 82 dogs was selected from a cross-sectional study

165

conducted in the municipality of Camaçari, located in the State of Bahia, North-

166

eastern Brazil (latitude: 12º 41' 51" S; longitude: 38º 19' 27" W) during 2015.

167

Dogs were examined and classified according to presence of clinical signs such

168

as: emaciation; anemia; periocular dermatitis; crusts or ulcers in the ears;

169

despigmentaion, hyperkeratosis or lesions in the muzzle; splenomegaly;

170

linfoadenomegaly; alopecia; exfoliative dermatitis; and onychogryphosis. Each

171

clinical sign was given a grade of 0, 1 or 2 depending on the intensity of the

172

clinical manifestation. Clinical score was calculated as the sum of grades for

173

each clinical sign, and could then range from 0 to 24 points. Infected animals

174

with a clinical score of ≤ 3 were classified as asymptomatic dogs and a clinical

7

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175

score of ≥ 4 were categorized as symptomatic dogs [32]. As controls dogs,

176

three healthy soronegative CVL dogs resident in an non endemic area for CVL

177

were used.

178

All samples were previously characterized by splenic aspirate culturing

179

and were used to validate the new duplex qPCR. The canine tissue samples

180

were collected from all 82 dogs as follows: 10 mL of blood by cephalic puncture,

181

500 µL of splenic aspirate by ultrasound guided aspiration, and a skin fragment

182

from the lateral scapular region using a sterile 3 mm punch (Kolplast, Brazil).

183

Both skin and splenic samples were collected after performing trichotomy under

184

cutaneous anesthesia with 500 µL of 1% lidocaine hydrochloride (Hypofarma,

185

São Geraldo, MG - Brazil). Samples of blood, splenic aspirate and skin were

186

obtained from CVL negative dogs and used as negative reaction controls. Blood

187

samples were preserved in EDTA-2Na tubes (Greiner bio-one, Kremsmunster,

188

Austria) and all other samples were stored in DNAase- and RNAase-free tubes

189

at -70 oC until DNA extraction.

190 191

3. Sample preparation

192

DNA from blood, splenic aspirate and skin samples was extracted using the

193

DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to manufacturer

194

protocols.

195

(MHOM/RI/WR-173) DNA was obtained from promastigotes cultivated in

196

Schneider’s Complete Medium supplemented with 20% bovine fetal serum at 24

197

°C [31]. L. infantum promastigotes were counted on Neubauer chambers and

198

adjusted to 4x107 parasites and sequentially washed in saline solution to remove

199

culture medium. The pellet formed was used to perform DNA extraction together

L.

infantum

(MHOM/BR2000/MERIVALDO)

or

L.

major

8

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200

with blood, splenic aspirate or skin from the control animals, using DNeasy Blood

201

& Tissue Kit (Qiagen, Hilden, Germany).

202 203 204

4. Duplex qPCR protocol We designed a duplex qPCR in which we simultaneously amplify and

205

detect L. infantum kDNA and a conserved region of the canine housekeeping

206

gene 18S rRNA (gb|DQ287955.1) Leishmania kDNA amplification and detection

207

used the following oligonucleotides [13]: LEISH-1, 5’-

208

AACTTTTCTGGTCCTCCGGGTAG-3’; LEISH-2, 5’-ACCCCCAGTTTCCCGCC-

209

3’; and LEISH-P, 5’-FAM-AAAAATGGGTGCAGAAAT-MGB/NFQ-3’ (Life

210

Technologies). For amplification and detection of the canine 18S rRNA

211

sequence, we used the following new primers and probe: 18SCanis_F, 5’-

212

TGCGAATGGCTCATTAAATC-3’; 18SCanis_R, 5’-

213

CGTCGGCATGTATTAGCTCT-3’; and 18SCanis_P, 5’-HEX-

214

TGGTTCCTTTGGTCGCTCGCT-BHQ1-3’ (Biosearch Technologies, CA, USA).

215

qPCR reactions contained the Multiplex PCR Mastermix

216

(IBMP/Fiocruz-PR, Curitiba, Brazil), 9 mM magnesium acetate (Mg(OAc)2) and

217

2 mg/mL BSA (molecular biology grade, Roche) as final concentrations. The

218

singleplex reaction for detection of Leishmania kDNA also contained 5 μL of

219

extracted DNA (varying from 50 to 500 ng, as measured by UV absorbance at

220

260 nm), 200 nM of LEISH-P, 900 nM of each primer LEISH-1 and LEISH-2,

221

and DNAse/RNAse-free H2O to 25 μL. The duplex reactions contained the

222

same reagents as the singleplex protocol, supplemented with 160 nM of each

223

primer 18SCanis_F and 18SCanis_R, and 40 nM of the 18SCanis_P.

9

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224

For clarity purposes, the terms “liquid” and “traditional” refer to

225

reactions performed using reagents stored in freezer (-20 oC), which have to be

226

manipulated in the traditional way (i.e., thawed before use). The terms “gelified”

227

and “ready-to-use” refer to reactions performed using reagents that can be store

228

above freezing temperatures, and do not need to be manipulated by the user

229

since they are preloaded into the reaction vessels.

230

Gelified reactions were produced by mixing qPCR reagents (enzyme,

231

buffer, salts, nucleotides, primers and probes), at final concentrations, with a

232

gelification solution [28,29,33]. This mixture was then aliquoted onto the 96-well

233

plates, and the plates were submitted to 3 cycles of vacuum (30 mBar) of 30

234

minutes each, at constant temperature (30 oC), as previously described [28,34].

235

Both liquid and gelified reactions were run in an ABI7500 Fast Real-Time PCR

236

System (Life Technologies) with the following temperature profile: 1x 50 oC/2

237

minutes; 1x 95 oC/10 minutes; 45x [95 oC/15s, 60 oC/60s].

238

Seven-point standard curves for each tissue type (blood, spleen or

239

skin) were prepared using DNA extracted from 2 x 105 L. infantum or L. major

240

promastigotes per microliter as starting material. The extracted DNA was 10-

241

fold serially diluted in Leishmania-negative tissue-specific canine DNA (10-100

242

ng/µl, as measured by UV absorbance at 260 nm) obtained from healthy

243

animals. All reactions were performed in triplicate and data are expressed in

244

terms of cycle threshold (Ct) mean values. For each tissue type, relevant Ct cut-

245

off values were calculated by Receiver-Operator Characteristic (ROC) curve

246

analysis to determine positivity for L. infantum or L. major DNA, as previously

247

described [31]. Ct cut-off values presenting 100% of specificity and the higher

248

sensitivity values were chosen for each tissue type. Quantification of

10

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249

Leishmania kDNA was performed using an absolute method by comparing the

250

mean Ct values obtained from each tissue type to the Ct values on the

251

corresponding seven-point standard curve.

252 253

5. Statistical Analysis

254

To prevent any bias, results of the molecular diagnostic techniques were

255

read without any knowledge of the other serological or parasitological results.

256

ROC analysis, used to establish the Ct cut-off value, was performed using

257

GraphPad Prism software v.5.0 (GraphPad Prism Inc., San Diego, CA). For each

258

tissue type analyzed, corresponding sensitivity and 95% confidence intervals (CI)

259

were calculated using splenic culturing as the reference standard. Comparisons

260

among singleplex and duplex results for each individual sample were evaluated

261

by the Wilcoxon signed rank sum test. The X2 or Fisher tests were used to

262

compare diagnostic methods results among all animals included in the study and

263

between assyntomatic and symptomatics dogs. McNemar’s test was used to

264

assess results among the same samples tested in singleplex relative to the

265

duplex protocol or in the regular format (“Liquid”) and the ready-to-use format

266

(“Gel”). A p-value below 0.05 was considered statistically significant.

267 268

Results

269

The existence of singleplex reactions is not a guarantee that the

270

corresponding duplex qPCR will exhibit the same characteristics, such as

271

efficiency and limit of detection. Losses of sensitivity and specificity may be due

272

to competition of targets for the enzyme or unspecific interactions between the

273

two sets of oligonucleotides or with the genomic targets. That is, the main issue

11

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274

in the development of multiplex PCR assays is the difficulty of adjusting two or

275

more different reactions occurring simultaneously without losing accuracy [35].

276

Therefore, we optimized the singleplex reaction described by Francino et al. [13]

277

into a duplex reaction using qPCR reagents produced at our own production

278

facility. Figure 1A shows the detection of 10-fold dilutions of L. infantum DNA

279

performed in the singleplex (blue lines) or duplex format (red lines). There is

280

virtually no difference between the detection of each dilution point. Singleplex and

281

duplex reactions exhibited efficiency of 101.84 and 105.47 and an equal limit of

282

detection of 0.05 parasites/µl, respectively. For clarity purposes, the detection of

283

the second target in the duplex reaction depicted in Figure 1A is shown only in

284

Figure 1B. Figure 1B depicts a typical duplex reaction which concomitantly

285

detects the L. infantum DNA dilutions (red lines) and the constitutive 18S gene

286

(blue lines). Detection of the 18S gene in these samples was possible because

287

the L. infantum promastigotes DNA was diluted in DNA extracted from

288

Leishmania-negative canine splenic aspirate, as described in Materials and

289

Methods. Mean Ct intervals for the detection of the conserved canine region for

290

each tissue were established as follows: 15.9-19.9 for blood, 16.6-19.0 for skin,

291

and 17.5-19.8 for splenic aspirate samples. All samples used in this study

292

presented mean Ct values within these ranges, thus considered suitable for

293

Leishmania diagnosis assessment.

294

Figure 2 shows a direct comparison between the detection cycle of L.

295

infantum DNA in canine blood samples analyzed with both the singleplex and the

296

duplex reaction. Figure 2A shows that some samples were detected in a higher

297

Ct in the duplex format, suggesting loss of sensitivity. Figure 2B shows that the

298

difference observed between the detection cycles with the singleplex and the

12

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299

duplex format ranged from -4.1 to +0.6, meaning that some samples were

300

detected 4.1 Cts higher in the duplex relative to the singleplex format. On the

301

other hand, some samples did not change their detection level, or were detected

302

earlier. However, the overall difference between reactions is negligible, averaging

303

a mean Ct value slightly higher than 1 (1.22) for the singleplex relative to the

304

duplex protocol (p=0.0248) (Figure 2B). Furthermore, it is important to highlight

305

that the observed difference did not affect qPCR positivity in our study,

306

irrespective which protocol was used (p=1.0, McNemar’s test). That is, all

307

samples considered positive by the singleplex reaction were also considered

308

positive by the duplex reaction.

309

Figure 3 shows the reportable dynamic range for L. infantum DNA

310

quantification by qPCR when the parasite’s DNA was diluted in DNA extracted

311

from Leishmania-negative blood, skin or splenic aspirates. Reactions performed

312

in all three DNA matrices exhibited a successful linear detection of up to 0.1

313

parasite per reaction. It should be noted that our reaction was able to detect 0.05

314

parasite per reaction (Figure 3, stars), although not always in the linear range of

315

detection.

316

DNA amplification efficiency can be affected by several parameters, such

317

as presence of inhibitors in the sample or suboptimal concentration of

318

oligonucleotides (primers and/or probe), and can be estimated by the slope of the

319

dilution curve detection. As described in Figures 1-3, we optimized the qPCR so

320

that the mean amplification efficiency calculated from at least five independent

321

standard curves was 96.8% for blood or skin samples (slope = -3.4±0.1 for each),

322

and 105.4% for splenic aspirate samples (slope = -3.2±0.2), with corresponding

323

R2 of 0.99 irrespective of the tissue.

13

324 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

We then used a convenience sample to test our duplex qPCR, so that we

325

were able to ran a ROC analysis, determine Ct cut-off values and maximize the

326

reaction’s sensitivity and specificity. Cut-off values for blood samples was

327

established at Ct 42.99, with prediction rates of 100% sensitivity (CI 73.4– 100)

328

and 100% specificity (CI 71.5–100); for skin samples, a Ct cut-off value of 40.0

329

provided prediction rates of 100% sensitivity (CI 73.5–100) and 100% specificity

330

(CI 69.2–100); finally, in splenic aspirate samples, the Ct cut-off value of 38.2

331

corresponded to prediction rates of 91.67% sensitivity (CI 61.5–99.8) and 100%

332

specificity (CI 73.5–100). ROC analysis for the three tissues revealed an area

333

under the curve of 1.0, indicating a high probability (p < 0.001) that any

334

randomly chosen positive sample would be correctly classified.

335

Although the duplex qPCR described here was designed for detection of L.

336

infantum DNA, we also analyzed it using DNA extracted from L. major, L.

337

amazonensis, or L. braziliensis promastigotes. Our results suggest that only L.

338

major DNA exhibits similar qPCR profile and limit of detection as L. infantum

339

DNA, while detection of DNA from 105 L. amazonensis promastigotes is at a

340

similar Ct than DNA from 100 L. infantum promastigotes. We also tested the

341

analytical specificity of the newly designed canine 18S rRNA oligonucleotides

342

and found no detectable qPCR amplification using DNA samples of Leishmania

343

spp (data not shown).

344

Next, blood, skin and splenic aspirate samples from 82 dogs were used to

345

evaluate our duplex qPCR protocol. All the dogs were previously characterized

346

by the reference method of splenic aspirate culturing, by which 42 were

347

considered positive and 40 negative. The results from each diagnostic technique

348

employed using different tissues are compared in Table 1. The qPCR of splenic

14

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349

aspirate samples was able to detect L. infantum DNA in 92.9% of the splenic

350

culturing from positive animals, followed by 50% when using skin samples, and

351

35.7% when using blood samples. Concerning these diagnostic techniques

352

results, no statistically significant differences were noticed. Regarding the clinical

353

status, duplex qPCR of splenic aspirate detected more positive samples than the

354

other detection methods, but not in a significant way, identifying 72% as positive

355

among asymptomatic and 77.8% among symptomatic dogs (Table 2). Among

356

dogs with negative splenic culturing tests, qPCR from splenic aspirate samples

357

was able to detect L. infantum DNA in 45% of these animals, while qPCR from

358

skin detected L. infantum DNA in 42.5% and qPCR from blood detected L.

359

infantum DNA in 27.5%. Results presented in Table 2 provide convincing

360

evidence of the high positivity rates derived from the qPCR technique in

361

comparison to parasitological and serological diagnostic methods.

362

Since qPCR reagents are temperature-sensitive, extreme care must be

363

taken to keep them below -20 oC during transportation and storage. To overcome

364

this hurdle, we applied the gelification technology to our reagents to store them

365

in a ready-to-use format, pre-loading them onto the plates inside the production

366

facility. Figure 4 shows that the reportable range for linear L. infantum DNA

367

detection is not significantly affected by the gelification process (Fig 4A). Although

368

the observed limit of detection for the gelified reaction was found to be 1

369

parasite/reaction, we were able to detect 0.1 parasite/reaction outside the linear

370

range of detection.

371

Interestingly, parameters such as amplification efficiency and linearity (R2)

372

obtained by the gelified duplex qPCR were comparable to those obtained with

373

the regular, liquid reaction format (Fig 3 and Fig 4A). Amplification efficiency for

15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

374

the detection of L. infantum DNA by the gelified qPCR in blood, skin or splenic

375

aspirate samples was 84.7%, 81.3%, and 82.4%, respectively. R2 was 0.96 for

376

all three reactions.

377

A direct comparison between the same samples tested with the regular

378

format (“Liquid”) and the ready-to-use format (“Gel”) shows that there is no

379

significant difference between them using McNemar’s test (Fig 4B). Although

380

some samples clearly had their detection affected by the gelification process, on

381

average the delay was less than 1 Ct in the detection by the gelified reaction

382

versus the liquid format. Figure 4C summarizes all the differences in Ct observed

383

for the detections in Figure 4B.

384 385 386

Discussion The successful output of a qPCR is directly correlated to the quality of

387

the sample and/or its extracted DNA. One straightforward strategy to control the

388

quality of the sample and, at the same time, the overall quality of the system

389

(reagents, instrument and analysis software) is to amplify a housekeeping gene

390

[22–26]. We developed a new reaction for the detection of the canine 18S rRNA

391

gene and combined it with a published reaction for detection of L. infantum [13]

392

into a new duplex qPCR that concomitantly detects the target and the internal

393

control gene. Our results demonstrate that the duplex qPCR format did not

394

affect the analytical sensitivity of L. infantum DNA amplification relative to the

395

single reaction format (Figures 1 and 2). The detection of less than one parasite

396

per reaction is possible because the target gene is present in multiple copies in

397

the genome, thus being dissolved into solution when the parasites are lysed.

16

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398

For Leishmania spp, the target gene is present at approximately 50 copies per

399

parasite genome [17].

400

The duplex protocol was successfully used for concomitant detection of

401

leishmania and canine DNA targets in blood, skin or splenic aspirate samples

402

(Figure 3). It is important to highlight that the detection of the highly abundant

403

18S gene did not affect the detection of leishmania DNA, even when the latter

404

was present in much lower amounts (Figure 1). Blood and skin are known to

405

contain qPCR inhibitors, such as hemoglobin, HSA (human serum albumin),

406

melanin and collagen [36], which were most likely removed during nucleic acid

407

extraction. Our results corroborate that hypothesis, since we did not observe

408

any meaningful difference between the qPCR, liquid or gelified, performed with

409

samples originated from spleen, blood or skin. Therefore, when we consider the

410

efficiency of the qPCR and the limit of detection, blood, skin or splenic aspirates

411

are equally good sources for detection of Leishmania DNA.

412

The present study did not attempt to extensively test the analytical

413

specificity of the new qPCR with respect to Leishmania DNA detection.

414

However, our preliminary assessments show that our reaction detects L. major

415

with the same sensitivity as L. infantum. Interestingly, our reaction would also

416

be able to detect the presence of L. amazonensis in a sample with high

417

parasitemia, because the detection of 105 L. amazonensis promastigotes is

418

similar to the detection of 100 L. infantum promastigotes.

419

Although qPCR is a powerful and sensitive technique, the absolute

420

requirement for maintaining freezing temperatures is a critical point and a major

421

hurdle for its adoption as a routine diagnostic tool. This is specially true for

422

Leishmania and other neglected tropical diseases detection because they

17

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423

usually are endemic to countries with deficient lab infrastructure, where a point

424

of care test would be most necessary. Therefore, almost as a sine qua non

425

condition, a point of care test should not be influenced by the

426

transportation/storage temperature [27]. Several technologies have been used

427

in the last years to overcome this obstacle, of which the gelification technology

428

stands out for being less harsh on the sensitive qPCR reagents [28,29]. Indeed,

429

pre-storage of gelified qPCR reagents on the reaction vessel has been

430

previously done, aiming the detection of Plasmodium spp [30, 31],

431

Campylobacter spp [28] or T. cruzi [31]. Our results show that the gelified qPCR

432

for detection of Leishmania DNA exhibits similar clinically-relevant linear

433

detection limit as the traditional liquid format, irrespective of the tissue used as

434

sample matrix (Figure 4). This format has previously been shown to allow qPCR

435

reagents to be stored at 4 oC for up to 12 months or at 22 oC for 1-3 months

436

without significant loss of sensitivity [28,29,34]. Furthermore, pre-loading the

437

reaction in a ready-to-use format directly into the reaction vessel decreases the

438

manipulation of reagents and minimizes the risk of operator mistakes.

439

We used our duplex qPCR to analyze 82 naturally-infected dogs and

440

observed that, among the positive animals, splenic aspirates are the most

441

effective tissue for detecting L. infantum infection, and should be preferred over

442

blood or skin if sanitary conditions allows proper collection. We believe that the

443

known spleen tropism of the parasite is the explanation for the higher positivity

444

in splenic samples [37]. However, we suggest that skin should be considered as

445

a viable alternative tissue for sample collection in environments with little-to-

446

none infrastructure or skilled personnel, mainly because the collection

447

procedure is less invasive and require less technical skills than splenic aspirate.

18

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448

In addition, since skin lesions are known to contain live parasites [38], skin

449

samples with active infection may have increased probability of Leishmania spp

450

detection, and possibly increased diagnostic positivity.

451

Although parasitology and serology are advantageous due to their

452

simplicity vis-à-vis molecular techniques, they are nonetheless limited and can

453

lead to misleading results, thereby neglecting the presence of Leishmania

454

infection in dogs [15,31]. In light of this consideration, we suggest that less

455

specific testing techniques should be used for screening and more specific

456

techniques such qPCR should be subsequently applied as a confirmatory test.

457

This protocol would greatly enhance the accuracy of LV diagnosis and lead to an

458

important improvement in control efforts.

459 460

Conclusion

461

We present a duplex qPCR able to detect Leishmania infantum or L. major

462

and canine genomic DNA in a single reaction. Detection of host DNA in the same

463

reaction strengthen CVL diagnosis by acting as an internal reaction control. The

464

duplex and singleplex qPCR have similar performance in detecting L. infantum

465

infection, with a clinically relevant detection limit, irrespective of the tissue tested

466

(blood, skin or splenic aspirate) or the reagents storage format (liquid or gelified).

467

Moreover, the duplex qPCR described herein offered greater sensitivity in

468

comparison to the methods routinely used for CVL diagnosis.

469

19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

470

List of abbreviations

471

VL – visceral leishmaniasis

472

CVL – canine visceral leishmaniasis

473

DPP® CVL – Dual Path Platform test for CVL diagnosis

474

EIE® CVL – enzyme-linked immunosorbent assay (ELISA) test for CVL

475

diagnosis

476

cPCR – conventional PCR

477

qPCR – quantitative PCR

478

kDNA – kinetopast DNA

479

Ct – cycle threshold

480

ROC – Receiver-Operator Characteristic

481

CI – confidence interval

482 483

Declarations

484

Ethics Statement

485

All canine tissue samples used in this study and their specific collection

486

procedures were approved by the CPqGM - FIOCRUZ Institutional Review

487

Board for Animal Experimentation under Permit Number 007/2013.

488 489

Consent for publication 20

490 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Not applicable.

491 492

Availability of data and materials

493

The datasets during and/or analysed during the current study available from the

494

corresponding author on reasonable request.

495 496

Competing interests

497

The authors declare that they have no competing interests.

498 499

Funding

500

This work was partially funded by FINEP project #01.13.0283.00 to M.A.K.

501

FINEP played no role in the design of the study and collection, analysis, and

502

interpretation of data and in writing the manuscript.

503 504

Authors’s contributions

505

RP and MS performed the qPCR experiments, analyzed the results, performed

506

the statistical analyses, and drafted the manuscript. LS, LP and JJ extracted the

507

DNA from the different tissues and performed the parasitological (splenic

508

culturing) and serological experiments (EIE and DPP). PV, DF, and MK

509

conceived and designed the study. AC analyzed the results, participated in the

21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

510

conception and design of the study, and in the writing of the manuscript. All

511

authors read and approved the final manuscript.

512 513

Acknowledgments

514

The authors are grateful for Dr. Daniela Pavoni’s critical reading of the

515

manuscript.

516 517

22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

518

References

519

1. Maia-Elkhoury ANS, Alves WA, Sousa-Gomes ML de, Sena JM de, Luna EA.

520

Visceral leishmaniasis in Brazil: trends and challenges. Cad. Saude Publica

521

2008;24:2941–7.

522

2. Lainson R, Rangel EF. Lutzomyia longipalpis and the eco-epidemiology of

523

American visceral leishmaniasis, with particular reference to Brazil: a review.

524

Memórias do Inst. Oswaldo Cruz 2005;100:811–27.

525

3. Molina R, Amela C, Nieto J, San-Andrés M, González F, Castillo JA, et al.

526

Infectivity of dogs naturally infected with Leishmania infantum to colonized

527

Phlebotomus perniciosus. Trans. R. Soc. Trop. Med. Hyg. 1994; 88:491–3.

528

4. Margonari C, Freitas CR, Ribeiro RC, Moura ACM, Timbó M, Gripp AH, et al.

529

Epidemiology of visceral leishmaniasis through spatial analysis, in Belo

530

Horizonte municipality, state of Minas Gerais, Brazil. Memórias do Inst.

531

Oswaldo Cruz 2006;101:31–8.

532

5. Salomón OD, Orellano PW. Lutzomyia longipalpis in Clorinda, Formosa

533

province, an area of potential visceral leishmaniasis transmission in Argentina.

534

Mem. Inst. Oswaldo Cruz. 2005;100:475–6.

535

6. Harhay MO, Olliaro PL, Costa DL, Costa CHN. Urban parasitology: Visceral

536

leishmaniasis in Brazil. Trends Parasitol. Elsevier Ltd; 2011;27:403–9.

537

7. Dantas-Torres F. The role of dogs as reservoirs of Leishmania parasites, with

538

emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia)

539

braziliensis. Vet. Parasitol. 2007;149:139–46.

540

8. Peixoto HM, de Oliveira MRF, Romero GAS. Serological diagnosis of canine

541

visceral leishmaniasis in Brazil: systematic review and meta-analysis. Trop.

542

Med. Int. Heal. 2015;20:334–52.

23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

543

9. Ministério da S. Manual Vigilancia Controle da Leishmaniose Visceral. 2006.

544

10. Ferreira E de C, de Lana M, Carneiro M, Reis AB, Paes DV, da Silva ES, et

545

al. Comparison of serological assays for the diagnosis of canine visceral

546

leishmaniasis in animals presenting different clinical manifestations. Vet.

547

Parasitol. 2007;146:235–41.

548

11. Solano-Gallego L, Morell P, Arboix M, Alberola J, Ferrer L. Prevalence of

549

Leishmania infantum infection in dogs living in an area of canine leishmaniasis

550

endemicity using PCR on several tissues and serology. J. Clin. Microbiol.

551

2001;39:560–3.

552

12. Silva ES, Gontijo CMF, Melo MN. Contribution of molecular techniques to

553

the epidemiology of neotropical Leishmania species. Trends Parasitol.

554

2005;21:550–2.

555

13. Francino O, Altet L, Sánchez-Robert E, Rodriguez a, Solano-Gallego L,

556

Alberola J, et al. Advantages of real-time PCR assay for diagnosis and

557

monitoring of canine leishmaniosis. Vet. Parasitol. 2006;137:214–21.

558

14. Manna L, Vitale F, Reale S, Caracappa S, Pavone LM, Morte R Della, et al.

559

Comparison of different tissue sampling for PCR-based diagnosis and follow-up

560

of canine visceral leishmaniosis. Vet. Parasitol. 2004;125:251–62.

561

15. Maia C, Nunes M, Cristóvão J, Campino L. Experimental canine

562

leishmaniasis: clinical, parasitological and serological follow-up. Acta Trop.

563

2010;116:193–9.

564

16. Otranto D, Paradies P, De Caprariis D, Stanneck D, Testini G, Grimm F, et

565

al. Toward diagnosing Leishmania infantum infection in asymptomatic dogs in

566

an area where leishmaniasis is endemic. Clin. Vaccine Immunol. 2009;16:337–

567

43.

24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

568

17. Lachaud L, Chabbert E, Dubessay P, Dereure J, Lamothe J, Dedet JP, et

569

al. Value of two PCR methods for the diagnosis of canine visceral leishmaniasis

570

and the detection of asymptomatic carriers. Parasitology 2002;125:197–207.

571

18. Manna L, Reale S, Vitale F, Gravino AE. Evidence for a relationship

572

between Leishmania load and clinical manifestations. Res. Vet. Sci.

573

2009;87:76–8.

574

19. Maia C, Ramada J, Cristóvão JM, Gonçalves L, Campino L. Diagnosis of

575

canine leishmaniasis: conventional and molecular techniques using different

576

tissues. Vet. J. 2009;179:142–4.

577

20. Reis AB, Martins-Filho OA, Teixeira-Carvalho A, Carvalho MG, Mayrink W,

578

França-Silva JC, et al. Parasite density and impaired biochemical/hematological

579

status are associated with severe clinical aspects of canine visceral

580

leishmaniasis. Res. Vet. Sci. 2006;81:68–75.

581

21. Barrouin-Melo SM, Larangeira DF, Trigo J, Aguiar PHP, Dos-Santos WLC,

582

Pontes-de- Carvalho L. Comparison between splenic and lymph node

583

aspirations as sampling methods for the parasitological detection of Leishmania

584

chagasi infection in dogs. Mem. Inst. Oswaldo Cruz 2004;99:195–7.

585

22. Gonçalves-de-Albuquerque SDC, Pessoa E Silva R, de Morais RCS,

586

Trajano-Silva LAM, Régis-da-Silva CG, Brandão-Filho SP, et al. Tracking false-

587

negative results in molecular diagnosis: proposal of a triplex-PCR based

588

method for leishmaniasis diagnosis. J. Venom. Anim. Toxins Incl. Trop. Dis.

589

2014;20:16.

590

23. Peleg O, Baneth G, Eyal O, Inbar J, Harrus S. Multiplex real-time qPCR for

591

the detection of Ehrlichia canis and Babesia canis vogeli. Vet. Parasitol.

592

2010;173:292–9.

25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

593

24. Piron M, Fisa R, Casamitjana N, López-Chejade P, Puig L, Vergés M, et al.

594

Development of a real-time PCR assay for Trypanosoma cruzi detection in

595

blood samples. Acta Trop. 2007;103:195–200.

596

25. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, et al.

597

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory

598

Testing. Clin. Microbiol. Rev.2006;19:165–256.

599

26. Melo MF, Moreira OC, Tenório P, Lorena V, Lorena-Rezende I, Júnior WO,

600

et al. Usefulness of real time PCR to quantify parasite load in serum samples

601

from chronic Chagas disease patients. Parasit. Vectors 2015;8:1–7.

602

27. Pai NP, Vadnais C, Denkinger C, Engel N, Pai M. Point-of-care testing for

603

infectious diseases: diversity, complexity, and barriers in low- and middle-

604

income countries. PLoS Med. 2012;9:e1001306.

605

28. P. M. F. S. Rosado, G. L. López, A. M. Seiz MD, Alberdi M. Method for

606

preparing stabilised reaction mixtures, which are totally or partially dried,

607

comprising at least one enzyme, reaction mixtures and kits containing said

608

mixtures. World Intellectual Property Organization (WIPO); 2002. p. 75.

609

29. Sun Y, Høgberg J, Christine T, Florian L, Monsalve LG, Rodriguez S, et al.

610

Pre-storage of gelified reagents in a lab-on-a-foil system for rapid nucleic acid

611

analysis. Lab Chip 2013;13:1509–14.

612

30. Grimaldi G, Teva A, Ferreira AL, dos Santos CB, Pinto I de-S, De-Azevedo

613

CT, et al. Evaluation of a novel chromatographic immunoassay based on Dual-

614

Path Platform technology (DPP® CVL rapid test) for the serodiagnosis of canine

615

visceral leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 2012;106:54–9.

616

31. Solcà MDS, Bastos LA, Guedes CES, Bordoni M, Borja LS, Larangeira DF,

617

et al. Evaluating the accuracy of molecular diagnostic testing for canine visceral

26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

618

leishmaniasis using latent class analysis. PLoS One. 2014; 9(7):e103635.

619

32. Solcà MS, Andrade BB, Abbehusen MMC, Teixeira CR, Khouri R,

620

Valenzuela JG, et al. Circulating Biomarkers of Immune Activation, Oxidative

621

Stress and Inflammation Characterize Severe Canine Visceral Leishmaniasis.

622

Sci. Rep. 2016;6:32619.

623

33. Iglesias N, Subirats M, Trevisi P, Ramírez-Olivencia G, Castán P, Puente S,

624

et al. Performance of a new gelled nested PCR test for the diagnosis of

625

imported malaria: comparison with microscopy, rapid diagnostic test, and real-

626

time PCR. Parasitol. Res. 2014;113:2587–91.

627

34. Graziani AG, Rampazzo RCP, Biondo CA, Costa MLN, Cereda M, Cocci A,

628

et al. A Portable Platform for Diagnostic of Tropical diseases: On-chip Real

629

Time PCR detection of Trypanosoma cruzi and Plasmodium spp. PLoS Negl.

630

Trop. Dis. 2016;submitted.

631

35. Markoulatos P, Siafakas N, Moncany M. Multiplex polymerase chain

632

reaction: a practical approach. J. Clin. Lab. Anal. 2002;16:47–51.

633

36. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors –

634

occurrence, properties and removal. J. Appl. Microbiol. 2012;113:1014–26.

635

37. Colmenares M, Kar S, Goldsmith-Pestana K, McMahon-Pratt D.

636

Mechanisms of pathogenesis: differences amongst Leishmania species. Trans.

637

R. Soc. Trop. Med. Hyg. 2002;96:S3–7.

638

38. Aslan H, Oliveira F, Meneses C, Castrovinci P, Gomes R, Teixeira C, et al.

639

New Insights Into the Transmissibility of Leishmania infantum From Dogs to

640

Sand Flies: Experimental Vector-Transmission Reveals Persistent Parasite

641

Depots at Bite Sites. J. Infect. Dis. 2016; 213:1752-61.

642

27

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643

Figure legends

644

Figure 1. Reportable range for L. infantum DNA quantification by qPCR.

645

Panel A shows the amplification plot of 10-fold serial dilutions of parasite DNA

646

(ranging from 0.1 to 105 parasites) detected using singleplex (blue lines) or duplex

647

(red lines) qPCR protocols. The thick horizontal red line represents the detection

648

threshold. The duplex qPCR was carried out using Leishmania infantum DNA

649

serially diluted into DNA extracted from leishmania-negative splenic aspirate.

650

Panel B shows the amplification plot the duplex reaction alone, depicting the

651

detection of L. infantum DNA (red lines) as well as the canine 18S gene (blue

652

lines). The thick horizontal red line represents the detection threshold for L.

653

infantum qPCR while the thick horizontal blue line represents the detection

654

threshold for the 18S qPCR. Each concentration of DNA was tested in triplicates.

655

Curves are representative of at least three independent experiments.

656 657

Figure 2. Comparison of threshold cycles between the singleplex and the

658

duplex qPCR for detection of Leishmania DNA. Panel A shows the

659

corresponding difference in Ct values obtained from the qPCR analysis of

660

individual samples tested by singleplex or duplex protocols. The same sample

661

was tested independently by both protocols, and the threshold cycles were

662

plotted and connected by dotted lines. Panel B plots the differences between Ct

663

values from the same sample using singleplex or duplex reactions, showing that

664

the mean difference is less than 1 Ct (thick horizontal line). Comparison among

665

singleplex and duplex results for each individual was evaluated by Wilcoxon

666

signed rank sum test (p<0.05).

667

28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

668

Figure 3. Comparison of dynamic ranges between L. infantum DNA

669

detection in blood, skin or splenic aspirates. Ct values obtained for duplex

670

qPCR reactions are plotted against known concentrations of parasite DNA

671

(ranging from 0.1 to 105 parasites) diluted in leishmania-negative DNA from

672

blood, skin or splenic aspirate. DNA representing 0.05 parasite’s genome were

673

stochastically detected, thus being shown as stars outside the linear regression.

674

Mean reaction efficiencies from at least three independent experiments were

675

respectively 96.8% for blood and skin samples,and 105.9% for splenic aspirate

676

samples, all three with a R2 of 0.99.

677 678

Figure 4. Detection of L. infantum DNA using a gelified, ready-to-use duplex

679

qPCR. Panel A shows 10-fold serial dilutions of L. infantum DNA (ranging from

680

0.5 to 105 parasites) diluted in leishmania-negative DNA from blood, skin or

681

splenic aspirates using the duplex qPCR in a gelified, ready-to-use format. Mean

682

reaction efficiencies from at least three independent experiments using blood,

683

skin or splenic aspirate samples were respectively 84.7 ± 2.5; 81.3 ± 1.1; and

684

82.4 ± 1.2, all three with a R2 of 0.96 or higher. Panel B shows a direct comparison

685

between the detection of a given sample by both reaction formats (liquid versus

686

gelified, “gel”). Panel C is a plot of the differences between the threshold cycles

687

of each individual detection performed in Panel B, showing that the mean

688

difference is less than 2 Ct (1.68).

689

29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

690

Tables

691

Table 1. Positive results of CVL diagnostic method (shown as %) performed on

692

canine samples from 82 animals, which were divided into two groups according

693

to splenic culturing result. Positivity n (%) Diagnostic method

Total

Splenic culturing positive (n=42)

Splenic culturing negative (n=40)

(n=82)

39 (92.9)

18 (45)

57 (69.5)

21 (50)

17 (42.5)

38 (46.3)

qPCR Blood

15 (35.7)

11 (27.5)

26 (31.7)

DPP® CVL

30 (71.4)

20 (50)

50 (61)

EIE® CVL

23 (54.8)

10 (25)

33 (40.2)

qPCR Splenic aspirate qPCR Skin

694

30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

695

Table 2. Positive results of CVL diagnostic method (shown in %) performed on

696

canine samples from 76 animals that tested positive in at least one method

697

employed. Animals were divided into asymptomatic or symptomatic groups

698

based on clinical score. Positivity n (%)

Diagnostic method

Asymptomatic dogs

Symptomatic dogs

Total

(n = 40)

(n=36)

(n= 76)

qPCR Splenic aspirate

29 (72.5)

28 (77.8)

57 (75)

qPCR Skin

19 (47.5)

19 (52.8)

38 (50)

qPCR Blood

12 (30)

14 (38.9)

26 (34.2)

DPP® CVL

28 (70)

22 (61.1)

50 (65.8)

EIE® CVL

16 (40)

17 (47.2)

33 (43.4)

Splenic culturing

22 (55)

20 (55.6)

42 (55.3)

699 700

31

Figure1

Click here to download Figure Figure 1 - qPCR curve.tiff

Figure2

Click here to download Figure Figure 2 - Singleplex multiplex.tiff

Figure3

Click here to download Figure Figure 3 - Blood Spleen and Skin LIQUID.tiff

Figure4

Click here to download Figure Figure 4abc - starred.tiff

91

CAPÍTULO 2 – Estudo exploratório para identificação de biomarcadores de gravidade de uma amostra de cães coletada durante um estudo de corte transversal em Camaçari-BA

Nesta segunda etapa, foi realizado um estudo exploratório para identificação de biomarcadores de gravidade de amostras coletadas durante um estudo de corte transversal da população canina de Camaçari-BA, uma área endêmica para LVC. A escolha de Camaçari para este estudo deve-se ao fato de que casos caninos e humanos de LV vem sendo diagnosticados nessa região nos últimos anos, tendo sido relatados por diferentes estudos (CUNHA et al., 1995; JULIÃO, 2004; BARBOZA et al., 2006; GOMES NETO, 2007; SILVA et al., 2010). Adicionalmente, o presente grupo de pesquisa realizou um estudo de corte transversal, nos anos de 2011 e 2012, identificando alta prevalência de LVC em diferentes bairros do município. Baseado nesses resultados prévios, para a realização desse estudo foram escolhidas regiões que apresentaram as maiores prevalências para LVC no período de 2011-2012, as localidades de Jauá, Pé de Areia, Barra do Jacuípe e Machadinho

com

46%,

51%,

51,3%

e

21,1%

de

prevalência

de

LVC,

respectivamente (BORDONI, 2014). O principal objetivo do presente estudo foi identificar diferentes possíveis biomarcadores, e correlacioná-los com a gravidade da infecção por L. infantum apresentada pelos animais examinados. A identificação de biomarcadores relacionados com a gravidade da LVC pode ser uma ferramenta interessante tanto para definir o prognóstico para o animal doente e direcionar seu tratamento, nos países onde este é permitido; assim como também para avaliar sua participação ativa no ciclo de transmissão da LV, uma vez que já é bem descrito na literatura que cães com a forma ativa e mais grave de LVC tendem a transmitir um maior número de parasitos para o flebotomíneo (DA COSTA-VAL et al., 2007; MICHALSKY et al., 2007; VERCOSA et al., 2008). Desta forma, neste estudo de corte transversal, foram avaliadas as hipóteses de associações entre a presença de biomarcadores de exposição à saliva do vetor e biomarcadores inflamatórios com a gravidade da infecção por L. infantum, que

92

podem revelar associações de interesse e poderão ser validadas no futuro em um estudo longitudinal que está sendo desenvolvido paralelamente ao presente estudo.

2.1 OBJETIVO GERAL •

Avaliar biomarcadores de exposição à saliva do vetor e identificar biomarcadores inflamatórios, correlacionando-os com a gravidade da LVC e carga parasitária.

2.2 OBJETIVOS ESPECÍFICOS •

Avaliar LJM 11/LJM 17 como biomarcadores imunológicos de exposição à saliva do vetor;



Realizar análise exploratória de diferentes biomarcadores inflamatórios, de ativação imune e de estresse oxidativo de LVC;



Avaliar a presença de correlação entre os biomarcadores imunológicos de exposição à saliva do vetor, inflamatórios, de ativação imune e de estresse oxidativo com a gravidade de LVC e carga parasitária.

93

2.3 Artigo científico publicado na revista Scientific Reports

Manuela S. Solcà, Bruno B. Andrade, Melissa Abbehusen, Clarissa Teixeira, Jesus G. Valenzuela, Shaden Kamhawi, Ricardo Khouri, Patrícia Torres Bozza, Deborah Bittencourt Mothé Fraga, Valeria Matos Borges, Patrícia Sampaio Tavares Veras, Claudia Ida Brodskyn. Circulating Biomarkers of Immune Activation, Oxidative Stress and Inflammation Characterize Severe Canine Visceral Leishmaniasis. 2016.

No presente estudo foram avaliados simultaneamente biomarcadores imunológicos de exposição à saliva do vetor, biomarcadores de inflamação, ativação imune e estresse oxidativo em cães com diferentes quadros clínicos de LVC, visando identificar biomarcadores associados com a gravidade da doença clínica. Foi realizado um estudo exploratório de amostras de cães coletadas durante um estudo de corte transversal, em Camaçari-BA. A análise identificou uma bioassinatura distinta em cães com diferentes manifestações clínicas, caracterizada por uma diminuição dos níveis de LTB-4 e de PGE-2 de acordo com o agravamento da doença, e um aumento de CXCL1 e CCL2. Além disso, utilizando uma combinação de 3 parâmetros diferentes (LTB-4, PGE-2 e CXCL1) fomos capazes de discriminar entre escores clínicos diferentes pela construção de uma curva ROC. Foi detectado também, que cães com escores clínicos elevados apresentaram-se, mais frequentemente, com negatividade para IgG anti-saliva e elevadas cargas parasitárias.

Este

estudo

permitiu

a

avaliação

e

identificação

de

vários

biomarcadores em cães, que podem ser importantes para auxiliar, na avaliação do curso da doença e prognóstico pelo médico veterinário que estiver atendendo um cão com LVC, além de futuramente poder ajudar na distinção entre cães resistentes ou susceptíveis direcionando estratégias de controle da LVC em áreas endêmicas.

www.nature.com/scientificreports

OPEN

Recei e : 2

a 2016

ccepte : 10 u ust 2016 Pu is e : 06 eptem er 2016

Circulating Biomarkers of Immune Activation, Oxidative Stress and Inflammation Characterize Severe Canine Visceral Leishmaniasis Manuela S. Solcà1, Bruno B. Andrade2,3, Melissa Moura Costa Abbehusen2, Clarissa R. Teixeira4, Ricardo Khouri2, Jesus G. Valenzuela5, Shaden Kamhawi5, Patrícia Torres Bozza6, Deborah Bittencourt Mothé Fraga1,7,8, Valeria Matos Borges2, Patrícia Sampaio Tavares Veras1,8,* & Claudia Ida Brodskyn2,9,* Clinical manifestations in canine visceral leishmaniasis (CVL) have not been clearly associated with immunological status or disease progression. We simultaneously assessed biomarkers of inflammation, immune activation, oxidative stress, and anti-sand fly saliva IgG concentrations in dog sera with different clinical manifestations to characterize a biosignature associated with CVL severity. In a cross-sectional exploratory study, a random population of 70 dogs from an endemic area in Brazil was classified according to CVL clinical severity and parasitological evaluation. A panel of biomarkers and anti–sand fly saliva IgG were measured in canine sera. Assessment of protein expression of profile biomarkers identified a distinct biosignature that could cluster separately animal groups with different clinical scores. Increasing severity scores were associated with a gradual decrease of LTB4 and PGE2, and a gradual increase in CXCL1 and CCL2. Discriminant analyses revealed that combined assessment of LTB4, PGE2 and CXCL1 was able to distinguish dogs with different clinical scores. Dogs with the highest clinical score values also exhibited high parasite loads and higher concentrations of anti-saliva antibodies. Our findings suggest CVL clinical severity is tightly associated with a distinct inflammatory profile hallmarked by a differential expression of circulating eicosanoids and chemokines. Visceral leishmaniasis (VL) is a widespread disease caused by the protozoan Leishmania infantum. This parasite is transmitted to humans and animals through the bite of the infected sand fly Lutzomyia longipalpis1,2. Dogs are considered the main urban reservoir of the parasite and its presence in the endemic area is known as a risk factor for the occurrence of human VL3,4. Clinical signs of canine VL (CVL) are non-specific, there is a widespread range of clinical manifestations varying from visceral to cutaneous presentation of the disease5,6, on the other hand some animals do not display any clinical signs during the course of infection7. The resistance and susceptibility to CVL is directly correlated with the development of Th1 (IFN- γ, IL-2 and TNF-α), or Th2 (IL-4, IL-5, IL-10, IL-13 and TGF-β) immune

1 a orat rio e ato o ia e iointer en o Instituto e es uisas on a o oni IO 0296 10 a a or Brazil. 2Laboratório Integrado de Microbiologia e Imunoregulação, Instituto de Pesquisas Gonçalo Moniz, FIOCRUZ, 0296 10 a a or ra i . 3 u tinationa Or ani ation etwor ponsorin rans ationa an pi emio o ica esearc O Initiati e un a o os i eira 00 0 080 a a or ra i . Fundação Oswaldo Cruz, iocru iau 6 128 eresina ra i . ector o ecu ar io o ection a orator of a aria an ector esearc ationa Institute of er an Infectious Diseases ationa Institutes of ea t 12 3 win roo ar wa oc i e D 208 2 . 6 a orat rio e Imunofarmaco o ia Instituto Oswa o ru io an uin os IO 210 0 900 io e aneiro ra i . Departamento e e icina eterin ria re enti a e ro u o nima sco a e e icina eterin ria e ootecnia ni ersi a e e era a a ia 01 0 110 a a or ra i . 8Instituto Nacional de i ncia e ecno o ia para Doen as ropicais I D 0110 160 a a or ra i . 9Instituto Nacional de Ciência e ecno o ia e In esti a o em Imuno o ia III I 0 03 900 o au o ra i . * ese aut ors contri ute e ua to t is wor . orrespon ence an re uests for materia s s ou e a resse to .I. . emai : ro s n a ia. ocru . r

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Figure 1. Distinct expression of immune and inflammatory markers in serum from dogs presenting with different VL clinical severity scores. (A) Hierarchical cluster analysis (Ward’s method) with bootstrap was performed to depict the overall expression profile of the indicated serum biomarkers in the different study groups. (B) Scatter plots of biomarkers displaying significant statistical differences (P < 0.005) between the study groups using Kruskal-Wallis test. Non-parametric linear trend ad hoc tests were employed to examine the variation of the biomarker levels following the clinical severity score. SOD levels did not exhibit linear trend, thus data were compared using the Dunn’s multiple comparisons test (**P < 0.01).

responses respectively, and the degree of immune activation is thought to directly impact the severity of disease8,9. Studies addressing the importance of other mediators, such as eicosanoids, Super Oxide Dismutase (SOD) and chemokines are few, especially in dogs10,11. Recently, our group has found the role played by LTB4 and PGE2 in modulating Leishmania infection in humans11,12. Experiments in vitro with human macrophages demonstrated that SOD increased the parasite burden in these cells due to the inhibition of reactive oxygen species (ROS)10. Knowledge of their role in CVL will further our understanding of the complex pathogenicity of the disease. Moreover, dogs constitute a model to study VL, since clinical signs in this specie have some similarities to those developed in humans13,14, allowing its use in the study of new targets for prophylactic and therapeutic strategies. It has been demonstrated that the production of anti-saliva antibodies in humans naturally exposed to Lu. longipalpis sand flies positively correlated with the development of delayed type hypersensitivity against Leishmania, and that in this setting, these antibodies are reported as a marker of protection against infection15. Notably, although dogs from endemic areas produce anti-saliva antibodies, there are no studies investigating whether these antibody levels correlate with protection, risk of disease development or transmission to sand flies. Nonetheless, due to the complexity of CVL, there are difficulties in establishing a clear association between clinical manifestations and immunologic status. Herein, we hypothesize that only a single parameter of cellular or humoral immune responses cannot clearly define disease severity. Integrated studies of multiple biomarkers are needed to better understand their role in the outcome of L. infantum infection. In this cross sectional exploratory study, we identified a distinct biosignature in dogs with different clinical scores where an increase in the severity of disease was characterized by a continuous decrease in levels of LTB4 and PGE2 and an increase in levels of CXCL1 and CCL2. Additionally, using 3 different parameters (LTB4, PGE2 and CXCL1) we were able to discriminate between different clinical score ranges through the construction of ROC curves. Moreover, there is an augment in the frequency of dogs displaying anti-saliva IgG and high parasite load along with the increase of the clinical score. This study allows the evaluation of multiple biomarkers in dogs, which could be important for CVL surveillance in endemic areas.

Results

Expression of immune and inflammatory markers. After diagnosing CVL in the canine random sample 21.4% (15/70) were found to be negative for CVL whereas 78.6% (55/70) animals were infected. Clinical score evaluation on the infected dogs classified 40% (22/55) dogs with subclinical disease, 38.2% (21/55) with mild disease, and 21.8% (12/55) with severe disease. All the biomarkers were analysed independently using univariate statistical analyses corrected for multiple observation, and only those ones that displayed significant differences among the different clinical groups were considered for the further analysis. A hierarchical clustering analysis of immune and inflammatory profiles in serum from dogs with different CVL clinical scores underlined a distinct biosignature associated with increased disease severity (Fig. 1). Remarkably, animals with higher severity scores (4–7 and >7) exhibited heightened serum concentrations of IL-10, CXCL1 and CCL2, whereas those with lower clinical scores (0–3) displayed increased levels of IL-6, IL-18 and CXCL10 relative to the average values of the entire study population (Fig. 1A). Infected dogs displayed reduced levels of several other biomarkers of inflammation and oxidative stress (Fig. 1A) when compared to uninfected ones. Amongst all the biomarkers, PGE2 and LTB4 values displayed a linear trend that decreased with disease severity (Fig. 1B). Conversely, we observed an upward linear trend in the amounts of CXCL1 and CCL2 with increasing clinical scores (Fig. 1B). In addition, dogs with a clinical score range from 4 to 7 displayed the highest serum levels of SOD, while those with the highest severity scores (>7) exhibited the lowest concentrations of this enzyme (Fig. 1B). Network analysis of the circulating biomarkers in dogs. We next examined the relationships between the biomarkers within each clinical group using network analysis based on statistically significant Spearman correlations (P < 0.05). We observed that the correlations profile exhibited distinct characteristics in each study group (Fig. 2A and Supplemental File 1). In addition, in all groups, most of the observed statistically significant correlations were positive (Fig. 2A and Supplemental File 1). Increased frequency of significant negative correlations was detected in the group of uninfected animals as well as in dogs with the highest severity scores (Fig. 2A). Of note, in these two groups of dogs, the majority of statistically significant correlations in the networks involving Scientific RepoRts | 6:32619 | DOI: 10.1038/srep32619

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Figure 2. Network analysis of the circulating biomarkers in dogs with VL reveals a distinct biosignature of VL clinical severity. (A) The network analysis (host interactome) shows statistically significant Spearman correlations (P < 0.05) between of the biomarkers measured. See Supplemental File 1 for additional details on the strength (r value) and level of significance (P-value) for each individual correlation. (B) Heatmap of the number of statistically significant correlations involving each biomarker measured in dogs with different VL disease severity is shown. Details on the numbers of correlations are shown is Supplemental File 1. (C) Comparisons of the network densities is shown (density calculations are described in Methods). Data was compared using permutation test. ns, non significant.

Figure 3. Using inflammatory markers to predict VL disease severity. (A–D) ROC curve analyses were performed to estimate in a quantitative way the performance of the different combinations of biomarkers used in the cluster analysis in segregating dogs diverging in VL clinical severity. AUC: area under the curve. *P < 0.05; **P < 0.01; ***P < 0.001.

LTB4 were negative associations (Fig. 2A). Moreover, following quantification of the number of significant correlations observed in each marker (node analysis), we found that the overall number of connections involving most of the markers was similar between the clinical groups (Fig. 2B and Supplemental File1). Interestingly, animals with the highest severity scores (>7) displayed increased participation of LTB4 while showing a decrease in the involvement of CCL2 in the network (Fig. 2B). Additional analyses revealed that the network densities were not significantly different between the groups (Fig. 2C).

ROC curve analyses using inflammatory markers to predict VL disease severity. ROC curve analyses were performed to estimate in a quantitative way the efficacy of different combinations of biomarkers in segregating dogs diverging in CVL clinical severity (Fig. 3). To simplify the analyses, the study groups were rearranged into: negative, score of 0–7 and score >7. We attempted to create a ROC curve combining the biomarkers that displayed a linear trend with disease severity score ranges (PGE2, LTB4, SOD, CXCL1 and CCL2, depicted in Fig. 1B). However, after testing different combinations (data not shown), the use of three biomarkers (PGE2, LTB4, and CXCL1) was shown to be the most efficient in distinguishing the clinical groups and therefore kept in the final ROC curve model. Figure 3A–C shows ROC curves for each marker PGE2, LTB4 and CXCL1, demonstrating that these three markers, when considered individually, have potential to distinguish the clinical groups. Notably, the combination of these biomarkers resulted in a better overall performance to discriminate the three clinical score categories (Fig. 3D). Associations between antibodies production against salivary recombinant proteins LJM11 and LJM17 and parasite load of infected dogs. The associations between antibody production against Scientific RepoRts | 6:32619 | DOI: 10.1038/srep32619

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Figure 4. Associations between antibodies production against salivary recombinant proteins LJM11 and LJM17 and parasite load of infected dogs. (A) Correlations between parasite load and antibodies production against salivary recombinant proteins LJM11 and LJM17 in negative and infected dogs showing different clinical signs (n = 70). Dotted lines on the X-axis represent the median value of parasite load within the group of infected dogs, while dotted lines on Y-axis indicate the cut-off value for antibodies production against LJM11 and LJM17. In (B), the correlations were stratified according to clinical score presented by the dogs. White areas designate the quadrants that include the dogs displaying values of parasite load below the median and negative antibodies production; light green area designate the quadrants that include the dogs displaying values of parasite load below the median and positive antibodies production; dark green areas designate the quadrants that include the dogs displaying values of parasite load above the median and positive antibodies production; and grey areas designate the quadrants that include the dogs displaying values of parasite load above the median and negative antibodies production. In (C) the percentage of individuals within each area were compared between the groups with different clinical score using a chi-square analysis. recombinant sand fly salivary proteins LJM11 and LJM17 and parasite load of infected dogs is presented in Fig. 4A–C. There was no correlation between antibody concentrations against these recombinant salivary proteins and parasite loads in dogs (Fig. 4A). We observed an increase in the frequency of dogs showing high parasite load and anti-saliva antibodies with the increase of severity disease (Fig. 4B,C dark green bars). Although it seems to be an increase in the frequency of dogs showing negative saliva serology and high parasite loads, the results were not statistically significant among the groups (Fig. 4B,C grey bars).

Discussion

In the present study, we evaluated different parameters associated to CVL severity, considering distinct groups of naturally infected dogs from a highly endemic area, in Camaçari, Bahia, Brazil. Our data revealed a distinct pattern of biomarkers correlated with the different clinical manifestations of CVL. We have also demonstrated that ROC curve analyses using combined biomarkers are able to distinguish dogs exhibiting different degrees of CVL severity. Furthermore, we identified that higher titers of antibodies to sand fly saliva is more frequent in highly parasitized dogs. Cross sectional analysis of serum levels of cytokines and chemokines as well as LTB4 and PGE2 in dogs suggest that the relationships between the biomarkers in the study groups, and especially those related to LTB4 and CCL2, could be directly involved in the pathogenesis of CVL. Levels of LTB4 and PGE2 displayed a significant decrease following a gradual increase in disease severity. Recently, our group has shown the importance of eicosanoids in modulating immune responses in Leishmania infection12. In addition, PGE2 has been shown to contribute to parasite proliferation11,16, whereas LTB4 increases macrophage activation and intracellular destruction17–19. Therefore, the balance between these two mediators seems to contribute to the control of Leishmania infection and inflammation. The decrease in the serum levels of both LTB4 and PGE2 found in our work indicate that dogs displaying severe disease may loose the ability to mount an efficient response to control the infection. SOD-1 has been found to play a deleterious role in Leishmania infection, increasing parasite burden in Leishmania-infected human macrophages10. In our samples, an increase in SOD was observed in the group of dogs with clinical score between 4 and 7, indicating that this enzyme could be related to the pathogenesis of disease. However, dogs with the highest clinical score displayed low levels of this enzyme, possibly reflecting exhaustion of the immune system. In the context of experimental or natural CVL infection, an up-regulation of chemokines expression in the spleen has been already described, although only CXCL10 and CCL5 were shown to be markedly elevated in oligosymptomatic dogs20. Concerning these chemokines, no significant differences in study groups were noticed. However, our findings showed a significant increase in CXCL1 and CCL2 serum levels with the increase of disease severity. CXCL1 is responsible for the recruitment of neutrophils while CCL2 is responsible for the recruitment of monocytes21,22. Results from literature have shown that dogs with CVL display neutropenia23,24 and that derangement in neutrophil numbers and function seems to be important features of CVL25,26. Moreover, Menezes-Souza et al.20 reported a higher expression of CCL2 in the skin of dogs with CVL that positively associated with a higher parasite density. The authors also reported an increase in the number of macrophages in the skin of symptomatic dogs displaying high parasitism. Our results as well as others studies suggest that these chemokines are recruiting immature or unresponsive macrophages, since animals with higher serum level of CXCL1 and CCL2 presented high levels of parasite in the spleen. We identified a signature of CVL characterized by the absence of inflammatory mediators such as IFN-γ, TNF-α, IL-2, IL-7, PGE2, LTB4, IL-15 and SOD in dogs with severe CVL. Although dogs with subclinical disease do not present many of the mediators described above, an observed increase in the levels of IL-6 and IL-18,

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www.nature.com/scientificreports/ suggests a restricted ability to control the infection. On the other hand, symptomatic dogs with a score higher than 7 showed a near absence of inflammatory mediators and an increase in IL-10, CCL2 and CXCL1 levels. Cytokines such as IFN- γ and TNF- α are considered the hallmark of protection in different clinical manifestations of leishmaniasis8. However, interactomes analysis showed that these protective cytokines do not have any correlation with the clinical status of the dogs, although in our study their levels decrease along the clinical score progression, but not in a significant way. ROC curve analysis ultimately compared the overall performance of the diverse combination of candidate biomarkers in distinguishing different clinical manifestations of CVL. The combination of PGE2, LTB4 and CXCL1 was the one that better discriminated among the groups of animals, pointing out the importance of these mediators in the follow up and prognostic of clinical manifestations in CVL. Salivary recombinant proteins are of value as markers of vector exposure. In humans and dogs, LJM11 and LJM17 emerged as potential markers of specific exposure to Lu. longipalpis27. Additionally, Gomes et al.15 positively correlates the appearance of an anti-saliva humoral response to an anti-L. infantum cell-mediated immunity raising the hypothesis that induction of immune response against sand fly saliva can facilitate the generation of a protective response against human VL. Thus, we can infer that anti-sand fly saliva antibodies can serve as an important epidemiological marker of vector exposure in endemic areas and even as a surrogate marker of protection. However, we observed an increase in the frequency of dogs with high parasite loads and anti-saliva antibodies with the increased of clinical scores. The strength of this dog study is the parasite load comparison together with the results of saliva serology. These type of data allowed the observation of the increased percentage of the highly infected and highly saliva exposed group along with disease severity. We speculate that dogs are likely more exposed to sand-fly bites in the endemic areas than humans, leading to an increase in the antibody production and higher chances of infection and elevated parasite load. This study evaluated multiple biomarkers in dogs and defined a biosignature related to different clinical manifestations of CVL. Characterization of disease severity in CVL is essential to prevent the spread of the infection, since symptomatic animals are better disseminators of parasites in endemic areas. Further studies are in progress to define a particular biosignature for dogs that transmit parasites to sand flies allowing for better surveillance in areas of CVL transmission.

Methods

A random sample of 70 dogs was selected from a cross-sectional study conducted in the municipality of Camaçari, located in the State of Bahia, North-eastern Brazil (latitude: 12° 41′ 51′′S; longitude: 38° 19′ 27′′W). This area is endemic for both VL and CVL, with a seroprevalence of CVL ranging from 20 to 40% in the canine population (unpublished data).

Study design.

This study was approved by the IGM - FIOCRUZ Institutional Review Board for Animal Experimentation under Permit Number 007/2013, within Brazilian Federal Law on Animal Experimentation (Law no. 11794), and following the guidelines for animal research established by the Oswaldo Cruz Foundation. All the dog owners signed an informed consent form, allowing the examination of the animals. Dogs were grouped according to CVL diagnosis and clinical score, as explained below.

Ethical aspects.

CVL diagnosis. Serum and splenic aspirate samples were collected as described before28 and used to perform

anti–sand fly saliva serology and biomarkers analyses. Parasitological evaluation of splenic aspirates was performed as previously described29, as well as the use of qPCR technique to assess positivity in the splenic aspirate28. The splenic parasite load was measured by qPCR technique as described elsewhere28. Anti-Leishmania antibodies were detected using the double pathway platform-screening test (DPP CVL, Bio-Manguinhos Unit, Rio de Janeiro, Brazil) followed by the confirmatory immunoassay (EIE CVL, Bio-Manguinhos Unit, Rio de Janeiro, Brazil). Dogs were considered infected if they present positive results in splenic culture or qPCR. Animals were considered uninfected if the above-mentioned tests were negative.

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All animals were clinically examined and classified according to the parameters described in Table 1. Each clinical sign was given a grade of 0, 1 or 2 depending on the intensity of the clinical manifestation. The clinical score was calculated as the sum of grades for each clinical sign with same weights. The composite score could then range from 0 to 24 points. Infected animals with a clinical score of ≤3 were classified as without clinical disease (subclinical); a clinical score of ≥4 < 7 were categorized as with mild disease and a clinical score of ≥7 was used to define severe disease.

Composite clinical severity score for CVL.

Immunoassays. Cytokine and chemokine levels in serum were measured using a pre-defined luminex-based

multiparametric kit (Milliplex Map Kit - canine cytokine magnetic bead panel, Life Technologies, Carlsbad, CA, USA). The markers examined were IFN-γ, IL-10, TNF-α, IL-2, IL-6, IL-7, IL-15, IL-8, CCL2, CXCL10, GM-CSF and CXCL1. Concentrations of PGE2, LTB4 and superoxide dismutase (SOD) were measured in serum samples from all the dogs using an enzyme-linked immunoassay (Cayman Chemical, Ann Harbor, MI, USA) as described previously10,12.

Anti–sand fly saliva serologic testing. Recombinant Lu. longipalpis salivary proteins were expressed in HEK cells and HPLC purified as previously described27. Anti–sand fly saliva serologic test ELISA was performed as described elsewhere27,30 with some adaptations for use in dogs. Antibody production against recombinant salivary proteins LJM11 and LJM17 was measured using the optical density values divided by the cut-off value of each experiment. Scientific RepoRts | 6:32619 | DOI: 10.1038/srep32619

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0

1

2

Normal or obese

Emaciate

Cachectic

Mucosa color

Normal

Anemic



Periocular dermatitis

Absent

Around one eye

Present in two eyes

Crust on ears

Absent

Present in one ear

Present in two ears

Ear Ulcers

Absent

Present in one ear

Present in two ears

Muzzle Depigmentation

Absent

In less than 1/3 of the muzzle

In more than 1/3 of the muzzle

Muzzle Hyperkeratosis

Absent

In less than 1/3 of the muzzle

In more than 1/3 of the muzzle Larger ulcerated lesion

Muzzle Lesions

Absent

Initial mucous lesion

Not palpable

Enlarged



Onychogryphosis

Absent

Slight enlargement

Excessive enlargement

Alopecia

Absent

Focal

In more than 1/3 of the body

Seborrheic dermatitis

Absent

Focal

In more than 1/3 of the body

Absent

One or two enlarged lymph nodes of the same pair

Enlarged lymph nodes of different pairs

Spleen size

Lymphadenomegaly

Table 1. VL clinical parameters employed to calculate the clinical score of each animal included in the study.

Statistical Analysis. Median values with interquartile ranges (IQR) were used as measures of central tendency. The chi-square test was employed to compare frequencies between the study groups. Hierarchical cluster analysis (Ward’s method) with bootstrap was performed to depict the overall expression profile of serum biomarkers in the negative and the different severity score groups. Significant statistical differences between groups of varying severity scores were evaluated using the Kruskal-Wallis test. Non-parametric linear trend ad hoc tests were employed to examine the variation of all biomarker levels following the clinical severity score. Biomarkers that did not exhibit linear trend were compared using the Dunn’s multiple comparisons test. Network analysis (host interactome) were generated from Spearman correlation matrices containing values of each biomarker measured in the serum samples, as described by Mendonça et al.31. The values were input in JMP 10.0 software (SAS, Cary, NC, USA). A heat map of the number of statistically significant correlations involving each biomarker was constructed for different severity score groups. Aiming to analyse the structure of the biomarker networks, the network density was calculated as described by Mendonça et al.32. Receiver-Operator Characteristic (ROC) curve analyses were used to test the power of some biomarkers (PGE2, LTB4, SOD, CLCX1 and CCL2) and their combination to distinguish dogs presenting with different stages of CVL clinical severity. For this analysis, the severity score groups 0–3 and 4–7 were combined. ROC curve analyses were performed using JMP 10.0 software. A p-value below 0.05 was considered statistically significant. Correlations between parasite load and antibodies production against salivary recombinant proteins LJM11 and LJM17 in negative and infected dogs showing different clinical signs were evaluated. The median values of parasite load and the cut-off value for antibodies production against LJM11 and LJM17 were used as measures of central tendency, differentiating high and low parameter levels. The X2 or Fisher tests were used to compare variables displayed as percentage.

References

1. Sherlock, I. A. Ecological interactions of visceral leishmaniasis in the state of Bahia, Brazil. Memorias do Instituto Oswaldo Cruz 91, 671–683 (1996). 2. Lainson, R. & Rangel, E. F. Lutzomyia longipalpis and the eco-epidemiology of American visceral leishmaniasis, with particular reference to Brazil: a review. Memorias do Instituto Oswaldo Cruz 100, 811–827, doi: /S0074-02762005000800001 (2005). 3. Molina, R. et al. Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, 491–493 (1994). 4. Bevilacqua, P. D., Paixão, H. H., Modena, C. M. & Castro, M. C. P. S. Urbanização da leishmaniose visceral em Belo Horizonte. Arq. Bras. Med. Vet. Zootec. 53, doi: 10.1590/S0102-09352001000100001 (2001). 5. Foglia Manzillo, V. et al. Prospective study on the incidence and progression of clinical signs in naive dogs naturally infected by Leishmania infantum. PLoS neglected tropical diseases 7, e2225, doi: 10.1371/journal.pntd.0002225 (2013). 6. Ciaramella, P. et al. A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum. The Veterinary record 141, 539–543 (1997). 7. Mancianti, F., Gramiccia, M., Gradoni, L. & Pieri, S. Studies on canine leishmaniasis control. 1. Evolution of infection of different clinical forms of canine leishmaniasis following antimonial treatment. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 566–567 (1988). 8. Reis, A. B., Giunchetti, R. C., Carrillo, E., Martins-Filho, O. A. & Moreno, J. Immunity to Leishmania and the rational search for vaccines against canine leishmaniasis. Trends in parasitology 26, 341–349, doi: 10.1016/j.pt.2010.04.005 (2010). 9. Barbosa, M. A. et al. Cytokine gene expression in the tissues of dogs infected by Leishmania infantum. Journal of comparative pathology 145, 336–344, doi: 10.1016/j.jcpa.2011.03.001 (2011). 10. Khouri, R. et al. SOD1 plasma level as a biomarker for therapeutic failure in cutaneous leishmaniasis. The Journal of infectious diseases 210, 306–310, doi: 10.1093/infdis/jiu087 (2014). 11. Araujo-Santos, T. et al. Prostaglandin E2/leukotriene B4 balance induced by Lutzomyia longipalpis saliva favors Leishmania infantum infection. Parasites & vectors 7, 601, doi: 10.1186/s13071-014-0601-8 (2014).

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www.nature.com/scientificreports/ 12. Franca-Costa, J. et al. Arginase I, polyamine, and prostaglandin E2 pathways suppress the inflammatory response and contribute to diffuse cutaneous leishmaniasis. The Journal of infectious diseases 211, 426–435, doi: 10.1093/infdis/jiu455 (2015). 13. Costa, D. J. et al. Experimental infection of dogs with Leishmania and saliva as a model to study Canine Visceral Leishmaniasis. PloS one 8, e60535, doi: 10.1371/journal.pone.0060535 (2013). 14. Moreno, J. & Alvar, J. Canine leishmaniasis: epidemiological risk and the experimental model. Trends in parasitology 18, 399–405 (2002). 15. Gomes, R. B. et al. Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity. The Journal of infectious diseases 186, 1530–1534, doi: 10.1086/344733 (2002). 16. Saha, A. et al. Prostaglandin E2 negatively regulates the production of inflammatory cytokines/chemokines and IL-17 in visceral leishmaniasis. J Immunol 193, 2330–2339, doi: 10.4049/jimmunol.1400399 (2014). 17. Tavares, N. M. et al. Understanding the mechanisms controlling Leishmania amazonensis infection in vitro: the role of LTB4 derived from human neutrophils. The Journal of infectious diseases 210, 656–666, doi: 10.1093/infdis/jiu158 (2014). 18. Serezani, C. H., Perrela, J. H., Russo, M., Peters-Golden, M. & Jancar, S. Leukotrienes are essential for the control of Leishmania amazonensis infection and contribute to strain variation in susceptibility. J Immunol 177, 3201–3208 (2006). 19. Chaves, M. M., Marques-da-Silva, C., Monteiro, A. P., Canetti, C. & Coutinho-Silva, R. Leukotriene B4 modulates P2X7 receptormediated Leishmania amazonensis elimination in murine macrophages. J Immunol 192, 4765–4773, doi: 10.4049/jimmunol.1301058 (2014). 20. Menezes-Souza, D. et al. Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis. PLoS neglected tropical diseases 6, e1566, doi: 10.1371/journal.pntd.0001566 (2012). 21. Bozic, C. R. et al. Expression and biologic characterization of the murine chemokine KC. J Immunol 154, 6048–6057 (1995). 22. Taub, D. D., Conlon, K., Lloyd, A. R., Oppenheim, J. J. & Kelvin, D. J. Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1 alpha and MIP-1 beta. Science 260, 355–358 (1993). 23. Freitas, J. C. et al. Clinical and laboratory alterations in dogs naturally infected by Leishmania chagasi. Revista da Sociedade Brasileira de Medicina Tropical 45, 24–29 (2012). 24. Geisweid, K., Mueller, R., Sauter-Louis, C. & Hartmann, K. Prognostic analytes in dogs with Leishmania infantum infection living in a non-endemic area. The Veterinary record 171, 399, doi: 10.1136/vr.100637 (2012). 25. Nicolato Rde, C. et al. Clinical forms of canine visceral Leishmaniasis in naturally Leishmania infantum-infected dogs and related myelogram and hemogram changes. PloS one 8, e82947, doi: 10.1371/journal.pone.0082947 (2013). 26. de Almeida Leal, G. G. et al. Immunological profile of resistance and susceptibility in naturally infected dogs by Leishmania infantum. Veterinary parasitology 205, 472–482, doi: 10.1016/j.vetpar.2014.08.022 (2014). 27. Teixeira, C. et al. Discovery of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi in Latin America. PLoS neglected tropical diseases 4, e638, doi: 10.1371/journal.pntd.0000638 (2010). 28. Solca, M. S. et al. Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis. PloS one 9, e103635, doi: 10.1371/journal.pone.0103635 (2014). 29. Barrouin-Melo, S. M. et al. Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniosis? A study on assymptomatic and polysymptomatic animals. Vet J 171, 331–339, doi: 10.1016/j.tvjl.2004.11.010 (2006). 30. Souza, A. P. et al. Using recombinant proteins from Lutzomyia longipalpis saliva to estimate human vector exposure in visceral Leishmaniasis endemic areas. PLoS neglected tropical diseases 4, e649, doi: 10.1371/journal.pntd.0000649 (2010). 31. Mendonca, V. R. et al. Unravelling the patterns of host immune responses in Plasmodium vivax malaria and dengue co-infection. Malar J 14, 315, doi: 10.1186/s12936-015-0835-8 (2015). 32. Mendonca, V. R., Queiroz, A. T., Lopes, F. M., Andrade, B. B. & Barral-Netto, M. Networking the host immune response in Plasmodium vivax malaria. Malar J 12, 69, doi: 10.1186/1475-2875-12-69 (2013).

Acknowledgements

The authors thank Andrezza Souza for technical and logistics support. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado da Bahia-FAPESB (N°SUS0036/2013 and PET0024/2013 to C.I.B.); grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (402670/2012-4 to C.I.B. and 400898/2013-6 to P.S.T.V.); grants from Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais (INCT-DT) (to D. B. M. F. and P. S. T. V.), from the Intramural Research Programs at the NIAID, NIH, USA (SK, JGV) and Instituto Nacional de Ciência e Tecnologia de Investigação em Imunologia (III-INCT) (to C. I. B.). C.I.B., P.S.T.V., V.M.B., P.T.B., are senior investigators of CNPq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Conceived and designed the experiments: M.S.S., C.R.T., R.K., P.T.B., D.B.M.F., V.M.B., P.S.T.V. and C.I.B. Performed the experiments: M.S.S., M.M.C.A., C.R.T. and R.K. Analysed the data: M.S.S., B.B.A., R.K., J.G.V., S.K., D.B.M.F., V.M.B., P.S.T.V. and C.I.B. Contributed reagents/materials/analysis tools: B.B.A., R.K., J.G.V., S.K., P.T.B., D.B.M.F., V.M.B., P.S.T.V. and C.I.B. Wrote the paper: M.S.S., B.B.A., C.R.T., R.K., J.G.V., S.K., P.T.B., D.B.M.F., V.M.B., P.S.T.V. and C.I.B.

Additional Information

Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Solcà, M. S. et al. Circulating Biomarkers of Immune Activation, Oxidative Stress and Inflammation Characterize Severe Canine Visceral Leishmaniasis. Sci. Rep. 6, 32619; doi: 10.1038/srep32619 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016

Scientific RepoRts | 6:32619 | DOI: 10.1038/srep32619

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Supplementary information Solcà et al., 2016

Spearman correlations and p-values of the host interactome NEGATIVE Variable 1 IL2 TNFa CCL2 IL 10 IL 15 IL 18 IL2 IL6 IL7 TNFa CCL2 IL 18 IL2 TNFa CCL2 IL2 TNFa CCL2 IL 15 IL 18 IL2 IL7 TNFa CCL2 IL 15 IL 18 IL2 TNFa CCL2 IL 15 IL2 IL6 CCL2 IL 10 LTB4 pg/ml IL2

Variable 2 CCL2 CCL2 GM-CSF GM-CSF GM-CSF GM-CSF GM-CSF GM-CSF GM-CSF GM-CSF IL 15 IL 15 IL 15 IL 15 IL 18 IL 18 IL 18 IL6 IL6 IL6 IL6 IL6 IL6 IL7 IL7 IL7 IL7 IL7 LTB4 pg/ml LTB4 pg/ml LTB4 pg/ml LTB4 pg/ml PGE2 pg/ml PGE2 pg/ml PGE2 pg/ml TNFa

R value 0,8528 0,743 0,8969 0,5591 0,8458 0,7523 0,9069 0,7265 0,8572 0,7794 0,849 0,7512 0,8459 0,9091 0,8981 0,7397 0,6427 0,8014 0,8909 0,7331 0,8768 0,8791 0,8395 0,8832 0,8768 0,8212 0,903 0,8904 -0,5576 -0,5624 -0,5293 -0,5916 -0,5827 -0,5252 0,5893 0,79

P value <.0001 0,0015 <.0001 0,0303 <.0001 0,0012 <.0001 0,0022 <.0001 0,0006 <.0001 0,0012 <.0001 <.0001 <.0001 0,0016 0,0098 0,0003 <.0001 0,0019 <.0001 <.0001 <.0001 <.0001 <.0001 0,0002 <.0001 <.0001 0,0308 0,0291 0,0425 0,0202 0,0226 0,0444 0,0208 0,0005

Subclinical 0-3 Variable 1 Variable 2 R value P value CCL2 IL6 0,9496 <.0001 IL7 IL6 0,9431 <.0001 IL2 IL6 0,9361 <.0001 IL2 IL7 0,9289 <.0001 TNFa IL7 0,9245 <.0001 CCL2 IL7 0,9228 <.0001 TNFa CCL2 0,8935 <.0001 TNFa IL6 0,8914 <.0001 IL 18 IL 15 0,8828 <.0001 CCL2 IL 15 0,8616 <.0001 IL 15 GM-CSF 0,8601 <.0001 IL 18 IL7 0,8583 <.0001 IL2 TNFa 0,854 <.0001 TNFa IL 18 0,8483 <.0001 IL 18 GM-CSF 0,8439 <.0001 IL2 CCL2 0,8422 <.0001 IL 15 IL7 0,8343 <.0001 TNFa GM-CSF 0,8256 <.0001 IL7 GM-CSF 0,8191 <.0001 TNFa IL 15 0,8173 <.0001 CCL2 IL 18 0,8144 <.0001 IL 15 IL6 0,7995 <.0001 IL2 IL 18 0,7968 <.0001 IL 18 IL6 0,7811 <.0001 CCL2 GM-CSF 0,7536 <.0001 IL6 GM-CSF 0,7526 <.0001 IL2 GM-CSF 0,7449 <.0001 IL2 IL 15 0,7178 0,0001 LTB4 pg/ml PGE2 pg/ml 0,6311 0,0012 CXCL1 IL8 0,5809 0,0037 CXCL10 LTB4 pg/ml 0,4697 0,0237 CXCL1 PGE2 pg/ml 0,4688 0,024 CXCL1 GM-CSF -0,4428 0,0344

CVL Moderate 4-7 Variable 1 Variable 2 R value P value IL2 GM-CSF 0,9199 <.0001 IL2 IL7 0,9012 <.0001 IL 18 IL 15 0,8767 <.0001 IL7 IL6 0,8532 <.0001 IL7 GM-CSF 0,8402 <.0001 IL 15 IL7 0,839 <.0001 IL 18 IL6 0,8377 <.0001 IL 18 IL7 0,8362 <.0001 TNFa IL7 0,8321 <.0001 TNFa GM-CSF 0,83 <.0001 IL 15 IL6 0,8277 <.0001 IL2 IL6 0,8277 <.0001 IL2 TNFa 0,8178 <.0001 TNFa IL6 0,8001 <.0001 TNFa IL 18 0,791 <.0001 IL2 IL 15 0,7791 <.0001 IL2 IL 18 0,7705 <.0001 IL6 GM-CSF 0,7471 <.0001 LTB4 pg/ml PGE2 pg/ml 0,7409 <.0001 IL 18 GM-CSF 0,7221 <.0001 IL 15 GM-CSF 0,7126 <.0001 TNFa IL 15 0,6603 0,0004 CCL2 IL6 0,588 0,0025 CXCL1 CXCL10 0,5673 0,0038 TNFa CCL2 0,5646 0,004 CCL2 CXCL1 0,5396 0,0065 CXCL1 IL 10 0,5254 0,0084 IL8 LTB4 pg/ml 0,5163 0,0098 CXCL1 IL6 0,5148 0,01 CCL2 IL 18 0,5099 0,0109 CXCL1 IL 18 0,4575 0,0246 CCL2 GM-CSF 0,4449 0,0294 IL2 CCL2 0,4347 0,0338 CCL2 IL7 0,4213 0,0403 CXCL10 IL 10 0,411 0,046

CVL Severe >7 Variable 1 Variable 2 CCL2 CXCL1 CCL2 IL8 CXCL1 IL8 CXCL10 IL 18 CXCL10 IL 10 GM-CSF LTB4 pg/ml IL 15 GM-CSF IL 15 IL7 IL 15 IL6 IL 15 INFg IL 15 LTB4 pg/ml IL 18 GM-CSF IL 18 IL 15 IL 18 IL7 IL 18 IL6 IL2 IL6 IL2 IL7 IL2 IL 15 IL2 GM-CSF IL2 IL 18 IL2 LTB4 pg/ml IL6 GM-CSF IL6 LTB4 pg/ml IL7 GM-CSF IL7 IL6 IL7 LTB4 pg/ml IL7 INFg INFg LTB4 pg/ml SOD U/ml LTB4 pg/ml TNFa IL 15 TNFa CCL2 TNFa CXCL1 TNFa GM-CSF TNFa IL8 TNFa IL7

R value 0,7088 0,6923 0,7692 0,5746 0,5654 -0,5691 0,9692 0,923 0,7721 0,6328 -0,5989 0,8852 0,809 0,6829 0,6424 0,9487 0,8796 0,8359 0,8349 0,6968 -0,6093 0,7351 -0,6155 0,8717 0,8374 -0,6357 0,5553 -0,663 -0,6929 0,6769 0,6648 0,6593 0,6354 0,6209 0,5635

P value 0,0067 0,0087 0,0021 0,04 0,044 0,0424 <.0001 <.0001 0,002 0,0203 0,0305 <.0001 0,0008 0,0101 0,0179 <.0001 <.0001 0,0004 0,0004 0,0081 0,0271 0,0042 0,0251 0,0001 0,0004 0,0195 0,0488 0,0135 0,0181 0,011 0,0132 0,0142 0,0196 0,0235 0,0449

Node Analysis marker GMCSF IL-8 IFNg TNFa IL-2 IL-7 PGE2 LTB4 IL-15 SOD IL-6 IL-18 CXCL10 IL-10 CXCL1 CCL2

negative

0-3 7 0 0 6 8 7 3 5 8 0 8 6 0 2 0 8



4-7 8 0 0 7 7 7 2 2 7 0 6 8 1 0 3 7

>7 7 1 0 7 7 7 1 2 6 0 8 8 2 2 5 7

7 1 3 7 6 8 0 7 8 1 7 9 2 1 3 3

106

DISCUSSÃO GERAL

No presente trabalho foram avaliados biomarcadores de exposição à saliva do vetor, biomarcadores parasitológicos e identificados biomarcadores inflamatórios que se correlacionaram com a gravidade da infecção por L. infantum em cães. A carga parasitária se mostrou um bom marcador parasitológico, havendo correlação desta com o estado clínico dos animais infectados. Da mesma forma, a produção de anticorpos contra as proteínas da saliva do vetor LJM11/LJM17 se correlacionou com a gravidade do quadro clínico dos animais. Dentre os biomarcadores inflamatórios avaliados, foi identificado um perfil caracterizado pelo aumento de CXCL1 e CCL2 e redução de LTB-4 e PGE-2 apresentado pelos animais com quadro clínico mais grave de LVC. É importante ressaltar que a classificação dos animais utilizando como base os sinais clínicos não é a melhor forma para determinar o estadiamento da LVC (SOLANO-GALLEGO et al., 2009). Diversos autores apontam que as alterações hematológicas e bioquímicas provocadas pela infecção do parasito são também importantes para determinar a gravidade da infecção no animal (TROPIA DE ABREU et al., 2011; FREITAS et al., 2012). Um parâmetro importante a ser avaliado é a função renal, uma vez que a LVC está associada a uma alta prevalência de doença renal crônica (COSTA et al., 2003). Solano-Gallego et al., (2011) em conjunto com outros participantes do grupo LeishVet, criaram um método de estadiamento da LVC baseado em 4 graus, desde doença leve até muito grave, baseado nas manifestações clínicas do animal, em conjunto com os achados hematológicos, bioquímicos e de urinálise. Em nosso estudo optamos por estadiar os cães baseado em presença e intensidade de sinais clínicos e da carga parasitária, e não uma avaliação mais completa dos animais, incluindo avaliações bioquímicas e hematológicas, principalmente, por razões de custo. A quantificação do número de parasitas nos tecidos dos animais infectados auxilia no monitoramento da progressão da doença clínica, ajudando a avaliar a gravidade da infecção nos animais (MANNA et al., 2008a), uma vez que os sinais clínicos apresentados pelos animais acometidos por L. infantum podem ser inespecíficos e podem não corresponder diretamente com a disseminação do parasita no organismo, como já foi demonstrado por Paiva Cavalcanti et al., (2015).

107

No presente trabalho (Capítulo 1), foi observada associação positiva significativa entre a intensidade da carga parasitária no baço e o número de sinais clínicos presentes nos cães. Os animais sem sinais clínicos ou os que apresentaram de 1-3 sinais clínicos tiveram cargas parasitárias menores no tecido esplênico, quando comparados com animais com quadro clínico de LVC mais grave (SOLCA et al., 2014). Esta correlação evidencia que a carga parasitária pode ser um bom biomarcador parasitológico para correlação com o quadro clínico dos animais. Esse resultado foi reforçado na segunda parte do estudo (Capítulo 2), no qual a carga parasitária também mostrou associação positiva, estatisticamente significativa, com a gravidade do quadro clínico da LVC (dado não apresentado no paper publicado de Solcà et al., 2016). O tecido esplênico apresentou maior taxa de positividade e maior carga parasitária nos cães infectados por Leishmania, quando comparado aos outros tecidos avaliados (Capítulo 1). Este achado pode ser explicado, devido ao baço ser um órgão importante de interação entre o sistema imune e os parasitos durante o curso da infecção (SANTANA et al., 2008). Além disso, já foi demonstrado que o baço mantém a infecção do parasito durante todo o curso da LVC (REIS et al., 2006a). Um avanço na forma de quantificar a carga parasitária foi obtido na presente tese, que consistiu no desenvolvimento e validação do protocolo de qPCR em configuração duplex em formato líquido e no formato em gel (ready-to-use), inicialmente estabelecido em protocolo singleplex (Capítulo 1) (FRANCINO et al., 2006; SOLCA et al., 2014). Esse avanço, abre a possibilidade de, não somente, podermos quantificar a carga parasitária dos animais infectados, como utilizá-lo como uma ferramenta diagnóstica de mais fácil utilização, que poderia ser útil para clínica e estudos científicos (ROURA et al., 2013). A detecção precisa da carga parasitária presente no cão infectado pela Leishmania é adicionalmente importante no monitoramento das infecções naturais, para acompanhamento de animais em tratamento e em acompanhamentos de cães avaliados em estudos experimentais (ROLÃO et al., 2004; PENNISI et al., 2005; MANNA et al., 2006; MANNA et al., 2008a; MANNA et al., 2008b). A detecção simultânea do gene 18S canino, no protocolo de qPCR desenvolvido, reforça a validade dos resultados desse método, principalmente para os resultados negativos. Naranjo et al., 2011 também empregaram como alvo para o

108

controle interno da reação o 18S rRNA, avaliando cães infectados por L. infantum, já outros autores empregaram outros alvos como beta actina (MANNA et al., 2008a; MANNA et al., 2009; MAIA et al., 2010) e beta globulina (QUARESMA et al., 2009). A confirmação da presença do DNA genômico nas amostras caninas, exclui a possibilidade de resultados falso negativos, pois é possível atestar que quando ocorre amplificação do DNA do hospedeiro, a ausência de amplificação do DNA de Leishmania não é devida a ausência de DNA ou presença de inibidores na amostra. O formato duplex da qPCR desenvolvida reforça ainda mais esse resultado, pois diminui o tempo de execução, combinando as duas reações de qPCR em uma, permitindo dessa forma a avaliação de amostras com pouco rendimento de DNA genômico após sua extração. Contudo, a qPCR continua um método complexo para ser inserido na rotina diagnóstica da LVC, principalmente como teste de triagem na saúde pública. Ademais, o tecido esplênico, apesar de sua elevada sensibilidade na detecção do parasito, apresenta uma coleta que demanda conhecimento e habilidade para execução, equipamentos específicos assim como medicamentos, sendo desta forma inviável de ser realizada em estudos epidemiológicos de saúde pública (GONTIJO e MELO, 2004; BARROUIN-MELO et al., 2006; CARVALHO et al., 2009; WATSON et al., 2011; SOLCA et al., 2014). No Brasil, a triagem para LVC é realizada por um teste rápido que apresenta uma série de vantagens como o fato de dispensar equipamentos laboratoriais, necessidade de uma amostra de coleta pouco invasiva e rapidez que fazem com que este seja o teste ideal a ser empregado na triagem dos cães em área endêmica em programas de saúde pública (BOARINO et al., 2005; GRIMALDI et al., 2012a). Todavia, a qPCR poderia ser empregada como teste confirmatório, principalmente no seu formato duplex ready to use, para uso em clínicas veterinárias, mas principalmente tem uma aplicabilidade para avaliação diagnóstica de LV em seres humanos, uma vez que alguns indivíduos infectados são também HIV positivos, não produzindo anticorpos específicos contra o parasito e sendo dificilmente detectados em testes sorológicos (ALVAR et al., 1997; ALVAR et al., 2008). Neste trabalho foi mostrado como a carga parasitária é um importante biomarcador para o monitoramento da infecção (Capítulo 1), desta forma a qPCR é uma ferramenta muito importante para pesquisa em LVC. A avaliação precisa da carga parasitária em infecções experimentais pode permitir avanços em estudos de

109

avaliação da patogênese da enfermidade, assim como, comparações confiáveis em estudos de avaliação da eficácia de drogas anti-Leishmania (ROURA et al., 2013). Pennisi et al., (2005) empregaram a qPCR para monitorar os efeitos do tratamento por

allopurinol

em

cães

experimentalmente

infectados

por

L.

infantum,

demonstrando como essa abordagem pode mostrar claramente a eficácia da droga, ao longo do tratamento, uma vez que foi possível por essa técnica demonstrar que o tratamento levou à diminuição do número de parasitas em diferentes tecidos dos animais. No Brasil, o tratamento da LVC é fiscalizado pelo MS, que não recomenda esta prática através da emissão de uma instrução normativa, sendo proibido o uso de medicamentos humanos para o tratamento dos animais (Portaria Interministerial n

o

1426, de 11 de julho de 2008). Contudo, existe uma série de outros

medicamentos, como allopurinol e miltefosina, que são empregados para tratar animais

infectados

(KOUTINAS

et

al.,

2001;

PENNISI

et

al.,

2005;

SARIDOMICHELAKIS et al., 2005; TORRES et al., 2011) associado ao uso de métodos repelentes. Ribeiro et al. (2013), pelas recomendações do grupo Brasileish sugerem que quando a opção for o tratamento, devem ser instituídos os protocolos que produzam cura clínica e redução da carga parasitária, que deve ser sustentada por meio de investigações clínicas e laboratoriais. Assim a qPCR pode ser importante também para clínicos veterinários que tenham interesse em tratar os animais acometidos por L. infantum e monitorar a progressão da doença de forma ambulatorial com uma ferramenta acurada e relevante. Uma perspectiva do presente estudo é a possibilidade de podermos acompanhar prospectivamente a infecção em animais subclínicos, para avaliar o desfecho clínico destes, validar os biomarcadores de gravidade encontrados e possivelmente encontrar outros biomarcadores indicativos de resistência ou susceptibilidade à infecção. Em nosso estudo, a utilização da qPCR duplex foi usada como uma ferramenta indispensável para detecção da infecção por L. infantum e quantificação precisa da carga parasitária dos animais e, com isso, avaliação da disseminação do parasito e realização de estudos de correlação da carga com os biomarcadores identificados (Capítulo 2). Em nosso estudo (Capítulo 2), foi observada associação entre maior frequência de cães com alta carga parasitária e presença de anticorpos anti-saliva

110

de flebotomíneos com maior gravidade da doença nos cães. Previamente, foi demonstrado em humanos, que a produção de anticorpos anti-saliva apresenta correlação positiva com o desenvolvimento de reação de hipersensibilidade tardia contra a Leishmania e constitui um potencial marcador de proteção contra a infecção parasitária (GOMES et al., 2002). No presente trabalho, mostramos que essa tendência parece ser oposta uma vez que os cães mais acometidos apresentaram maior frequência de positividade para anticorpos anti-saliva no soro. Anteriormente, foi demonstrado que cães da área endêmica produzem anticorpos anti-saliva (TEIXEIRA et al., 2010), contudo não existem estudos que investiguem se a frequência e os níveis desses anticorpos se correlacionam com o risco de desenvolvimento de doença ou transmissibilidade para os flebotomíneos em cães. Adicionalmente, foi identificada uma tendência dos animais com produção de anticorpos anti-saliva apresentarem maior carga parasitária, o que pode indicar uma maior exposição destes animais ao inseto vetor. Vale ressaltar que este é um estudo transversal, onde poucas inferências podem ser assumidas, sendo, portanto, necessário um estudo de acompanhamento para determinar se a exposição prévia à saliva do vetor pode influenciar no curso da infecção por L. infantum. A identificação de um biomarcador que permita avaliar a exposição dos animais ao vetor e que se correlacione com o desfecho clínico poderá auxiliar no entendimento do ciclo da LV e LVC nas áreas endêmicas. Nesta segunda etapa foi identificada uma bioassinatura distinta em cães com diferentes

manifestações

clínicas,

caracterizada

por

menores

níveis

de

biomarcadores como LTB-4 e PGE-2 (Capítulo 2) em animais com quadro grave de LVC. Já foi demonstrado que a diminuição dos níveis de PGE-2 contribui para a proliferação parasitária (ARAUJO-SANTOS et al., 2014; SAHA et al., 2014) enquanto que LTB-4 aumenta a ativação de macrófagos e a destruição intracelular do parasita (CHAVES et al., 2014; TAVARES et al., 2014). Portanto, o equilíbrio entre esses dois mediadores parece contribuir para o controle da infecção por Leishmania. Araujo-Santos et al., (2014) demonstraram que o equilíbrio na relação PGE-2 / LTB-4 pode ser importante para a modulação da resposta imune provocada pela saliva do vetor, permitindo o aumento da viabilidade do parasita, bem como a carga parasitária dentro dos leucócitos durante os primeiros momentos de exposição a L. infantum.

111

Ademais,

no

presente

trabalho,

foi

encontrada

uma

bioassinatura

caracterizada também por maiores níveis de CXCL1 e CCL2 nos cães apresentando doença mais grave (Capítulo 2). CXCL1 é uma quimiocina expressa por macrófagos, neutrófilos e células epiteliais, e tem atividade quimioatratora de neutrófilos (ABBAS, LICHTMAN e PILLAI, 2012). Estudos sugeriram que CXCL1 pode ser específica para a inflamação de origem infecciosa, já tendo sido detectada significativamente aumentada no sobrenadante de células mononucleares caninas estimuladas com lipopolissacárideos (KARLSSON et al., 2016). CCL2 por sua vez, é referida como proteína quimioatratora de monócitos, recruta monócitos, células T de memória e células dendríticas para os locais de inflamação produzidos por lesão tecidual ou infecção (ABBAS, LICHTMAN e PILLAI, 2012). Até o presente momento, nenhum estudo procurou avaliar a associação destas quimiocinas com a gravidade da infecção por L. infantum em cães, sendo no futuro interessante realizar uma correlação com os níveis dessas quimiocinas e com os achados laboratoriais de leucograma para averiguar sua influência na quantidade de monócitos e neutrófilos circulantes. É bem descrito na literatura a importância de outras citocinas como IFN-y e TNF-α para ativar macrófagos e matar os parasitas (REIS et al., 2010). Estas citocinas são consideradas como marcadores de proteção em diferentes manifestações clínicas de leishmaniose. A IL-10, uma citocina anti-inflamatória, é considerada uma das citocinas responsáveis pela gravidade da doença, uma vez (SOLANO-GALLEGO et al., 2006; YABSLEY et al., 2008)que seus níveis já foram encontrados aumentados nos sobrenadantes de cultura celular de pacientes com LV, contribuindo para a imunossupressão observada nesses indivíduos (SAHA et al., 2007). Entretanto, nossos resultados não demonstram nenhuma correlação entre essas citocinas e o estádio clínico dos cães, embora os seus níveis sejam mais baixos em cães com menor gravidade do quadro clínico, mas não de forma significativa. Vale salientar que não pesquisamos a presença de coinfecção nos cães utilizados em nosso estudo. Os agentes causadores de hemoparasitoses mais comuns em cães são Ehrlichia canis, Babesia canis e Anaplasma platys (GUILLEN LLERA et al., 2002; BEUGNET e MARIE, 2009; MEKUZAS et al., 2009; VASCELLARI et al., 2016). Estes são patógenos intracelulares que utilizam monócitos-macrófagos, hemácias e plaquetas como células hospedeiras (GREENE,

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2006). A epidemiologia destas hemoparasitoses caninas e da LVC sobrepõe-se em muitas áreas do mundo, tropicais e não tropicais, uma vez que a atividade dos vetores (carrapatos e flebotomíneos) e os períodos de transmissão desses patógenos são semelhantes (BEUGNET, CHALVET-MONFRAY e LOUKOS, 2009; MENN, LORENTZ e NAUCKE, 2010; OTRANTO e DANTAS-TORRES, 2010). O aspecto mais importante de avaliar a presença de outros patógenos em estudos de patogênese e resposta imune é que a maioria destes também se dissemina da pele para o baço, fígado e medula óssea, da mesma forma que L. infantum e ativam mecanismos imunológicos do hospedeiro que induzem uma gama de respostas imunopatológicas semelhantes (MEKUZAS et al., 2009; VASCELLARI et al., 2016). Já foi relatada a produção de citocinas pró-inflamatórias, como o TNF-α por parte dos linfócitos T CD8+ em animais infectados por E. canis (ISMAIL et al., 2004), adicionalmente já foi demonstrado que a imunopatogenia da erliquiose canina pode estar relacionada à̀ produção descontrolada de TNF-α pelos linfócitos T CD8+ na fase aguda da infecção e, em menor grau, durante a fase assintomática da infecção (HARRUS et al., 1997; HARA et al., 2004). Apesar de não termos encontrado níveis expressivos aumentados dessas citocinas, no presente estudo, a presença de coinfecções pode levar a um sinergismo na resposta imune específica da LVC (MEKUZAS et al., 2009; CARVALHO, 2015), assim no estudo longitudinal em progressão decidimos realizar essa pesquisa para poder discriminar realmente quais os biomarcadores específicos da infecção por L. infantum. O estudo exploratório realizado foi muito importante uma vez que permitiu avaliar diferentes possíveis biomarcadores, e correlacioná-los com a gravidade da infecção por L. infantum apresentada pelos animais examinados. Estudos transversais como esse, permitem uma análise mais ampla de redes de causalidade na qual são elaboradas hipóteses que podem ser validadas em estudos longitudinais. Adicionalmente, estudos transversais são mais rápidos, mais baratos, mais fáceis em termos logísticos (FRONTEIRA, 2013). Contudo, vale salientar que em um estudo de corte transversal o animal está sendo avaliado pontualmente quanto a sua situação clínica, não sendo possível distinguir por exemplo, um animal subclínico resistente de um animal subclínico em fase de latência. Desta forma, é importante que os biomarcadores identificados no estudo transversal sejam validados sucessivamente em um estudo longitudinal, o que está sendo desenvolvido. Neste estudo longitudinal será possível confirmar os dados deste

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trabalho e avaliar quais biomarcadores estão relacionados com a progressão da enfermidade e seus diferentes desfechos clínicos, uma vez que o estudo de coorte permite distinguir um cão resistente de um animal em fase de latência, que irá desenvolver a forma clínica da LVC, pois avaliamos a progressão da infecção e da doença nos animais acompanhados. Os cães assintomáticos, em fase de latência, são importantes alvos de estudo, pois quando estes são mantidos na área endêmica podem participar da dinâmica de transmissão da LV, uma vez que os cães sintomáticos geralmente são removidos e os animais latentes permanecerão e poderão desenvolver LVC (BURATTINI et al., 1998; NUNES et al., 2008). Assim, a identificação precoce de marcadores de progressão de cães assintomáticos que estão em fase de latência, nos quais ocorrerá o aparecimento de sinais clínicos, permitirá intervenções mais rápidas e eficazes no controle da LV no Brasil. Assim como, a identificação de um conjunto de biomarcadores dentre os animais que não desenvolvem o quadro clínico da doença e que poderão ser relacionados com a resistência a LVC, podendo ajudar a identificar esses animais no futuro, em áreas endêmicas, e indicar a eutanásia quando o cão tiver um papel na transmissão da LV. A identificação dos biomarcadores de progressão da doença pode ser importante para uma melhor compreensão da patogênese da doença, e, futuramente, direcionar o prognóstico e manejo terapêutico dos animais acometidos, bem como novos alvos para estratégias profiláticas e terapêuticas. Assim, neste trabalho foi possível concluir que existem biomarcadores parasitológicos como a carga parasitária, biomarcadores imunológicos e inflamatórios como CXCL1, CCL2, LTB-4, PGE-2, além da produção de anticorpos específicos contra as proteínas LJM11 e LJM17 da saliva do vetor, que são capazes de diferenciar animais infectados por L. infantum de acordo com seus diferentes quadros clínicos.

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REFERÊNCIAS

ABBAS, A. K.; LICHTMAN, A. H.; PILLAI, S. H. I. V. Imunologia Celular e Molecular. 7. ed. Rio de Janeiro: Elsevier, 2012. ABDELADHIM, M.; KAMHAWI, S.; VALENZUELA, J. G. What's behind a sand fly bite? The profound effect of sand fly saliva on host hemostasis, inflammation and immunity. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, v. 28, p. 691-703, dec. 2014. ALEIXO, J. A. et al. Atypical American visceral leishmaniasis caused by disseminated Leishmania amazonensis infection presenting with hepatitis and adenopathy. Transaction of Royal Society of Tropical Medicine and Hygiene, v. 100, n. 1, p. 79-82, jan. 2006. ALEXANDER, J.; RUSSELL, D. G. The interaction of Leishmania species with macrophages. Advances in Parasitology, v. 31, p. 175-254, 1992. ALEXANDER, J.; SATOSKAR, A. R.; RUSSELL, D. G. Leishmania species: models of intracellular parasitism. Journal of Cell Science, v. 112 Pt 18, p. 2993-3002, sep. 1999. ALVAR, J. et al. The relationship between leishmaniasis and AIDS: the second 10 years. Clinical Microbiology Reviews, v. 21, n. 2, p. 334-359, apr. 2008. ALVAR, J. et al. Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clinical Microbiology Reviews, v. 10, n. 2, p. 298-319, apr. 1997. ALVAR, J. et al. Canine leishmaniasis. Advances in Parasitology, v. 57, p. 1-88, 2004. ALVAR, J. et al. Leishmaniasis worldwide and global estimates of its incidence. PloS One, v. 7, n. 5, p. e35671, 2012. ALVES, W. A.; BEVILACQUA, P. D. Reflexões sobre a qualidade do diagnóstico da leishmaniose visceral canina em inquéritos epidemiológicos: o caso da epidemia de Belo Horizonte, Minas Gerais, Brasil, 1993-1997. Cadernos de Saúde Pública, v. 20, n. 1, p. 259-265, 2004. AMORIM, L. C. A. Biomarkers for evaluating exposure to chemical agents present in the environment. Revista Brasileira de Epidemiologia, v. 6, n. 2, p. 158-170, 2003. ANDRADE, B. B. et al. Heme impairs prostaglandin E2 and TGF-beta production by human mononuclear cells via Cu/Zn superoxide dismutase: insight into the pathogenesis of severe malaria. Journal of Immunology, v. 185, n. 2, p. 1196-1204, jul. 2010.

115

ANDRADE, B. B.; BARRAL-NETTO, M. Biomarkers for susceptibility to infection and disease severity in human malaria. Memorias do Instituto Oswaldo Cruz, v. 106 Suppl 1, p. 70-78, aug. 2011. ANDRADE, B. B. et al. Role of sand fly saliva in human and experimental leishmaniasis: current insights. Scandinavian Journal of Immunology, v. 66, n. 23, p. 122-127, aug.-sep. 2007. ANTINORI, S. et al. Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in HIV-infected and HIV-uninfected patients: a single-center, 8year experience in Italy and review of the literature. Clinical Infectious Diseases, v. 44, n. 12, p. 1602-1610, jun. 2007. ANTOINE, J. C. et al. Parasitophorous vacuoles of Leishmania amazonensisinfected macrophages maintain an acidic pH. Infection and Immunity, v. 58, n. 3, p. 779-787, mar. 1990. AOUN, O. et al. Canine leishmaniasis in south-east of France: screening of Leishmania infantum antibodies (western blotting, ELISA) and parasitaemia levels by PCR quantification. Veterinary Parasitology, v. 166, n. 1-2, p. 27-31, dec. 2009. AQUINO, D. M. et al. Epidemiological study of the association between antiLutzomyia longipalpis saliva antibodies and development of delayed-type hypersensitivity to Leishmania antigen. The American Journal of Tropical Medicine and Hygiene, v. 83, n. 4, p. 825-827, oct. 2010. ARAUJO-SANTOS, T. et al. M. Prostaglandin E2/leukotriene B4 balance induced by Lutzomyia longipalpis saliva favors Leishmania infantum infection. Parasites & Vectors, v. 7, p. 601, 2014. BANETH, G. et al. Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one. Trends in Parasitology, v. 24, n. 7, p. 324-330, jul. 2008. BARBOZA, D. C. P. M. et al. Estudo de coorte em áreas de risco para leishmaniose visceral canina, em municípios da Região Metropolitana de Salvador, Bahia, Brasil. Revista Brasileira de Saúde e Produção Animal, v. 7, n. 2, p. 152-163, 2006. BARRAL, A. et al. Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. The American journal of Tropical Medicine and Hygiene, v. 44, n. 5, p. 536-546, may. 1991. BARROUIN-MELO, S. M. et al. Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniosis? A study on assymptomatic and polysymptomatic animals. The Veterinary Journal, v. 171, n. 2, p. 331-339, mar. 2006. BARROUIN-MELO, S. M. et al. Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs. Memorias do Instituto Oswaldo Cruz, v. 99, n. 2, p. 195197, mar. 2004.

116

BASTIEN, P.; PROCOP, G. W.; REISCHL, U. Quantitative real-time PCR is not more sensitive than "conventional" PCR. Journal of Clinical Microbiology, v. 46, n. 6, p. 1897-900, jun. 2008. BATES, P. A. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. International Journal for Parasitology, v. 37, n. 10, p. 1097-1106, aug. 2007. BELKAID, Y. et al., D. L. Development of a natural model of cutaneous leishmaniasis: powerful effects of vector saliva and saliva preexposure on the longterm outcome of Leishmania major infection in the mouse ear dermis. The Journal of Experimental Medicine, v. 188, n. 10, p. 1941-1953, nov. 16 1998. BENDERITTER, T. et al. Glomerulonephritis in dogs with canine leishmaniasis. Annals of Tropical Medicine and Parasitology, v. 82, n. 4, p. 335-341, aug. 1988. BEUGNET, F.; CHALVET-MONFRAY, K.; LOUKOS, H. FleaTickRisk: a meteorological model developed to monitor and predict the activity and density of three tick species and the cat flea in Europe. Geospatial Health, v. 4, n. 1, p. 97113, nov. 2009. BEUGNET, F.; MARIE, J. L. Emerging arthropod-borne diseases of companion animals in Europe. Veterinary Parasitology, v. 163, n. 4, p. 298-305, aug. 2009. BEVILACQUA, P. D. et al. Urbanização da leishmaniose visceral em Belo Horizonte. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v. 53, n. 1, 2001. BIOMARKERS DEFINITIONS WORKING GROUP. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clinical Pharmacology & Therapeutics, v. 69, n. 3, p. 89-95, mar. 2001. BOARINO, A. et al. Development of recombinant chimeric antigen expressing immunodominant B epitopes of Leishmania infantum for serodiagnosis of visceral leishmaniasis. Clinical and Diagnostic Laboratory Immunology, v. 12, n. 5, p. 647-53, May 2005. BORDONI, G. M. Prevalência, distribuição e identificação de prováveis fatores de risco para Leishmaniose Visceral canina em Camaçari - BA. 2014. Dissertação (Mestrado em Biotecnologia em Saúde e Medicina Investigativa). Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador. BORJA, L. S. et al. Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors. Veterinary Parasitology, v. 229, p. 110-117, oct. 2016. BRASIL. Portaria Interministerial. 1426 1 de julho de 2008. BRASIL. Ministério da Saúde, S. D. V. E. S. Departamento de Vigilância das Doenças Transimissíveis. Nota técnica conjunta n°01/2011. Brasília, DF., 2011.

117

BRASIL. Ministério da Saúde e Secretaria de Vigilância em Saúde Epidemiológica. D. D. V. N. Manual de Vigilância e Controle da Leishmaniose Visceral. Brasília, 2014. BURATTINI, M. et al. Modelling the dynamics of leishmaniasis considering human, animal host and vector populations. Journal of Biological Systems, v. 4, p. 337356, 1998. CAMARGO-NEVES, V. L. et al. Use of spatial analysis tools in the epidemiological surveillance of American visceral leishmaniasis, Aracatuba, Sao Paulo, Brazil, 19981999. Cadernos de Saude Pública, v. 17, n. 5, p. 1263-1267, sep.-oct. 2001. CAMPINO, L. et al. Infectivity of promastigotes and amastigotes of Leishmania infantum in a canine model for leishmaniosis. Veterinary Parasitology, v. 92, n. 4, p. 269-275, oct. 2000. CARVALHO, D. et al. An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs. Pesquisa Veterinária Brasileira, v. 29, n. 2, p. 120-124, 2009. CARVALHO, R. M. D. A. Estudo da coinfecção Leishmania infantum e Ehrlichia canis em cães numa área endêmica para leishmaniose visceral canina. 2015. 79 f. Tese (Doutorado)- Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz, Salvador, 2015. CAVALCANTI, A. S. et al. Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum-naturally infected dogs. PloS One, v. 10, n. 4, p. e0123009, 2015. CHARLAB, R. et al. Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis. PNAS, Proceedings of the National Academy of Sciences, v. 96, n. 26, p. 15155-15160, dec. 21 1999. CHAVES, M. M. et al. Leukotriene B4 modulates P2X7 receptor-mediated Leishmania amazonensis elimination in murine macrophages. Journal of Immunology, v. 192, n. 10, p. 4765-7473, may. 2014. CIARAMELLA, P. et al. A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum. The Veterinary Record, v. 141, n. 21, p. 539-43, Nov 22 1997. COLLIN, N. et al. Sand fly salivary proteins induce strong cellular immunity in a natural reservoir of visceral leishmaniasis with adverse consequences for Leishmania. PLoS Pathogens, v. 5, n. 5, p. e1000441, may. 2009. CORREA, A. P. et al. Evaluation of transformation growth factor beta1, interleukin10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi. Veterinary Parasitology, v. 143, n. 34, p. 267-274, feb. 2007.

118

COSTA, F. A. et al. Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis. Veterinary Pathology, v. 40, n. 6, p. 677-684, nov. 2003. COURA-VITAL, W. et al. Humoral and cellular immune responses in dogs with inapparent natural Leishmania infantum infection. The Veterinary Journal, v. 190, n. 2, p. e43-7, nov. 2011. COURTENAY, O. et al. Infectiousness in a cohort of brazilian dogs: why culling fails to control visceral leishmaniasis in areas of high transmission. The Journal of Infectious Diseases, v. 186, n. 9, p. 1314-1320, nov. 2002. CUNHA, S. et al. Visceral leishmaniasis in a new ecological niche near a major metropolitan area of Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene, v. 89, n. 2, p. 155-158, mar.-apr. 1995. DA COSTA-VAL, A. P. et al. Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity. The Veterinary Journal, v. 174, n. 3, p. 636-643, nov. 2007. DA SILVA, E. S. et al. Diagnosis of canine leishmaniasis in the endemic area of Belo Horizonte, Minas Gerais, Brazil by parasite, antibody and DNA detection assays. Veterinary Research Communications, v. 30, n. 6, p. 637-643, aug. 2006. DAUGSCHIES, A.; JOACHIM, A. Eicosanoids in parasites and parasitic infections. Advances in Parasitology, v. 46, p. 181-240, 2000. DE ALMEIDA FERREIRA, S. et al. Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil. PLoS Neglected Tropical Diseases, v. 6, n. 4, p. e1596, apr. 2012. DE OLIVEIRA, C. I. et al. Challenges and perspectives in vaccination against leishmaniasis. Parasitology International, v. 58, n. 4, p. 319-324, dec. 2009. DE PAIVA-CAVALCANTI, M. et al. Leishmaniases diagnosis: an update on the use of immunological and molecular tools. Cell & Bioscience, v. 5, p. 31, 2015. DESCOTEAUX, A.; TURCO, S. J. Glycoconjugates in Leishmania infectivity. Biochimica et Biophysica Acta, v. 1455, n. 2-3, p. 341-352, oct. 1999. DESJEUX, P. Leishmaniasis. Nature Reviews Microbiology, v. 2, n. 9, p. 692, sep. 2004. DOS-SANTOS, W. L. et al. Associations among immunological, parasitological and clinical parameters in canine visceral leishmaniasis: Emaciation, spleen parasitism, specific antibodies and leishmanin skin test reaction. Veterinary Immunology and Immunopathology, v. 123, n. 3-4, p. 251-259, jun. 2008. DOURADO, Z. F. et al. Panorama histórico do diagnóstico Laboratorial da leishmaniose visceral até o surgimento dos testes imunocromatográficos (rk39). Revista de Patologia Tropical, v. 36, n. 3, p. 205-214, 2007.

119

DYE, C. The logic of visceral leishmaniasis control. The American Journal of Tropical medicine and hygiene, v. 55, n. 2, p. 125-130, aug. 1996. ESPY, M. J. et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clinical Microbiology Reviews, v. 19, n. 1, p. 165-256, jan. 2006. FEITOSA, M. M. et al. Aspectos clínicos de cães com leishmaniose visceral no município de Araçatuba- São Paulo (Brasil). Clínica Veterinária, v. 28 n. 1, p. 36-44, 2003. FERREIRA, E. D. et al. Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations. Veterinary Parasitology, v. 146, n. 3-4, p. 235-241, may. 2007. FERRER, L. M. Clinical aspects of canine leishmaniasis. In: PROCEEDINGS OF THE INTERNATIONAL CANINE LEISHMANIASIS FORUM. Barcelona, Spain. Canine Leishmaniasis: an update, 1999. p. 6-10. FISA, R. et al. Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates. Veterinary Parasitology, v. 99, n. 2, p. 105-111, aug. 2001. FOGLIA MANZILLO, V. et al. Prospective study on the incidence and progression of clinical signs in naive dogs naturally infected by Leishmania infantum. PLoS Neglected Tropical Diseases, v. 7, n. 5, p. e2225, 2013. FRAGA, D. B. et al.The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs. PLoS Neglected Tropical Diseases, v. 10, n. 1, p. e0004333, jan. 2016. FRANCA-COSTA, J. et al. Arginase I, polyamine, and prostaglandin E2 pathways suppress the inflammatory response and contribute to diffuse cutaneous leishmaniasis. The Journal of Infectious Diseases, v. 211, n. 3, p. 426-435, feb. 2015. FRANCINO, O. et al. Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis. Veterinary Parasitology, v. 137, n. 3-4, p. 214221, apr. 2006. FRANKE, C. R. et al. Impact of the El Nino/Southern Oscillation on visceral leishmaniasis, Brazil. Emerging Infectious diseases Journal, v. 8, n. 9, p. 914-917, sep. 2002. FREITAS, J. C. et al. Clinical and laboratory alterations in dogs naturally infected by Leishmania chagasi. Revista da Sociedade Brasileira de Medicina Tropical, v. 45, n. 1, p. 24-29, feb. 2012. FRONTEIRA, I. Observational studies in the era of evidence based medicine: short review on their relevance, taxonomy and designs. Acta Médica Portuguesa, v. 26, n. 2, p. 161-170, mar.-apr. 2013.

120

GALLETTI, E. et al. Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantum in dog specimens. Research in Veterinary Science, v. 91, n. 2, p. 243-245, oct. 2011. GEISWEID, K. et al. Prognostic analytes in dogs with Leishmania infantum infection living in a non-endemic area. The Veterinary Record, v. 171, n. 16, p. 399, oct. 2012. GIUNCHETTI, R. C. et al. Histopathological and immunohistochemical investigations of the hepatic compartment associated with parasitism and serum biochemical changes in canine visceral leishmaniasis. Research in Veterinary Science, v. 84, n. 2, p. 269-277, apr. 2008. GOMES NETO, C. M. B. Pesquisa sobre o envolvimento do marsupial Didelphis albiventris Lund, 1840 (Didelphimorphia, Didelphidae) e de cães domiciliados na epidemiologia da leishmaniose visceral no município de Camaçari, localidade de Barra do Pojuca, Bahia. 2007. Dissertação (Mestrado em Ciência Animal nos Trópicos)- Universidade Federal da Bahia, Salvador, 2007. GOMES, R. et al. Immunity to sand fly salivary protein LJM11 modulates host response to vector-transmitted leishmania conferring ulcer-free protection. The Journal of Investigative Dermatology, v. 132, n. 12, p. 2735-2743, dec. 2012. GOMES, R. B. et al. Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity. The Journal of Infectious Diseases, v. 186, n. 10, p. 1530-1534, nov. 2002. GOMES, Y. M. et al. Diagnosis of canine visceral leishmaniasis: biotechnological advances. The Veterinary Journal, v. 175, n. 1, p. 45-52, Jan 2008. GONCALVES-DE-ALBUQUERQUE S. DA, C. et al. Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis. Journal of Venomous Animals and Toxins including Tropical Diseases, v. 20, p. 16, 2014. GONTIJO, C. M. F.; MELO, M. N. Leishmaniose Visceral no Brasil: quadro atual, desafios e perspectivas. Revista Brasileira de Epidemiologia, v. 7, n. 3, p. 338349, 2004. GRAMICCIA, M. Recent advances in leishmaniosis in pet animals: epidemiology, diagnostics and anti-vectorial prophylaxis. Veterinary Parasitology, v. 181, n. 1, p. 23-30, sep. 2011. GREENE, C. E. Infectious Diseases of the Dog and Cat. 3rd. Edinburgh: Elsevier Saunders, 2006. GRIMALDI, G., JR.; TESH, R. B. Leishmaniases of the New World: current concepts and implications for future research. Clinical Microbiology Reviews, v. 6, n. 3, p. 230-250, jul. 1993. GRIMALDI, G., JR. et al. Evaluation of a novel chromatographic immunoassay based on Dual-Path Platform technology (DPP(R) CVL rapid test) for the serodiagnosis of

121

canine visceral leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, v. 106, n. 1, p. 54-59, jan. 2012a.

GRIMALDI, G., JR. et al. The effect of removing potentially infectious dogs on the numbers of canine Leishmania infantum infections in an endemic area with high transmission rates. The American Journal of Tropical Medicine and Hygiene, v. 86, n. 6, p. 966-971, jun. 2012b. GUARGA, J. L. et al. Canine leishmaniasis transmission: higher infectivity amongst naturally infected dogs to sand flies is associated with lower proportions of T helper cells. Research in Veterinary Science, v. 69, n. 3, p. 249-253, dec. 2000. GUARGA, J. L. et al. Evaluation of a specific immunochemotherapy for the treatment of canine visceral leishmaniasis. Veterinary Immunology and Immunopathology, v. 88, n. 1-2, p. 13-20, sep. 2002. GUILLEN LLERA, J. L.; LOPEZ GARCIA, M. L.; MARTIN REINOSO, E.; DE VIVAR GONZALEZ, R. Differential serological testing by simultaneous indirect immunofluorescent antibody test in canine leishmaniosis and ehrlichiosis. Veterinary parasitology, v. 109, n. 3-4, p. 185-190, nov. 2002. GUILPIN, V. O. et al. Maxadilan, the vasodilator/immunomodulator from Lutzomyia longipalpis sand fly saliva, stimulates haematopoiesis in mice. Parasite Immunology, v. 24, n. 8, p. 437-446, aug. 2002. HARA, T. et al. Excessive production of tumor necrosis factor-alpha by bone marrow T lymphocytes is essential in causing bone marrow failure in patients with aplastic anemia. European Journal of Haematology, v. 73, n. 1, p. 10-16, jul. 2004. HARRUS, S. et al. Canine monocytic ehrlichiosis: a retrospective study of 100 cases, and an epidemiological investigation of prognostic indicators for the disease. The Veterinary Record, v. 141, n. 14, p. 360-363, oct. 1997. IKEDA-GARCIA, F. A. et al. Evaluation of renal and hepatic functions in dogs naturally infected by visceral leishmaniasis submitted to treatment with meglumine antimoniate. Research in Veterinary Science, v. 83, n. 1, p. 105-108, aug. 2007. ISMAIL, N. et al. Overproduction of TNF-alpha by CD8+ type 1 cells and downregulation of IFN-gamma production by CD4+ Th1 cells contribute to toxic shock-like syndrome in an animal model of fatal monocytotropic ehrlichiosis. Journal of Immunology, v. 172, n. 3, p. 1786-1800, feb. 2004. JULIÃO, F. S. Estudo epidemiológico de focos de leishmaniose visceral canina na Região Metropolitana de Salvador, Bahia, Brasil. 2004. Dissertação (Mestrado em Medicina Veterinária Tropical)- Universidade Federal da Bahia, Salvador, 2004. JULIÃO, F. S. et al. Investigação de áreas de risco como metodologia complementar ao controle da leishmaniose visceral canina. Pesquisa Veterinaria Brasileira, v. 27, n. 8, p. 319-324, 2007.

122

KAMHAWI, S. The biological and immunomodulatory properties of sand fly saliva and its role in the establishment of Leishmania infections. Microbes and Infection / Institut Pasteur, v. 2, n. 14, p. 1765-1773, nov. 2000. KAMHAWI, S. Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends in Parasitology, v. 22, n. 9, p. 439-445, sep. 2006. KAR, K. Serodiagnosis of leishmaniasis. Critical Reviews in Microbiology, v. 21, n. 2, p. 123-152, 1995. KARLSSON, I. et al. Multiplex cytokine analyses in dogs with pyometra suggest involvement of KC-like chemokine in canine bacterial sepsis. Veterinary Immunology and Immunopathology, v. 170, p. 41-46, Feb. 2016. KAYE, P.; SCOTT, P. Leishmaniasis: complexity at the host-pathogen interface. Nature Reviews Microbiology, v. 9, n. 8, p. 604-615, Aug. 2011. KEENAN, C. M. et al. Visceral leishmaniasis in the German shepherd dog. I. Infection, clinical disease, and clinical pathology. Veterinary Pathology, v. 21, n. 1, p. 74-79, Jan. 1984. KHOURI, R. et al. SOD1 plasma level as a biomarker for therapeutic failure in cutaneous leishmaniasis. The Journal of Infectious Diseases, v. 210, n. 2, p. 306310, Jul. 2014. KILLICK KENDRICK, R.; RIOUX, J. A. Intravectorial cycle of Leishmania in sandflies. Annales de Parasitologie Humaine et Comparee, v. 66 Suppl 1, p. 71-74, 1991. KILLICK-KENDRICK, R. The biology and control of phlebotomine sand flies. Clinics in Dermatology, v. 17, n. 3, p. 279-289, May-Jun. 1999. KOUTINAS, A. F. et al. Clinical considerations on canine visceral leishmaniasis in Greece: a retrospective study of 158 cases (1989-1996). Journal of the American Animal Hospital Association, v. 35, n. 5, p. 376-383, Sep-Oct. 1999. KOUTINAS, A. F. et al. A randomised, blinded, placebo-controlled clinical trial with allopurinol in canine leishmaniosis. Veterinary Parasitology, v. 98, n. 4, p. 247-261, Jul. 2001. KUHLS, K. et al. Comparative microsatellite typing of new world Leishmania infantum reveals low heterogeneity among populations and its recent old world origin. PLoS Neglected Tropical Diseases, v. 5, n. 6, p. e1155, Jun. 2011. LAGE, R. S. et al. Analysis of the cytokine profile in spleen cells from dogs naturally infected by Leishmania chagasi. Veterinary Immunology and Immunopathology, v. 115, n. 1-2, p. 135-145, Jan. 2007. LAINSON, R.; RANGEL, E. F. Lutzomyia longipalpis and the eco-epidemiology of American visceral leishmaniasis, with particular reference to Brazil: a review. Memorias do Instituto Oswaldo Cruz, v. 100, n. 8, p. 811-827, Dec. 2005.

123

LAINSON, R.; SHAW, J. J. Epidemiology and ecology of leishmaniasis in LatinAmerica. Nature, v. 273, n. 5664, p. 595-600, Jun. 1978. LEANDRO, C. et al. Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis. Veterinary Immunology and Immunopathology, v. 79, n. 3-4, p. 273-284, May. 2001. LEITE, R. S. et al. PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples. Veterinary Parasitology, v. 170, n. 3-4, p. 201206, Jun. 2010. LÉVEILLÉ, R. et al. Complications after ultrasound-guided biopsy of abdominal structures in dogs and cats: 246 cases (1984-1991). Journal of the American Veterinary Medical Association, v. 203, n. 3, p. 413-415, Aug. 1993. LIPOLDOVA, M.; DEMANT, P. Genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis. Nature Reviews Genetics, v. 7, n. 4, p. 294305, Apr. 2006. LOMBARDO, G. et al. Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques. Veterinary Parasitology, v. 184, n. 1, p. 10-17, Feb. 2012. LOPEZ, R. et al. Circulating immune complexes and renal function in canine leishmaniasis. Zentralblatt Fur Veterinarmedizin. Reihe B. Journal of Veterinary Medicine. Series B, v. 43, n. 8, p. 469-474, Oct. 1996. MADEIRA, M. F. et al. Parasitological diagnosis of canine visceral leishmaniasis: is intact skin a good target? Research in Veterinary Science, v. 87, n. 2, p. 260-262, Oct. 2009. MAIA, C. et al. Molecular detection of Leishmania infantum in naturally infected Phlebotomus perniciosus from Algarve region, Portugal. Journal of Vector Borne Diseases, v. 46, n. 4, p. 268-272, Dec. 2009. MAIA, C.; CAMPINO, L. Methods for diagnosis of canine leishmaniasis and immune response to infection. Veterinary Parasitology, v. 158, n. 4, p. 274-87, Dec 20 2008. MAIA, C. et al. Experimental canine leishmaniasis: clinical, parasitological and serological follow-up. Acta Tropica, v. 116, n. 3, p. 193-199, Dec. 2010. MAIA-ELKHOURY, A. N. et al. Visceral leishmaniasis in Brazil: trends and challenges. Cadernos de saude publica / Ministerio da Saude, Fundacao Oswaldo Cruz, Escola Nacional de Saude Publica, v. 24, n. 12, p. 2941-2947, Dec. 2008. MANNA, L. et al. Leishmania DNA quantification by real-time PCR in naturally infected dogs treated with miltefosine. Annals of the New York Academy of Sciences, v. 1149, p. 358-360, Dec. 2008a.

124

MANNA, L. et al. Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Veterinary Parasitology, v. 142, n. 3-4, p. 271-280, Dec. 2006. MANNA, L. et al. Evidence for a relationship between Leishmania load and clinical manifestations. Research in Veterinary science, v. 87, n. 1, p. 76-78, Aug. 2009. MANNA, L. et al. Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol. The Veterinary Journal, v. 177, n. 2, p. 279282, Aug. 2008b. MANNA, L. et al. Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis. Veterinary Parasitology, v. 125, n. 3-4, p. 251-262, Nov. 2004. MARGONARI, C. et al. Epidemiology of visceral leishmaniasis through spatial analysis, in Belo Horizonte municipality, state of Minas Gerais, Brazil. Memorias do Instituto Oswaldo Cruz, v. 101, n. 1, p. 31-38, Feb. 2006. MARTINEZ, V. et al. Canine leishmaniasis: the key points for qPCR result interpretation. Parasites & Vectors, v. 4, p. 57, 2011. MARZOCHI, M. C. et al. Canine visceral leishmaniasis in Rio de Janeiro, Brazil. Clinical, parasitological, therapeutical and epidemiological findings (1977-1983). Memorias do Instituto Oswaldo Cruz, v. 80, n. 3, p. 349-357, Jul-Sep. 1985. MAURICIO, I. L. et al. Genomic diversity in the Leishmania donovani complex. Parasitology, v. 119 ( Pt 3), p. 237-246, Sep. 1999. MAURICIO, I. L.; STOTHARD, J. R.; MILES, M. A. The strange case of Leishmania chagasi. Parasitology Today, v. 16, n. 5, p. 188-9, May 2000. MEKUZAS, Y. et al. Ehrlichia canis and Leishmania infantum co-infection: a 3-year longitudinal study in naturally exposed dogs. Clinical Microbiology and Infection, v. 15 Suppl 2, p. 30-31, Dec. 2009. MENDONCA, L. et al. Clinical aspects of visceral leishmaniasis in naturally infected dogs in the city of Teresina, Piauí. Revista Brasileira Parasitologia Veterinária, v. 8, n. 1, p. 23-25, 1999. MENEZES-SOUZA, D. et al. Cytokine and transcription factor profiles in the skin of dogs naturally infected by Leishmania (Leishmania) chagasi presenting distinct cutaneous parasite density and clinical status. Veterinary Parasitology, v. 177, n. 12, p. 39-49, Apr. 2011. MENEZES-SOUZA, D. et al. Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis. PLoS Neglected Tropical Diseases, v. 6, n. 4, p. e1566, 2012. MENN, B.; LORENTZ, S.; NAUCKE, T. J. Imported and travelling dogs as carriers of canine vector-borne pathogens in Germany. Parasites & Vectors, v. 3, p. 34, Apr. 2010.

125

MICHALSKY, E. M. et al. Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies. Veterinary Parasitology, v. 147, n. 1-2, p. 67-76, Jun. 2007. MIRÓ, G. et al. Canine leishmaniosis--new concepts and insights on an expanding zoonosis: part two. Trends in Parasitology, v. 24, n. 8, p. 371-377, Aug. 2008. MOLINA, R. et al. Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus. Transactions of the Royal Society of Tropical Medicine and Hygiene, v. 88, n. 4, p. 491-493, Jul-Aug. 1994. MOLYNEUX, D. H.; ASHFORD, R. W. The biology of Trypanosoma and Leishmania, parasites of man and domestic animals. 4. John St, London: Taylor & Francis, Ltd, 1983. MONTEIRO, M. C. et al. Effect of salivary gland extract of Leishmania vector, Lutzomyia longipalpis, on leukocyte migration in OVA-induced immune peritonitis. European Journal of Immunology, v. 35, n. 8, p. 2424-2433, Aug 2005. MOREIRA, M. A. et al. Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs. Veterinary Parasitology, v. 145, n. 3-4, p. 245-52, Apr. 2007. MORENO, J.; ALVAR, J. Canine leishmaniasis: epidemiological risk and the experimental model. Trends in Parasitology, v. 18, n. 9, p. 399-405, Sep. 2002. MORENO, J. et al. The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy. Veterinary Immunology and Immunopathology, v. 71, n. 3-4, p. 181-195, Nov. 1999. NARANJO, C. et al. Evaluation of the presence of Leishmania spp. by real-time PCR in the lacrimal glands of dogs with leishmaniosis. The Veterinary Journal, Nov. 2011. NASCIMENTO, M. S. et al. Naturally Leishmania infantum-infected dogs display an overall impairment of chemokine and chemokine receptor expression during visceral leishmaniasis. Veterinary Immunology and Immunopathology, v. 153, n. 3-4, p. 202-208, Jun. 2013. NEVES, D. P. Parasitologia Dinâmica. 2. São Paulo: Atheneu, 2006. NICOLE, C. H.; COMTE, C. H. Recherche sur le kala-azar enterprises a l’Institut Pasteur de Tunis. IV: origine canine kala-azar. Archive de l’Institute Pasteur Tunis, v. 3, p. 59-62, 1908. NOLI, C.; SARIDOMICHELAKIS, M. N. An update on the diagnosis and treatment of canine leishmaniosis caused by Leishmania infantum (syn. L. chagasi). The Veterinary Journal, v. 202, n. 3, p. 425-435, Dec. 2014. NUNES, C. M. et al. Dog culling and replacement in an area endemic for visceral leishmaniasis in Brazil. Veterinary Parasitology, v. 153, n. 1-2, p. 19-23, May. 2008.

126

OLIVA, G. et al. Incidence and time course of Leishmania infantum infections examined by parasitological, serologic, and nested-PCR techniques in a cohort of naive dogs exposed to three consecutive transmission seasons. Journal of Clinical Microbiology, v. 44, n. 4, p. 1318-1322, Apr. 2006. OLIVEIRA, C. D. L. et al. Spatial distribuition of human and canine visceral leishmaniasis in Belo Horizonte, Minas Gerais state, Brazil. Cadernos de saude publica / Ministerio da Saude, Fundacao Oswaldo Cruz, Escola Nacional de Saude Publica, v. 17, p. 1231-1239, 2001. OLIVEIRA, L. C. et al. Seroprevalence and risk factors for canine visceral leishmaniasis in the endemic area of Dias D'Avila, State of Bahia, Brazil. Revista da Sociedade Brasileira de Medicina Tropical, v. 43, n. 4, p. 400-404, Jul-Aug. 2010. OTRANTO, D.; DANTAS-TORRES, F. Canine and feline vector-borne diseases in Italy: current situation and perspectives. Parasites & Vectors, v. 3, p. 2, Jan. 2010. PAIVA-CAVALCANTI, M.; REGIS-DA-SILVA, C. G.; GOMES, Y. M. Comparison of real-time PCR and conventional PCR for detection of Leishmania (Leishmania) infantum infection: a mini-review. The Journal of Venomous Animals and Toxins including Tropical Diseases, v. 16, n. 4, p. 537-542, 2010. PELEG, O. et al. Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli. Veterinary Parasitology, v. 173, n. 3-4, p. 292-299, Oct. 2010. PENNISI, M. G. et al. Real-time PCR in dogs treated for leishmaniasis with allopurinol. Veterinary Research Communications, v. 29 Suppl 2, p. 301-303, Aug. 2005. PINELLI, E. et al. J. Leishmania infantum-specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex-restricted manner. European Journal of Immunology, v. 25, n. 6, p. 15941600, Jun. 1995. PINELLI, E. et al. Cellular and humoral immune responses in dogs experimentally and naturally infected with Leishmania infantum. Infection and Immunity, v. 62, n. 1, p. 229-235, Jan. 1994. PINELLI, E. et al. Compensation for decreased expression of B7 molecules on Leishmania infantum-infected canine macrophages results in restoration of parasitespecific T-cell proliferation and gamma interferon production. Infection and Immunity, v. 67, n. 1, p. 237-243, Jan. 1999. PIRON, M. et al. Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples. Acta Tropica, v. 103, n. 3, p. 195-200, Sep. 2007. PRATES, D. B. et al. New Insights on the Inflammatory Role of Lutzomyia longipalpis Saliva in Leishmaniasis. Journal of Parasitology Research, v. 2012, p. 643029, 2012.

127

PRATES, D. B. et al. Lutzomyia longipalpis saliva drives apoptosis and enhances parasite burden in neutrophils. Journal of Leukocyte Biology, v. 90, n. 3, p. 575582, Sep. 2011. QUARESMA, P. F. et al. Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR. Acta tropica, v. 111, n. 3, p. 289-294, Sep. 2009. QUINNELL, R. J.; COURTENAY, O. Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis. Parasitology, v. 136, n. 14, p. 1915-1934, Dec. 2009. RAFATI, S. et al. Protective vaccination against experimental canine visceral leishmaniasis using a combination of DNA and protein immunization with cysteine proteinases type I and II of L. infantum. Vaccine, v. 23, n. 28, p. 3716-3725, May. 25 2005. REALE, S. et al. Detection of Leishmania infantum in dogs by PCR with lymph node aspirates and blood. Journal of Clinical Microbiology, v. 37, n. 9, p. 2931-2935, Sep. 1999. REIS, A. B. et al. Immunity to Leishmania and the rational search for vaccines against canine leishmaniasis. Trends in Parasitology, v. 26, n. 7, p. 341-349, Jul. 2010. REIS, A. B. et al. Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis. Research in Veterinary Science, v. 81, n. 1, p. 68-75, Aug. 2006a. REIS, A. B. et al. Systemic and compartmentalized immune response in canine visceral leishmaniasis. Veterinary Immunology and Immunopathology, v. 128, n. 1-3, p. 87-95, Mar. 2009. REIS, A. B. et al. Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi. Clinical and Experimental Immunology, v. 146, n. 2, p. 303-311, Nov. 2006b. REITHINGER, R.; DUJARDIN, J. C. Molecular diagnosis of leishmaniasis: current status and future applications. Journal of Clinical Microbiology, v. 45, n. 1, p. 2125, Jan. 2007. REY, L. Parasitologia. 4. Rio de Janeiro, RJ: Guanabara & Koogan, 2008. RHALEM, A. et al. Analysis of immune responses in dogs with canine visceral leishmaniasis before, and after, drug treatment. Veterinary Immunology and Immunopathology, v. 71, n. 1, p. 69-76, Oct. 1999. RIBEIRO, J. M.; FRANCISCHETTI, I. M. Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annual Review of Entomology, v. 48, p. 73-88, 2003.

128

RIBEIRO, V. M. et al. Control of visceral leishmaniasis in Brazil: recommendations from Brasileish. Parasites & Vectors, v. 6, n. 1, p. 8, Jan. 2013. ROLÃO, N. et al. Quantification of Leishmania infantum parasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzymelinked immunosorbent assay. The Journal of Parasitology, v. 90, n. 5, p. 11501154, Oct. 2004. ROMICH, J. A. Understanding Zoonotic Diseases. 1. Delmar Cengage Learning, 2008. ROURA, X. et al. Prognosis and monitoring of leishmaniasis in dogs: a working group report. The Veterinary Journal, v. 198, n. 1, p. 43-47, Oct. 2013. RUSSELL, D. G.; WRIGHT, S. D. Complement receptor type 3 (CR3) binds to an Arg-Gly-Asp-containing region of the major surface glycoprotein, gp63, of Leishmania promastigotes. The Journal of Experimental Medicine, v. 168, n. 1, p. 279-292, Jul. 1988. SAHA, A. et al. Prostaglandin E2 negatively regulates the production of inflammatory cytokines/chemokines and IL-17 in visceral leishmaniasis. Journal of Immunology, v. 193, n. 5, p. 2330-2339, Sep. 2014. SAHA, S. et al. IL-10- and TGF-beta-mediated susceptibility in kala-azar and postkala-azar dermal leishmaniasis: the significance of amphotericin B in the control of Leishmania donovani infection in India. Journal of Immunology, v. 179, n. 8, p. 5592-5603, Oct. 2007. SALDARRIAGA, O. A. et al. Immunogenicity of a multicomponent DNA vaccine against visceral leishmaniasis in dogs. Vaccine, v. 24, n. 11, p. 1928-1940, Mar. 2006. SAMUELSON, J.; LERNER, E.; TESH, R.; TITUS, R. A mouse model of Leishmania braziliensis braziliensis infection produced by coinjection with sand fly saliva. The Journal of experimental medicine, v. 173, n. 1, p. 49-54, Jan. 1991. SANTANA, C. C. et al. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: a study on naturally infected dogs. Parasite Immunology, v. 30, n. 10, p. 515-524, Oct. 2008. SARIDOMICHELAKIS, M. N. Advances in the pathogenesis of canine leishmaniosis: epidemiologic and diagnostic implications. Veterinary Dermatology, v. 20, n. 5-6, p. 471-489, Oct. 2009. SARIDOMICHELAKIS, M. N. et al. Periodic administration of allopurinol is not effective for the prevention of canine leishmaniosis (Leishmania infantum) in the endemic areas. Veterinary Parasitology, v. 130, n. 3-4, p. 199-205, Jun. 2005. SHERLOCK, I. A. Ecological interactions of visceral leishmaniasis in the state of Bahia, Brazil. Memorias do Instituto Oswaldo Cruz, v. 91, n. 6, p. 671-683, NovDec. 1996.

129

SILVA, D. A. et al. Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program. Veterinary Parasitology, v. 179, n. 1-3, p. 257-261, Jun. 2011. SILVA, E. S. et al. Visceral leishmaniasis in the Metropolitan Region of Belo Horizonte, State of Minas Gerais, Brazil. Memorias do Instituto Oswaldo Cruz, v. 96, n. 3, p. 285-291, Apr. 2001. SILVA, F. T. S. et al. Aspectos clínicos da leishmaniose visceral canina no distrito de Monte Gordo, Camaçari (BA). Revista Baiana de Saúde Pública, v. 34, n. 4, p. 783-795, 2010. SILVA, K. R. et al. Scoring clinical signs can help diagnose canine visceral leishmaniasis in a highly endemic area in Brazil. Memorias do Instituto Oswaldo Cruz, v. 112, n. 1, p. 53-63, Jan. 2017. SINAN/SVS/MS. Casos confirmados de Leishmaniose Visceral, Brasil, Grandes Regiões e Unidades Federadas. 1990 a 2015. 2017. SOLANO-GALLEGO, L. et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Veterinary Parasitology, v. 165, n. 1-2, p. 1-18, Oct. 2009. SOLANO-GALLEGO, L. et al. A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Veterinary Research, v. 37, n. 2, p. 231-244, Mar-Apr. 2006. SOLANO-GALLEGO, L. et al. LeishVet guidelines for the practical management of canine leishmaniosis. Parasites & Vectors, v. 4, p. 86, 2011. SOLANO-GALLEGO, L. et al. Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology. Journal of Clinical Microbiology, v. 39, n. 2, p. 560-563, Feb. 2001. SOLANO-GALLEGO, L. et al. Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis. Veterinary Parasitology, v. 147, n. 3-4, p. 315-319, Jul. 2007. SOLCÀ, M. D. S. et al. Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs. Veterinary Parasitology, v. 184, n. 2-4, p. 133-40, Mar 23 2012. SOLCÀ, M. S. Uso de PCR no diagnóstico da leishmaniose visceral canina: uma abordagem comparativa de diferentes protocolos e tecidos. 2012. 94 (Master). Patologia, Universidade Federal da Bahia, Salvador, BA, Brasil. SOLCÀ, M. S. et al. Circulating Biomarkers of Immune Activation, Oxidative Stress and Inflammation Characterize Severe Canine Visceral Leishmaniasis. Scientific Reports - Nature, v. 6, p. 32619, Sep. 2016.

130

SOLCA, M. S. et al. Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis. PloS One, v. 9, n. 7, p. e103635, 2014. SOUZA, A. P. et al. Using recombinant proteins from Lutzomyia longipalpis saliva to estimate human vector exposure in visceral Leishmaniasis endemic areas. PLoS Neglected Tropical Diseases, v. 4, n. 3, p. e649, 2010. STRAUSS-AYALI, D.; BANETH, G.; JAFFE, C. L. Splenic immune responses during canine visceral leishmaniasis. Veterinary Research, v. 38, n. 4, p. 547-564, Jul-Aug. 2007. STRAUSS-AYALI, D. et al. Polymerase chain reaction using noninvasively obtained samples, for the detection of Leishmania infantum DNA in dogs. The Journal of Infectious Diseases, v. 189, n. 9, p. 1729-1733, May. 2004. STRIMBU, K.; TAVEL, J. A. What are biomarkers? Current Opinion in HIV and AIDS, v. 5, n. 6, p. 463-466, Nov. 2010. SUNDAR, S.; RAI, M. Laboratory diagnosis of visceral leishmaniasis. Clinical and Diagnostic Laboratory Immunology, v. 9, n. 5, p. 951-958, Sep. 2002. TALAMAS-ROHANA, P. et al. Lipophosphoglycan from Leishmania mexicana promastigotes binds to members of the CR3, p150,95 and LFA-1 family of leukocyte integrins. Journal of Immunology, v. 144, n. 12, p. 4817-4824, Jun. 1990. TAVARES, N. M. et al. Understanding the mechanisms controlling Leishmania amazonensis infection in vitro: the role of LTB4 derived from human neutrophils. The Journal of Infectious Diseases, v. 210, n. 4, p. 656-666, Aug. 2014. TEIXEIRA, C. et al. Discovery of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi in Latin America. PLoS Neglected Tropical Diseases, v. 4, n. 3, p. e638, 2010. TORRES, M. et al. Long term follow-up of dogs diagnosed with leishmaniosis (clinical stage II) and treated with meglumine antimoniate and allopurinol. The Veterinary Journal, v. 188, n. 3, p. 346-51, Jun. 2011. TRAVI, B. L. et al. Clinical, parasitologic, and immunologic evolution in dogs experimentally infected with sand fly-derived Leishmania chagasi promastigotes. The American Journal of Tropical Medicine and Hygiene, v. 81, n. 6, p. 994-1003, Dec. 2009. TRONCARELLI, M. Z. et al. Leishmania spp. and/or Trypanosoma cruzi diagnosis in dogs from endemic and nonendemic areas for canine visceral leishmaniasis. Veterinary Parasitology, v. 164, n. 2-4, p. 118-123, Oct. 2009. TROPIA DE ABREU, R. et al. Influence of clinical status and parasite load on erythropoiesis and leucopoiesis in dogs naturally infected with Leishmania (Leishmania) chagasi. PloS One, v. 6, n. 5, p. e18873, 2011.

131

VASCELLARI, M. et al. Exposure to vector-borne pathogens in candidate blood donor and free-roaming dogs of northeast Italy. Parasites & Vectors, v. 9, n. 1, p. 369, Jun. 2016. VENTURIN, G. L. et al. M1 polarization and the effect of PGE2 on TNF-alpha production by lymph node cells from dogs with visceral leishmaniasis. Parasite Immunology, v. 38, n. 11, p. 698-704, Nov. 2016. VERCOSA, B. L. et al. Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis. BMC Veterinary Research, v. 4, p. 45, 2008. VINHAS, V. et al. Human anti-saliva immune response following experimental exposure to the visceral leishmaniasis vector, Lutzomyia longipalpis. European Journal of Immunology, v. 37, n. 11, p. 3111-3121, Nov. 2007. VOULDOUKIS, I. et al. Canine visceral leishmaniasis: successful chemotherapy induces macrophage antileishmanial activity via the L-arginine nitric oxide pathway. Antimicrobial Agents and Chemotherapy, v. 40, n. 1, p. 253-256, Jan. 1996. WATSON, A. T. et al. Safety and correlation of test results of combined ultrasoundguided fine-needle aspiration and needle core biopsy of the canine spleen. Veterinary Radiology & Ultrasound: the Official Journal of the American College of Veterinary Radiology and the International Veterinary Radiology Association, v. 52, n. 3, p. 317-22, May-Jun. 2011. WHO. Epidemiological situation Leishmaniasis. http://www.who.int/leishmaniasis/en/ >.

2017.

Disponível em: <

XU, X. et al. Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection. The Journal of Biological Chemistry, v. 286, n. 37, p. 32383-32393, Sep. 2011. YABSLEY, M. J. et al. Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada. Veterinary Parasitology, v. 151, n. 2-4, p. 279-285, Feb. 2008.

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Colaborações em artigos científicos • Anexo 1 – Colaboração em artigo científico publicado o Fraga, D. M. B. et al., 2016 - The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs. PLoS Neglected Tropical Diseases. • Anexo 2 – Colaboração em artigo científico publicado o Borja, L. S. et al., 2016 - Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors. Veterinary Parasitology.

RESEARCH ARTICLE

The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs Deborah Bittencourt Mothé Fraga1,2,3☯, Luciano Vasconcellos Pacheco1☯, Lairton Souza Borja1, Pétala Gardênia da Silva Estrela Tuy4, Leila Andrade Bastos1, Manuela da Silva Solcà1, Leila Denise Alves Ferreira Amorim4, Patrícia Sampaio Tavares Veras1,3* 1 Laboratório de Patologia e Biointervenção, Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, Salvador, Bahia, Brazil, 2 Departamento de Medicina Veterinária Preventiva e Produção Animal, Escola de Medicina Veterinária e Zootecnia, Universidade Federal da Bahia, Salvador, Bahia, Brazil, 3 Instituto de Ciência e Tecnologia de Doenças Tropicais, INCT-DT, Bahia, Brazil, 4 Instituto de Matemática, Departamento de Estatística, Universidade Federal da Bahia, Bahia, Brazil OPEN ACCESS Citation: Fraga DBM, Pacheco LV, Borja LS, Tuy PGdSE, Bastos LA, Solcà MdS, et al. (2016) The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs. PLoS Negl Trop Dis 10(1): e0004333. doi:10.1371/journal.pntd.0004333 Editor: Albert Picado, Barcelona Institute for Global Health, SPAIN Received: July 4, 2015 Accepted: December 6, 2015 Published: January 5, 2016 Copyright: © 2016 Fraga et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Instituto de Ciência e Tecnologia em Doenças Tropicais (INCTDT- http://inct.cnpq.br/ -Grant number: 576269/20085). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

☯ These authors contributed equally to this work. * [email protected]

Abstract Visceral Leishmaniasis (VL) has spread to many urban centers worldwide. Dogs are considered the main reservoir of VL, because canine cases often precede the occurrence of human cases. Detection and euthanasia of serologically positive dogs is one of the primary VL control measures utilized in some countries, including Brazil. Using accurate diagnostic tests can minimize one undesirable consequence of this measure, culling false-positive dogs, and reduce the maintenance of false-negative dogs in endemic areas. In December 2011, the Brazilian Ministry of Health replaced the ELISA (EIE CVL) screening method and Indirect Immunofluorescence Test (IFI CVL) confirmatory method with a new protocol using the rapid DPP CVL screening test and EIE CVL confirmatory test. A study of diagnostic accuracy of these two protocols was done by comparing their performance using serum samples collected from a random sample of 780 dogs in an endemic area of VL. All samples were evaluated by culture and real time PCR; 766 out of the 780 dogs were tested using the previous protocol (IFI CVL + EIE CVL) and all 780 were tested using the current protocol (DPP CVL + EIE CVL). Performances of both diagnostic protocols were evaluated using a latent class variable as the gold standard. The current protocol had a higher specificity (0.98 vs. 0.95) and PPV (0.83 vs. 0.70) than the previous protocol, although sensitivity of these two protocols was similar (0.73). When tested using sera from asymptomatic animals, the current protocol had a much higher PPV (0.63 vs. 0.40) than the previous protocol (although the sensitivity of either protocol was the same, 0.71). Considering a range of theoretical CVL prevalences, the projected PPVs were higher for the current protocol than for the previous protocol for each theoretical prevalence value. The findings presented herein show that

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the current protocol performed better than previous protocol primarily by reducing false-positive results.

Author Summary Visceral Leishmaniasis (VL) is a major public health problem. Its control is based on detection and culling of positive dogs, treatment of human cases and vector control. Canine cases often precede the occurrence of human cases; hence, disease control in dogs is important. Use of accurate diagnostic tests is required to avoid culling false-positive dogs and to minimize the number of false-negative dogs that are maintained in endemic areas. In December 2011, the Brazilian Ministry of Health changed the diagnostic protocol for canine VL (CVL). In the present study, the accuracy of this current protocol was compared to the previous one using serum samples of 780 dogs from an endemic area of VL. The findings revealed that the current protocol performed better than the previous protocol primarily by reducing false-positive results. Considering different theoretical prevalence values, the current protocol misdiagnosed fewer dogs than the previous one.

Introduction Visceral leishmaniasis (VL) is a major public health problem worldwide. This disease in Brazil and Europe is caused by the protozoan parasite Leishmania infantum, which is transmitted to humans by the bite of sandflies from the genus Lutzomyia [1]. Dogs are considered the main reservoir of urban VL since: i) these animals harbor high parasitism in skin that offers a high capacity of parasite transmission to sandflies, ii) humans and dogs coexist in close proximity and iii) canine cases generally precede the occurrence of VL in humans [1–4]. The identification and euthanasia of serologically positive dogs is one of the primary VL control strategies recommended by the governments of some countries, such as Brazil. The use of accurate diagnostic tests for canine VL (CVL) can reduce failures on VL control program by minimizing maintenance of false-negative animals and culling of false-positive dogs that impact on euthanasia controversial measure, subsequently, decreasing dog owners’ compliance and society disagreement. More accurate tests could also reduce the number of false-negative dogs that are maintained in endemic areas [4]. CVL is typically diagnosed by parasitological, serological and molecular tests. In December 2011, the program of the Brazilian Ministry of Health for monitoring and control of leishmaniasis replaced the enzyme-linked immunosorbent assay (EIE CVL) screening method and the indirect immunofluorescence assay (IFI CVL) confirmatory test with a new serodiagnostic protocol for CVL composed of the Dual Path Platform (DPP CVL) screening test and the EIE CVL confirmatory test [5]. The evaluation of sensitivity and specificity revealed low values for previous protocol that detects infection by determining seropositivity in dogs. This low performance is probably due to undesirable preservation of blood samples normally collected onto filter papers. This simple procedure for sample collection is performed easily and facilitates sample storage and transportation. However, it often submits the biological specimens to stress conditions that might damage samples and lead to unreliable test results [2,6–8]. Additionally, the low sensitivity and specificity offered by the old protocol can be explained by further reasons: i) both screening EIE CVL and confirmatory IFI CVL tests have been performed using blood samples that were collected in endemic areas and then sent to reference laboratories, where the tests were performed,

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ii) EIE CVL and IFI CVL tests are time-consuming techniques, whereas IFI CVL has an additional difficulty to be standardized and interpreted depending on the ability of the observer to detect the antigen-antibody reaction by fluorescence microscope. This may lead to misinterpretation of the results and may compromise IFI CVL reproducibility in different laboratories. DPP CVL is a rapid test based on a multi-epitope, recombinant chimeric protein (rK28) resulted from fusion of L. infantum genes: k9, single repeat units of k39 and k26 [9] that has been adopted as the screening method in a new protocol established by the Brazilian government. DPP CVL rapid test is an immunochromatographic assay that offers several advantages: i) rK28 was proven to provide very high levels of sensitivity and specificity for canine VL [9], ii) DPP CVL has a great potential for facilitating faster decision, since it is a point-of-care screening test that gives result within 15 minutes, iii) DPP CVL in association with the confirmatory test EIE CVL give results within 15 days, in comparison to previous protocol (EIE CVL + IFI CVL) that results were only liberated after a lengthy time interval that varied from one to two months. Thus, the incorporation of this rapid test into the current protocol accelerates the implementation of the control measures in endemic areas. In addition, this procedure uses only small blood samples and does not require specialized equipment and supplies [10]. The use of tests presenting low accuracy has serious epidemiological consequences: falsenegative dogs are undetected thereby maintaining the parasite life cycle in endemic areas, and detection of false-positive dogs results in excessive dog culling. The lack of a reliable gold standard test for CVL hinders the assessment of diagnostic protocol performance and can result in misinterpretation of diagnostic test accuracy [11–16]. Indeed, although the common used gold-standard, culturing of L. infantum, is highly specific, its low sensitivity [17] hampers the evaluation of other diagnostic techniques. In light of this limitation, latent class analysis (LCA) has been shown to be a valuable alternative to the classical validation approach of using parasitological methods as gold standards [18,19]. LCA is based on the theory that the observed results of different imperfect tests for the same disease are influenced by a latent common variable that cannot be directly measured, but can reflect accurately the true disease status. Previous studies employing LCA have accurately assessed serological [20–24] and molecular [12,25] diagnostic methods. Despite the advantages of DPP CVL [10,14,26,27] for CVL diagnosis, few studies have assessed its performance [28,29]. To the best of our knowledge, the present study is an initial attempt designed to compare the accuracy of the current (DPP CVL and EIE CVL) and previous protocol (EIE CVL and IFI CVL) for CVL diagnosis employing a latent class variable as the reference standard. Serum samples were obtained during a cross-sectional study performed in an endemic area for VL in Brazil.

Methods Ethics Statement All experimental procedures involving dogs were carried out according to the Brazilian Federal Law on Animal Experimentation (Law no. 11794), the guidelines for animal research established by the Oswaldo Cruz Foundation (FIOCRUZ) and the Brazilian Ministry of Health Manual for the Surveillance and Control of VL [4]. The Institutional Review Board approved the present study for Animal Experimentation (CEUA, protocol no. 015/2009). Dog owners who agreed to participate in the study signed a Free, Prior and Informed Consent (FPIC) form.

Study Area A cross-sectional study was conducted in the municipality of Camaçari, located in the State of Bahia in Northeastern Brazil. Using district sketches of households throughout 36 districts in

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Camaçari obtained from the Zoonosis Control Center, a sample of domiciled dogs was randomly selected, during the years of 2011 and 2012. The sample size was calculated using Epi Info 3.5.1 (The Centers for Disease Control and Prevention—CDC, USA) based on estimates of the canine population (15,820 dogs) derived from an anti-rabies vaccination campaign and an expected CVL prevalence of 20% (5% margin of error, 95% confidence interval).

Sampling Dogs were classified as asymptomatic or symptomatic based on the presence or absence of the following clinical signs: emaciation, alopecia, anemia, conjunctivitis, dehydration, dermatitis, erosion, ulcerations, lymphadenopathy, and onychogryphosis. They were classified as asymptomatic when presented 0 until 3 signs or symptomatic when presented more than 3 signs. Blood and splenic aspirate samples were obtained for CVL diagnosis from each dog at the same time. Blood was collected by venipuncture in sterile tubes to obtain serum. All serum samples were stored at -20°C until serological testing. Splenic aspirate samples were obtained using a puncture technique previously described by Barrouin-Melo and collaborators (2006) [30], and modified by Solcà and collaborators (2014) for ultrasound-guided collection. All 780 splenic samples were evaluated by culture and real time PCR; 766 out of the 780 serum samples were tested using the previous protocol (IFI CVL + EIE CVL) and all 780 were tested using the current protocol (DPP CVL + EIE CVL) (S1 Fig).

Parasitological Testing Splenic aspirate samples were cultivated in Novy-Mac Neal-Nicolle (NNN) medium supplemented with 20% FBS (Fetal Bovine Serum, Gibco BRL, New York, USA) and 100 μg/mL of gentamicin. The cultures were maintained at 24°C for four weeks and examined weekly for the presence of parasites [31].

Serological Tests All serological diagnostic test kits for CVL (DPP CVL, EIE CVL and IFI CVL Bio-Manguinhos) were used in accordance with manufacturer’s recommendations.

DNA Extraction DNA was extracted from splenic aspirate samples using DNeasy Blood & Tissue kit from Qiagen (Hilden, Germany), in accordance with manufacturer’s recommendations. DNA concentrations were determined using a digital spectrophotometer (Nanodrop—ND-1000 Thermo Scientific, Wilmington, USA), then aliquoted at a concentration of 30 ng/μL and stored at -20°C until real time PCR amplification.

Real Time PCR DNA extracted from splenic aspirate samples was amplified using real time PCR technique, in accordance with the protocol established by Francino and collaborators (2006) [32] and modified by Solcà and collaborators (2014). Control samples were added in all of the real time PCR experiments. As positive controls were used splenic aspirate samples from two dogs that had previously been identified in an endemic area as positive for Leishmania infection and as negative controls were employed splenic aspirates of two healthy dogs from the municipality of Pelotas, Rio Grande do Sul, Brazil, an area non-endemic for CVL.

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Statistical Analysis All test readers executing and reading the index tests had prior training and great experience in CVL diagnosis. All diagnostic testing was carried out under blinded conditions, which means that test readers interpreted the results obtained from each diagnostic technique for a given sample without knowledge of the other tests’ results. The interpretation of the results using the previous and current diagnostic protocols classified dogs as positive when both tests (screening and confirmatory) presented positive results. Epi Info 3.5.1 (The Centers for Disease Control and Prevention—CDC, Atlanta, USA) and STATA 12.0 (StataCorp LP, Texas, USA) software programs were used to analyze results. LCA was performed to define a latent class variable to evaluate the accuracy of the diagnostic tests and employed as previously described in Solcà and collaborators (2014). Latent variable modeling used the results of the following diagnostic techniques as indictor variables: serological (EIE CVL, DPP CVL and IFI CVL Bio-Manguinhos), parasitological (culture of splenic samples), and molecular (real time PCR of splenic aspirate) tests. We chose a two-class latent class model based on goodness of fit criteria, such as the Akaike information criterion (AIC) and Bayes information criterion (BIC). We also used the Lo-Mendel-Rubin test and the entropy for model evaluation [33]. MPlus version 5 software was used to implement LCA [34]. The performance of the diagnostic tests and protocols was estimated using the latent class variable as the reference standard. Diagnostic performance was calculated in 2 x 2 contingency tables of positive and negative test results, using the command diagt in Stata. We determined specificity, positive predictive values (PPV), negative predictive values (NPV) and diagnostic accuracy with 95% exact binomial confidence intervals (CI). Diagnostic accuracy was calculated as the number of true positive + number of true negative/total number of tested serum samples. Differences among diagnostic protocols regarding their performance (sensitivity and specificity) were assessed using McNemar chi-square test (p-value < 0.05), for all dogs and for two categories of disease status based on symptomatology. The number of animals considered as false negative and false positive was also calculated for each of the diagnostic techniques evaluated, considering as true positive those dogs that were positive according to the latent class variable.

Results From April 2011 until July 2012, 780 dogs pure and mixed-breed with estimated ages from 1 to 10 years old, were enrolled in the study. According to the presence of clinical signs of CVL, 47.8% dogs were asymptomatics and 54.2% symptomatics. Five diagnostic tests were used to determine the proportion that tested positive in this random population. The IFI CVL yielded the highest percentage of positivity (36%), whereas the splenic aspirate culture yielded the lowest percentage of positivity (13.1%). Among the remaining tests, the EIE CVL, real time PCR and DPP CVL tests were positive in 24.9%, 22.4% and 16.9% of the dogs, respectively (Fig 1 and Table 1). Using LCA, 14.1% of the 780 dogs were classified as positive (Table 1). Evaluation of LCA entropy showed that a high accuracy in the classification of dogs by LCA was achieved, with value of 0.97. A posteriori average probabilities that dogs were properly classified in the latent classes "positive" and "negative" were, respectively, 95% and 99%. Moreover, the test of LoMendel-Rubin indicated that the model with two classes produced better results than that with only one class (p < 0.01). These results are supported by the analysis of AIC and BIC (AIC = 3025.996, BIC = 3077.249). The real time PCR and culture techniques yielded the highest sensitivities, 0.97 and 0.90, respectively, when the latent class variable served as the reference standard. Among the three

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Fig 1. Percent of positive results of five CVL diagnostic tests performed on canine sera samples from an endemic area of Camaçari. doi:10.1371/journal.pntd.0004333.g001

serological tests, IFI CVL and DPP CVL had the highest sensitivity (0.86) and EIE CVL (0.79) (Table 2). Regarding specificity, culture was found to be the most specific (1.00), followed by DPP CVL (0.94), then real time PCR (0.90), EIE CVL (0.84) and IFI CVL (0.73). When the latent class variable was considered as the reference test, the PPV of culture was 1.00. Among the other four techniques, DPP CVL had the highest PPV (0.71), followed by real time PCR (0.61), EIE CVL (0.45) and IFI CVL (0.37). Likewise, among serological tests, DPP CVL (0.98), followed by IFI CVL (0.97) and EIE CVL (0.96) showed the highest NPV (Table 2). The measures of diagnostic accuracy of the current diagnostic protocol were then compared to those of the previous protocol (Table 3). Both protocols had equally high sensitivity (>0.72; McNemar’s chi-square test, p = 0.051600) and NPV (0.96), whereas the new protocol consistently had a higher specificity (>0.97, p = 0.0078) and PPV (>0.83). The diagnostic accuracy was higher when current diagnostic protocol was compared to the previous protocol (0.94 vs. 0.92). Table 1. Prevalence of latent classes and conditional probabilities according to the LCA model for CVL diagnoses. Latent classes Technique

Frequency Positive n = 780 (%)

Positive n = 110 (14.1%)

Negative n = 670 (85.9%)

IFI CVL

36.0

85.9

26.6

EIE CVL

24.9

79.2

15.8

Real time PCR

22.4

96.2

10.1

DPP CVL

16.9

84.1

5.6

Culture

13.1

88.1

0.0

Conditional probabilities (%)

doi:10.1371/journal.pntd.0004333.t001

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Table 2. Performance of diagnostic tests considering the latent class variable as the gold standard. Diagnostic tests

Sensitivity

Specificity

PPV

NPV

DPP CVL

0.86 (0.78–0.92)

0.94 (0.92–0.97)

0.71 (0.63–0.79)

0.98 (0.96–0.99)

EIE CVL

0.79 (0.70–0.86)

0.84 (0.81–0.87)

0.45 (0.38–0.52)

0.96 (0.94–0.98)

IFI CVL

0.86 (0.78–0.92)

0.73 (0.69–0.77)

0.37 (0.31–0.43)

0.97 (0.95–0.98)

Real time PCR

0.97(0.92–0.99)

0.90 (0.87–0.92)

0.61 (0.54–0.68)

1.00(0.99–1.00)

Culture

0.90 (0.83–0.95)

1.00 (0.99–1.00)

1.00 (0.96–1.00)

0.98 (0.97–0.99)

doi:10.1371/journal.pntd.0004333.t002

Table 3. Performance of current and previous protocols for CVL diagnosis, considering the latent class variable as the gold standard. Diagnostic tests Previous protocol

Current protocol

Sensitivity

Specificity*

PPV

NPV

Accuracy

EIE CVL + IFI CVL (Asymptomatic dogs)

0.71 (0.42–0.92)

0.96 (0.93–0.98)

0.40 (0.21–0.61)

0.99 (0.97–1.00)

0.95

EIE CVL + IFI CVL (Symptomatic dogs)

0.74 (0.64–0.82)

0.94 (0.90–0.96)

0.79 (0.69–0.87)

0.92 (0.88–0.95)

0.89

EIE CVL + IFI CVL (Total)

0.73 (0.64–0.81)

0.95 (0.93–0.96)

0.70 (0.61–0.78)

0.96 (0.94–0.97)

0.92

DPP CVL + EIE CVL (Asymptomatic dogs)

0.71 (0.42–0.92)

0.98 (0.96–0.99)

0.63 (0.35–0.85)

0.99 (0.97–1.00)

0.97

DPP CVL + EIE CVL (Symptomatic dogs)

0.73 (0.63–0.82)

0.97 (0.94–0.98)

0.88 (0.78–0.94)

0.92 (0.89–0.95)

0.91

DPP CVL + EIE CVL (Total)

0.73 (0.63–0.81)

0.98 (0.96–0.99)

0.83 (0.74–0.90)

0.96 (0.94–0.97)

0.94

*The specificity of previous and current protocol was statically different, based on McNemar test (p = 0.0078). doi:10.1371/journal.pntd.0004333.t003

Comparing the performance of current protocol (DPP CVL + EIE CVL) to that of DPP CVL alone revealed that sensitivity showed higher value for DPP CVL (0.86) than that for the current protocol (0.73), although PPV showed a slight lower value for DPP CVL (0.71) compared to PPV for the current protocol (0.83). When the dogs were categorized according to the presence of clinical signs of CVL (Table 3), the sensitivity of both diagnostic protocols was similar in asymptomatic and symptomatic dogs. However, in symptomatic dogs, the new protocol had higher specificity and PPV (0.97 and 0.88, respectively) than the previous protocol (0.94 and 0.79, respectively). In addition, in asymptomatic dogs, the PPV of the current protocol was significantly higher, by 22.5%, than that of the previous protocol (p = 0.0078). Also, difference was observed in diagnostic accuracy of protocols when they were used in symptomatic dogs (0.91 vs. 0.89) and asymptomatic dogs (0.97 vs. 0.95). To generalize the better performance of current protocol to other settings, the PPV and NPV were calculated for the current and previous protocol accordingly to different theoretical values of CVL prevalence (Table 4). For each estimated prevalence value, the current protocol was estimated to yield higher PPVs, ranging from 0.23 to 0.99, whereas the projected PPVs for the previous protocol ranged from 0.13 to 0.98. Regarding NPV, both protocols yielded similar projected values, ranging from 0.47 to 1.00.

Discussion The present study primarily demonstrated that the DPP CVL + EIE CVL protocol, in comparison with the EIE CVL + IFI CVL protocol, performed better for the serodiagnosis of CVL. The adoption of this new protocol offered several advantages, due to inclusion of the rapid DPP

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Table 4. Estimates of PPV and NPV of current and previous protocols for CVL diagnosis, considering the latent class variable as the gold standard, by theoretical values of CVL prevalence. Previous protocol

Current protocol

Prevalence (%)

PPV

NPV

PPV

NPV

1

0.13

1.00

0.23

1.00

5

0.43

0.99

0.62

0.99

10

0.61

0.97

0.77

0.97

15

0.71

0.95

0.84

0.95

20

0.78

0.93

0.88

0.94

30

0.86

0.89

0.93

0.89

50

0.93

0.78

0.97

0.78

80

0.98

0.47

0.99

0.47

doi:10.1371/journal.pntd.0004333.t004

CVL screening test, which can be performed easily and quickly and does not require specialized equipment and personnel [27,28]. Several authors previously discussed that the lack of a perfect gold standard test for CVL hampers the evaluation of diagnostic tests for CVL [12,13]. Previous studies have proven that LCA is effective for evaluating diagnostic tests’ performance [12,20,21,35–38]. Herein, using a latent class variable as the reference standard, we were able to comprehensively compare two protocols for serodiagnosis of CVL using serum samples collected from 780 randomly selected dogs from an endemic area of VL. The use of LCA had an additional advantage: we were able to evaluate the performance of both real time PCR and culture. Very few studies have evaluated the performance of real time PCR, and most studies that evaluated the performance of CVL diagnostic techniques used culture as the gold standard [10,14,26,28]. Using LCA, we found that real time PCR and culture were the most sensitive techniques. Among the serological tests evaluated, DPP CVL had the best performance. Although IFI CVL had the highest sensitivity, it was the least specific, as previously described by de Santis and collaborators (2013) and Laurenti and collaborators (2014). Regarding the performance measure that has epidemiological relevance, the PPV, the DPP CVL had the highest PPV among the serological tests evaluated as previously described by da Silva and collaborators (2013). In addition to evaluating each individual test for CVL diagnosis, we compared the performance of previous and current protocols employed in Brazil. The usefulness of protocols was evaluated by determining PPVs and NPVs of each protocol. The better individual performance of the DPP CVL was reflected in the 13% higher PPV of the current protocol for CVL detection compared to the previous protocol. Both protocols yielded a NPV of 0.96, suggesting that when these protocols have negative results it is highly probable that serum are from dogs that are actually uninfected. By contrast, the current diagnostic protocol provides a greater PPV (0.83) than that of the previous protocol (0.70), indicating that the current protocol provides a greater level of assertiveness in diagnosing positive dogs. Although the new protocol showed a higher specificity and PPV than the previous one, the sensitivity is still limited (around 0.73) in both protocols, meaning that the maintenance of false-negative dogs in endemic areas still represents a public health concern and more efforts should be done to try to find out better protocols or new antigens to reduce the maintenance of infected dogs in areas of zoonotic transmission. Considering questions rose about the wisdom to diagnose CVL using DPP CVL + EIE CVL instead of DPP CVL alone, the comparison of performances showed that a higher sensitivity value (0.86) and lower PPV (0.71) for DPP CVL compared to DPP CVL + EIE CVL (0.73 and

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0.83, respectively) that might cause detection of false positive dogs. Mostly to avoid this, a confirmatory test, EIE CVL, has been associated to DPP CVL in the current protocol.” When current protocol is applied for diagnosing asymptomatic and symptomatic dogs, it showed similar performance for sensitivity (0.71, 0.73) and specificity (0.98, 0.97), respectively. While, the level of NPV (0.99) was greater, the level of PPV (0.63) was much lower for asymptomatic dogs in comparison to NPV (0.92) and PPV (0.88) for symptomatic animals. In accordance to this results, Otranto and collaborators (2009) showed that recently exposed or newly infected dogs might not be detected by serological tests, since these false-negative animals do not seroconvert soon after infection or they may develop a cellular type of immune response that are not detected using serological tests. In addition to this difficulty, no appropriate gold standard for Leishmania infection detection in asymptomatic dogs was established, highlighting the necessity for the development of new tests to improve diagnosis of asymptomatic dog. Across a range of plausible prevalence, the theoretical expectation for PPV varied among 0.13 to 0.98 for previous protocol, and 0.23 to 0.99 for current protocol. PPV and NPV of a diagnostic test are known to be influenced by the prevalence of a given disease in a population. Thus, as disease becomes more prevalent the probability of subjects to test positive in diagnostic tests will be higher among sick individuals. In the present study, the analysis using different theoretical prevalence revealed that the current protocol has high performance irrespective of disease prevalence. In accordance, higher PPVs provided by DPP CVL + EIE CVL for diagnosing CVL have additional advantages since in endemic countries, regardless of the prevalence of CVL, the current protocol compared to previous one would better discriminate truly uninfected dogs from those that have risky to be infected. In summary, our findings show that the current protocol for diagnosis of CVL implemented in Brazil has an excellent accuracy (0.91 for symptomatic dogs and 0.97 for asymptomatic), due to its greater specificity values and PPV. Because of the simplicity of test procedures and rapidity of results, the data presented herein strongly support the idea that the introduction of DPP CVL into the diagnostic CVL protocol contribute to improve CVL diagnosis that can have consequent effects that impact positively on disease control.

Supporting Information S1 Checklist. Standards for the Reporting of Diagnostic Accuracy Studies (STARD) checklist for reporting of studies of diagnostic accuracies. (DOC) S1 Fig. STARD flowchart. Standards for the Reporting of Diagnostic Accuracy Studies (STARD) description of the experimental design to calculate accuracy of CVL serodiagnostic protocols. (TIFF)

Acknowledgments The authors would like to thank the staff of the Zoonosis Control Center of Camaçari for their assistance in conducting the study. We especially thank Gilmar Cerqueira Pereira, Adriana O. Almeida and Sandra M. S. Passos for their help in the endemic area. Additionally, the authors acknowledge Bio-Manguinhos for providing the DPP CVL, EIE CVL and IFI CVL diagnostic tests. Finally, we are grateful to BioMed Proofreading LLC for providing English revision, consulting services and manuscript edition by native English-speaking experts.

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Author Contributions Conceived and designed the experiments: DBMF LVP PSTV. Performed the experiments: DBMF LVP LSB LAB MdSS. Analyzed the data: DBMF LVP PGdSET LDAFA. Contributed reagents/materials/analysis tools: PGdSET LDAFA PSTV. Wrote the paper: DBMF LVP LDAFA PSTV.

References 1.

Alvar J, Canavate C, Molina R, Moreno J, Nieto J (2004) Canine leishmaniasis. Adv Parasitol 57: 1– 88. PMID: 15504537

2.

Bevilacqua PD, Paixão HH, Modena CM, Castro MCPS (2001) Urbanização da leishmaniose visceral em Belo Horizonte. Arq Bras Med Vet Zootec 53.

3.

Quinnell RJ, Courtenay O (2009) Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis. Parasitology 136: 1915–1934. doi: 10.1017/S0031182009991156 PMID: 19835643

4.

Brasil (2010) Manual de Vigilância e Controle da Leishmaniose Visceral. In: Epidemiológica DdVn, editor. Brasília: Ministério da Saúde e Secretaria de Vigilância em Saúde.

5.

MS, CGDT, CGLAB, DEVIT, SVS (2011) Technical Note joint N° 01/2011 "Enlightenment on replacement of the diagnostic protocol of canine visceral leishmaniasis".

6.

Figueiredo FB, Madeira MF, Nascimento LD, Abrantes TR, Mouta-Confort E, et al. (2010) Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples. Rev Inst Med Trop Sao Paulo 52: 193–196. PMID: 21748226

7.

Lira RA, Cavalcanti MP, Nakazawa M, Ferreira AG, Silva ED, et al. (2006) Canine visceral leishmaniosis: a comparative analysis of the EIE-leishmaniose-visceral-canina-Bio-Manguinhos and the IFI-leishmaniose-visceral-canina-Bio-Manguinhos kits. Vet Parasitol 137: 11–16. PMID: 16446034

8.

Silva DA, Madeira MF, Teixeira AC, de Souza CM, Figueiredo FB (2011) Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program. Vet Parasitol 179: 257–261. doi: 10.1016/j.vetpar.2011.01.048 PMID: 21349644

9.

Boarino A, Scalone A, Gradoni L, Ferroglio E, Vitale F, et al. (2005) Development of recombinant chimeric antigen expressing immunodominant B epitopes of Leishmania infantum for serodiagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol 12: 647–653. PMID: 15879027

10.

Grimaldi G Jr., Teva A, Ferreira AL, dos Santos CB, Pinto IS, et al. (2012) Evaluation of a novel chromatographic immunoassay based on Dual-Path Platform technology (DPP(R) CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis. Trans R Soc Trop Med Hyg 106: 54–59. doi: 10.1016/j. trstmh.2011.10.001 PMID: 22137538

11.

Ferroglio E, Zanet S, Mignone W, Poggi M, Trisciuoglio A, et al. (2013) Evaluation of a rapid device for serological diagnosis of Leishmania infantum infection in dogs as an alternative to immunofluorescence assay and Western blotting. Clin Vaccine Immunol 20: 657–659. doi: 10.1128/CVI.00719-12 PMID: 23446218

12.

Solca Mda S, Bastos LA, Guedes CE, Bordoni M, Borja LS, et al. (2014) Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis. PLoS One 9: e103635. doi: 10.1371/journal.pone.0103635 PMID: 25076494

13.

Rodriguez-Cortes A, Ojeda A, Francino O, Lopez-Fuertes L, Timon M, et al. (2010) Leishmania infection: laboratory diagnosing in the absence of a "gold standard". Am J Trop Med Hyg 82: 251–256. doi: 10.4269/ajtmh.2010.09-0366 PMID: 20134001

14.

de Santis B, Santos EGB, de Souza CdSF, Chaves SAdM (2013) Performance of DPP TM immunochromathographic rapid test (IRT) for canine visceral leishmaniasis: comparison with other serological methods in suspected dogs from Cuiabá, Mato Grosso State, Brazil. Braz J Vet Res Anim Sci 50: 198–205.

15.

Schallig HD, Cardoso L, Hommers M, Kroon N, Belling G, et al. (2004) Development of a dipstick assay for detection of Leishmania-specific canine antibodies. J Clin Microbiol 42: 193–197. PMID: 14715752

16.

Solano-Gallego L, Villanueva-Saz S, Carbonell M, Trotta M, Furlanello T, et al. (2014) Serological diagnosis of canine leishmaniosis: comparison of three commercial ELISA tests (Leiscan, ID Screen and Leishmania 96), a rapid test (Speed Leish K) and an in-house IFAT. Parasit Vectors 7: 111. doi: 10. 1186/1756-3305-7-111 PMID: 24655335

17.

Solcà MdS, Guedes CE, Nascimento EG, Oliveira GG, dos Santos WL, et al. (2012) Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs. Vet Parasitol 184: 133–140. doi: 10.1016/j.vetpar.2011.08.026 PMID: 21917379

18.

Rindskopf D, Rindskopf W (1986) The value of latent class analysis in medical diagnosis. Stat Med 5: 21–27. PMID: 3961312

PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004333

January 5, 2016

10 / 11

Diagnosis of Canine Visceral Leishmaniasis

19.

Hui SL, Walter SD (1980) Estimating the error rates of diagnostic tests. Biometrics 36: 167–171. PMID: 7370371

20.

Boelaert M, Rijal S, Regmi S, Singh R, Karki B, et al. (2004) A comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis. Am J Trop Med Hyg 70: 72–77. PMID: 14971701

21.

Boelaert M, Aoun K, Liinev J, Goetghebeur E, Van der Stuyft P (1999) The potential of latent class analysis in diagnostic test validation for canine Leishmania infantum infection. Epidemiol Infect 123: 499– 506. PMID: 10694163

22.

Langhi DM Jr., Bordin JO, Castelo A, Walter SD, Moraes-Souza H, et al. (2002) The application of latent class analysis for diagnostic test validation of chronic Trypanosoma cruzi infection in blood donors. Braz J Infect Dis 6: 181–187. PMID: 12204185

23.

Girardi E, Angeletti C, Puro V, Sorrentino R, Magnavita N, et al. (2009) Estimating diagnostic accuracy of tests for latent tuberculosis infection without a gold standard among healthcare workers. Euro Surveill 14.

24.

Koukounari A, Webster JP, Donnelly CA, Bray BC, Naples J, et al. (2009) Sensitivities and specificities of diagnostic tests and infection prevalence of Schistosoma haematobium estimated from data on adults in villages northwest of Accra, Ghana. Am J Trop Med Hyg 80: 435–441. PMID: 19270295

25.

Ibironke O, Koukounari A, Asaolu S, Moustaki I, Shiff C (2012) Validation of a new test for Schistosoma haematobium based on detection of Dra1 DNA fragments in urine: evaluation through latent class analysis. PLoS Negl Trop Dis 6: e1464. doi: 10.1371/journal.pntd.0001464 PMID: 22235360

26.

da Silva DA, Madeira Mde F, Abrantes TR, Filho CJ, Figueiredo FB (2013) Assessment of serological tests for the diagnosis of canine visceral leishmaniasis. Vet J 195: 252–253. doi: 10.1016/j.tvjl.2012. 06.010 PMID: 22789627

27.

Schubach EY, Figueiredo FB, Romero GA (2014) Accuracy and reproducibility of a rapid chromatographic immunoassay for the diagnosis of canine visceral leishmaniasis in Brazil. Trans R Soc Trop Med Hyg 108: 568–574. doi: 10.1093/trstmh/tru109 PMID: 25015665

28.

Laurenti MD, de Santana Leandro MV Jr., Tomokane TY, De Lucca HR, Aschar M, et al. (2014) Comparative evaluation of the DPP((R)) CVL rapid test for canine serodiagnosis in area of visceral leishmaniasis. Vet Parasitol 205: 444–450. doi: 10.1016/j.vetpar.2014.09.002 PMID: 25257505

29.

Coura-Vital W, Ker HG, Roatt BM, Aguiar-Soares RD, Leal GG, et al. (2014) Evaluation of change in canine diagnosis protocol adopted by the visceral leishmaniasis control program in Brazil and a new proposal for diagnosis. PLoS One 9: e91009. doi: 10.1371/journal.pone.0091009 PMID: 24608904

30.

Barrouin-Melo SM, Larangeira DF, de Andrade Filho FA, Trigo J, Juliao FS, et al. (2006) Can spleen aspirations be safely used for the parasitological diagnosis of canine visceral leishmaniosis? A study on assymptomatic and polysymptomatic animals. Vet J 171: 331–339. PMID: 16490717

31.

Barrouin-Melo SM, Larangeira DF, Trigo J, Aguiar PH, dos-Santos WL, et al. (2004) Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs. Mem Inst Oswaldo Cruz 99: 195–197. PMID: 15250475

32.

Francino O, Altet L, Sanchez-Robert E, Rodriguez A, Solano-Gallego L, et al. (2006) Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis. Vet Parasitol 137: 214– 221. PMID: 16473467

33.

Collins LM, Lanza ST (2010) Latent Class and Latent Transition Analysis with Applications in the Social, Behavioural, and Health Sciences. New Jersey, USA: Wiley.

34.

Muthén LK, Muthén BO (1998–2010) MPlus Statistical Analysis With Latent Variables User’s Guide. Los Angeles, CA: Muthén & Muthén.

35.

Canavate C, Herrero M, Nieto J, Cruz I, Chicharro C, et al. (2011) Evaluation of two rK39 dipstick tests, direct agglutination test, and indirect fluorescent antibody test for diagnosis of visceral leishmaniasis in a new epidemic site in highland Ethiopia. Am J Trop Med Hyg 84: 102–106. doi: 10.4269/ajtmh.2011. 10-0229 PMID: 21212210

36.

Machado de Assis TS, Rabello A, Werneck GL (2012) Latent class analysis of diagnostic tests for visceral leishmaniasis in Brazil. Trop Med Int Health 17: 1202–1207. doi: 10.1111/j.1365-3156.2012. 03064.x PMID: 22897740

37.

Pan-ngum W, Blacksell SD, Lubell Y, Pukrittayakamee S, Bailey MS, et al. (2013) Estimating the true accuracy of diagnostic tests for dengue infection using bayesian latent class models. PLoS One 8: e50765. doi: 10.1371/journal.pone.0050765 PMID: 23349667

38.

Wu HM, Cordeiro SM, Harcourt BH, Carvalho M, Azevedo J, et al. (2013) Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis. BMC Infect Dis 13: 26. doi: 10.1186/1471-2334-13-26 PMID: 23339355

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Contents lists available at ScienceDirect

Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Research paper

Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors Lairton Souza Borja a , Orlando Marcos Farias de Sousa a , Manuela da Silva Solcà a , Leila Andrade Bastos a , Marcelo Bordoni a , Jairo Torres Magalhães b , Daniela Farias Larangeira b , Stella Maria Barrouin-Melo b , Deborah Bittencourt Mothé Fraga a,b,c , Patrícia Sampaio Tavares Veras a,c,∗ a Laboratório de Patologia e Biointervenc¸ão, Centro de Pesquisa Gonc¸alo Moniz–FIOCRUZ, Rua Waldemar Falcão, 121, Candeal, 40296-710 Salvador, Bahia, Brazil b Escola de Medicina Veterinária, Universidade Federal da Bahia, Avenida Adhemar de Barros, 500, Ondina, 40170-110 Salvador, Bahia, Brazil c Instituto Nacional de Ciência e Tecnologia em Doenc¸as Tropicais, INCT – DT, Bahia, Brazil

a r t i c l e

i n f o

Article history: Received 13 July 2016 Received in revised form 29 September 2016 Accepted 2 October 2016 Keywords: Canine infectiousness Parasite load Xenodiagnosis

a b s t r a c t The sand fly Lutzomyia longipalpis is primarily responsible for the transmission of visceral leishmaniasis (VL) in the New World, and dogs are considered to be the main urban reservoir of this disease. In order to improve the efficacy of control measures, it is essential to assess the transmission capacity of Leishmania infantum to the sand fly vector by naturally infected dogs. The present study investigated the existence of correlations between canine clinical presentation and the intensity of parasite load in the blood, skin and spleen of naturally infected dogs. In addition, we also attempted to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs. A total of 23 dogs were examined and classified according to clinical manifestation of canine VL. Blood samples, splenic aspirate and skin biopsies were collected and parasite DNA was quantified by qPCR. Canine capacity to infect Lu. longipalpis with parasites was evaluated by xenodiagnosis and parasite loads were measured five days after feeding. No significant differences were observed with respect to canine clinical manifestation and the parasite loads detected in the blood, skin and spleen samples obtained from naturally infected dogs. Regardless of clinical canine visceral leishmaniasis (CVL) presentation and the degree of parasite burden, almost half of the dogs successfully infected sandflies with parasites, albeit to a low number of sandflies with correspondingly low parasite loads. Parasite loads in both canine blood and skin were shown to be positively correlated with the canine infectiousness to the sand fly vector, and positive correlations were also observed with respect to these tissues and the sand fly infection rate, as well as the parasite load detected in sandflies following xenodiagnosis. In conclusion, this indicates that parasite loads in both blood and skin can function as potentially reliable markers of canine capacity to infect sand fly vector. © 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Visceral leishmaniasis (VL) is a severe systemic disease that affects humans, dogs and other mammalian vertebrates (WHO, 2010). In the New World, VL generally results from infection by the

∗ Corresponding author at: Laboratório de Patologia e Biointervenc¸ão, Centro de Pesquisa Gonc¸alo Moniz–FIOCRUZ, Rua Waldemar Falcão, 121, Candeal, 40296-710 Salvador, Bahia, Brazil. E-mail addresses: [email protected], [email protected] (P.S.T. Veras).

protozoan parasite Leishmania infantum (syn. L. chagasi), which is transmitted to mammalian hosts mainly by the Lu. longipalpis sand fly vector (WHO, 2010). Abundant evidence indicate that domestic dogs are the main reservoir of VL in urban areas, which is supported by reports of canine epidemics preceding outbreaks in humans in endemic regions (Alvar et al., 2004; Bevilacqua et al., 2001). Due to variability in the immune response to infection, dogs with visceral leishmaniasis (CVL) can present as either asymptomatic or with a progressively symptomatic form of the disease (Ciaramella et al., 1997; Rallis et al., 2005). In general, the capa-

http://dx.doi.org/10.1016/j.vetpar.2016.10.004 0304-4017/© 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

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bility to transmit parasites to sandflies has been associated with disease severity. Several studies have reported that most symptomatic dogs are capable of transmitting parasites to sandflies in endemic areas, and that these animals are more infectious than asymptomatic dogs, confirming their importance in the transmission cycle of VL (Courtenay et al., 2002; da Costa-Val et al., 2007; Guarga et al., 2000). By contrast, other studies have demonstrated that a given dog’s capacity to transmit parasites to sand fly vectors is independent of its clinical CVL presentation (asymptomatic, oligosymptomatic or polysymptomatic) (Guarga et al., 2000; Molina et al., 1994). Unfortunately, none of these studies has attempted to elucidate parameters for distinguishing dogs that are capable of transmitting parasites to sand fly vectors from those that are incapable of transmission. Since VL control measures in Brazil are based on serological diagnosis and the euthanasia of seropositive dogs (Ministério da Saúde et al., 2006), to increase the efficacy of such measures, it is essential to ascertain infectiousness of naturally infected dogs. Parasite transmission occurs by the contact of sand fly proboscides with the skin and blood of an infected host vertebrate during blood-feeding (Bates, 2007). We hypothesized that a given infected dog would infect sandflies with L. infantum in accordance with the intensity of its tissue (skin or blood) parasite burden. To test this hypothesis, we quantified parasite load in the skin and blood of naturally infected dogs presenting a range of clinical manifestations and correlated the intensity of parasite load in these tissues with a given dog’s capacity to infect sandflies with parasites.

2. Materials and methods 2.1. Ethical considerations All experiments involving canine specimens were performed in compliance with Brazilian federal law for animal experimentation (Law 11794), in conformity with the Oswaldo Cruz Foundation (FIOCRUZ) animal experimentation guidelines, and according to the instructions outlined in the Brazilian Ministry of Health’s manual for the surveillance and control of VL. The present study was approved by the Institutional Review Board (CEUA protocol no. 015/2009) of the Gonc¸alo Moniz Research Center in Bahia, Brazil (CPqGM–FIOCRUZ/BA). Dog owners who agreed to participate in the study signed a Free, Prior and Informed Consent (FPIC) form.

2.2. Canine specimens A total of 23 dogs were recruited directly from their owners during the period of April 2011 and September 2012, some of whom reside in endemic regions within the State of Bahia, Brazil: (i) 15 animals were pets from the municipality of Dias D’Ávila, (ii) 6 from the municipality of Camac¸ari and (iii) 2 dogs domiciled in Salvador. Dogs were included in the study if they tested positive for CVL by aspirate spleen culture or qPCR of blood, skin or spleen tissue. All dogs were clinically examined by a veterinarian and subsequently classified in accordance with the intensity of the most common clinical signs of CVL, including weight loss, alopecia, cutaneous lesions, conjunctivitis, onychogryphosis and lymphadenopathy. Thereafter, each detected clinical sign was categorized in grades of 0, 1, 2 or 3 in accordance with its intensity. Clinical scores were then calculated by summing the grades assigned for each clinical sign present in infected dogs. Animals were classified as without clinical manifestation (subclinical infection, clinical score 0–3) or with clinical disease (symptomatic infection, clinical score 3 + ). The Municipal Center for Zoonotic Disease Control received notification regarding all positive dogs.

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2.3. Sample collection To perform parasitological diagnosis and determine parasite loads, blood collection, spleen aspiration and skin biopsies were performed. Samples of peripheral blood were collected by venipuncture (minimum of 5 mL) then aliquoted and stored at −80 ◦ C. The dogs were anesthetized with acepromazine (0.1 mg/kg iv, Vetnil, Brazil) and splenic aspirate samples were obtained using a puncture technique previously described by Barrouin-Melo et al. (2006), which was modified to incorporate guidance by a portable ultrasound device (Solca Mda et al., 2014). Lidocaine solution (0.1%) was used as local anesthesia to perform cutaneous punch biopsies. Skin samples with a diameter of 4 mm were collected from the internal surface of a single lesion-free ear. 2.4. Parasitological testing and serological evaluation Splenic aspirate samples were collected as explained above and culturing was performed as previously described (BarrouinMelo et al., 2006; Solca Mda et al., 2014). Briefly, splenic aspirates were cultured for a period of four weeks in Novy–MacNeal–Nicolle (NNN) biphasic medium, supplemented with 20% FBS (Fetal Bovine Serum, Gibco BRL, New York, USA) and 50 !g/mL of gentamicin to avoid contamination (Sigma Chemical Co., St. Louis, MO) at 23 ◦ C (Barrouin-Melo et al., 2006). Microscopy for parasite detection was performed at weekly intervals for no less than four weeks. Each splenic culture was prepared in duplicate and double-checked to avoid misidentification. The serological evaluation was realized usind the immunochromatographic rapid test DPP® LVC (Bio-Manguinhos, Brazil). The diagnostic test procedures with DPP® LVC was realized in accordance with manufacturer recommendations. 2.5. Xenodiagnosis All 23 animals were submitted to xenodiagnosis no later than 10 days following CVL diagnosis and tissue sample collection. Adult Lu. longipalpis were obtained from closed colonies at the Laboratory of Veterinary Infectious Diseases at the Federal University of Bahia and the other at the Gonc¸alo Moniz Research Center (FIOCRUZ-BA). Both sand fly colonies were originally formed by sandflies captured in the endemic municipality of Ipecaetá, located in northeastern Bahia. Insects from 8 to 12 generations were used in xenodiagnosis experimentation. To carry out this procedure, sandflies, including both 30–40 females and 10–15 males, were starved for 24 h, then placed in transparent PVC cylinders covered with fine mesh over one end. Each dog had a cylinder affixed to one of its ears without lesions or scabs, so as to maintain contact for 40 min, thus allowing the insects to feed on the dogs’ blood. Xenodiagnosis was only considered successful when an estimated 70% or more of the sandflies were observed taking a blood meal. In only one xenodiagnosis procedure, the estimated percentage was lower than 70%, thus the procedure was considered invalid, and was repeated the next day. All sandflies were subsequently kept for a period of five days and allowed to feed on a saturated glucose solution. After five days, all surviving sandflies were frozen at −80 ◦ C until DNA extraction. qPCR was then performed to determine the presence of parasite DNA and quantify parasite load. The capacity of naturally infected dogs to infect sand fly vectors with L. infantum was assessed by considering multiple factors: (1) canine infectiousness to sandflies, estimated as the percentage of dogs that successfully infected sandflies with L. infantum during xenodiagnosis; (2) sand fly infection rate, estimated as the number of sandflies that were successfully infected during xenodiagnosis; and (3) the average parasite load detected in successfully infected sandflies following xenodiagnosis.

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2.6. DNA extraction from canine tissue and sandflies DNA extraction from splenic aspirates, skin biopsies (4 mm), and whole blood (200 !L) samples was performed using the DNeasy Blood & Tissue kit in accordance with the manufacturer’s instructions (Qiagen, Hilden, Germany). The females of Lu. longipalpis used in xenodiagnosis were individually transferred to 1.5 mL microcentrifuge tubes and DNA extraction was performed as previously described (Michalsky et al., 2007). DNA concentration and quality were then assessed and each sample was aliquoted and stored at −20 ◦ C. 2.7. Leishmania kinetoplast DNA (kDNA) Quantification To determine parasite load in canine samples and sandflies, Leishmania DNA was quantified by qPCR assays employing an amplification procedure previously described (Francino et al., 2006) and modified by Solcà and collaborators (Solca Mda et al., 2014). The taqMan-MGB probe and PCR primers were designed to target conserved DNA regions of the kinetoplast minicircle DNA from L. infantum to obtain a 120-bp amplicon. The qPCR amplification protocol (Solca Mda et al., 2014) employed the following primers: forward primer 5′ -AACTTTTCTGGTCCTCCGGGTAG-3′ (Leish-1) and reverse primer 5′ -ACCCCCAGTTTCCCGCC-3′ (Leish2), both at a final concentration of 900 nM. A fluorogenic probe 5′ -AAAAATGGGTGCAGAAAT-3′ was used for detection, synthesized using a FAM reporter molecule attached to the 5′ end, as well as a MGB-NFQ quencher linked to the 3′ -end (Perkin-Elmer Applied Biosystems) at a final concentration of 200 nM. Parasite load was determined by comparing cycle threshold (CT) values derived from a standard curve, which was obtained from DNA amplification using 10-fold serial dilutions of Leishmania DNA performed in triplicate, ranging from 105 to 10−1 parasites. In order to minimize interplate variation, the averaged CT values for each sample were normalized based on a common fluorescence detection baseline value. A CT value was recorded at the point at which its fluorescence signal crossed the established detection baseline. In order to overcome the limitations caused by melanin present in skin samples submitted to PCR, all steps leading up to DNA amplification were performed in the presence of bovine serum albumin (2.5 !g/each reaction) (Sigma Chemical) to prevent the inhibition of PCR (Giambernardi et al., 1998). Two separate receiver operating characteristic (ROC) curves were plotted to obtain the optimal CT cut-off point for considering either a canine or sand fly sample as positive. First, to determine the CT cut-off point for Leishmania DNA in canine samples, the amplification results from 20 Leishmania-negative samples and 20 Leishmania-positive samples were plotted on a ROC curve. Next, for Leishmania DNA detection in sand fly samples, a ROC curve was constructed using the amplification results from 40 sandflies used in the xenodiagnosis of two confirmed negative animals (noninfected dogs from a non-endemic area with negative results in serology by DPP® LVC, screening test recommended by Brazilian Health Ministry, and negative results in PCR and culture of spleen aspirate) and 12 male sandflies reared in a laboratory spiked with Leishmania DNA (positive controls). Finally, a range of CT cut-off points across each ROC curve were analyzed for sensitivity and specificity to determine its optimal CT cut-off value. Canine tissue and sand fly samples were considered positive when CT values were less than or equal to its corresponding ROC curve CT cut-off value. To evaluate dog DNA integrity, amplification of the 18S rRNA gene (Perkin-Elmer Applied Biosystems) was used as an internal reference of genomic canine DNA. Only canine DNA samples that successfully amplified the 18S rRNA gene were included. Predeveloped TaqMan assay reagents were used in accordance with

manufacturer’s recommendations. The slope of the standard curve for the 18S rRNA gene corresponded to −3.406 (SD ± 0.415) and an average slope was determined from CT values obtained from five independent experiments with a corresponding correlation coefficient (r2 ) of 0.993 (SD ± 0.002). The results of parasite loads in infected dogs are expressed as the number of parasites per 10 mg of DNA of canine host tissue. 2.8. Statistical analysis Differences in parasite loads among dogs with different clinical manifestations, as well as parasite loads in infected sandflies used for xenodiagnosis, were evaluated using the Mann-Whitney test. Fisher’s exact test was used to compare infectiousness of dogs with presenting different clinical manifestation. Spearman’s rank correlation coefficient was used to conduct correlation analysis. All p values < 0.05 were considered to be statistically significant. 3. Results 3.1. Study group Of the 23 dogs included in this study, 13 were classified as dogs with subclinical infection and 10 as with symptomatic infection (Table 1). Cultures of splenic aspirate were positive in nine animals (39%), 6 with subclinical infection (46%) and 3 with symptomatic infection (30%) while serology by DPP was positive in eight (35%), 3 with subclinical infection (23%) and 5 with symptomatic infection (50%). All dogs tested positive by qPCR with respect to at least one of the tissue types evaluated, including 18 positive skin samples (78.2%), 10 with subclinical infection, 77%; 8 with symptomatic infection, 80%, 18 splenic aspirate samples (78.2%), 11 with subclinical infection (75%) and 7 with symptomatic infection (70%), and eight blood samples (35%), 4 with subclinical infection (31%) and 4 with symptomatic infection (40%). 3.2. Parasite load in tissue samples from infected dogs with diverse clinical manifestations of CVL The median values found for parasite load in each evaluated canine tissue type were: 316 parasites in skin and 1335 in splenic aspirate (Table 1). Since in 15 out of 23 dogs, parasite DNA was not detected in blood samples, the median parasite load resulted in 0. Considered only samples from dogs that qPCR tested positive (n = 8), the median parasite load resulted in 309. No significant differences were found when comparing detected parasite load values within a given tissue type with respect to clinical manifestations of CVL (dogs with subclinical or symptomatic infection) (Fig. 1). With respect to correlation testing considering parasite burden among the three tissue types evaluated, positive correlations were observed between parasite load in the blood and spleen (Spearman’s; r = 0.57, P < 0.001), blood and skin (Spearman’s; r = 0.64, p < 0.0001), and spleen and skin (Spearman’s; r = 0.57, p < 0.001). 3.3. Capacity of naturally infected dogs to infect sand fly vectors with parasites An assessment of the capacity of naturally infected dogs to infect sand fly vectors with parasites revealed that among the 653 sandflies used in xenodiagnosis, only 74 (11.4%) tested positive for Leishmania DNA by qPCR (Table 1). Canine infectiousness to sandflies, calculated as the percentage of dogs that successfully transmitted parasites to sandflies during xenodiagnosis, was similar in dogs with subclinical infection (54%; 07/13) and symptomatic infection (40%; 4/10) (p = 0.39). Further analysis of these dogs’ capacity to infect sandflies showed that 44% of the infected animals

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Table 1 Evaluation of naturally infected dogs with different clinical manifestations according to positivity in diagnostic tests for Leishmania infection and the capacity to infect sand fly vector. Clinical classification/Dog no. Clinical Score Diagnostic Test Type Clinical classification/Dog no. DPP

Canine capacity of infect sandflies

Aspirate Spleen Culture qPCR (Leishmania kDNA) Skin

Subclinical infection 01 02 03 04 05 06 07 08 09 10 11 12 13 Symptomatic infection 14 15 16 17 18 19 20 21 22 23 Total positivity rate

0 0 0 0 2 1 2 1 1 3 1 2 2 5 7 6 8 10 12 10 17 8 7

− − − − + − − − − − + + − − − − + − + + + − + 08/23 (35%)

+ − + − + − − − − + − + +

89 0 1238 0 18,020,075 0 24 66 645 1553 1014 97 59,187,691

− − − − − − − + + + 09/23 (39.1%)

316 67 0 42,632 147 737,615 0 3,451,889,073 30,625,119 22,904,007 18/23 (78.2%)

Infectiousness Infection rate Parasite load detected

Spleen

Blood

274 0 0 0 6315 0 0 118 0 0 0 0 509

1335 113 660 447 339,826 18437 0 0 628 966 26,274 602 17,105

0 0 0 0 0 58 0 151194 344 179 08/23 (35%)

0 0 0 0 9382 0 5640 0 0 1 17,602 0 2198 0 17,863,788 1 19,058,029 1 3440,041 1 18/23 (78.2%) 11/23 (47.8%)

1 0 0 1 1 0 0 1 1 1 0 0 1

01/22 (4.5%) 0/31 (0%) 0/42 (0%) 02/32 (6.25%) 01/36 (2.7%) 00/39 (0%) 0/14 (0%) 01/23 (4.3%) 1/41 (2.4%) 25/31 (80.6%) 0/25 (0%) 0/28 (0%) 01/28 (3.5%) 00/32 (0%) 0/23 (0%) 00/25 (0%) 0/25 (0%) 01/24 (4.1%) 0/13 (0%) 0/23 (0%) 28/36 (77.7%) 11/29 (38%) 02/28 (7.1%) 74/653 (11.3%)

3 0 0 3 45 0 0 4 3 689 0 0 3 0 0 0 0 8 0 0 29774 197 970 1338

Fig. 1. Blood, skin and spleen samples parasite load of infected dogs according to clinical classification. Blood (A), skin (B), and spleen aspirate samples (C) were obtained from dogs classified with a different form of clinical manifestations of CVL: subclinical infection or symptomatic infection. Parasite load was detected using qPCR in all skin, blood, and splenic aspirate samples. Each plot represents the detected parasite load in an individual dog.

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Fig. 2. Sand fly infection rate of naturally infected dogs according to clinical classification. Sand fly infection rates, calculated as the number of successfully infected sandflies following xenodiagnostic procedures, distributed according to clinical manifestation.

were capable of successfully infect sand fly vector with parasites, although 80% of these dogs infected to a relatively small number of sandflies (n = 1 to 2). Sand fly infection rate, which measures the number of sandflies that were positively infected following xenodiagnosis, remained similar between dogs with subclinical infection (9%) and with symptomatic infection (13%) dogs (Fig. 2). Parasite load quantification performed in each sand fly submitted to xenodiagnosis revealed that parasite load ranged from 1 to 5971 parasites in the 74 infected sandflies, with a median value of 28 parasites, the 25% percentile was 4 and 75% percentile was 112.5 parasites. The median parasite loads among sandflies that became successfully infected was 10, after feeding on dogs with subclinical infection, and 84, after feeding on dogs with symptomatic infection. No differences were found when comparing the average parasite loads in infected sandflies, irrespective of the clinical classification of the animals on which the vectors fed (Fig. 3A). Fig. 3B illustrates individual sand fly parasite loads five days after xenodiagnosis. Most sandflies showed low parasite loads and, notably, a single highly symptomatic dog (score = 17, dog no 21) was found to be responsible for the highest parasite loads detected in sandflies (total: 29,774 parasites counted in 28 infected sandflies) (Fig. 3B). Parasite load values in the dogs’ blood and skin were shown to be positively correlated with canine infectiousness to sandflies (blood r = 0.65, p < 0.001; skin r = 0.42, p = 0.05) (Fig. 4A and B), yet no correlations were found with respect to spleen tissue (r = 0.27, p = 0.21). Interestingly, positive correlations were also found between parasite load in blood (r = 0.59 P < 0.01) or skin (r = 0.43, P ≤ 0.05) and sand fly infection rate (Fig. 4C and D). Finally, a positive correlation was additionally found between parasite load in canine blood (r = 0.66, P < 0.001) and skin (r = 0.54, P < 0.01) and parasite burden in sandflies following xenodiagnosis (Fig. 5A and B). No correlations were found when comparing the parasite loads detected in splenic aspirate samples (Fig. 5C) to the parasite burden found in vectors five days after feeding. 4. Discussion The present study represents an initial attempt to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs with a range of clinical presentations. Almost half of the dogs were found to be capable of infect sand fly vectors with parasites, regardless of their clinical classification and corresponding clinical score. This finding supports the notion

that a particular canine clinical manifestation of VL is not indicative of a dog’s capacity to infect sand fly vectors with parasites. Remarkably, among these infective dogs, most (82%) infected to a relatively limited number of sandflies (between 1 and 2), which corresponds to approximately 10% of the total number of sandflies used in xenodiagnosis procedures. These results are consistent with those obtained by Courtenay et al. (2002), who similarly showed that most of the infective dogs studied infected no more than 10% of the sandflies employed. Taken together, these findings would seem to suggest that, in a given endemic area, one would expect a low number of infective dogs to successfully infect a proportionately low number of sandflies with parasites. The present study utilized qPCR to detect parasite DNA in vectors five days following xenodiagnosis procedures, with low parasite burden found in the vast majority of sandflies. This may be explained by the choice to assess parasite load in the vectors at only five days post-xenodiagnosis. Undoubtedly, by quantifying the number of infective metacyclic forms of promastigotes under direct observation at seven days or later following blood-feeding, sensitivity would be enhanced. Additionally, this important information would provide a notion of how many infective parasites could be successfully transmitted to another mammalian host in a subsequent sand fly blood feeding. Another possibility to improve sensitivity with respect to this procedure is performing xenodiagnosis in animal lesion sites, as previously described by Aslan et al. (2016) in an experimental model of CVL. Future studies should strive to not only comprehensively address the proper site on the animal’s skin to perform xenodiagnosis in naturally infected dogs, but also quantify parasite burden, in the sandflies that were successfully blood-fed, at time points seven days or later following blood-feeding. The low infection rates and low parasite loads detected in sandflies in the present study support the notion that most vectors do not significantly impact the urban VL transmission cycle. Interestingly, we found a very high parasite load in the blood and skin of one of the dogs we evaluated, which was able to infect a high number of sandflies (78%) resulting in high parasite loads (5.9 to 10 × 103 parasites) at five days after feeding. We recognize that since our dog population represents a convenience sampling, the present findings are not indicative of the actual transmission cycle seen in a given dog population within an endemic area. Nonetheless, we speculate that a low number of highly infected dogs would likely have a more significant impact on parasite transmission to sandflies, while higher numbers of less-infected dogs should present less risk of transmissibility in the urban VL transmission cycle in endemic areas. In the present report we also found that neither canine infectiousness nor sand fly infection rates have been found associated to CVL clinical manifestations similar to other authors (Guarga et al., 2000; Laurenti et al., 2013; Magalhaes-Junior et al., 2016; Molina et al., 1994). Different from us, other authors have demonstrated that both the capacity of dogs infect sandflies and sand fly infection rates are positively correlated with the presence of clinical signs in infected dogs (da Costa-Val et al., 2007; Vercosa et al., 2008). These discrepancies in the literature may be due to the lack of standardization regarding xenodiagnoses. Xenodiagnosis is considered the only way to accurately identify which dogs are infective to sand fly vectors (Guarga et al., 2000; Michalsky et al., 2007; Molina et al., 1994), yet, due to its inherent limitations, this technique is unable to improve the effectiveness of programs designed to control leishmaniasis in endemic areas. Thus, the identification of tissues in which parasite load is positively correlated with canine infectiousness is paramount. Deane and Deane (1954) showed that the skin of asymptomatic dogs in an endemic area can harbor Leishmania parasites, indicating this tissue type could be a potential marker of infectiousness. Interest-

L.S. Borja et al. / Veterinary Parasitology 229 (2016) 110–117

115

Fig. 3. Parasite load in infected sandflies fed on naturally infected dogs with a range of clinical classifications. (A) Mean parasite loads in sandflies measured 5 days after being fed on naturally infected dogs, which were classified according to clinical VL presentation. (B) Transmission capacity of infective dogs, measured as the parasite load in each sand fly 5 days following xenodiagnosis, according to clinical manifestation of CVL.

Fig. 4. Parasite load in the blood and skin of naturally infected dogs in association with canine capacity to infect sand fly: distribution of parasite load in blood (A) and skin (B) of non-infective and infective dogs. Correlation between parasite load in blood (C) and skin (D) of dogs with sand fly infection rate following xenodiagnosis. Parasite load was detected by qPCR in blood and skin samples and it is represented as the number of parasites measured in 10 mg of DNA. Each plot represents the detected parasite load in an individual dog. Relevant Spearman correlation coefficients (r) and p values are shown.

ingly, the present report we found positive correlations between parasite loads in both skin and blood with respect to canine infectiousness to the sand fly vector, as well as the sand fly infection rate and parasite load in sand fly vectors following xenodiagnosis. Furthermore, De Amorim et al. (2011) and Courtenay et al. (2002) also demonstrated an association between high parasite load in dog skin and canine infectiousness. These authors considered a high parasite

load in dog skin to be a strong predictor of canine infectiousness to sandflies (Courtenay et al., 2014). Moreover, Vercosa et al. (2008) demonstrated a positive association between canine infectiousness to sandflies and an elevated degree of blood parasitism in dogs, which is in agreement with our finding of a positive correlation between parasite load in canine blood and dog capacity to infect sandflies. Taken together, these findings support the notion that

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Fig. 5. Evaluation of parasite load in infected sandflies in relation to parasite load in different tissues of naturally infected dogs. Each plot represents the mean parasite load detected in sandflies 5 days following xenodiagnosis in accordance with the parasite load detected in canine tissue type: blood (A), skin (B) and splenic aspirate (C). Relevant Spearman correlation coefficients (r) and p values for each tissue type are shown in the respective plots.

parasite load in both canine skin and blood could potentially serve as reliable markers of the canine capacity to infect sand fly vector. Acknowledgments The authors would like to thank Dr. Flávia W. Cruz McBride for assistance in obtaining negative control samples in Pelotas. We are grateful to Andris K. Walter for assistance with manuscript revision and consulting services. This work was supported in part by Instituto de Ciência e Tecnologia em Doenc¸as Tropicais (INCT-DT- Grant number: 576269/2008-5) and Fundac¸ão de Amparo a Pesquisa da Bahia (FAPESB – Grant number: SUS0011/2010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Alvar, J., Canavate, C., Molina, R., Moreno, J., Nieto, J., 2004. Canine leishmaniasis. Adv. Parasitol. 57, 1–88. Amorim, I.F., Silva, S.M., Figueiredo, M.M., Moura, E.P., Castro, R.S., Lima, T.K., Gontijo Nde, F., Michalick, M.S., Gollob, K.J., Tafuri, W.L., 2011. Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis. PLoS One 6, e27679. Aslan, H., Oliveira, F., Meneses, C., Castrovinci, P., Gomes, R., Teixeira, C., Derenge, C.A., Orandle, M., Gradoni, L., Oliva, G., Fischer, L., Valenzuela, J.G., Kamhawi, S., 2016. New insights into the transmissibility of Leishmania infantum from dogs to sand flies: experimental vector-transmission reveals persistent parasite depots at bite sites. J. Infect. Dis. 213, 1752–1761. Barrouin-Melo, S.M., Larangeira, D.F., Santos, S.O., Chagas-Junior, A.D., Paixao, M., Aguiar, P.H., dos-Santos, W.L., Pontes-de-Carvalho, L., 2006. A standardized cytological and immunochemical method for the analysis of fine-needle spleen

aspirates: assessment of leukocyte population changes in canine visceral leishmaniosis. Vet. Immunol. Immunopathol. 111, 251–261. Bates, P.A., 2007. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int. J. Parasitol. 37, 1097–1106. Bevilacqua, P.D., Paixao, H.H., Modena, C.M., Castro, M.C.P.S., 2001. Urbanization of visceral leishmaniose in belo horizonte, Brazil. Arq. Bras. Med. Vet. Zootec. 53, 1–8. Ciaramella, P., Oliva, G., Luna, R.D., Gradoni, L., Ambrosio, R., Cortese, L., Scalone, A., Persechino, A., 1997. A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum. Vet. Rec. 141, 539–543. Courtenay, O., Quinnell, R.J., Garcez, L.M., Shaw, J.J., Dye, C., 2002. Infectiousness in a cohort of brazilian dogs: why culling fails to control visceral leishmaniasis in areas of high transmission. J. Infect. Dis. 186, 1314–1320. Courtenay, O., Carson, C., Calvo-Bado, L., Garcez, L.M., Quinnell, R.J., 2014. Heterogeneities in Leishmania infantum infection: using skin parasite burdens to identify highly infectious dogs. PLoS Negl. Trop. Dis. 8, e2583. da Costa-Val, A.P., Cavalcanti, R.R., de Figueiredo Gontijo, N., Michalick, M.S., Alexander, B., Williams, P., Melo, M.N., 2007. Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity. Vet. J. 174, 636–643. Deane, L.M., Deane, M.P., 1954. Isolation of leishmaniae in the viscera and the skin of a fox in the kala-azar endemic zone in Sobral, Ceara. Hospital (Rio J) 45, 419–421. Francino, O., Altet, L., Sanchez-Robert, E., Rodriguez, A., Solano-Gallego, L., Alberola, J., Ferrer, L., Sanchez, A., Roura, X., 2006. Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis. Vet. Parasitol. 137, 214–221. Giambernardi, T.A., Rodeck, U., Klebe, R.J., 1998. Bovine serum albumin reverses inhibition of RT-PCR by melanin. Biotechniques 25, 564–566. Guarga, J.L., Lucientes, J., Peribanez, M.A., Molina, R., Gracia, M.J., Castillo, J.A., 2000. Experimental infection of Phlebotomus perniciosus and determination of the natural infection rates of Leishmania infantum in dogs. Acta Trop. 77, 203–207. Laurenti, M.D., Rossi, C.N., Matta, V.L., Tomokane, T.Y., Corbett, C.E., Secundino, N.F., Pimenta, P.F., Marcondes, M., 2013. Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector. Vet. Parasitol. Magalhaes-Junior, J.T., Mota, T.F., Porfirio-Passos, G., Larangeira, D.F., Franke, C.R., Barrouin-Melo, S.M., 2016. Xenodiagnosis on dogs with visceral leishmaniasis:

L.S. Borja et al. / Veterinary Parasitology 229 (2016) 110–117 canine and sand fly aspects related to the parasite transmission. Vet. Parasitol. 223, 120–126. Michalsky, E.M., Rocha, M.F., da Rocha Lima, A.C., Franca-Silva, J.C., Pires, M.Q., Oliveira, F.S., Pacheco, R.S., dos Santos, S.L., Barata, R.A., Romanha, A.J., Fortes-Dias, C.L., Dias, E.S., 2007. Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies. Vet. Parasitol. 147, 67–76. Ministério da Saúde, Secretaria de Vigilância em Saúde, Departamento de Vigilância Epidemiológica, 2006. Manual de Vigilância e Controle da Leishmaniose Visceral, 2 edition. Ministério da Saúde, Brasil. Molina, R., Amela, C., Nieto, J., San-Andres, M., Gonzalez, F., Castillo, J.A., Lucientes, J., Alvar, J., 1994. Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus. Trans. R. Soc. Trop. Med. Hyg. 88, 491–493. Rallis, T., Day, M.J., Saridomichelakis, M.N., Adamama-Moraitou, K.K., Papazoglou, L., Fytianou, A., Koutinas, A.F., 2005. Chronic hepatitis associated with canine leishmaniosis (Leishmania infantum): a clinicopathological study of 26 cases. J. Comp. Pathol. 132, 145–152.

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Solca Mda, S., Bastos, L.A., Guedes, C.E., Bordoni, M., Borja, L.S., Larangeira, D.F., da Silva Estrela Tuy, P.G., Amorim, L.D., Nascimento, E.G., de Sa Oliveira, G.G., dos-Santos, W.L., Fraga, D.B., Veras, P.S., 2014. Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis. PLoS One 9, e103635. Vercosa, B.L., Lemos, C.M., Mendonca, I.L., Silva, S.M., de Carvalho, S.M., Goto, H., Costa, F.A., 2008. Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis. BMC Vet. Res. 4, 45. WHO, 2010. Control of the leishmaniases. World Health Organization technical report series, xii–xiii, 1–186, back cover.

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