Development of quantitative PCR and digital PCR for the quantification of Leishmania infantum in dogs.

Leishmaniasis is a zoonotic disease with worldwide distribution. In the Americas, the causative agent of the visceral form is the protozoa Leishmania (Leishmania) infantum. Transmission to the host or vertebrate reservoir occurs through the bite of infected arthropod females like Lutzomyia longipalpis. The epidemiological connection between the infection in dogs and humans generate constant studies about the relationship between the parasite and the canine host, including the development of methods and tests for the detection and quantification ofLeishmania (L.) infantum. Both conventional PCR (cPCR) and quantitative PCR (qPCR) can be used in the diagnosis of the parasite. Dropet Digital PCR (ddPCR) is another useful tool. Knowing the parasite load and its relationship with the clinical signs of naturally infected dogs is useful in research development and for establishing treatments that reduce the transmission of the disease. In this study, thirty-nine clinical samples of spleen from dogs naturaly infected by L. infantum were collected after necropsy. Two molecular tools were used to quantify the parasite load (qPCR and ddPCR) and there was 100% agreement in the results of the them. The tools developed in this work are important for the detection of L. infantum in dogs and humans. Droplet Digital PCR does not require a standard curve and is easy to standardize. In such manner, this new tool can generate more in-depth information in the broad debate about parasitic loads and the pathogenesis of leishmaniasis.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil.

BACKGROUND: Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES: In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS: A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS: We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS: To the best of our knowledge, this is the first detection of LRV2 in the New World.

Accuracy of diagnostic tests for American tegumentary leishmaniasis: a systematic literature review with meta-analyses.

OBJECTIVES: To analyse the accuracy of American tegumentary leishmaniasis (ATL) diagnostic methods and evaluate the quality of the existing publications by means of a systematic review. METHODS: Diagnostic tests evaluated in at least two studies with common reference standards were included in the sensitivity and/or specificity meta-analyses. Quality and susceptibility to bias were analysed using the QUADAS-2 and STARD tools. RESULTS: The title and abstract of 3387 publications were evaluated after deduplication resulting from database searches. Thirty-eight studies were included in the review, and 26 of them had results inserted in meta-analyses. The diagnostic methods with the highest pooled sensitivity values were ELISA, polymerase chain reaction (PCR), indirect immunofluorescence reaction and Montenegro's intradermal reaction. Cytometry was assessed in only two studies and presented 100% sensitivity in both. Smear slide microscopy and histopathology showed low pooled values of sensitivity. For specificity, the highest pooled values were identified for PCR. High values were also identified for ELISA, except for studies in which the reference standard for defining negative participants included individuals with Chagas' disease or paracoccidioidomycosis, which also occurred for cytometry. IFR had lower specificities than ELISA. There was a predominance of case-control designs of phase 1 or 2 and only four studies were strongly recommended as evidence generators. Several reference standards were adopted, and different methods were assessed in a small number of studies. CONCLUSION: PCR showed the highest accuracy for the diagnosis of ATL, and its use should be encouraged in clinical practice. ELISA is recommended for the screening of suspected individuals, but the possibility of cross-reactions should be considered. New validation studies for the tests evaluated in few publications and studies of phase 3 with appropriate methods are needed.

OBJECTIFS: Analyser l'exactitude des méthodes de diagnostic de la leishmaniose tégumentaire américaine (LTA) et évaluer la qualité des publications existantes au moyen d'une analyse systématique. MÉTHODES: Les tests diagnostiques évalués dans au moins deux études avec des étalons de référence communs ont été inclus dans les méta-analyses de sensibilité et/ou de spécificité. La qualité et la sensibilité au biais ont été analysées à l'aide des outils QUADAS-2 et STARD. RÉSULTATS: Le titre et le résumé de 3387 publications ont été évalués après déduplication résultant de recherches dans la base de données. 38 études ont été incluses dans la revue et 26 d'entre elles ont eu des résultats inclus dans des méta-analyses. Les méthodes de diagnostic avec les valeurs de sensibilité poolées les plus élevées étaient ELISA, la réaction en chaîne par polymérase (PCR), la réaction d'immunofluorescence indirecte et la réaction intradermique du Monténégro. La cytométrie a été évaluée dans seulement deux études et présentait une sensibilité de 100% dans les deux. La microscopie et l'histopathologie sur lame de frottis ont montré de faibles valeurs poolées de sensibilité. Pour la spécificité, les valeurs poolées les plus élevées ont été identifiées pour la PCR. Des valeurs élevées ont également été identifiées pour l'ELISA, à l'exception des études dans lesquelles la norme de référence pour définir les participants négatifs incluait des individus atteints de la maladie de Chagas ou de paracoccidioïdomycose, qui s'est également produite pour la cytométrie. L'IFR avait des spécificités plus faibles que l'ELISA. Il y avait une prédominance des essais cas-témoins de phases 1 ou 2 et seules quatre études étaient fortement recommandées comme génératrices de preuves. Plusieurs normes de référence ont été adoptées et différentes méthodes ont été évaluées dans un petit nombre d'études. CONCLUSION: la PCR a montré la plus grande exactitude pour le diagnostic de l'ATL et son utilisation doit être encouragée dans la pratique clinique. L'ELISA est recommandé pour le dépistage des personnes suspectées, mais la possibilité de réactions croisées doit être considérée. De nouvelles études de validation des tests évalués dans quelques publications et des études de phase 3 avec des méthodes appropriées sont nécessaires.

IgG avidity index and complete blood count as biomarkers of clinical disease in naturally infected dogs with Leishmania infantum.

Canine visceral leishmaniosis (CVL), a parasitic disease caused by Leishmania infantum, may evolve to a chronic condition and lead to death. Evaluation of infected dogs is important to establish the clinical and laboratory parameters involved in the evolution of the disease. The objectives of the present study were to discriminate a canine population (n = 52) into sub-clinical and clinically affected dogs based on signs and scores, to evaluate the hematological, biochemical, histopathological and parasitological parameters of the two dog groups, and to analyze the results by multivariate regression analysis with the aim of establishing biomarkers of CVL clinical disease. The most common signs observed in the clinically affected dogs (n = 29) were hyperkeratosis, weight loss, onychogryphosis, pale mucosa and lymphadenomegaly. In the multivariate analysis, animals presenting high IgG avidity index and low red blood, lymphocyte and eosinophil counts, and low serum urea concentration had an increased probability of being classified as clinically affected (p < 0.05). All five parameters were considered to be strong biomarkers for monitoring the clinical disease, while IgG avidity percentage was strongly correlated with the number of clinical signs and could function as an indicator of the duration of infection. This is the first report on the application of IgG avidity and of multivariate regression analysis in establishing associations between the clinical signs of CVL and host biomarkers. Since avidity index (AI) percentages were strongly correlated with the number of clinical signs, it could be useful in clinical practice for auxiliary diagnosis of CVL and monitoring disease progression. A limitation of this study is the lack of information on co-infections by Anaplasma platys, Babesia canis vogeli, Ehrlichia canis and Hepatozoon canis. Therefore future studies should evaluate the influence of such co-infections on the associations studied using multivariate methods with larger samples.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil

BACKGROUND Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS To the best of our knowledge, this is the first detection of LRV2 in the New World.

FIRST CASE OF HUMAN INFECTION BY Bertiella studeri (Blanchard, 1891) Stunkard,1940 (Cestoda; Anoplocephalidae) IN BRAZIL.

Cestodes of the Bertiella genus are parasites of non-human primates found in Africa, Asia, Oceania and the Americas. Species Bertiella studeri and Bertiella mucronata could, accidentally, infect human beings. The infection occurs from ingestion of mites from the Oribatida order containing cysticercoid larvae of the parasite. The objective of this report is to register the first case of human infection by Bertiella studeri in Brazil. Proglottids of the parasite, found in the stool sample of a two-and-a-half-year-old child, were fixed, stained and microscopically observed to evaluate its morphological characteristics. Eggs obtained from the proglottids were also studied. The gravid proglottids examined matched the description of the genus Bertiella. The eggs presented a round shape, with the average diameter of 43.7 µm, clearly showing the typical pyriform apparatus of B. studeri. The authors concluded that the child was infected with Bertiella studeri,based on Stunkard's (1940) description of the species. This is the fifth case of human Bertiellosis described in Brazil through morphometric analysis of the parasite, the third in Minas Gerais State and the first diagnosed case of Bertiella studeri in Brazil.

FIRST CASE OF HUMAN INFECTION BY Bertiella studeri (Blanchard, 1891) Stunkard,1940 (Cestoda; Anoplocephalidae) IN BRAZIL / Primeiro caso de infecção humana por Bertiella studeri (Blanchard, 1891) Stunkard,1940 (Cestoda; Anoplocephalidae) no Brasil

SUMMARY Cestodes of the Bertiella genus are parasites of non-human primates found in Africa, Asia, Oceania and the Americas. Species Bertiella studeri and Bertiella mucronatacould, accidentally, infect human beings. The infection occurs from ingestion of mites from the Oribatida order containing cysticercoid larvae of the parasite. The objective of this report is to register the first case of human infection by Bertiella studeri in Brazil. Proglottids of the parasite, found in the stool sample of a two-and-a-half-year-old child, were fixed, stained and microscopically observed to evaluate its morphological characteristics. Eggs obtained from the proglottids were also studied. The gravid proglottids examined matched the description of the genus Bertiella. The eggs presented a round shape, with the average diameter of 43.7 µm, clearly showing the typical pyriform apparatus of B. studeri. The authors concluded that the child was infected with Bertiella studeri,based on Stunkard's (1940) description of the species. This is the fifth case of human Bertiellosis described in Brazil through morphometric analysis of the parasite, the third in Minas Gerais State and the first diagnosed case of Bertiella studeriin Brazil.

RESUMO Os cestódeos do gênero Bertiellasão parasitos de primatas não humanos, os quais são encontrados na África, Ásia, Austrália, Oceania e Américas. As espécies Bertiella studeri e Bertiella mucronata podem, eventualmente, vir a infectar os seres humanos e a infecção acontece pela ingestão acidental de ácaros da ordem Oribatida infectados com larvas cisticercóides do parasito. O objetivo deste estudo foi relatar o primeiro caso humano por Bertiella studeri no Brasil. Proglotes do parasito, encontrado na amostra de fezes de uma criança com idade de 2,5 anos, foram fixados, corados e observados ao microscópio para avaliar as suas características morfológicas. Ovos, obtidos a partir dos proglotes também foram estudados. As proglotes grávidas examinadas estavam de acordo com a descrição do gênero Bertiella. Os ovos apresentam forma arredondada com diâmetro médio de 43,7 µm, demonstrando claramente aparelho piriforme típico de B. studeri. Os autores concluíram que a criança estava infectada com Bertiella studeri, de acordo com a descrição da espécie por Stunkard (1940). Este é o quinto caso de Bertiellose humana descrita no Brasil por meio de análises morfométricas do parasito, o terceiro em Minas Gerais e o primeiro caso de diagnóstico por Bertiella studeri no Brasil.