Molecular survey of Leishmania spp. in skin samples of capybaras (Hydrochoerus hydrochaeris) from different areas of Brazil / Investigação molecular de Leishmania spp. em amostras de pele de capivaras (Hydrochoerus hydrochaeris) de diferentes regiões do Brasil

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the genus Leishmania, with some species of rodents being incriminated as reservoirs. The capybara is the largest extant rodent species in the world and is widely distributed in South America. The occurrence of infection by Leishmania spp. was investigated in capybaras captured in Brazil during 2015­2019 from established populations in five highly anthropic areas of the state of São Paulo and two natural areas of the states of Mato Grosso and Mato Grosso do Sul. A total of 186 individuals were captured and subjected to abdominal skin biopsy. All skin samples were Leishmania kDNA-negative, suggesting that capybaras have no role in the transmission cycles of Leishmania species in the studied areas despite the well-known role of other rodents in the life cycle of Leishmania spp.(AU)

As leishmanioses compreendem um espectro de doenças causadas por protozoários do gênero Leishmania e algumas espécies de roedores são incriminadas como reservatórios de Leishmania spp. As capivaras compreendem a maior espécie de roedores existentes e são amplamente distribuídas na América do Sul. Para investigar a ocorrência de infecção por Leishmania spp. em capivaras, durante os anos de 2015-2019 capivaras foram capturadas em cinco áreas antrópicas do estado de São Paulo e em duas áreas naturais dos estados do Mato Grosso e do Mato Grosso do Sul, todos esses ambientes com populações de capivaras estabelecidas. Um total de 186 indivíduos foram capturados e submetidos à biópsia de pele abdominal. Todas as amostras de pele foram negativas para o alvo kDNA, assim, os dados sugerem que nas áreas estudadas as capivaras não têm papel no ciclo de transmissão de espécies de Leishmania spp., apesar do papel bem conhecido de outros roedores no ciclo de vida de Leishmania spp.(AU)

Detección de Leishmania (V) guyanensis en ejemplares de Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) recolectados en pecaríes de collar (Pecari tajacu) / Detection of Leishmania (V) guyanensis in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) collected from Pecari tajacu

Resumen Introducción. En estudios previos se detectó la presencia de Leishmania infantum en Rhipicephalus sanguineus, lo cual planteaba la posibilidad de que R. sanguineus transmitiera la leishmaniasis a una variedad de huéspedes. Objetivo. Identificar Leishmania (Viannia) spp. en garrapatas recolectadas en animales silvestres de una zona endémica para leishmaniasis. Materiales y métodos. Se hicieron 81 extracciones individuales de ADN en las garrapatas recogidas de tres tapires o dantas (Tapirus terrestres) y tres pecaríes de collar (Pecari tajacu) cazados en Madre de Dios, Perú. Las garrapatas recolectadas se identificaron taxonómicamente y se prepararon para la identificación del cinetoblasto (kDNA) de Leishmania (Viannia) spp. mediante reacción en cadena de la polimerasa (PCR), así como de la especie de Leishmania mediante PCR de fusión de alta resolución (High Resolution Melt, HRM). Resultados. Se detectó el kDNA de Leishmania (V) spp. en tres garrapatas silvestres de R. (Boophilus) microplus, Canestrini, 1888, recolectadas en un pecarí de collar cazado en la selva de Madre de Dios. El análisis mediante HRM-PCR evidenció que una de las muestras positivas de kDNA tenía una curva compatible con L. (V) guyanensis. Conclusión. Los resultados evidenciaron la presencia de ADN de L. (V) guyanensis en R. (Boophilus) microplus, probablemente adquirida después de picar al pecarí. Es importante hacer nuevos estudios para aclarar la participación de R. (Boophilus) microplus en la transmisión de la leishmaniasis.

Abstract Introduction: Previous studies identified the presence of Leishmania infantum in Rhipicephalus sanguineus and indicated the possibility that it could transmit leishmaniasis to a variety of hosts. Objective: To identify parasites of Leishmania (Viannia) spp. in ticks collected from wild animals in an endemic area for leishmaniasis. Materials and methods: We performed 81 individual DNA extractions from ticks collected from three Tapirus terrestris and three Pecari tajacu in Madre de Dios, Perú. Ticks were taxonomically identified and they were subsequently prepared to identify Leishmania (Viannia) spp. kDNA by PCR and the species of Leishmania by HRM-PCR. Results: Leishmania (Viannia) kDNA was detected in three wild ticks of the species R. microplus, collected from a collard peccary (P. tajacu) hunted in the forests of Madre de Dios. The HRM-PCR showed that one of the positive samples had a kDNA curve compatible with L. (V) guyanensis. Conclusion: The results showed the presence of L. (V) guyanensis DNA in R. microplus possibly acquired after biting a collarde peccary. Therefore, it is important to design future studies to clarify R. microplus involvement in the transmission of leishmaniasis.

USEFULNESS OF kDNA PCR IN THE DIAGNOSIS OF VISCERAL LEISHMANIASIS REACTIVATION IN CO-INFECTED PATIENTS / Utilidade da kDNA PCR no diagnostico de reativacao de leishmaniose visceral em pacientes co-infectados sintomaticos

SUMMARY It is important to develop new methods for diagnosing relapses in the co-infection of visceral leishmaniasis (VL) and HIV to enable earlier detection using less invasive methods. We report a case of a co-infected patient who had relapses after VL treatment, where the qualitative kDNA PCR showed a good performance. The kDNA PCR seems to be a useful tool for diagnosing VL and may be a good marker for predicting VL relapses after treatment of co-infected patients with clinical symptoms of the disease. .

RESUMO É importante a pesquisa de novos métodos laboratoriais para o diagnóstico de recidivas em casos de co-infecção leishmaniose visceral (LV) e vírus da imunodeficiência humana (HIV), que permitam o diagnóstico precoce das recidivas, utilizando métodos menos invasivos. Descrevemos aqui, o caso de paciente co-infectada que apresentou recidivas após o tratamento da LV e onde a PCR qualitativa demonstrou bom desempenho. A kDNA PCR parece ser ferramenta útil para o diagnóstico de recidivas de LV após o tratamento em pacientes co-infectados com sintomas clínicos da doença. .

Desenvolvimento de sistemas baseados em Nested-PCR convencional e em único tubo para o diagnóstico de leishmaniose visceral / Development systems based on nested-PCR and conventional single tube for the diagnosis of visceral leishmaniasis

A leishmaniose visceral ocorre em países dos cinco continentes e quando não tratada pode levar a óbito. Para se evitar esse desfecho, são essenciais o diagnóstico precoce e tratamento adequado. Com o objetivo de contribuir na pesquisa de novos diagnósticos para leishmaniose visceral, esse trabalho propôs desenvolver sistemas baseados em Nested-PCR convencional e em único tubo para o diagnóstico de leishmaniose visceral. A partir de uma revisão na literatura em busca de alvos moleculares utilizados no diagnóstico dessa parasitose, foram selecionados os alvos subunidade menor do RNA ribossômico (ssu rRNA) e espaçador transcrito interno 1 (ITS-1), que compõem o DNA do agente etiológico Leishmania infantum, para o desenvolvimento das nested-PCR. Foi também escolhido o alvo kDNA, o mais aplicado nas abordagens de PCR, para comparações com os sistemas desenvolvidos. Após otimizar todas as PCR com DNA genômico de L. infantum, esses sistemas foram avaliados em amostras de sangue, soro e urina de indivíduos com suspeita de leishmaniose visceral dos hospitais de referência da cidade do Recife - PE. Para utilização da urina, foram avaliados quatro protocolos de extração de DNA e identificou-se que a extração por fenol-clorofórmio, com modificações, foi a de melhor desempenho. Na avaliação com amostras biológicas, as PCR simples e nested-PCR com os alvos ssu rRNA e ITS-1 não tiveram boa sensibilidade ao se usar sangue, e não foram capaz de amplificar DNA do parasito em soro e urina. Esses sistemas desenvolvidos não podem ser usados para o diagnóstico da leishmaniose visceral. No entanto, a kDNAPCR apresentou bons resultados quando avaliada com urina. Mais estudos devem ser feitos para avalia-la como um diagnóstico seguro para esse tipo de amostra biológica. Esse trabalho representa um ponto de início para posteriores estudos que objetivem o aprimoramento e validação da nested-PCR único tubo para o diagnóstico da leishmaniose visceral.

The use of polymerase chain reaction to confirm diagnosis in skin biopsies consistent with american tegumentary leishmaniasis at histopathology: a study of 90 cases / Uso da reação em cadeia da polimerase em biópsias de pele para confirmar o diagnóstico histopatológico consistente com leishmaniose tegumentar americana: um estudo de 90 casos

BACKGROUND: Cutaneous leishmaniasis is a chronic, infectious disease caused by protozoa of the genus leishmania. The incidence of this disease is high in Brazil, with 19,746 new cases having been detected in 2008. The presence of amastigotes in the cytoplasm of histiocytes constitutes diagnosis of the disease; however, their presence is rarely found in late lesions, making histological diagnosis difficult. Polymerase chain reaction has been shown to represent a highly sensitive and specific technique for the diagnosis of cutaneous leishmaniasis. OBJECTIVES: To use polymerase chain reaction to evaluate paraffin-embedded skin biopsies with histopathological features consistent with cutaneous leishmaniasis. MATERIAL AND METHODS: Polymerase chain reaction amplification of a 120-base-pair fragment of Leishmania kinetoplast DNA (kDNA) minicircles was performed on 90 skin biopsies. The male/female ratio was 75/15. Mean age was 32.36 years, with a median of 31 years, range 4-72 years. Samples were histologically compatible with cutaneous leishmaniasis but a definitive diagnosis could not be made since amastigotes were not found. All cases were histologically classified according to the patterns described by de Magalhães. RESULTS: According to the de Magalhães classification, the most common histological pattern was type IV (exudative granulomatous reaction), which was found in 65.6 percent of cases (56/90), followed by type I (exudative cellular reaction) in 21.1 percent of cases (19/90) and type III (exudative and necrotic granulomatous reaction) in 12.2 percent of cases (11/90). Leishmania DNA was found in 96.7 percent of the biopsies (87/90). CONCLUSION: Polymerase chain reaction performed by amplifying kDNA is able to confirm a diagnosis of cutaneous leishmaniasis with a high degree of sensitivity in cases in which histopathology is consistent with a diagnosis of cutaneous leishmaniasis but not definitive.

FUNDAMENTOS: Cutaneous leishmaniasis is a chronic, infectious disease caused by protozoa of the genus leishmania. The incidence of this disease is high in Brazil, with 19,746 new cases having been detected in 2008. The presence of amastigotes in the cytoplasm of histiocytes constitutes diagnosis of the disease; however, their presence is rarely found in late lesions, making histological diagnosis difficult. Polymerase chain reaction has been shown to represent a highly sensitive and specific technique for the diagnosis of cutaneous leishmaniasis. OBJECTIVES: To use polymerase chain reaction to evaluate paraffin-embedded skin biopsies with histopathological features consistent with cutaneous leishmaniasis. MATERIAL AND METHODS: Polymerase chain reaction amplification of a 120-base-pair fragment of Leishmania kinetoplast DNA (kDNA) minicircles was performed on 90 skin biopsies. The male/female ratio was 75/15. Mean age was 32.36 years, with a median of 31 years, range 4-72 years. Samples were histologically compatible with cutaneous leishmaniasis but a definitive diagnosis could not be made since amastigotes were not found. All cases were histologically classified according to the patterns described by de Magalhães. RESULTS: According to the de Magalhães classification, the most common histological pattern was type IV (exudative granulomatous reaction), which was found in 65.6 por cento of cases (56/90), followed by type I (exudative cellular reaction) in 21.1 por cento of cases (19/90) and type III (exudative and necrotic granulomatous reaction) in 12.2 por cento of cases (11/90). Leishmania DNA was found in 96.7 por cento of the biopsies (87/90). CONCLUSION: Polymerase chain reaction performed by amplifying kDNA is able to confirm a diagnosis of cutaneous leishmaniasis with a high degree of sensitivity in cases in which histopathology is consistent with a diagnosis of cutaneous leishmaniasis but not definitive.

Leishmania (Viannia) lainsoni (Kinetoplastida: Trypanosomatidae), a divergent Leishmania of the Viannia subgenus: a mini review

Leishmania (Viannia) lainsoni is the Leishmania species that presents the most distinct biological (morphology, growth in axenic culture medium), biochemical (enzymatic electrophoresis profile), and molecular biology characteristics, when compared to other species of the Viannia subgenus. Development of promastigote forms of this parasite attached to the wall of the pyloric and hind gut regions of sand fly vectors is a solid characteristic that allows its positioning in the Viannia subgenus. However, taxonomic data from biochemical and molecular techniques on this Leishmania species are still not conclusive. It is evident the difficulty in taxonomically positioning this borderline Leishmania species. In this review we present the data accumulated since L. (Viannia) lainsoni has been described and we discuss its position in the Viannia subgenus.

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5 percent when compared to the histopathology test which was 61.9 percent. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Parasite persistence in treated chagasic patients revealed by xenodiagnosis and polymerase chain reaction

Polymerase chain reaction (PCR) was compared with xenodiagnosis performed 20 years after trypanocidal chemotherapy to investigate parasite clearance. Eighty-five seropositive individuals for Chagas disease presenting a positive xenodiagnosis were treated with specific drugs; 37 in the acute phase and 48 in the chronic phase. Fifteen chronic assymptomatic patients received a placebo. Treatment in the acute phase led to PCR negative results in 73 percent of the cases, while xenodiagnosis was negative in 86 percent. In the chronic phase, PCR was negative in 65 percent of the patients and 83 percent led to xenodiagnosis negative results. Regarding the untreated group (placebo), 73 percent gave negative results by xenodiagnosis, of which 36 percent were positive by PCR. Individuals that were considered seronegative (n=10), presented unequivocally negative results in the PCR demonstrating the elimination of parasite DNA. Seventeen individuals had their antibodies titers decreased to such a level that the final results were considered as doubtful and 16 of them presented negative PCR. The molecular method represents a clear advantage over conventional techniques to demonstrate persistent infections in Chagas disease patients that underwent chemotherapy

Mexican Trypanosoma cruzi stocks: analysis of minicircle kDNA homologies by cross-hybridization

Homologies of minicircle kDNA of 27 Mexican stocks were studied by cross-hybridization with four kDNA probes derived from three reference stocks belonging to groups Trypanosoma cruzi I (SO34 cl4 and Silvio) and T. cruzi II (MN) and one Mexican stock. High homologies were only observed with Silvio (six stocks) and Mexican probes (11 stocks). After 30 min exposure (low homology) additional stocks were recognized with SO34 cl4 (three stocks) and Silvio (six stocks) probes; with the Mexican probe only five stocks remained non-reactive. All the stocks were typed by isoenzyme (16 loci) and Mexican stocks belonged to T. cruzi I. Hybridization patterns were not strictly correlated with the observed clustering and cross-hybridization of kDNA minicircles is not available to distinct Mexican stocks.

Characterization of Trypanosoma cruzi populations by several molecular markers supports a clonal mode of reproduction

Chilean T. cruzi populations from different endemic areas and transmission cycles were characterized at several biochemical levels, to mention: hybridization with kinetoplast DNA probes, molecular karyotype, isoenzyme studies and kinetoplast DNA restriction fragment length polymorphism. Furthermore, an immunological approach with immune sera from Balb/c infected mice with different T. cruzi populations was used to differentiate among parasite types by the in vitro complement-mediated lysis assay. Parasite grouping by the above described methods allows to classify T. cruzi populations on a very defined number, suggesting that they have a clonal structure