Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama

BACKGROUND The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.

Identification of Leishmania species by high-resolution DNA dissociation in cases of American cutaneous leishmaniasis

Abstract Background American cutaneous leishmaniasis is an infectious dermatosis caused by protozoa of the genus Leishmania, which comprises a broad spectrum of clinical manifestations depending on the parasite species involved in the infections and the immunogenetic response of the host. The use of techniques for amplification of the parasites DNA based on polymerase chain reaction polymerase chain reaction and the recent application of combined techniques, such as high-resolution DNA dissociation, have been described as a viable alternative for the detection and identification of Leishmania spp. in biological samples. Objectives To identify the Leishmania species using the polymerase chain reaction high-resolution DNA dissociation technique in skin biopsies of hospital-treated patients, and compare with results obtained by other molecular identification techniques. Methods A retrospective study assessing patients with suspected American cutaneous leishmaniasis seen at a hospital in São Paulo/Brazil was conducted. The paraffin blocks of 22 patients were analyzed by polymerase chain reaction high-resolution DNA dissociation to confirm the diagnosis and identify the species. Results Of the 22 patients with suspected American cutaneous leishmaniasis, the parasite was identified in 14, comprising five cases (35.6%) of infection by L. amazonensis, four (28.5%) by L. braziliensis, two (14.4%) by L. amazonensis + L. infantum chagasi, two (14.4%) by L. guyanensis, and one (7.1%) by Leishmania infantum chagasi. In one of the samples, in which the presence of amastigotes was confirmed on histopathological examination, the polymerase chain reaction high-resolution DNA dissociation technique failed to detect the DNA of the parasite. Study limitations The retrospective nature of the study and small number of patients. Conclusions The method detected and identified Leishmania species in paraffin-embedded skin biopsies with a sensitivity of 96.4% and could be routinely used in the public health system.

Lutzomyia umbratilis from an area south of the Negro River is refractory to in vitro interaction with Leishmania guyanensis

BACKGROUND Lutzomyia umbratilis, the vector for Leishmania guyanensis in northern South America, has been found naturally infected with L. guyanensis only in areas north of the Negro and Amazon rivers. While populations of this sand fly species are also found in areas south of these rivers, these populations have never been reported to be infected and/or transmitting L. guyanensis. However, no studies on the corresponding host-parasite interactions are available. OBJECTIVES This study evaluated the interaction between Lu. guyanensis promastigotes and field-collected Lu. umbratilis sand flies from Rio Preto da Eva and Manacapuru, which are located to the north and south, respectively, of the Negro River. METHODS Procyclic and metacyclic attachment was quantified using an in vitro system. FINDINGS Low attachment of parasites to the midguts of insects collected from Manacapuru was detected. Conversely, greater binding of metacyclic parasites was observed in the midguts of insects collected from Rio Preto da Eva, and this attachment was more pronounced than that observed for procyclics (p < 0.03). MAIN CONCLUSIONS The Lu. umbratilis population from an area south of the Negro River has lower in vitro interaction with L. guyanensis. The higher attachment of L. guyanensis to midguts of insects from Rio Preto da Eva may suggest better vector competence. These findings are in accordance with previously reported epidemiological information of American cutaneous leishmaniasis (ACL) transmission in the Amazon.

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.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis / Correlação entre a presença de Leishmania RNA Vírus 1 e as características clínicas da leishmaniose de mucosa nasal

ABSTRACT INTRODUCTION: Mucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. OBJECTIVE: To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. METHODS: A cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. RESULTS: Out of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. CONCLUSION: Correlation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area.

RESUMO Introdução: A leishmaniose de mucosa (LM) é uma forma clínica grave da leishmaniose. Fatores complexos ligados ao parasita e ao hospedeiro são atribuídos ao desenvolvimento das lesões de mucosa. Leishmania RNA Vírus 1 (LRV1) pode subverter a resposta imune, podendo ser o principal determinante da gravidade da doença e deve ser pesquisado. Objetivo: Estudar a existência de diferenças clínicas entre pacientes portadores de LM com endosimbiose por LRV1 e as que não possuem. Métodos: Foi realizado um estudo de coorte histórica com corte transversal com avaliação clínica, detecção da Leishmania por técnica de PCR, classificação da espécie e pesquisa de LRV1. Foram incluídos na análise da pesquisa somente os pacientes com diagnóstico confirmado de LM com PCR positivo, com lesão de mucosa nasal. Resultados: Dos 37 pacientes, 30 (81,1%) foram diagnosticados com L. braziliensis, 5 (13,5%) com L. guyanensis e 2 (5,4%) com infecção mista de L. braziliensis e L. guyanensis. O vírus LVR1 estava presente em 26 casos (70,3%). Conclusão: A correlação entre o fenótipo clínico e a presença do LRV1 não foi constatada, porém a frequência do vírus é duas vezes maior em lesão de mucosa do que encontrado em trabalho, da mesma região, sobre lesão cutânea.

American tegumentary leishmaniasis - a case of therapeutic challenge

American Tegumentary Leishmaniasis (ATL) is a chronic, non-contagious, infectious disease affecting millions of people worldwide. The timely and proper treatment is of great importance to prevent the disease from progressing to destructive and severe forms. Treatment for ATL recommended by the Brazilian Ministry of Health is similar for the whole country, regardless of the species of Leishmania. It is known that the response to treatment may vary with the strain of the parasite, the immune status of the patient and clinical form. We report the case of a healthy patient, coming from Manaus, Amazonas state, Brazil, who presented resistance to treatment with N-methyl-glutamine and liposomal amphotericin B, only being healed after using pentamidine.

An improved purification procedure for Leishmania RNA virus (LRV)

Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations.

Combining diagnostic procedures for the management of leishmaniasis in areas with high prevalence of Leishmania guyanensis / Procedimentos diagnósticos combinados no manejo da leishmaniose em áreas com alta prevalência de Leishmania guyanensis

BACKGROUND: The Amazon region corresponds to approximately 40 percent of the cases of leishmaniasis in Brazil. We report a prospective study with 180 patients conducted in a health care unit that diagnoses 10 percent of the cases of leishmaniasis in the Brazilian Amazon. The study addresses how a combination of procedures improves diagnosis in areas with high prevalence of Leishmania guyanensis. OBJECTIVES: to evaluate diagnostic methods in areas with high prevalence of Leishmania guyanensis. METHODS: All subjects were amastigote-positive by direct microscopic examination of lesion scarifications. We conducted skin biopsy and histopathology, polymerase chain reaction and parasite cultivation. RESULTS: Polymerase chain reaction detected almost ninety percent of infections when two amplification protocols were used (mini-exon and HSP-70). HSP-70 specific polymerase chain reaction matched the sensitivity of parasite cultivation plus histopathology. CONCLUSION: The best combination was polymerase chain reaction plus histopathology, which increased diagnostic sensitivity to 94 percent. Species discrimination by polymerase chain reaction disclosed prevalence of human infections with Leishmania guyanensis of 94 percent and with Leishmania braziliensis of 6 percent for this region.

FUNDAMENTOS: O Amazonas corresponde a aproximadamente 40 por cento dos casos de leishmaniose do país. Nós reportamos um estudo prospectivo com 180 pacientes de uma unidade de saúde que diagnostica 10 por cento dos casos de leishmaniose da amazônia brasileira, com combinação de métodos diagnóstico em área de alta prevalência de Leishmania guyanensis. OBJETIVOS: avaliar métodos diagnóstico da Leishmaniose em área endêmica para Leishmania Amazonensis. MÉTODOS: Todos os pacientes tiveram exame direto positivo com presença de amastigotas. Foi feita também biópsia cutânea, com realização de exame histológico, reação em cadeia da polimerase e cultura. RESULTADO: A reação em cadeia da polimerase detectou aproximadamente 90 por cento de infecção quando foram usados duas técnicas de amplificação (mini-exon and HSP-70). A reação em cadeia da polimerase com HSP-70 foi mais sensível que a cultura associada à histopatologia. CONCLUSÃO: A melhor combinação foi a reação em cadeia da polimerase com histopatologia, com sensibilidade de 94 por cento. A discrimanação das espécies causadoras de infecção humana nessa região mostrou Leishmania guyanensis em 94 por cento dos casos e Leishmania brasiliensis em 6 por cento.