First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare.

BACKGROUND: Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. OBJECTIVES: Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. METHODS: Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. FINDINGS: The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. MAIN CONCLUSIONS: The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

HRM Accuracy and Limitations as a Species Typing Tool for Leishmania Parasites.

High Resolution Melting Analysis (HRM) has been pointed out as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to parasite load-dependent accuracy. In light of these findings and considering the importance of parasite load determination for clinical and research purposes, we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis.

Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania.

First report of Leishmania (Viannia) lindenbergi causing tegumentary leishmaniasis in the Brazilian western Amazon region.

Tegumentary Leishmaniasis (TL) in the Brazilian Amazon region is associated with several Leishmania species. In this report, we describe two cases of TL related to Leishmania lindenbergi occurring in different locations of Rondônia state. After clinical diagnosis, lesion samples were collected for parasitological diagnoses via direct microscopic visualization, parasite isolation, and PCR. PCR reactions were positive in both clinical samples. Parasite isolation was possible for both patients, and isolates were submitted to species identification by isoenzyme electrophoresis and DNA sequencing. This report is the first to describe human infections caused by L. lindenbergi since the initial description and record of human infection by this species in 2002.

First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare

BACKGROUND Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. OBJECTIVES Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. METHODS Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. FINDINGS The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. MAIN CONCLUSIONS The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

Ensaios moleculares quantitativos e baseados em temperatura de dissociação do DNA para diagnóstico específico da infecção por Leishmania spp

A leishmaniose é uma doença negligenciada, presente na Europa, Ásia, África e nas Américas, abrangendo infecções assintomáticas e duas formas clínicas principais: Leishmaniose Visceral (LV) e Leishmaniose Tegumentar (LT). É causada por protozoários do gênero Leishmania, transmitidos pela fêmea de flebotomíneos vetores. Sabe-se que as diferentes espécies de Leishmania estão associadas às diferentes formas clínicas da doença e ao seu prognóstico. O diagnóstico da leishmaniose é feito a partir da combinação de dados clínicos, epidemiológicos e laboratoriais, mas principalmente através do isolamento e cultivo dos parasitos, que pode ser seguido de ensaio de isoenzimas para identificação da espécie. Este processo, contudo, tem baixa eficiência, além de demandar tempo e recursos que podem ser minimizados com a utilização de abordagem molecular diretamente em material clínico. A técnica de PCR em tempo real (qPCR) tem sido amplamente utilizada para detecção da infecção por Leishmania e para a quantificação da sua carga parasitária, por se tratar de uma metodologia simples, rápida e extremante sensível, capaz de detectar concentrações muito baixas de parasitos presentes em uma amostra A análise de curvas de dissociação por High Resolution Melting (HRM) tem como principal vantagem permitir investigar toda a região amplificada pelos iniciadores e tem sido utilizada para a identificação de espécies por apresentar uma sensibilidade capaz de identificar diferenças de até um nucleotídeo entre sequências de DNA. A realização de um estudo que estabeleça uma metodologia capaz de detectar e identificar espécies de Leishmania, e que inclua uma validação analítica para uma padronização adequada e permita a determinação da carga parasitária em paralelo, para possibilitar a associação desses dados, ainda se faz necessária. Assim, o objetivo deste trabalho é estabelecer uma metodologia baseada em qPCR e HRM, que dispense o isolamento e cultivo do parasito, e que permita a detecção, identificação de espécies de Leishmania e a quantificação da carga parasitária diretamente em material clínico. Para isso, as metodologias de qPCR e HRM foram padronizadas com cepas de referência e posteriormente validadas com amostras clínicas de pacientes de Rondônia, Brasil, diagnosticados com leishmaniose cutânea, sendo realizada a quantificação da carga parasitária por qPCR e a identificação da espécie envolvida por HRM. Os resultados apontam que o alvo HSP70 é melhor indicado para determinar a carga parasitária de Leishmania. Além disso, estabelecemos um algoritmo para a identificação das principais espécies de Leishmania de relevância clínica por HRM, a partir da combinação de dois alvos moleculares, HSP70 e COI.