PCR primers designed for new world Leishmania: A systematic review.

Studies of the primers that were designed to detect New World Leishmania were systematically reviewed to report the characteristics of each target, detection limit, specificity of the primers designed and diagnostic sensibility. The papers identified in the databases PubMed and Web of Science involved 50 studies. Minicircle is the most applied target in molecular research for diagnosis, due to its high sensitivity in detecting Leishmania in different clinical samples, a characteristic that can be partially attributed to the higher number of copies of the minicircle per cell. The other molecular targets shown in this review were less sensitive to diagnostic use because of the lower number of copies of the target gene per cell, but more specific for identification of the subgenus and/or species. The choice of the best target is an important step towards the result of the research. The target allows the design of primers that are specific to the genus, subgenus or a particular species and also imparts sensitivity to the method for diagnosis. The findings of this systematic review provide the advantages and disadvantages of the main molecular targets and primers designed for New World Leishmania, offering information so that the researcher can choose the PCR system best suited to their research need. This is a timely and extremely thorough review of the primers designed for New World Leishmania.

Multiplex Polymerase Chain Reaction for Detection of Leishmania Sp. in Sandflies of the Paraná River Islands, Southern Brazil.

Leishmaniases are classified as tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL). Brazil is among the countries with the highest number of TL and VL cases. This study was undertaken to standardize the multiplex polymerase chain reaction (PCR) for the detection of the genus Leishmania in sandflies of endemic regions, on islands in the Upper Paraná River, northwestern Paraná. The sandflies were collected on 10 islands, from November 2012 to November 2014, with Falcão light traps, identified and conserved in tubes containing isopropanol, for subsequent DNA extraction. Two pairs of primers were used for multiplex PCR: A1/A2 and 5Llcac/3Llcac. Nyssomyia neivai was the predominant species of the collected specimens. A total of 3870 samples of female sandflies were analyzed and submitted to multiplex PCR, for the validation of the technique. All pools showed the 220 bp fragment for sandfly DNA detection, but no ∼120 bp fragment of Leishmania DNA was found. Although no natural infection of Ny. neivai by Leishmania was found in this study, the interaction of sandflies with Leishmania and its natural reservoirs continues in these Paraná River islands, despite the low diversity of the sandfly fauna. Some of these islands have permanent residents and are frequented by tourists.

American tegumentary leishmaniasis: diagnostic and treatment challenges in a clinical case.

This clinical case presents a patient with a raised and ulcerative lesion with erythematous edges in the mouth, on the lower lip that was unsuccessfully treated as herpes labialis. Clinical data and laboratory tests (Montenegro skin test, indirect immunofluorescence, direct parasite search and polymerase chain reaction) led to the diagnosis of American tegumentary leishmaniasis caused by Leishmania (Viannia) sp. Treatment with pentavalent antimonial (Glucantime®) for 120 days was not effective and administration of amphotericin B for 30 days resulted in wound healing. Glucantime® treatment protocol was longer than the recommended by the Brazilian Ministry of Health in the handbook of mucosal leishmaniasis. This suggests that amphotericin B should have been administered earlier, preventing the psychological and social problems faced by the patient. This study reports a rare clinical case of primary mucosal leishmaniasis on the lip that had a delayed diagnosis, highlighting the precariousness in the management of disease and showing that, despite the importance of leishmaniasis in Brazil, it is still neglected by health professionals.

New primers for the detection Leishmania species by multiplex polymerase chain reaction.

Leishmaniasis is caused by protozoa of the Leishmania genus, which is divided into subgenus Viannia and Leishmania. In humans, the course of infection largely depends on the host-parasite relationship and primarily of the infective species. The objective of the present study was to design specific primers to the identification of Leishmania species using multiplex PCR. Four primers were designed, based on the GenBank sequences of the kDNA minicircle, amplifying 127 bp for subgenus Viannia, 100 bp for L. amazonensis, and 60 bp for Leishmania donovani complex and L. major. None of the primers amplified Trypanosoma cruzi or L. mexicana. The limit of detection of multiplex PCR was 2 × 10-5 parasites for L. braziliensis, 2 x 10-3 parasites for L. amazonensis, and 1.4 × 10-3 parasites for L. infantum. The high sensitivity of multiplex PCR was confirmed by the detection of parasites in different biological samples, including lesion scrapings, spleen imprinting of a hamster, sandflies, and blood. The multiplex PCR that was developed herein presented good performance with regard to detecting and identifying the parasite in different biological samples and may thus be useful for diagnosis, decision making with regard to the proper therapeutic approach, and determining the geographic distribution of Leishmania species.

Detection of Leishmania (Viannia) DNA in leucocytes from the blood of patients with cutaneous leishmaniasis.

INTRODUCTION: Cutaneous leishmaniasis (CL) is a serious and global public health issue, with the potential of developing a mucosal form, occurring as subclinical cases, and showing recurrence despite previous treatment. METHODS: Polymorphonuclear and mononuclear DNA obtained from 49 patients was subjected to polymerase chain reaction for detection of Leishmania (Viannia). RESULTS: DNA was detected in mononuclear cells from two patients with active primary lesions positive for CL, with infection periods of 3 and 6 months, respectively. CONCLUSIONS: The DNA of Leishmania (Viannia) indicates probable parasite dissemination possibly explaining subclinical case emergence, lesion recurrence, and mucosal lesion appearance.

Avaliação de sódio, potássio e cloretos em diferentes tipos de amostras empregando uma metodologia ISE direto / Evaluation of sodium, potassium and chloride in different types of samples using Direct Method ISE

A tecnologia do eletrodo íon-seletivo (ISE) direto determina a concentração de eletrólitos no componente plasmático do sangue total ou em outros tipos de amostras não diluídas. Este estudo objetivou determinar a ocorrência de diferenças significativas entre os resultados de sódio, potássio e cloretos obtidos por um analisador de gases sanguíneos / eletrólitos integrados, método ISE direto entre amostras de soro e de sangue total arterial. Foi feito um estudo retrospectivo, prospectivo, quantitativo, por meio de busca de dados em arquivo do Laboratório de Análises Clínicas do Hospital Universitário Oeste do Paraná (HUOP). Nesta pesquisa, foram incluídas 206 amostras de pacientes admitidos no HUOP de diferentes setores, de ambos os gêneros e com idade variada. Todos os resultados foram comparados pelo Teste de Wilcoxon com um p<= 0,005 indicando significância estatística. Observou-se diferença significativa entre os valores dos constituintes potássio (p=0,0003) e sódio (p=0,035) obtidos a partir do soro e sangue total arterial pelo método ISE direto. Porém não houve diferença significativa entre os valores de cloretos obtidos a partir desses dois tipo de amostras.