ABSTRACT Background: American cutaneous leishmaniasis is a commonly neglected, vector-borne tropical parasitic disease that is a major public health concern in Brazil. Leishmania (Viannia) braziliensis is the main species associated with the disease. Accurate diagnosis is based on epidemiological surveillance, clinical assessment, and laboratory testing. Leishmania (V.) braziliensis has been detected in several wild and synanthropic mammals. Their epidemiological role has not been entirely elucidated. This study aimed to assess potential L. braziliensis infections in asymptomatic domestic animals, by molecular and serological testing in endemic areas, in the metropolitan region of Recife. Methods: Blood samples and conjunctival fluids were collected from 232 animals (canids, felids, equines, and caprines) for the detection of L. braziliensis using molecular tests (conventional and real-time polymerase chain reaction [PCR and qPCR]). For immunological detection, blood samples from 115 dogs were assessed using enzyme-linked immunosorbent assay. Results: Real-time quantitative PCR showed positive results for blood and conjunctival samples in all investigated species. The results of the blood and conjunctival samples were 68.2% and 26.9% in Canis familiaris, 100% and 41.7% in Felis catus, 77.3% and 30.8% in Equus caballus/Equus asinus, and 50% and 33.3% in Capra hircus samples, respectively. Conclusions: Results from this study adds valuable information to our understanding of the role of asymptomatic domestic animals, L. braziliensis life cycle, and American cutaneous leishmaniasis in Northeast Brazil.

ABSTRACT Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.

Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

RESUMORelato de caso de paciente masculino de 45 anos, agricultor, residente na zona da mata do Estado de Pernambuco, diagnosticado com HIV em 1999 e em uso de ARV. Em 2005 foi registrada a primeira ocorrência de LV através do diagnóstico parasitológico a partir do aspirado da medula óssea. À admissão no hospital apresentava-se com febre, hepatoesplenomegalia, perda de peso e diarréia. Desde então houve a ocorrência de mais sete episódios de LV, tendo ocorrido em media, um evento a cada ano (2005-2012 exceto em 2008). O paciente apresentou, em 2011, um quadro cutâneo disseminado, sendo realizada biopsia de pele que evidenciou formas amastigotas de Leishmania no exame histopatológico. Em 2005, o tratamento foi realizado com antimoniato de N-metil-glucamina e anfotericina B desoxicolato, mas desde 2006, devido à toxicidade, o medicamento de escolha foi a anfotericina B lipossomal. Como recomendado pelo Ministério da Saúde, esse relato reforça a necessidade de que os casos de HIV residentes em área endêmica de LV deverão ter inserido em seu protocolo de acompanhamento essa parasitose, principalmente após o primeiro episódio.

SUMMARYReport of a 45-year-old male farmer, a resident in the forest zone of Pernambuco, who was diagnosed with human immunodeficiency virus (HIV) in 1999 and treated using antiretroviral (ARV) drugs. In 2005, the first episode of visceral leishmaniasis (VL), as assessed by parasitological diagnosis of bone marrow aspirate, was recorded. When admitted to the hospital, the patient presented fever, hepatosplenomegaly, weight loss, and diarrhea. Since then, six additional episodes of VL occurred, with a frequency rate of one per year (2005-2012, except in 2008). In 2011, the patient presented a disseminated skin lesion caused by the amastigotes of Leishmania, as identified by histopathological assessment of skin biopsy samples. In 2005, he was treated with N-methyl-glucamine-antimony and amphotericin B deoxycholate. However, since 2006 because of a reported toxicity, the drug of choice was liposomal amphotericin B. As recommended by the Ministry of Health, this report emphasizes the need for HIV patients living in VL endemic areas to include this parasitosis in their follow-up protocol, particularly after the first infection of VL.