ABSTRACT
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.
Subject(s)
Humans , DNA, Protozoan/urine , Leishmania/genetics , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/urine , Polymerase Chain Reaction/methods , Reproducibility of Results , DNA, Protozoan/isolation & purification , Sensitivity and Specificity , Leishmania/isolation & purificationABSTRACT
Canine visceral leishmaniasis (CVL) is difficult to diagnosis, mainly due to the presence of asymptomatic animals, the diversity of clinical symptoms and the difficulty in obtaining diagnostic evidence of high sensitivity and specificity. The purpose of this study was to diagnose CVL in urinary sediment of 70 dogs of different breeds, sexes and ages from the veterinary hospital of the Federal University of Piauí and Zoonosis Control Center of Teresina, Brazil. The serological tests were TR DPP® for CVL and enzyme-linked immunosorbent assay (ELISA) for CVL, parasitological exams of bone marrow and lymph nodes and urine sediment cultures. Leishmania was detected in the bone marrow and/or lymph node of 61.0% of the animals (43/70), and urine sediment culture was positive in 9.30% (4/43) of these animals. In the serological exams, 70.0% (49/70) were reactive using the DPP and 78.2% (55/70) were reactive using ELISA. The goal of this study was to diagnose the presence of L. (infantum) chagasi in a culture of urinary sediment.
A leishmaniose visceral canina (LVC) é uma doença de difícil diagnóstico. Principalmente devido à presença de animais assintomáticos, a diversidade da sintomatologia clínica apresentada e também pela dificuldade em se obter uma prova diagnóstica que reúna alta sensibilidade e especificidade. O objetivo deste trabalho foi relatar a presença de L. (infantum) chagasi em meio de cultura, utilizando-se sedimento urinário. Foram utilizados neste experimento, 70 cães provenientes do Hospital Veterinário Universitário da Universidade Federal do Piauí e do Centro de Controle de Zoonoses de Teresina, com raça, sexo e idade variada. Foram realizados exames sorológicos: TR DPP® Leishmaniose Visceral Canina (DPP) e Ensaio Imunoenzimático Leishmaniose Visceral Canina (ELISA), exames parasitológicos de amostras de medula e/ou linfonodo e cultura de sedimento urinário. Em 61,0% (43/70) dos animais estudados, observou-se presença de Leishmania em medula e/ou linfonodo, e destes 9,30% (4/43) foram positivos na cultura de sedimento urinário. Nos exames sorológicos, 70,0% (49/70) dos animais apresentavam-se reativos no DPP e 78,2% (55/70) no ELISA. Pode-se concluir, neste estudo, que é possível diagnosticar a LVC por meio da cultura de sedimento urinário.
Subject(s)
Animals , Dogs , Dog Diseases/diagnosis , Dog Diseases/parasitology , Dog Diseases/urine , Leishmania infantum/isolation & purification , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/urine , Leishmaniasis, Visceral/veterinary , Urine/parasitologyABSTRACT
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. .
Subject(s)
Humans , DNA, Protozoan/urine , Leishmania infantum/genetics , Leishmaniasis, Visceral/parasitology , Specimen Handling/methods , Leishmania infantum/isolation & purification , Leishmaniasis, Visceral/urine , Polymerase Chain ReactionABSTRACT
Neste estudo foi analisada a distribuição de isotipos de imunoglobulinas IgG1, IgG2 e IgE envolvidas na resposta a Leishmania no soro e a expressão de mRNA para IFN-g e IL-4 in situ no baço de cães naturalmente infectados com L.infantum com diferentes perfis de susceptibilidade ou resistência à doença. A atividade sérica de anticorpos do isotipo IgG1 contra Leishmania tendeu a ser maior nos animais com teste cutêneo da Leishmanina negativo e com parasitismo esplênico (grupo infectado potencialmente susceptível, 0,424±0,401) que nos outros grupos: com teste cutêneo da Leishmanina positivo e ausência de parasitismo esplênico (grupo infectado potencialmente resistente, 0,226±0,114), ou com ambos teste cutêneo da Leishmanina e cultura esplênica positivos (grupo em fase indeterminada da doença, 0,234±0,125) ou com ambos os parâmetros negativos (grupo potencialmente não infectado, 0,159±0,044). Essa diferença não foi, contudo estatisticamente significante (ANOVA, P=0,1450). IgG2 específica anti-Leishmania foi maior (ANOVA P=0,0001)...
We analyzed the distribution immunoglobulin isotypes IgG1, IgG2 and IgE involved in the response to Leishmania and the mRNA expression for IFN-g and IL-4 in situ in the spleen of dogs naturally infected with IgG1 antibody anti-Leishmania serum activity was higher in animals with negative Leishmanin skin test and splenic parasitism (infected and potencially susceptible to VL group, 0,424±0,401) than in other groups: positive Leishmanin skin test and negative splenic parasitism (infected and potentially resistant to VL group, 0,226±0,114), or both positive Leishmanin skin test and slpenic parasitism (infected with undefined susceptibility status group, 0234±0,125) or both negative parameters (non-infected group, 0159±0,044). This difference however was not statistically significant (ANOVA, P=0145)...