Parasitology
A urine-based polymerase chain reaction method for the diagnosis of visceral leishmaniasis in immunocompetent patients

https://doi.org/10.1016/j.diagmicrobio.2007.09.001Get rights and content

Abstract

In the Mediterranean basin and Middle East, including Iran, visceral leishmaniasis (VL), also known as kala-azar, is caused by Leishmania donovani infantum. For the first time, the use of urine samples for the diagnosis of VL in immunocompetent patients has been used in this study. Based on its high sensitivity and specificity, as well as simplicity, this approach can serve as a valuable tool in the diagnosis of VL. We studied 60 urine samples from 60 individuals, 30 of which were patients with VL confirmed by parasitology, serology, or molecular methods, 5 were from healthy individuals, and 25 were from patients with cutaneous leishmaniasis, malaria, brucellosis, and hydatid cyst. Out of 30 samples from confirmed VL immunocompetent patients, 29 were positive (sensitivity, 96.8%) by polymerase chain reaction (RV1 and RV2 primers), and all the remaining 30 samples either from healthy individuals or patients with other diseases were negative (specificity, 100%). High sensitivity, specificity, and simplicity of the test can serve as a valuable tool in the diagnosis of VL.

Introduction

Visceral leishmaniasis (VL), also known as kala-azar, is a potentially fatal disease caused by obligate intracellular protozoan parasites of the genus Leishmania, in particular, Leishmania donovani complex, which includes 3 species: L. donovani, Leishmania infantum, and Leishmania chagasi (Mauricio et al., 2000). VL still remains a serious health problem especially in countries of tropical and subtropical regions where the disease is endemic.

Because successful control of the disease requires efficient and reliable methods of diagnosis, enormous efforts have been directed toward developing such tests. However, detection of the parasites in aspirates from lymph nodes, bone marrow, and spleen still remains the “gold standard” method (Antinori et al., 2007, Zijlstra et al., 1992). Considering the fact that these techniques are invasive, painful, and hazardous and need skilled personnel, immunologic and polymerase chain reaction (PCR)-based methods have been developed, which have their own limitations. For example, available serodiagnostic techniques based on anti-Leishmania antibody detection are not entirely satisfactory because they do not discriminate between disease and asymptomatic infection (Sarkari et al., 2005). As for direct agglutination test (DAT), cost, multiple steps, incubation, and antigenic variations are the limiting factors (Shyam and Rai, 2002).

The PCR-based methods have previously been used on bone marrow, lymph node, and spleen aspirates (Costa et al., 1996, Mathis and Deplazes, 1995, Piarroux et al., 1996, Adhya et al., 1995, Andresen et al., 1997, Osman et al., 1997, Piarroux et al., 1994), as well as peripheral blood and serum samples (Hu et al., 2000).

Recently, L. infantum DNA was detected by real-time PCR in urine from different groups of dogs with clinical leishmaniasis. Leishmania-positive PCR was found in 47% (20/43) of the urine from dogs with leishmaniasis, but the percentages of positive Leishmania PCR were 85% (11/13) in dogs with renal insufficiency (Solano-Gallego et al., 2007).

Renal involvement in human being suffering from VL has been reported by many people Efstratiadis et al. (2006), Chaigne et al. (2004), ElShafie et al. (2006), Salgado Filho et al. (2003), Romero Maroto et al. (1995), Prasad et al. (1992). Leishmania bodies in kidney were also detected in light and electron microscopy in a patient from Iran (Kumar et al., 2004). Kala-azar were also reported in patients with kidney transplant who received their organs from endemic area of VL (Ivanovski et al., 2005). These reports indicate the presence of Leishmania amastigote or particles of parasites in kidney. It seems Leishmania particles or parasites can be detected in urine samples in acute phase of disease. We designed a research on urine of kala-azar patients for diagnostic purpose.

Section snippets

Sample preparation

Thirty urine samples were collected from suspected VL patients with clinical symptoms who were admitted in Shiraz University of Medical Sciences hospitals, Shiraz, Iran, from January until August 2006. These patients confirmed having VL by parasitology (observation of amastigote in bone marrow aspiration), serology (IFA ≤1/128), or PCR (specific primers of Lin17 and lin4 as described by Azizi et al., 2006) methods. For control, 5 samples were collected from healthy individuals, 15 samples from

Results

Out of 30 samples from confirmed VL immunocompetent patients, 29 were positive by PCR, and all the remaining 30 samples either from healthy individuals or patients with other diseases were negative, which shows a sensitivity of 96.8% (29/30) and a specificity of 100% because there were no cross-reactions with the control samples. Representative positive and negative sample results are seen in Fig. 1.

Discussion

VL is a major health problem in countries of tropical and subtropical regions and is targeted for control under the World Health Organization Special Program for Research and Training in Tropical Disease. Because the effective control and treatment of the disease relies basically on early diagnosis, various tests have already been developed to serve this goal. Demonstration of the causative parasites in aspirates from lymph nodes, bone marrow, and spleen, which still remains the gold standard

Acknowledgments

We would like to thank the Office of Vice Chancellor for Research of Shiraz University of Medical Sciences, Shiraz, Iran, for financial support of this study.

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