Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment.

Faria Hossain,Alessandro Picone,Malcolm S Duthie,Aarthy C Vallur,Md Anik Ashfaq Khan,Steven G Reed,Randall F Howard,Dinesh Mondal,Prakash Ghosh,Farhat Afrin

doi:10.1371/journal.pone.0185606

Abstract

Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL.

Highlights

Visceral leishmaniasis (VL) is a systemic disease and the most severe clinical form of the vector borne disease complex Leishmaniasis
Laboratory tests were performed at International Centre for Diarrheal Disease Research, Bangladesh. 40 patients presenting with characteristic symptoms such as fever for more than two weeks, splenomegaly and/or hepatomegaly and positive rK39 RDT, with no history of VL, were selected as VL cases. 10 patients with previous history of VL who developed recurring symptoms after 6 months but within 1 year of treatment and who were positive by splenic smear microscopy were selected as Relapsed VL (RVL) cases. 20 patients diagnosed and treated for VL in the past were included as cured VL cases. 40 patients showing characteristic skin rash and with a history of VL, who were treated for VL in the past and were positive for rK39 RDT, were selected as Post Kala-azar Dermal Leishmaniasis (PKDL) cases
The novel real time PCR assay detected as low as 10 fg of Leishmania donovani genomic DNA per reaction, a quantity that corresponds to 0.1 parasite (Fig 1A and Table 3)

Summary

Introduction

Visceral leishmaniasis (VL) is a systemic disease and the most severe clinical form of the vector borne disease complex Leishmaniasis. During VL, Leishmania parasites infect predominantly macrophages of the visceral organs, usually spleen, liver and bone marrow [1]. Each year approximately 300,000 VL cases occur globally, with over 20,000 case fatalities [2]. VL is endemic in large areas of the tropics, subtropics and the Mediterranean Basin, where it is variably known as Kala-azar, Black fever or Dumdum fever. The causative agent of VL in East Africa and the Indian subcontinent is L. donovani, whereas in Europe, North Africa and Latin America it is L. infantum [3]. In the Indian sub-continent, the disease is anthroponotic and is transmitted exclusively by the sandfly vector Phlebotomus argentipes [4,5]

Methods

Results

Conclusion