Research Article: Quantitative PCR in Epidemiology for Early Detection of Visceral Leishmaniasis Cases in India

Date Published: December 11, 2014

Publisher: Public Library of Science

Author(s): Medhavi Sudarshan, Toolika Singh, Abhishek Kumar Singh, Ankita Chourasia, Bhawana Singh, Mary E. Wilson, Jaya Chakravarty, Shyam Sundar, Alain Debrabant.

Abstract: IntroductionStudies employing serological, DTH or conventional PCR techniques suggest a vast proportion of Leishmania infected individuals living in regions endemic for Visceral Leishmaniasis (VL) remain asymptomatic. This study was designed to assess whether quantitative PCR (qPCR) can be used for detection of asymptomatic or early Leishmania donovani infection and as a predictor of progression to symptomatic disease.MethodsThe study included 1469 healthy individuals living in endemic region (EHC) including both serology-positive and -negative subjects. TaqMan based qPCR assay was done on peripheral blood of each subject using kDNA specific primers and probes.ResultsA large proportion of EHC 511/1469 (34.78%) showed qPCR positivity and 56 (3.81% of 1469 subjects) had more than 1 calculated parasite genome/ml of blood. However, the number of individuals with parasite load above 5 genomes/ml was only 20 (1.36% of 1469). There was poor agreement between serological testing and qPCR (k = 0.1303), and 42.89% and 31.83% EHC were qPCR positive in seropositive and seronegative groups, respectively. Ten subjects had developed to symptomatic VL after 12 month of their follow up examination, of which eight were initially positive according to qPCR and among these, five had high parasite load.DiscussionThus, qPCR can help us to detect significant early parasitaemia, thereby assisting us in recognition of potential progressors to clinical disease. This test could facilitate early intervention, decreased morbidity and mortality, and possibly interruption of disease transmission.

Partial Text: The Leishmania spp. parasites of humans are endemic in 98 countries, and more than 350 million people are at risk of infection [1]. Leishmaniasis is a neglected tropical disease, and the most severe form visceral leishmaniasis (VL, also known as kala-azar) is fatal if untreated. VL is primarily an anthroponotic infection caused by Leishmania donovani in India, transmitted by the sand fly vector Phelobotomus argentipes[2], [3].The state of Bihar in India accounts for 90% of cases in the country [4]. A majority of infected individuals do not develop clinical illness [5], [6], [7]. According to a serology-based epidemiological survey, the prevalence of asymptomatic Leishmania donovani infection in Bihar is 110 per 1,000 persons, and the rate of progression to symptomatic VL is 17.85 per 1,000 persons [8]. The kinetics of parasite amplification during the progression from infection to disease is as yet uncharacterized. We have recently shown that a highly quantitative qPCR test of blood can track the decrease in parasite load during successful treatment of infection [9]. The current study was based on the hypothesis that the number or the kinetics of circulating parasites in asymptomatically infected individuals, as measured by qPCR, might provide the most sensitive early indicator of infected subjects apt to progress to full blown disease.

A flow chart of the progression of results is shown in Fig. 1. Serological results were interpreted in light of our original description of the first serosurvey. Cutoff values for positive serology were chosen considering results from study of negative control unexposed Indian subjects, positive control subjects with acute or successfully treated VL, and recommendations from the serological test manufacturers [20]. Considering conversion of either the DAT or the rK39 ELISA as a seroloconversion, 401 subjects converted from seronegative to seropositive between the first and the second serosurvey, whereas the remaining 1068 individuals remained seronegative.

In this study Leishmania DNA was detected in large proportions of both seropositive and seronegative endemic healthy groups (Fig. 1). In contrast, to nonendemic healthy who were negative for the test. Similar findings were reported in a study of L. infantum infection, a cause of VL in the Mediterranean and in Latin America [17]. In one of our earlier study we showed that the parasite load in individuals with acute symptomatic VL due to L. donovani was at least 20, and at day 30 of treatment was >1.12 genome equivalents/ml [9]. In other study we found 5 parasite genome/ml of blood as the threshold value to differentiate asymptomatic from symptomatic [18]. Mary et al. cited a persistent level of more than 1 parasite/ml as a risk for relapse of L. infantum disease [17]. Our ability to quantify the parasite load in asymptomatic individuals led us to examine a potential threshold for progression to active infection in previously uninfected individuals.



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