Quantitative PCR in Epidemiology for Early Detection of Visceral Leishmaniasis Cases in India Medhavi Sudarshan 1 , Toolika Singh 1 , Abhishek Kumar Singh 1 , Ankita Chourasia 1 , Bhawana Singh 1 , Mary E. Wilson 2 , Jaya Chakravarty 1 , Shyam Sundar 1 * 1 Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, 2 Departments of Internal Medicine and Microbiology, University of Iowa and the VA Medical Center, Iowa City, Iowa, United States of America Abstract Introduction: Studies 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. Methods: The 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. Results: A 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. Discussion: Thus, 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. Citation: Sudarshan M, Singh T, Singh AK, Chourasia A, Singh B, et al. (2014) Quantitative PCR in Epidemiology for Early Detection of Visceral Leishmaniasis Cases in India. PLoS Negl Trop Dis 8(12): e3366. doi:10.1371/journal.pntd.0003366 Editor: Alain Debrabant, US Food and Drug Administration, United States of America Received April 18, 2014; Accepted October 23, 2014; Published December 11, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The study was funded in part by National Institute of Allergy and Infectious disease (NIAID), DMID funding mechanism: Tropical Medicine Research Center Grant number: P50AI074321. Partial funding was also derived from NIH grant R01 AI076233 (MEW). Authors Medhavi Sudarshan and Bhawana Singh got financial support from Council of Scientific and Industrial Research (CSIR), New Delhi, India. Toolika Singh and Abhishek Singh got financial support from Indian Council of Medical Research(ICMR), New Delhi, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: drshyamsundar@hotmail.com Introduction 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 asymp- tomatic 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. Alternate techniques to detect parasites in persons with VL include direct histological examination and/or culture of bone marrow and splenic aspirates. However these methods are not feasible for screening methods or epidemiological research due to their invasive nature. Serological methods are simple, non-invasive means of detecting specific antibodies, but it is already shown that there is a lack of correlation between serology and nucleic acid PLOS Neglected Tropical Diseases | www.plosntds.org 1 December 2014 | Volume 8 | Issue 12 | e3366