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Objectives Recent epidemiological reports indicate that asymptomatic human infections with Leishmania donovani, the causative agent of visceral leishmaniasis or Kala-azar ( KA), occur frequently in India. We explored markers of infection. Methods Blood samples were collected from 286 healthy subjects from 16 villages in the Muzaffarpur district of Bihar. These individuals were classified into three groups: (i) persons with no history of KA and living in a house where no KA cases were previously reported, (ii) persons with no history of KA but living in a house where KA cases were diagnosed at the time of sampling or in the past, and (iii) successfully treated KA patients. Each sample was tested using a Leishmania-specific PCR to detect Leishmania DNA, and two serological tests to demonstrate anti- Leishmania antibodies: the Direct Agglutination Test and rK39 ELISA. Results PCR positivity was similar among the three groups (20-25%). In contrast, among treated patients, the percentage of serologically positive individuals was roughly five times that of healthy individuals with no KA history, as measured with either test. Living in a house where KA had been reported did not affect seropositivity. Conclusion A significant proportion of asymptomatic infections of Leishmania exist in endemic regions. Using a combination of molecular and serological tests increases the capacity to detect infections at different stages. Further work is required to understand the kinetics of the markers. [ABSTRACT FROM AUTHOR]