Human subjects and ethical clearance

Blood samples were collected from (n = 46) asymptomatic, (n = 48) symptomatic VL patients and (n = 59) endemic control individuals at the Kala‐azar Medical Research Center (Muzaffarpur, Bihar, India). Active VL cases were confirmed based on clinical signs, including fever (> 2 weeks), splenomegaly, positive serology for rK39 rapid diagnostic test and by microscopic demonstration of Leishmania amastigotes in splenic or bone marrow aspirate smears. Clinical data from these patients are summarised in Table 1. Asymptomatic healthy subjects living in endemic regions were tested for anti‐leishmania antibodies by direct agglutination test (DAT) and rK39 ELISA. A positive and negative control (filter paper pooled eluates from VL patients and non‐endemic healthy controls, NEHC) were run in each rK39‐ELISA, and the positive control was used as a reference to calculate a relative value of positivity of each sample, expressed as percentage positivity (PP). In the first sero‐survey, subjects positive by both DAT and rK39‐ELISA (≥ 1:1600 and ≥ 14PP, respectively), and highly seropositive by one or both assays (≥ 1:25 600 and ≥ 23 PP) and who met the inclusion criteria, were invited to KAMRC within 14 days of identification. All asymptomatic subjects were monitored monthly for 24 months after enrolment in the study to observe any development of active VL. This work was conducted with ethical approval (No. Dean/2017/EC/185 dated 24/10/2017) obtained from Institutional Review Committees of Banaras Hindu University, Varanasi, India. Each study patient was informed both verbally and in writing (in English and Hindi) about the nature of the study, the anticipated risks and benefits, the discomforts to which the patient will be exposed, and their right to discontinue participation at any time of their own free will. Written informed consent was obtained from all participants, and where participants were below 18 years of age, written informed consent was obtained from their legal guardian. We excluded pregnant women or lactating mothers, subjects having a vaccination within 30 days, and hepatitis B or C positive subjects. All subjects were HIV‐negative and above 12 years of age.

Demographic and clinical information on study participants

ND, Assay not done.