Latent infection with Leishmania donovani in highly endemic villages in Bihar, India

Abstract

Introduction: Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4-10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.

Methods: We selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.

Results: We enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).

Discussion: Although infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.

Figure 1. DAT titers at baseline.

Serologic titers are graphed for the 12,634 subjects without prior or current VL, measured by the DAT test. Numbers of subjects with positive DAT tests at the indicated dilution are shown. The two graphs show the same data on different scales, so that both large and small titer peaks can be appreciated.

Figure 2. rK39 optical densities at baseline.

Serologic titers are graphed for the 12,634 subjects without prior or current VL, measured by rK39 ELISA. The data are expressed as percentage of positive (pp), calculated as the (subject OD/positive control OD)×100 control for each subject. The left and the right figure show the same data on different scales, so that range of OD titers below 45 can be appreciated.

Figure 3. Median titer of positive DAT (left) or rK39 (right) tests by age.

Among individuals who had a positive DAT response over or equal to 1∶1600 or a positive rK39 response over or equal to 14 pp, the median DAT titer (left) or rK39 pp (right) plus 95% confidence intervals are plotted for each age group.

Figure 4. Median DAT and rK39 titers among 6-months cohorts of former VL patients in function of the time that elapsed since diagnosis (n = 118).

Figure 5. Probability of VL, sero conversion, and sero-prevalence by age group.

Cumulative VL incidence over the 2½ year period preceding the first round sero-survey, probability of DAT and rK39 positivity during the first round sero-survey, and probability of sero-conversion on either DAT or rK39 during the 12-months interval between the first and second round survey have been plotted by age.