Inaccuracy of enzyme-linked immunosorbent assay using soluble and recombinant antigens to detect asymptomatic infection by Leishmania infantum

Abstract

Background: One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period.

Methodology: Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve.

Findings: The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10).

Conclusions: Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised.

Figure 1. PCR and hybridization products to identify L. donovani complex in blood samples.

A: PCR products obtained with primers specific for the L. donovani complex (Piarroux, 1993); molecular size markers (lane 1), positive control (lane 2-kDNA extracted from cultured L.infantum), VL patient (lane 3), asymptomatic (lanes 4;7;9), health individuals (lane 5–6); negative control (lane 8- no DNA). B: PCR products obtained with primers for genus Leishmania (Degrave et al 1994); molecular size markers (lane 1), VL patient (lane 2), positive control (lane 3-kDNA extracted from cultured L. chagasi); healthy individual (lane 4), asymptomatic (lanes 5–7, 9–10), negative control (lanes 8 and 11- no DNA). C: Dot-blots obtained using specific probe for the L. donovani complex, hybridized with PCR products.

Figure 2. Comparison of absorbances between cohort's participants, positive and negative controls, using three different antigens.

A: L. amazonensis. B: L.infantum. C, rK39. Healthy = 22 negative controls; Cohort = 136 inhabitant of endemic area; VL = 8 positive controls (patients with visceral leishmaniasis). Differences statistically significant were sign (p<0.05, p<0.001, Kruskall Wallis and Dunn's test).