Inaccuracy of Enzyme-Linked Immunosorbent Assay Using Soluble and Recombinant Antigens to Detect Asymptomatic Infection by Leishmania infantum.

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. Author Summary: Visceral leishmaniasis or kala-azar in Brazil, caused by a parasite protozoon, is transmitted to humans by sandflies and has wild and domestic dogs as reservoirs. The disease can become chronic, characterized by fever, weight loss, and enlargement of the liver and spleen. However, the infection in humans can be asymptomatic, and the importance of this phase is unknown. The low levels of immune response and the small number of circulating parasites in the blood are the main problems in identifying the asymptomatic phase. In this study we evaluated the performance of the serology tests compared with molecular tests to identify asymptomatic infection in individuals identified in an urban area of Brazil. Also included were non-infected individuals (healthy controls) and patients (diseased controls). Blood was collected and examined by different serological methods and compared with molecular techniques. The presence of the parasite was confirmed by all molecular techniques and should be the method used to identify true infection. The serology methods showed low sensitivity in detecting the infection. We concluded that the serologic methods are inaccurate in identifying asymptomatic infection when compared to molecular techniques and could underestimate the infection in population studies. [ABSTRACT FROM AUTHOR]