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

Abstract

BackgroundOne 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.MethodologyBlood 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.FindingsThe 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).ConclusionsSerological 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.

Antileishmania Immunological Tests for Asymptomatic Subjects Living in a Visceral Leishmaniasis-Endemic Area in Brazil

“…22 Our results agree with other investigators who clinically followed up asymptomatic individuals in Brazil who tested seropositive by ELISArK39, and we found that none of them developed the disease. 4,23,24 One aspect to be considered is the observation that, in the first study, the tests presented a specificity ranging from 96% to 100% using subjects from a non-endemic area as a reference. As a consequence, there was a false-positive rate of up to 4%.…”

“…4,13,25 The comparison of tests may provide some additional information that contributes to the selection of the serological tests that best discriminates between infected and uninfected subjects. In this respect, ELISA assays, which are easily performed compared with IIFT and MST, would be an interesting option for this diagnosis.…”

“…However, the diagnosis of asymptomatic infection with L. chagasi is still a matter of controversy and requires larger studies. 4 The standardization and validation of diagnostic methods for VL usually include patients with the classical clinical presentation of the disease and controls. The gold-standard techniques used for the detection of the parasite are not justified in asymptomatic cases of the disease, because they may pose a risk or excessive discomfort to the individual without providing evident benefit.…”

Anti-Leishmania infantum IgG Antibody Avidity in Visceral Leishmaniasis

“…PCR has shown higher efficacy than that of serological tests in the identification of asymptomatic cases of VL (12). However, this technique also detected positive results for subjects living in areas of endemicity but who did not progress to classic disease.…”

“…Some investigators suggested a good sensitivity of ELISA using the recombinant K39 antigen (ELISA-rK39) in the diagnosis of cases of active VL but not cases of inapparent infection (9,10). Other studies found asymptomatic subjects with positive ELISA-rK39 results, but follow-up did not reveal progression of these cases to classic VL (11)(12)(13).…”