Development and Validation of a PCR-ELISA for the Diagnosis of Symptomatic and Asymptomatic Infection by<i>Leishmania (Leishmania) infantum</i>

Abstract

A <i>kDNA</i> PCR enzyme-linked immunosorbent assay (<i>kDNA</i> PCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL) was developed. The detection limit of the reaction, precision measurements, and cut-off of the <i>kDNA</i> PCR-ELISA were defined in a proof-of-concept phase. A reference strain of <i>Leishmania (Leishmania) infantum</i> and a bank of 14 peripheral blood samples from immunocompetent patients with VL were characterized using techniques considered gold standards, and 11 blood samples obtained from healthy individuals of an endemic area were also assessed. Phase II evaluation determined the performance of the assay in peripheral blood samples from 105 patients with VL (adults and children), 25 patients with <i>Leishmania</i>/HIV coinfection, 40 healthy individuals, and 33 asymptomatic individuals living in endemic areas. The <i>kDNA</i> PCR-ELISA exhibited satisfactory precision, with a detection limit of 0.07 fg of DNA from <i>L</i>. <i>(L</i>.<i>) infantum</i> and 1 parasite/mL blood. The overall sensitivity of the assay for all groups studied was 100% (95% confidence interval [CI]: 97.1-100%), and the specificity was 95% (95% CI: 83.5-98.6%). The <i>kDNA</i> PCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use in endemic countries may help monitor control interventions.