Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection of <i>Leishmania</i> spp. in Human Serum

Abstract

To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results.

Keywords

• leishmania
• visceral
• Kala Azar
• real-time PCR
• test comparison
• in-house