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 Leishmania spp. in Human Serum.

Konstantin Tanida,Beatrice Nickel,Egbert Tannich,Alexandra Veit,Patrick Leander Scheid,Andreas Hahn,Sven Poppert,Ralf Matthias Hagen,Hagen Frickmann,Carsten Balczun,Ulrike Loderstädt

doi:10.3390/pathogens10070826

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.

Highlights

As reviewed recently [1], the diagnosis of causative agents of visceral leishmaniasis is mainly based on microscopical, serological, and nucleic acid amplification testing (NAT) procedures
For a disease with high parasitemia, such as symptomatic visceral leishmaniasis, it is even discussed that NAT might be too sensitive, so it will detect asymptomatic, self-limiting infections without clinical implications as well [3]
The examination of the initial samples from visceral leishmaniasis patients resulted in nearly 100% sensitivity for a real-time PCR targeting kinetoplast DNA

Summary

Introduction

As reviewed recently [1], the diagnosis of causative agents of visceral leishmaniasis is mainly based on microscopical, serological, and nucleic acid amplification testing (NAT) procedures. For a disease with high parasitemia, such as symptomatic visceral leishmaniasis, it is even discussed that NAT might be too sensitive, so it will detect asymptomatic, self-limiting infections without clinical implications as well [3]. As known from study results from Brazil, ratios of asymptomatic infections to clinically apparent visceral leishmaniasis may range from 6.5:1 up to 18:1 [4,5,6], so infection events do not necessarily require medical treatment. Even in the case of clinically relevant disease, the persistence of parasites in well-vascularized lymphatic nodes in spite of successfully cured infections will be no exemption [7], limiting the value of real-time PCR for therapy control. Scientists within the US armed forces medical service were able to demonstrate such asymptomatic infections in deployed soldiers in a recently published assessment [9]

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Conclusion