Technical Evaluation of qPCR Multiplex Assays for the Detection of Ixodes ricinus-Borne Pathogens

Abstract

Background: The extent to which infections with Ixodes ricinus-borne pathogens (TBPs), other than Borrelia burgdorferi s. l. and tick-borne encephalitis virus (TBEV), cause disease in humans remains unclear. One of the reasons is that adequate diagnostic modalities are lacking in routine or research settings. Methods: We evaluated the analytical specificity, sensitivity and robustness of qPCR assays for the detection of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Spiroplasma ixodetis, several Babesia species and Spotted Fever Rickettsia species as well as Bartonella species in human samples. Results: The qPCRs were found to perform well, given the difficulties of dealing with microorganisms for which confirmed patient materials are scarce or non-existent, a hurdle that was partially overcome by using synthetic controls. Spiking blood samples with the tested microorganisms showed that the detection of the TBPs was not inhibited by the presence of blood. The acceptable sensitivity when multiplexing the different pathogens, the good inter-assay variability and the absence of cross-reactivity make them potentially suitable as human diagnostics. Conclusions: The qPCRs evaluated in this study are technically suitable for the laboratory diagnostic assessment of clinical samples for infection with tick-borne pathogens. However, clinical validation and independent confirmation are still needed, pending the availability of sufficient human samples for testing in different laboratories.

Ixodiphagus hookeri (Hymenoptera: Encyrtidae) and Tick-Borne Pathogens in Ticks with Sympatric Occurrence (and Different Activities) in the Slovak Karst National Park (Slovakia), Central Europe

“…Subsequently, all qPCR-positive samples from OspA/flaB duplex qPCR were examined using conventional PCR to amplify the intergenic spacer region (IGS) of B. burgdorferi s.l., according to Coipan et al [39], and sequenced. Three multiplex qPCR assays were used for the detection of Anaplasma spp., Babesia spp., Bartonella, and Rickettsia spp., as published by Azagi et al [40]. The selected positive PCR products, with amplified fragments of wasp-and/or pathogen-related genes, were purified using the NucleoSpin Gel and PCR Cleanup kit (Macherey-Nagel GmbH & Co., Duren, Germany) and Sanger sequenced in both directions using the corresponding PCR primers in the Laboratory of Biomedical Microbiology and Immunology (University of Veterinary Medicine and Pharmacy in Košice, Slovakia) and/or by Eurofins Genomics (Ebersberg, Germany).…”