TY - JOUR
T1 - Technical Evaluation of qPCR Multiplex Assays for the Detection of Ixodes ricinus-Borne Pathogens
AU - Azagi, Tal
AU - Hoeve-Bakker, B. J. A.
AU - Jonker, Mark
AU - Roelfsema, Jeroen H.
AU - Sprong, Hein
AU - Kerkhof, Karen
N1 - Funding Information: This research was funded by The Netherlands Organization for Health Research and Development (ZonMw, project number 52200-30-07), which peer-reviewed the grant application, and by the Dutch Ministry of Health, Welfare, and Sports. HS is supported by the NorthTick, European Union, European Regional Development Fund, in the North Sea Region Programme. Publisher Copyright: © 2022 by the authors.
PY - 2022/11/1
Y1 - 2022/11/1
N2 - 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.
AB - 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.
KW - Anaplasma phagocytophilum
KW - Babesia divergens
KW - Babesia microti
KW - Bartonellaspp
KW - Candidatus Neoehrlichia mikurensis
KW - Rickettsia helvetica
KW - Rickettsia stenos group
KW - Spiroplasma ixodetis
KW - Taqman
KW - multiplex qPCR
UR - http://www.scopus.com/inward/record.url?scp=85141757940&partnerID=8YFLogxK
U2 - https://doi.org/10.3390/microorganisms10112222
DO - https://doi.org/10.3390/microorganisms10112222
M3 - Article
C2 - 36363814
SN - 2076-2607
VL - 10
JO - Microorganisms
JF - Microorganisms
IS - 11
M1 - 2222
ER -