Transfusion-associated tick-borne pathogens; Development and evaluation of Babesia spp. real-time PCR and detection of Neoehrlichia mikurensis DNA in blood donors

Abstract

Tick borne diseases are emerging in Norway. People living in the Kristiansand area located on the southern coastline are at high risk of contracting a tick-borne disease since ticks are prevalent. The Ixodes ricinus ticks transmit pathogens causing diseases as Lyme borreliosis and tick-borne encephalitis (TBE), both well-known infections in this region. Among the less recognized tick-borne pathogens that may infect and cause disease in humans are Babesia species (spp.) and Neoehrlichia mikurensis causing babesiosis and neoehrlichiosis, respectively. N. mikurensis are frequently (< 25%) detected in ticks in the southern part of Norway, whereas Babesia spp. are infrequently (< 1%) detected. Babesiosis may be transmitted from an infected human by transfusion of blood component, and in the USA the risk of such transmission is high. Neoehrlichiosis in healthy individuals are often asymptomatic and bacteria in blood seem to be persistent for a longer time, potentially posing a risk of transferring disease to the recipients by infected blood components. The impact of babesiosis and neoehrlichiosis in transfusion medicine and public health in Europe is not yet identified.

At Sørlandet Hospital, a laboratory developed real-time PCR detecting N. mikurensis is established in the routine for human diagnostics. However, a sensitive Babesia spp. specific real-time assay for use in research and diagnostics is lacking. As a part of this study, a real-time Babesia spp. PCR was developed for detection of the most common human pathogenic Babesia spp. in Europe. In a second part of the study, the prevalence of N. mikurensis infection in healthy blood donors was investigated. Blood donors at Sørlandet Hospital, Kristiansand were retrospectively analysed for the presence of N. mikurensis DNA by real-time PCR in the blood and the PCR positive donors were retested to examine if infection was persisting.

A Babesia spp. qualitative duplex real-time PCR assay targeting the COX1 gene for detection of B. divergens, B. venatorum and B. microti, was designed and verified. The assay performance in preliminary validation was good, i.e., sensitivity was high and the amplification efficiency was good. Moreover, no negative cross-reactivity was observed with other pathogen tested, blood matrix (including human DNA) did not interfere with the assay and all Babesia spp. tested were detected.

Among 499 blood donors from Sørlandet Hospital, Kristiansand 9% had detectable low levels of N. mikurensis DNA in the blood. After retesting 1-7 month later, 69% of them was still infected. Persistent infection over months among donors is more likely than reinfection. A high infection rate of N. mikurensis in blood donors indicates a risk of transferring infection to the recipients of blood component, and further research is needed.