Development of microsphere-based molecular and serodiagnostics and their clinical utility

Microbial infections share many symptoms in common, rendering diagnosis difficult solely on clinical grounds. Thus, rapid, cost-effective and reliable tests are necessary for the diagnosis of infectious diseases. While the traditional diagnosis is mostly confined to detection of one pathogen at a time, a multiplex array could be a feasible alternative to improve the efficiency in the detection of infections. The Luminex xMAP-based high-throughput platform can provide simultaneous analysis of multiple analytes from the same sample by utilising differentially dyed microspheres. In this thesis, I developed xMAP-based Suspension Immuno Assays (SIAs) for the determination of IgG antibodies, IgM antibodies, as well as the avidity of IgG against the human cytomegalovirus (HCMV), Toxoplasma gondii (T. gondii) or human parvovirus B19 (B19V). Moreover, I also developed xMAP-based multiplex DNA assays for 13 human polyomaviruses (HPyVs). Primary infections by HCMV, T. gondii and B19V during pregnancy can result in severe consequences to the foetus. The serological status of the mother is critically important in counselling and recognition of infections. Hereby, I developed and evaluated SIAs for IgG, IgM and IgG-avidity against these three important pathogens. Diagnostic performances of the new assays were assessed with more than 1000 well-characterised serum samples. All the newly developed assays exhibited excellent performance compared to corresponding high-quality reference methods. The positive and negative percent agreements of the antibody-SIAs in comparison with high-standard reference assays were 92-100% and 95-100%, respectively. Kappa efficiencies between SIAs and corresponding reference assays were 0.94-1. Intra-assay and inter-assay coefficients of variations ranged between 2-12% and 1-19%, respectively. Among clinical samples from individuals with primary infection, the IgM- and IgG-SIAs served as highly sensitive screening means for detection of acute infections and immune status; and IgG-avidity-SIAs as a highly specific confirmatory approach for separation of primary infections from long-term B-cell immunity. On the other hand, during the past 12 years, a dozen viruses have joined the known family members of HPyVs. Serological studies have shown that HPyV infections occur at young age and most of the viruses circulate widely in the general population. Although HPyV infections are generally asymptomatic, severe complications can arise due to virus reactivations in immunocompromised or elderly individuals. HPyVs can persist lifelong after primary infection; however, their tissue specificities, persistence sites and transmission routes are still unclear. Also, the clinical manifestations of HPyVs with regard to immune suppression are largely unidentified. To this end, I developed xMAP-based multiplex DNA assays for all 13 HPyVs known before 2017, by using primer pairs and probes targeting the respective HPyV major capsid protein VP1 genes. The xMAP-based multiplex assays allowed for simultaneous detection of all the HPyVs with detection limits of 1-100 copies/µl. At high copies (105 copies/µl) each of the 13 target sequences were identified correctly with no cross-reactions. With this novel and specific assay, the extent to which the lymphoid system plays a role in the HPyV infection and persistence was assessed. The frequency of occurrence of HPyV viral genomes was explored in 78 lymphoid tissues from children and adults with tonsillar diseases. HPyV-DNA was found in 17.9% (14/78) of tonsils: JC polyomavirus (JCPyV, n=1), WU polyomavirus (WUPyV, n=3), Merkel cell polyomavirus (MCPyV, n=1), human polyomavirus 6 (HPyV6, n=6), trichodysplasia spinulosa polyomavirus (TSPyV, n=3). The observed frequent occurrence of HPyVs in human tonsils suggests the lymphoid tissue plays an important role as a potential transmission route and a location of persistence for these viruses. However, whether or not the undetected HPyVs share the same infection route requires more investigation with different sample types. Furthermore, to determine the occurrences in skin and clinical associations of HPyVs, I studied their genoprevalences in biopsies of premalignant [squamous cell carcinoma in situ (SCCis) or actinic keratosis (AK)] lesional vs. benign skin from 126 liver transplant recipients (LiTRs); as well as in healthy skin of 80 immunocompetent adults. Multiplex screening was followed by singleplex qPCRs of positive samples, for reference and quantification of the viral DNAs. In total, five dermal HPyVs – MCPyV, HPyV6, human polyomavirus 7, TSPyV, and Lyon IARC polyomavirus (LIPyV) – were found in 26.2% (58/221) skin biopsies. The prevalences and quantities of MCPyV in premalignant vs. benign skin of LiTRs were similar to those in healthy skin of controls. TSPyV was found in a single skin lesion at very low copies. The other three HPyVs occurred exclusively in benign skin. Overall, in 10 out of 12 SCCis/AK patients the viral DNA findings in skin were alike. Thereby, the occurrences of HPyVs in the skin of LiTRs and controls speak against a role for any of HPyVs in SCC development. The work presented in this thesis shows that the xMAP-based serological approaches exhibit excellent diagnostic performances compared to corresponding conventional methods. Moreover, the developed xMAP-based multiplex PCR for 13 HPyVs could be applied successfully in a variety of clinical materials. Altogether, the newly developed systems provide a powerful tool for medical diagnosis and research.