Multiplex clinical viral detection in formalin-fixed paraffin-embedded tissue samples Abstract: Transplantation is increasingly common and successful; however, high levels of immunosuppression remain a near-universal precondition for success. As such, solid organ and hematopoietic stem cell transplantation, as well as other conditions associated with immunosuppression, remain complicated by high rates of opportunistic infections. Of these, viral etiologies are the most difficult to diagnose due to substantial clinical/histologic overlap with non-viral processes as well as a lack of assays capable of detecting viruses in affected tissues. Indeed, virus diagnostics examining affected tissue directly are currently limited to immunohistochemistry and in situ hybridization, both of which suffer from severely limited clinical and analytic sensitivity/accuracy. As such, physicians often must rely on single-analyte serologic or singleplex nucleic acid assays of questionable applicability that are associated with substantial detection bias. There is thus an urgent need for multiplexed tissue-based viral diagnostics. Here we propose to develop a highly specific, highly sensitive method to simultaneously detect and quantify 55 clinically relevant viruses within formalin-fixed paraffin-embedded (FFPE) biopsy tissue, which is the standard specimen for histopathologic analysis. Our approach utilizes complementary long padlock probes (cLPPs) to selectively capture and quantitatively amplify defined genomic regions from clinically relevant viruses with very low off-target reactivity. Subsequently, reciprocal paired-end sequencing (Illumina MiSeq) will produce an accurate and high coverage representation of the targeted viral repertoire from which species/strain identity is assigned. Moreover, quantitative cLPP target capture allows viral copy number to be determined by simple read counting and normalization to internal human calibration targets, allowing discrimination between pathologic (high copy) and bystander (low copy) viruses. We will determine our methods' capture efficiency and accuracy, false discovery rate, and analytical sensitivity by using 100 well- characterized commercial and clinical viral isolates currently available in our laboratory. Additionally, clinical diagnostic feasibility will be established using 100 FFPE biopsies from various clinical contexts with either objective evidence for or clinical suspicion of viral infection in comparison to 100 similar biopsis with established non-viral etiologies (GVHD, rejection, inflammatory bowel disease, etc.). Overall, this technology will significantly improve our ability to diagnose and treat viral infectin in transplant patients while simultaneously reducing turnaround time and cost.
Multiplex clinical viral detection in formalin-fixed paraffin-embedded tissue samples Project Narrative: Opportunistic infections remain major sources of transplant-attributable morbidity and mortality, accounting for ~20% of all transplant patient deaths. In such infections, early and definitive diagnosis is critical to treatment outcome, but viral etiologies - the most common and clinically problematic - often remain diagnoses of exclusion or inference even after costly and time-consuming diagnostic evaluation. Here we propose the development, validation, and demonstration of clinical feasibility for a highly specific, highly sensitive method to simultaneously detect and quantify >50 clinically relevant viruses within FFPE biopsy tissue.
