Clinical next-generation sequencing assay combining full-length gene amplification and shotgun sequencing for the detection of CMV drug resistance mutations.

Cytomegalovirus (CMV) causes severe systemic and tissue-invasive disease in immunocompromised patients, particularly solid organ and hematopoietic stem cell transplant recipients. While antiviral drugs offer promising efficacy, clinical management is complicated by the high frequency of drug resistance-associated mutations. The most commonly encountered mutations occur in the genes encoding for the drug targets: UL54 (DNA polymerase), UL56 (terminase complex), and UL97 (phosphotransferase), conferring resistance to ganciclovir/cidofovir/foscarnet, letermovir, and ganciclovir/maribavir, respectively. Currently, standard practice for detecting drug resistance is sequencing-based genotypic analysis by commercial reference laboratories with strictly prescribed sample requirements and reporting parameters that can often restrict testing in a highly vulnerable population. In order to circumvent these limitations, we developed a dual-step next-generation sequencing (NGS)-based clinical assay that utilizes full-length gene amplification by long-range PCR followed by shotgun sequencing for mutation analysis. This laboratory-developed test (LDT) achieved satisfactory performance with 96.4% accuracy, 100% precision, and an analytical sensitivity of 300IU/mL with 20% allele frequency. Highlighted by two clinical cases, our NGS LDT was able to provide critical results from patient specimens with viral loads <500IU/mL and volumes <0.5 mL - conditions otherwise unacceptable by reference laboratories. Here, we describe the development and implementation of a robust NGS LDT that offers greater testing flexibility and sensitivity to accommodate a more diverse patient population.

Translocation of Viable Gut Microbiota to Mesenteric Adipose Drives Formation of Creeping Fat in Humans.

A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of "creeping fat" (CrF), defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study, we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections, and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates, suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli, which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages, leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria.