Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses.

BACKGROUND: Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. METHODS: We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. RESULTS: We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. CONCLUSIONS: This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.

Deficient butyrate metabolism in the intestinal microbiome is a potential risk factor for recurrent kidney stone disease.

Intestinal microbiome dysbiosis is a known risk factor for recurrent kidney stone disease (KSD) with prior data suggesting a role for dysfunctional metabolic pathways other than those directly utilizing oxalate. To identify alternative mechanisms, the current study analyzed differences in the metabolic potential of intestinal microbiomes of patients (n = 17) and live-in controls (n = 17) and determined their relevance to increased risk for KSD using shotgun metagenomic sequencing. We found no differences in the abundance of genes associated with known oxalate degradation pathways, supporting the notion that dysfunction in other metabolic pathways plays a role in KSD. Further analysis showed decreased abundance of key enzymes involved in butyrate biosynthesis in patient intestinal microbiomes. Furthermore, de novo construction of microbial genomes showed that the majority of genes significantly enriched in non-stone formers are affiliated with Faecalibacterium prausnitzii, a major butyrate producer. Specifically pertaining to butyrate metabolism, the majority of abundant genes mapped back to F. prausnitzii, Alistipes spp., and Akkermansia muciniphila. No differences were observed in ascorbate or glyoxylate metabolic pathways. Collectively, these data suggest that impaired bacterial-associated butyrate metabolism may be an oxalate-independent mechanism that contributes to an increased risk for recurrent KSD. This indicates that the role of the intestinal microbiome in recurrent KSD is multi-factorial, which is representative of the highly intertwined metabolic nature of this complex environment. Future bacteria-based treatments must not be restricted to targeting only oxalate metabolism.

Treatment of HCV with direct-acting antivirals on reducing mortality related to extrahepatic manifestations: a large population-based study in British Columbia, Canada.

Background: HCV infection is associated with mortality due to extrahepatic manifestations (EHM). Sustained virologic response (SVR) following direct-acting antiviral (DAA) therapy has been linked to decreased all-cause and liver-related mortality. However, evidence regarding the impact of DAA on EHM-related deaths is lacking. This study aimed to assess the impact of DAA and SVR on EHM-related mortality. Methods: The British Columbia Hepatitis Testers Cohort comprises ∼1.7 million people tested for HCV between 1990 and 2015 and is linked with administrative health data. Among individuals diagnosed with HCV by 12/31/2020, those who received at least one DAA treatment were matched to those who never received treatment by the year of their first HCV RNA positive date. We compared three groups: treated & SVR, treated & no-SVR, and untreated; and generated EHM mortality rates and incidence curves. To account for differences in baseline characteristics, we used inverse probability of treatment weights (IPTW). IPTW-weighted multivariable cause-specific Cox regression models were adjusted for competing risk and confounders. Findings: Study population included 12,815 treated (12,287 SVR, 528 no-SVR) and 12,815 untreated individuals (median follow-up 3.4 years, IQR 2.9). The untreated group had the highest EHM mortality rate (30.9 per 1000 person-years [PY], 95% CI 29.2-32.8), followed by the treated & no-SVR group (21.2 per 1000 PY, 95% CI 14.9-30.1), while the treated & SVR group had the lowest EHM mortality rate (7.9 per 1000 PY, 95% CI 7.1-8.7). In the multivariable model, EHM mortality in the treated & SVR group was significantly decreased (adjusted cause-specific hazard ratio [acsHR] 0.20, 95% CI 0.18-0.23). The treated & SVR group had significant reductions in mortality related to each of the EHMs (78-84%). Interpretation: Treatment of HCV with DAA was associated with significant reductions in EHM-related mortality. These findings emphasize the critical importance of timely diagnosis and treatment of HCV to prevent deaths associated with EHM, and have important implications for clinical practice and public health. Funding: This work was supported by the BC Centre for Disease Control and the Canadian Institutes of Health Research (CIHR) [Grant # NHC-348216, PJT-156066, and PHE-337680]. DJ has received Doctoral Research Award (#201910DF1-435705-64343) from the Canadian Institutes of Health Research (CIHR) and Doctoral fellowship from the Canadian Network on Hepatitis C (CanHepC). CanHepC is funded by a joint initiative of the Canadian Institutes of Health Research (CIHR) (NHC-142832) and the Public Health Agency of Canada (PHAC).

SinoNasal Microbiota Transfer to treat recalcitrant chronic rhinosinusitis: A case series.

KEY POINTS: SinoNasal Microbiota Transfer (SNMT) was safe with immediate benefit in all recipients, with sustained improvement in two of three recipients for up to 180 days. The addition of antimicrobial photodynamic therapy worsened chronic rhinosinusitis. These promising SNMT results warrant further study of safety and efficacy.

Bifidobacterium longum modifies a nutritional intervention for stunting in Zimbabwean infants.

Child stunting is an indicator of chronic undernutrition and reduced human capital. However, it remains a poorly understood public health problem. Small-quantity lipid-based nutrient supplements (SQ-LNS) have been widely tested to reduce stunting, but have modest effects. The infant intestinal microbiome may contribute to stunting, and is partly shaped by mother and infant histo-blood group antigens (HBGA). We investigated whether mother-infant fucosyltransferase status, which governs HBGA, and the infant gut microbiome modified the impact of SQ-LNS on stunting at age 18 months among Zimbabwean infants in the SHINE Trial ( NCT01824940 ). We found that mother-infant fucosyltransferase discordance and Bifidobacterium longum reduced SQ-LNS efficacy. Infant age-related microbiome shifts in B. longum subspecies dominance from infantis , a proficient human milk oligosaccharide utilizer, to suis or longum , proficient plant-polysaccharide utilizers, were partly influenced by discordance in mother-infant FUT2+/FUT3- phenotype, suggesting that a "younger" microbiome at initiation of SQ-LNS reduces its benefits on stunting.