Digital Droplet PCR to Track SARS-CoV-2 Outbreak in a Hospital Transitional Care Unit.

We describe an outbreak of SARS-CoV-2 on a transition unit composed of elderly patients awaiting placement. Environmental and patient sample analyses using digital droplet PCR (ddPCR) suggested possible fomite transmission and a high viral burden source from a few individual patients. This outbreak illustrates challenges inherent to this specific patient population.

SARS-CoV-2 Viral Load Quantification, Clinical Findings and Outcomes in Children Seeking Emergency Department Care: Prospective Cohort Study.

We compared the perfomance of SARS-CoV-2 reverse transcriptase real-time polymerase chain reaction (RT-PCR) to droplet digital PCR (ddPCR). 95% and 40% of positive and negative RT-PCR specimens, respectively, were positive on ddPCR yielding sensitivities of 84% (95% CI: 74, 91) and 97% (95% CI: 89, 99), for RT-PCR and ddPCR, respectively. We found that SARS-CoV-2 RT-PCR testing in children has a concerning false-negative rate at lower nucleocapsid gene copy numbers.

The Role of Subgenomic RNA in Discordant Results From Reverse Transcription-Polymerase Chain Reaction Tests for COVID-19.

CONTEXT.­: Reverse transcription-polymerase chain reaction (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when 1 RT-PCR target is positive for SARS-CoV-2 and 1 RT-PCR target is negative for SARS-CoV-2 within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. OBJECTIVE.­: To understand the role of subgenomic RNA transcripts in discordant results from RT-PCR tests for COVID-19. DESIGN.­: A panel of 6 droplet digital PCR assays was designed to quantify the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity. Eleven clinical nasopharyngeal swabs were also tested with this panel. RESULTS.­: In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. The same trends in the relative abundance of copies across different targets observed in infected cells were observed in clinical samples, although trends were more pronounced in infected cells. CONCLUSIONS.­: This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by these data.

SARS-CoV-2 variant detection with ADSSpike.

The SARS-CoV-2 coronavirus pandemic has been an unprecedented challenge to global pandemic response and preparedness. With the continuous appearance of new SARS-CoV-2 variants, it is imperative to implement tools for genomic surveillance and diagnosis in order to decrease viral transmission and prevalence. The ADSSpike workflow was developed with the goal of identifying signature SNPs from the S gene associated with SARS-CoV-2 variants through amplicon deep sequencing. Seventy-two samples were sequenced, and 30 mutations were identified. Among those, signature SNPs were linked to 2 Zeta-VOI (P.2) samples and one to the Alpha-VOC (B.1.17). An average depth of 700 reads was found to properlycorrectly identify all SNPs and deletions pertinent to SARS-CoV-2 mutants. ADSSpike is the first workflow to provide a practical, cost-effective, and scalable solution to diagnose SARS-CoV-2 VOC/VOI in the clinical laboratory, adding a valuable tool to public health measures to fight the COVID-19 pandemic for approximately $41.85 USD/reaction.

Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production.

Paraffinic n-alkanes (>C17) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffin n-octacosane (C28H58). Compared with that in controls, the consumption of n-octacosane was coupled to methane production, demonstrating its biodegradation under these conditions. Smithella was postulated to be an important C28H58 degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identified assA gene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from other Smithella organisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays for assA demonstrated significant increases in the abundance and expression of this gene in C28H58-degrading cultures compared with that in controls, suggesting n-octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C28H58-degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery.IMPORTANCE Understanding the methanogenic biodegradation of different classes of hydrocarbons has important applications for effective fuel-contaminated site remediation and for improved recovery from oil reservoirs. Previous studies have clearly demonstrated that short-chain alkanes (C17) that comprise many fuel mixtures. Using an enrichment culture derived from a freshwater fuel-contaminated site, we demonstrate that the model waxy alkane n-octacosane can be biodegraded under methanogenic conditions by a presumed Smithella phylotype. Compared with that of controls, we show an increased abundance and expression of the assA gene, which is known to be important for anaerobic n-alkane metabolism. Metabolite analyses revealed the presence of a range of α,ω-dicarboxylic acids found only in n-octacosane-degrading cultures, a novel finding that lends insight as to how anaerobic communities may access waxes as growth substrates in anoxic environments.