ABSTRACT
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.
ABSTRACT
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.
Subject(s)
COVID-19 , COVID-19/diagnosis , Humans , RNA , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2/geneticsABSTRACT
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.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/epidemiology , Child , Emergency Service, Hospital , Humans , Prospective Studies , RNA, Viral/analysis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral LoadABSTRACT
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.