Summary (Abstract)BackgroundFecal shedding of SARS-CoV-2 has raised concerns about transmission through fecal microbiota transplantation (FMT) procedures. While many tests have been authorized for diagnosis of COVID-19 using respiratory samples, no fully validated stool tests for detection of SARS-CoV-2 are currently available. We sought to adapt and validate an available test specifically for detection of SARS-CoV-2 in human stool.MethodsStool samples were spiked with inactivated SAR-CoV-2 virus for development and validation of the assay. A modified version of the CDC rRT-PCR SARS-CoV-2 test was used for detection of virus. Analytical sensitivity, assay reproducibility, and sample stability under a variety of storage conditions were assessed. We also performed the assay on stool samples collected from known COVID positive individuals.FindingsThe lower limit of detection (LoD) of the assay was found to be 3000 viral RNA copies per gram of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Samples were relatively stable in all buffers tested at both 4°C and ambient temperature, with the exception of storage in STAR buffer at ambient temperature. Assay sensitivity was slightly diminished in low-copy-number samples after a single freeze-thaw cycle at −80°C. Thirty contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, we detected SARS-CoV-2 RNA in the stool of known COVID-19 positive individuals using this method.InterpretationThis is a sensitive, reproducible, and validated assay for detection of SARS-CoV-2 RNA in human stool with potential uses in FMT donor screening, sewage monitoring, and further research into the impact of fecal shedding on the epidemiology of this pandemic.FundingNational Institute for Allergy and Infectious Diseases, NIH. Center for Biologics Evaluation and Research, FDA.Research in ContextEvidence before this studySince the onset of the COVID-19 pandemic, multiple studies have documented shedding of SARS-CoV-2 RNA in feces and considered the potential for fecal-oral transmission of this virus. This potential risk led to the U.S. Food and Drug Administration issuing a safety alert that contained the recommendation that no stool donated after December 1, 2019 be used for manufacture of Fecal Microbiota for Transplantation (FMT) products in the United States until such a time as sufficient screening procedures could be put in place to mitigate this risk.Added value of this studyHere, we report the development and validation of an assay specifically meant for the detection of SARS-CoV-2 RNA in the stool of healthy individuals. While studies have reported detection of viral RNA in stool previously, this is the first publication of a validated assay designed for this purpose.Implications of all the available evidenceThe work presented here provides a validated SARS-CoV-2 stool assay with potential application to FMT donor screening protocols, sewage monitoring protocols, as well as research studies assessing the role of stool shedding and transmission on the epidemiology of COVID-19.
Treatment of choice in ART therapy for HIV-1 viremia remains an issue as all treatments interrupt HIV repopulation albeit at different reproductive points. The issue was addressed by grouping patients into two-tiered HIV level design of High >50,000 and Low <50,000 plasma cps/ml. The High group with 3 patients had combined class ART using NRTI plus NNRTI or PI regimens until viremia was reduced to <75 cps/ml and then PI alone. The Low group with 11 patients had PIMT regimen. CD4 T cell levels were monitored throughout. ART efficacy was assessed as total number of months of controlled (<75 cps/ml) viral infection level vs. number of months of uncontrolled levels of (>75 cps/ml). In that adherence/non-adherence to regimen is a confound, it was also assessed by rate of suppression: HIVj-HIVk/ HVj-HVn. In the High group, one patient attained long term controlled HIV viremia over 56 months vs. 3 months of uncontrolled viremia while the other two patients (21 vs. 25; 9 vs. 11) did not. Corresponding values in Group 2 ranged from 6 months to 139 months vs. <5 months A t-test for correlated means (with control for duration differences) showed significant difference: t=4.10, α=<0.01, df=10. Suppression rates, both within and across groups, were from: 0.99-1.0 of cps/ml. Accordingly, PI monotherapy can maintain long term viremia suppression for viral levels of <50,000 cps/ml and ART is operative within an inferred functionally closed boundary system as no host characterization including CD4 T cell level affected ART outcome.
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