Gut Dysbiosis and IL-21 Response in Patients with Severe COVID-19.

BACKGROUND: The disease severity, ranging from being asymptomatic to having acute illness, and associated inflammatory responses has suggested that alterations in the gut microbiota may play a crucial role in the development of chronic disorders due to COVID-19 infection. This study describes gut microbiota dysbiosis in COVID-19 patients and its implications relating to the disease. DESIGN: A cross sectional prospective study was performed on thirty RT-PCR-confirmed COVID-19 patients admitted to the All India Institute of Medical Sciences, Bhopal, India, between September 10 and 20, 2020. Ten healthy volunteers were recruited as the control group. IFN, TNF, and IL-21 profiling was conducted using plasma samples, and gut bacterial analysis was performed after obtaining the metagenomics data of stool samples. RESULTS: Patients with a variable COVID-19 severity showed distinct gut microflora and peripheral interleukin-21 levels. A low Firmicute/Bacteroidetes ratio, caused by the depletion of the fibre-utilizing bacteria, F. prausnitzii, B. Plebius, and Prevotella, and an increase in Bacteroidetes has associated gut microbiota dysbiosis with COVID-19 disease severity. CONCLUSIONS: The loss of the functional attributes of signature commensals in the gut, due to dysbiosis, is a predisposing factor of COVID-19 pathophysiology.

Testing of four-sample pools offers resource optimization without compromising diagnostic performance of real time reverse transcriptase-PCR assay for COVID-19.

Quick identification and isolation of SARS-CoV-2 infected individuals is central to managing the COVID-19 pandemic. Real time reverse transcriptase PCR (rRT-PCR) is the gold standard for COVID-19 diagnosis. However, this resource-intensive and relatively lengthy technique is not ideally suited for mass testing. While pooled testing offers substantial savings in cost and time, the size of the optimum pool that offers complete concordance with results of individualized testing remains elusive. To determine the optimum pool size, we first evaluated the utility of pool testing using simulated 5-sample pools with varying proportions of positive and negative samples. We observed that 5-sample pool testing resulted in false negativity rate of 5% when the pools contained one positive sample. We then examined the diagnostic performance of 4-sample pools in the operational setting of a diagnostic laboratory using 500 consecutive samples in 125 pools. With background prevalence of 2.4%, this 4-sample pool testing showed 100% concordance with individualized testing and resulted in 66% and 59% reduction in resource and turnaround time, respectively. Since the negative predictive value of a diagnostic test varies inversely with prevalence, we re-tested the 4-sample pooling strategy using a fresh batch of 500 samples in 125 pools when the prevalence rose to 12.7% and recorded 100% concordance and reduction in cost and turnaround time by 36% and 30%, respectively. These observations led us to conclude that 4-sample pool testing offers the optimal blend of resource optimization and diagnostic performance across difference disease prevalence settings.

Evaluation of pooled sample analysis strategy in expediting case detection in areas with emerging outbreaks of COVID-19: A pilot study.

Timely diagnosis of COVID-19 infected individuals and their prompt isolation are essential for controlling the transmission of SARS-CoV-2. Though quantitative reverse transcriptase PCR (qRT-PCR) is the method of choice for COVID-19 diagnostics, the resource-intensive and time-consuming nature of the technique impairs its wide applicability in resource-constrained settings and calls for novel strategies to meet the ever-growing demand for more testing. In this context, a pooled sample testing strategy was evaluated in the setting of emerging disease outbreak in 3 central Indian districts to assess if the cost of the test and turn-around time could be reduced without compromising its diagnostic characteristics and thus lead to early containment of the outbreak. From 545 nasopharyngeal and oropharyngeal samples received from the three emerging districts, a total of 109 pools were created with 5 consecutive samples in each pool. The diagnostic performance of qRT-PCR on pooled sample was compared with that of individual samples in a blinded manner. While pooling reduced the cost of diagnosis by 68% and the laboratory processing time by 66%, 5 of the 109 pools showed discordant results when compared with induvial samples. Four pools which tested negative contained 1 positive sample and 1 pool which was positive did not show any positive sample on deconvolution. Presence of a single infected sample with Ct value of 34 or higher, in a pool of 5, was likely to be missed in pooled sample analysis. At the reported point prevalence of 4.8% in this study, the negative predictive value of qRT-PCR on pooled samples was around 96% suggesting that the adoption of this strategy as an effective screening tool for COVID-19 needs to be carefully evaluated.

Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling.

BACKGROUND & OBJECTIVES: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. METHODS: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed. RESULTS: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ctvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. INTERPRETATION & CONCLUSIONS: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.