Extraction-free clinical detection of SARS-CoV-2 virus from saline gargle samples using Hamilton STARlet liquid handler.

As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput SARS-CoV-2 multiplex polymerase chain reaction (PCR) testing in order to minimize cost and technologist time. This was achieved through elimination of nucleic acid extraction and automation of sample handling on a widely available robotic liquid handler, Hamilton STARlet. A customized barcode scanning script for reading the sample ID by the Hamilton STARlet's software system was developed to allow primary tube sampling. Use of pre-frozen SARS-CoV-2 assay reaction mixtures reduced assay setup time. In both validation and live testing, the assay produced no false positive or false negative results. Of the 1060 samples tested during validation, 3.6% (39/1060) of samples required retesting as they were either single gene positive, had internal control failure or liquid aspiration error. Although the overall turnaround time was only slightly faster in the automated workflow (185 min vs 200 min), there was a 76% reduction in hands-on time, potentially reducing staff fatigue and burnout. This described process from sample self-collection to automated direct PCR testing significantly reduces the total burden on healthcare systems in terms of human resources and reagent requirements.

Evaluation of observed and unobserved self-collection of saline gargle samples for the detection of SARS-CoV-2 in outpatients.

The diagnostic sensitivity of observed and unobserved self-collected saline gargle samples for the molecular detection of SARS-CoV-2 in adults and school-aged children was evaluated against a reference standard of health care worker collected nasopharyngeal flocked swab. A total of 46 participants had a positive nasopharyngeal swab sample; of these, 10 were in the observed phase and 36 were in the unobserved phase. Only one matching saline gargle sample tested negative and this was in the unobserved phase, giving an overall sensitivity of 98%. Average viral target Ct values were higher in the saline gargle samples. RNaseP Ct values were lower in unobserved collected samples compared to observed collected samples. Unobserved self-collection of saline gargle samples is a promising outpatient testing method for COVID-19 diagnosis. The self-collection method has potential to simplify the diagnostic cycle and facilitate implementation of COVID-19 testing, particularly in settings with limited access to health care workers.

Development and validation of a new triplex real-time quantitative reverse Transcriptase-PCR assay for the clinical detection of SARS-CoV-2.

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.

Self-Collected Saline Gargle Samples as an Alternative to Health Care Worker-Collected Nasopharyngeal Swabs for COVID-19 Diagnosis in Outpatients.

We assessed the performance, stability, and user acceptability of swab-independent self-collected saliva and saline mouth rinse/gargle sample types for the molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in adults and school-aged children. Outpatients who had recently been diagnosed with COVID-19 or were presenting with suspected COVID-19 were asked to have a nasopharyngeal (NP) swab collected and provide at least one self-collected sample type. Participants were also asked about sample acceptability using a five-point Likert scale. For those previously diagnosed with COVID-19, all samples underwent real-time PCR testing using a lab-developed assay, and the majority were also tested using an FDA-authorized assay. For those presenting with suspected COVID-19, only those with a positive nasopharyngeal swab sample went on to have other samples tested. Saline mouth rinse/gargle and saliva samples were tested daily at time zero, day 1, and day 2 to assess nucleic acid stability at room temperature. Fifty participants (aged 4 to 71 years) were included; of these, 40 had at least one positive sample and were included in the primary sample yield analysis. Saline mouth rinse/gargle samples had a sensitivity of 98% (39/40), while saliva samples had a sensitivity of 79% (26/33). Both saline mouth rinse/gargle and saliva samples showed stable viral RNA detection after 2 days of room temperature storage. Mouth rinse/gargle samples had the highest (mean, 4.9) and health care worker (HCW)-collected NP swabs had the lowest acceptability scores (mean, 3.1). In conclusion, saline mouth rinse/gargle samples demonstrated higher combined user acceptability ratings and analytical performance than saliva and HCW-collected NP swabs. This sample type is a promising swab-independent option, particularly for outpatient self-collection in adults and school-aged children.