Comparison of the methods for isolation and detection of SARS-CoV-2 RNA in municipal wastewater.

Introduction: Coronavirus SARS-CoV-2 is a causative agent responsible for the current global pandemic situation known as COVID-19. Clinical manifestations of COVID-19 include a wide range of symptoms from mild (i.e., cough, fever, dyspnea) to severe pneumonia-like respiratory symptoms. SARS-CoV-2 has been demonstrated to be detectable in the stool of COVID-19 patients. Waste-based epidemiology (WBE) has been shown as a promising approach for early detection and monitoring of SARS-CoV-2 in the local population performed via collection, isolation, and detection of viral pathogens from environmental sources. Methods: In order to select the optimal protocol for monitoring the COVID-19 epidemiological situation in region Turiec, Slovakia, we (1) compared methods for SARS-CoV-2 separation and isolation, including virus precipitation by polyethylene glycol (PEG), virus purification via ultrafiltration (Vivaspin®) and subsequent isolation by NucleoSpin RNA Virus kit (Macherey-Nagel), and direct isolation from wastewater (Zymo Environ Water RNA Kit); (2) evaluated the impact of water freezing on SARS- CoV-2 separation, isolation, and detection; (3) evaluated the role of wastewater filtration on virus stability; and (4) determined appropriate methods including reverse transcription-droplet digital PCR (RT-ddPCR) and real-time quantitative polymerase chain reaction (RT-qPCR) (targeting the same genes, i.e., RdRp and gene E) for quantitative detection of SARS-CoV-2 in wastewater samples. Results: (1) Usage of Zymo Environ Water RNA Kit provided superior quality of isolated RNA in comparison with both ultracentrifugation and PEG precipitation. (2) Freezing of wastewater samples significantly reduces the RNA yield. (3) Filtering is counterproductive when Zymo Environ Water RNA Kit is used. (4) According to the specificity and sensitivity, the RT-ddPCR outperforms RT-qPCR. Discussion: The results of our study suggest that WBE is a valuable early warning alert and represents a non-invasive approach to monitor viral pathogens, thus protects public health on a regional and national level. In addition, we have shown that the sensitivity of testing the samples with a nearer detection limit can be improved by selecting the appropriate combination of enrichment, isolation, and detection methods.

Sensitive SARS-CoV-2 detection, air travel Covid-19 testing, variant determination and fast direct PCR detection, using ddPCR and RT-qPCR methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monitoring in air traffic is important in the prevention of the virus spreading from abroad. The gold standard for SARS-CoV-2 detection is RT-qPCR; however, for early and low viral load detection, a much more sensitive method, such as droplet digital PCR (ddPCR), is required. Our first step was to developed both, ddPCR and RT-qPCR methods, for sensitive SARS-CoV-2 detection. Analysis of ten swab/saliva samples of five Covid-19 patients in different stages of disease showed positivity in 6/10 samples with RT-qPCR and 9/10 with ddPCR. We also used our RT-qPCR method for SARS-CoV-2 detection without the need of RNA extraction, obtaining results in 90-120 minutes. We analyzed 116 self-collected saliva samples from passengers and airport staff arriving from abroad. All samples were negative by RT-qPCR, while 1 was positive, using ddPCR. Lastly, we developed ddPCR assays for SARS-CoV-2 variants identification (alpha, beta, gamma, delta/kappa) that are more economically advantageous when compared to NGS. Our findings demonstrated that saliva samples can be stored at ambient temperature, as we did not observe any significant difference between a fresh sample and the same sample after 24 hours (p = 0.23), hence, saliva collection is the optimal route for sampling airplane passengers. Our results also showed that droplet digital PCR is a more suitable method for detecting virus from saliva, compared to RT-qPCR. Keywords: COVID-19; RT-PCR; ddPCR; SARS-CoV-2; nasopharyngeal swab; saliva.

Comparison of SARS-CoV-2 Detection by Rapid Antigen and by Three Commercial RT-qPCR Tests: A Study from Martin University Hospital in Slovakia.

The global pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having a tremendous impact on the global economy, health care systems and the lives of almost all people in the world. The Central European country of Slovakia reached one of the highest daily mortality rates per 100,000 inhabitants in the first 3 months of 2021, despite implementing strong prophylactic measures, lockdowns and repeated nationwide antigen testing. The present study reports a comparison of the performance of the Standard Q COVID-19 antigen test (SD Biosensor) with three commercial RT-qPCR kits (vDetect COVID-19-MultiplexDX, gb SARS-CoV-2 Multiplex-GENERI BIOTECH Ltd. and Genvinset COVID-19 [E]-BDR Diagnostics) in the detection of infected individuals among employees of the Martin University Hospital in Slovakia. Health care providers, such as doctors and nurses, are classified as "critical infrastructure", and there is no doubt about the huge impact that incorrect results could have on patients. Out of 1231 samples, 14 were evaluated as positive for SARS-CoV-2 antigen presence, and all of them were confirmed by RT-qPCR kit 1 and kit 2. As another 26 samples had a signal in the E gene, these 40 samples were re-isolated and subsequently re-analysed using the three kits, which detected the virus in 22, 23 and 12 cases, respectively. The results point to a divergence not only between antigen and RT-qPCR tests, but also within the "gold standard" RT-qPCR testing. Performance analysis of the diagnostic antigen test showed the positive predictive value (PPV) to be 100% and negative predictive value (NPV) to be 98.10%, indicating that 1.90% of individuals with a negative result were, in fact, positive. If these data are extrapolated to the national level, where the mean daily number of antigen tests was 250,000 in April 2021, it points to over 4700 people per day being misinterpreted and posing a risk of virus shedding. While mean Ct values of the samples that were both antigen and RT-qPCR positive were about 20 (kit 1: 20.47 and 20.16 for Sarbeco E and RdRP, kit 2: 19.37 and 19.99 for Sarbeco E and RdRP and kit 3: 17.47 for ORF1b/RdRP), mean Ct values of the samples that were antigen-negative but RT-qPCR-positive were about 30 (kit 1: 30.67 and 30.00 for Sarbeco E and RdRP, kit 2: 29.86 and 31.01 for Sarbeco E and RdRP and kit 3: 27.47 for ORF1b/RdRP). It confirms the advantage of antigen test in detecting the most infectious individuals with a higher viral load. However, the reporting of Ct values is still a matter of ongoing debates and should not be conducted without normalisation to standardised controls of known concentration.