Routine, Cost-Effective SARS-CoV-2 Surveillance Testing Using Pooled Saliva Limits Viral Spread on a Residential College Campus.

Routine testing for SARS-CoV-2 is rare for institutes of higher education due to prohibitive costs and supply chain delays. During spring 2021, we routinely tested all residential students 1 to 2 times per week using pooled, RNA-extraction-free, reverse transcription quantitative PCR (RT-qPCR) testing of saliva at a cost of $0.43/sample with same-day results. The limit of detection was 500 copies/ml on individual samples, and analysis indicates 1,000 and 2,500 copies/ml in pools of 5 and 10, respectively, which is orders of magnitude more sensitive than rapid antigen tests. Importantly, saliva testing flagged 83% of semester positives (43,884 tests administered) and was 95.6% concordant with nasopharyngeal diagnostic results (69.0% concordant on the first test when the nucleocapsid gene (N1) cycle threshold (CT) value was >30). Moreover, testing reduced weekly cases by 59.9% in the spring despite far looser restrictions, allowing for more normalcy while eliminating outbreaks. We also coupled our testing with a survey to clarify symptoms and transmissibility among college-age students. While only 8.5% remained asymptomatic throughout, symptoms were disparate and often cold-like (e.g., only 37.3% developed a fever), highlighting the difficulty with relying on symptom monitoring among this demographic. Based on reported symptom progression, we estimate that we removed 348 days of infectious individuals by routine testing. Interestingly, viral load (CT value) at the time of testing did not affect transmissibility (R2 = 0.0085), though those experiencing noticeable symptoms at the time of testing were more likely to spread the virus to close contacts (31.6% versus 14.3%). Together, our findings support routine testing for reducing the spread of SARS-CoV-2. Implementation of cost- and resource-efficient approaches should receive strong consideration in communities that lack herd immunity. IMPORTANCE This study highlights the utility of routine testing for SARS-CoV-2 using pooled saliva while maintaining high sensitivity of detection (under 2,500 copies/ml) and rapid turnaround of high volume (up to 930 samples in 8 h by two technicians and one quantitative PCR [qPCR] machine). This pooled approach allowed us to test all residential students 1 to 2 times per week on our college campus during the spring of 2021 and flagged 83% of our semester positives. Most students were asymptomatic or presented with symptoms mirroring common colds at the time of testing, allowing for removal of infectious individuals before they otherwise would have sought testing. To our knowledge, the total per-sample consumable cost of $0.43 is the lowest to date. With many communities still lagging in vaccination rates, routine testing that is cost-efficient highlights the capacity of the laboratory's role in controlling the spread of SARS-CoV-2.

In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity.

As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources.

OBJECTIVES: To establish the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2. METHODS: The most efficient pool size was determined to be five specimens using a web-based application. From this analysis, 25 experimental pools were created using 50 µL from one SARS-CoV-2 positive nasopharyngeal specimen mixed with 4 negative patient specimens (50 µL each) for a total volume of 250 µL. Viral RNA was subsequently extracted from each pool and tested using the CDC SARS-CoV-2 RT-PCR assay. Positive pools were consequently split into individual specimens and tested by extraction and PCR. This method was also tested on an unselected group of 60 nasopharyngeal specimens grouped into 12 pools. RESULTS: All 25 pools were positive with cycle threshold (Ct) values within 0 and 5.03 Ct of the original individual specimens. The analysis of 60 specimens determined that 2 pools were positive followed by identification of 2 individual specimens among the 60 tested. This testing was accomplished while using 22 extractions/PCR tests, a savings of 38 reactions. CONCLUSIONS: When the incidence rate of SARS-CoV-2 infection is 10% or less, group testing will result in the saving of reagents and personnel time with an overall increase in testing capability of at least 69%.

Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene Allplex™ 2019-nCoV assay.

BACKGROUND: The fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction. RESULTS: Optimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (Ct values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at Ct values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at - 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection. CONCLUSION: We successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.

A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection.

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.

Optimized and scalable synthesis of magnetic nanoparticles for RNA extraction in response to developing countries' needs in the detection and control of SARS-CoV-2.

Ecuador is one of the most affected countries, with the coronavirus disease 2019 (COVID-19) infection, in Latin America derived from an ongoing economic crisis. One of the most important methods for COVID-19 detection is the use of techniques such as real time RT-PCR based on a previous extraction/purification of RNA procedure from nasopharyngeal cells using functionalized magnetic nanoparticles (MNP). This technique allows the processing of ~ 10,000 tests per day in private companies and around hundreds per day at local Universities guaranteeing to reach a wide range of the population. However, the main drawback of this method is the need for specialized MNP with a strong negative charge for the viral RNA extraction to detect the existence of the SARS-CoV-2 virus. Here we present a simplified low cost method to produce 10 g of nanoparticles in 100 mL of solution that was scaled to one litter by parallelizing the process 10 times in just two days and allowing for the possibility of making ~ 50,000 COVID-19 tests. This communication helps in reducing the cost of acquiring MNP for diverse biomolecular applications supporting developing country budgets constraints and chemical availability specially during the COVID-19 International Health Emergency.

Optimising triage procedures for patients with cancer needing active anticancer treatment in the COVID-19 era.

BACKGROUND: Immunosuppression induced by anticancer therapy in a COVID-19-positive asymptomatic patient with cancer may have a devastating effect and, eventually, be lethal. To identify asymptomatic cases among patients receiving active cancer treatment, the Federico II University Hospital in Naples performs rapid serological tests in addition to hospital standard clinical triage for COVID-19 infection. METHODS: From 6 to 17 April 2020, all candidates for chemotherapy, radiotherapy or target/immunotherapy, if negative at the standard clinical triage on the day scheduled for anticancer treatment, received a rapid serological test on peripheral blood for COVID-19 IgM and IgG detection. In case of COVID-19 IgM and/or IgG positivity, patients underwent a real-time PCR (RT-PCR) SARS-CoV-2 test to confirm infection, and active cancer treatment was delayed. RESULTS: Overall 466 patients, negative for COVID-19 symptoms, underwent serological testing in addition to standard clinical triage. The average age was 61 years (range 25-88 years). Most patients (190, 40.8%) had breast cancer, and chemotherapy with or without immunotherapy was administered in 323 (69.3%) patients. Overall 433 (92.9%) patients were IgG-negative and IgM-negative, and 33 (7.1%) were IgM-positive and/or IgG-positive. Among the latter patients, 18 (3.9%), 11 (2.4%) and 4 (0.9%) were IgM-negative/IgG-positive, IgM-positive/IgG-negative and IgM-positive/IgG-positive, respectively. All 33 patients with a positive serological test, tested negative for RT-PCR SARS-CoV-2 test. No patient in our cohort developed symptoms suggestive of active COVID-19 infection. CONCLUSION: Rapid serological testing at hospital admission failed to detect active asymptomatic COVID-19 infection. Moreover, it entailed additional economic and human resources, delayed therapy administrationand increased hospital accesses.

Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: A reliable, faster and economical method.

BACKGROUND: Corona virus disease 2019 (COVID-19) which initially started as a cluster of pneumonia cases in the Wuhan city of China has now become a full-blown pandemic. Timely diagnosis of COVID-19 is the key in containing the pandemic and breaking the chain of transmission. In low- and middle-income countries availability of testing kits has become the major bottleneck in testing. Novel methods like pooling of samples are the need of the hour. OBJECTIVE: We undertook this study to evaluate a novel protocol of pooling of RNA samples/elutes in performance of PCR for SARS CoV-2 virus. STUDY DESIGN: Extracted RNA samples were randomly placed in pools of 8 on a 96 well plate. Both individual RNA (ID) and pooled RNA RT-qPCR for the screening E gene were done in the same plate and the positivity for the E gene was seen. RESULTS: The present study demonstrated that pool testing with RNA samples can easily detect even up to a single positive sample with Ct value as high as 38. The present study also showed that the results of pool testing is not affected by number of positive samples in a pool. CONCLUSION: Pooling of RNA samples can reduce the time and expense, and can help expand diagnostic capabilities, especially during constrained supply of reagents and PCR kits for the diagnosis of SARS-CoV-2 infection.

The cost-effectiveness of conducting three versus two reverse transcription-polymerase chain reaction tests for diagnosing and discharging people with COVID-19: evidence from the epidemic in Wuhan, China.

OBJECTIVES: The objectives were to evaluate the effectiveness of conducting three versus two reverse transcription-PCR (RT-PCR) tests for diagnosing and discharging people with COVID-19 with regard to public health and clinical impacts by incorporating asymptomatic and presymptomatic infection and to compare the medical costs associated with the two strategies. METHODS: A model that consisted of six compartments was built. The compartments were the susceptible (S), the asymptomatic infective (A), the presymptomatic infective (L), the symptomatic infective (I), the recovered (R), and the deceased (D). The A, L and I classes were infective states. To construct the model, several parameters were set as fixed using existing evidence and the rest of the parameters were estimated by fitting the model to a smoothed curve of the cumulative confirmed cases in Wuhan from 24 January 2020 to 6 March 2020. Input data about the cost-effectiveness analysis were retrieved from the literature. RESULTS: Conducting RT-PCR tests three times for diagnosing and discharging people with COVID-19 reduced the estimated total number of symptomatic cases to 45| 013 from 51 144 in the two-test strategy over 43 days. The former strategy also led to 850.1 quality-adjusted life years (QALYs) of health gain and a net healthcare expenditure saving of CN¥49.1 million. About 100.7 QALYs of the health gain were attributable to quality-adjusted life day difference between the strategies during the analytic period and 749.4 QALYs were attributable to years of life saved. CONCLUSIONS: More accurate strategies and methods of testing for the control of COVID-19 may reduce both the number of infections and the total medical costs. Increasing the number of tests should be considered in regions with relatively severe epidemics when existing tests have moderate sensitivity.

Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs.

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.