A rapid and low-cost protocol for the detection of B.1.1.7 lineage of SARS-CoV-2 by using SYBR Green-based RT-qPCR.

BACKGROUND: The new SARS-CoV-2 variant VOC (202012/01), identified recently in the United Kingdom (UK), exhibits a higher transmissibility rate compared to other variants, and a reproductive number 0.4 higher. In the UK, scientists were able to identify the increase of this new variant through the rise of false negative results for the spike (S) target using a three-target RT-PCR assay (TaqPath kit). METHODS: To control and study the current coronavirus pandemic, it is important to develop a rapid and low-cost molecular test to identify the aforementioned variant. In this work, we designed primer sets specific to the VOC (202012/01) to be used by SYBR Green-based RT-PCR. These primers were specifically designed to confirm the deletion mutations Δ69/Δ70 in the spike and the Δ106/Δ107/Δ108 in the NSP6 gene. We studied 20 samples from positive patients, detected by using the Applied Biosystems TaqPath RT-PCR COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. 16 samples displayed an S-negative profile (negative for S target and positive for N and ORF1ab targets) and four samples with S, N and ORF1ab positive profile. RESULTS: Our results emphasized that all S-negative samples harbored the mutations Δ69/Δ70 and Δ106/Δ107/Δ108. This protocol could be used as a second test to confirm the diagnosis in patients who were already positive to COVID-19 but showed false negative results for S-gene. CONCLUSIONS: This technique may allow to identify patients carrying the VOC (202012/01) or a closely related variant, in case of shortage in sequencing.

Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater.

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.

Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction.

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.

Active testing of groups at increased risk of acquiring SARS-CoV-2 in Canada: costs and human resource needs.

BACKGROUND: Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely passive, which impedes epidemic control. We defined active testing strategies for SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) for groups at increased risk of acquiring SARS-CoV-2 in all Canadian provinces. METHODS: We identified 5 groups who should be prioritized for active RT-PCR testing: contacts of people who are positive for SARS-CoV-2, and 4 at-risk populations - hospital employees, community health care workers and people in long-term care facilities, essential business employees, and schoolchildren and staff. We estimated costs, human resources and laboratory capacity required to test people in each group or to perform surveillance testing in random samples. RESULTS: During July 8-17, 2020, across all provinces in Canada, an average of 41 751 RT-PCR tests were performed daily; we estimated this required 5122 personnel and cost $2.4 million per day ($67.8 million per month). Systematic contact tracing and testing would increase personnel needs 1.2-fold and monthly costs to $78.9 million. Conducted over a month, testing all hospital employees would require 1823 additional personnel, costing $29.0 million; testing all community health care workers and persons in long-term care facilities would require 11 074 additional personnel and cost $124.8 million; and testing all essential employees would cost $321.7 million, requiring 25 965 added personnel. Testing the larger population within schools over 6 weeks would require 46 368 added personnel and cost $816.0 million. Interventions addressing inefficiencies, including saliva-based sampling and pooling samples, could reduce costs by 40% and personnel by 20%. Surveillance testing in population samples other than contacts would cost 5% of the cost of a universal approach to testing at-risk populations. INTERPRETATION: Active testing of groups at increased risk of acquiring SARS-CoV-2 appears feasible and would support the safe reopening of the economy and schools more broadly. This strategy also appears affordable compared with the $169.2 billion committed by the federal government as a response to the pandemic as of June 2020.

Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR.

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 107 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.

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.

Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification.

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

Evaluating the efficiency of specimen pooling for PCR-based detection of COVID-19.

In the age of a pandemic, such as the ongoing one caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the world faces a limited supply of tests, personal protective equipment, and factories and supply chains are struggling to meet the growing demands. This study aimed to evaluate the efficacy of specimen pooling for testing of SARS-CoV-2 virus, to determine whether costs and resource savings could be achieved without impacting the sensitivity of the testing. Ten previously tested nasopharyngeal and throat swab specimens by real-time polymerase chain reaction (PCR), were pooled for testing, containing either one or two known positive specimens of varying viral concentrations. Specimen pooling did not affect the sensitivity of detecting SARS-CoV-2 when the PCR cycle threshold (Ct) of original specimen was lower than 35. In specimens with low viral load (Ct > 35), 2 of 15 pools (13.3%) were false negative. Pooling specimens to test for Coronavirus Disease 2019 infection in low prevalence (≤1%) areas or in low risk populations can dramatically decrease the resource burden on laboratory operations by up to 80%. This paves the way for large-scale population screening, allowing for assured policy decisions by governmental bodies to ease lockdown restrictions in areas with a low incidence of infection, or with lower-risk populations.

COVID-19 polymerase chain reaction testing before endoscopy: an economic analysis.

BACKGROUND AND AIMS: The novel coronavirus disease 2019 (COVID-19) pandemic has limited endoscopy utilization, causing significant health and economic losses. We aim to model the impact of polymerase chain reaction (PCR) testing into resuming endoscopy practice. METHODS: We performed a retrospective review of endoscopy utilization during the COVID-19 pandemic for a baseline reference. A computer model compared 3 approaches: strategy 1, endoscopy for urgent indications only; strategy 2, testing for semiurgent indications; and strategy 3, testing all patients. Analysis was made under current COVID-19 prevalence and projected prevalence of 5% and 10%. Primary outcomes were number of procedures performed and/or canceled. Secondary outcomes were direct costs, reimbursement, personal protective equipment used, and personnel infected. Disease prevalence, testing accuracy, and costs were obtained from the literature. RESULTS: During the COVID-19 pandemic, endoscopy volume was 12.7% of expected. Strategies 2 and 3 were safe and effective interventions to resume endoscopy in semiurgent and elective cases. Investing 22 U.S. dollars (USD) and 105 USD in testing per patient allowed the completion of 19.4% and 95.3% of baseline endoscopies, respectively. False-negative results were seen after testing 4700 patients (or 3 months of applying strategy 2 in our practice). Implementing PCR testing over 1 week in the United States would require 13 and 64 million USD, with a return of 165 and 767 million USD to providers, leaving 65 and 325 healthcare workers infected. CONCLUSIONS: PCR testing is an effective strategy to restart endoscopic practice in the United States. PCR screening should be implemented during the second phase of the pandemic, once the healthcare system is able to test and isolate all suspected COVID-19 cases.

Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19.

Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.