Role of Hologic® Panther AptimaTM SARS-CoV-2 assay in the detection of SARS-CoV-2: screening or diagnostic technique?

During the multiple waves of COVID-19 suffered all over the world, having a rapid and sensitive diagnostic test has become a priority for microbiology laboratories. The AptimaTM SARS-CoV-2 transcription-mediated amplification (TMA) assay running on the Panther system (Hologic) was presented as a very good option to cover this need. To evaluate this system, 570 respiratory samples were included in the study and were processed both by the Panther (Hologic) system and by qRT-PCR (Thermo Fisher Science, Waltham, USA), current assay for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A high number of false positives (n=76) was obtained with Panther system (Hologic), but the number of false positives decreases as the relative light units (RLU) value increases. These results show that this technique can be a good option for sample screening but checking for positive results should be mandatory, especially those with low RLU values.

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR.

BACKGROUND: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. METHODS: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. RESULTS: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. CONCLUSION: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

In Silico Evaluation of SARS-CoV-2 K417N, L452R, and E484K Detection Assays Against Omicron Variants.

The study's objective was to assess whether the performance of the DIAGNOVITAL SARS-CoV-2 Mutation Detection Assays is affected by Omicron mutations. In silico evaluation of 67,717 Variant of Concern, Variant of Interest sequences and 6,612 sequences of the Omicron variants involving BA1., BA2., BA3 sub-lineages downloaded from the GISAID database by 17 December 2021, were performed. The sequences were aligned according to the reference genome MN908947.3 using MAFFT multiple sequence alignment software version 7. Our findings showed that among 6,612 Omicron, 41 Spike gene mutations with a frequency of ≥70% were identified. Some of the Omicron mutations (R408S, N440K, G446S, Q493S, Q498R) could affect the diagnostic performance of K417N, L452R, and E484K assays against the Omicron sub-lineages. However, L452R and K417N mutation tests allow differentiation of the Delta and Omicron variants mutation profile. The COVID-19 pandemic lasted longer than expected, and the rapid modification of diagnostic kits seems necessary to combat the pandemic.

Ozone Efficacy for the Disinfection of Ambulances Used to Transport Patients during the COVID-19 Pandemic in Peru.

We assessed the disinfection efficacy of an ozone generator prototype in ambulances used to transport patients with coronavirus disease (COVID-19). This research consisted of three stages: in vitro tests using microbial indicators, such as Candida albicans, Escherichia coli, Staphylococcus aureus and Salmonella phage, which were experimentally inoculated onto polystyrene crystal surfaces within a 23 m3 enclosure. They were then exposed to ozone at a 25 ppm concentration using the ozone generator (Tecnofood SAC) portable prototype, and the decimal reduction time (D) was estimated for each indicator. The second stage involved the experimental inoculation of the same microbial indicators on a variety of surfaces inside conventional ambulances. The third stage consisted of exploratory field testing in ambulances used to transport patients with suspected COVID-19. During the second and third stages, samples were collected by swabbing different surfaces before and after 25 ppm ozonisation for 30 min. Results suggested that ozone was most effective on Candida albicans (D = 2.65 min), followed by Escherichia coli (D = 3.14 min), Salmonella phage (D = 5.01 min) and Staphylococcus aureus (D = 5.40 min). Up to 5% of the microbes survived following ozonisation of conventional ambulances. Of the 126 surface samples collected from ambulances transporting patients with COVID-19, 7 were positive (5.6%) for SARS-related coronavirus as determined on reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Ozone exposure from the ozone generator prototype inside ambulances at a concentration of 25 ppm for 30 min can eliminate gram positive and negative bacteria, yeasts, and viruses.

Actionable wastewater surveillance: application to a university residence hall during the transition between Delta and Omicron resurgences of COVID-19.

Wastewater surveillance has gained traction during the COVID-19 pandemic as an effective and non-biased means to track community infection. While most surveillance relies on samples collected at municipal wastewater treatment plants, surveillance is more actionable when samples are collected "upstream" where mitigation of transmission is tractable. This report describes the results of wastewater surveillance for SARS-CoV-2 at residence halls on a university campus aimed at preventing outbreak escalation by mitigating community spread. Another goal was to estimate fecal shedding rates of SARS-CoV-2 in a non-clinical setting. Passive sampling devices were deployed in sewer laterals originating from residence halls at a frequency of twice weekly during fall 2021 as the Delta variant of concern continued to circulate across North America. A positive detection as part of routine sampling in late November 2021 triggered daily monitoring and further isolated the signal to a single wing of one residence hall. Detection of SARS-CoV-2 within the wastewater over a period of 3 consecutive days led to a coordinated rapid antigen testing campaign targeting the residence hall occupants and the identification and isolation of infected individuals. With knowledge of the number of individuals testing positive for COVID-19, fecal shedding rates were estimated to range from 3.70 log10 gc ‧ g feces-1 to 5.94 log10 gc ‧ g feces-1. These results reinforce the efficacy of wastewater surveillance as an early indicator of infection in congregate living settings. Detections can trigger public health measures ranging from enhanced communications to targeted coordinated testing and quarantine.

20-Month monitoring of SARS-CoV-2 in wastewater of Curitiba, in Southern Brazil.

The COVID-19 pandemic resulted in the collapse of healthcare systems and led to the development and application of several approaches of wastewater-based epidemiology to monitor infected populations. The main objective of this study was to carry out a SARS-CoV-2 wastewater based surveillance in Curitiba, Southern Brazil Sewage samples were collected weekly for 20 months at the entrance of five treatment plants representing the entire city and quantified by qPCR using the N1 marker. The viral loads were correlated with epidemiological data. The correlation by sampling points showed that the relationship between the viral loads and the number of reported cases was best described by a cross-correlation function, indicating a lag between 7 and 14 days amidst the variables, whereas the data for the entire city presented a higher correlation (0.84) with the number of positive tests at lag 0 (sampling day). The results also suggest that the Omicron VOC resulted in higher titers than the Delta VOC. Overall, our results showed that the approach used was robust as an early warning system, even with the use of different epidemiological indicators or changes in the virus variants in circulation. Therefore, it can contribute to public decision-makers and health interventions, especially in vulnerable and low-income regions with limited clinical testing capacity. Looking toward the future, this approach will contribute to a new look at environmental sanitation and should even induce an increase in sewage coverage rates in emerging countries.

Establishment of wastewater-based SARS-CoV-2 monitoring system over two years: Case studies in South Korea.

With the global COVID-19 pandemic, wastewater surveillance has received a considerable attention as a method for the early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater treatment plant (WWTP) and sewer systems. For the first time in Korea, this study utilized the wastewater surveillance technique to monitor the COVID-19 outbreak. Sampling efforts were carried out at the WWTPs in the capital city of Korea, Seoul, and Daegu the place where the first severe outbreak was reported. The RNA of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been extracted from the collected wastewater influent and primary sewage sludge samples. The outcomes were contrasted with the COVID-19 cases in the WWTPs served area. Additionally, whole transcriptome sequencing was used to compare the microbial community alterations before and after the COVID-19 outbreak and SARS-CoV-2 variations. The results demonstrated that the changes in SARS-CoV-2 RNA concentrations in the influent and sludge matched the trends of reported COVID-19 cases, especially sludge showed high-resolution data, which is well-matched when fewer COVID-19 cases (0-250) are reported. Interestingly, one month before the clinical report, we found that the SARS-CoV-2 Beta variant (South Africa, B.1.351) in the wastewater. In addition, the Aeromonas bacterial species was dominated (21.2%) among other bacterial species in wastewater after the COVID-19 outbreak, suggesting a potential indirect microbial indicator of the COVID-19 outbreak.

Monitoring influenza and respiratory syncytial virus in wastewater. Beyond COVID-19.

Wastewater-based surveillance can be a valuable tool to monitor viral circulation and serve as an early warning system. For respiratory viruses that share similar clinical symptoms, namely SARS-CoV-2, influenza, and respiratory syncytial virus (RSV), identification in wastewater may allow differentiation between seasonal outbreaks and COVID-19 peaks. In this study, to monitor these viruses as well as standard indicators of fecal contamination, a weekly sampling campaign was carried out for 15 months (from September 2021 to November 2022) in two wastewater treatment plants that serve the entire population of Barcelona (Spain). Samples were concentrated by the aluminum hydroxide adsorption-precipitation method and then analyzed by RNA extraction and RT-qPCR. All samples were positive for SARS-CoV-2, while the positivity rates for influenza virus and RSV were significantly lower (10.65 % for influenza A (IAV), 0.82 % for influenza B (IBV), 37.70 % for RSV-A and 34.43 % for RSV-B). Gene copy concentrations of SARS-CoV-2 were often approximately 1 to 2 logarithmic units higher compared to the other respiratory viruses. Clear peaks of IAV H3:N2 in February and March 2022 and RSV in winter 2021 were observed, which matched the chronological incidence of infections recorded in the Catalan Government clinical database. In conclusion, the data obtained from wastewater surveillance provided new information on the abundance of respiratory viruses in the Barcelona area and correlated favorably with clinical data.

COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR.

The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and requires a lot of resources. Sample pooling methods improve the efficiency of mass screening and consume less reagents by increasing the capacity of testing and reducing the number of experiments performed, and are therefore especially suitable for under-developed countries with limited resources. Here, we propose a simple, reliable pooling strategy for COVID-19 testing using clinical nasopharyngeal (NP) and/or oropharyngeal (OP) swabs. The strategy includes the pooling of 10 NP/OP swabs for extraction and subsequent testing via quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), and may also be applied to the screening of other pathogens.

SARS-CoV-2 RT-qPCR Ct values in saliva and nasopharyngeal swab samples for disease severity prediction.

Background: Comparison of clinical value of RT-qPCR-based SARS-CoV-2 tests performed on saliva samples (SSs) and nasopharyngeal swab samples (NPSs) for prediction of the COVID-19 disease severity. Methods: Three paired SSs and NPSs collected every 3 days from 100 hospitalised COVID-19 patients during 2020 Jul-2021 Jan were tested by RT-qPCR for the original SARS-CoV-2 virus and compared to 150 healthy controls. Cases were divided into mild+moderate (Cohort I, N = 47) and severe disease (Cohort II, N = 53) cohorts and compared. Results: SARS-CoV-2 was detected in 65% (91/140) vs. 53% (82/156) of NPSs and 49% (68/139) vs. 48% (75/157) of SSs collected from Cohort I and II, respectively, resulting in the total respective detection rates of 58% (173/296) vs. 48% (143/296) (P = 0.017). Ct values of SSs were lower than those of NPSs (mean Ct = 28.01 vs. 30.07, P = 0.002). Although Ct values of the first SSs were significantly lower in Cohort I than in Cohort II (P = 0.04), it became negative earlier (mean 11.7 vs. 14.8 days, P = 0.005). Multivariate Cox proportional hazards regression analysis showed that Ct value ≤30 from SSs was the independent predictor for severe COVID-19 (HR = 10.06, 95% CI: 1.84-55.14, P = 0.008). Conclusion: Salivary RT-qPCR testing is suitable for SARS-CoV-2 infection control, while simple measurement of Ct values can assist in prediction of COVID-19 severity.

Multiplex Assays Enable Simultaneous Detection and Identification of SARS-CoV-2 Variants of Concern in Clinical and Wastewater Samples

The targeted screening and sequencing approaches for COVID-19 surveillance need to be adjusted to fit the evolving surveillance objectives which necessarily change over time. We present the development of variant screening assays that can be applied to new targets in a timely manner and enable multiplexing of targets for efficient implementation in the laboratory. By targeting the HV69/70 deletion for Alpha, K417N for Beta, K417T for Gamma, and HV69/70 deletion plus K417N for sub-variants BA.1, BA.3, BA.4, and BA.5 of Omicron, we achieved simultaneous detection and differentiation of Alpha, Beta, Gamma, and Omicron in a single assay. Targeting both T478K and P681R mutations enabled specific detection of the Delta variant. The multiplex assays used in combination, targeting K417N and T478K, specifically detected the Omicron sub-variant BA.2. The limits of detection for the five variants of concern were 4-16 copies of the viral RNA per reaction. Both assays achieved 100% clinical sensitivity and 100% specificity. Analyses of 377 clinical samples and 24 wastewater samples revealed the Delta variant in 100 clinical samples (nasopharyngeal and throat swab) collected in November 2021. Omicron BA.1 was detected in 79 nasopharyngeal swab samples collected in January 2022. Alpha, Beta, and Gamma variants were detected in 24 wastewater samples collected in May-June 2021 from two major cities of Alberta (Canada), and the results were consistent with the clinical cases of multiple variants reported in the community. © 2023 The Authors. Published by American Chemical Society.

Comparing artificial intelligence based diagnosis with expert results in SARS-COV-2 RT-qPCR

Objectives: Reverse transcription and real-time polymerase chain reaction (RT-qPCR) based on the SARS-CoV-2 viral RNA demonstration is the gold standard in diagnosis. Data files obtained from PCR devices should be analysed by a specialist physician and results should be transferred to Laboratory Information Management System (LIMS). CAtenA Smart PCR (Ventura, Ankara, Turkiye) program is a local bioinformatics software that assess PCR data files with artificial intelligence, submits to expert approval and transfers the approved results to LIMS. The aim of this study is to investigate its accuracy and matching success rate with expert analysis. Method(s): A total of 9400 RT-qPCR test results studied in Ankara Provincial Health Directorate Public Health Molecular Diagnosis Laboratory were compared with respect to expert evaluation and CAtenA results. Result(s): It was determined that the preliminary evaluation results of the CAtenA matched 86% of the negative and 90% of the positive results provided by expert analysis. 987 tests which CAtenA determined as inconclusive and suggested repeating PCR were found either negative or positive by expert analysis. A significant difference between positive and negative matching success rates and artificial intelligence (AI) based software overall accuracy was found and associated with the missed tests of the AI. Conclusion(s): As a result, it was suggested there is a low risk of confirming false positive results without expert analysis and test repetitions would cause losing time along with extra test costs. It was agreed that the PCR analysis used in CAtenA should be improved particularly in terms of test repetitions.Copyright © 2023 by Prusa Medical Publishing.

Self-perception of Voice Handicap in COVID 19 Patients.

Voice abnormalities were reported in patients during the course of COVID-19 infection. This study aims to evaluate the effect of COVID 19 infection on the self-perception of voice handicap in positive COVID 19 patients in addition to investigating the factors that might correlate with voice handicap if present. Voice handicap index-10 was filled in by 200 patients that were confirmed to be COVID 19 positive based on the RT-qPCR and symptomatology of the disease. The result showed that about 65.5% had mild degree of COVID 19 and 27.5% had moderate degree. Dysphonia was reported by 19% of the patients when questioned about voice symptoms. Dysphonia was detected in 35% of them by auditory perceptual assessment. Symptoms of Dyspnea, dysphonia, headache were significantly correlated with total and subtotal scores of Voice handicap index. COVID 19 infection has a negative impact on some of the patients? self- perception of voice handicap on the functional, physical and emotional domains. Age and degree of COVID 19 severity were correlating with the patients? self -perception of voice handicap.

Comparison of diagnostic performance of RT-qPCR, RT-LAMP and IgM/IgG rapid tests for detection of SARS-CoV-2 among healthcare workers in Brazil.

BACKGROUND: COVID-19 has become a major public health problem after the outbreak caused by SARS-CoV-2 virus. Great efforts to contain COVID-19 transmission have been applied worldwide. In this context, accurate and fast diagnosis is essential. METHODS: In this prospective study, we evaluated the clinical performance of three different RNA-based molecular tests - RT-qPCR (Charité protocol), RT-qPCR (CDC (USA) protocol) and RT-LAMP - and one rapid test for detecting anti-SARS-CoV-2 IgM and IgG antibodies. RESULTS: Our results demonstrate that RT-qPCR using the CDC (USA) protocol is the most accurate diagnostic test among those evaluated, while oro-nasopharyngeal swabs are the most appropriate biological sample. RT-LAMP was the RNA-based molecular test with lowest sensitivity while the serological test presented the lowest sensitivity among all evaluated tests, indicating that the latter test is not a good predictor of disease in the first days after symptoms onset. Additionally, we observed higher viral load in individuals who reported more than 3 symptoms at the baseline. Nevertheless, viral load had not impacted the probability of testing positive for SARS-CoV-2. CONCLUSION: Our data indicates that RT-qPCR using the CDC (USA) protocol in oro-nasopharyngeal swabs samples should be the method of choice to diagnosis COVID-19.

Comparable detection of nasopharyngeal swabs and induced sputum specimens for viral nucleic acid detection of suspected novel coronavirus (SARS-Cov-2) patients in Fayoum governorate, Egypt.

Background: The most commonly utilized samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) are nasopharyngeal swabs (NPS) and oropharyngeal swabs. However, there are some drawbacks. For SARS-CoV-2 detection, induced sputum might be analyzed and may be equivalent to pharyngeal swabs. This study was done to assess the potential superiority of induced sputum over NPS for SARS-CoV-2 detection. Sixty symptomatic COVID-19 patients who attended Fayoum University Hospitals in Fayoum Governorate, Egypt, were included in this cross-sectional descriptive study. Paired NPS and induced sputum samples were collected from each subject on the third and tenth days after symptoms began for RT-qPCR SARS-COV2 diagnosis. Results: At day 3, 52 (86.7%) of NPS and 48 (80.00%) of induced sputum specimens had positive RT-qPCR results with a significant statistical difference (P = 0.001). At day 10, 41 induced sputum samples (68.3%) were negative, while 19 (31.7%) were positive. Only three (5.0%) of the 19 positive induced sputum samples tested positive for NPS. NPS samples had a higher viral load than induced sputum samples at day 3 [25 (41.7%) vs. 23 (38.3%)]. At day 10, induced sputum samples had a higher viral load than NPS [9 (15.0%) vs. 6 (10.0%)]. A statistically significant positive correlation between the viral load value of the NPS and the induced sputum sample at day 3 (r = 0.497, p = 0.00) denoting similarity in the results of the two types of samples. By ROC analysis, the highest area under the curve for the overall CT value of the induced sputum was (0.604), with a statistically significant difference (p value = 0.0418). Conclusion: In the early stages of the disease, induced sputum and NPS tests had comparable results, but NPS yielded more false negative results later in the disease course than an induced sputum sample, which yielded higher sample positivity and viral load than NPS. Furthermore, induced sputum collection is a straightforward, non-invasive, and risk-free method. As a result, induced sputum could be useful for COVID-19 confirmation in patients with radiologically or epidemiologically suspected COVID-19 who have a negative NPS or in difficult-to-diagnose COVID-19 patients.

Wastewater-Based Epidemiology of SARS-CoV-2: Assessing Prevalence and Correlation with Clinical Cases.

Wastewater-based epidemiology has been recognized as a tool to monitor the progress of COVID-19 pandemic worldwide. The study presented herein aimed at quantitating the SARS-CoV-2 RNA in the wastewaters, predicting the number of infected individuals in the catchment areas, and correlating it with the clinically reported COVID-19 cases. Wastewater samples (n = 162) from different treatment stages were collected from three wastewater treatment plants (WWTPs) from Mumbai city during the 2nd surge of COVID-19 (April 2021 to June 2021). SARS-CoV-2 causing COVID-19, was detected in 76.2% and 4.8% of raw and secondary treated (n = 63 each) wastewater samples respectively while all tertiary treated samples (n = 36) were negative. The quantity of SARS-CoV-2 RNA determined as gene copies/100 mL varied among all the three WWTPs under study. The gene copy numbers thus obtained were further used to estimate the number of infected individuals within the population served by these WWTPs using two published methods. A positive correlation (p < 0.05) was observed between the estimated number of infected individuals and clinically confirmed COVID-19 cases reported during the sampling period in two WWTPs. Predicted infected individuals calculated in this study were 100 times higher than the reported COVID-19 cases in all the WWTPs assessed. The study findings demonstrated that the present wastewater treatment technologies at the three WWTPs studied were adequate to remove the virus. However, SARS-CoV-2 genome surveillance with emphasis on monitoring its variants should be implemented as a routine practice to prepare for any future surge in infections.

Clinical evaluation of anterior nasal cavity swab specimens by a rapid antigen test using a GLINE-2019-nCoV Ag Kit to diagnose COVID-19.

The promising diagnostic performance of rapid antigen tests (RATs) using non-invasive anterior nasal (AN) swab specimens to diagnose COVID-19 has been reported. A large number of RATs are commercially available; however, the careful assessment of RATs is essential prior to their implementation in clinical practice. We evaluated the clinical performance of the GLINE-2019-nCoV Ag Kit as a RAT using AN swabs in a prospective, blinded study. Adult patients who visited outpatient departments and received SARS-CoV-2 tests between August 16 and September 8, 2022, were eligible for this study. Patients who were aged under 18 years and patients without appropriate specimens were excluded. Two sets of AN and nasopharyngeal (NP) swabs were collected from all patients. Each set of specimens was tested by the RAT and quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Of the 138 recruited patients, 84 were positive and 54 were negative by RT-qPCR using NP swabs. The positive agreement rate between RT-qPCR using NP swabs and RAT using AN swabs was 78.6% (95% confidence interval [CI], 68.3%-86.8%), the negative agreement rate was 98.1% (95% CI, 90.1%-99.9%), and the overall agreement rate was 86.2% (95% CI, 79.3%-91.5%), with a κ coefficient of 0.73. The positive agreement rate in the early phase (≤3 days from symptom onset) was >80%, but this fell to 50% in the late phase (≥4 days). This study demonstrates that the GLINE-2019-nCoV Ag Kit using AN swabs has good clinical performance and might be a reliable alternative method for diagnosing COVID-19.

Performance Evaluation of Different RT-PCR Kits for the Direct Detection of SARS-CoV-2 in Preheated Specimens

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created high demand for molecular kits and consumables for mass screening of suspected individuals. Direct real-time polymerase chain reaction (RT-PCR) assay without nucleic acid extraction has several advantages in saving testing time and cost and helps in the rapid reporting of SARS-CoV-2. The present study evaluated the analytical performance of four SARS-CoV-2 RT-PCR for direct RT-PCR testing using preheated specimens.Methods A total of 100 clinical specimens were selected and divided into three different groups: (1) group I: 20 SARS-CoV-2 positive specimens with high viral load, viz., low Ct values (< 30 Ct), (2) group II: 50 SARS-CoV-2 positive specimens with low viral load, viz., high Ct values (> 30 Ct), and (3) group III: 30 SARS-CoV-2 negative specimens. Specimens were heat-inactivated at 70 >= C for 10 minutes and cooled down at 4 >= C and were evaluated for standard and direct RT-PCR method by using ViralDtect-II Multiplex Real-Time PCR kit, TaqPath COVID-19 Combo kit, COVIDsure Pro Multiplex RT-PCR kit, and Hi-PCR Coronavirus (COVID-19) Multiplex Probe PCR kit.Results Results showed that except ViralDtect-II kit, the other three TaqPath COVID-19 Combo kit, COVIDsure Pro kit, and Hi-PCR Coronavirus (COVID-19) RT-PCR kit were able to amplify all the SARS-CoV-2 genes in the direct RT-PCR method using preheated specimens. In group I specimens, 100% sensitivity was observed in all three RT-PCR kits. In group II specimens, COVIDsure Pro kit was found to be superior among other kits.Conclusion Direct RT-PCR method during pandemic situation is valuable and cost effective for the detection of SARS-CoV-2. All three TaqPath COVID-19 Combo kit, COVIDsure Pro kit, and Hi-PCR Coronavirus (COVID-19) RT-PCR kit can be used for direct RT-PCR method and COVIDsure Pro kit performance was found to be superior among all.

Exploration on wastewater-based epidemiology of SARS-CoV-2: Mimic relative quantification with endogenous biomarkers as internal reference.

Wastewater-based epidemiology has become a powerful surveillance tool for monitoring the pandemic of COVID-19. Although it is promising to quantitatively correlate the SARS-CoV-2 RNA concentration in wastewater with the incidence of community infection, there is still no consensus on whether the viral nucleic acid concentration in sewage should be normalized against the abundance of endogenous biomarkers and which biomarker should be used as a reference for the normalization. Here, several candidate endogenous reference biomarkers for normalization of SARS-CoV-2 signal in municipal sewage were evaluated. The human fecal indicator virus (crAssphage) is a promising candidate of endogenous reference biomarker for data normalization of both DNA and RNA viruses for its intrinsic viral nature and high and stable content in sewage. Without constructing standard curves, the relative quantification of sewage viral nucleic acid against the abundance of the reference biomarker can be used to correlate with community COVID-19 incidence, which was proved via mimic experiments by spiking pseudovirus of different concentrations in sewage samples. Dilution of pseudovirus-seeded wastewater did not affect the relative abundance of viral nucleic acid, demonstrating that relative quantification can overcome the sewage dilution effects caused by the greywater input, precipitation and/or groundwater infiltration. The process of concentration, recovery and detection of the endogenous biomarker was consistent with that of SARS-CoV-2 RNA. Thus, it is necessary to co-quantify the endogenous biomarker because it can be not only an internal reference for data normalization, but also a process control.