Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

From Biowaste to Lab-Bench: Low-Cost Magnetic Iron Oxide Nanoparticles for RNA Extraction and SARS-CoV-2 Diagnostics.

The gold standard for diagnostics of SARS-CoV-2 (COVID-19) virus is based on real-time polymerase chain reaction (RT-PCR) using centralized PCR facilities and commercial viral RNA extraction kits. One of the key components of these kits are magnetic beads composed of silica coated magnetic iron oxide (Fe2O3 or Fe3O4) nanoparticles, needed for the selective extraction of RNA. At the beginning of the pandemic in 2019, due to a high demand across the world there were severe shortages of many reagents and consumables, including these magnetic beads required for testing for SARS-CoV-2. Laboratories needed to source these products elsewhere, preferably at a comparable or lower cost. Here, we describe the development of a simple, low-cost and scalable preparation of magnetic nanoparticles (MNPs) from biowaste and demonstrate their successful application in viral RNA extraction and the detection of COVID-19. These MNPs have a unique nanoplatelet shape with a high surface area, which are beneficial features, expected to provide improved RNA adsorption, better dispersion and processing ability compared with commercial spherical magnetic beads. Their performance in COVID-19 RNA extraction was evaluated in comparison with commercial magnetic beads and the results presented here showed comparable results for high throughput PCR analysis. The presented magnetic nanoplatelets generated from biomass waste are safe, low-cost, simple to produce in large scale and could provide a significantly reduced cost of nucleic acid extraction for SARS-CoV-2 and other DNA and RNA viruses.

SARS-CoV-2 molecular testing and whole genome sequencing following RNA recovery from used BinaxNOW COVID-19 antigen self tests.

Widespread use of over-the-counter rapid diagnostic tests for SARS-CoV-2 has led to a decrease in availability of clinical samples for viral genomic surveillance. As an alternative sample source, we evaluated RNA isolated from BinaxNOW swabs stored at ambient temperature for SARS-CoV-2 rRT-PCR and full viral genome sequencing. 81 of 103 samples (78.6%) yielded detectable RNA, and 46 of 57 samples (80.7 %) yielded complete genome sequences. Our results illustrate that SARS-CoV-2 RNA extracted from used Binax test swabs provides an important opportunity for improving SARS-CoV-2 genomic surveillance, evaluating transmission clusters, and monitoring within-patient evolution.

Trends in SARS-CoV-2 cycle threshold values in the Czech Republic from April 2020 to April 2022.

The inability to predict the evolution of the COVID-19 epidemic hampered abilities to respond to the crisis effectively. The cycle threshold (Ct) from the standard SARS-CoV-2 quantitative reverse transcription-PCR (RT-qPCR) clinical assay is inversely proportional to the amount of SARS-CoV-2 RNA in the sample. We were interested to see if population Ct values could predict future increases in COVID-19 cases as well as subgroups that would be more likely to be affected. This information would have been extremely helpful early in the COVID-19 epidemic. We therefore conducted a retrospective analysis of demographic data and Ct values from 2,076,887 nasopharyngeal swab RT-qPCR tests that were performed at a single diagnostic laboratory in the Czech Republic from April 2020 to April 2022 and from 221,671 tests that were performed as a part of a mandatory school surveillance testing program from March 2021 to March 2022. We found that Ct values could be helpful predictive tools in the real-time management of viral epidemics. First, early measurement of Ct values would have indicated the low viral load in children, equivalent viral load in males and females, and higher viral load in older individuals. Second, rising or falling median Ct values and differences in Ct distribution indicated changes in the transmission in the population. Third, monitoring Ct values and positivity rates would have provided early evidence as to whether prevention measures are effective. Health system authorities should thus consider collecting weekly median Ct values of positively tested samples from major diagnostic laboratories for regional epidemic surveillance.

SARS-CoV-2 viral RNA detection using the novel CoVradar device associated with the CoVreader smartphone app.

The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (∼3 h/test), cost (∼€1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.

Insights from a workplace SARS-CoV-2 specimen collection program, with genomes placed into global sequence phylogeny.

In 2020, the Department of Energy established the National Virtual Biotechnology Laboratory (NVBL) to address key challenges associated with COVID-19. As part of that effort, Pacific Northwest National Laboratory (PNNL) established a capability to collect and analyze specimens from employees who self-reported symptoms consistent with the disease. During the spring and fall of 2021, 688 specimens were screened for SARS-CoV-2, with 64 (9.3%) testing positive using reverse-transcriptase quantitative PCR (RT-qPCR). Of these, 36 samples were released for research. All 36 positive samples released for research were sequenced and genotyped. Here, the relationship between patient age and viral load as measured by Ct values was measured and determined to be only weakly significant. Consensus sequences for each sample were placed into a global phylogeny and transmission dynamics were investigated, revealing that the closest relative for many samples was from outside of Washington state, indicating mixing of viral pools within geographic regions.

Optimization and Improvement of qPCR Detection Sensitivity of SARS-CoV-2 in Saliva.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major public health threat globally, especially during the beginning of the pandemic in 2020. Reverse transcription-quantitative PCR (RT-qPCR) is utilized for viral RNA detection as part of control measures to limit the spread of COVID-19. Collecting nasopharyngeal swabs for RT-qPCR is a routine diagnostic method for COVID-19 in clinical settings, but its large-scale implementation is hindered by a shortage of trained health professionals. Despite concerns over its sensitivity, saliva has been suggested as a practical alternative sampling approach to the nasopharyngeal swab for viral RNA detection. In this study, we spiked saliva from healthy donors with inactivated SARS-CoV-2 from an international standard to evaluate the effect of saliva on viral RNA detection. On average, the saliva increased the cycle threshold (CT) values of the SARS-CoV-2 RNA samples by 2.64 compared to the viral RNA in viral transport medium. Despite substantial variation among different donors in the effect of saliva on RNA quantification, the outcome of the RT-qPCR diagnosis was largely unaffected for viral RNA samples with CT values of <35 (1.55 log10 IU/mL). The saliva-treated viral RNA remained stable for up to 6 h at room temperature and 24 h at 4°C. Further supplementing protease and RNase inhibitors improved the detection of viral RNA in the saliva samples. Our data provide practical information on the storage conditions of saliva samples and suggest optimized sampling procedures for SARS-CoV-2 diagnosis. IMPORTANCE The primary method for detection of SARS-CoV-2 is using nasopharyngeal swabs, but a shortage of trained health professionals has hindered its large-scale implementation. Saliva-based nucleic acid detection is a widely adopted alternative, due to its convenience and minimally invasive nature, but the detection limit and direct impact of saliva on viral RNA remain poorly understood. To address this gap in knowledge, we used a WHO international standard to evaluate the effect of saliva on SARS-CoV-2 RNA detection. We describe the detection profile of saliva-treated SARS-CoV-2 samples under different storage temperatures and incubation periods. We also found that adding protease and RNase inhibitors could improve viral RNA detection in saliva. Our research provides practical recommendations for the optimal storage conditions and sampling procedures for saliva-based testing, which can improve the efficiency of COVID-19 testing and enhance public health responses to the pandemic.

Evaluation of the AdvanSure One-Stop COVID-19 Plus Kit for SARS-CoV-2 Detection Using a Streamlined RNA Extraction Method.

Real-time reverse transcription (rRT)-PCR, which is the reference standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, generally involves a time-consuming and costly RNA extraction step prior to amplification. We evaluated the performance of the AdvanSure One-Stop COVID-19 Plus Kit (LG Chem, Seoul, Korea), a novel rRT-PCR assay that can detect SARS-CoV-2 within 90 minutes using a streamlined RNA extraction method. In total, 509 nasopharyngeal swab (NPS) specimens (SARS-CoV-2 positive: N=205; SARS-CoV-2 negative: N=304) previously tested using the PowerChek SARS-CoV-2 Real-time PCR Kit (Kogene Biotech, Seoul, Korea) were tested using the AdvanSure assay. The limit of detection (LOD) of the AdvanSure assay was determined using serially diluted inactivated SARS-CoV-2. The positive and negative percent agreements between the AdvanSure and PowerChek assays were 99.5% (204/205) and 99.3% (302/304), respectively. The LODs of the AdvanSure assay for SARS-CoV-2 nucleocapsid and spike/RNA-dependent RNA polymerase genes were 672 and 846 copies/mL, respectively. The results show that the performance of the AdvanSure assay is comparable to that of the PowerChek assay used for routine SARS-CoV-2 testing, suggesting that the AdvanSure assay is a useful diagnostic tool for rapid and accurate detection of SARS-CoV-2 infection.

Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus.

Since the outbreak of coronavirus 2019 (COVID-19), detection technologies have been attracting a great deal of attention in molecular diagnosis applications. In particular, the droplet digital PCR (ddPCR) has become a promising tool as it offers absolute quantification of target nucleic acids with high specificity and sensitivity. In recent years, the combination of the isothermal amplification strategies has made ddPCR a popular method for on-site testing by enabling amplification at a constant temperature. However, the current isothermal ddPCR assays are still challenging due to inherent non-specific amplification. In this paper, we present a multiplexed droplet digital recombinase polymerase amplification (MddRPA) with precise initiation of the reaction. First, the reaction temperature and dynamic range of reverse transcription (RT) and RPA were characterized by real-time monitoring of fluorescence intensities. Using a droplet-based microfluidic chip, the master mix and the initiator were fractionated and rapidly mixed within well-confined droplets. Due to the high heat transfer and mass transfer of the droplets, the precise initiation of the amplification was enabled and the entire assay could be conducted within 30 min. The concentrations of target RNA in the range from 5 copies per µL to 2500 copies per µL could be detected with high linearity (R2 > 0.999). Furthermore, the multiplexed detection of three types of human coronaviruses was successfully demonstrated with high specificity (>96%). Finally, we compared the performance of the assay with a commercial RT-qPCR system using COVID-19 clinical samples. The MddRPA assay showed a 100% concordance with the RT-qPCR results, indicating its reliability and accuracy in detecting SARS-CoV-2 nucleic acids in clinical samples. Therefore, our MddRPA assay with rapid detection, precise quantification, and multiplexing capability would be an interesting method for molecular diagnosis of viral infections.

Rapid and Accurate Detection of SARS-CoV-2 Using an iPad-Controlled, High-Throughput, Portable, and Multiplex Hive-Chip Platform (HiCube).

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the most effective measures to control the coronavirus disease 2019 (COVID-19) pandemic. However, there is still lack of an ideal detection platform capable of high sample throughput, portability, and multiplicity. Herein, by combining Hive-Chip (capillary microarray) and reverse transcriptional loop-mediated isothermal amplification (RT-LAMP), we developed an iPad-controlled, high-throughput (48 samples at one run), portable (smaller than a backpack), multiplex (monitoring 8 gene fragments in one reaction), and real-time detection platform for SARS-CoV-2 detection. This platform is composed of a portable Hive-Chip device (HiCube; 32.7 × 29.7 × 20 cm, 5 kg), custom-designed software, and optimized Hive-Chips. RT-LAMP primers targeting seven SARS-CoV-2 genes (S, E, M, N, ORF1ab, ORF3a, and ORF7a) and one positive control (human RNase P) were designed and prefixed in the Hive-Chip. On-chip RT-LAMP showed that the limit of detection (LOD) of SARS-CoV-2 synthetic RNAs is 1 copy/µL, and there is no cross-reaction among different target genes. The platform was validated by 100 clinical samples of SARS-CoV-2, and the results were highly consistent with those of the traditional real-time PCR assay. In addition, on-chip detection of 6 other respiratory pathogens showed no cross-reactivity. Overall, our platform has great potential for fast, accurate, and on-site detection of SARS-CoV-2.

A Portable RT-LAMP/CRISPR Machine for Rapid COVID-19 Screening.

The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf® PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary.

A triple-target reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid and accurate detection of SARS-CoV-2 virus.

The spreading of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across the world has impacted people's health and lives worldwide in recent years. Rapid and accurate diagnosis is crucial for curbing the pandemic of coronavirus disease 2019 (COVID-19). Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has great potential for SARS-CoV-2 detection but fails to completely replace conventional PCR due to the high false-positive rate (FPR). We proposed a triple-target RT-LAMP method for dual-signal, sensitive, and simultaneous detection of conserved genes of SARS-CoV-2. Multiple LAMP primer sets were designed for N, E, and M genes and their amplification efficacy were screened. Then, using artificial plasmids and RNA, the optimal primer set for each gene was examined on specificity, sensitivity, and detection range. The RT-LAMP initiated by these primer sets exhibited better specificity and sensitivity than that of RT-qPCR, and the triple-target RT-LAMP could determine different variants of SARS-CoV-2. By testing 78 artificial RNA samples, the total FPR of triple-target RT-LAMP was eliminated compared with that of mono-target RT-LAMP. The triple-target RT-LAMP method precisely identified throat swab specimens through colorimetry and fluorescent signals within 60 min, and the limit of detection (LOD) was as low as 187 copies/reaction. In the future, the triple-target RT-LAMP can be applied to in-field and on-site diagnosis of symptomatic and asymptomatic virus carriers.

COVID-19 detection using AIE-active iridium complexes.

The highly contagious COVID-19, caused by the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed using reverse transcription polymerase chain reaction (RT-PCR). However, despite being highly sensitive, RT-PCR is also time consuming and quite complex, which limits its use for point-of-care (POC) testing. We have developed a simple single-step fluorescence assay for SARS-CoV-2 RNA detection based on the principle of aggregation-induced emission (AIE) using iridium complexes. Our smartly designed iridium probes fluorescently "turn-on" in the presence of SARS-CoV-2 RNA and give specific results at room temperature within 10 min. The lower limit of detection (LOD) is 1.84 genome copies per reaction, and the sensitivity and specificity of the assay in 20 clinical samples are found to be 90% and 80%, respectively.

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.

A loop-mediated isothermal amplification-enabled analytical assay for the detection of SARS-CoV-2: A review.

The number of words: 4645, the number of figures: 4, the number of tables: 1The outbreak of COVID-19 in December 2019 caused a global pandemic of acute respiratory disease, and with the increasing virulence of mutant strains and the number of confirmed cases, this has resulted in a tremendous threat to global public health. Therefore, an accurate diagnosis of COVID-19 is urgently needed for rapid control of SARS-CoV-2 transmission. As a new molecular biology technology, loop-mediated isothermal amplification (LAMP) has the advantages of convenient operation, speed, low cost and high sensitivity and specificity. In the past two years, rampant COVID-19 and the continuous variation in the virus strains have demanded higher requirements for the rapid detection of pathogens. Compared with conventional RT-PCR and real-time RT-PCR methods, genotyping RT-LAMP method and LAMP plus peptide nucleic acid (PNA) probe detection methods have been developed to correctly identified SARS-CoV-2 variants, which is also why LAMP technology has attracted much attention. LAMP detection technology combined with lateral flow assay, microfluidic technology and other sensing technologies can effectively enhance signals by nucleic acid amplification and help to give the resulting output in a faster, more convenient and user-friendly way. At present, LAMP plays an important role in the detection of SARS-CoV-2.

A simplified viral RNA extraction method based on magnetic nanoparticles for fast and high-throughput detection of SARS-CoV-2.

The ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based methods are being widely applied for clinical diagnosis, timely and accurate diagnosis towards COVID-19 causing virus, the SARS-CoV-2, is still limited due to labor-intensive and time-consuming operations. Herein, we report a new viral RNA extraction method based on poly-(amino ester) with carboxyl group (PC)-coated magnetic nanoparticles (pcMNPs) for the sensitive detection of SARS-CoV-2. This method combines the lysis and binding steps into one step, and refines multiple washing steps into one step, giving a turnaround time of less than 9 min. Furthermore, the extracted pcMNP-RNA complexes can be directly introduced into subsequent RT-PCR reactions without elution. This simplified viral RNA method could be well adapted in fast manual and automated high-throughput nucleic acids extraction protocols suitable for different scenarios. A high sensitivity down to 100 copies/mL and a linear correlation between 100 and 106 copies/mL of SARS-CoV-2 pseudovirus particles are achieved in both protocols. Benefitting from the simplicity and excellent performances, this new method can dramatically improve the efficiency and reduce operational requirements for the early clinical diagnosis and large-scale SARS-CoV-2 nucleic acid screening.

Comparative evaluation of saliva and nasopharyngeal swab for SARS-CoV-2 detection using RT-qPCR among COVID-19 suspected patients at Jigjiga, Eastern Ethiopia.

BACKGROUND: Nasopharyngeal swab (NPS) remains the recommended sample type for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) diagnosis. However, the collection procedure causes discomfort and irritation to the patients, lowering the quality of the sample and exposing healthcare workers to risk. Furthermore, there is also a shortage of flocked swabs and personnel protective equipment in low-income settings. Therefore, this necessitates an alternative diagnostic specimen. The purpose of this study was to evaluate the performance of saliva against NPS for SARS-CoV-2 detection using RT-qPCR among COVID-19 suspected patients at Jigjiga, Eastern Ethiopia. METHODS: Comparative cross-sectional study was conducted from June 28 to July 30, 2022. A total of 227 paired saliva and NPS samples were collected from 227 COVID-19 suspected patients. Saliva and NPS samples were collected and transported to the Somali Regional Molecular Laboratory. Extraction was conducted using DaAn kit (DaAn Gene Co., Ltd China). Veri-Q RT-qPCR was used for amplification and detection (Mico BioMed Co, Ltd, Republic of Korea). The data were entered into Epi-data version 4.6 and analyzed using SPSS 25. McNemar's test was used to compare the detection rate. Agreement between NPS and saliva was performed using Cohen's Kappa. The mean and median of cycle threshold values were compared using paired t-tests and the correlation between cycle threshold values was measured using Pearson correlation coefficient. P value < 0.05 was considered statistically significant. RESULTS: The overall positivity rate of SARS-CoV-2 RNA was 22.5% (95% CI 17-28%). Saliva showed higher sensitivity (83.8%, 95% CI, 73-94.5%) than NPS (68.9%, 95% CI 60.8-76.8%). The specificity of saliva was 92.6% (95% CI, 80.6% - 100%) compared to NPS (96.7%, 95% CI, 87% - 100%). The positive, negative, and overall percent agreement between NPS and saliva was 83.8%, 92.6%, and 91.2% respectively (κ = 0.703, 95% CI 0.58-0.825, P = 0.00). The concordance rate between the two samples was 60.8%. NPS showed a higher viral load than saliva. There was low positive correlation between the cycle threshold values of the two samples (r = 0.41, 95% CI -1.69 to -0.98, P >0.05). CONCLUSION: Saliva showed a higher detection rate for SARS-CoV-2 molecular diagnosis than NPS and there was significant agreement between the two specimens. Therefore, saliva could be suitable and easily obtainable alternative diagnostic specimen for SARS-CoV-2 molecular diagnosis.

Wastewater concentrations of human influenza, metapneumovirus, parainfluenza, respiratory syncytial virus, rhinovirus, and seasonal coronavirus nucleic-acids during the COVID-19 pandemic: a surveillance study.

BACKGROUND: Respiratory disease is a major cause of morbidity and mortality; however, surveillance for circulating respiratory viruses is passive and biased. Wastewater-based epidemiology has been used to understand SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV) infection rates at a community level but has not been used to investigate other respiratory viruses. We aimed to use wastewater-based epidemiology to understand community viral respiratory infection occurrence. METHODS: A retrospective wastewater-based epidemiology surveillance study was carried out at a large wastewater treatment plant located in California, USA. Using droplet digital RT-PCR, we measured RNA concentrations of influenza A and influenza B viruses, RSV A and RSV B, parainfluenza (1-4) viruses, rhinovirus, seasonal coronaviruses, and metapneumovirus in wastewater solids three times per week for 17 months (216 samples) between Feb 1, 2021, and June 21, 2022. Novel probe-based RT-PCR assays for non-influenza viral targets were developed and validated. We compared viral RNA concentrations to positivity rates for viral infections from clinical specimens submitted to California Sentinel Clinical Laboratories (sentinel laboratories) to assess concordance between the two datasets. FINDINGS: We detected RNA from all tested viruses in wastewater solids. Human rhinovirus (median concentration 4300 [0-9500] copies per gram dry weight) and seasonal human coronaviruses (35 000 [17 000-56 000]) were found at the highest concentrations. Concentrations of viral RNA correlated significantly and positively with positivity rates of associated viral diseases from sentinel laboratories (tau 0·32-0·57, p<0·0009); the only exceptions were influenza B and RSV A, which were rarely detected in wastewater solids. Measurements from wastewater indicated coronavirus OC43 dominated the seasonal human coronavirus infections whereas parainfluenza 3 dominated among parainfluenza infections during the study period. Concentrations of all tested viral RNA decreased noticeably after the omicron BA.1 surge suggesting a connection between changes in human behaviour during the surge and transmission of all respiratory viruses. INTERPRETATION: Wastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a localised, community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community. Results from wastewater can be available within 24 h of sample collection, generating real time information to inform public health responses, clinical decision making, and individual behaviour modifications. FUNDING: CDC Foundation.

A simple algorithm based on initial Ct values predicts the duration to SARS-CoV-2 negativity and allows more efficient test-to-release and return-to-work schedules.

Especially during global pandemics but also in the context of epidemic waves, the capacity for diagnostic quantitative reverse transcription-polymerase chain reactions (qRT-PCRs) rapidly becomes a limiting factor. The aim of the study was to optimize retesting regimens for test-to-release from isolation and return-to-work applications. For this purpose, we investigated the association between Ct values at the first diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the period until test negativity was reached, or at least until the Ct value exceeded 30, which is considered to indicate the transition to a non-infectious state. We included results from the testing of respiratory material samples for the detection of SARS-CoV-2 RNA, tested from March 1, 2020 to January 31, 2022. Lower initial Ct values were associated with longer periods of SARS-CoV-2 RNA positivity. Starting with Ct values of <20, 20 to 24.99, 25 to 29.99, 30 to 34.99, and ≥35, it took median intervals of 20 (interval: 14-25), 16 (interval: 10-21), 12 (interval: 7-16), 7 (interval: 5-14), and 5 (interval: 2-7) days, respectively, until the person tested negative. Accordingly, a Ct threshold of 30 was surpassed after 13 (interval: 8-19), 9 (interval: 6-14), 7 (interval: 6-11), 6 (interval: 4-10), and 3 (interval: 1-6) days, respectively, in individuals with aforementioned start Ct values. Furthermore, the time to negativity was longer for adults versus children, wild-type SARS-CoV-2 variant versus other variants of concern, and in patients who were treated in the intensive care units. Based on these data, we propose an adjusted retesting strategy according to the initial Ct value in order to optimize available PCR resources.