Strategies for Increasing the Throughput of Genetic Screening: Lessons Learned from the COVID-19 Pandemic within a University Community.

Amidst the COVID-19 pandemic, the Polytechnic University of Setúbal (IPS) used its expertise in molecular genetics to establish a COVID-19 laboratory, addressing the demand for community-wide testing. Following standard protocols, the IPS COVID Lab received national accreditation in October 2020 and was registered in February 2021. With the emergence of new SARS-CoV-2 variants and safety concerns for students and staff, the lab was further challenged to develop rapid and sensitive diagnostic technologies. Methodologies such as sample-pooling extraction and multiplex protocols were developed to enhance testing efficiency without compromising accuracy. Through Real-Time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) analysis, the effectiveness of sample pooling was validated, proving to be a clear success in COVID-19 screening. Regarding multiplex analysis, the IPS COVID Lab developed an in-house protocol, achieving a sensitivity comparable to that of standard methods while reducing operational time and reagent consumption. This approach, requiring only two wells of a PCR plate (instead of three for samples), presents a more efficient alternative for future testing scenarios, increasing its throughput and testing capacity while upholding accuracy standards. The lessons learned during the SARS-CoV-2 pandemic provide added value for future pandemic situations.

A hybrid demultiplexing strategy that improves performance and robustness of cell hashing.

Cell hashing, a nucleotide barcode-based method that allows users to pool multiple samples and demultiplex in downstream analysis, has gained widespread popularity in single-cell sequencing due to its compatibility, simplicity, and cost-effectiveness. Despite these advantages, the performance of this method remains unsatisfactory under certain circumstances, especially in experiments that have imbalanced sample sizes or use many hashtag antibodies. Here, we introduce a hybrid demultiplexing strategy that increases accuracy and cell recovery in multi-sample single-cell experiments. This approach correlates the results of cell hashing and genetic variant clustering, enabling precise and efficient cell identity determination without additional experimental costs or efforts. In addition, we developed HTOreader, a demultiplexing tool for cell hashing that improves the accuracy of cut-off calling by avoiding the dominance of negative signals in experiments with many hashtags or imbalanced sample sizes. When compared to existing methods using real-world datasets, this hybrid approach and HTOreader consistently generate reliable results with increased accuracy and cell recovery.

Multi-well plate lid for single-step pooling of 96 samples for high-throughput barcode-based sequencing.

High-throughput transcriptomics is of increasing fundamental biological and clinical interest. The generation of molecular data from large collections of samples, such as biobanks and drug libraries, is boosting the development of new biomarkers and treatments. Focusing on gene expression, the transcriptomic market exploits the benefits of next-generation sequencing (NGS), leveraging RNA sequencing (RNA-seq) as standard for measuring genome-wide gene expression in biological samples. The cumbersome sample preparation, including RNA extraction, conversion to cDNA and amplification, prevents high-throughput translation of RNA-seq technologies. Bulk RNA barcoding and sequencing (BRB-seq) addresses this limitation by enabling sample preparation in multi-well plate format. Sample multiplexing combined with early pooling into a single tube reduces reagents consumption and manual steps. Enabling simultaneous pooling of all samples from the multi-well plate into one tube, our technology relies on smart labware: a pooling lid comprising fluidic features and small pins to transport the liquid, adapted to standard 96-well plates. Operated with standard fluidic tubes and pump, the system enables over 90% recovery of liquid in a single step in less than a minute. Large scale manufacturing of the lid is demonstrated with the transition from a milled polycarbonate/steel prototype into an injection molded polystyrene lid. The pooling lid demonstrated its value in supporting high-throughput barcode-based sequencing by pooling 96 different DNA barcodes directly from a standard 96-well plate, followed by processing within the single sample pool. This new pooling technology shows great potential to address medium throughput needs in the BRB-seq workflow, thereby addressing the challenge of large-scale and cost-efficient sample preparation for RNA-seq.

Sampling technique to pool genetic materials of microorganism communities in blue-swimming crab processing plant industry.

The crab and seafood processing industry must fulfill standard requirements for sanitation, hygiene, and good manufacturing methods to ensure the safety of the products and free from foodborne bacteria. However, equipment and processing unit surfaces are challenging to clean optimally, which can cause persistent bacteria to emerge. Eliminating persistent bacteria is the latest challenge in the fish processing industry for optimal disinfection, preventing cross-contamination, and controlling foodborne outbreaks. Microbiological testing in industry has been limited to selective culture-media techniques; thus, a rapid, sensitive, accurate, and routine applicable analytical method is urgently needed. The significant reduction in the costs of high-throughput sequencing technologies supports the possibility of routine applications in the industry. This study aimed to determine the profile of the microbial community on the surface of the production room and blue-swimming crab processing unit equipment using short-read metagenomic techniques. The analysis included the stages of sampling, bacterial incubation, bacterial DNA isolation, sequencing, and bioinformatics analysis. The first important step to increase the possibility of routine adoption in the seafood industry is to reduce the cost, complexity, and time required to complete the analysis. Therefore, in this protocol, we generate a scalable, flexible, cost-effective, and auditable workflow.•Collection of bacterial samples by swabbing the surface of the equipment using a sterile cotton swab and sterile cloth, which is easy to apply and follow in the blue-swimming crab processing plant industry.•Effective and efficient sample-pooling is an important step in identifying bacterial communities by metagenomic analysis.

ADSP: An adaptive sample pooling strategy for diagnostic testing.

BACKGROUND: We often must conduct diagnostic tests on a massive volume of samples within a limited time during outbreaks of infectious diseases (e.g., COVID-19,screening) or repeat many times routinely (e.g., regular and massive screening for plant virus infections in farms). These tests aim to obtain the diagnostic result of all samples within a limited time. In such scenarios, the limitation of testing resources and human labor drives the need to pool individual samples and test them together to improve testing efficiency. When a pool is positive, further testing is required to identify the affected individuals; whereas when a pool is negative, we conclude all individuals in the pool are negative. How one splits the samples into pools is a critical factor affecting testing efficiency. OBJECTIVE: We aim to find the optimal strategy that adaptively guides users on optimally splitting the sample cohort into test-pools. METHODS: We developed an algorithm that minimizes the expected number of tests needed to obtain the diagnostic results of all samples. Our algorithm dynamically updates the critical information according to the result of the most recent test and calculates the optimal pool size for the next test. We implemented our novel adaptive sample pooling strategy into a web-based application, ADSP (https://ADSP.uvic.ca). ADSP interactively guides users on how many samples to be pooled for the current test, asks users to report the test result back and uses it to update the best strategy on how many samples to be pooled for the next test. RESULTS: We compared ADSP with other popular pooling methods in simulation studies, and found that ADSP requires fewer tests to diagnose a cohort and is more robust to the inaccurate initial estimate of the test cohort's disease prevalence. CONCLUSION: Our web-based application can help researchers decide how to pool their samples for grouped diagnostic tests. It improves test efficiency when grouped tests are conducted.

scSNPdemux: a sensitive demultiplexing pipeline using single nucleotide polymorphisms for improved pooled single-cell RNA sequencing analysis.

BACKGROUND: Here we present scSNPdemux, a sample demultiplexing pipeline for single-cell RNA sequencing data using natural genetic variations in humans. The pipeline requires alignment files from Cell Ranger (10× Genomics), a population SNP database and genotyped single nucleotide polymorphisms (SNPs) per sample. The tool works on sparse genotyping data in VCF format for sample identification. RESULTS: The pipeline was tested on both single-cell and single-nuclei based RNA sequencing datasets and showed superior demultiplexing performance over the lipid-based CellPlex and Multi-seq sample multiplexing technique which incurs additional single cell library preparation steps. Specifically, our pipeline demonstrated superior sensitivity and specificity in cell-identity assignment over CellPlex, especially on immune cell types with low RNA content. CONCLUSIONS: We designed a streamlined pipeline for single-cell sample demultiplexing, aiming to overcome common problems in multiplexing samples using single cell libraries which might affect data quality and can be costly.

High-throughput screening of LogD by using a sample pooling approach based on the traditional shake flask method.

A new approach for screening LogD is presented. The method is based on the shake flask method combined with rapid generic LC-MS/MS bioanalysis by using a sample pooling approach that enables high-throughput screening of LogD or LogP in the drug discovery stage. The method is evaluated by a comparison of measured LogD between single and pooled compounds for a test set of structurally diverse compounds with a wide range of LogD values (from -0.04 to 6.01). Test compounds include 10 commercially available drug standards along with 27 new chemical entities. A good correlation (RMSE = 0.21, R2 = 0.9879) of LogD between the single and pooled compounds was obtained, suggesting that at least 37 compounds can be simultaneously measured with acceptable accuracy. The sample pooling method significantly reduced the number of bioanalysis samples as compared to the single compound measurement by the conventional shake flask method. The impact of DMSO content on LogD measurement was also investigated and the result demonstrated that at least 0.5% DMSO was tolerated in this method. The current new development will facilitate the drug discovery process by more rapidly assessing the LogD or LogP of drug candidates.

Evaluation of pooling strategy of SARS-CoV-2 RT-PCR in limited resources setting in Egypt at low prevalence.

Sample pooling testing for SARS-COV-2 can be an effective tool in COVID-19 screening when resources are limited, yet it is important to assess the performance before implementation as pooling has its limitations. Our objective was to assess the efficacy of pooling samples for coronavirus 2019 (COVID-19) compared to an individual analysis by using commercial platforms for nucleic acid testing. A total of 2200 nasopharyngeal swabs for SARS-COV-2 were tested individually and in pools of 4, 8, and 10. The cycle threshold (Ct) values of the positive pooled samples were compared to their corresponding individual positive samples. In pool size 10 samples, an estimated increase of 3-Ct was obtained, which led to false negative results in low viral load positive samples. Pooling SARS COV-2 samples is an effective strategy of screening to increase laboratories' capacity and reduce costs without affecting diagnostic performance. A pool size of 8 is recommended.

Performance and Cost-effectiveness of Pooling COVID-19 Samples Using Real time Polymerase Chain Reaction Test.

BACKGROUND: Rapid detection of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) by real-time polymerase chain reaction (RT-PCR) is the most reliable method used worldwide. Although the incidence of the disease has increased globally, the limited availability of PCR kits has become the major bottleneck for the diagnosis of COVID positive patients. METHODS: Random samples were pooled for two months in group of two-five and tested for SARS-CoV-2. If the pool was negative, all individuals in the pool were reported negative. If the pool was positive, then the individual samples were retested to identify the positive individual. RESULTS: The mean cycle threshold (Ct) value of pooled samples was not significantly different with that of individual samples for N, ORF-1ab and E genes. Also, pooling saved more than 60% of reagents, time and effort, workforce and cost. CONCLUSIONS: In this study, the positivity rate was around 5% and saving of reagent, cost, time and manpower was more than 60%.

Rapid SARS-CoV-2 Virus Enrichment and RNA Extraction for Efficient Diagnostic Screening of Pooled Nasopharyngeal or Saliva Samples for Dilutions Up to 1:100.

As COVID-19 transmission control measures are gradually being lifted, a sensitive and rapid diagnostic method for large-scale screening could prove essential for monitoring population infection rates. However, many rapid workflows for SARS-CoV-2 detection and diagnosis are not amenable to the analysis of large-volume samples. Previously, our group demonstrated a technique for SARS-CoV-2 nanoparticle-facilitated enrichment and enzymatic lysis from clinical samples in under 10 min. Here, this sample preparation strategy was applied to pooled samples originating from nasopharyngeal (NP) swabs eluted in viral transport medium (VTM) and saliva samples diluted up to 1:100. This preparation method was coupled with conventional RT-PCR on gold-standard instrumentation for proof-of-concept. Additionally, real-time PCR analysis was conducted using an in-house, ultra-rapid real-time microfluidic instrument paired with an experimentally optimized rapid protocol. Following pooling and extraction from clinical samples, average cycle threshold (CT) values from resultant eluates generally increased as the pooling dilution factor increased; further, results from a double-blind study demonstrated 100% concordance with clinical values. In addition, preliminary data obtained from amplification of eluates prepared by this technique and analyzed using our portable, ultra-rapid real-time microfluidic PCR amplification instrument showed progress toward a streamlined method for rapid SARS-CoV-2 analysis from pooled samples.

Evaluation of the influenza and respiratory syncytial virus (RSV) targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay: sample pooling increases testing throughput.

During the COVID-19 pandemic, sample pooling has proven an effective strategy to overcome the limitations of reagent shortages and expand laboratory testing capacity. The inclusion of influenza and respiratory syncytial virus (RSV) in a multiplex tandem PCR platform with SARS-CoV-2 provides useful diagnostic and infection control information. This study aimed to evaluate the performance of the influenza and RSV targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay, including the effect of pooling samples on target detection. RSV target detection in clinical samples was compared to the Cepheid Xpert Xpress Flu/RSV assay as a reference standard. Samples were then tested in pools of four and detection rates were compared. Owing to the unavailability of clinical samples for influenza, only the effect of sample pooling on simulated samples was evaluated for these targets. RSV was detected in neat clinical samples with a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.5% compared to the reference standard, demonstrating 99.7% agreement. This study demonstrates that sample pooling by four increases the average Ct value by 2.24, 2.29, 2.20 and 1.91 cycles for the target's influenza A, influenza A typing, influenza B and RSV, respectively. The commercial AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay was able to detect influenza and RSV at an intermediate concentration within the limit of detection of the assay. Further studies to explore the applicability of sample pooling at the lower limit of detection of the assay is needed. Nevertheless, sample pooling has shown to be a viable strategy to increase testing throughput and reduce reagent usage. In addition, the multiplexed platform targeting various respiratory viruses assists with public health and infection control responses, clinical care, and patient management.

Simple Two-step, High Yield Protocol for Isolation and Amplification of Bacteriophages Against Methicillin-resistant Staphylococcus Aureus (MRSA).

Bacteriophages are bacteria-targeting viruses that may prove useful as therapeutic agents against multidrug-resistant bacterial strains. Though phage therapy is a century-old concept, there is very limited progress on its therapeutic application due to the rapid expansion of antibiotics portfolios in the last few decades. However, the emergence of multidrug-resistant organisms has brought our attention back to bacteriophages. The first step towards developing effective phage therapy against multidrug-resistant bacteria is isolation, amplification, and purification of specific bacteriophages. There are many reported protocols for isolating host-specific bacteriophages from the environment. However, most of them are complex, multistep, low-yielding, resource-intensive protocols, requiring elaborate laboratory setup. We have demonstrated a simple two-step, high-yielding protocol for isolating and amplifying bacteriophages against methicillin-resistant Staphylococcus aureus (MRSA). We have shown that mixing various environmental samples (i.e., sample pooling) and phage amplification at two different temperatures significantly enhance the yield of MRSA phages. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of water sample filtrate for isolation of bacteriophages Basic Protocol 2: Bacterial strain and culture conditions Basic Protocol 3: Native bacteriophage count in water sample filtrate Basic Protocol 4: Isolation and enrichment of MRSA-specific bacteriophages Basic Protocol 5: Quantification of bacteriophages by drop cast method Basic Protocol 6: Effect of incubation temperature and heat shock on bacteriophage yield.

Implementation of the compulsory universal testing scheme in Hong Kong: Mathematical simulations of a household-based pooling approach.

This study aims to propose a pooling approach to simulate the compulsory universal RT-PCR test in Hong Kong and explore the feasibility of implementing the pooling method on a household basis. The mathematical model is initially verified, and then the simulation is performed under different prevalence rates and pooled sizes. The simulated population is based in Hong Kong. The simulation included 10,000,000 swab samples, with a representative distribution of populations in Hong Kong. The samples were grouped into a batch size of 20. If the entire batch is positive, then the group is further divided into an identical group size of 10 for re-testing. Different combinations of mini-group sizes were also investigated. The proposed pooling method was extended to a household basis. A representative from each household is required to perform the RT-PCR test. Results of the simulation replications, indicate a significant reduction (p < 0.001) of 83.62, 64.18, and 48.46% in the testing volume for prevalence rate 1, 3, and 5%, respectively. Combined with the household-based pooling approach, the total number of RT-PCR is 437,304, 956,133, and 1,375,795 for prevalence rates 1, 3, and 5%, respectively. The household-based pooling strategy showed efficiency when the prevalence rates in the population were low. This pooling strategy can rapidly screen people in high-risk groups for COVID-19 infections and quarantine those who test positive, even when time and resources for testing are limited.

Implementation of Sample Pooling Procedure Using a Rapid SARS-CoV-2 Diagnostic Real-Time PCR Test Performed Prior to Hospital Admission of People with Intellectual Disabilities.

Reliability, accuracy, and timeliness of diagnostic testing for SARS-CoV-2 infection have allowed adequate public health management of the disease, thus notably helping the timely mapping of viral spread within the community. Furthermore, the most vulnerable populations, such as people with intellectual disability and dementia, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions, lower health maintenance, and a propensity for rapid community spread. This led to an urgent need for reliable in-house rapid testing to be performed prior to hospital admission. In the present study, we describe a pooling procedure in which oropharyngeal and nasopharyngeal swabs for SARS-CoV-2 detection (performed prior to hospital admission using rapid RT-PCR assay) are pooled together at the time of sample collection. Sample pooling (groups of 2-4 samples per tube) allowed us to significantly reduce response times, consumables, and personnel costs while maintaining the same test sensitivity.

Drug metabolic stability in early drug discovery to develop potential lead compounds.

Knowledge of the metabolic stability of a new drug substance eliminated by biotransformation is essential for envisaging the pharmacokinetic parameters required for deciding drug dosing and frequency. Strategies aimed at modifying lead compounds may improve metabolic stability, thereby reducing the drug dosing frequency. Replacement of selective hydrogens with deuterium can effectively enhance the drug's metabolic stability by increasing the biological half-life. Further, cyclization, change in ring size, and chirality can substantially improve the metabolic stability of drugs. The microsomal t1/2 approach for measuring drug in vitro intrinsic clearance by automated LC-MS/MS offers sensitive high-throughput screens with reliable data. The obtained in vitro intrinsic clearance from metabolic stability data helps predict the drug's in vivo total clearance using different scaling factors and hepatic clearance models. This review summarizes all the recent approaches and technological advancements in metabolic stability studies for narrowing down the potential lead compounds in drug discovery. Further, we summarized the potential pitfalls and assumptions made during the in vivo intrinsic clearance estimation from in vitro intrinsic clearance.

Sample-Pooling Strategy for SARS-CoV-2 Detection among Students and Staff of the University of Sannio.

Since the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, it has been clear that testing large groups of the population was the key to stem infection and prevent the effects of the coronavirus disease of 2019, mostly among sensitive patients. On the other hand, time and cost-sustainability of virus detection by molecular analysis such as reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) may be a major issue if testing is extended to large communities, mainly asymptomatic large communities. In this context, sample-pooling and test grouping could offer an effective solution. Here we report the screening on 1195 oral-nasopharyngeal swabs collected from students and staff of the Università degli Studi del Sannio (University of Sannio, Benevento, Campania, Italy) and analyzed by an in-house developed multiplex RT-qPCR for SARS-CoV-2 detection through a simple monodimensional sample pooling strategy. Overall, 400 distinct pools were generated and, within 24 h after swab collection, five positive samples were identified. Out of them, four were confirmed by using a commercially available kit suitable for in vitro diagnostic use (IVD). High accuracy, sensitivity and specificity were also determined by comparing our results with a reference IVD assay for all deconvoluted samples. Overall, we conducted 463 analyses instead of 1195, reducing testing resources by more than 60% without lengthening diagnosis time and without significant losses in sensitivity, suggesting that our strategy was successful in recognizing positive cases in a community of asymptomatic individuals with minor requirements of reagents and time when compared to normal testing procedures.

High-sensitivity SARS-CoV-2 group testing by digital PCR among symptomatic patients in hospital settings.

BACKGROUND: Worldwide demand for SARS-CoV-2 RT-PCR testing is still high as testing remains central to follow the disease spread and vaccine efficacy. Group testing has been proposed as a solution to expand testing capabilities but sensitivity concerns may limit its impact on the management of the pandemic. Digital PCR (RT-dPCR) has been shown to be highly sensitive and could help by providing larger testing capabilities without compromising sensitivity. METHODS: We implemented RT-dPCR based COVID-19 group testing on a commercially available system and assay (naica® system from Stilla Technologies) and investigated the sensitivity of the method in real life conditions of a university hospital in Paris, France, in May 2020. We tested the protocol in a direct comparison with reference RT-PCR testing on 448 samples split into groups of 8, 16 and 32 samples for RT-dPCR analysis. RESULTS: Individual RT-PCR testing identified 25/448 positive samples. Using 56 groups of 8, RT-dPCR identified 23 groups as positive, corresponding to 26 positive samples by individual PCR (positive percentage agreement 95.2% [95% confidence interval: 76.2-99.9%]) and including 2 samples not detected by individual RT-PCR but confirmed positive by further investigation. 15 of 28 groups of 16 tested positive, corresponding to 25 positive samples by individual PCR (positive percentage agreement 87.5% [95% confidence interval: 61.7-98.4%]). 14 groups of 32 were fully concordant with individual PCR testing but will need to be confirmed on larger datasets. CONCLUSIONS: Our proposed approach of group testing by digital PCR has similar diagnostic sensitivity compared to individual RT-PCR testing for group up to 16 samples. This approach reduces the quantity of reagent needed by up to 80% while reducing costs and increasing capabilities of testing up to 10-fold.

Critical Aspects Concerning the Development of a Pooling Approach for SARS-CoV-2 Diagnosis Using Large-Scale PCR Testing.

The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy.

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.

Evaluation of automated molecular tests for the detection of SARS-CoV-2 in pooled nasopharyngeal and saliva specimens.

BACKGROUND: Pooling of samples for SARS-CoV-2 testing in low-prevalence settings has been used as an effective strategy to expand testing capacity and mitigate challenges with the shortage of supplies. We evaluated two automated molecular test systems for the detection of SARS-CoV-2 RNA in pooled specimens. METHODS: Pooled nasopharyngeal and saliva specimens were tested by Qiagen QIAstat-Dx Respiratory SARS-CoV-2 Panel (QIAstat) or Cepheid Xpert Xpress SARS-CoV-2 (Xpert), and the results were compared to that of standard RT-qPCR tests without pooling. RESULTS: In nasopharyngeal specimens, the sensitivity/specificity of the pool testing approach, with 5 and 10 specimens per pool, were 77%/100% (n = 105) and 74.1%/100% (n = 260) by QIAstat, and 97.1%/100% (n = 250) and 100%/99.5% (n = 200) by Xpert, respectively. Pool testing of saliva (10 specimens per pool; n = 150) by Xpert resulted in 87.5% sensitivity and 99.3% specificity compared to individual tests. Pool size of 5 or 10 specimens did not significantly affect the difference of RT-qPCR cycle threshold (CT ) from standard testing. RT-qPCR CT values obtained with pool testing by both QIAstat and Xpert were positively correlated with that of individual testing (Pearson's correlation coefficient r = 0.85 to 0.99, p < 0.05). However, the CT values from Xpert were significantly stronger (p < 0.01, paired t test) than that of QIAstat in a subset of SARS-CoV-2 positive specimens, with mean differences of -4.3 ± 2.43 and -4.6 ± 2 for individual and pooled tests, respectively. CONCLUSION: Our results suggest that Xpert SARS-CoV-2 can be utilized for pooled sample testing for COVID-19 screening in low-prevalence settings providing significant cost savings and improving throughput without affecting test quality.