Testing Individual Samples Research Articles

Pooling of six respiratory samples for the detection of SARS-CoV-2: A validation and cost study in a cohort in Lima, Peru

BackgroundThe continuous evolution of the SARS-CoV-2 pandemic has led to a high demand for diagnostic testing and major shortages in testing materials, especially in low- and middle-income countries. As an alternative to testing individual samples, pooling of respiratory samples has been suggested. Previous studies have assessed performance of pooling, mainly using nasopharyngeal samples for the detection of SARS-CoV-2, but few studies have examined the performance of pooling the more practical nasal swabs or saliva samples. ObjectiveTo evaluate the sensitivity, specificity, and potential cost reduction of pooling of nasal swab (NS) and saliva (SL) samples for detection of SARS-CoV-2 in a community-based cohort study in Lima, Peru. Study designA prospective cohort study was conducted in a community setting in San Juan de Lurigancho, Lima-Peru. NS and SL samples were collected from 132 participants twice-a-week for a 2-month period. Pools of 2 to 12 samples of the same type, from participants of the same household, were tested by RT-PCR. After pooled testing, all individual samples from positive pools and all individual samples from randomly chosen negative pools were evaluated. For assessment of diagnostic performance, pool testing results were compared with results from individual testing, which served as reference, and concordance in pooled and individual test detections was evaluated. Laboratory costs for both types of samples and testing were compared. ResultsA total of 2008 NS and 2002 SL samples were collected from 132 study participants. We tested 329 NS and 333 SL pools. The mean pool size for NS and SL pools was 6.22 (SD = 0.92) and 6.39 (SD = 1.71), respectively. Using individual testing as reference, NS pooling of 6 had a sensitivity and specificity of 94% and 100%, respectively, with kappa of 0.97 (CI 95%: 0.93–1.00). The corresponding values for SL pooling of 6 were 83%, 100%, and 0.90 (CI 95%: 0.83–0.97). Compared with individual testing, pooling resulted in a cost reduction of 74.8% for NS and 72.4% for SL samples. ConclusionsPooling easy-to-collect respiratory samples, especially NS, demonstrated very high diagnostic performance for detection of SARS-CoV-2 with substantial cost savings. This approach could be considered in large population screening programs, especially in LMIC.

Evaluation of sample pooling for the detection of SARS-CoV-2 in a resource-limited setting, Dominican Republic

IntroductionCOVID-19 is a worldwide public health threat. Diagnosis by RT-PCR has been employed as the standard method to confirm viral infection. Sample pooling testing can optimize the resources by reducing the workload and reagents shortage, and be useful in laboratories and countries with limited resources. This study aims to evaluate SARS-CoV-2 detection by sample pooling testing in comparison with individual sample testing. Materials and methodsWe created 210 pools out of 245 samples, varying from 4 to 10 samples per pool, each containing a positive sample. We conducted detection of SARS-CoV-2-specific RdRp/E target sites. ResultsPooling of three samples for SARS-CoV-2 detection might be an efficient strategy to perform without losing RT-PCR sensitivity. ConclusionsConsidering the positivity rate in Dominican Republic and that larger sample pools have higher probabilities of obtaining false negative results, the optimal sample size to perform a pooling strategy shall be three samples.

Novel application of one-step pooled molecular testing and maximum likelihood approaches to estimate the prevalence of malaria parasitaemia among rapid diagnostic test negative samples in western Kenya

BackgroundDetection of malaria parasitaemia in samples that are negative by rapid diagnostic tests (RDTs) requires resource-intensive molecular tools. While pooled testing using a two-step strategy provides a cost-saving alternative to the gold standard of individual sample testing, statistical adjustments are needed to improve accuracy of prevalence estimates for a single step pooled testing strategy.MethodsA random sample of 4670 malaria RDT negative dried blood spot samples were selected from a mass testing and treatment trial in Asembo, Gem, and Karemo, western Kenya. Samples were tested for malaria individually and in pools of five, 934 pools, by one-step quantitative polymerase chain reaction (qPCR). Maximum likelihood approaches were used to estimate subpatent parasitaemia (RDT-negative, qPCR-positive) prevalence by pooling, assuming poolwise sensitivity and specificity was either 100% (strategy A) or imperfect (strategy B). To improve and illustrate the practicality of this estimation approach, a validation study was constructed from pools allocated at random into main (734 pools) and validation (200 pools) subsets. Prevalence was estimated using strategies A and B and an inverse-variance weighted estimator and estimates were weighted to account for differential sampling rates by area.ResultsThe prevalence of subpatent parasitaemia was 14.5% (95% CI 13.6–15.3%) by individual qPCR, 9.5% (95% CI (8.5–10.5%) by strategy A, and 13.9% (95% CI 12.6–15.2%) by strategy B. In the validation study, the prevalence by individual qPCR was 13.5% (95% CI 12.4–14.7%) in the main subset, 8.9% (95% CI 7.9–9.9%) by strategy A, 11.4% (95% CI 9.9–12.9%) by strategy B, and 12.8% (95% CI 11.2–14.3%) using inverse-variance weighted estimator from poolwise validation. Pooling, including a 20% validation subset, reduced costs by 52% compared to individual testing.ConclusionsCompared to individual testing, a one-step pooled testing strategy with an internal validation subset can provide accurate prevalence estimates of PCR-positivity among RDT-negatives at a lower cost.

Effectiveness Of Pcr Pool Testing For Screening Of Covid-19 Infection In Pakistan.

We tested the utility of mini-pool PCR testing for the rational use of PCR consumables in screening for CoViD-19. After pilot experiments, 3-samples pool size was selected. One step RT-PCR was performed. The samples in the mini-pool having COVID gene amplification were tested individually. 1548 samples tested in 516 mini-pools resulted 396 mini-pools as negative and 120 as positive. Upon individual testing, 110 samples tested positive and 9 were inconclusive. 876 PCR reactions were performed to test 1548 samples, saving 43% PCR reagents. Centres with low prevalence resulted in most saving on reagents (50%), while centres with high prevalence resulted in more test reactions. Testing of individual samples resulted in delays in reporting. Pooling can increase lab capacity, however, pooling delays results and cause degradation of samples.

Evaluation of detection of severe acute respiratory syndrome coronavirus-2 by chip-based real-time polymerase chain reaction test (truenat™ beta CoV) in multi-sample pools

Introduction: Systematic testing for Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) using molecular diagnostic tools to identify individuals with coronavirus disease 2019 (COVID-19) infection, and tracing their primary and secondary contacts is important to curb its spread. With resource limitations on testing individual samples, testing of pooled samples provides alternative approach to increase testing capacity. Present aimed at assessing the detection of SARS-CoV-2 RNA in pooled samples using chip-based real-time polymerase chain reaction Test (Truenat™ Beta CoV). Materials and Methods: Pooled sample size of five was used from laboratory confirmed COVID-19 positive and negative samples. SARS-CoV-2 positive nasopharyngeal specimens of known samples from high, medium, low, and very low viral load were mixed with SARS-CoV-2 negative nasopharyngeal specimens of known samples in 1:4 ratio, followed by analysis using Truenat. Furthermore, each sample in that pool was tested individually. Pooled sample testing was also done on the samples of unknown status. Results: The results of the present study showed cycle threshold (Ct) values of pooled sample with SARS-CoV-2 positive RNA of high, medium, low, and very low viral load were 16.8, 24.22, 28.2, and 33.43, compared to Ct values of individual samples of 16.43, 22.0, 28.00, and 33.00, respectively. Conclusion: These results suggest that the Ct values of pooled samples were in agreement with Ct values of individual samples indicating the validity of pooled sample testing for screening SARS-CoV-2 using Truenat. The following core competencies are addressed in this article: Medical knowledge, Patient care and procedural skills systems-based practice, Practice-based learning and improvement.

RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

Evaluation of SARS-CoV-2 genome detection by real-time PCR assay using pooled specimens.

Objective: To evaluate SARS-CoV-2 genome detection using pooled samples by RT-qPCR assay, compared to individual samples. Method: At first all samples were tested individually using two commercial methods targeting ORF1ab, NP and E genes. Then, four experimental groups of samples were pooled and evaluated using the same detection methods. Findings: Compared to the individual sample testing, the sample pooling conserved the sensitivity of the detection in all groups of pooled samples when the Ct value in single test was lower than 33. Conclusion: Specimen pooling may fail to detect positive samples with high Ct values. However, in scarcity of reagents or in population surveys, it could be considered as an alternative method in low prevalence settings.

Characteristics (sensitivity and specificity) of herd-level diagnostic tests for Mycobacterium avium subspecies paratuberculosis in cattle — A systematic review

Diagnostic tests for Johne’s disease in cattle are characterised by poor sensitivity and often imperfect specificity at the animal level. Because farmers and veterinarians have limited assurance or confidence from results of testing individual animals for Mycobacterium avium subsp. paratuberculosis (MAP), control programmes based on whole herd-level testing provide the best framework for classifying herds. At the herd level, there is a diverse range of testing options for MAP based on both direct and indirect testing of individual and pooled samples. The most common measures of herd test performance, herd sensitivity (HSe) and herd specificity (HSp), are important for decision-making in herd test selection, for estimating prevalence and as inputs for simulation studies. This systematic review investigated the results of herd test evaluations for MAP in cattle, through a comprehensive search of the literature and a systematic four-stage screening process to identify relevant publications.Forty-six publications with relevant results were eligible for inclusion in the final review, containing evaluations of whole-herd ELISA serological testing, bulk milk tank ELISA, culture, PCR and phage testing, pooled faecal testing and environmental sample testing. Data extracted from each publication included sample populations, methods of analysis, reference tests, cut-off points, HSe and HSp. Direct comparisons between the reported HSe and HSp estimates of different studies is challenging due to the variations in herd prevalence and test protocols used. The data in this systematic review will benefit decision-makers and researchers and highlights knowledge gaps requiring further research.

Pooling Nasopharyngeal Swab Specimens to Improve Testing Capacity for SARS-CoV-2 by Real-Time RT-PCR

BackgroundThe detection of SARS-CoV-2 using qRT-PCR with the pooling of samples can reduce workload and costs especially when the prevalence rate of COVID-19 in a population is low.To analyse the effect of pooling samples on the sensitivity of RT-qPCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we compared the cycle threshold (Ct) values of pools of 5 and 10 that tested positive with Ct values of individual samples that tested positive in that pool. Twenty positive nasopharyngeal (NP) specimens with low to high viral load were selected and pooled individually with four and nine negative NP.ResultsIn NP specimens, the sensitivity of pools of 5 and 10 were 90 and 85%, compared to individual sample testing, respectively. The RT-qPCR sensitivity of pools of 5 and 10 against individual testing were not significantly different (p > 0.05). Detection of positive samples with low Ct values (< 36) was consistently achieved in pools of 5 and 10. However, there were higher false negatives when samples with high ct values (> 36) were pooled and tested. The mean Ct values obtained with the 5-sample pooled testing exceeded individual sample testing by 1.85 ± 1.09 cycles, while Ct values obtained with the 10-sample pooling exceeded individual sample testing by 3.4 ± 1.65 cycles.ConclusionsIn a low prevalence setting, testing capacity can be increased by pooling 5 or 10 samples, but the risk of additional false negatives needs to be considered.

Evaluation of sample pooling for the detection of SARS-CoV-2 in a resource-limited setting, Dominican Republic

IntroductionCOVID-19 is a worldwide public health threat. Diagnosis by RT-PCR has been employed as the standard method to confirm viral infection. Sample pooling testing can optimize the resources by reducing the workload and reagents shortage, and be useful in laboratories and countries with limited resources. This study aims to evaluate SARS-CoV-2 detection by sample pooling testing in comparison with individual sample testing. Materials and methodsWe created 210 pools out of 245 samples, varying from 4 to 10 samples per pool, each containing a positive sample. We conducted detection of SARS-CoV-2-specific RdRp/E target sites. ResultsPooling of three samples for SARS-CoV-2 detection might be an efficient strategy to perform without losing RT-PCR sensitivity. ConclusionsConsidering the positivity rate in Dominican Republic and that larger sample pools have higher probabilities of obtaining false negative results, the optimal sample size to perform a pooling strategy shall be three samples.

Pool Testing as a Strategy for Prevention of SARS-CoV-2 Outbreaks in Schools: Protocol for a Feasibility Study.

BackgroundSchool closures are a widely implemented strategy for limiting infection spread in the current COVID-19 pandemic. The negative impact of school closures on children and young people is increasingly apparent, however.ObjectiveWe aim to evaluate the feasibility of an infection monitoring program in schools to enable targeted quarantining to replace school closures. The program is currently being implemented in two model schools in Magdeburg, Germany, within the framework of the Study of Coronavirus Outbreak Prevention in Magdeburg Schools (Studie zur Ausbruchsvermeidung von Corona an Magdeburger Schulen [STACAMA]).MethodsFive pupils per class are pseudorandomly selected twice a week and asked to provide a gargle sample over a 16-week evaluation period. RNA is extracted from each sample individually in a laboratory and pooled according to school class for real-time reverse transcription polymerase chain reaction (rRT-PCR) analysis. Immediate individual sample testing will be carried out in the case of a positive pool test. Individual RNA extraction prior to pooling and application of rRT-PCR result in high test sensitivity. Testing will be performed in strict adherence to data protection standards. All participating pupils will receive a 16-digit study code, which they will be able to use to access their testResultsWhen the study commenced on December 2, 2020, 520 (52%) pupils and their families or guardians had consented to study participation. The study was suspended after four test rounds due to renewed school closures resulting from rising regional infection incidence. Testing resumed when schools reopened on March 8, 2021, at which time consent to participation was provided for 54% of pupils. We will quantitatively and qualitatively evaluate the logistics and acceptability of the program.ConclusionsThe findings from this study should inform the design of infection surveillance programs in schools based on gargle samples and a PCR-based pool testing procedure, enabling the identification of aspects that may require adaptation before large-scale implementation. Our focus on each step of the logistics and on the experiences of families should enable a robust assessment of the feasibility of such an approach.International Registered Report Identifier (IRRID)DERR1-10.2196/28673

Speciation of rare earth elements in acid mine drainage precipitates by sequential extraction

Recent efforts have shown that acid mine drainage precipitates (AMDp) may be a promising source of rare earth elements and critical materials (REE/CM). To better understand this resource, several particle characterization studies were conducted on AMDp samples from three AMD treatment sites in Northern and Central Appalachia. This work included the determination of particle size, size distribution, morphology, mineralogy, chemical speciation, and rare earth content. While the particle characteristics and mineralogy varied considerably from site to site, sequential extraction tests showed that the largest portion of REEs (45% to 75%) were found in soluble phases bound to Fe-Mn oxides. Carbonates (5% to 16%) and insoluble residues (4% to 24%) were also found to be significant in some samples, suggesting that different AMD chemistry and AMD treatment approaches can lead to substantial variation in the deportment of REEs to the various chemical phases. Altogether, the results suggest that chemical extraction of REEs from AMDp can be achieved under mild conditions with high recovery values; however, the significant site-by-site variation will necessitate detailed testing of individual samples to determine the overall economic viability.

Evaluation of pooling of samples for testing SARS-CoV- 2 for mass screening of COVID-19

BackgroundThe current pandemic of the SARS-CoV-2 virus, widely known as COVID-19, has affected millions of people around the world. The World Health Organization (WHO) has recommended vigorous testing to differentiate SARS-CoV-2 from other respiratory infections to aid in guiding appropriate care and management. Situations like this have demanded robust testing strategies and pooled testing of samples for SARS-CoV-2 virus has provided the solution to mass screening of people for COVID-19. A pooled testing strategy can be very effective in testing when resources are limited, yet it comes with its own limitations. These benefits and limitations need critical consideration when it comes to testing highly infectious diseases like COVID-19.MethodsThis study evaluated the pooled testing of nasopharyngeal swabs for SARS-COV-2 by comparing the sensitivity of individual sample testing with 4-and 8-pool sample testing. Median cycle threshold (Ct) values were compared, and the precision of pooled testing was assessed through an inter- and intra-assay of pooled samples. Coefficient of variance was calculated for inter- and intra-assay variability.ResultsThe sensitivity becomes considerably lower when the samples are pooled. There is a high percentage of false negative reports with larger sample pool size and when the patient viral load is low or weak positive samples. High variability was seen in the intra- and inter-assay, especially among weak positive samples and when more number of samples are pooled together.ConclusionAs COVID − 19 infection numbers and need for testing remain high, we must meticulously evaluate the testing strategy for each country depending on its testing capacity, infrastructure, economic strength, and need to determine the optimal balance on the cost-effective strategy of resource saving and risk/ cost of missing positive patients.

Systematic Review of Pooling Sputum as an Efficient Method for Xpert MTB/RIF Tuberculosis Testing during the COVID-19 Pandemic.

GeneXpert-based testing with Xpert MTB/RIF or Ultra assays is essential for tuberculosis diagnosis. However, testing may be affected by cartridge and staff shortages. More efficient testing strategies could help, especially during the coronavirus disease pandemic. We searched the literature to systematically review whether GeneXpert-based testing of pooled sputum samples achieves sensitivity and specificity similar to testing individual samples; this method could potentially save time and preserve the limited supply of cartridges. From 6 publications, we found 2-sample pools using Xpert MTB/RIF had 87.5% and 96.0% sensitivity (average sensitivity 94%; 95% CI 89.0%–98.0%) (2 studies). Four-sample pools averaged 91% sensitivity with Xpert MTB/RIF (2 studies) and 98% with Ultra (2 studies); combining >4 samples resulted in lower sensitivity. Two studies reported that pooling achieved 99%–100% specificity and 27%–31% in cartridge savings. Our results show that pooling may improve efficiency of GeneXpert-based testing.

The Application of Sample Pooling for Mass Screening of SARS-CoV-2 in an Outbreak of COVID-19 in Vietnam.

ABSTRACTWe sampled nasal–pharyngeal throat swabs from 96,123 asymptomatic individuals at risk of SARS-CoV-2 infection, and generated 22,290 pools at collection, each containing samples from two to seven individuals. We detected SARS-CoV-2 in 24 pools, and confirmed the infection in 32 individuals after resampling and testing of 104 samples from positive pools. We completed the testing within 14 days. We would have required 64 days to complete the screening for the same number of individuals if we had based our testing strategy on individual testing. There was no difference in cycle threshold (Ct) values of pooled and individual samples. Thus, compared with individual sample testing, our approach did not compromise PCR sensitivity, but saved 77% of the resources. The present strategy might be applicable in settings, where there are shortages of reagents and the disease prevalence is low, but the demand for testing is high.

Genetic variability of milk proteins in two cattle breeds of Piedmont region and the potential effects on milk quality

In a scenario of climate change and risk of losing biodiversity, the safeguard of locally adapted breeds and their genetic reservoirs assumes a key role. Furthermore, these breeds are linked to their rearing territory maintenance also through the nutritional, economic or social value of their products. This study focussed on the analysis of milk protein genetic variability of the Piedmontese, the most abundant beef Italian breed, and the Barà-Pustertaler breed, through the testing of individual milk samples by isoelectrofocusing. Allelic and haplotypes frequencies were analysed and the presence of rare variants (CSN1S1*G, 0.7%, and CSN2*C, 0.7%, in Barà and Piedmontese breeds, respectively) was detected. The analysed breeds showed allelic frequencies close to the one found in breeds with good cheesemaking aptitude, like the Italian Brown and Reggiana breeds (e.g. CSN2*B, 20.7% and 16.4%, and CSN3*B, 41,1% and 35%, in Piedmontese and Barà breeds, respectively), and are a good reservoir of alleles that are decreasing (e.g. CSN2*B) in Italian Holstein. Moreover, they have higher frequencies of interesting haplotypes (e.g. BA1B, 8% and 11.2%, and BA2B, 20.8 and 13.1% in Piedmontese and Barà breeds, respectively) that could positively influence composition, nutritional, and technological properties of milk. These breeds could be used to further investigate the effects of variants and haplotypes that could help support the evolution and the changing demands of the dairy sector. Highlights In order to address the problem of climate change effects on animal biodiversity, local breeds should be safeguarded and valorised. The analysed breeds have discrete frequencies of milk protein variants and casein haplotypes associated with good technological properties. Knowing the genetic background of non-dairy commercial breeds is the first step to preserve potentially useful genetic variability.

Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling

ObjectivesLimited testing capacity has characterized the ongoing coronavirus disease 2019 (COVID-19) pandemic in Spain, hampering timely control of outbreaks and opportunities to reduce the escalation of community transmission. This study investigated the potential to use sample pooling, followed by one-step retrotranscription and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to increase testing capacity for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). MethodsVarious pool sizes (five, 10 and 15 samples) were evaluated prior to RNA extraction followed by standard RT-qPCR for the diagnosis of COVID-19. The pool size achieving reproducible results with individual sample testing was subsequently used to assess nasopharyngeal samples in a tertiary hospital in August 2020. ResultsA pool size of five samples had higher sensitivity compared with pool sizes of 10 and 15 samples, showing a mean cycle threshold (Ct) shift of 3.5 [standard deviation (SD) 2.2] between the pooled test and positive samples in the pool. Next, a pool size of five was used to test a total of 895 pools (4475 prospective samples) using two different RT-qPCR kits. The Real Accurate Quadruplex corona-plus PCR Kit (PathoFinder) reported the lowest mean Ct shift [2.2 (SD 2.4)] between the pool and individual samples. This strategy enables detection of individual positive samples in positive pools with Ct of 16.7–39.4. ConclusionsGrouping samples into pools of five for RT-qPCR resulted in an increase in SARS-CoV-2 testing capacity with minimal loss of sensitivity compared with testing each sample individually.

Saliva sample pooling for the detection of SARS-CoV-2.

As the battle against coronavirus disease 2019 pandemic continues, an increase in workload and medical expenses have been a concern to the health care system worldwide. Developing a measure that helps to conserve the health care resource is, therefore, highly desirable, and the pooling of the specimens for testing is one of the attractive strategies. Recently, we showed that saliva could be a potential alternative specimen for the detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by real‐time polymerase chain reaction (RT‐PCR). In the present study, we performed the pooling of saliva specimens for testing by SARS‐CoV‐2 RT‐PCR. We showed that the saliva pool of either 5 or 10 samples, by allowing the detection of either gene in the pool at an increased cycle threshold cutoff value, further performing individual sample testing in the positive pools did not compromise the detection of SARS‐CoV‐2.

Bayesian estimation of diagnostic accuracy of fecal culture and PCR-based tests for the detection of Salmonella enterica in California cull dairy cattle.

Epidemiological studies of low prevalence disease problems are often hindered by the high cost of diagnostic testing. The objective of this study was to evaluate PCR screening of both individual and pooled fecal samples from culled dairy cows for the invA gene of Salmonella followed by culture to determine if the sensitivity and specificity were comparable to the results from traditional culture methods applied to individual samples. Cows from six different dairies were sampled in all four seasons. A total of 240 individual cow fecal samples, 24 fecal pools and 24 pools of 24-hour tetrathionate enrichment broth were tested. Diagnostic sensitivity of PCR screening followed by culture of PCR positive or indeterminate samples (i.e PCR-CUL method) was lower than that of culture (CUL) when applied to individual fecal samples (94.8%, 99.5%), however the specificity was comparable (99.6% and 97.7% respectively). For pools of five fecal samples and pools of five, 24 h tetrathionate broth samples, the specificity of both tests were comparable (∼98%); however, their sensitivity was only comparable in pooled fecal samples (∼93%) but greater for culture compared to PCR-CUL in pooled broth samples (∼99% versus ∼93%). Compared to culture results from testing of individual fecal samples, testing pooled fecal samples by culture had a relative sensitivity of 74% and relative specificity of 96%, testing pooled fecal samples by PCR-CUL resulted in relative sensitivity of 90% and relative specificity of 96%. Testing of pooled 24-hour enrichment broth by PCR-CUL increased the relative sensitivity and specificity to 100%. PCR testing followed by culture of positive or indeterminate samples is a time saving alternative to traditional methods. In addition, pooling of samples may be a useful method for decreasing cost if study aims can accommodate a moderate loss of relative sensitivity.

Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling.

Background & objectives:Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India.Methods:Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed.Results:A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ct values ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools.Interpretation & conclusions:Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.