Pooled Sample Testing Research Articles

Effect of pooled tracheal sample testing on the probability of Mycoplasma hyopneumoniae detection

Tracheal pooling for Mycoplasma hyopneumoniae (M. hyopneumoniae) DNA detection allows for decreased diagnostic cost, one of the main constraints in surveillance programs. The objectives of this study were to estimate the sensitivity of pooled-sample testing for the detection of M. hyopneumoniae in tracheal samples and to develop probability of M. hyopneumoniae detection estimates for tracheal samples pooled by 3, 5, and 10. A total of 48 M. hyopneumoniae PCR-positive field samples were pooled 3-, 5-, and 10-times using field M. hyopneumoniae DNA-negative samples and tested in triplicate. The sensitivity was estimated at 0.96 (95% credible interval [Cred. Int.]: 0.93, 0.98) for pools of 3, 0.95 (95% Cred. Int: 0.92, 0.98) for pools of 5, and 0.93 (95% Cred. Int.: 0.89, 0.96) for pools of 10. All pool sizes resulted in PCR-positive if the individual tracheal sample Ct value was < 33. Additionally, there was no significant decrease in the probability of detecting at least one M. hyopneumoniae-infected pig given any pool size (3, 5, or 10) of tracheal swabs. Furthermore, this manuscript applies the probability of detection estimates to various real-life diagnostic testing scenarios. Combining increased total animals sampled with pooling can be a cost-effective tool to maximize the performance of M. hyopneumoniae surveillance programs.

Statistical Tests for Proportion Difference in One-to-Two Matched Binary Diagnostic Data: Application to Environmental Testing of Salmonella in the United States

Pooled sample testing is an effective strategy to reduce the cost of disease surveillance in human and animal medicine. Testing pooled samples commonly produces matched observations with dichotomous responses in medical and epidemiological research. Although standard approaches exist for one-to-one paired binary data analyses, there is not much work on one-to-two or one-to-N matched binary data in the current statistical literature. The existing Miettinen’s test assumes that the multiple observations from the same matched set are mutually independent. In this paper, we propose exact and asymptotic tests for one-to-two matched binary data. Our methods are markedly different from the previous studies in that we do not rely on the mutual independence assumption. The emphasis on the interdependence of observations within a matched set is inherent and attractive in both human health and veterinary medicine. It can be applied to all kinds of diagnostic studies with a one-to-two matched data structure. Our methods can be generalized to the one-to-N matched case. We discuss applications of the proposed methods to the environmental testing of salmonella in the United States.

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR.

The declaration of the end of the Public Health Emergency for COVID-19 on May 11th, 2023, has shifted the global focus led by WHO and CDC towards monitoring the evolution of SARS-CoV-2. Augmenting these international endeavors with local initiatives becomes crucial to not only track the emergence of new variants but also to understand their spread. We present a cost-effective digital PCR-based pooled sample testing methodology tailored for early variant surveillance. Using 1200 retrospective SARS-CoV-2 positive samples, either negative or positive for Delta or Omicron, we assessed the sensitivity and specificity of our detection strategy employing commercial TaqMan variant probes in a 1:9 ratio of variant-positive to variant-negative samples. The study achieved 100% sensitivity and 99% specificity in 10-sample pools, with an Area Under the Curve (AUC) exceeding 0.998 in ROC curves, using distinct commercial TaqMan variant probes. The employment of two separate TaqMan probes for both Delta and Omicron establishes dual validation routes, emphasizing the method's robustness. Although we used known samples to model realistic emergence scenarios of the Delta and Omicron variants, our main objective is to demonstrate the versatility of this strategy to identify future variant appearances. The utilization of two divergent variants and distinct probes for each confirms the method's independence from specific variants and probes. This flexibility ensures it can be tailored to recognize any subsequent variant emergence, given the availability of its sequence and a specific probe. Consequently, our approach stands as a robust tool for tracking and managing any new variant outbreak, reinforcing our global readiness against possible future SARS-CoV-2 waves.

Loss of Caprine Arthritis Encephalitis Virus (CAEV) Herd Accreditation: Characteristics, Diagnostic Approach, and Specific Follow-Up Scenarios on Large Dairy Goat Farms.

The retrovirus causing caprine arthritis encephalitis (CAE), a slowly progressive inflammatory disease in goats, belongs to the group of small ruminant lentiviruses (SRLVs) which cause lifelong infections that ought to be avoided for animal welfare as well as economic reasons. SRLV accreditation has been in place for forty years in The Netherlands and is based on the screening of small ruminant sera for specific antibodies. This paper evaluates 38 dairy goat herds that lost CAEV accreditation between 2012 and 2022. The characteristics of these herds are discussed, and specific follow-up scenarios, depending on desired goals, are introduced. The herd size of the participating herds varies from approximately 400 to 4600 adult dairy goats. The larger herds tended to be more prone to lose herd accreditation and had more difficulties regaining accreditation. Possible routes of introduction are lined up. The Royal GD's tailor-made approach and advice to support livestock farmers with herds that have lost CAE accreditation are discussed in detail. Specific emphasis is placed on the strategic deployment of various diagnostic tests (such as antibody ELISAs and PCR) in different media, such as (pooled) sera, (bulk)milk and tissue samples. Special attention is paid to the added value of retrospective bulk milk testing or the specific testing of groups based on housing and management, which enables the investigation of the moment of viral introduction and route of transmission into a herd. Furthermore, the prospective implementation of bulk milk and strategic pooled milk sample testing in the Dutch SRLV accreditation programs intensifies surveillance and enables the taking of swift action to prevent further transmission within and between herds. An appeal is made to share experiences to improve programs collectively, and to start research into the underlying mechanisms.

Pooling Rectal, Pharyngeal, and Urine Samples to Detect Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium Using Multiplex Polymerase Chain Reaction Is as Effective as Single-Site Testing for Men Who Have Sex With Men.

Screening for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) at pharyngeal, urogenital, and anorectal sites is recommended for men who have sex with men (MSM). Pooling samples is a promising technique, but no data are available when pooled screening also includes Mycoplasma genitalium (MG). The main objective of this study was to examine the sensitivity of pooled samples for detecting CT, NG, and MG in MSM using nucleic acid amplification versus single-site testing. In this multicenter study, MSM with a positive result for CT, NG, or MG were recalled to the clinic for treatment and were asked to participate in this study. Separate samples were sent to a central virological department that proceeded to form the pooled samples. Testing was performed using the multiplex real-time polymerase chain reaction Allplex STI Essential Assay (Seegene, Seoul, Korea), which can simultaneously detect 7 pathogens. A total of 130 MSM with at least 1 positive test for CT, NG, or MG were included. A total of 25.4% had a coinfection. The sensitivities of pooled-sample testing were 94.8% for CT, 97.0% for NG, and 92.3% for MG. Pooling failed to detect 8 infections, but pooled-sample analysis missed detecting only samples with a low bacterial load (cycle threshold >35). Pooling samples from MSM to detect CT, NG, and MG is as sensitive as individual-site testing for these 3 pathogens using the Allplex assay. Missed infections with a very low bacterial load could have a low impact on further transmission. Clinical Trials Registration. NCT03568695.

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.

Mathematical Model and Optimization Methods of Wide-Scale Pooled Sample Testing for COVID-19

Currently, coronavirus disease 2019 (COVID-19) has become the most severe infectious disease affecting the world, which has spread around the world to more than 200 countries in 2020. Until the number of COVID-19 vaccines is insufficient, nucleic acid testing is considered as an effective way to screen virus carriers and control the spread of the virus. Considering that the medical resources and infection rates are different across various countries and regions, if all infected areas adopt the traditional individual nucleic acid testing method, the workload will be heavy and time-consuming. Therefore, this will not lead to the control of the pandemic. After Wuhan completed a citywide nucleic acid testing in May 2020, China basically controlled the spread of COVID-19 and entered the post-epidemic period. Since then, although some cities in China, such as Qingdao, Xinjiang, Beijing, and Dalian, have experienced a local epidemic resurgence, the pandemic was quickly suppressed through wide-scale pooled nucleic acid testing methods. Combined with the successful experience of mass nucleic acid testing in China, this study introduces two main pooled testing methods used in two cities with a population of more than ten million people, Wuhan’s “five-in-one” and Qingdao’s “ten-in-one” rapid pooled testing methods. This study proposes an improved method for optimising the second round of “ten-in-one” pooled testing, known as “the pentagram mini-pooled testing method”, which speeds up the testing process (as a result of reducing the numbers of testing by 40%) and significantly reduces the cost. Qingdao’s optimised “ten-in-one” pooled testing method quickly screens out the infections by running fewer testing samples. This study also mathematically examines the probabilistic principles and applicability conditions for pooled testing of COVID-19. Herein, the study theoretically determines the optimal number of samples that could successfully be combined into a pool under different infection rates. Then, it quantitatively discusses the applicability and principles for choosing the pooled testing instead of individual testing. Overall, this research offers a reference for other countries with different infection rates to help them in implementing the mass testing for COVID-19 to reduce the spread of coronavirus.

Pooled sample testing for COVID-19 diagnosis: Evaluation of bi-directional matrix pooling strategies

In the on-going COVID-19 pandemic, pooled testing of samples by RT-PCR has been recommended at certain scenarios to increase labs’ testing capacity and reduce cost of testing. This paper describes the evaluation of bi-directional matrix pooling strategies with clinical samples in a 5 × 5 and 10 × 10 matrix. Nasopharyngeal swab samples in viral transport medium (VTM) previously tested (positive or negative) by real time RT-PCR for SARS-CoV-2 were used for these experiments. Ten sets of 5 × 5 (250 samples) and ten sets of 10 × 10 (1000 samples) pooling of samples in both directions was done with known positive samples introduced at random positions. Extracted nucleic acid was tested for SARS-CoV-2 E-gene by RT-PCR. Sensitivity or concordance and feasibility of matrix pooling were assessed in comparison to direct RT-PCR testing. In comparison to direct testing, the overall concordance was 86.6% for 5 × 5 pooling, 73.3% for 10 × 10 with 200 µL extraction volume and 86.6% for 10 × 10 with 400 µL extraction volume. Bi-directional matrix pooling can be adopted with advantage over conventional direct or pool testing for COVID-19 by RT-PCR under the following conditions: i) sample positivity rate of ≤ 5%, ii) matrix pool size of 8–10 samples, iii) use of min. 40 µL VTM from each sample and iv) utilization of automated liquid handling equipment, if available, for sample addition to avoid human errors.

Modified self-obtained pooled sampling to screen for Chlamydia trachomatis and Neisseria gonorrhoeae infections in men who have sex with men

ObjectivesTo assess whether pooled sample testing with nucleic acid amplification tests was a potential alternative to three single-site sample testing to screen for Chlamydia trachomatis and Neisseria gonorrhoeae infections in...

Evaluating the efficiency of specimen (sample) pooling for real-time PCR based diagnosis of COVID-19

PurposeThis study is aims at evaluating the efficacy and sensitivity of specimen pooling for testing of SARS-CoV-2 virus to determine the accuracy, resource savings, and identification of borderline positive cases without impacting the accuracy of the testing. MethodThis study was conducted between August and October 2020, we performed COVID-19 testing by RT-PCR on the samples from varying prevalence of rural population (non-hot spot) referred to COVID laboratory, in the first step, the samples were collated into pools of 5 or 10. These pools were tested by RT-PCR. Negative pools were reported as negative whereas positive pools of 5 and 10 were then de-convoluted and each sample was tested individually. ResultsIn the present study, we tested 1580 samples in 158 pools of 10 and 17,515 samples in 3503 pools of 5. Among 10 samples pool, 11 (13%) pools flagged positive in the first step. In the second step, among 11 pools (110 samples) de-convoluted strategy was followed in which 10 individual samples came positive. Among 5 samples pool, 164 (13%) pools flagged positive in the first step. In the second step, among 164 pools (820 samples) de-convoluted strategy was followed in which 171 individual samples came positive. The pooled sample testing strategy saves substantial resources and time during surge testing and enhanced pandemic surveillance. This approach requires around 76%–93% fewer tests in low to moderate prevalence settings and group sizes up to 5–10 in a population, compared to individual testing. ConclusionPooled sample RT- PCR analysis strategies can save substantial resources and time for COVID-19 mass testing in comparison with individual testing without compromising the quality of outcome of the test. In particular, the pooled sample approach can facilitate mass screening in the early asymptomatic stages of COVID-19 infections.

Pooled-sample testing for detection of Mycoplasma hyopneumoniae during late experimental infection as a diagnostic tool for a herd eradication program

Early and accurate detection of Mycoplasma hyopneumoniae infection in live pigs is a critical component to measure the success of disease eradication strategies. However, the imperfect sensitivity of in vivo diagnostic tools, change in sensitivity over the course of infection, and expected low prevalence level at the end of an eradication program create a challenging diagnostic scenario. Here, the individual and pool sensitivities for detection of M. hyopneumoniae during the chronic phase of infection was determined using deep tracheal catheter samples, the in vivo sample type with the highest reported diagnostic sensitivity. Fifty samples from known infected pigs collected at 113 days post-M. hyopneumoniae intra-tracheal inoculation, were diluted in known negative samples to form pools of 1:3 and 1:5. Samples were tested for M. hyopneumoniae by a species-specific PCR. Ninety-eight percent (49/50) of individual samples, 84 % (42/50) of pools of 1:3, and 82 % (41/50) of 1:5 were detected positive for M. hyopneumoniae. To apply the sensitivity estimates for detection of M. hyopneumoniae in a low prevalence scenario, sample sizes with associated sample collection costs were calculated for individual and pooled testing using algorithms within the program EpiTools One-Stage Freedom Analyses. Assumptions included a ≥95 % population sensitivity, infinite population size, prevalence levels of ≥0.5 %, ≥1 %, ≥2 %, ≥3 %, ≥4 %, or ≥5 %, 100 % specificity, along with the mean and lower confidence limit of the individual or pool sensitivity for each pool size, when appropriate. For instance, following completion of a herd eradication program, if a low risk approach is targeted, sample size estimates for ≥2 % prevalence using the lower limit of the diagnostic or pool sensitivity 95 %CI may be followed. If samples were to be tested individually, 167 individuals would be sampled at a cost of 6,012 USD. If pooled by 3, 213 would be sampled (testing cost 3,266 USD), and for pools of 5, 220 individuals would be sampled (testing cost 2,464 USD). Population sensitivity was also calculated for a range of testing scenarios. Our study indicated that pooling samples by 3 or 5 was a cost-effective method for M. hyopneumoniae detection in low prevalence scenarios. Cost-effective detection was evidenced despite the increased sample collection costs associated with large sample sizes in order to offset decreased testing sensitivity attributable to pooling. The post-eradication sample collection scheme, combined with pooling, suggested lower cost options than individual sampling for testing to be applied at the end of an eradication program, without significantly compromising the likelihood of detection.

Intricacies in characterizing positivity in pooled sample testing for SARS-CoV-2.

The unprecedented demand for testing for the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 has led to an acute shortage and limited availability of test reagents for which pooling of samples has been recommended in areas with low prevalence. Considering the possibility of dilution factor in pool testing, an attempt was made to find out possibility of any true positive samples in pools with late amplification. The study was conducted on samples received from various collection centers in different districts of Odisha as well as from patients attending the screening clinic or admitted in COVID ward of the hospital. Nasal/nasopharyngeal/throat swabs received in viral transport media in cold chain were subjected to Real-time polymerase chain reaction (RT-PCR) testing in a Biosafety Laboratory level-2 by including uniform volume of four units (samples) per pool. All confirmed and probable positive pools in screening assay were de-convoluted and individual samples tested for confirmatory assay. Inclusion of an additional criteria of probable positive pool (Ct value >35 with non-sigmoid amplification curve or showing a line of amplification towards the end of the cycle) yielded 39 (15.5%) more true positive samples out of a total of 251 positive samples that would otherwise have been missed if only the classical criteria of positive (Ct within 35 with proper sigmoid curve) had been considered. The study highlights the importance of considering any indication of late amplification in the RT-PCR test to label a pool as positive to avoid missing any true positive sample in the pool.

Feasibility, efficiency & effectiveness of pooled sample testing strategy (pooled NAAT) for molecular testing of COVID-19.

Background & objectives:During the current COVID-19 pandemic, a large number of clinical samples were tested by real-time PCR. Pooling the clinical samples before testing can be a good cost-saving and rapid alternative for screening large populations. The aim of this study was to compare the performance characteristics, feasibility and effectiveness of pooling nasal swab and throat swab samples for screening and diagnosis of SARS-CoV-2.Methods:The pool testing was applied on a set of samples coming from low COVID-19 positivity areas. A total of 2410 samples were tested in pools of five samples each. A total of five pools of five samples each were generated and tested for E gene.Results:Of the total of 482 pools (2410 samples) 24 pools flagged positive. Later on pool de-convolution, a total of 26 samples were detected as positive for COVID-19, leading to positivity of about one per cent in the test population. For the diagnosis of individual samples, the pooling strategies resulted in cost savings of 75 per cent (5 samples per pool).Interpretation & conclusions:It was observed that testing samples for COVID-19 by reverse transcription (RT)- PCR after pooling could be a cost-effective method which would save both in manpower and cost especially for resource-poor countries and at a time when test kits were short in supply.

RT-PCR Screening Tests for SARS-CoV-2 with Saliva Samples in Asymptomatic People: Strategy to Maintain Social and Economic Activities while Reducing the Risk of Spreading the Virus

The year 2020 will be remembered for the coronavirus disease 2019 (COVID-19) pandemic, which continues to affect the whole world. Early and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is fundamental to combat the disease. Among the current diagnostic tests, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) is the most reliable and frequently used method. Herein, we discuss the interpretation of RT-qPCR results relative to viral infectivity. Although nasopharyngeal swab samples are often used for RT-qPCR testing, they require collection by trained medical staff. Saliva samples are emerging as an inexpensive and efficient alternative for large-scale screening. Pooled-sample testing of saliva has been applied for mass screening of SARS-CoV-2 infection. Current policies recommend isolating people with borderline cycle threshold (Ct) values (35<Ct <40), despite these Ct values indicating minimal infection risk. We propose the new concept of a "social cut-off" Ct value and risk stratification based on the correlation of Ct with infectivity. We also describe the experience of RT-qPCR screening of saliva samples at our institution. It is important to implement a scientific approach to minimize viral transmission while allowing economic and social activities to continue.

Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens.

There are more than 350 real‐time polymerase chain reaction (RT‐PCR) coronavirus disease‐2019 (COVID‐19) testing kits commercially available but these kits have not been evaluated for pooled sample testing. Thus, this study was planned to compare and evaluate seven commercially available kits for pooled samples testing. Diagnostic accuracy of (1) TRUPCR SARS‐CoV‐2 Kit (Black Bio), (2) TaqPath RT‐PCR COVID‐19 Kit (Thermo Fisher Scientific), (3) Allplex 2019‐nCOV Assay (Seegene), (4) Patho detect COVID‐19 PCR kit (My Lab), (5) LabGun COVID‐19 RT‐PCR Kit (Lab Genomics, Korea), (6) Fosun COVID‐19 RT‐PCR detection kit (Fosun Ltd.), (7) Real‐time Fluorescent RT‐PCR kit for SARS CoV‐2 (BGI) was evaluated on precharacterised 40 positive and 10 negative COVID‐19 sample pools. All seven kits detected all sample pools with low C t values (<30); while testing weak positive pooled samples with high C t value (>30); the TRUPCR Kit, TaqPath Kit, Allplex Assay, and BGI RT‐PCR kit showed 100% sensitivity, specificity, and accuracy. However, the Fosun kit, LabGun Kit, and Patho detect kit could detect only 90%, 85%, and 75% of weakly positive samples, respectively. We conclude that all seven commercially available RT‐PCR kits included in this study can be used for routine molecular diagnosis of COVID‐19. However, regarding performing pooled sample testing, it might be advisable to use those kits that performed best regarding positive identification in samples' pool, that is TRUPCR SARS‐CoV‐2 Kit, TaqPath RT‐PCR COVID‐19 Kit, Allplex 2019‐nCOV Assay, and BGI Real‐time RT‐PCR kit for detecting SARS CoV‐2.

Considerations for Group Testing: A Practical Approach for the Clinical Laboratory.

Group testing, also known as pooled sample testing, was first proposed by Robert Dorfman in 1943. While sample pooling has been widely practiced in blood-banking, it is traditionally seen as anathema for clinical laboratories. However, the ongoing COVID-19 pandemic has re-ignited interest for group testing among clinical laboratories to mitigate supply shortages. We propose five criteria to assess the suitability of an analyte for pooled sample testing in general and outline a practical approach that a clinical laboratory may use to implement pooled testing for SARS-CoV-2 PCR testing. The five criteria we propose are: (1) the analyte concentrations in the diseased persons should be at least one order of magnitude (10 times) higher than in healthy persons; (2) sample dilution should not overly reduce clinical sensitivity; (3) the current prevalence must be sufficiently low for the number of samples pooled for the specific protocol; (4) there is no requirement for a fast turnaround time; and (5) there is an imperative need for resource rationing to maximise public health outcomes. The five key steps we suggest for a successful implementation are: (1) determination of when pooling takes place (pre-pre analytical, pre-analytical, analytical); (2) validation of the pooling protocol; (3) ensuring an adequate infrastructure and archival system; (4) configuration of the laboratory information system; and (5) staff training. While pool testing is not a panacea to overcome reagent shortage, it may allow broader access to testing but at the cost of reduction in sensitivity and increased turnaround time.

Pooled Sample Testing with Clustering (For Efficient COVID-19 Screening)

Testing medical samples is expensive and pooling samples can reduce costs. But what is the optimal setup? What is the optimal pool size? We consider the simplest and most practical pooling technique and demonstrate how the correlation between samples reduces the testing cost and increases the size of the optimal pool. Then we create a simple clustering model using a Dirichlet Process that could be used as a template for multiple SIR models.

Effectiveness of Sample Pooling Strategies for SARS-CoV-2 Mass Screening by RT-PCR: A Scoping Review.

The ongoing COVID-19 pandemic has hugely impacted the economy of many countries, and there is an acute shortage of diagnostic resources. With the exponential increase in the number of cases and necessity to screen large number of people, there is a steep increase in the demand for diagnostic kits. Pooled-sample testing is a promising strategy to screen a large population rapidly with limited resources. The aim of this work was to compile a cohesive literature review of the effectiveness and accuracy of pooled-sample testing in the detection of SARS-CoV-2 and critically analyze its limitations. Medline, Google Scholar, Embase, and preprint servers (e.g., bioRxiv) were searched for literature on pooled testing for diagnosis of COVID-19, and out of initial 60 articles/reports, nine original articles were retained. Optimal pool size (number of samples in a pool) seemed to be dependent on factors like prevalence or rate of positivity in community. In low-prevalence localities pool size of around 30 seemed to be effective as observed by some authors. All the researchers had found significant reduction in number of tests (depending on pool size, stages, and pooling design), leading to conservation of resources. Pooling can be done with extracted RNA eluate or directly with patient’s sample before extraction. This leads to further reduction in consumables, time and manpower. Risk of false negativity in samples with high-threshold cycle (i.e., low-viral load) value was a concern. Some researchers suggest adding few additional cycles to lower the chances of missing positive cases with low-Ct value. Lower limit of detection (LoD) of RT-PCR kits, that is, sensitivity of kits was another factor to consider. Thus, in a country like India, given the economic benefit and scarcity of resources, pooling strategy can be very effective, especially in low-prevalence areas and in low-risk contacts.

Recommendations for sample pooling on the Cepheid GeneXpert® system using the Cepheid Xpert® Xpress SARS-CoV-2 assay.

The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. In pooled sample testing, multiple samples are combined (or pooled) together and tested as a single unit. If the pool is positive, the individual samples can then be individually tested to identify the positive case(s). Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the limit of detection of this assay was comparable to laboratory-developed reverse-transcription quantitative PCR SARS-CoV-2 tests, with observed detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were pooled in groups of six. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted based on public health needs. The suggested number of samples per pool, or the pooling depth, is unique for each point-of-care testing site and can be determined by the positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.

Evaluation of pooled sample analysis strategy in expediting case detection in areas with emerging outbreaks of COVID-19: A pilot study.

Timely diagnosis of COVID-19 infected individuals and their prompt isolation are essential for controlling the transmission of SARS-CoV-2. Though quantitative reverse transcriptase PCR (qRT-PCR) is the method of choice for COVID-19 diagnostics, the resource-intensive and time-consuming nature of the technique impairs its wide applicability in resource-constrained settings and calls for novel strategies to meet the ever-growing demand for more testing. In this context, a pooled sample testing strategy was evaluated in the setting of emerging disease outbreak in 3 central Indian districts to assess if the cost of the test and turn-around time could be reduced without compromising its diagnostic characteristics and thus lead to early containment of the outbreak. From 545 nasopharyngeal and oropharyngeal samples received from the three emerging districts, a total of 109 pools were created with 5 consecutive samples in each pool. The diagnostic performance of qRT-PCR on pooled sample was compared with that of individual samples in a blinded manner. While pooling reduced the cost of diagnosis by 68% and the laboratory processing time by 66%, 5 of the 109 pools showed discordant results when compared with induvial samples. Four pools which tested negative contained 1 positive sample and 1 pool which was positive did not show any positive sample on deconvolution. Presence of a single infected sample with Ct value of 34 or higher, in a pool of 5, was likely to be missed in pooled sample analysis. At the reported point prevalence of 4.8% in this study, the negative predictive value of qRT-PCR on pooled samples was around 96% suggesting that the adoption of this strategy as an effective screening tool for COVID-19 needs to be carefully evaluated.