Pooled nasopharyngeal swab collection in a single vial for the diagnosis of SARS CoV-2 infection: An effective cost saving method

BackgroundPool testing is one of the strategy to expedite testing capacities while simultaneously conserving various diagnostic kits, reagents and consumables and time. In the present study, we investigated potential role of combined specimen collection technique for the diagnosis of SARS-CoV-2 virus infection where five nasopharyngeal swabs were collected from different individuals and pooled together in a single viral transport medium (VTM).Material and methodsThis pilot study was conducted on different cohorts of Delhi state. Two nasopharyngeal swabs were collected from each enrolled individual. One swab was put into VTM vial to be further used for individual swab testing (ID). The other swab was put into a fresh VTM for pool swab collection. Each pool comprised five swabs collected from five different patients in one VTM vial. Both IDs and pools were tested in parallel for the detection of SARS-CoV-2 using real time PCR.ResultsA total of 46 pools were collected from 230 enrolled individuals.Among 230 ID tested, 60 were found to be positive for both E and RdRp gene. Among 46 pools, 25 pools included all negatives samples and remaining 21 pools included one or more positives. Comparing ID with pool results, overall concordance was seen in 42 pools (91.3%). Four pools showed false positive results as all included samples on ID testing were found to be negative. Considering ID results as reference, swab pool showed 100% sensitivity, 84% specificity, 84% positive predictive value and 100% negative predictive value.ConclusionThe pooling of swab strategy could be beneficial only among asymptomatic in low prevalence areas.     

Laboratory testing of SARS‐CoV,MERS‐CoV,and SARS‐CoV‐2 (2019‐nCoV): Current status,challenges, and countermeasures

Emerging and reemerging infectious diseases are global public concerns. With the outbreak of unknown pneumonia in Wuhan, China in December 2019, a new coronavirus, SARS‐CoV‐2 has been attracting tremendous attention. Rapid and accurate laboratory testing of SARS‐CoV‐2 is essential for early discovery, early reporting, early quarantine, early treatment, and cutting off epidemic transmission. The genome structure, transmission, and pathogenesis of SARS‐CoV‐2 are basically similar to SARS‐CoV and MERS‐CoV, the other two beta‐CoVs of medical importance. During the SARS‐CoV and MERS‐CoV epidemics, a variety of molecular and serological diagnostic assays were established and should be referred to for SARS‐CoV‐2. In this review, by summarizing the articles and guidelines about specimen collection, nucleic acid tests (NAT) and serological tests for SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2, several suggestions are put forward to improve the laboratory testing of SARS‐CoV‐2. In summary, for NAT: collecting stool and blood samples at later periods of illness to improve the positive rate if lower respiratory tract specimens are unavailable; increasing template volume to raise the sensitivity of detection; putting samples in reagents containing guanidine salt to inactivate virus as well as protect RNA; setting proper positive, negative and inhibition controls to ensure high‐quality results; simultaneously amplifying human RNase P gene to avoid false‐negative results. For antibody test, diverse assays targeting different antigens, and collecting paired samples are needed.     

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.     

Diagnostic efficacy of anti‐SARS‐CoV‐2 IgG/IgM test for COVID‐19: A meta‐analysis

The serological testing of anti‐SARS‐CoV‐2 immunoglobulin G (IgG) and/or IgM is widely used in the diagnosis of COVID‐19. However, its diagnostic efficacy remains unclear. In this study, we searched for diagnostic studies from the Web of Science, PubMed, Embase, CNKI, and Wanfang databases to calculate the pooled diagnostic accuracy measures using bivariate random‐effects model meta‐analysis. As a result, 22 from a total of 1613 articles, including 2282 patients with SARS‐CoV‐2 and 1485 healthy persons or patients without SARS‐CoV‐2, were selected for a meta‐analysis. Pooled sensitivity, specificity, and area under curve of the summary receiver operator curve (SROC) were: (a) 0.85 (95% confidence interval [CI]: 0.79‐0.90), 0.99 (95% CI: 0.98‐1.00), and 0.99 (95% CI: 0.97‐0.99) for anti‐SARS‐CoV‐2 IgG and (b) 0.74 (95% CI: 0.65‐0.81), 0.99 (95% CI: 0.97‐1.00), and 0.95 (95% CI: 0.93‐0.97) for IgM. A subgroup analysis among detection methods indicated the sensitivity of IgG and IgM using enzyme‐linked immunosorbent assay were slightly lower than those using gold immunochromatography assay (GICA) and chemiluminescence immunoassay (P > .05). These results showed that the detection of anti‐SARS‐CoV‐2 IgG and IgM had high diagnostic efficiency to assist the diagnosis of SARS‐CoV‐2 infection. And, GICA might be used as the preferred method for its accuracy and simplicity.     

Sample pooling for SARS-CoV-2 RT-PCR screening

ObjectiveTo evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of coronavirus disease 2019 (COVID-19) by using different commercial platforms for nucleic acid extraction and amplification.MethodsA total of 3519 nasopharyngeal samples received at nine Spanish clinical microbiology laboratories were processed individually and in pools (342 pools of ten samples and 11 pools of nine samples) according to the existing methodology in place at each centre.ResultsWe found that 253 pools (2519 samples) were negative and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 PCR tests. For 29 pools (made out of 290 samples), we found discordant results when compared to their correspondent individual samples, as follows: in 22 of 29 pools (28 samples), minor discordances were found; for seven pools (7 samples), we found major discordances. Sensitivity, specificity and positive and negative predictive values for pooling were 97.10% (95% confidence interval (CI), 94.11–98.82), 100%, 100% and 99.79% (95% CI, 99.56–99.90) respectively; accuracy was 99.80% (95% CI, 99.59–99.92), and the kappa concordant coefficient was 0.984. The dilution of samples in our pooling strategy resulted in a median loss of 2.87 (95% CI, 2.46–3.28) cycle threshold (C_t) for E gene, 3.36 (95% CI, 2.89–3.85) C_t for the RdRP gene and 2.99 (95% CI, 2.56–3.43) C_t for the N gene.ConclusionsWe found a high efficiency of pooling strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA testing across different RNA extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity and positive and negative predictive values.     

Serum pooling for rapid expansion of anti-SARS-CoV-2 antibody testing capacity

ObjectivesExamine possible pooling strategies designed to expand SARS-CoV-2 serological testing capacity.MethodsNegative pools were assessed to determine optimal optical density (OD) cutoffs, followed by spiking weak or strong positive samples to assess initial assay performance. Samples were then randomly subjected to pool and individual testing approaches.ResultsSingle positive specimens consistently converted pools of 5, 10, or 20 into positive outcomes. However, weaker IgG-positive samples failed to similarly convert pools of 50 to a positive result. In contrast, a stronger individual positive sample converted all pools tested into positive outcomes. Finally, examination of 150 samples configured into pools of 5, 10, 20 or 50 accurately predicted the presence of positive or negative specimens within each pool.ConclusionsThese results suggest that pooling strategies may allow expansion of serological testing capacity. While limitations exist, such strategies may aid in large-scale epidemiological screening or identification of optimal convalescent plasma donors.     

Real-life validation of a sample pooling strategy for screening of hepatitis C

ObjectivesTo test a real-life sample pooling screening strategy which contributes to increasing the diagnostic capacity of clinical laboratories and expanding access to massive screening of hepatitis C.MethodsAfter evaluating the sensitivity of the pooling strategy for seven different commercial assays which are used to determine the concentration of hepatitis C virus (HCV)-RNA in the plasma or serum, consecutive samples submitted for HCV diagnosis during the first 3 weeks of November 2021 were tested for HCV antibodies and, in parallel and in a blinded way, were pooled into 100 samples and tested for HCV-RNA. When the result was positive, a strategy to un-mask the positive(s) pool(s), which needed up to 15 total HCV-RNA tests, was used.ResultsAll platforms were able to detect the presence of HCV-RNA in a single sample from a patient with viremic HCV present in pools of up to at least 10 000 HCV-RNA-free samples. A total of 1700 samples (17 pools) were analysed, with an overall prevalence of anti-HCV and HCV-RNA of 0.24%. After pooling, we could detect all samples previously detected using standard diagnosis tests (reflex testing) with a specificity and sensitivity of 100% (CI, 99.78–100%). Given the median current prices of anti-HCV and HCV-RNA on the market in Spain as well as personnel costs, testing using the pooling strategy would have resulted in a save of 3320€.ConclusionsHere, we demonstrated that by improving cost effectiveness, with no loss of sensitivity and specificity, the strategy of pooling samples may serve as an appropriate tool for use in large-scale screening of HCV.     

Pooling cervical swabs and testing by ligase chain reaction are accurate and cost-saving strategies for diagnosis of Chlamydia trachomatis

Specimen pooling to achieve efficiency when testing urine specimens for Chlamydia trachomatis nucleic acids has been suggested. We pooled endocervical swabs from 1,288 women and also tested individual swabs by ligase chain reaction (LCR). Out of 53 positive specimens, pools of 4 or 8 specimens missed two positives, providing 96.2% accuracy compared to individual test results. Dilution and positive-control spiking experiments showed that negative specimens with inhibitors of LCR in the pool reduced the signal. Conversely, two extra positives, detected only through pooling, were negative by individual testing but became positive after storage, suggesting that fresh positive specimens with labile inhibitors may be positive in a pool because of dilution of inhibitors. For this population of women with a 4% prevalence of C. trachomatis infection, substantial savings in cost of reagents (55 to 63%) and technologist time (50 to 63%) made pooling strategies a desirable alternative to individual testing.     

Seropositivity of Anti-SARS CoV2 IgG antibodies in health care workers of an Indian tertiary care hospital during COVID-19

PurposeHealth care workers [HCW] are at a higher risk of infection SARS CoV2 infection due to frequent and close contact to patients with COVID-19.MethodsSerum samples from 500 HCW's were tested for SARS CoV2 IgG antibodies in October 2020. A questionnaire was used to collect demographic and clinical data. All these HCWs were tested for COVID-19, in 2nd week of September 2020, as a hospital policy.ResultsAnti SARS CoV2 antibodies were detected in 128/ 500 [25.6%] HCWs. A total of 195/ 500 [39%] enrolled cases had already tested positive for Covid-19 at least once in last six months by RT-PCR. Sixty eight percent of HCWs with previous COVID-19 positivity by RT- PCR tested positive for Anti SARS CoV2 antibodies, whereas only 2.76% of asymptomatic HCWs tested positive. Of 121 anti SARS-CoV-2 IgG positive persons, 70 [57.85%] had CT value ?< ?25. Low CT value and asymptomatic cases had a strong reverse statistically significant association with SARS CoV2 IgG antibody positivity.ConclusionsWe report that sero-conversion rate in HCWs is similar to that in general population suggesting that preventive practices used in hospitals are satisfactory. Cases with low viral counts in respiratory sample and asymptomatic cases have lower rate of seroconversion.     

Optimizing screening for acute human immunodeficiency virus infection with pooled nucleic acid amplification tests

Recent studies have shown the public health importance of identifying individuals with acute human immunodeficiency virus infection (AHI); however, the cost of nucleic acid amplification testing (NAAT) makes individual testing of at-risk individuals prohibitively expensive in many settings. Pooled NAAT (or group testing) can improve efficiency and test performance of testing for AHI, but optimizing the pooling algorithm can be difficult. We developed simple, flexible biostatistical models of specimen pooling with NAAT for the identification of AHI cases; these models incorporate group testing theory, operating characteristics of biological assays, and a model of viral dynamics during AHI. Pooling algorithm sensitivity, efficiency (test kits used per individual specimen evaluated), and positive predictive value (PPV) were modeled and compared for three simple pooling algorithms: two-stage minipools (D2), three-stage hierarchical pools (D3), and square arrays with master pools (A2m). We confirmed the results by stochastic simulation and produced reference tables and a Web calculator to facilitate pooling by investigators without specific biostatistical expertise. All three pooling strategies demonstrated improved efficiency and PPV for AHI case detection compared to individual NAAT. D3 and A2m algorithms generally provided better efficiency and PPV than D2; additionally, A2m generally exhibited better PPV than D3. Used selectively and carefully, the simple models developed here can guide the selection of a pooling algorithm for the detection of AHI cases in a wide variety of settings.     

Screening for HBV, HCV and HIV genomes in blood donations: shortcomings of pooling revealed by a multicentre study simulating real-time testing.

This study was undertaken in order to determine whether screening of viremic blood donations by testing of pooled donor samples could constitute a technically feasible transfusional safety measure. A pilot study of real-time simulation, on a day-to-day basis, of screening of three viral genomes (hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV)) was conducted by five French Blood Centers on plasma samples collected from blood donors and studied within undiluted samples and within sample pools of various sizes. This study was carried out within time conditions compatible with the release of platelets. For the detection of HCV and HIV genomes, the five laboratories achieved a sensitivity that decreased with the size of the sample pool. Four were successful in detecting all undiluted samples. In the 1/10 diluted samples, four failed to detect one HIV or HCV sample. In the 1/100 diluted samples, all laboratories failed to detect one or more HIV or HCV samples. For HBV genome, no participating laboratories detected all of the samples of the panel, even undiluted samples, and the sample pooling considerably affected sensitivity. The improvement and standardization of assays needs to be attained, and training of laboratories appears to be a step crucial for routine screening of viral genomes in blood donations.     

Evaluation of human immunodeficiency virus seroprevalence in population surveys using pooled sera.

The pooling of individual serum samples to determine human immunodeficiency virus (HIV) seropositivity was examined to assess whether testing pooled sera was technically feasible, cost-effective, and accurate for estimating seroprevalence in large population surveys. The sensitivities and specificities of three commercially available HIV enzyme-linked immunosorbent assay (ELISA) kits were tested using 65 serum pools of 15 individual serum samples each (975 total serum samples) at two different dilutions. With pooled sera, the Organon Teknika Bio-EnzaBead ELISA at half the dilution recommended by the manufacturer showed the best agreement with ELISA and Western blot results of individual sera. In subsequently testing 92 pools, each containing 15 individual serum samples from a population of American patients attending a sexually transmitted diseases clinic, the estimated seroprevalence was 5.27 compared with 4.93% in a test of 1,380 individual serum samples and 5.19% in a test of 4,028 individual serum samples from the same population. In an evaluation of 1,380 African patients using 10 serum samples per pool, the estimated seroprevalence was 5.79 compared with 6.16% in a test of individual sera. These results indicate that ELISA testing with pooled sera is highly sensitive and specific and appears to be a cost-effective means for estimating HIV seroprevalence in large population-based surveys.     

Use of colony pools for diagnosis of enterotoxigenic Escherichia coli diarrhea.

Diagnosis of enterotoxigenic Escherichia coli diarrhea was made in 109 adult males with an acute dehydrating cholera-like syndrome in Dacca, Bangladesh, by testing 10 colonies isolated from admission stool specimens for production of heat-labile and heat-stable toxins. Toxin testing of one colony yielded a diagnosis in 92% of the cases, testing of two colonies yielded a diagnosis in 95% of the cases, testing of a pool of 5 colonies yielded a diagnosis in 95% of the cases, and testing of a pool of 10 colonies yielded a diagnosis in 96% of the cases. From stool cultures obtained on subsequent days, toxin testing of individual colonies and pools revealed diminished efficacy of pooling with decreasing numbers of enterotoxin-positive isolates in the pool. To detect the presence of enterotoxigenic E. coli in stools, toxin testing of 5 individual isolates and a pool of 10 colonies was found to be almost as effective as the testing of 10 individual isolates.