key: cord-291729-4l4v9jxd authors: de Salazar, Adolfo; Aguilera, Antonio; Trastoy, Rocio; Fuentes, Ana; Alados, Juan Carlos; Causse, Manuel; Galán, Juan Carlos; Moreno, Antonio; Trigo, Matilde; Pérez-Ruiz, Mercedes; Roldán, Carolina; José Pena, Ma; Bernal, Samuel; Serrano-Conde, Esther; Barbeito, Gema; Torres, Eva; Riazzo, Cristina; Cortes-Cuevas, Jose Luis; Chueca, Natalia; Coira, Amparo; Sanchez-Calvo, Juan M.; Marfil, Eduardo; Becerra, Federico; Gude, María José; Pallarés, Ángeles; Pérez Del Molino, María Luisa; García, Federico title: Sample pooling for SARS-COV-2 RT-PCR screening date: 2020-09-10 journal: Clin Microbiol Infect DOI: 10.1016/j.cmi.2020.09.008 sha: doc_id: 291729 cord_uid: 4l4v9jxd OBJECTIVE: To evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of COVID-19, by using different commercial platforms for nucleic acid extraction and amplification. METHODS: 3519 nasopharyngeal samples received at 9 Spanish clinical microbiology laboratories were processed individually and in pools (342 pools of 10 samples and 11 pools of 9 samples) according to the existing methodology in each of the centres. RESULTS: We 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: in 22/29 pools (28 samples), minor discordances were found; for seven pools (7 samples), we found major discordances. Sensitivity, specificity, positive and negative predictive values for pooling were 97.10% (CI95%; 94.11-98.82), 100%, 100% and 99.79% (CI95%; 99.56-99.90) respectively; accuracy was 99.80% (CI95%; 99.59-99.92) and kappa concordant coefficient was 0.984. The dilution of samples in our pooling strategy resulted into a median loss of 2.87 (CI95%; 2.46-3.28) CTs for E gene, 3.36 (CI95%; 2.89-3.85) CTs for RdRP gene and 2.99 (CI95%; 2.56-3.43) CTs for N gene. CONCLUSION: we show a high efficiency of pooling strategies for 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. SARS-CoV-2 pandemic has posed an immense challenge for the National Health Systems of the affected countries. In the absence of a vaccine or effective treatment, molecular diagnosis is the only tool to contain the pandemic, identifying the transmitting infected patients, and proceed to their isolation to avoid new infections. Due to its high demand, testing opportunities may have been hampered in some scenarios, as a consequence of the lack of reagent supplies, and their limited production. The current challenge for SARS-CoV-2 diagnosis is the great demand of testing that we are facing in the new test and trace era. Clinical laboratories must plan to increase their analytical capacity to face these new public health challenges that will allow, by means of massive analysis, to identify all infected persons, proceed with their isolation and trace their contacts. This undoubtedly constitutes a challenge due to the high number of diagnostic processes required and the limited resources available in the face of a disease with a variable incubation period, an uncertain viral dynamic [1, 2] and an unknown number of asymptomatic carriers who can transmit the infection. Dorfman in 1943 [3] introduced the strategy of mixing samples in a single test into clinical diagnosis, and this strategy has been helpful in correctly identifying all infected individuals using fewer diagnostic tests [4, 5] . The diagnosis of SARS-CoV-2 infection is fundamentally based on real-time RT-PCR, which is the reference technique (6, 7) . This is a robust technology with high sensitivity and specificity and has already been used in the pooling strategy of samples for the screening of HIV, HBV and HCV (8) (9) (10) (11) , where it has proven to be cost-effective and efficient in surveillance and diagnosis (detection) for prevalence below 30%, regardless of the population studied. Therefore, the combination of pooled tests and patients with low risk of infection is considered a practical and effective method to analyse large quantities of samples without compromising precision, especially when it comes to centralized models with automated systems. In SARS-CoV-2 infection, the data on the best strategy for the detection of cases by grouping of samples and how they influence the sensitivity of the RT-PCR analysis are limited, therefore, it is necessary to investigate the effect of the number of samples, especially with commercially available assays [12] [13] [14] [15] [16] [17] . Our objective in this study has been to evaluate the efficacy of sample pooling in a multicentre way compared to the individual analysis for the detection of COVID-19 by using different commercial platforms available for genomic extraction and amplification by RT -PCR in real time. J o u r n a l P r e -p r o o f Between March and May 2020, nasopharyngeal swabs (n = 3519) were collected from patients or health professionals and sent to the virology laboratories of the participating centres (Supplementary table 1) . Nasopharyngeal and pharyngeal swab were collected at a time and both swabs were placed in the same tube with transport media. Several viral transport mediums were used: Vircell Transport Medium (Vircell -(Granada, Spain)), δswab® Transport Medium (Deltalab -(Rubí, Spain)) and UTM®: Universal Transport (Copan -(Brescia, Italy). Samples were processed on the first 24 hours upon reception. Nine or ten individual samples were pooled, and screening was performed using reverse transcriptase-polymerase chain reaction targeting the same target as for individual samples. Pooled testing was performed at each of the participating sites in the study. Pooling was performed by hand, after inactivation of each sample that made part of the pool. Samples for pooling were selected randomly, according to availability at each site during the study period. For both individual testing and pooled analysis, samples were inactivated 1:1 in lysis buffer and processed according to the existing methodology in each laboratory, which is summarized in Table 1 . Major discordance was defined as a negative pool result when at least one of the individual samples showed Ct values <35 for one or more SARS-COV-2 genes. Minor discordant calls were considered when at least one individual sample had a Ct value >35 in one or two of the SARS-COV-2 genes assayed and the pool scored negative. The performance characteristics such as sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and relative efficiency was calculated comparing the individually analysed sample (gold standard) with respect to the sample result analysed within the pool. Statistics were performed on R Studio and Graph Pad Prism V8 software. This study was approved by "Comité de Ética de la Investigación con medicamentos de Galicia (CEIm-G)" review board. Given the deidentified nature of testing, individual patient consent was not required for this study. The study included 3519 samples from 9 different sites in Spain. We analyzed all samples individually and also in parallel, pooled into 353 groups of samples (342 pools of 10 samples and 11 pools of 9 samples). Two hundred and forty-one (6.85%) of the samples were positive. We found that 253 pools, made up of 2519 samples, were negative (242 pools of 10 samples and 11 pools of 9 samples); 99 pools, made up of 990 samples, were positive (99 pools of 10 samples). One pool, made of 10 samples, was invalid. Therefore, our pooling strategy would have saved 2167 (86%) PCR tests. A description of positive and negative pools can be found in table 2. Overall, 323 pools with 3219 samples showed concordant results with the individual samples analyzed (224 pools with 2229 samples negative and 99 pools with 990 samples that included at least one positive sample). For 29 pools (290 samples) we found discordant results compared to individual samples. In 22 pools minor discordances were found and in 7 pools we found major discordances. A detailed description of the discordances can be found in Supplementary Table 2 , where it can be seen that the pools with minor discordances included 24 samples from patients who had a prior positive SARS-CoV 2 test and were submitted for PCR testing at least 20 days after to evaluate RNA clearance. When only major discordances were considered for the analysis, sensitivity, specificity, positive and negative predictive values for pooling were 97.10% (CI95%; 94.11-98.82), 100%, 100% and 99.79% (CI95%; 99.56-99.90) respectively. Accuracy was also 99.80% (CI95%; 99.59-99.92) and kappa concordant coefficient was 0.984. These data are detailed in Table 3 . When all discordances were considered for the analysis, sensitivity, specificity, positive and negative predictive values for pooling were 85.48% (CI95%; 80.39-89.67), 100%, 100% and 98.94% (CI95%; 98.57-99.22) respectively. Accuracy was 99.00% (CI95%; 98.62-99.30) and kappa concordant coefficient was 0.916. These data are detailed in Table 4 . Supplementary Table 3 shows the number and rate of discrepancies across the different tests and targets used at the participating sites. The lowest rate of discrepancies was observed for the Viasure SARS-CoV-2 Real Time PCR (CerTest) (n=3; Here we report on the high efficiency of pooling strategies for 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. We believe that our results may help clinical laboratories to respond to the clinical need on SARS-CoV-2 testing. Although sample pooling strategy works for other pathogens that are diagnosed by RT-PCR, for SARS-CoV-2 infection, there is still limited data in the literature regarding surveillance and detection strategies by grouping samples [12] [13] [14] [15] [16] [17] . In our study we have evaluated the efficacy of sample pooling in a multicentre way compared to the individual analysis for the detection of COVID-19 by using different commercial platforms available for genomic extraction and amplification by RT-PCR in real time. Within a 6.8% positive rate, we have obtained excellent numbers in sensitivity, specificity, and positive and negative predictive values, both in a scenario for which only major discordances were considered (97.10%, 100%, 100%, 99.79%, respectively) and also when minor discordances were counted (85.48%. 100%, 100% and 98.94%). As expected, and as other authors have also shown [18] , the dilution of samples in our pooling strategy resulted into a median loss of 2.87 CTs for E gene, 3.36 CTs for RdRP gene and 2.99 CTs for N gene. This drop in the sensitivity was responsible for most of the discordances found in our study, that were mainly observed for samples with the lowest positivity signals, always with CTs very close to 40, and very frequently in only one gene of the two-three that were included in the tests. Although special attention on RT-PCR false negative results must be paid [19] , it is also known that most of the positive results obtained from just one gene targeted and with CTs >35 correspond to non-viable/noninfectious particles that are still detected by RT-PCR [20] . In addition, false positive results yielding CTs>35 may also be expected. Our study's main limitation was the variability upon extraction and amplification methods used, and the number of samples included in the different pools tested; however, this limitation in the design may turn into its main strength given the consideration that even in this scenario our results were excellent. In the sample pooling strategy, it is a priority to determine the group size in which the maximum precision is maintained in the analysis performed, since, due to the dilution of the sample, this procedure can decrease the sensitivity of the molecular assays of RT-Realtime PCR "Optimization of group size in pool testing strategy for SARS-CoV-2: A simple mathematical model" [21] . For this reason, before systematically implementing a sample grouping strategy, it is important to consider these characteristics (detection limit, sensitivity and specificity of the test) together with the expected prevalence; in this regard, there are already applications that allow it to be calculated (https://www.chrisbilder.com/shiny/); in addition, and in depth mathematical analysisof pool testing by Cherif et al. is also available [22] . The main advantages of the pooling strategy are that it allows using the same standard protocols of commercial reagents, with no need of additional training, equipment or materials, and consequently it can be implemented immediately to expand the detection and surveillance capabilities of COVID-19. Another limitation to our study is that samples were pooled by hand. We are currently evaluating the automation of pooling samples, as this is a pre-analytical critical step to be solved to handle the great number of samples that the pooling strategy will allow to process. Result reporting is another critical step that needs to be addressed. Finally, we did not collect information on clinical data/symptoms; we believe that a great number of patients, if not all, would be symptomatic, as asymptomatic screening has not been a part of clinical practice in Spain during the pandemic first wave. It should be noted that pooling as a screening strategy will not completely eliminate the need for individual diagnostic tests, which will be essential when community transmission intensifies. In our study, even in the setting of a 6.86% prevalence, out of 3519 samples analysed, we would have saved a total of 2167 PCR tests, with a great saving in time, costs and personnel. Within the current epidemiological situation, in which prevalence has significantly decreased but the high demand on PCR testing continues, this efficiency measures would help clinical laboratories in alleviating the workload in order to prepare for future outbreaks. In summary, we show a high efficiency of pooling strategies for 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. Whether pooling may be used highly relies on SARS-COV-2 prevalence, rather on the fact that patients are asymptomatic (prevalence may be high if community transmission has been reached) or symptomatic. Specific recommendations for pooling testing have become available very recently from FDA (https://www.fda.gov/newsevents/press-announcements/coronavirus-covid-19-update-fda-issues-first-emergencyauthorization-sample-pooling-diagnostic). As we believe our findings may be essential to expand clinical laboratories capabilities in the very near future, we recommend to validate this strategy in each specific setting of extraction and amplification reagents before its introduction for clinical management, specially to ensure that sensitivity of the assay and especially the false negative rates are acceptable. 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