key: cord-1003124-7f1ffybq authors: KHODARE, A.; Padhi, A.; Gupta, E.; Agarwal, R.; Dubey, S.; Sarin, S. K. title: Optimal size of sample pooling for RNA pool testing: an avant-garde for scaling up SARS CoV 2 testing date: 2020-06-14 journal: nan DOI: 10.1101/2020.06.11.20128793 sha: e161b910c1411b6d44fb63dbb5534dda132d44cd doc_id: 1003124 cord_uid: 7f1ffybq Introduction-Timely diagnosis is essential for the containment of the disease and breaks in the chain of transmission of SARS-CoV-2. The present situation demands countries to scale up their testing and design innovative strategies to conserve diagnostic kits and reagents. The pooling of samples saves time, manpower, and most importantly diagnostic kits and reagents. In the present study, we tried to define the pool size that could be applied with acceptable confidence for testing. Material and methods- We used repeatedly tested positive clinical sample elutes having different levels of SARS CoV 2 RNA and negative sample elutes to prepare seven series of 11 pools each, having pool sizes ranging from 2 to 48 samples to estimate the optimal pool size. Each pool had one positive sample elute in different compositions. All the pools were tested by SARS CoV 2 RT-qPCR. Results- Out of the 77 pools, only 53 (68.8%) were found positive. The sensitivity of pools of 2 to 48 samples was decreased from 100% (95% CL; 98.4-100) to 41.41% (95% CL; 34.9-48.1). The maximum size of the pool with acceptable sensitivity (>95%) was found to be of 6 samples. For the pool size of 6 samples, the sensitivity was 97.8% and the efficiency of pooling was 0.38. Conclusion- The pooling of samples is a practical way for scaling up testing and ultimately containing the further spread of the COVID-19 pandemic. p a n d e m i c . What started as a cluster of pneumonia cases in Wuhan city of China has become now a fullblown pandemic, the first of its kind due to the coronavirus [1] . This pandemic popularly known as COVID-19 (coronavirus disease 2019) is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) [2] . As of 3 rd June 2020, COVID-19 had globally caused 6, 287, 771 confirmed cases and 379, 941 deaths affecting over 200 countries [3] . Timely diagnosis is essential for the containment of the disease and breaks in the chain of transmission of SARS-CoV-2. The laboratory diagnosis of SARS-CoV-2 is based primarily on nucleic acid amplification test (NAAT) like real-time reverse transcriptase PCR (RT-qPCR). As the number of COVID-19 cases is increasing every day the availability of diagnostic kits and reagents has emerged as a major bottleneck in the laboratory testing of SARS-CoV-2 [4] . The currently available testing strategies mainly focus on symptomatic individuals. But detecting the carrier or asymptomatic individuals holds the key in containing the spread of the infection into the community. Earlier the infected person is identified, sooner the spread of the infection can be contained and the surveillance machinery can be activated for contact tracing and ultimately break in the chain of transmission of the virus [5] . Densely and heavily populated countries like India and other developing countries have abysmally low test rates per capita in the world [6] and the number of cases is expected to shoot up post lockdown. So the present situation demands countries to scale up their testing and design innovative strategies to conserve diagnostic kits and reagents. The pooling of diagnostic tests, which has previously been applied in other infectious diseases [7] [8] , is one such method where samples are mixed and tested at a single pool, and subsequent individual tests are made only if the pool tests positive. Pooling saves time, manpower, and most importantly diagnostic kits and reagents. In the present study we tried to define the pool size that could be applicable to a particular population with acceptable confidence and avoiding drop of positive cases which is a major risk of pool testing. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. C o m b i n e d The copyright holder for this preprint this version posted June 14, 2020. ( T I B M O L B I O L ) . R e a c t i o n s w e r e h e a t e d t o 5 5 o C f o r 5 m i n u t e s f o r r e v e r s e t r a n s c r i p t i o n , d e n a t u r e d i n 9 5 Primers and probes for the RT-qPCR [10] Gene Oligonucleotide Sequence is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 14, 2020. . Known positive and negative sample elutes were used for making of pools, but for calculation, it was considered that if following a pooling algorithm after obtaining results of . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. u t i l i z i n g A g P a t h - I D T M O n e - S t e p R T - P C R R e a g e The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.11.20128793 doi: medRxiv preprint pool testing, the same previously obtained results will get after individual testing of samples. The result of individually tested samples was considered the gold standard for the calculation of sensitivity, specificity, negative predictive value (NPV), and positive predictive value. to 30 th April 2020 using the same PCR reagents used for pool testing. (Table-2) . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. NPV of pool testing-The NPV was defined as the probability that an individual specimen identified as negative at the end of a pooling algorithm was truly negative. The overall negative predictive value was found to be 97.8%. The NPV for a pool size of 2 and 4 was 100% and found no false negative results but for the pool sizes of 6, 8, and 10, NPV gradually decreased from 97.2 % to 95.45 % due to gradual increase in false negative results. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.11.20128793 doi: medRxiv preprint For pool sizes from 12 to 48, NPV was progressively increased due to stable false negative results but increasing pool size. PPV of pool testing-The PPV was defined as the probability that a specimen identified as positive at the end of a pooling algorithm was truly positive. The PPV of pooling was 100% and no false positive results were found. The efficiency of a pooling algorithm was defined as the expected number of NAATs required per individual specimen evaluated. An efficiency of sample pooling was calculated 0.38 using the online calculator considering the two-stage Dorfman mini-pool strategy of pool testing with the conservative predictions of a sensitivity of 95%, a specificity of 99%, 4% prevalence of SARS-CoV-2 infection and pool size of 6 samples. ((http://www.bios.unc.edu/~mhudgens/optimal.pooling.b.htm) Most positive pools reach the threshold at a later Ct as they are further diluted. A pool size of 6 samples having a sensitivity of 97.8% and NPV of 97.2% was considered acceptable for the pooling of sample elutes for RT-qPCR. While individual level nucleic acid amplification testing (NAAT) remains the gold standard for the diagnosis of COVID-19 infection, a limited supply of diagnostic kits and reagents remains the major bottleneck in expediting testing of COVID-19 in the community especially during surge testing [11] . Furthermore, mass testing of samples should be done to control the COVID-19 pandemic at the earliest [12] . Hence a testing method where a large number of samples can be tested and consuming minimal testing kits and reagent is the need of the hour. In this study effect of pool size on detection of SARS CoV 2 and its accuracy in the . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.11.20128793 doi: medRxiv preprint population was assessed. We found that a single clinical sample with SARS-CoV-2 RNA can be consistently detected in a pool of a maximum of four samples. The Ct values of individually tested RNA elute and that of the pooled group were in sync. As evident from figure 2 pooling in this pattern leads to a gradual increase in the threshold cycle of the pooled group. Furthermore, additional amplification cycles could lower the detection limit allowing better detection of pools having samples of higher Ct values [13] . The pooling of samples is essentially important in monitoring the infection in cohesive groups such as quarantine facilities, health care workers, community surveillance, and diagnosing the asymptomatic cases. The infection load in these groups may be low but even a single positive case amongst such groups can activate the surveillance system and quarantine the affected group and prevent the further spread in the community. The pooling is better suited for low prevalence populations and it could fasten the testing over a very short time that would be able to identify new hot spot areas for infection before becoming condition worst. The pooling of samples can also be done prior to RNA extraction that is at the time of sample collection, putting the nasopharyngeal/ oropharyngeal swabs into a common viral transport media (VTM) and after sample collection by pooling of VTM. By doing so, the major bottleneck of RNA extraction can be removed. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 14, 2020. . n u c l e i c a c i d s o t h a t t h e s e n s i t i v i t y d o e s n o t g e t c o m p r o m i s e d . W e d e t e r m i n e d t h e p o o l s i z e b y c o n s i d e r i n g o f a m o u n t o f v i r a l n u c l e i c a c i d ( a s C t v a l u e i s u s u a l l y p r o p o r t i o n a l t o t h e a m o u n t o f n u c l e i c a c i d ) t h a t i s m o r e p r e v a l e n t i n t h e c o m m u n i t y d u r i n g t h i s p a n d e m i all component individual specimens are tested (the second of the two stages) [16] . . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.11.20128793 doi: medRxiv preprint As the COVID-19 pandemic continues spreading there is tremendous stress on logistics. Hence pooling of samples is a practical way for scaling up testing and ultimately containing the further spread of the COVID-19 pandemic. This study suggests that a pool size of 6 samples would be optimal with acceptable confidence . sample 20 sample 24 sample 32 sample 48 sample 1 25 TD TD TD TD TD TD TD TD TD TD TD 2 29 TD TD TD TD TD TD TD TD TD TD TD 3 31 TD TD TD TD TD TD TD TD TD TD TD 4 33 TD TD TD TD TD TD TD TD TD TD TND 5 35 TD TD TD TD TND TND TND TND TND TND TND 6 38 TD TD TD TND TND TND TND TND TND TND TND 7 39 TD TD TND TND TND TND TND TND TND TND TND TND TND TND TND TND TND TND TND TND . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 14, 2020. . . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 14, 2020. . https://doi.org/10.1101/2020.06.11.20128793 doi: medRxiv preprint A Novel Coronavirus from Patients with Pneumonia in China Naming the coronavirus disease (COVID-19) and the virus that causes it The Laboratory Diagnosis of COVID-19 Infection: Current Issues and Challenges COVID-19) Situation Summary [Internet]. Centers for Disease Control and Prevention2020 People power: How India is attempting to slow the coronavirus Evaluation of the pooling of swabs for real-time PCR detection of low titre shedding of low pathogenicity avian influenza in turkeys Estimating Community Prevalence of Ocular Chlamydia trachomatis Infection using Pooled Polymerase Chain Reaction Testing Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR faces such a shortage of coronavirus test-kits that people say getting one is like "winning the lottery Covid-19 mass testing facilities could end the epidemic rapidly Evaluation of COVID-19 RT-qPCR test in multi-sample pools Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: a reliable, faster and economical method Presymptomatic SARS-CoV-2 Infections and Transmission in a Skilled Nursing Facility The Detection of Defective Members of Large Populations We acknowledge all the technical staff involved in testing and ICMR for providing the Primers and probes for the conduct of RT-q PCR.