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Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling.
Indian J Med Res. 2020 Jul & Aug; 152(1 & 2):88-94.IJ

Abstract

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 Ctvalues ≤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.

Authors+Show Affiliations

Divsion of Epidemiology & Communicable Diseases, Indian Council of Medical Research, New Delhi, India.Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India.Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.ICMR-National Institute of Virology, Kerala Unit, Alappuzha, Kerala, India.Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India.Regional Medical Research Centre, Dibrugarh, Assam, India.ICMR-National Institute of Virology, Bangalore Unit, Bengaluru, Karnataka, India.ICMR-Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India.Department of Microbiology, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India.Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India.ICMR-Regional Medical Research Centre, Bhubaneswar, Odisha, India.ICMR-National Institute of Virology, Kerala Unit, Alappuzha, Kerala, India.Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India.Department of Microbiology, All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, India.Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India.ICMR-Regional Medical Research Centre, Bhubaneswar, Odisha, India.ICMR-Regional Medical Research Centre, Bhubaneswar, Odisha, India.ICMR-National Institute of Virology, Bangalore Unit, Bengaluru, Karnataka, India.Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India.Regional Medical Research Centre, Dibrugarh, Assam, India.Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India.ICMR-Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India.ICMR-National Institute of Virology, Kerala Unit, Alappuzha, Kerala, India.ICMR-Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India.Department of Health Research, Ministry of Health & Family Welfare, New Delhi, India.Department of Health Research, Ministry of Health & Family Welfare, New Delhi, India.Department of Health Research, Ministry of Health & Family Welfare, New Delhi, India.Divsion of Epidemiology & Communicable Diseases, Indian Council of Medical Research, New Delhi, India.Divsion of Epidemiology & Communicable Diseases, Indian Council of Medical Research, New Delhi, India.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32893844

Citation

Praharaj, Ira, et al. "Pooled Testing for COVID-19 Diagnosis By Real-time RT-PCR: a Multi-site Comparative Evaluation of 5- & 10-sample Pooling." The Indian Journal of Medical Research, vol. 152, no. 1 & 2, 2020, pp. 88-94.
Praharaj I, Jain A, Singh M, et al. Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling. Indian J Med Res. 2020;152(1 & 2):88-94.
Praharaj, I., Jain, A., Singh, M., Balakrishnan, A., Dhodapkar, R., Borkakoty, B., Ashok, M., Das, P., Biswas, D., Kalawat, U., Turuk, J., Sugunan, A. P., Prakash, S., Singh, A. K., Barathidasan, R., Subhadra, S., Sabat, J., Manjunath, M. J., Kanta, P., ... Gupta, N. (2020). Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling. The Indian Journal of Medical Research, 152(1 & 2), 88-94. https://doi.org/10.4103/ijmr.IJMR_2304_20
Praharaj I, et al. Pooled Testing for COVID-19 Diagnosis By Real-time RT-PCR: a Multi-site Comparative Evaluation of 5- & 10-sample Pooling. Indian J Med Res. 2020 Jul & Aug;152(1 & 2):88-94. PubMed PMID: 32893844.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling. AU - Praharaj,Ira, AU - Jain,Amita, AU - Singh,Mini, AU - Balakrishnan,Anukumar, AU - Dhodapkar,Rahul, AU - Borkakoty,Biswajyoti, AU - Ashok,Munivenkatappa, AU - Das,Pradeep, AU - Biswas,Debasis, AU - Kalawat,Usha, AU - Turuk,Jyotirmayee, AU - Sugunan,A P, AU - Prakash,Shantanu, AU - Singh,Anirudh K, AU - Barathidasan,Rajamani, AU - Subhadra,Subhra, AU - Sabat,Jyotsnamayee, AU - Manjunath,M J, AU - Kanta,Poonam, AU - Mudhigeti,Nagaraja, AU - Hazarika,Rahul, AU - Mishra,Hricha, AU - Abhishek,Kumar, AU - Santhalembi,C, AU - Dikhit,Manas Ranjan, AU - Vijay,Neetu, AU - Narayan,Jitendra, AU - Kaur,Harmanmeet, AU - Giri,Sidhartha, AU - Gupta,Nivedita, PY - 2020/9/8/pubmed PY - 2020/9/30/medline PY - 2020/9/7/entrez KW - COVID-19 KW - Concordance KW - E KW - SARS-CoV-2 KW - gene KW - pooling KW - real-time RT-PCR KW - sensitivity SP - 88 EP - 94 JF - The Indian journal of medical research JO - Indian J Med Res VL - 152 IS - 1 & 2 N2 - 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 Ctvalues ≤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. SN - 0971-5916 UR - https://www.unboundmedicine.com/medline/citation/32893844/Pooled_testing_for_COVID_19_diagnosis_by_real_time_RT_PCR:_A_multi_site_comparative_evaluation_of_5__&_10_sample_pooling_ L2 - http://www.ijmr.org.in/article.asp?issn=0971-5916;year=2020;volume=152;issue=1;spage=88;epage=94;aulast=Praharaj DB - PRIME DP - Unbound Medicine ER -