• Ira Praharaj, Amita Jain, Mini Singh, Anukumar Balakrishnan, Rahul Dhodapkar, Biswajyoti Borkakoty, Munivenkatappa Ashok, Pradeep Das, Debasis Biswas, Usha Kalawat, Jyotirmayee Turuk, A P Sugunan, Shantanu Prakash, Anirudh K Singh, Rajamani Barathidasan, Subhra Subhadra, Jyotsnamayee Sabat, M J Manjunath, Poonam Kanta, Nagaraja Mudhigeti, Rahul Hazarika, Hricha Mishra, Kumar Abhishek, C Santhalembi, Manas Ranjan Dikhit, Neetu Vijay, Jitendra Narayan, Harmanmeet Kaur, Sidhartha Giri, and Nivedita Gupta.
• Divsion of Epidemiology & Communicable Diseases, Indian Council of Medical Research, New Delhi, India.
• Indian J Med Res. 2020 Jul 1; 152 (1 & 2): 889488-94.

Background & ObjectivesPublic 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.MethodsTen 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.ResultsA 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 & ConclusionsResults 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.

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