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Year : 2020  |  Volume : 152  |  Issue : 1  |  Page : 88-94

Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling

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

Correspondence Address:
Nivedita Gupta
Division of Epidemiology & Communicable Diseases, Indian Council of Medical Research, Ansari Nagar, New Delhi 110 029
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmr.IJMR_2304_20

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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.

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