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

• Published in: Indian journal of medical research (New Delhi, India : 1994) Vol. 152; no. 1; pp. 88 - 94
• Main Authors: Praharaj, Ira, Jain, Amita, Singh, Mini, Balakrishnan, Anukumar, Dhodapkar, Rahul, Borkakoty, Biswajyoti, Ashok, Munivenkatappa, Das, Pradeep, Biswas, Debasis, Kalawat, Usha, Turuk, Jyotirmayee, Sugunan, A, Prakash, Shantanu, Singh, Anirudh, Barathidasan, Rajamani, Subhadra, Subhra, Sabat, Jyotsnamayee, Manjunath, M, Kanta, Poonam, Mudhigeti, Nagaraja, Hazarika, Rahul, Mishra, Hricha, Abhishek, Kumar, Santhalembi, C, Dikhit, Manas, Vijay, Neetu, Narayan, Jitendra, Kaur, Harmanmeet, Giri, Sidhartha, Gupta, Nivedita
• Format: Journal Article
• Language: English
• Published: India Wolters Kluwer India Pvt. Ltd 01-01-2020; Medknow Publications and Media Pvt. Ltd; Scientific Scholar; Wolters Kluwer - Medknow
• Subjects: Betacoronavirus - genetics; Betacoronavirus - isolation & purification; Betacoronavirus - pathogenicity; Clinical Laboratory Techniques; Comparative analysis; Coronavirus Infections - diagnosis; Coronavirus Infections - epidemiology; Coronavirus Infections - genetics; Coronavirus Infections - virology; Coronaviruses; COVID-19; COVID-19 diagnostic tests; COVID-19 Testing; COVID-19 Vaccines; Diagnosis; Diagnostic Tests, Routine - methods; Female; Humans; India - epidemiology; Male; Original; Pandemics; Pneumonia, Viral - diagnosis; Pneumonia, Viral - epidemiology; Pneumonia, Viral - genetics; Pneumonia, Viral - virology; Real-Time Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral - genetics; RNA, Viral - isolation & purification; SARS-CoV-2; Serologic Tests; Severe acute respiratory syndrome coronavirus 2; Specimen Handling; Testing laboratories; Viral Load - genetics; India; COVID-19; SARS-CoV-2; pooling; E; gene; Concordance; sensitivity; real-time RT-PCR
• ISSN: 0971-5916; 0975-9174
• DOI: 10.4103/ijmr.IJMR_2304_20

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.