Interpretation of pooled sample testing of COVID-19 for efficient use of resources

Sir,

We read with interest the article by Prahraj et al1. The authors have concluded that the pooling of five COVID-19 suspected samples for SARS-CoV-2 detection by real-time reverse transcription polymerase chain reaction (RT-PCR) may be an acceptable strategy without much loss of sensitivity (88%) even for low viral loads compared to higher number of false negatives with 10-sample pools. Based on the current evidence, we would like to offer our additional inputs on the interpretation of the five-sample pool testing strategy for efficient detection of cases to limit missing out of positive cases.

In India, the first case of COVID-19 was reported on January 30, 2020 from Kerala2. Following which, a seropositivity rate of 0.73 per cent was observed (6,468,388 adult infections) during May 11 to June 4, 20203. As of now (April 13, 2021), a total of 13,689, 453 cases with 1.25 per cent mortality is reported4. The percentage of positivity is still low (7.0%). Hence, the pooling strategy of five samples will be more helpful in country like India having a large population with minimal resources.

Prahraj et al1, have shown that an average Ct value obtained with the five-sample pools exceeds an individual sample testing by 2.18±1.86 cycles, while Ct value obtained with 10-sample pooling exceeds an individual sample testing by 3.81±2.26 cycles. At ICMR-Regional Medical Research Centre, Gorakhpur, India, a total of 206,232 samples were tested for SARS-CoV-2, with around 2.5 per cent positivity. Ct value of 100 pools of five-sample pools and their respective individual samples were analyzed. It was observed that an average Ct value obtained with the five-sample pooled testing exceeded an individual sample testing by 2.62±1.40 and 2.76±1.63 with E and ORF1ab genes, respectively (unpublished data), which was concordant with the findings of Prahraj et al1.

Hence, while determining the positive pools for segregation, the cut-off value should be adjusted as RT-PCR kit recommended Ct cut-off value plus four cycles in case of five-sample pools for interpretation of the real-time RT-PCR results. This recommendation may help reduce the possibility of missing COVID-19–positive cases. An elucidation from the authors about the results and interpretations taking into account the above observations will benefit the scientific community.