Intricacies in characterizing positivity in pooled sample testing for SARS‐CoV‐2

Abstract

The unprecedented demand for testing for the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 has led to an acute shortage and limited availability of test reagents for which pooling of samples has been recommended in areas with low prevalence. Con-

SARS-CoV-2 RT-qPCR testing of pooled saliva samples: a case study of 824 asymptomatic individuals and a questionnaire survey in Japan

“…Their study highlighted the importance of catching late amplification to avoid missing positive samples. When carefully reviewing the PCR curves from pooled testing for case 2, we observed late amplification patterns at the end of the reaction for N1, as described by Mohanty et al (64).…”

“…Their study highlighted the importance of catching late amplification to avoid missing positive samples. When carefully reviewing the PCR curves from pooled testing for case 2, we observed late amplification patterns at the end of the reaction for N1, as described by Mohanty et al(64).We also encountered a relatively high frequency of PCR inhibition in individual tests, defined as Ct values of IC being unmeasurable or above 40, or by no sigmoid PCR amplification pattern. Saliva can contain inhibitors that impair nucleic acid amplification…”

“…Mohanty et al showed that considering late amplification while interpreting the results of pooled samples allowed the identification of additional positives (64). They first used criteria for positivity (Ct within 35 with a sigmoid curve) for 4-sample pools, but also included an additional class of ‘probably positive’ (Ct>35 with non-sigmoid amplification curve, or increased amplification at the end of the reaction).…”

“…In both of the positive cases in our study, Ct values in individual testing were higher than 30, and 10-sample pools failed to produce any Ct values in case 2. Given the decrease in sensitivity in 10-sample pools, and with the two individual positive cases having high Ct values, it is probable that this case would have been undetected in a 10-sample pool if we had not performed simultaneous individual testing.Mohanty et al showed that considering late amplification while interpreting the results of pooled samples allowed the identification of additional positives(64). They first used criteria for positivity (Ct within 35 with a sigmoid curve) for 4-sample pools, but also included an additional class of 'probably positive' (Ct >35 with non-sigmoid amplification curve, or increased amplification at the end of the reaction).…”

SARS-CoV-2 RT-qPCR testing of pooled saliva samples: A case study of 824 asymptomatic individuals and a questionnaire survey in Japan

“…Mohanty et al showed that considering late amplification while interpreting the results of pooled samples allowed the identification of additional positives [ 61 ]. They first used criteria for positivity (Ct within 35 with a sigmoid curve) for 4-sample pools, but also included an additional class of ‘probably positive’ (Ct > 35 with non-sigmoid amplification curve, or increased amplification at the end of the reaction).…”