Challenges and issues of SARS-CoV-2 pool testing

We read with interest Stefan Lohse and colleagues' Correspondence about sample pooling for testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in asymptomatic people.¹ Some of the findings Lohse and colleagues report do not seem to be consistent with other research results2, 3 nor our experiences.

In panels C and D of the figure in Lohse and colleagues' letter,¹ which show the three pooled samples, there is one positive sample in 30 negative samples in each pool, and the pooled samples show lower Ct values than do single samples, which suggests the RNA concentration increased after pooling. Considering that concentrations of RNA had been reduced to 1/31 in the pooled specimens, the Ct values were expected to increase by five compared with single samples. However, in figures C and D, the actual Ct values of the pooled specimens were approximately six values lower than expected, corresponding to a 60-fold increase in RNA concentration.⁴ By contrast, we found that when testing pooling of 50 nasopharyngeal and oropharyngeal samples, Ct values (RdRp gene) increased with pool size (appendix).

Lohse and colleagues attribute the decreased Ct values to the carrier effect from a higher RNA content in the pool; however, we did not observe a similar phenomenon in 600 tests. Lohse and colleagues did not describe clearly whether the experiment was done with media pooling or swab pooling in a single tube. To our knowledge, the NucliSens easyMAG instrument does not use carrier RNA or DNA for extraction, and there was no evidence to support the carrier phenomenon in the Correspondence.

During our experiments, we observed a few instances wherein the Ct value decreased despite an increased pool size. However, the changes in Ct value and pooling size were small and so could probably be explained by random variation in the PCR or pooling process with small volumes. By contrast, the difference between the expected and observed Ct values in Loshe and colleagues' study was large and so could not be attributed to random variation. A decrease in Ct value after pooling with negative specimens might cause a false-positive result and would be regarded as contamination in a clinical setting.