Challenges and issues of SARS-CoV-2 pool testing.

Stefan Lohse and colleagues described a sample pooling strategy for testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via RT-PCR to meet the unprecedented demand for laboratory testing. Lohse and colleagues evaluated a range of pool sizes (four to 30 samples per pool) in asymptomatic people. The additional time to deconvolute the larger pools yielding a positive result into sub-pools precludes the use of this strategy in patients with severe acute respiratory illness and high-risk contacts. Moreover, most of the studies on pooled sample testing have not discussed the crucial technical points. Here, we raise certain technical issues pertaining to SARS-CoV-2 pool testing.

First, sample collection for SARS-CoV-2 testing in field settings is done without supervision and dependent on the skill of the people doing the test. The crucial pre-analytical variable in SARS-CoV-2 testing is the amount of host RNA (detected by RNAse P). Before we adopted pool testing in our laboratory, approximately 3% of individual samples tested showed no RNAse P amplification, indicating inadequate sample collection, which was resolved on repeat sample collection. Such a sample would have been missed in pooled testing and might have been reported as negative despite an inadequate amount of clinical material.

Second, Lohse and colleagues attributed the lower Ct values of pools than of single samples to the carrier effect of the higher RNA content in pools. If the same hypothesis is applicable to the adequacy of a sample, then inadequate samples in a large pool will be falsely reported as negative.

Third, different RNA extraction kits recommend different volumes of sample, ranging from 140 μL to 200 μL. It is not clear how Lohse and colleagues addressed the issues of total amount of pooled sample and the minimum amount of each sample to be added in pool sizes ranging from four to 30 samples. In a large pool of up to 30 samples, if we take 5–10 μL of each sample, there is every chance of missing borderline-positive single samples.

In our laboratory, as suggested by the Indian Council of Medical Research, we are testing four samples in a pool. Some pools have been inadequate (RNAse P not detected), which was resolved when individual testing was attempted. Absence of RNAse P in a pool might be due to an inhibitory effect of concentrated RNA samples on reverse transcription. Large-scale validation of SARS-CoV-2 sample pooling strategies addressing these technical issues is needed to reach a consensus strategy.