key: cord-0869883-d71jwqbi
authors: Lee, Jaehyeon; Kim, So Yeon; Sung, Heungsup; Lee, Sang Won; Lee, Hyukmin; Roh, Kyoung Ho; Yoo, Cheon Kwon; Hong, Ki Ho
title: Challenges and issues of SARS-CoV-2 pool testing
date: 2020-07-14
journal: Lancet Infect Dis
DOI: 10.1016/s1473-3099(20)30516-8
sha: 07d0bbd1d5c6e9977202144ca8e706cad6c209ef
doc_id: 869883
cord_uid: d71jwqbi

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If the large pool is positive, the three subpools are reanalysed, and then the individual samples of the positive subpool. In our analyses during March 13-21, 2020, testing of 1191 samples required only 267 tests to detect 23 positive individuals (prevalence 1·93%). The rate of positive tests was 4·24% in our institution during this period.

These data suggest that pooling of up to 30 samples per pool can increase test capacity with existing equipment and test kits and detects positive samples with sufficient diagnostic accuracy. We must mention that borderline positive single samples might escape detection in large pools. We see these samples typically in convalescent patients 14-21 days after symptomatic infection. The pool size can accommodate different infection scenarios and be optimised according to infra structure constraints. 

We read with interest Stefan Lohse and colleagues' Correspondence about sample pooling for testing for severe acute respiratory syndrome coronavirus 2 (SARSCoV2) in asymptomatic people. 1 Some of the findings Lohse and colleagues report do not seem to be consistent with other research results 2,3 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 60fold 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 In our laboratory, as suggested by the Indian Council of Medical Research, 3 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. 4 Largescale validation of SARSCoV2 sample pooling strategies addressing these technical issues is needed to reach a consensus strategy.

We declare no competing interests. First, sample collection for SARSCoV2 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 SARSCoV2 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 borderlinepositive single samples. 2 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.

Stefan Lohse and colleagues 1 described a sample pooling strategy for testing for severe acute respiratory syndrome coronavirus 2 (SARSCoV2) via RTPCR to meet the unprecedented demand for laboratory testing. Lohse Published Online July 14, 2020 https://doi.org/10.1016/ S14733099(20) 304631 We read with interest the Corres pondence by Stefan Lohse and colleagues, 1 who evaluated the practicability of pool testing for severe acute respiratory syndrome coronavirus 2 (SARSCoV2). Pooling of samples yields considerable savings of test kits when the prevalence of infection is low because pools with allnegative samples can be discarded with a single test. Lohse and colleagues' findings suggest that pooling up to 30 samples is technically feasible with currently used and

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