key: cord-0914100-vxl0s0uy
authors: Poon, Kok‐Siong; Tee, Nancy Wen‐Sim
title: Detecting SARS‐CoV‐2 RNA in fecal specimens: The practical challenges
date: 2021-06-08
journal: J Med Virol
DOI: 10.1002/jmv.27071
sha: 663569d271dc690638ab108f167abacff5af1426
doc_id: 914100
cord_uid: vxl0s0uy

nan

From the perspective of a molecular diagnostic laboratory, detection of SARS-CoV-2 RNA in fecal specimens is more technically challenging than in respiratory swabs due to additional specimen preparation steps before nucleic acid extraction. The presence of impurities in the fecal specimens is known to exert different degrees of inhibitory effect on PCR. 9,10 Different pretreatment methods with varied efficiency on removing impurities can also affect the detection of SARS-CoV-2 in fecal samples. 11 Fecal samples with low amounts of viral RNA may be reported falsely as negative due to potential PCR inhibition. With a dilution of the nucleic acid extract, potential inhibitory effect may be reduced or totally removed should there be a presence of PCR inhibitors in the purified nucleic acids. As a precautionary step, the laboratory usually performs PCR on the diluted nucleic acid alongside the "neat" extracts. In laboratory practice, validity of PCR results relies on the presence and performance of internal controls, which are assay dependent. These controls may be human gene targets found endogenously in the specimens, or exogenous controls, either spiked into PCR master mix, or during nucleic acid extraction to also control for extraction efficiency.

Although dilution to remove PCR inhibitors may reduce false negativity in samples with a high viral titer, it is of limited usefulness in samples with low amounts of viral RNA because dilution reduces the amount of target for detection in the PCR assay. In the study by Fumian et al., 1 the cycle threshold cut-off was set at 38 cycles for viral targets. Assuming a similar setting, a 10-fold dilution of nucleic acid is expected to delay the cycle threshold by a value of 3.3, which will significantly reduce the assay's ability to detect the target. In view of these caveats, we would recommend an "inconclusive result" to avoid issuing a potentially falsenegative report, if a negative result is obtained after sample dilution for a fecal sample showing initial signs of PCR inhibition.

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