There are several potential reasons why someone who is infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent of Covid-19, may test negative by PCR. These including the threshold for detection of virus (which can vary among different manufacturers from as low as 100 viral copies/ml to >6,000 copies/ml),1 timing of the sample collection with respect to infection stage (lowest false-negative rate [~20%] on day 3 of symptoms or 8 days post-infection),2 specimen storage and transport and, particularly in the case of nasopharyngeal specimens, the adequacy of the sample obtained. 3
Suboptimal specimen collection from nasopharynx has long been suspected as an explanation for false-negative PCR tests in patients who subsequently have a positive test or are highly suspected of having Covid-19, but without any good support data. Until now…
A clever study looked at the presence of human DNA recovered from nasopharyngeal swabs as a marker for adequate specimen collection quality and found that human DNA levels were significantly lower in samples from patients with confirmed or suspected Covid-19 that yielded negative results compared to those of representative pool of samples submitted for Covid-19 testing.3
Interestingly, major commercial assays do not include any internal controls that ensure adequate sampling before testing for SARS-CoV2.
A typical microbiology lab can reject a sputum culture if gram-stain suggests poor quality specimen (eg, saliva only) but it looks like no similar rule exists for nasopharyngeal PCR tests for SARS-CoV-2 through commercial labs. Apparently, the US-CDC diagnostic panel does include a human RNAseP RNA-specific primer/probe set but the interpretation criteria for this control may also be too liberal.3
For these reasons, in patients highly suspected of having Covid-19 but with a negative initial PCR test, a repeat test on the same day or next 2 days is recommended.4
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References
- Prinzi A. False negatives and refinfections: the challenges of SARS-CoV-2 RT-PCR testing. Available at https://asm.org/Articles/2020/April/False-Negatives-and-Reinfections-the-Challenges-of Accessed October 5, 2020.
- Kucirka LM, Lauer SA, Laeyendecker O, et al. Variation in false-negative rate of reverse transcriptase polymerase chain reaction-based SARS-CoV-2 tests by time since exposure. Ann Intern Med 2020 May 13:M20-1495. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240870/
- Kinloch NN, Ritchie G, Brumme CJ, et al. Suboptimal biological sampling as a probable cause of false-negative COVID-19 diagnostic test results. J Infect Dis 2020;222:899-902. https://academic.oup.com/jid/article/222/6/899/5864227
- Green DA, Zucker J, Westbade LF, et al. Clinical performance of SARS-CoV-2 molecular testing. J Clin Microbiol 2020. DOI:10.1128/JCM.00995-20. https://jcm.asm.org/content/58/8/e00995-20
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