RT-PCR in covid-19.

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The knowledge about sars-cov-2 is continuously evolving with new development and discovery every passing day. A clear understanding of the nature of the tests and interpretation of their findings is important. This blog will describe how to interpret reverse transcriptase–polymerase chain reaction (RT-PCR) and how the results may vary over time.


As of now the most commonly used and reliable test for diagnosis of COVID-19 has been the RT-PCR test. This test is performed with nasopharyngeal swabs or other upper respiratory tract specimens, including throat swab or, more recently, saliva. 


The PCR test uses various  RNA gene targets  with most common tests targeting 1 or more of the envelope (env), nucleocapsid (N), spike (S), RNA-dependent RNA polymerase (RdRp), and ORF1 genes. Various comparison studies have shown that the sensitivities of the tests to individual genes are comparable except the RdRpSARSr (Charité) primer probe, which has a slightly lower sensitivity.

In most of the cases of symptomatic cases of covid-19 the viral RNA in the nasopharyngeal swab as measured by the cycle threshold (Ct) becomes detectable as early as day 1 of symptoms and peaks within the first week of symptom onset. 

The Ct is the number of replication cycles of PCR required to produce a fluorescent signal. Lower the Ct value higher viral RNA loads. A Ct value less than 40 is clinically reported as PCR positive. 

This positivity starts to decline by week 3 and subsequently becomes undetectable. 

In severely ill  patients the Ct values are lower than the Ct values of mild cases, and PCR positivity may persist beyond 3 weeks after illness onset when most mild cases will yield a negative result. 

However, a “positive” PCR result reflects only the detection of viral RNA and does not necessarily indicate presence of viable virus.

In some cases, viral RNA has been detected by RT-PCR even beyond week 6 following the first positive test. A few cases have also been reported positive after 2 consecutive negative PCR tests performed 24 hours apart. It is unclear if this is a testing error, reinfection, or reactivation. Or it may be non-viable viral antigens and can be better identifies after a viral culture.

It indicates that health care workers can return to work, if “at least 3 days (72 hours) have passed since recovery defined as resolution of fever without the use of fever-reducing medications and improvement in respiratory symptoms (e.g., cough, shortness of breath); and, at least 10 days have passed since symptoms first appeared.

THIS STRATEGY IS BASED FOLLOWING EVIDENCES:

Available data indicate that shedding of SARS-CoV-2 RNA in upper respiratory specimens declines after onset of symptoms. At 10 days after illness onset, recovery of replication-competent virus in viral culture (as a proxy of the presence of infectious virus) is decreased and approaches zero. Although persons may produce PCR-positive specimens for up to 6 weeks (Xiao, 2020), it remains unknown whether these PCR-positive samples represent the presence of infectious virus. After clinical recovery, many patients do not continue to shed SARS-CoV-2 viral RNA. Among recovered patients with detectable RNA in upper respiratory specimens, concentrations of RNA after 3 days are generally in ranges where virus has not been reliably cultured by CDC.

Why are some recovered covid-19 cases test positive in PCR again?

Although no definitive cause is identified, this might be due to persistence of non-viable viral antigen in the respiratory tract which the PCR cannot differentiate from viable or due to the presence of viable virus which is not able to replicate.  


The timeline of PCR positivity is different in specimens other than nasopharyngeal swab. PCR positivity declines more slowly in sputum and may still be positive after nasopharyngeal swabs are negative. In a study of 205 patients with confirmed COVID-19 infection, RT-PCR positivity was highest in bronchoalveolar lavage specimens (93%), followed by sputum (72%), nasal swab (63%), and pharyngeal swab (32%).

Specificity of most of the RT-PCR tests is 100% and occasional false positive results may occur due to technical errors and reagent contamination. 

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