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Real-time RT-PCR in COVID detection: issues affecting the results – PMC.COVID Testing Frequently Asked Questions For Patients

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“The ICMR does not allow RT-PCR for asymptomatic negatives, but that is for hospitals, hotspots and containment zones only. It is believed that. RT-PCR at initial presentation in patients with suspected COVID can miss infected patients. Antibody titers of SARS-CoV-2 assays are linked. If your PCR test is negative, you’ll get a text message to say that the test did not detect COVID This means that the virus was not.
 
 

Fact check: The CDC’s PCR test will be withdrawn after Dec. 31.PCR positives: what do they mean? – The Centre for Evidence-Based Medicine

 
The PCR test used by MIT, like other PCR tests, is very unlikely to return a false positive. If the test comes back positive, we can be sure. RT-PCR at initial presentation in patients with suspected COVID can miss infected patients. Antibody titers of SARS-CoV-2 assays are linked. Very rarely a PCR test may not find traces of SARS-CoV-2, even though the person has been infected with the virus. This is what we call a ‘false negative’.

 

Why rt pcr test is done – none:. Negative PCR test result (COVID-19 not detected)

 

The new PMC design is here! Learn more about navigating our updated article layout. The PMC legacy view will also be available for a limited time. Federal government websites often end in. The site is secure. Since December , there has been considerable challenge regarding the use of nucleic acid test or clinical characteristics of infected patients as the reference standard to make a definitive diagnose of COVID patients.

As the early diagnosis of COVID is critical for prevention and control of this pandemic, clinical characteristics cannot alone define the diagnosis of COVID, especially for patients presenting early-onset of symptoms.

Along with the advancement in medical diagnosis, nucleic acid detection-based approaches have become a rapid and reliable technology for viral detection. An important issue with the real-time RT-PCR test is the risk of eliciting false-negative and false-positive results.

Thus, a negative result does not exclude the possibility of COVID infection and should not be used as the only criterion for treatment or patient management decisions. Several factors have been proposed to be associated with the inconsistency of real-time RT-PCR [ 5 ].

It is expected that this could provide beneficial information for the comprehension of the limitations of the obtained results and to improve diagnosis approaches and control of the disease.

Genetic diversity and rapid evolution of this novel coronavirus have been observed in different studies [ 6 , 7 ]. Although it was attempted to design the real-time RT-PCR assay as precisely as possible based on the conserved regions of the viral genomes, variability causing mismatches between the primers and probes and the target sequences can lead to decrease in assay performance and potential false-negative results.

In this regard, multiple target gene amplification could be used to avoid invalid results. All of them behind the laboratory practice standard and personnel skill in the relevant technical and safety procedures explain some of the false-negative results.

According to the natural history of the COVID and viral load kinetics in different anatomic sites of the patients, sampling procedures largely contribute to the false-negative results. Optimum sample types and timing for peak viral load during infections caused by SARS-CoV-2 remain to be fully determined. A study has reported sputum as the most accurate sample for laboratory diagnosis of COVID, followed by nasal swabs, while throat swabs were not recommended for the diagnosis [ 8 ].

They also suggested the detection of viral RNAs in bronchoalveolar lavage fluid BALF for the diagnosis and monitoring of viruses in severe cases. However, gathering of BALF needs both a suction tool and an expert operator, in addition to being painful to the patients.

While BALF samples are not practical for the routine laboratory diagnosis and monitoring of the disease, collection of other samples such as sputum, nasal swab, and throat swab is rapid, simple, and safe.

To avoid inconsistent results, it would be better to use different specimen types stool and blood besides respiratory specimen during different stages. It is worth noting that samples should be obtained by dacron or polyester flocked swabs and should reach the laboratory as soon as possible after collection. False-negative results may occur due to the presence of amplification inhibitors in the sample or insufficient organisms in the sample rising from inappropriate collection, transportation, or handling.

Viral load kinetics of SARS-CoV-2 infection have been described in two patients in Korea, suggesting a different viral load kinetics from that of previously reported other coronavirus infections [ 9 ].

In the first patient, the virus was detected from upper respiratory tract URT and lower respiratory tract LRT specimens on days 2 and 3 of symptom onset, respectively. On day 5, the viral load was increased from day 3 in the LRT specimen. Finally, the assay became undetectable for two consecutive days from day 14 LRT specimen and day 15 URT specimen , respectively. However, the initial viral loads were relatively lower than those of patient 1 in whom the test was performed on day 2 of symptom onset.

However, it was interpreted as negative due to high Ct value of the RdRp gene Ct value of These findings indicate the different viral load kinetics of SARS-coV-2 in different patients, suggesting that sampling timing and period of the disease development play an important role in real-time RT-PCR results.

In accordance, the negative template control NTC sample should be negative, showing no fluorescence growth curves that cross the threshold line. The occurrence of false positive with one or more of the primer and probe NTC reactions is indicative of sample contamination. Importantly, the internal control should be included to help identify the specimens containing substances that may interfere with the extraction of nucleic acid and PCR amplification. Because of the several risks to patients in the event of a false-positive result, all clinical laboratories using this test must follow the standard confirmatory testing and reporting guidelines based on their proper public health authorities.

In conclusion, according to the mentioned reasons, the results of real-time RT-PCR tests must be cautiously interpreted. In the case of real-time RT-PCR negative result with clinical features suspicion for COVID, especially when only upper respiratory tract samples were tested, multiple sample types in different time points, including from the lower respiratory tract if possible, should be tested. Proper sampling procedures, good laboratory practice standard, and using high-quality extraction and real-time RT-PCR kit could improve the approach and reduce inaccurate results.

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.

Expert Rev Mol Diagn. Published online Apr Alireza Tahamtan a and Abdollah Ardebili b. Author information Article notes Copyright and License information Disclaimer. Received Mar 14; Accepted Apr This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID pandemic or until permissions are revoked in writing.

This article has been cited by other articles in PMC. Associated Data Data Citations [[cited Mar 23;]]. Expert opinion In conclusion, according to the mentioned reasons, the results of real-time RT-PCR tests must be cautiously interpreted.

Funding Statement This paper was not funded. Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. Reviewers disclosure Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.

References 1. Recent advances and perspectives of nucleic acid detection for coronavirus. J Pharm Anal. DOI: Int J Mol Sci. November 23; 17 11 :E Simultaneous detection of severe acute respiratory syndrome, middle east respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR. Arch Virol. J Med Virol. February 25 [Epub ahead of print] DOI: Understanding the influence factors in viral nucleic acid test of novel coronavirus nCoV.

Chin J Lab Med. Phan T. Infect Genet Evol. February 21; 81 Clin Infect Dis. March 4:ciaa [Epub ahead of print] DOI: Laboratory diagnosis and monitoring the viral shedding of nCoV infections. J Korean Med Sci. February 24; 35 7 :e