Viral RNA detection by real-time polymerase reaction (RT-PCR) is considered as the gold standard and the most commonly used and the recommended test for SARS-CoV-2 infection. Figure 1 depicts the viral and serological kinetics of SARS-CoV-2 infection.

Fig1. Viral and serological kinetics in SARS-CoV-2

Clinical specimens: RT-PCR for diagnosis is done from respiratory samples, such as nasal/ pharyngeal or nasopharyngeal swab, sputum, or bronchoalveolar lavage (BAL). Viral RNA also has been detected in stool, saliva, blood and urine samples. However, these samples are not the recommended clinical specimen for diagnosis because of their poor positivity and inconsistent result. Of the respiratory samples, BAL has shown highest sensitivity (93%) followed by sputum (72%), nasal swab (63%), and pharyngeal swab (32%). Practically, nasopharyngeal or oropharyngeal samples are most commonly used. Swabs are collected in viral transport medium. All samples need to be transported to the laboratory in cold chain. Samples should be stored at 4°C till tested.

RT-PCR steps: Samples are subjected to lysis for inactivation of the virus followed by nucleic acid extraction, reverse transcription to generate complementary DNA (cDNA) and then amplification of the cDNA for detection of the virus target genes by using target specific primers and probes.

Target genes: Most of the currently available RT-PCR kits for COVID-19 are multiplex RT PCR having the detection system for more than one target gene in one run. A variety of target genes are used by different manufacturers, mostly consisting of genes of structural proteins like envelop (E), nucleocapsid (N) or spike (S) or non-structural proteins such as RNA-dependent RNA polymerase (RdRp), and ORF1 genes. E gene target is mostly used as the screening gene indicating the positivity for Sarbecovirus, whereas other genes are used as the specific target genes for SARS CoV-2. The sensitivity of all the target genes is comparable except the rdRp gene which has shown lower sensitivity. Assay having more than one target gene is more sensitive than the assay having only one target gene detection system.

Cycle threshold: Cycle threshold value is the cycle at which the fluorescence signal crosses the defined background threshold. The cycle threshold (Ct) value less than 40 is clinically reported as PCR positive. However, different commercially available approved RT-PCT kits for COVID-19 have different Ct cut-off for consideration as positive. Indian Council of Medical Research (ICMR) has recommended cut-off of 35 Ct value as positive. This is based on various study results that in samples of Ct>35, virus is non-cultivable indicating the non-viability of the virus and as RT-PCR detects viral RNA which can be positive even in sample containing non-viable virus.

Sensitivity: The clinical sensitivity of various RT-PCR assays is reported from 70–>90%. The analytical sensitivity depicting the limit of detection has been found to be as low as <5 log10 copies/mL. The positivity of RT-PCR is affected by various factors. False negative RT PCR could be due to low viral load in the patient, too early or too late stage of infection, early viral clearance, viral replication predominantly occurring in lower respiratory tract, RT-PCR and nucleic acid extraction kit variation, type and number of target genes used in RT-PCR assay, type of clinical specimen, quality of specimen, transport and storage system, etc. Variation in any of these factors may lead to false negative RT-PCR result.

Specificity: The specificity of RT-PCR is often 100% as the currently used primer probes have been tested as specific for SARS-CoV-2 virus with no cross-reactivity with other viruses including other human coronaviruses. However, false positive may occur due to reagent contamination or carry over contamination.

Result: The result of RT-PCR can occur as: Positive, negative, inconclusive or invalid.

Positive RT-PCR is often taken as per the cut-off Ct value of the assay used. Both Ct value and the appearance of proper sigmoid amplification curve should be taken into account to label the sample as positive. However, after several studies have shown the non-viability of virus in samples with Ct >35, Ct <35 is presently widely used. The cut-off Ct to consider positive may vary as per the policy of different countries or different health agency.

Negative: Sample showing no amplification or Ct value beyond the defined cut off are considered as negative.

Inconclusive: Samples labelled in this category are basically border line positive samples, where both the genes are not within the defined cut-off of Ct value or the sigmoid curve is not proper. The criteria of inconclusive is different in different RT-PCR kits used.

Invalid: Non-amplification of the in-built internal control in the reagent assay is considered as invalid. This could be due to inadequate sample, presence of PCR inhibitor in clinical specimen, failure of extraction or failure of amplification process.

Clinical implication of COVID-19 RT-PCR results: RT-PCR positivity depends on the viral kinetics. Several studies have shown the lowest Ct value at the symptom onset. However, many studies have shown lower Ct from 5 days before to 10 days after the onset of symptom. Majority become positive by day 3 or day 4. Most of the mild cases become negative by day 8–day 10 of symptom onset. In symptomatic cases, RT-PCR usually become negative during second week of illness. In patients with steroid treatment, viral clearance may take longer duration, so RT-PCR may remain positive for a longer time than average duration. The RT-PCR status in a proportion of COVID-19 positive cases may follow a positive-negative pattern for 3–6 weeks as observed in several studies in hospitalized patients.

In hospital admitted patients after day 10 of illness, the Ct value usually becomes more than 30 which is considered as non-transmissible as virus from those samples have been found to be non-cultivable. Hence, after 10 days of symptom onset and when the patient become afebrile for 3 consecutive days without taking antipyretic, patient is considered as non-infectious irrespective of RT-PCR positivity status as in majority shows high Ct value.

The quality of the conventional RT-PCR test result demands a stringent quality control. Involvement of several steps in this makes the test procedure time consuming and cumber some. The major drawback of the molecular assay is high turn around time. It needs batch testing. It takes minimum 4–6 hours which may be more depending on the number of samples to be tested in one batch, infrastructure available. Several FDA approved high throughput platforms are available for RT PCR testing for COVID-19. Cobas 6800 is one such platform which is widely used.

Cobas 6800 and 8800 instruments are fully automated RT-PCR testing platforms. It consists of sample supply module, transfer module, processing module and analytic module. For detection of SARS-CoV-2, the system uses two target genes; ORF1, a non-structural region and E gene for pan Sarbecovirus detection, a target from a conserved region. The overall agreement with other standard RT-PCR system is reported as 96–98%.

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مواضيع ذات صلة

Laboratory diagnosis of SARS-CoV-2 : Point of Care Molecular Test

marijuana (THC) test (Cannabinoid or cannabis testing, Tetrahydrocannabinol [THC] testing)

lung cancer molecular testing (AKT1, ALK, BRAF, Epidermal growth factor [EGFR], HER2, KRAS, MEK1, MET, NRAS, PIK3CA, RET, and ROS1)

kidney stone analysis (Urinary stone analysis, Renal calculus analysis)

Variability and Standardization of CSF Examination

Lab Diagnosis of Yellow fever

hexosaminidase (Hexosaminidase A, Hex A, Total hexosaminidase, Hexosaminidase A and B)

hemoglobin electrophoresis (Hgb electrophoresis)

growth hormone stimulation test (GH provocation test, Insulin tolerance test [ITT], Arginine test, Glucagon Stimulation test)

Markers of Bone Resorption: Amino and Carboxy-Terminal Telopeptides of Collagen Type I

Markers of Bone Resorption: Collagen Cross-Links

Bone Formation Markers : Osteocalcin