Evaluation of the relationship between the cycle threshold (Ct) of RTqPCR and COVID-19 clinical symptoms in outpatients

Abstract

The gold standard diagnosis (GSDP) for COVID-19 is amplification of viral RNA by retrotranscription and real-time polymerase chain reaction (RT-qPCR), using a nasopharyngeal swab sample (NPS), processed with a traditional RNA extraction. Regarding the sample, some substitutions have been used in the diagnostic process, such as saliva and nasopharyngeal secretion samples with auto collection without the need to use a swab to reduce the time and cost of these tests and mainly to reduce the biological risk of health personnel. The data used in RT-qPCR for its positivity or negativity is the value of the threshold cycle (Ct), and according to some studies this data can provide an important tool as a prognostic value in the clinical epidemiological context of the patient, influencing the therapeutic decision making of health personnel, with a possible positive impact on the clinical evolution of patients; however, several of the published studies show low or in some cases no significant relationship between the Ct value and the clinical evolution of the patient. OBJECTIVE: To evaluate the relationship between RTqPCR cycling threshold (Ct) and clinical COVID-19 in outpatients. METHODOLOGY: 479 samples from patients who met the inclusion criteria were processed, amplification was performed by two already standardized RT-qPCR protocols of SARS CoV2 E and RdRp genes, 4 positive samples were sequenced by NGS for diagnostic confirmation.  Factors related (signs and symptoms) to SARS-CoV-2 viral load were evaluated. Viral load was determined based on RT-qPCR reaction and threshold value (CT). Two groups were stratified; high viral load (Ct<30) and low viral load (Ct>30). The data were recorded in an excel spreadsheet and subsequently analyzed with the statistical software R 4.4.1. from Project for Statistical Computing. Values of p≤0.05 were considered significant, the strength of the association was measured with the Odds Ratio and its confidence intervals (95% CI). RESULTS: Of 479 samples processed, 87 samples were positive, the highest severity(N:3) and lethality(N:1) variables clustered in Ct< 30, only one severe patient clustered in Ct>30. The rest (N:84) without mortality or severity clustered similarly in both groups Ct<30 and Ct>30, in all cases with a weak or no relationship significance with respect to the Ct threshold (p ≥ 0.05). Similarly, no significant impact of the vaccine on the Ct value was observed. CONCLUSION: The usefulness of Ct as a predictor of severity of COVID-19 and for other respiratory viral infections should be subjected to more rigorous studies, meanwhile given the available information and the results of the present study confirm that the usefulness of the Ct threshold in the clinical prognosis of COVID-19 patients is accompanied by certain limitations. In any case, if the Ct value is used as a prognostic factor in COVID-19 patients, it should be done with caution and awareness of the limitations.