Comparing studies of SARS-CoV-2 viral loads requires caution.

To the editor,

Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), research groups around the world are unraveling key factors of the associated disease, coronavirus disease 2019 (COVID‐19). In light of this, many studies have sought to elucidate predictors for COVID‐19 severity to guide clinical management and prognosis of the disease. With this in mind, a growing body of evidence suggests that severe cases of COVID‐19 are linked with pronounced cytokine storm, high levels of C‐reactive protein, d‐dimer, immunoglobulin G, total antibodies, lymphopenia, lymphocyte dysfunction and activation, monocyte and granulocyte abnormalities. , , , Moreover, many studies have evaluated the association between viral load and COVID‐19 severity with controversial findings. , , , , , , , , In summary, a cumulative body of data obtained during the COVID‐19 pandemic course has demonstrated high, little, or no statistical correlation between viral load and severity in COVID‐19 patients. Taken together, these results published until now demonstrate that this is a question that remains unclear and undefined.

Throughout the COVID‐19 pandemic, many studies have suggested that the high viral load was associated with a higher risk of severe disease in COVID‐19 patients. , In one of the first reports assessing the relationship between viral load and COVID‐19 severity, Liu et al.  analyzed the C t values in patients classified with mild and severe disease using 76 respiratory specimens. After quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) analysis, the results demonstrated that the viral load in nasopharyngeal specimens of COVID‐19 severe cases was around 60 times higher than mild cases, and this positive correlation was maintained during the first 12 days of infection. In a similar study, SARS‐CoV‐2 RNA viral shedding was evaluated in 3497 samples (serum, respiratory, stool, and urine) from 96 consecutively admitted COVID‐19 patients in a hospital in Zhejiang province, China. Viral load in respiratory specimens, with exception of stool and serum, of individuals with severe disease was higher than in individuals with mild disease. In severe ill patients, male gender and old age was associated with longer viral shedding. Similarly, these findings also corroborate with outcomes reported by other research teams across the world. , , ,

In contrast, several reports have pointed that the high viral load was not associated with a higher risk of severe disease in COVID‐19 patients. , , For instance, a multicenter cross‐sectional retrospective study was conducted by Abdulrahman et al. using data obtained from Bahrain's National COVID‐19 Task force's centralized database to explore whether a correlation exists between viral load and COVID‐19 severity. A multivariable logistic regression was applied to assess for a correlation using data from a total of 1057 admitted COVID‐19 cases. In summary, the results showed that the C t values obtained from RT‐qPCR showed no statistical significance for an association with the requirement for oxygenation on admission among COVID‐19 patients. In the midst of the COVID‐19 pandemic, what factors have led to this controversial association between viral load and COVID‐19 severity? A probable answer is the use of RT‐qPCR C t values instead of true quantitative determinations. ,

At present, most studies just considered the C t value for analysis viral load among COVID‐19 patients, instead of the number of RNA copies/ml. In fact, C t values are correlated with the amount of viral RNA in a sample. However, C t values cannot be directly compared across RT‐qPCR assays and, therefore, they must be interpreted with caution. Notably, the exclusive use of C t value to assess viral load can represent a bias during the statistical analysis since many technical issues that might impact and alter the C t value during RT‐qPCR reactions—including differences in protocols, threshold values, viral target, primers, enzymes, and research kits, calibration of RT‐qPCR machine, type of biological samples, and period of sample collection, which means that the C t value not represent the best parameter to assess viral load in COVID‐19 patients. With this in mind, I suggest that further studies should consider a combination of C t values and RNA copies/ml for viral load analysis among COVID‐19 patients. This new perspective, combined with the evaluation of host‐related factors (e.g., age, sex, comorbidities, etc.) , will be critical to understand the real impact of SARS‐CoV‐2 viral load on COVID‐19 disease severity.

CONFLICT OF INTEREST

The author declares that there is no conflict of interest.

AUTHOR CONTRIBUTIONS

Severino Jefferson Ribeiro da Silva conceived the work, wrote the original manuscript, and reviewed the final version of the submitted manuscript.