The relationship between COVID‐19 viral load and disease severity: A systematic review

Abstract Introduction Patients with COVID‐19 may present different viral loads levels. However, the relationship between viral load and disease severity in COVID‐19 is still unknown. Therefore, this study aimed to systematically review the association between SARS‐CoV‐2 viral load and COVID‐19 severity. Methods The relevant studies using the keywords of “COVID‐19” and “viral load” were searched in the databases of PubMed, Scopus, Google Scholar, and Web of Science. A two‐step title/abstract screening process was carried out and the eligible studies were included in the study. Results Thirty‐four studies were included from the initial 1015 records. The vast majority of studies have utilized real‐time reverse transcription‐polymerase chain reaction of the nasopharyngeal/respiratory swabs to report viral load. Viral loads were commonly reported either as cycle threshold (C t) or log10 RNA copies/ml. Conclusion The results were inconclusive about the relationship between COVID‐19 severity and viral load, as a similar number of studies either approved or opposed this hypothesis. However, the studies denote the direct relationship between older age and higher SARS‐CoV‐2 viral load, which is a known risk factor for COVID‐19 mortality. The higher viral load in older patients may serve as a mechanism for any possible relationships between COVID‐19 viral load and disease severity. There was a positive correlation between SARS‐CoV‐2 viral load and its transmissibility. Nonetheless, further studies are recommended to precisely characterize this matter.

Since its emergence, the SARS-CoV-2 has undergone multiple mutations resulting in weaker or even or more dangerous variants of the virus. SARS-CoV-2 continuously evolves and potentially becomes more transmissible or fatal with each mutation. 2 Four variants of SARS-CoV-2 have been declared as the "variants of concern" by the WHO so far, which cause COVID- 19. A. Alpha variant: Alpha variant, or the lineage B.1.1.7, is the first SARS-CoV-2 variant and can be substituted by 23 mutations. As a consequence of the mutation, the transmissibility of the virus increased by about 50% as compared to the wild strain, making it more infectious with more severe complications 14 ; B. Beta variant: These mutations enhance the ability of the virus to attach to the human cells more easily in comparison with the previous variants 15 ; C. Gamma variant: Gamma variant caused widespread infection in early 2021 and is currently considered as a "variant of concern" 16 ; D. Delta variant: The Delta variant is more infectious and each infected person can transmit the virus to seven or more people. 17 For the clinical management of COVID-19 disease, it is substantial to quantify the viral load of the blood. 18 Viral load indicates active viral proliferation and is used to identify the severe viral infections of the respiratory tract and monitor the disease progression and treatment. 19 The viral load can be obtained from the patient's viral RNA with a certain concentration (the value that exceeds the threshold) by testing the value of the C t cycle threshold of the reverse transcription-polymerase chain reaction (RT-PCR). The lower the C t values than a patient's sample, the higher the viral load. 20 The relationship between the viral load and severity of disease in COVID-19 patients has not yet been fully understood. The investigation demonstrated that patients with COVID-19 who have been treated in the intensive care unit with a severe illness have a relatively higher viral load. A study also suggested that in large hospital groups, a high viral load is associated with an increased risk of death. 21 Thus, the study of the correlation between COVID-19 viral load and the progression of the disease and the treatment and prevention of COVID-19 helps to science promotion significantly. 22 A Chinese study working on the association of viral load with the development of COVID-19 found that patients with more viral load had fewer lymphocytes but more neutrophils. 23 In another study that examined the relationship between viral load and disease severity with COVID-19 clinical results, viral load in severe disease was much higher than in mild or asymptomatic disease. 24 However, conflicts exist regarding the effects of SARS-CoV-2 viral load on disease severity. Therefore, the present study systematically reviewed the association between SARS-CoV-2 viral load and COVID-19 severity.

| Data sources
Relevant articles were systematically searched from the keywords "COVID-19" and "viral load" in the online databases of PubMed, Science Direct, Scopus, and Web of Science. All the relevant literature published from December 2019 to August 2021 was retrieved and further screened using EndNote.

| Study objectives
The principal aim was to investigate the relationship between the COVID-19 viral load and its severity. However, the relationship between viral load and COVID-19 infectivity as well as the patients' age and viral load was also discussed.

| Study selection and inclusion/ exclusion criteria
We conducted a two-phase screening process; first, the studies were evaluated based on their title and abstract, and then the eligible ones were screened based on their full texts. We included peer-reviewed articles that studied the association between SARS-CoV-2 viral load and the COVID-19 disease severity or mortality. The selected articles were cross-examined by other researchers to avoid duplication.
The exclusion criteria were as follows: -Literature with no available full-texts including the conference papers and abstracts; -Literature with the main focus of nonhuman experiments of any kind like in vitro studies, animal trials, or literature without justifying details; -Reviews, systematic reviews, or meta-analyses; -Case reports.

| Data extraction
Two independent investigators summarized and extracted the following information from the included publications: The first author's ID (Reference), year, and type of publication (e.g., cross-sectional study), country of study, the sample size of the study, patient mean age and gender, sampling site, measured viral load, and disease outcome; the data were further gathered in a specifically designed sheet and organized into tables.

| Quality/risk of bias assessment
We used the Newcastle-Ottawa Scale to assess the quality of the studies. 25 This scale yields a total score out of 9 to the studies based on their selection, comparability, and exposure/outcomes.

| RESULTS
The search strategies resulted in 1015 records, being 928 remaining after removing the duplicates. Of which, 753 records were excluded in the title/abstract screening, and 175 full texts were reviewed. Finally, 34 studies met the eligibility criteria to be included after full-text screening ( Figure 1). Most of the studies were from China (n = 7); three studies per following countries: Japan, Spain, Turkey, and the USA; two studies per following countries: Italy, South Korea, and Switzerland; and one study for the following countries: Brazil, Czech Republic, England, France, Germany, India, Israel, and Singapore ( Table 1). The studies had overall acceptable quality, all of them scoring 4 and above ( Table 2).
Most of the studies included adults and had a similar share of men and women. The vast majority of the studies have utilized real-time RT-PCR of the nasopharyngeal/respiratory swabs to report viral load. Viral load was usually reported in two categories; cycle threshold DADRAS ET AL. | 3 of 25 (C t ) and log 10 RNA copies/ml. Studies have reported viral load in several groups: mild, moderate, and severe patients, symptomatic versus asymptomatic patients, and groups sorted by age. The results were inconsistent; while some studies found a significant relationship between SARS-CoV-2 viral load and severity of illness, other studies were against it (Table 1).

| DISCUSSION
SARS-CoV-2, the new coronavirus accountable for COVID-19, was first detected in China in late 2019 and then spread out globally. The WHO declared this disease a public health emergency of international concern on January 30, 2020. Although having the potential of causing severe pneumonia, SARS-CoV-2 can also involve various organs and cause physical symptoms such as fever, cough, and dyspnea, as well as psychological and gastrointestinal symptoms. Several interventions and measures have been implemented to restrict the spread of the virus and control the situation, such as community education, border controls, lockdown, social distancing, wearing masks in public, hand hygiene, and schools shut down. These public health efforts not only slowed down SARS-CoV-2 transmission but also caused a decrease of mortality rate. 1,12 In the present study, the main hypothesis along with two minor ones was discussed against the similar available studies. The main hypothesis recommended a potential relationship between the viral load and the severity of the disease. The minor hypotheses, which were also frequently reported in the included studies, are the relation between the age and the viral load as well as the relation between viral load and virus transmissibility. Symptoms included in the table were aimed to represent the severity of the diseases and the included comorbidities were to avoid the bias of imposture relation between severity and the viral load.
For both hypotheses, the key method of measuring the viral loads was the RT-PCR. Viral nucleic acid detection by RT-PCR assays is the gold standard for the diagnosis of COVID-19. Using this technique, we can gain an indirect viral load value (C t ) easily and immediately after diagnosis. 33 The main hypothesis could be explained by the association between viral load and inflammatory factors that are also clearly connected with the disease severity. It is well-known that excessive release of proinflammatory cytokines and chemokines contributes to the severity of clinical outcomes in various infections. Therefore, our findings that the plasma concentrations of IFN-α, IFN-γ, IP-10, MIG, and IL-6 were elevated in the severe and critical cases at 5-10 days from symptom onset suggest that the higher plasma concentrations of proinflammatory cytokines after approximately a week from symptom beginning may have a role in the enhancement of severity. Intriguingly, a recent longitudinal study showed that plasma IFN-α continued to be high in patients with severe COVID- 19 Similar to our findings, He et al., 34 have identified that higher viral load was positively associated with COVID-19 severity. This finding highlights the importance of monitoring the viral kinetics to identify patients at greater risk of progressing to severe pneumonia. Similarly, Guo et al., 58 have found that the upper respiratory tract viral RNA load of SARS-CoV-2 at the time of hospital admission is an independent predictive factor of COVID-19. However, there were some studies with inconsistent results. The study performed by Hasanoglu et al., 59 is an example of this controversy. They demonstrated that asymptomatic patients have higher SARS-CoV-2 viral loads than symptomatic patients and unlike in the few study in the literature, a major decrease in viral load of nasopharyngeal/oropharyngeal samples was observed with increasing disease severity. Similarly, Cho et al. 31 have found that both severity and recovery from these symptoms have no associations with the viral load of SARS-CoV-2. Le Borgne et al., 44 have also found that respiratory viral load measurement on the first nasopharyngeal swab (by RT-PCR) during initial ED management is neither a predictor of severity nor a predictor of mortality in SARS-CoV-2 infection.
To support our minor hypotheses suggesting the association between viral load and patient's age, the findings from the study by To et al.,52 suggested no relationships between severity of disease and viral load; their study only showed that the median viral load was 1 log 10 higher in severe cases than in mild cases, but on the other hand, they found a direct connection between age and viral load. Similarly, Shlomai et al. 48 have found that low viral load was independently associated with reduced risk for mechanical ventilation and mortality; and interestingly, patients' age also correlated positively with the viral load. Aydin et al. 27 found that viral load detected in saliva in the early symptomatic period of infection may have a prognostic value in showing the course of the disease in patients over 45-year-old. Overall, the studies found a positive correlation between patients' age and viral load. This finding might be a rationale for any possible relationship between viral load and increased disease severity, as older age is related to worse COVID-19 outcomes. 11 It also raises the alarm that older patients may be more likely to transmit the virus.
In the present study, the final hypothesis suggesting the association between viral load and the COVID-19 infectivity could be supported by the findings of Kawasuji et al.'s study, which suggested that a high nasopharyngeal viral load may contribute to the secondary transmission of COVID-19. 39 Similarly, Sarkar et al. found that 84% of cases had low viral load and practically will spread the virus even to very few their contacts, demonstrating the connection between viral load and transmission. 47 Buder et al. have also reported similar results that merely having no symptoms is not enough for recognizing whether the patients have the ability of transmission or not. They found that SARS-CoV-2 positively correlated with the infectivity of the patients, regardless of whether they are symptomatic or not. 30 Therefore, viral load is probably one of the factors influencing SARS-CoV-2 transmission.
There are some limitations in the present study. First and most important, a meta-analysis was not conducted due to the significant heterogeneity that existed between the included studies. Furthermore, there were few studies on some of the discussed matters and this may decrease the validity and reliability of reported outcomes. However, this study may provide relevant insights for future research to conduct original studies and/or metaanalyses to precisely determine the relationship between viral load and disease severity, and, in addition, to explore further discussed topics in this review, such as the correlation between age and SARS-CoV-2 viral load.

| CONCLUSION
We have discussed three different hypotheses related to the viral load of COVID-19. The results were inconclusive about the relationship between COVID-19 severity and viral load, as a similar number of studies either approved or opposed this hypothesis. However, the included studies found a positive association between age and viral load. The higher viral load also appeared to be associated with the higher transmissibility of the disease. Nevertheless, such findings require careful meta-analyses to be confirmed.