The year 2020 brought a completely different endeavor to the entire medical world. While the doctors were dealing with a large number of COVID-19 patients on one hand, they tried to carry out studies to reveal the virus characteristics on the other. Although we have left half of the year behind and encountered millions of patients, many points of the SARS-CoV-2 virus have still not been elucidated. Virus dynamics in different hosts and different samples and their relationship with disease severity have not been clearly revealed.
In the current literature, although there are studies with a relatively good sample size, differing results are reported regarding the PCR positivity rates [2,3,4,5,6]. In this study, SARS-CoV-2 was detected in different samples from 60 COVID-19 patients. PCR positivity rates were 80%, 50%, 13.3%, 8.3%, and 1.7% for NP+OP, saliva, oral cavity, rectal, and urine samples, respectively. Wang et al. reported that PCR positivity rate for nasal swaps, pharyngeal swaps, and feces were 63%, 32%, and 29%, respectively . Their study consisted of 1070 specimens from 205 patients with different severity and found 1% blood positivity and no urine sample positivity. We found no blood sample positivity, but 1 PCR-positive urine sample (1.7%) collected from an asymptomatic patient. In some studies, no PCR positivity in the urine samples was detected, despite the existence of viremia [4, 6, 10]. There are too many points that need to be clarified about the extrapulmonary replication of SARS-CoV-2 virus and time dependent changes of PCR positivity. Chen et al. reported that SARS-CoV-2 RNA was detected in the blood (6 out of 57 patients) and anal swabs (11 out of 28 patients) . They concluded a quite different point from our study, that the presence of virus outside of the respiratory tract like blood and anal swab is related with severe disease. However, we found PCR positivity in anal and urine samples of asymptomatic patients. Zheng et al. reported that the PCR positivity rate in stool samples did not differ between patients with mild disease and patients with severe disease and they detected only one PCR positivity in a critically ill patient among 180 urine samples . Note that, although there is some evidence that a higher viral load can be found in the stool of some patients, this does not provide substantial evidence for the use of anal swab samples as can also be seen in this study results where only 5 out of 60 patients’ samples were found positive.
Another issue regarding PCR positivity is its relationship with infectivity. PCR positivity does not always indicate infectivity. Studies demonstrated that despite positive PCR results, patients are less likely to be infectious after 1 week [13, 14]. Wölfel et al. reported that all viral cultures were negative after day 8 . In addition to all this points, the infectivity may be affected by the presence of neutralizing SARS-CoV-2 antibodies.
Since the beginning of the pandemic, there are controversial views about the infectiousness of asymptomatic patients. It is reported that approximately 40–45% of patients infected with SARS-CoV-2 will remain asymptomatic . Surprisingly in our study, viral loads of asymptomatic patients were found to be significantly higher (p = 0.0141). Zou et al. evaluated SARS-CoV-2 viral load in 18 COVID-19 patients’ upper respiratory specimens. They included only one asymptomatic patient and found that NP+OP sample’s viral load of asymptomatic patient was similar to that in the symptomatic patients . The period of the disease when samples for the PCR test were collected is important both in terms of PCR positivity and viral load. This correlation makes it difficult to compare the results of the studies. In a study including 31 adult patients who were virologically confirmed COVID-19 but were asymptomatic on admission, it is reported that the cycle threshold values of asymptomatic patients were significantly higher than those of asymptomatic patients who developed symptoms after admission to hospital while having similar duration of viral shedding . In our study, all PCR and viral load analysis were performed on admission to hospital for all patients.
In a study from China, He et al. evaluated temporal patterns of viral shedding in 94 laboratory-confirmed COVID-19 patients and reported that infectivity started from 2.3 days before symptom onset, peaked at 0.7 days before symptom onset, and declined quickly within 7 days . Therefore, patients actually begin to be infectious before the symptom onset and isolation. Probably, this is one of the important points that makes the control of the epidemic difficult. Asymptomatic patients maybe another important point. Although asymptomatic patients are intuitively thought to be less infectious due to producing less aerosols since they do not cough as much as symptomatic patients, their high levels of viral load may indicate a higher than expected infectiousness. It must also be kept in mind that asymptomatic patients are rarely detected and isolated. Therefore, their exact role in the pandemic might be more important than initially thought.
There are a limited number of studies in the literature evaluating the relationship between viral load and disease severity. Shi et al. found that pneumonia cases had the lowest viral loads, followed by non-pneumonia cases, and severe pneumonia cases having the highest with no statistically significant differences . Zheng et al. reported that viral load of respiratory samples was significantly higher in severe patients than patients with mild disease . But the respiratory samples in their study were sputum and saliva. They found that in the mild disease group, viral load was higher during the early phase, peaked at the second week from disease onset, and declined afterwards. However, they also found that the viral load remained high in the severe disease group. We found a significant decrease in viral load with increased disease severity. To interpret the studies correctly, it is absolutely necessary to consider when the samples are taken. In our study, patients were in relatively early phase of the disease since the median time from illness onset to admission was 3 (1–14) days. The reason for low viral load in severe patients can be interpreted as late admission to hospital. However, it is not the case in our study, since no significant difference was found in admission day from symptom onset of patients belonging to different severity groups (p = 0.805). To et al. reported no difference between median viral loads of severe and mild cases . As seen in these examples, there are studies that find the viral load in severe patients lower, higher, or equal (i.e., no significant difference) compared to the mild patients.
In our country, mean age of COVID-19 cases is 42 years . Mean age of patients in our study was 33.9 years. According to the published data, younger patients are more likely to be asymptomatic than older patients . Studies demonstrated that older age and male gender is associated with severe disease [22,23,24]. But little is known about correlation of viral load with age and gender. We found that gender is not a factor affecting PCR positivity and viral load. But the viral load had a significant negative trend with increasing age. Zheng et al. reported that duration of viral shedding was significantly longer in men than in women and increased with older age . To et al. found similar results with Zheng et al. and concluded that older age is associated with higher viral load .
The strengths of our study were evaluating 6 different sample types (OP+NP, oral cavity, saliva, rectal, urine, and blood) of patients with different ages and clinics, revealing the relationship between disease course and SARS-COv-2 viral load, and differences in viral loads of asymptomatic and symptomatic patients. Our study has some limitations. First, we performed the PCR and viral load tests only on admission. Serial sampling would be much better to see the dynamics of viral loads in different tissues and viral shedding patterns. Second, we did not evaluate lower respiratory samples. Third, not all PCR-positive samples have undergone viral load analysis. Finally, higher number of patients would have strengthened the study.
In conclusion, this study demonstrates that asymptomatic patients have higher SARS-CoV-2 viral loads than symptomatic patients and unlike in the few study in the literature, a significant decrease in viral load was observed with increasing disease severity. Factors associated with poor prognosis like low lymphocyte count, bilateral ground-glass opacity in chest CT, and older age are found to be significantly correlated with low SARS-CoV-2 viral load. COVID-19 is a complicated puzzle with pieces of many colors and shapes. Further virologic and immunological studies are urgently needed to put all the pieces together and see the big picture.