Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China, January-March 2020: retrospective cohort study

Abstract

Introduction

A novel human coronavirus first detected during an unexplained cluster of pneumonia cases in Wuhan, China in December 2019 has spread globally.^{1 2} As of 22 March 2020, the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (genus Betacoronavirus, family Coronaviridae) has been reported in 190 countries with more than 300 000 confirmed cases and 14 510 deaths.³ A predominant number of cases has occurred in China,⁴ with early clinical characterisation showing that 13.8% of those infected developed severe disease, and death occurred in 2.3% of the cases.

Viral load measurements from tissue samples are indicative of active virus replication and are routinely used to monitor severe viral respiratory tract infections, including clinical progression, response to treatment, cure, and relapse.^{5 6 7} One study described changes in viral loads in samples from the upper respiratory tract of 18 patients with coronavirus disease 2019 (covid-19, an infectious disease caused by SARS-CoV-2), showing that the viral loads were equally high among asymptomatic patients and those with symptoms.⁸ However, the viral load dynamics in lower respiratory tract and other tissue samples and the relation between viral load and disease severity is unknown—information that are important for the formulation of disease control strategies and clinical treatment.

We systematically estimated the viral loads in more than 3000 samples collected from 96 patients after admission who were infected with SARS-CoV-2, and analysed the temporal change in viral loads and the correlation between viral loads in different sample types and disease severity.

Methods

Results

Table 1 shows the clinical characteristics of 96 patients with confirmed covid-19: 22 with mild disease and 74 with severe disease. The median age was 55 years (interquartile range 44.3-64.8). Of the patients infected in Wuhan, a significantly higher proportion were in the severe group (35%) than in the mild group (9%). Hypertension (36%) and diabetes mellitus (11%) were the most common underlying disease. Most of the patients developed fever (89%) and cough (56%). Overall, 78 (81%) patients received glucocorticoids and 33 (34%) antibiotic treatment. All patients received antiviral treatment comprising interferon α inhalation, lopinavir-ritonavir combination, arbidol, favipiravir, and darunavir-cobicistat combination. Among them, 63 (66%) started antiviral treatment within five days from illness onset and 29 (30%) more than five days after illness onset. Thirty (41%) patients with severe disease were admitted to the intensive care unit. By 20 March, all patients tested negative for SARS-CoV-2, nine (9% of all patients) patients with severe disease were still in hospital, and no deaths had occurred. Supplementary figure S1 shows the outcome among patients infected with SARS-CoV-2, and supplementary table S1 the laboratory findings.

Correlation between viral duration in different sample types and disease severity

The median duration of virus in stool samples (22 days, interquartile range 17-31 days) was significantly longer than in respiratory (18 days, 13-29 days; P=0.02) and serum samples (16 days, 11-21 days; P<0.001) (fig 1). In the respiratory samples, the median duration of virus in patients with severe disease (21 days, 14-30 days) was significantly longer than in patients with mild disease (14 days, 10-21 days; P=0.04) (fig 1), whereas no significant difference was observed in the duration of virus between stool and serum samples among patients with different disease severities (fig 1). Supplementary figures S3-S5 show the duration of virus in different sample types in each patient.

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Fig 1

Duration of detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by sample types and disease severity. Coloured bars represent medians and black bars represent interquartile ranges

Correlation between viral load in different sample types and disease severity

Viral load differed significantly by sample type, with respiratory samples showing the highest, followed by stool samples, and serum samples showing the lowest (fig 2). In respiratory samples, patients with severe disease had significantly higher viral loads than patients with mild disease (fig 2). Viral loads in stool and serum samples showed no significant difference between patients with mild disease and patients with severe disease (fig 2).

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Fig 2

Comparison of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load by sample types and disease severity. Coloured bars represent medians and black bars represent interquartile ranges

Using a loess regression analysis, we found that in the mild group, the viral load in respiratory samples was greater during the initial stages of the disease, reached a peak in the second week from disease onset, and was followed by lower loads (fig 3). In the severe group, however, the viral load in respiratory samples continued to be high during the third and fourth weeks after disease onset (fig 3). The viral load of stool samples was highest during the third and fourth weeks after disease onset (fig 3).

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Fig 3

Smooth lines were fitted using loess method to explore the variation of viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across the days since symptoms onset in respiratory samples from patients with mild and severe disease and in stool samples

Factors associated with duration of virus and viral load

We found that types and timeliness of antiviral treatments had no overall effect on the duration of the virus and viral load. In the severe group, the duration of the virus was significantly higher in patients treated with glucocorticoids continuously for more than 10 days than in patients treated with glucocorticoids continuously for less than 10 days, whereas different treatments had no effect on viral load (supplementary table S2). When patients with severe disease were stratified, the duration of the virus was significantly longer in men than in women, and significantly longer in patients older than 60 years than younger (fig 4).

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Fig 4

Association between sex and age and duration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Coloured bars represent medians and black bars represent interquartile ranges

Discussion

We have systematically described the clinical characteristics of 96 patients with covid-19 and described the dynamic changes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) loads and disease progression in 3497 samples of multiple types, revealing the interaction between SARS-CoV-2 replication and clearance by host defence mechanisms. The median duration of virus in respiratory samples was 18 days, which was consistent with the median duration of 20 days for Middle East respiratory syndrome (MERS).¹² Peak viral shedding in respiratory specimens of patients with severe acute respiratory syndrome (SARS) occurred after about 10 to 12 days from symptom onset,^{13 14} which is similar to the peak observed for SARS-CoV-2 in our study. Consistent with earlier reports of SARS-CoV-2,¹⁵ we found differences in the viral load in patients with different disease severities, those with severe disease showing a significantly higher viral load than those with mild disease, which suggests that viral load can be used to assess prognosis.

Studies have found that the peak load of SARS-CoV-2 in upper respiratory tract specimens was during the early stages of the disease^{8 16}; however, we found that the duration of virus shedding in lower respiratory tract samples was longer, and peak viral shedding occurred after about two weeks from symptom onset. These findings are important for effective control and prevention of the epidemic as it suggests strict management of the whole disease process in patients with SARS-CoV-2. In this study, we also found that the viral load in patients with severe disease was significantly higher than in patients with mild disease, suggesting that high viral load might be a risk factor for severe disease.

Active replication of SARS in the gut has been shown through live virus isolation.¹⁷ During 2003, the prevalence of SARS RNA in stool samples was so high that testing of stool was proposed as a reliable and sensitive way to routinely diagnose the disease,^{18 19} whereas MERS RNA was found in only 15% of stool samples, with a low RNA concentration.²⁰ In this study, we detected SARS-CoV-2 in the stool samples from 59% of patients and found that the duration of virus was longer and viral load peaked later in stool samples compared with respiratory samples. Based on this study, we think the role of faecal excretion in the spread of SARS-CoV-2 cannot be ignored; however, the importance of high detection in stool samples in the prevention and control of the SARS-CoV-2 epidemic requires comprehensive and careful evaluation. We rarely found SARS-CoV-2 RNA in urine samples in this study, although viral RNA detection rates of up to 50% have been found in the urine of patients with SARS.^{18 19}

A clear difference between SARS and SARS-CoV-2 was in the detection of viral RNA in serum. Evidence has been found of SARS virus replicating in circulating lymphocytes, monocytes, macrophages, and dendritic cells, albeit at low levels.^{21 22 23} In some studies, up to 79% of serum samples were found to contain SARS RNA during the first week of illness, and around 50% during the second week.^{24 25 26} The rates were similar in MERS.²⁰ In this study, we found that the detection rate of SARS-CoV-2 in serum was only 41%.

At present, the therapeutic effect of glucocorticoids and antiviral drugs in patients with SARS-CoV-2 is unclear.^{27 28} We found that the duration of treatment with glucocorticoids was positively correlated with viral duration in patients with severe disease. As we did not analysis the type and dose of antiviral drugs and glucocorticoids, however, we cannot evaluate the effect of antiviral drugs and glucocorticoids. Monitoring the effectiveness of antiviral drugs and glucocorticoids needs to be validated by multicentre randomised studies.

A sex dependent increase in disease severity after infection with pathogenic coronavirus was reported for both SARS and MERS,^{29 30} and this was also found for SARS-CoV-2.³¹ In this study, we found that the duration of virus was significantly longer in men than in women. Our results shed light on the causes of disease severity in men in terms of the duration of the virus. In addition to differences in immune status between men and women, it has also been reported to be related to differences in hormone levels.³² In this study, we also found a correlation between age and duration of virus, which partly explains the high rate of severe illness in patients older than 60 years. This is partly because of immunosenescence.³³ Another reason is that older people have higher levels of angiotensin converting enzyme 2 in their alveoli,³⁴ which is thought to be a receptor for novel coronaviruses.

Acknowledgments

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Notes

Contributors: TL and YC contributed equally to this paper and are joint corresponding authors. SZ, JF, FY, and BF are joint first authors. The corresponding and first authors conceived and designed the study. All authors selected the articles and extracted data. TL and YC were co-principal investigators. They designed and supervised the study and wrote the grant application (assisted by SZ). KX, XL, GW, JZ, QF, HC, YQ, and JS had roles in recruitment, data collection, and clinical management. SZ, JF, FY, BF, BL, QZ, GX, SL, RW, XY, WC, QW, DZ, YL, RG, ZM, SL, YX, YG, JZ, and HY did clinical laboratory testing and analysis. SZ, FY, BF, YC, and TL drafted the manuscript. All authors reviewed and revised the manuscript and approved the final version. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. TL and YC are the guarantors.

Funding: This work was supported by the China National Mega-Projects for Infectious Diseases (grant Nos 2017ZX10103008 and 2018ZX10101001) and the National Natural Science Foundation of China (grant Nos 81672014 and 81702079). The research was designed, conducted, analysed, and interpreted by the authors entirely independently of the funding sources.

Competing interests: All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: support from the China National Mega-Projects for Infectious Diseases and the National Natural Science Foundation of China, for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.

Ethical approval: This study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committee of the First Affiliated Hospital, Zhejiang University School of Medicine (2020IIT A0107).

Patient consent: Obtained.

Data sharing: No additional data available.

The lead authors and manuscript’s guarantors affirm that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.

Dissemination to participants and related patient and public communities: No study participants were involved in the preparation of this article. The results of the article will be summarised in media press releases from the Zhejiang University and presented at relevant conferences.

References