key: cord-0898138-3kwib1o3
authors: Shi, Fengjuan; Wu, Tao; Zhu, Xiaojuan; Ge, Yiyue; Zeng, Xiaoyan; Chi, Ying; Du, Xuefei; Zhu, Liguo; Zhu, Fengcai; Zhu, Baoli; Cui, Lunbiao; Wu, Bin
title: Association of viral load with serum biomakers among COVID-19 cases
date: 2020-04-30
journal: Virology
DOI: 10.1016/j.virol.2020.04.011
sha: 9001205b71d89c7e661971fae90494f49f21ecd4
doc_id: 898138
cord_uid: 3kwib1o3

ABSTRACTS Since SARS-CoV-2 spreads rapidly around the world, data have been needed on the natural fluctuation of viral load and clinical indicators associated with it. We measured and compared viral loads of SARS-CoV-2 from pharyngeal swab, IgM anti-SARS-CoV-2, CRP and SAA from serum of 114 COVID-19 patients on admission. Positive rates of IgM anti-SARS-CoV-2, CRP and SAA were 80.7%, 36% and 75.4% respectively. Among IgM-positive patients, viral loads showed different trends among cases with different severity, While viral loads of IgM-negative patients tended to increase along with the time after onset. As the worsening of severity, the positive rates of CRP and SAA also showed trends of increase. Different CRP/SAA type showed associations with viral loads in patients in different severity and different time after onset. Combination of the IgM and CRP/SAA with time after onset and severity may give suggestions on the viral load and condition judgment of COVID-19 patients.

Since SARS-CoV-2 spreads rapidly around the world, data have been needed on the 31 natural fluctuation of viral load and clinical indicators associated with it. We measured 32

and compared viral loads of SARS-CoV-2 from pharyngeal swab, IgM anti-SARS-33

CoV-2, CRP and SAA from serum of 114 COVID-19 patients on admission. Positive 34 rates of IgM anti-SARS-CoV-2, CRP and SAA were 80.7%, 36% and 75.4% SARS-CoV-2 has been the focus of worldwide attention. In February, the World Health Organization has declared it a public health emergency of international 48 concern. Within three months, as of March 31 th , SARS-CoV-2 has spreaded around 49 the world with 750890 confirmed cases and 36405 deaths 1 . The number is still 50 increasing. 51

Since the discovery of SARS-CoV-2, scientists and doctors from various 52 countries have done a lot of work in the past three months. The genome variations 2, 3 , 53 epidemiological 4 and clinical characteristics 5 and other investigative findings 6 have 54 been previously described, but the natural fluctuation of viral loads among different 55 COVID-19 cases before antiviral therapy were still not well presented 7, 8, 9, 10, 11 . The 56 clinical indicators associated with the load changes at different stages of the illness 57 are also largely unknown. 58

Here, we explored the association of viral load with IgM anti-SARS-CoV-2 (IgM), 59 C-reactive protein (CRP) and Serum amyloid A (SAA) among COVID-19 patients on 60 admission, to find the clues that may facilitate the prevention and treatment of this 61 infectious disease. 62

As of February 4 th , samples collected on admission from Jiangsu Province of 65

China were sent to our laboratory to confirm the infection of SARS-CoV-2. Real-time 66 reverse-transcriptase polymerase-chain-reaction (RT-PCR) assay (BioGerm, China) 67 was used to detect SARS-CoV-2 nucleic acids from pharyngeal swab samples. Only 68 laboratory confirmed cases were included in this study. Clinical data were obtained 69 from patients' medical records. According to the severity of pneumonia based on 70 radiologic assessments 12 , these patients were classified into non-pneumonia group, 71 pneumonia group and severe pneumonia group in this study. The days after onset was the interval between the date of sampling when on admission and the date of 73 onset. All data were cross-checked. 74

IgM Anti-SARS-CoV-2, CRP and SAA were measured with dry fluorescence 76 immunoassay (Lansionbio, China) from the serum of the COVID-19 cases. According 77 to the manufacturer's instruction, IgM concentrations ≥0.04 AU/mL, CRP 78 concentrations >10µg/mL, and SAA concentrations ≥10µg/mL were judged as 79 positive and marked as IgM(+), CRP(+) and SAA(+) respectively. Each sample was 80 confirmed twice. 81

The viral loads of SARS-CoV-2 were measured as the copy number of the N 83 gene from pharyngeal swabs of the COVID-19 cases. Briefly, based on the SARS-84

CoV-2 genome sequence in Genbank (Accession Number: MN908947), SARS-CoV-85 2 N gene was amplified with primers containing T7 promoter sequence: 5'-86 ACTCGTTAATACGACTCACTATAGGGAAAGGCCAACAACAACAAGG-3' (Forward) 87 and 5'-AGTCTGCGGTAAGGCTTGAGTT-3' (Reverse), and in vitro transcribed with 88 T7 RNA polymerase (TaKaRa, China), then purified and quantified, and finally ten-89 fold diluted ranging from 10 7 to10 1 RNA copies/μL to make RNA standards 13 . The N 90 gene from pharyngeal swabs of the COVID-19 cases was amplified 14 along with the 91 RNA standards (TaKaRa, China). The viral loads were calculated from mean Ct 92 values of three repeats from each patient and the standard curve generated from the 93 Figure S1 ) 94

Continuous variables with normal distribution were analyzed with one way 96 ANOVA or student t-test. Other continuous variables were analyzed with Kruskal-97

Wallis test. Categorical variables were analyzed using Chi-square test, and Fisher 98 exact test when the data were limited. SPSS 19.0 software and GraphPad 7.0 were 99 used for statistical analysis. 100

Of all 125 cases with both serum and pharyngeal swab collected on admission in 103 this study, 11(8.8%) with hemolytic serum were excluded. In the final 114 patients, 32 104 cases were categorized into non-pneumonia group, 74 into pneumonia group and 8 105 into severe pneumonia group on admission (Table 1 ). The median age was 43.5 106 years, and 48.3% were females, with a median time after onset of 4 days. Age and 107 days after onset differed significantly among the three groups (both P<0.05). The 108 positive rates of IgM anti-SARS-CoV-2, CRP and SAA were 80.7%, 36% and 75.4% 109 respectively. Along with the worsening of severity, the positive rates of CRP and SAA 110 tended to increase with significant differences in CRP (P<0.01). 111 

As shown in Figure 1A , the mean viral load/mL (log10) was lower in pneumonia 113 cases (5.15), followed by non-pneumonia cases (5.22), and highest in severe 114 pneumonia cases (5.58), but no significant differences were found. Also, no statistical 115 significance was found between male and female cases with the same severity 116 (mean, 5.36, 5.20 and 5.36 for male cases; 5.06, 5.10 and 5.81 for female case, 117 respectively, in Figure 1B ). But within the female cases, the mean viral load in severe 118 pneumonia patients was higher and significantly differed from non-pneumonia 119 patients (P<0.001) and pneumonia patients (P<0.05). Mean viral loads tended to 120 increase along with the age of patients, and no severe pneumonia patient was found 121 younger than 29 years old in this study, but all the differences were not significant 122 ( Figure 1C) . 123 According to the time after onset, we divided the 114 patients into five groups 130 (day 0-1, day 2-3, day 4-5, day 6-8 and day 9-12). For the IgM(+) patients, the mean 131 viral load/mL (log10) in non-pneumonia cases was 5.27 in day 2-3 group, dropped 132 from 5.40 in day 0-1 group. The value decreased to 4.91 in day 4-5, and increased to 133 5.09 in day 6-8 ( Figure 2 ). The value in pneumonia cases slightly increased to 5.34 in 134 day 2-3 group from 5.27 in day 0-1. It was maintained at 5.25 in day 4-5, decreased 135 to 4.89 in day 6-8 group, and showed a lower level in day 9-12 group (4.60). The 136 value in severe cases maintained high among the groups (all>5.25 ). For the IgM(-) 137 patients, it tended to increase along with the increasing days. Significance was found 138 when compared it in day 9-12 with in day 6-9 (P<0.05). The two IgM(-) patients day 139 9-12 were both with pneumonia, and their mean viral load/mL (log10) also differed 140 significantly from IgM(+) pneumonia patients in day 9-12 (P<0.001). 141

Patients. The number of patients tested on each day is shown above the plot. rose to 5.52 on day 2-3 (P<0.05). The value was dropped to 5.07 on day 4-5 , and maintained at 4.99 on day 6-8. Then it increased about 10 fold in patients of day 9-12 171 from cases in day 6-8 (6.12 vs 5.00, P<0.05 ), and about 100 fold more than that of 172 patients 9-12 days after onset in CRP(-)/SAA(-) group (6.12 vs 4.08). The two CRP (-173 )/SAA(+) patients were both pneumonia cases with IgM (-), while the two CRP(-174 )/SAA(-) patients were both pneumonia cases with IgM (+). 175

IgM is a specific indicator produced early of infectious diseases, and can be 177 used for early diagnosis 15 and has some protective effects. CRP is a non-specific 178 indicator for the early stage of infection, mainly but not limited to bacterial diseases 16 . 179 Current research has found that this indicator is related to COVID-19 severe cases 17 . 180 SAA is another non-specific indicator for the early stage of infection, mainly but not 181 limited to viral diseases 18 . The association between this indicator and the COVID-19 182 has not been reported 5, 19 . In this study, both CRP and SAA showed trends of indicate the viral load of the patient is still at a high level, and the treatment plan may 215 need to be changed to control the viral load. 216 Compared with 62.5% of severe pneumonia patients had CRP(+)/SAA(+), we 217 did not find any severe cases in CRP(-)/SAA(-) group. We also found that in CRP (-218 )/SAA(-) group, the two patients of 9-12 days after onset had a relative viral load 219 about 10 fold lower than that of patients of 6-8 days. Considering that the two 220 patients was both IgM(+) and the antiviral effect of IgM, CRP(-)/SAA(-) with IgM(+) 221 might indicate that the patients' condition is relatively optimistic. Interestingly, in 222 CRP(-)/SAA(+) group, another two patients with an onset of 9-12 days had a relative 223 viral load about 10 fold higher than that of patients with onset of 6-8 days. Since the 224 two patients were both negative for IgM antibodies, CRP(-)/SAA(+) with IgM(-) might 225

indicate that the patients' condition were not in good directions. Due to our sample 226 size is not big enough, the viral load of CRP(-)/SAA(-) with IgM(-) and CRP(-)/SAA (+) 227 with IgM(+) patients was not observed at 9-12 days. However, these clues still allow 228 us to see the possibility of judging the condition of COVID-19 patients by combining 229

IgM and CRP/SAA. If these hypotheses are tested with a larger population on and 230 after admission, combining these three indicators will facilitate the judgment and 231 treatment of COVID-19 patients. 232

Combination of the IgM and CRP/SAA with some conventional clinical 234 information, including time after onset and the type of severity, may give suggestions 235 on the viral load and condition judgment of COVID-19 patients. More samples and 236 researches are needed to confirm these clues. Taking into account these indicators 237 had wide range of clinical applications, the confirmation of these clues will greatly 238 help us to prevent and control the SARS-CoV-2 pandemic. 

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☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: