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      The kinetics of viral load and antibodiesto SARS-CoV-2

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          Abstract

          Objectives

          To understand persistence of the virus in body fluids and immune response of infected host to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), an agent of coronavirus disease 2019 (COVID-19).

          Methods

          We determined the kinetics of viral load in several body fluids through real time reverse transcription polymerase chain reaction (rRT-PCR), serum antibodies of IgA, IgG and IgM by enzyme linked immunosorbent assay (ELISA), and neutralizing antibodies by microneutralization assay in 35 COVID-19 cases from two hospitals in Guangdong, China.

          Results

          We found higher viral loads and prolonged shedding of virus RNA in severe cases of COVID-19 in nasopharyngeal (1.3×10 6 vs 6.4×10 4, p<0.05; 7∼8w) and throat (6.9×10 6 vs 2.9×10 5, p<0.05; 4∼5w), while comparable in sputum samples (5.5×10 6 vs 0.9×10 6, p<0.05; 4∼5w). Viraemia was rarely detected (2.8%, n=1/35). We detected early seroconversion of IgA and IgG at 1 st week after illness onset (day 5, 5.7%, n=2/35). Neutralizing antibodies were produced in the second week, and observed in all 35 included cases after 3 rd week illness onset. The levels of neutralizing antibodies correlated with IgG (r s=0.85, p<0.05; kappa=0.85) and IgA (r s=0.64, p<0.05; kappa=0.61) in severe, but not mild cases (IgG, r s=0.42, kappa=0.33; IgA, r s=0.32, kappa=0.22). No correlation with IgM in either severe (r s=0.17, kappa=0.06) or mild cases (r s=0.27, kappa=0.15) was found.

          Conclusions

          We revealed a prolonged shedding of virus RNA in upper respiratory tract, and evaluated the consistency production of IgG, IgA, IgM and neutralizing antibodies in COVID-19 cases.

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          Most cited references10

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          SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients

          To the Editor: The 2019 novel coronavirus (SARS-CoV-2) epidemic, which was first reported in December 2019 in Wuhan, China, and has been declared a public health emergency of international concern by the World Health Organization, may progress to a pandemic associated with substantial morbidity and mortality. SARS-CoV-2 is genetically related to SARS-CoV, which caused a global epidemic with 8096 confirmed cases in more than 25 countries in 2002–2003. 1 The epidemic of SARS-CoV was successfully contained through public health interventions, including case detection and isolation. Transmission of SARS-CoV occurred mainly after days of illness 2 and was associated with modest viral loads in the respiratory tract early in the illness, with viral loads peaking approximately 10 days after symptom onset. 3 We monitored SARS-CoV-2 viral loads in upper respiratory specimens obtained from 18 patients (9 men and 9 women; median age, 59 years; range, 26 to 76) in Zhuhai, Guangdong, China, including 4 patients with secondary infections (1 of whom never had symptoms) within two family clusters (Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org). The patient who never had symptoms was a close contact of a patient with a known case and was therefore monitored. A total of 72 nasal swabs (sampled from the mid-turbinate and nasopharynx) (Figure 1A) and 72 throat swabs (Figure 1B) were analyzed, with 1 to 9 sequential samples obtained from each patient. Polyester flock swabs were used for all the patients. From January 7 through January 26, 2020, a total of 14 patients who had recently returned from Wuhan and had fever (≥37.3°C) received a diagnosis of Covid-19 (the illness caused by SARS-CoV-2) by means of reverse-transcriptase–polymerase-chain-reaction assay with primers and probes targeting the N and Orf1b genes of SARS-CoV-2; the assay was developed by the Chinese Center for Disease Control and Prevention. Samples were tested at the Guangdong Provincial Center for Disease Control and Prevention. Thirteen of 14 patients with imported cases had evidence of pneumonia on computed tomography (CT). None of them had visited the Huanan Seafood Wholesale Market in Wuhan within 14 days before symptom onset. Patients E, I, and P required admission to intensive care units, whereas the others had mild-to-moderate illness. Secondary infections were detected in close contacts of Patients E, I, and P. Patient E worked in Wuhan and visited his wife (Patient L), mother (Patient D), and a friend (Patient Z) in Zhuhai on January 17. Symptoms developed in Patients L and D on January 20 and January 22, respectively, with viral RNA detected in their nasal and throat swabs soon after symptom onset. Patient Z reported no clinical symptoms, but his nasal swabs (cycle threshold [Ct] values, 22 to 28) and throat swabs (Ct values, 30 to 32) tested positive on days 7, 10, and 11 after contact. A CT scan of Patient Z that was obtained on February 6 was unremarkable. Patients I and P lived in Wuhan and visited their daughter (Patient H) in Zhuhai on January 11 when their symptoms first developed. Fever developed in Patient H on January 17, with viral RNA detected in nasal and throat swabs on day 1 after symptom onset. We analyzed the viral load in nasal and throat swabs obtained from the 17 symptomatic patients in relation to day of onset of any symptoms (Figure 1C). Higher viral loads (inversely related to Ct value) were detected soon after symptom onset, with higher viral loads detected in the nose than in the throat. Our analysis suggests that the viral nucleic acid shedding pattern of patients infected with SARS-CoV-2 resembles that of patients with influenza 4 and appears different from that seen in patients infected with SARS-CoV. 3 The viral load that was detected in the asymptomatic patient was similar to that in the symptomatic patients, which suggests the transmission potential of asymptomatic or minimally symptomatic patients. These findings are in concordance with reports that transmission may occur early in the course of infection 5 and suggest that case detection and isolation may require strategies different from those required for the control of SARS-CoV. How SARS-CoV-2 viral load correlates with culturable virus needs to be determined. Identification of patients with few or no symptoms and with modest levels of detectable viral RNA in the oropharynx for at least 5 days suggests that we need better data to determine transmission dynamics and inform our screening practices.
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            Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

            Summary Background Coronavirus disease 2019 (COVID-19) causes severe community and nosocomial outbreaks. Comprehensive data for serial respiratory viral load and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not yet available. Nasopharyngeal and throat swabs are usually obtained for serial viral load monitoring of respiratory infections but gathering these specimens can cause discomfort for patients and put health-care workers at risk. We aimed to ascertain the serial respiratory viral load of SARS-CoV-2 in posterior oropharyngeal (deep throat) saliva samples from patients with COVID-19, and serum antibody responses. Methods We did a cohort study at two hospitals in Hong Kong. We included patients with laboratory-confirmed COVID-19. We obtained samples of blood, urine, posterior oropharyngeal saliva, and rectal swabs. Serial viral load was ascertained by reverse transcriptase quantitative PCR (RT-qPCR). Antibody levels against the SARS-CoV-2 internal nucleoprotein (NP) and surface spike protein receptor binding domain (RBD) were measured using EIA. Whole-genome sequencing was done to identify possible mutations arising during infection. Findings Between Jan 22, 2020, and Feb 12, 2020, 30 patients were screened for inclusion, of whom 23 were included (median age 62 years [range 37–75]). The median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was 5·2 log10 copies per mL (IQR 4·1–7·0). Salivary viral load was highest during the first week after symptom onset and subsequently declined with time (slope −0·15, 95% CI −0·19 to −0·11; R 2=0·71). In one patient, viral RNA was detected 25 days after symptom onset. Older age was correlated with higher viral load (Spearman's ρ=0·48, 95% CI 0·074–0·75; p=0·020). For 16 patients with serum samples available 14 days or longer after symptom onset, rates of seropositivity were 94% for anti-NP IgG (n=15), 88% for anti-NP IgM (n=14), 100% for anti-RBD IgG (n=16), and 94% for anti-RBD IgM (n=15). Anti-SARS-CoV-2-NP or anti-SARS-CoV-2-RBD IgG levels correlated with virus neutralisation titre (R 2>0·9). No genome mutations were detected on serial samples. Interpretation Posterior oropharyngeal saliva samples are a non-invasive specimen more acceptable to patients and health-care workers. Unlike severe acute respiratory syndrome, patients with COVID-19 had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. This finding emphasises the importance of stringent infection control and early use of potent antiviral agents, alone or in combination, for high-risk individuals. Serological assay can complement RT-qPCR for diagnosis. Funding Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, and Sanming Project of Medicine.
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              Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections

              The clinical features and immune responses of asymptomatic individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have not been well described. We studied 37 asymptomatic individuals in the Wanzhou District who were diagnosed with RT-PCR-confirmed SARS-CoV-2 infections but without any relevant clinical symptoms in the preceding 14 d and during hospitalization. Asymptomatic individuals were admitted to the government-designated Wanzhou People's Hospital for centralized isolation in accordance with policy1. The median duration of viral shedding in the asymptomatic group was 19 d (interquartile range (IQR), 15-26 d). The asymptomatic group had a significantly longer duration of viral shedding than the symptomatic group (log-rank P = 0.028). The virus-specific IgG levels in the asymptomatic group (median S/CO, 3.4; IQR, 1.6-10.7) were significantly lower (P = 0.005) relative to the symptomatic group (median S/CO, 20.5; IQR, 5.8-38.2) in the acute phase. Of asymptomatic individuals, 93.3% (28/30) and 81.1% (30/37) had reduction in IgG and neutralizing antibody levels, respectively, during the early convalescent phase, as compared to 96.8% (30/31) and 62.2% (23/37) of symptomatic patients. Forty percent of asymptomatic individuals became seronegative and 12.9% of the symptomatic group became negative for IgG in the early convalescent phase. In addition, asymptomatic individuals exhibited lower levels of 18 pro- and anti-inflammatory cytokines. These data suggest that asymptomatic individuals had a weaker immune response to SARS-CoV-2 infection. The reduction in IgG and neutralizing antibody levels in the early convalescent phase might have implications for immunity strategy and serological surveys.
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                Author and article information

                Journal
                Clin Microbiol Infect
                Clin. Microbiol. Infect
                Clinical Microbiology and Infection
                Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.
                1198-743X
                1469-0691
                6 September 2020
                6 September 2020
                Affiliations
                [1 ]Guangdong Provincial Institute of Public Health, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China
                [2 ]The First Hospital of Foshan, Foshan, China
                [3 ]The Guangdong Second Provincial General Hospital, Guangzhou, China
                [4 ]Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China
                []Corresponding authors: Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 510300, P.R. China. Tel.: +86(020)31051236.
                Author notes
                [∗∗ ]Corresponding authors: The Guangdong Seconded Provincial General Hospital, Guangzhou 510317, P.R. China.
                [#]

                These authors contributed equally to this work.

                Article
                S1198-743X(20)30527-9
                10.1016/j.cmi.2020.08.043
                7474805
                32898715
                cfb6cc2e-1763-43f7-8d34-ac68c7816938
                © 2020 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 30 April 2020
                : 27 August 2020
                : 31 August 2020
                Categories
                Article

                Microbiology & Virology
                covid-19,iga,igg,igm,neutralizing antibodies,sars-cov-2,viral load
                Microbiology & Virology
                covid-19, iga, igg, igm, neutralizing antibodies, sars-cov-2, viral load

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