Serum SARS-COV-2 Nucleocapsid Protein: A Sensitivity and Specificity Early Diagnostic Marker for SARS-COV-2 Infection

Summary Background A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. Methods All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by WHO and the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. Findings By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0–58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0–13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. Interpretation The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. Funding Ministry of Science and Technology, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technology Commission.

Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

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.