ABSTRACT
Background: Acute respiratory infection caused by RNA viruses is still one of the main diseases all over the world such as SARS CoV 2 and Influenza A virus. mNGS was a powerful tool for ethological diagnosis. But there were some challenges during mNGS implementation in clinical settings such as time consuming manipulation and lack of comprehensive analytical validation. Methods: We set up CATCH that was a mNGS method based on RNA and DNA hybrid tagmentation via Tn5 transposon. Seven respiratory RNA viruses and three subtypes of Influenza A virus had been used to test capabilities of CATCH on detection and quantification. Analytical performance of SARS CoV 2 and Influenza A virus had been determined with reference standards. We compared accuracy of CATCH with quantitative real time PCR by using clinical 98 samples from 64 COVID19 patients. Results: We minimized the library preparation process to 3 hours and handling time to 35 minutes. Duplicate filtered RPM of 7 respiratory viruses and 3 Influenza A virus subtypes were highly correlated with viral concentration. LOD of SARS CoV 2 was 39.2 copies/test and of Influenza A virus was 278.1 copies/mL. Comparing with quantitative real time PCR, the overall accuracy of CATCH was 91.4%. Sensitivity was 84.5% and specificity was 100%. Meanwhile, there were significant difference of microbial profile in oropharyngeal swabs among critical, moderate patients and healthy controls. Conclusion: Although further optimization is needed before CATCH can be rolled out as a routine diagnostic test, we highlight the potential impact of it advancing molecular diagnostics for respiratory pathogens.
Subject(s)
COVID-19 , Respiratory Tract Infections , Severe Acute Respiratory SyndromeABSTRACT
Background. COVID-19 is a pandemic with no specific antiviral treatments or vaccines. The urgent needs for exploring the neutralizing antibodies from patients with different clinical characteristics are emerging. Methods. A total of 117 blood samples were collected from 70 COVID-19 inpatients and convalescent patients. The presence of neutralizing antibody was determined with a modified cytopathogenic assay based on live SARS-CoV-2. The dynamics of neutralizing antibody levels at different with different clinical characteristics were analyzed. Results. The seropositivity rate reached up to 100.0% within 20 days since onset, and remained 100.0% till day 41-53. The total GMT was 1:163.7 (95% CI, 128.5 to 208.6), and the antibody level was highest during day 31-40 since onset, and then decreased slightly. Individual differences in changes of antibody levels were observed among 8 representative convalescent patients. In multivariate GEE analysis, patients at age of 31-60 and 61-84 had a higher antibody level than those at age of 16-30 ({beta}=1.0518, P=0.0152; {beta}=1.3718, P=0.0020). Patients with a worse clinical classification had a higher antibody titer ({beta}=0.4639, P=0.0227). Conclusions. The neutralizing antibodies were detected even at the early stage of disease, and a significant response showed in convalescent patients. Moreover, changes on antibody levels ware individual specific.
Subject(s)
COVID-19ABSTRACT
WHO has declared COVID-19 a pandemic with more than 300,000 confirmed cases and more than 14,000 deaths. There is urgent need for accurate and rapid diagnostic kits. Here we report the development and validation of a COVID-19/SARS-CoV-2 S1 serology ELISA kit for the detection of total anti-virus antibody (IgG+IgM) titers in sera from either the general population or patients suspected to be infected. For indirect ELISA, CHO-expressed recombinant full length SARS-CoV-2-S1 protein with 6*His tag was used as the coating antigen to capture the SARS-CoV-2-S1 antibodies specifically. The specificity of the ELISA kit was determined to be 97.5%, as examined against total 412 normal human sera including 257 samples collected prior to the outbreak and 155 collected during the outbreak. The sensitivity of the ELISA kit was determined to be 97.5% by testing against 69 samples from hospitalized and/or recovered COVID-19 patients. The overall accuracy rate reached 97.3%. Most importantly, in one case study, the ELISA test kit was able to identify an infected person who had previously been quarantined for 14 days after coming into contact with a confirmed COVID-19 patient, and discharged after testing negative twice by nucleic acid test. With the assays developed here, we can screen millions of medical staffs in the hospitals and people in residential complex, schools, public transportations, and business parks in the epidemic centers of the outbreaks to fish out the "innocent viral spreaders", and help to stop the further spreading of the virus.