ABSTRACT
Background: Effective therapeutics and vaccines for coronavirus disease 2019 (COVID-19) are currently lacking because of the mutation and immune escape of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on the propagation characteristics of SARS-CoV-2, rapid and accurate detection of complete virions from clinical samples and the environment is critical for assessing infection risk and containing further COVID-19 outbreaks. However, currently applicable methods cannot achieve large-scale clinical application because of factors such as the high viral load, cumbersome virus isolation steps, demanding environmental conditions, and long experimental periods. In this study, we developed an immunomolecular detection method combining capture of the viral spike glycoprotein with monoclonal antibodies and nucleic acid amplification via quantitative reverse transcription PCR to rapidly and accurately detect complete virions. Results: : After constructing a novel pseudovirus, screening for specific antibodies, and optimizing the detection parameters, the assay achieved a limit of detection of 9 × 10 2 transduction units/mL of viral titer with high confidence (~95%) and excellent stability against human serum and common virus/pseudovirus. The coefficients of variation were 1.0–2.0% for intra-assay and inter-assay analyses, respectively. Compared with reverse transcription-PCR, the immunomolecular method more accurately quantified complete virions. SARS-CoV-2/pseudovirus was more stable on plastic and paper than on aluminum and copper in the detection of SARS-CoV-2 pseudovirus under different conditions, and complete virions were detected up to 96 h after they were applied to these surfaces (except for copper), although the titer of the virions was greatly reduced. Conclusions: : Convenient, inexpensive, and accurate complete virus detection can be applied in many fields, such as monitoring the infectivity of convalescent and post-discharge patients and assessing high-risk environments (isolation rooms, operating rooms, patient living environments, and cold chain logistics). This method can also be used to detect intact virions such as those of hepatitis B and C viruses, human immunodeficiency virus, influenza, and the partial pulmonary virus, which may further improve the accuracy of diagnoses and facilitate individualized and precise treatments.
Subject(s)
Coronavirus Infections , Immunologic Deficiency Syndromes , COVID-19 , Hepatitis BABSTRACT
Background We aim to investigate the profile of acute antibody response in COVID-19 patients, and provide proposals for the usage of antibody test in clinical practice. Methods A multi-center cross-section study (285 patients) and a single-center follow-up study (63 patients) were performed to investigate the feature of acute antibody response to SARS-CoV-2. A cohort of 52 COVID-19 suspects and 64 close contacts were enrolled to evaluate the potentiality of the antibody test. Results The positive rate for IgG reached 100% around 20 days after symptoms onset. The median day of serocon-version for both lgG and IgM was 13 days after symptoms onset. Seroconversion of IgM occurred at the same time, or earlier, or later than that of IgG. IgG levels in 100% patients (19/19) entered a platform within 6 days after seroconversion. The criteria of IgG seroconversion and [≥] 4-fold increase in the IgG titers in sequential samples together diagnosed 82.9% (34/41) of the patients. Antibody test aided to confirm 4 patients with COVID-19 from 52 suspects who failed to be confirmed by RT-PCR and 7 patients from 148 close contacts with negative RT-PCR. Conclusion IgM and IgG should be detected simultaneously at the early phase of infection. The serological diagnosis criterion of seroconversion or [≥] 4-fold increase in the IgG titer is suitable for a majority of COVID-19 patients. Serologic test is helpful for the diagnosis of SARS-CoV-2 infection in suspects and close contacts.
Subject(s)
COVID-19ABSTRACT
A respiratory illness has been spreading rapidly in China, since its outbreak in Wuhan city, Hubei province in December 2019. The illness was caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical manifestations related to SARS-CoV-2 infection ranged from no symptom to fatal pneumonia. World Health Organization (WHO) named the diseases associated with SARS-CoV-2 infection as COVID-19. Real time RT-PCR is the only laboratory test available till now to confirm the infection. However, the accuracy of real time RT-PCR depends on many factors, including sampling location and of methods, quality of RNA extraction and training of operators etc. Variations in these factors might significantly lower the sensitivity of the detection. We developed a peptide-based luminescent immunoassay to detect IgG and IgM. Cut-off value of this assay was determined by the detection of 200 healthy sera and 167 sera from patients infected with other pathogens than SARS-CoV-2. To evaluate the performance of this assay, we detected IgG and IgM in the 276 sera from confirmed patients. The positive rate of IgG and IgM were 71.4% (197/276) and 57.2% (158/276) respectively. By combining with real time RT-PCR detection, this assay might help to enhance the accuracy of diagnosis of SARS-CoV-2 infection.