Prevalence of non-pneumonic infections with SARS-correlated virus
Authors: Patrick CY Woo, Susanna KP Lau, Kwok-yung Yuen
Journal: The Lancet (2004)

Authors' reply Sir—The technique of using an ELISA and confirmation by western-blot assays has been widely applied for serodiagnosis of viral infections.1Centers for Disease Control and PreventionInterpretation and use of the Western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections.MMWR Morb Mortal Wkly Rep. 1989; 38: 1-7PubMed Google Scholar, 2Van der Poel CL Cuypers HTM Reesink HW et al.Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay.Lancet. 1991; 337: 317-319Summary PubMed Scopus (489) Google Scholar In our (His)6-tagged recombinant nucleocapsid protein and spike polypeptide antibody assays, the purified proteins were checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with antibodies against His, confirming that the single prominent band seen after Coomassie blue staining corresponded to the immunoreactive band reacting to antibody against His. This finding inferred that no major residual E coli proteins existed in the purified antigens used in the antibody assays. After initial screening by the nucleocapsid ELISA, potentially false-positive results caused by cross-reaction with minute amounts of residual E coli proteins, not detectable by Coomassie blue staining, were eliminated by the nucleocapsid western-blot assay. Potentially false-positive findings due to antibodies against the nucleocapsid proteins of other coronaviruses were further eliminated by the spike western-blot assay. Because of concerns from readers and reviewers that serum samples from patients with other coronavirus infections might also react with the spike polypeptide despite the very low aminoacid identity between the spike protein of SARS coronavirus and those of human coronaviruses OC43 and 229E, we did additional analyses in collaboration with Robert Couch (Baylor College of Medicine, Houston, TX, USA). Results showed that none of the 21 and seven pairs of serum samples from patients with OC43 and 229E infections, respectively, were positive by the nucleocapsid ELISA, nucleocapsid western blot, and spike western blot, although four showed false-positive results on the ELISA, three of which also showed bands by the nucleocapsid western blot (unpublished data). These findings might explain the false-positive results detected by the nucleocapsid ELISA and western-blot assay obtained in our seroprevalence study. Interestingly, one of the 28 serum samples also tested positive by the SARS immunofluorescence assay (unpublished data), indicating that this assay might not be more specific than the ELISA and western-blot assays. The apparent difference in seroprevalence among different studies could be due to the different sensitivities of the assays used and the samples collected in the studies. Serum samples from SARS patients that tested seronegative by the immunofluorescence assay have tested positive by the nucleocapsid ELISA, with clear-cut positive absorbance values, and confirmation by the western blot assays (unpublished data). This finding indicates that the nucleocapsid ELISA might be able to catch additional cases that the immunofluorescence assay has missed. We suggest that non-pneumonic SARS-virus infections, which could be caused by lower viral loads, might induce milder antibody responses. These milder responses may be missed, or interpreted as "false-positives", by seroprevalence studies that include the immunofluorescence assay. As for the different sampling populations, it is interesting to see that seroprevalence varies from 0·009% to 1·8%.3Chinese University of Hong KongTerritory-wide SARS seroprevalence study suggests Hong Kong is not a human reservoir for SARS coronavirus. Chinese University of Hong Kong, Shatin2004http://www.cuhk.edu.hk/ipro/pressrelease/040315e.htmGoogle Scholar, 4Zheng BJ Wong KH Zhou J et al.SARS-related virus predating SARS outbreak, Hong Kong.Emerg Infect Dis. 2004; 10: 176-178Crossref PubMed Scopus (93) Google Scholar In the study that showed a seroprevalence of 1·8%, all 17 positive samples were confirmed with the viral neutralisation assay. Although the heterogeneous nature of our study population did not allow a perfect comparison of the relative rates of non-pneumonic SARS-virus infections and SARS-virus pneumonia in the whole population, comparison in the paediatric and adult group (healthy blood donor) subpopulations separately also showed higher rates of non-pneumonic SARS-virus infections than SARS-virus pneumonia (p<0·001 in both groups). Further studies, using the same antibody test, should be done in different populations to determine their relative prevalence of SARS-virus infections. Prevalence of non-pneumonic infections with SARS-correlated virusPatrick Woo and colleagues (Mar 13, p 841)1 suggest that non-pneumonic severe acute respiratory syndrome (SARS)-virus infections are more common than SARS-virus pneumonia in Hong Kong on the basis of a serological survey of about 1000 non-pneumonic healthy individuals of different groups and periods. We query the validity of the authors' experimental approach, which involved western blots only, on the confirmation of the ELISA positive samples. Full-Text PDF Prevalence of non-pneumonic infections with SARS-correlated virusPatrick Woo and colleagues1 use an IgG antibody test combined with western-blot analysis to demonstrate the existence of subclinical or non-pneumonic infections with SARS virus and the prevalence of such cases compared with pneumonic SARS cases. However, the four patients identified as having non-pneumonic SARS were among a group of 33 positive by the authors' ELISA, of whom 26 were also positive by nucleocapsid western blot. Thus, 22 samples turned out to be positive for antibodies against SARS virus nucleocapsid protein, but negative for antibodies against SARS virus spike protein. Full-Text PDF Prevalence of non-pneumonic infections with SARS-correlated virusPatrick Woo and colleagues1 provide evidence of subclinical or non-pneumonic infections with the virus thought to cause SARS and propose that non-pneumonic infections are more common than SARS virus pneumonia in Hong Kong. However, I worry that the method used by Woo and colleagues could have led to a misleading concept. Although the authors claim that they used western blot to confirm the specificity of antibodies against SARS-correlated virus, the method they used, which was based on a recombinant nucleocapsid protein and a recombinant spike polypeptide blotted onto membranes to probe corresponding antibodies, is actually a dot-blot ELISA. Full-Text PDF Prevalence of non-pneumonic infections with SARS-correlated virusPatrick Woo and colleagues' findings1 are certainly pertinent to health-care-system planning for potential SARS outbreaks. However, I wish to express my concern over the comparison of a figure reported as a rate for the study population with the cumulative incidence of pneumonic SARS for the Hong Kong population. Full-Text PDF