Diagnosis of Severe Acute Respiratory Syndrome (SARS)

1. Diagnosis of Severe Acute Respiratory Syndrome (SARS) • Clinical characteristics of illness • Epidemiologic link to SARS, i.e. ,travel or contact • Laboratory evidence of SARS-CoV infection • Type of specimen • Timing of specimen collection • Laboratory assay to detect infection

2. Association Between SARS-CoV and SARS Disease • Infection in SARS patients – virus and serology • CDC • Germany, Canada, Hong Kong, Taiwan, Singapore, France, China, Thailand, etc. • Neg-serology in non-SARS patients (~1000 sera at CDC, 280 HK) • Virus in lung tissue/BAL specimen • Identical sequences different outbreaks (c/w point source) • US; Hong Kong, Thailand, Singapore, Taiwan, Vietnam, Canada, Germany • Pulmonary pathology in monkey model • Questions – histopathology and rate of positivity

3. SARS Etiologic Agent Order: Nidovirales Family: Coronaviridae • Torovirus • Coronavirus • Grp I • Grp II • Grp III

4. Coronaviruses, Hosts and Diseases Antigenic Group Virus HostRespiratoryEntericOther IHCoV-229E human X TGEV pig X PRCoV pig X FIPV cat X X X FECoV cat X CCoV dog X IIHCoV-OC43 human X ?? MHV mouse X X X RCoV rat X X HEV pig X X BCoV cattle X X III IBV chicken X X TCoV turkey X

5. SARS-CoV Genome Organizationand mRNA Synthesis A E ORF 1a M N S ORF 1b 5,000 10,000 15,000 20,000 25,000 30,000 1 C B 2 1 3 20,001 25,000 30,000 kB X1 E X3 9.0 M N 6.0 5.0 S 4.0 X5 X2 X4 3.0 2.5 2.0 1.5 8.3 kb RNA 2 4.5 kb RNA 3 1.0 3.4 kb RNA 4 0.5 2.5 kb RNA 5 1.7 kb RNA 6

6. Laboratory Diagnosis of SARS Infection • Type and timing of specimen collection (we need to know more) • Type of assays • Sensitivity • Specificity • Interpretation of results

7. Laboratory Assays for SARS • Detection of virus • EM in clinical specimens (CoV-like particles) • Isolation of virus • Detection of viral antigens (IHC for tissue, ?sensitivity of IFA or ELISA for respiratory specimens) • Detection of viral RNA (PCR) • Respiratory secretions • Stool specimens • Urine specimens • Tissue – lung and kidney • Detection of SARS-specific antibody • IFA • ELISA • Neutralization

8. Real-Time RT-PCR (TaqMan)

9. Characteristics of SARS-CoV PCR • Limited experience/data • Specimens • Upper respiratory maybe ~50% positivity in acute-phase specimens • Stool possibly higher sensitivity later in illness, e.g., 10-14 days • Sputum/BAL probably higher rate of positivity • Other specimens, urine, blood, tissues, ? • Interpretation of Results • Negative -- does not rule out SARS-CoV infection • Positive – possibility of false positive (test error/contamination)

10. SARS CoV Antibody Assays

11. Characteristics of SARS-CoV Antibody Assays • Limited data • Very low or absent antibody in controls and persons without acute SARS • Acute sera may be positive as early as 6 days after onset of sera • Convalescent sera should be positive by 21 days after onset • Interpretation of results • Single positive sera indicative of acute infection • Later diagnostic rise in antibody between acute- and convalescent-phase sera or IgM antibodies may be need to diagnose acute infection • Convalescent-phase serum specimen collected >21 days after onset of illness is required to confirm lack of antibody response to infection • False positive/false negative results

12. Other Tests for SARS Infection • Limited data • Tissue culture isolation • Electron microscopy for CoV-like particles • Immunohistologic or in situ studies of tissue specimens • IFA or ELISA assays SARS-CoV antigens in respiratory or other specimens(? Likely to be sufficiently sensitive)

13. Conclusions • SARS is a clinical and epidemiologic diagnosis • Laboratory testing can diagnose SARS-CoV infection during the acute illness • Laboratory testing can not rule out infection until the convalescent phase of illness • Dual infections, e.g., SARS-CoV and human metapneumovirus, can occur in patients with SARS