User:Shawndouglas/sandbox/sublevel1

Severe acute respiratory syndrome, otherwise known as SARS, arose in South China in late 2002. Caused by the SARS coronavirus (SARS-CoV) and believed to have originated from horseshoe bats, SARS eventually was contained in the summer of 2003. The last known infection was in April 2004, due to a laboratory accident. During that time, the following sample collection and test procedures evolved from the related outbreaks (note that this is only a summary; consult the cited literature directly for full details):

• Determine that the patient is indicating clinical and/or epidemiological evidence of SARS (meets case definitions). As Knobler et al. put it: "SARS-CoV testing should be considered if no alternative diagnosis is identified 72 hours after initiation of the clinical evaluation and the patient is thought to be at high risk for SARS-CoV disease (e.g., is part of a cluster of unexplained pneumonia cases)."

• Collect multiple specimen types at different time points of the patient's illness. Respiratory and plasma or serum specimens should be collected early into the first week of illness. Respiratory samples should be from nasopharyngeal aspirates and swabs of the upper respiratory tract or, in some cases, fluids from the lower respiratory tract using bronchoalveolar lavage, tracheal aspiration, or a pleural tap. (Sputum can also be collected.) Whole blood (5 to 10 ml) is collected into either a serum separator tube for blood serum or EDTA tube for blood plasma. Stool samples are also of import early on for virus isolation or detection and are useful in at least the first and second weeks of the illness. Blood serum is useful in weeks two and three for detecting a rising titre. Additionally, the literature also makes reference to methods of collecting specimens postmortem.

• Conduct testing. At the time, the two primary test types used were enzyme immunoassay (EIA; today more commonly known as ELISA) for detection of serum antibody and reverse transcription polymerase chain reaction (RT-PCR) for detection of the virus' RNA. The U.S. Centers for Disease Control and Prevention (CDC) had this to say about these tests in May 2004:

Both the EIA and the RT-PCR tests are sensitive and highly specific for SARS-CoV. The ability to diagnose SARS-CoV infection in a patient is often limited, however, by either the low concentration of virus in most clinical specimens (RT-PCR assays) or the time it takes a person to mount a measurable antibody response to SARS-CoV (serologic assays). The likelihood of detecting infection is increased if multiple specimens (e.g., stool, serum, respiratory tract specimens) are collected at several times during the course of illness.

The literature also makes reference to an immunofluorescence assay (IFA) for detecting antibody, with the CDC calling its results "essentially identical to those for the EIA for SARS antibody." Tangentially, isolation of SARS-CoV in cell culture from a clinical specimen is also referenced, though such activity is reserved for Biosafety Level 3 (BSL-3) laboratories.

• Confirm the results. Laboratory confirmation is based on one of 1. initial local lab detection and subsequent national reference lab confirmation of a validated serology-based test detection; 2. isolation of SARS-CoV in cell culture with subsequent confirmation from a validated test; or 3. initial local lab detection and subsequent national reference lab confirmation of SARS-CoV RNA from a validated RT-PCR test which used either two clinical specimens from different sources or two same-source clinical specimens from two different days.

Additionally, in the case of serology, one of the following must be true:

1. SARS-CoV serum antibodies are detected in a single serum specimen; or,
2. a "four-fold or greater increase in SARS-CoV antibody titer between acute- and convalescent-phase serum specimens tested in parallel" is detected; or,
3. a "negative SARS-CoV antibody test result on acute-phase serum and positive SARS-CoV antibody test result on convalescent-phase serum tested in parallel" is detected.

Of note is the World Health Organization's (WHO) January 2004 cautionary message about serological diagnostics in not only SARS-CoV but other types of coronaviruses. At that time, they showed a level of unsurety in regards to how coronaviruses elicited serological cross-reactions and generated antigenic recall. They also preached caution in interpreting serological results in non-epidemic periods and when no viral sequence data are available. Finally, they also mentioned the added difficulties of rate cases when coinfection with a related human coronavirus occurs, "although the use of expressed proteins in Western blots may help to sort this out." More than 15 years later, Loeffelholz and Tang put this concept into clearer terms, indicating that while "serological assays are not routinely used for diagnosis of [human coronavirus] infections due to the lack of commercial reagents," they still have important value "for understanding the epidemiology of emerging [human cornaviruses], including the burden and role of asymptomatic infections," as well as for antibody detection of novel and emerging coronaviruses.

• Arrange for confirmatory testing to be performed by an appropriate test site in the case of a positive RT-PCR test.

• Report to state or local health departments details of patients radiographically confirmed with pneumonia with at least one SARS-CoV risk factor for exposure, clusters of healthcare workers with unexplained pneumonia, and any positive SARS-CoV test results. Additional international reporting of SARS by WHO Member States in regards to probable and laboratory-confirmed cases is also requested.

• Send off for an additional verification by an external member of the WHO's SARS Reference and Verification Laboratory Network before internationally announcing results as a laboratory-confirmed case.