Dr. Cao then described a number of the diagnostics that are currently available for SARS:
• RT-PCR assays convert viral RNA into complementary DNA, which is then amplified and detected. The technique requires specialized equipment, often suffers from low sensitivity, and contamination may cause false positives (Di et al., 2005). Since the technique looks for the virus directly instead of waiting for an immune response, it can be used earlier in an infection than other methods, but it cannot be used for retrospective tests.
• Enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies can look for antigen to the SARS coronavirus nucleocapsid protein (Di et al., 2005). As antigen appears before antibodies, the test is useful for early diagnosis. However, as the antigen becomes undetectable 20 days post onset of symptoms, the test, which does not require viral culture, cannot be used for retrospective work.
• Dr. Cao showed that immunofluorescence assays (IFA) could detect SARS antibodies in 90 percent of the patients 15 days after infection.
• ELISA assays using whole viral lysate as the antigen, like IFA assays, can detect SARS antibodies in serum. Dr. Cao reported that the technique has a higher false positive rate than the comparatively more difficult IFA.
• ELISA assays using recombinant nucleocapsid protein as the antigen can detect SARS in 68 percent of patients 6-10 days after the start of infection and in 90 percent of patients after 10-61 days (Shi et al., 2003). Unlike ELISA with whole viral lysate or IFA, viral culture is not required as the needed protein can be expressed in E. coli. Dr. Cao developed the technique in April 2003, and China’s State Food and Drug Administration certified it soon after. Antibodies to nucleocapsid protein persist for months (Liu et al., 2004), allowing the assay to be used for retrospective work.
Dr. Cao also shared some biosafety and biosecurity concerns and advice. First, as SARS-coronavirus-like viruses are circulating in the Chinese and Slovenian horseshoe bat populations and the Nigerian leaf-nosed bat population, disease reemergence is a concern. Second, the 2004 laboratory leak in Beijing that resulted in 8 cases of SARS, including one death, and the SARS LAIs in laboratory workers in Singapore and Taiwan illustrate the importance of rigorously following safety procedures. To that end, Dr. Cao argued that SARS coronavirus and infected tissues must be manipulated only in BSL-3 labs by well-trained scientists and technicians. While serum tests can be done in BSL-2 labs in BSCs, he cautioned that sera from SARS patients should be incubated at 56Â°C for 30 minutes to inactivate the virus.
Dr. Cao also presented data showing the role of nucleocapsid protein in the immune response to SARS coronavirus.
Crimean-Congo Hemorrhagic Fever: Pakistan’s Perspective
Birjees Mazhar Kazi (National Institute of Health, Islamabad-Pakistan) described the tests and the accompanying precautions the National Institute of Health in Islamabad, Pakistan uses for CCHF diagnosis.
Dr. Kazi explained that due to the prevalence of the tick vector, Pakistan experiences CCHF outbreaks and that the National Institute of Health, Pakistan tracks reported cases. Current diagnostic tools include immunoglobulin M (IgM) capture ELISA using a kit from Biological Diagnostic Supplies Limited, RT-PCR using a published protocol (Schwarz et al., 1996), and genetic sequencing. Dr. Kazi noted that although the National Institute of Health, Pakistan has the infrastructure and trained personnel for DNA sequencing, reagents for this