III. Diagnostic Assays
Supplement F: Laboratory Guidance
Public Health Guidance for Community-Level Preparedness and Response to Severe Acute Respiratory Syndrome (SARS) Version 2/3
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Among the several types of assays used to detect SARS-CoV, RT-PCR and antibody assays are the most commonly used.
A. Real-Time RT-PCR Assays
Many laboratories have developed SARS-CoV real-time RT-PCR assays, which have several advantages over traditional RT-PCR assays. Because real-time RT-PCR assays use internal probes as well as amplification primers, they can be designed to be very specific for SARS-CoV RNA (or cDNA). They can also be very sensitive, with consistent detection limits of between 1 and 10 SARS-CoV RNA copies per reaction. Real-time PCR assays can be performed faster than traditional RT-PCR assays and, because they operate as closed systems, with reduced risk of contamination in the laboratory. Finally, real-time RT-PCR assays can give an accurate estimate of the quantity of virus present in a sample.
As with all PCR assays, interpretation of RT-PCR tests must account for the possibility of false-negative and false-positive results. False-negative results can arise from poor sample collection or degradation of the viral RNA during shipping or storage. Application of appropriate assay controls that identify poor-quality samples can help avoid most false-negative results. A more difficult problem is the apparently low titer of SARS-CoV shed in specimens collected early in illness, which may make it difficult to confirm a diagnosis.
The most common cause of false-positive results is contamination with previously amplified DNA. The use of real-time RT-PCR helps mitigate this problem by operating as a contained system. A more difficult problem is the cross-contamination that can occur between specimens during collection, shipping, and aliquoting in the laboratory. Liberal use of negative control samples in each assay and a well-designed plan for confirmatory testing can help ensure that laboratory contamination is detected and that specimens are not inappropriately labeled as SARS-CoV positive.
In the absence of SARS-CoV transmission worldwide, the probability that a positive test result will be a “false positive” is high. To decrease the possibility of a false-positive result, testing should be limited to patients with a high index of suspicion for having SARS-CoV disease. For information on the indications for SARS-CoV testing, see Clinical Guidance on the Identification and Evaluation of Possible SARS-CoV Disease among Persons Presenting with Community-Acquired Illness.
In addition, any positive specimen should be retested in a reference laboratory to confirm that the specimen is positive. To be confident that a positive PCR specimen indicates that the patient is infected with SARS-CoV, a second specimen should also be confirmed positive. Finally, all laboratory results should be interpreted in the context of the clinical and epidemiologic information available for the patient.
B. Antibody Assays
The most commonly used serologic assays are based on cultured SARS-CoV antigen as either inactivated whole virus lysate for EIA or inactivated virus in cells fixed for IFA. These assays have proven to be highly specific, with no cross-reactivity with paired serum specimens from patients infected with the other known human coronaviruses (229E and OC43) or from healthy blood donors and other persons without clinical or epidemiologic evidence of SARS-CoV disease.
Antibody assays have been the most reliable indicators of SARS-CoV infection when applied to convalescent-phase serum specimens collected >28 days after onset of illness. Since previous SARS-CoV infection is still rare in most populations, demonstration of SARS-CoV-specific antibodies in a single serum specimen is sufficient for diagnosis. However, demonstration of a four-fold or greater increase in antibody titer or conversion from a negative to a positive result between acute- and convalescent-phase serum specimens provides additional confidence that SARS-CoV is linked to any corresponding illness. In some patients, antibody becomes detectable within 8 to 10 days, and most have detectable antibody by 2 weeks. However, some persons do not develop detectable antibodies until 28 days after onset of illness. Although false-positive SARS-CoV serology results are much less likely than false-positive PCR results, guidelines for confirmatory testing similar to those outlined for RT-PCR still apply. Neutralization antibody assays can also be used to detect infection. IgM assays and assays using the S or N proteins as antigens have been developed and are under evaluation.
C. Other Assays
Among the other methods used to detect SARS-CoV are: isolation in cell culture, electron microscopy for CoV-like particles, and immunohistologic or in situ probe hybridization studies on tissue specimens. These methods are less likely to detect SARS-CoV infection than are RT-PCR or antibody assays. Although isolation of SARS-CoV in cell culture represents a definitive diagnosis, it is not recommended for routine detection as it lacks sensitivity compared to RT-PCR and also requires more restrictive Biosafety Level (BSL) 3 conditions.
Diagnostic assays for other respiratory pathogens may be helpful in differentiating SARS-CoV disease from other illnesses, but SARS patients can sometimes be infected with SARS-CoV and another respiratory pathogen simultaneously.