Emergencies preparedness, response

SARS: Availability and use of laboratory testing

24 April 2003

Researchers in several countries are working towards developing fast and accurate laboratory tests for the SARS. However, until those tests have been adequately field tested and shown to be reliable, SARS diagnosis remains dependant on the clinical findings of an atypical pneumonia not attributed to another cause and a history of exposure to a suspect or probable case of SARS or their respiratory secretions and other bodily fluids. This requirement is reflected in the current WHO case definitions for suspect or probable SARS. However several countries (Canada, France, Germany, Hong Kong SAR, Japan, the Netherlands, Singapore, United Kingdom and the United States of America) are testing samples for suspected and probable SARS cases in research settings.

Interpretation of test results

Positive test results indicate that SARS patients are, or recently were, infected with the SARS virus. Specificity of the different tests still needs to be established.

Negative test results: A negative SARS virus test does not mean that the patient does not have SARS. The reasons for negative test results in a patient with SARS include the following:
- The patient is not infected with the SARS virus; the illness is caused by another infectious agent (virus, bacterium, fungus) or non-infective cause.
- Test results are incorrect (“false-negative”). Current tests need to be further developed to improve sensitivity.
- Specimens were not collected at a time when the virus or its genetic material was present (pertains to PCR and cell culture). The virus and its genetic material may be present for a brief period only, depending on the type of specimen tested.
- Specimens were collected early in the course of the illness and before antibodies had been produced (pertains to ELISA and immunoflourescence assays).

Sampling for research: To enhance the future understanding of the SARS disease process, WHO recommends that clinicians collect and store sequential samples from patients with SARS for testing when diagnostic tests become readily available. This is particularly important for the first case(s) recognized in countries that have not previously reported SARS.

Guidelines on sample handling of suspected or probable SARS patients, can be found in WHO biosafety guidelines for handling of SARS specimens.

Status of laboratory tests currently under development

1 Antibody tests
- ELISA (Enzyme Linked ImmunoSorbant Assay) detects antibodies in the serum of SARS patients reliably as from day 21 after the onset of clinical symptoms and signs.
- Immunofluorescence Assays detect antibodies in serum of SARS patients after about day 10 of illness onset. This is a reliable test requiring the use of fixed SARS virus, an immunofluorescence microscope and an experienced microscopist. Positive antibody tests indicate that the patient was infected with the SARS virus.

2 Molecular tests (PCR)
PCR can detect genetic material of the SARS virus in various specimens (blood, stool, respiratory secretions or body tissue). Primers, which are the key pieces for a PCR test, have been made publicly available by WHO network laboratories on the WHO web site. The primers have since been used by numerous countries around the world. A ready-to-use PCR test kit containing primers and positive and negative control has been developed. Testing of the kit by network members is expected to quickly yield the data needed to assess the test’s performance, in comparison with primers developed by other WHO network laboratories. Existing PCR tests are very specific but lack sensitivity. That means that negative tests can’t rule out the presence of the SARS virus in patients. Various WHO network laboratories are working on their PCR protocols and primers to improve their reliability.

Laboratories performing SARS specific PCR tests should adopt quality control procedures and identify a partner laboratory in their country or among the WHO collaborating research laboratories to cross-check their positive findings.

Laboratories performing SARS specific PCR tests should adopt strict criteria for confirmation of positive results, especially in low prevalence areas, where the positive predictive value might be lower:

- The PCR procedure should include appropriate negative and positive controls in each run, which should yield the expected results:

  • 1 negative control for the extraction procedure and 1 water control for the PCR run
  • 1 positive control for extraction and PCR
  • the patient sample spiked with a weak positive control to detect PCR inhibitory substances (inhibition control)

- If a positive PCR result has been obtained, it should be confirmed by:

  • repeating the PCR starting from the original sample
  • having the same sample tested in a second laboratory.

Amplifying a second genome region could further increase test specificity.

3 Cell culture
Virus in specimens (such as respiratory secretions, blood or stool) from SARS patients can also be detected by infecting cell cultures and growing the virus. Once isolated, the virus must be identified as the SARS virus with further tests. Cell culture is a very demanding test, but the only means to show the existence of a live virus.