Laboratory diagnosis of SARS-coronavirus infectieons

disease focus
S ARS
Laboratory diagnosis of SARScoronavirus
infections
by Karen Sonnenberg
as published in CLI September 2004
SARS-coronavirus (SARS-CoV) is the infectious agent responsible for the recent epidemic outbreak of severe acute respiratory
syndrome (SARS). In an impressively short time after the discovery of the virus, molecular and serological tests were
developed. Laboratory diagnosis using these tests became extremely important in identifying or confirming infections with
SARS-CoV. PCR-based tests have been shown to be powerful tools for detecting SARS-CoV early after disease onset, whereas
from day 10 after the onset of symptoms, the detection of specific antibodies is the preferred diagnostic approach.
The first cases of SARS appeared in southern China in November 2002. In
March 2003 the causative agent of this disease was identified as a novel
coronavirus. Since its first reported occurrence in humans, the virus has
infected more than 8000 people and caused more than 750 deaths. Between
February 2003 and June 2003 cases of the disease were predominantly
reported in Asia, but were also reported in North America and Europe.
SARS-CoV is spread mainly by close person-to-person contact, for example,
through respiratory secretions from coughing or sneezing. The virus
can also spread from surfaces or objects contaminated with infectious
droplets. The incubation period for SARS is typically 2-7 days, after which
patients develop high fever (>38°C) and respiratory symptoms, mainly
pneumonia. About 10 to 20 percent of patients have diarrhoea. Other
symptoms are headache, an overall feeling of discomfort and body aches.
The overall mortality from SARS is around 10%, with levels as high as 50%
in the over-65 age group. Currently there is no specific and effective therapy
or method of prevention for SARS.
The initial diagnosis of SARS was based on clinical and epidemiological
criteria. During the initial outbreak, molecular and serological tests for
detecting infections with the SARS-CoV were developed. Direct or indirect
identification of the virus by these diagnostic tests has become an important
means of diagnosing the disease, of understanding the pathways of
transmission, and of studying SARS epidemiology.
SARS case definitions
SARS cases are classified as suspect or probable based on clinical, epidemiological
and laboratory criteria [1] defined by the World Health
Organisation (WHO) [Figure 2]. A suspected case of SARS that is positive
for SARS coronavirus by one or more assays [Table 1] is classified as a
probable case.
Laboratory methods for confirmation of suspected cases: PCR for
SARS-CoV
The polymerase chain reaction (PCR) allows direct detection of SARS-CoV
genetic material in various patient specimens, such as blood, stool, respiratory
secretions or body tissues. Positive PCR results are very specific and
mean that there is genetic material (RNA) of the SARS-CoV in the sample.
This does not necessarily mean that the active virus is present, or that it is
present in a quantity great enough to infect another person.
Negative PCR results cannot exclude the presence of the SARS-CoV in a
patient. Besides the possibility of obtaining false-negative test results, specimens
may not have been collected at a time when sufficient virus or its
genetic material was present. The sensitivity of PCR tests for SARS depends
both on the type of specimen and the time of testing during the course of
the illness. To date the
optimal type of sample
to use at different times
after the onset of symptoms
has not yet been
determined. Sensitivity
can be increased if multiple
specimens are tested.
The specificity of PCR
tests for SARS is excellent
if the technical procedures
used follow
quality control guidelines.
False positive
results may arise as a
result of technical problems
(e.g. laboratory
Figure 1. SARS-CoV structure.
contamination), so every
positive PCR test should be verified.
In contrast with many other viral respiratory tract diseases, the viral load is
unusually low in the early symptomatic phase of SARS. Depending on the
specimen, the viral load of SARS-
CoV in SARS patients reaches its
peak level at approximately day 10
after the onset of the disease [2,
3]. Nasopharyngeal aspirates
(NPA), throat swabs or sputum
samples appear to be the most
useful clinical specimens in the
first 5 days of illness, but as the
disease progresses viral RNA can
be detected more readily in stool
specimens [4, 5].
Seroconversion determined by
ELISA or IFA
Antibodies against SARS-CoV
become detectable with high sensitivity
around 10 days after the
onset of infection, but they can be
undetectable prior to this by current
testing methods. Positive
antibody test results indicate that
there has been an infection with
SARS-CoV. Seroconversion from
Figure 2. SARS case definition scheme
(WHO).

S ARS
as published in CLI September 2004
Laboratory methods
A. Confirmed positive
PCR for SARS-CoV
B. Seroconversion by
ELISA or IFA
C. Virus isolation
WHO recommendations on interpretation of laboratory results
At least two different clinical specimens (e.g. nasopharyngeal
and stool)
OR
the same clinical specimen collected on two or more days
during the course of the illness (e.g. two or more nasopharyngeal
aspirates)
OR
two different assays or repeat PCR using the original clinical
sample on each occasion of testing
Negative antibody test on acute serum followed by positive
antibody test on convalescent serum
OR
four-fold or greater rise in antibody titre between acute and
convalescent phase sera tested in parallel
Isolation in cell culture of SARS-CoV from any specimen
AND
PCR confirmation using a validated method
Table 1. Laboratory methods for confirmation of suspected cases.
negative to positive, or a four-fold rise in antibody titre in the serum of a
convalescent patient compared with that patient’s serum during acute illness,
denotes a recent infection. A negative serological result 21 days after
onset of symptoms indicates absence of SARS-CoV infection. Cross-reactions
with antibodies to other agents (including the human coronaviruses
HCoV-229E and HCoV-OC43) are not known. Several serological studies
with SARS patient sera using immunofluorescence tests (IIFT) and/or
ELISA showed sensitivities between 92 and 99% [3, 4, 6, 7]. A comparison
of IIFT, ELISA and PCR [Figure 5] showed that PCR predominantly
enables fresh infections to be identified. In later stages of the illness (∼10
days after onset) antibody determination using IIFT or ELISA is the most
reliable method for identifying infections with SARS-CoV.
Virus isolation
The presence of the infectious virus can be detected by inoculating suitable
cell cultures (e.g. Vero cells) with patient specimens (e.g. respiratory
secretions, blood or stool) and propagating the virus in vitro. Cell culture
is a very demanding test. Once isolated, the virus must be identified as
SARS-CoV using further tests (predominantly nucleic acid-based).
Positive results indicate the presence of living SARS-CoV in the sample.
Negative cell culture results do not necessarily exclude SARS, as discussed
previously.
Differential diagnosis
According to the WHO SARS case definition, a case should be excluded if
an alternative diagnosis can fully explain the illness. For example, influenza
viruses, parainfluenza viruses, Chlamydia pneumoniae, Mycoplasma
pneumoniae and Legionella pneumophila can also cause atypical pneumonia.
Positive laboratory test results for these agents may serve as exclusion
criteria.
Figure 5. Comparison of Euroimmun IIFT, ELISA and PCR results from 34 sera of SARS
patients.
Every laboratory confirmation
of SARS should
be undertaken in a
national or regional reference
laboratory and
reported to the WHO.
The WHO encourages
each country to designate
a laboratory at national
level for the investigation
and shipment of specimens
from possible SARS Figure 3. PCR amplification of SARS-CoV nucleic acids.
patients. Furthermore, members of the WHO network laboratories [8] have
agreed to test samples of suspected or probable SARS patients from countries
which may not have the laboratory capacity (PCR technology and biosafety
level 3). Guidelines for the safe handling of SARS specimens are also
described on the WHO web site [9].
References
1. World Health Organisation. Case definitions for surveillance of severe acute respiratory
syndrome (SARS). www.who.int/csr/sars/casedefinition/en
2. Peiris JS, Chu CM, Cheng
VC, et al. Clinical progression
and viral load in a community
outbreak of coronavirus-associated
SARS
pneumonia: a prospective
study. Lancet 2003; 361:
1767-72
3. Tang P, Louie M,
Richardson SE, Smieja M,
Simor AE, Jamieson F, et al.
Interpretation of diagnostic
laboratory tests for severe
acute respiratory syndrome:
the Toronto experience.
CMAJ 2004; 170(1): 47-54.
4. Chan KH, Poon LL, Cheng
VC, Guan Y, Hung IF, Kong J,
Yam LY, Seto WH, Yuen KY
and Peiris JS. Detection of
SARS coronavirus in patients
with suspected SARS. Emerg
Infect Dis 2004; 10(2): 294-9
5. World Health Organisation. Use of laboratory methods for SARS diagnosis.
www.who.int/csr/sars/labmethods/en
6. Poon LL, Wong OK, Luk W, Yuen KY, Peiris JS and Guan Y. Rapid diagnosis of a
coronavirus associated with severe acute respiratory syndrome (SARS). Clin Chem
2003; 49: 953-5
7. Rainer TH, Cameron PA, Smit D, Ong KL, Hung AN, Nin DC, et al. Evaluation of
WHO criteria for identifying patients with severe acute respiratory syndrome out of
hospital: prospective observational study. BMJ 2003; 326: 1354-8
8. World Health Organisation. WHO collaborative multi-centre research project on
Severe Acute Respiratory Syndrome (SARS) diagnosis. www.who.int/csr/sars/project/en
9. World Health Organisation. WHO post-outbreak biosafety guidelines for handling
of SARS-CoV specimens and cultures.
www.who.int/csr/sars/biosafety2003_12_18/en
The author
Karen Sonnenberg, M Eng.
EUROIMMUN AG
Seekamp 31
23560 Luebeck, Germany
Phone: +49 451 5855 535
Fax: +49 451 5855 591
E-mail: k.sonnenberg@euroimmun.de
Figure 4. Euroimmun IIFT: antibodies against SARS-
CoV.

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http://www.euroimmun.com
CLI September 2004
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