The first cases of SARS appeared in southern China in November 2002. InMarch 2003 the causative agent of this disease was identified as a novelcoronavirus. Since its first reported occurrence in humans, the virus hasinfected more than 8000 people and caused more than 750 deaths. BetweenFebruary 2003 and June 2003 cases of the disease were predominantlyreported in Asia, but were also reported in North America and Europe.

SARS-CoV is spread mainly by close person-to-person contact, for exam-ple, through respiratory secretions from coughing or sneezing. The viruscan also spread from surfaces or objects contaminated with infectiousdroplets. The incubation period for SARS is typically 2-7 days, after whichpatients develop high fever (>38°C) and respiratory symptoms, mainlypneumonia. About 10 to 20 percent of patients have diarrhoea. Othersymptoms 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 thera-py or method of prevention for SARS.

The initial diagnosis of SARS was based on clinical and epidemiologicalcriteria. During the initial outbreak, molecular and serological tests fordetecting infections with the SARS-CoV were developed. Direct or indirectidentification of the virus by these diagnostic tests has become an impor-tant means of diagnosing the disease, of understanding the pathways oftransmission, and of studying SARS epidemiology.

SARS case definitionsSARS cases are classified as suspect or probable based on clinical, epidemi-ological and laboratory criteria [1] defined by the World HealthOrganisation (WHO) [Figure 2]. A suspected case of SARS that is positivefor SARS coronavirus by one or more assays [Table 1] is classified as aprobable case.

Laboratory methods for confirmation of suspected cases: PCR forSARS-CoVThe polymerase chain reaction (PCR) allows direct detection of SARS-CoVgenetic material in various patient specimens, such as blood, stool, respira-tory secretions or body tissues. Positive PCR results are very specific andmean 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 ispresent in a quantity great enough to infect another person.

Negative PCR results cannot exclude the presence of the SARS-CoV in apatient. Besides the possibility of obtaining false-negative test results, spec-imens may not have been collected at a time when sufficient virus or itsgenetic material was present. The sensitivity of PCR tests for SARS dependsboth on the type of specimen and the time of testing during the course of

the illness. To date theoptimal type of sampleto use at different timesafter the onset of symp-toms has not yet beendetermined. Sensitivitycan be increased if mul-tiple specimens are test-ed.The specificity of PCRtests for SARS is excel-lent if the technical pro-cedures used followquality control guide-lines. False positiveresults may arise as aresult of technical prob-lems (e.g. laboratorycontamination), so everypositive PCR test should be verified.

In contrast with many other viral respiratory tract diseases, the viral load isunusually low in the early symptomatic phase of SARS. Depending on thespecimen, the viral load of SARS-CoV in SARS patients reaches itspeak level at approximately day 10after the onset of the disease [2,3]. Nasopharyngeal aspirates(NPA), throat swabs or sputumsamples appear to be the mostuseful clinical specimens in thefirst 5 days of illness, but as thedisease progresses viral RNA canbe detected more readily in stoolspecimens [4, 5].

Seroconversion determined byELISA or IFA Antibodies against SARS-CoVbecome detectable with high sen-sitivity around 10 days after theonset of infection, but they can beundetectable prior to this by cur-rent testing methods. Positiveantibody test results indicate thatthere has been an infection withSARS-CoV. Seroconversion from

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Laboratory diagnosis of SARS-coronavirus infections

SARS-coronavirus (SARS-CoV) is the infectious agent responsible for the recent epidemic outbreak of severe acute respira-

tory 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, where-

as from day 10 after the onset of symptoms, the detection of specific antibodies is the preferred diagnostic approach.

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by Karen Sonnenberg

Figure 2. SARS case definition scheme(WHO).

Figure 1. SARS-CoV structure.

as published in CLI September 2004

negative to positive, or a four-fold rise in antibody titre in the serum of aconvalescent patient compared with that patient’s serum during acute ill-ness, denotes a recent infection. A negative serological result 21 days afteronset of symptoms indicates absence of SARS-CoV infection. Cross-reac-tions with antibodies to other agents (including the human coronavirusesHCoV-229E and HCoV-OC43) are not known. Several serological studieswith SARS patient sera using immunofluorescence tests (IIFT) and/orELISA showed sensitivities between 92 and 99% [3, 4, 6, 7]. A comparisonof IIFT, ELISA and PCR [Figure 5] showed that PCR predominantlyenables fresh infections to be identified. In later stages of the illness (∼10days after onset) antibody determination using IIFT or ELISA is the mostreliable method for identifying infections with SARS-CoV.Virus isolationThe presence of the infectious virus can be detected by inoculating suit-able cell cultures (e.g. Vero cells) with patient specimens (e.g. respiratorysecretions, blood or stool) and propagating the virus in vitro. Cell cultureis a very demanding test. Once isolated, the virus must be identified asSARS-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 discussedpreviously.

Differential diagnosisAccording to the WHO SARS case definition, a case should be excluded ifan alternative diagnosis can fully explain the illness. For example, influen-za viruses, parainfluenza viruses, Chlamydia pneumoniae, Mycoplasmapneumoniae and Legionella pneumophila can also cause atypical pneumo-nia. Positive laboratory test results for these agents may serve as exclusioncriteria.

Every laboratory confir-mation of SARS shouldbe undertaken in anational or regional refer-ence laboratory andreported to the WHO.The WHO encourageseach country to designatea laboratory at nationallevel for the investigationand shipment of speci-mens from possible SARSpatients. Furthermore, members of the WHO network laboratories [8] haveagreed to test samples of suspected or probable SARS patients from countrieswhich may not have the laboratory capacity (PCR technology and biosafetylevel 3). Guidelines for the safe handling of SARS specimens are alsodescribed on the WHO web site [9].References1. World Health Organisation. Case definitions for surveillance of severe acute respi-ratory syndrome (SARS). www.who.int/csr/sars/casedefinition/en 2. Peiris JS, Chu CM, ChengVC, et al. Clinical progres-sion and viral load in a com-munity outbreak of coron-avirus-associated SARSpneumonia: a prospectivestudy. Lancet 2003; 361:1767-72 3. Tang P, Louie M,Richardson SE, Smieja M,Simor AE, Jamieson F, et al.Interpretation of diagnosticlaboratory tests for severeacute respiratory syndrome:the Toronto experience.CMAJ 2004; 170(1): 47-54.4. Chan KH, Poon LL, ChengVC, Guan Y, Hung IF, Kong J,Yam LY, Seto WH, Yuen KYand Peiris JS. Detection ofSARS coronavirus in patientswith suspected SARS. EmergInfect Dis 2004; 10(2): 294-95. 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 acoronavirus associated with severe acute respiratory syndrome (SARS). Clin Chem2003; 49: 953-57. Rainer TH, Cameron PA, Smit D, Ong KL, Hung AN, Nin DC, et al. Evaluation ofWHO criteria for identifying patients with severe acute respiratory syndrome out ofhospital: prospective observational study. BMJ 2003; 326: 1354-88. World Health Organisation. WHO collaborative multi-centre research project onSevere Acute Respiratory Syndrome (SARS) diagnosis. www.who.int/csr/sars/proj-ect/en9. World Health Organisation. WHO post-outbreak biosafety guidelines for handlingof SARS-CoV specimens and cultures.www.who.int/csr/sars/biosafety2003_12_18/en

The authorKaren Sonnenberg, M Eng.EUROIMMUN AGSeekamp 3123560 Luebeck, GermanyPhone: +49 451 5855 535Fax: +49 451 5855 591E-mail: k.sonnenberg@euroimmun.de

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Table 1. Laboratory methods for confirmation of suspected cases.

Figure 3. PCR amplification of SARS-CoV nucleic acids.

Laboratory methods

A. Confirmed positive PCR for SARS-CoV

B. Seroconversion byELISA or IFA

C. Virus isolation

WHO recommendations on interpretation of laboratory results

At least two different clinical specimens (e.g. nasopharyngealand stool) OR the same clinical specimen collected on two or more daysduring the course of the illness (e.g. two or more nasopha-ryngeal aspirates)ORtwo different assays or repeat PCR using the original clinicalsample on each occasion of testing

Negative antibody test on acute serum followed by positive antibody test on convalescent serumORfour-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 specimenANDPCR confirmation using a validated method

as published in CLI September 2004

Figure 5. Comparison of Euroimmun IIFT, ELISA and PCR results from 34 sera of SARSpatients.

Figure 4. Euroimmun IIFT: antibodies against SARS-CoV.

http://www.euroimmun.com

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