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Preface

Summary

1. Timeline

2. Virology

3. Transmission

4. Epidemiology

5. Prevention

6. Case Definition

7. Diagnostic Tests

8. Clinical Presentation and Diagnosis

9. SARS Treatment

10. Pediatric SARS



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7. Diagnostic Tests

Wolfgang Preiser, Christian Drosten

Introduction

Despite the initial rapid progress in the discovery of the causative agent (see Chapter 2: Virology) and the early development of diagnostic tests, further progress in the establishment of laboratory tests for SARS has been slower than originally expected.

While various molecular (PCR-based) assays have been developed by different groups around the world, and although one such assay is available commercially, results of these tests should still not be used to rule out a suspected case of SARS, according to current WHO recommendations.

The continual lack of a rapid laboratory test to aid the diagnosis of suspected cases of SARS makes this area a priority for further research efforts (WHO, Update 71).

In many viral diseases, virus shedding is greatest during the early symptomatic phase, i.e. around, and immediately following the onset of symptoms. Unfortunately, virus excretion is comparatively low during the initial phase of SARS. It peaks in respiratory specimens and in stools at around day 10 after the onset of the clinical illness. In order to make an early diagnosis, it is therefore necessary to use highly sensitive tests that are able to detect the low levels of viral genome present during the first days of illness.

Because presently available tests are not generally able to detect the small amounts of SARS coronavirus (SARS-CoV) initially shed, they do not yet play a role in patient management and case control, as SARS patients may be capable of infecting others during the initial phase and therefore need to be reliably detected and quickly isolated (WHO, Update 71).

The results of the first clinical studies on SARS are now available and able to shed light on the clinical usefulness of various tests on different patient samples at different time points. In one series, IgG seroconversion was documented in 93% of patients at a mean of 20 days; about 50 % of patients had seroconverted at around 15 days after the onset of symptoms (Peiris).

In the same study, SARS-associated coronavirus RNA was detected in nasopharyngeal aspirates by RT-PCR in 20 patients (32%) at initial presentation (mean 3.2 days after the onset of illness) and in 68% at day 14 (Peiris). Quantification revealed that the viral load peaked on day 10 with a mean geometric value of 1.9*107 copies per ml, compared to values of 2.3*105 copies per ml and 9.8*104 copies per ml on days 5 and 15, respectively (Peiris).

Furthermore, viral RNA was detected in 97% of stool samples collected later in the illness (a mean of 14.2 days after onset). Similarly, viral RNA was detected in 42% of urine samples collected at a mean of 15.2 days after the onset of symptoms (Peiris).

The authors therefore conclude that although viral RNA detection in the nasopharyngeal aspirate has a sensitivity of only 32% at presentation, testing of multiple nasopharyngeal and fecal samples is able to increase the predictive value of the RT-PCR assay (Peiris).

Laboratory tests

Due to the efforts of the WHO-led international multi-center collaborative network of laboratories testing for SARS, tests for the novel coronavirus have been developed with unprecedented speed (SARS: Laboratory diagnostic tests - 29 April 2003; http://www.who.int/csr/sars/diagnostictests/e n/). Samples from suspected and probable SARS cases have been tested for SARS-CoV for some time in several countries, including Canada, France, Germany, Hong Kong SAR, Italy, Japan, the Netherlands, Singapore, the United Kingdom and the United States of America.

Nevertheless, until standardized reagents for virus and antibody detection become available and methods have been adequately field tested, the diagnosis of SARS remains based on clinical and epidemiological findings. The revised case definition from May 1, 2003, (see: http://www.who.int/csr/sars/casedefinition/en/) includes laboratory results for the first time: a suspected case of SARS, that is positive for SARS-CoV in one or more assays, should be reclassified as a probable case. At present there are no defined criteria for SARS-CoV test results to confirm or reject the diagnosis of SARS.

Positive laboratory test results for other known agents that are able to cause atypical pneumonia such as Legionella pneumophila, influenza and parainfluenza viruses, Mycoplasma pneumoniae etc. may serve as exclusion criteria: according to the case definition, a case should be excluded if an alternative diagnosis can fully explain the illness. However, the possibility of dual infection must not be ruled out completely.

Molecular tests

SARS-CoV-specific RNA can be detected in various clinical specimens such as blood, stool, respiratory secretions or body tissues by the polymerase chain reaction (PCR). A number of PCR protocols developed by members of the WHO laboratory network are available on the WHO website (http://www.who.int/csr/sars/primers/en/). Furthermore, a 5-nuclease RT-PCR test kit containing primers and positive and negative controls, developed by the Bernhard Nocht Institute (http://www.bni-hamburg.de/; Drosten et al.), is available commercially (http://www.artus-biotech.de). An inactivated standard preparation is also available for diagnostic purposes through the European Network for Imported Viral Infections (ENIVD; http://www.enivd.de). ENIVD is also preparing an international external quality assessment scheme for SARS-CoV assays.

Despite their sometimes high sensitivity, the existing PCR tests cannot rule out, with certainty, the presence of the SARS virus in patients (Peiris, McIntosh, Poon). On the other hand, contamination of samples in laboratories might lead to false positive results. Stringent guidelines on laboratory quality control and confirmatory testing have therefore been issued by the WHO (http://www.who.int/csr/sars/labmethods/en/).

A valid positive PCR result indicates that there is genetic material (RNA) from the SARS-CoV in the sample. It does not mean, however, that the virus present is infectious, or that it is present in a large enough quantity to infect another person.

Negative PCR results do not exclude SARS. Besides the possibility of obtaining incorrect, false-negative test results (e.g. through lack of sensitivity), specimens may not have been collected at a time when the virus or its genetic material was present.

Currently, efforts are underway to improve the sensitivity of PCR assays to increase their clinical usefulness. One approach is to amplify another gene of SARS-CoV than the hitherto used polymerase gene; due to the unique transcription strategy of coronaviruses, a PCR targeting the nucleoprotein may have a higher sensitivity. While evaluations of such a PCR are ongoing, the protocol is already available from the Bernhard Nocht Institute.

Virus isolation

The presence of the infectious virus can be detected by inoculating suitable cell cultures (e.g., Vero cells) with patient specimens (such as respiratory secretions, blood or stool) and propagating the virus in vitro. Once isolated, the virus must be identified as SARS-CoV using further tests. Cell culture is a very demanding test, but currently (with the exception of animal trials) the only means to show the existence of a live virus. It has to be performed under at least biosafety safety level (BSL) 3 conditions (see below). Positive cell culture results indicate the presence of live SARS-CoV in the sample tested. Negative cell culture results do not exclude SARS (see negative PCR test result).

Antibody detection

Various methods provide a means for the detection of antibodies produced in response to infection with SARS-CoV. Different types of antibodies (IgM and IgG) appear and change in level during the course of infection. They can be undetectable in the early stages of infection. IgG usually remains detectable after resolution of the illness.

The following test formats are being developed:

  • Enzyme-linked immunosorbent assay (ELISA): a test which detects a mixture of IgM and IgG antibodies in the serum of SARS patients and reliably yields positive results at around day 21 after the onset of illness.
  • Immunofluorescence assay (IFA): This requires the use of SARS-CoV-infected cells fixed on a microscope slide; patient antibodies bind to viral antigens and are in turn detected by immunofluorescent-labelled secondary antibodies against human IgG or IgM or both, using an immunofluorescence microscope. IFA typically yields a positive result after about day 10 after the onset of illness. Results may be quantified by using serial titrations of patient sera. A SARS-CoV IFA manufactured by Euroimmun AG (Seekamp 31, D-23560 Lübeck, Germany; http://www.euroimmun.de) is now available commercially for the detection of IgG and IgM antibodies against SARS-CoV.
  • Neutralization test (NT): This test assesses and quantifies, by means of titration, the ability of patient sera to neutralize the infectivity of SARS-CoV on cell culture. NT is therefore likely to be the best correlate of immunity. However, due to the use of the infectious virus it is limited to institutions with BSL-3 facilities.

Interpretation

Positive antibody test results indicate previous infection with SARS-CoV. Seroconversion from negative to positive or a four-fold rise in the antibody titer from acute to convalescent serum indicates a recent infection. A negative antibody test result later than 21 days after the onset of illness is likely to indicate that no infection with SARS-CoV has taken place. There seems to be no background seroprevalence against SARS-CoV in the control populations screened so far. Antibody testing allows the indirect diagnosis of SARS-CoV infection and is unsuitable during the acute illness; it has the advantage of being rather independent of the sample type and timing, in contrast to other virus detection methods.

Limitations

All tests for SARS-CoV available so far have limitations. Extreme caution is therefore necessary when management decisions are to be based on virological test results. For more details, see the WHO Update 39, "Caution urged when using diagnostic tests": http://www.who.int/csr/sarsarchive/2003_04_ 25/en/. In particular, false negative test results (due to low sensitivity, unsuitable sample type, or time of sampling, etc.) may give a false sense of security; in the worst case, they could allow persons carrying the SARS virus, and therefore capable of infecting others, to escape detection.

To aid in the better understanding of SARS, the WHO recommends that sequential samples be stored from patients with suspected or probable SARS - and also close contacts who are not ill themselves - for future use. This is particularly important for the first case(s) recognized in countries that have not previously reported SARS. Data on the clinical and contact history should also be collected in order to obtain a better understanding of the shedding pattern of the virus and the period of transmissibility. Such patient samples should be suitable for viral culture, PCR, antigen detection, immunostaining and/or serological antibody assays. For details, refer to "Sampling for Severe Acute Respiratory Syndrome (SARS) diagnostic tests", http://www.who.int/csr/sars/sampling/en/). The WHO also encourages each country to designate a reference laboratory for investigation and/or referral of specimens from possible SARS patients.

Biosafety considerations

So far, not a single case of a laboratory-associated SARS-CoV infection has been reported. Nevertheless, the WHO has issued biosafety guidelines for the handling of clinical specimens associated with SARS cases and materials derived from laboratory investigations of SARS (on April 25, 2003; see http://www.who.int/csr/sars/biosafety2003 _04_25/en/). Suitable measures must be taken to prevent the potential spread by droplets, air, and/or contaminated surfaces and objects, with particular emphasis on avoiding the unguarded production of aerosols.

For routine diagnostic testing of serum and blood samples, manipulations involving known inactivated (lysed, fixed or otherwise treated) virus particles and/or incomplete, non-infectious portions of the viral genome, routine examination of mycotic and bacterial cultures, and final packaging of specimens (already in a sealed, decontaminated primary container) for transport to diagnostic laboratories for additional testing, BSL-2 facilities with appropriate BSL-2 work practices are deemed sufficient. Any procedure that may generate aerosols should be performed in a biological safety cabinet, and laboratory workers should wear eye protection and a surgical mask in addition to standard protective equipment such as gloves, etc.

In vitro cell culture of the etiologic agent and manipulations involving growth or concentration of the etiologic agent require BSL-3 facilities and BSL-3 work practices.

The current Dangerous Goods Regulations (2003) of the International Air Transport Association (IATA) allow specimens known or suspected of containing the SARS agent to be transported as UN 3373 "Diagnostic Specimens" when they are transported for diagnostic or investigational purposes. Specimens transported for any other purpose, and cultures prepared for the deliberate generation of pathogens, must be transported as UN 2814, and marked as: "Infectious substance, affecting humans (Severe Acute Respiratory Syndrome virus)". All specimens that are to be transported (UN 3373 or UN 2814) must be packaged in triple packaging consisting of three packaging layers.

Further detailed information about containment facilities and biosafety practices can be found in the WHO Laboratory Biosafety Manual, 2nd revised edition, available from the WHO website (http://www.who.int/c sr/resources/publications/biosafety/Labbiosafety.pdf).

Outlook

In addition to allowing the rapid diagnosis of SARS infection, the availability of diagnostic tests will help to address important questions such as the period of virus shedding (and communicability) during convalescence, the presence of virus in different body fluids and excreta, and the presence of virus shedding during the incubation period.

Until a certain degree of standardization and quality assurance has been achieved for the SARS-CoV laboratory tests, test results must be used with utmost caution in clinical situations. It is strongly advisable to closely check on updated recommendations by the WHO and relevant national organizations regarding the availability and use of such tests. If in doubt, advice should be sought from reference laboratories (see http://www.who.int/csr/sars/labmethods/en/).< /P>

Table, Figures

Table 1: Currently (July 2003) available diagnostic tests for the SARS-associated coronavirus.

Detection method

Clinical material/ specimen

Technical details

Diagnostic significance

Virus detection

Virus isolation on cell culture

Respiratory tract samples: sputum, BAL

Suitable cell lines: Vero; biosafety level 3 facility required

Indicates presence of infectious virus; negative result does not preclude SARS!

Polymerase chain reaction (PCR)

Respiratory tract samples: sputum, BAL, throat swab, throat washing, stool

Different primer sequences and protocols available from the WHO website *

Indicates presence of viral genome, not necessarily of infectious virus; negative result does not preclude SARS! *

Antibody detection

Immunfluorescence assay (IFA)

Serum

For detection of specific IgG or IgM antibodies or both

IgM IFA usually positive from day 10 after the onset of symptoms

Enzyme-linked immunosorbent assay (ELISA)

Serum

May be designed to detect specific IgG or IgM antibodies or both

Usually positive from day 21 after the onset of symptoms

Neutralization test (NT)

Serum

Requires BSL-3 facility ("live" virus)

Under investigation; study use only

See also: "Severe Acute Respiratory Syndrome (SARS): Laboratory diagnostic tests" (http://www.who.int/csr/sars/diagnostictests/e n/)

*see "PCR primers for SARS developed by the WHO Network Laboratories" (http://www.who.int/csr/sars/primers/en/) and "Recommendations for laboratories testing by PCR for presence of SARS coronavirus - RNA" (http://www.who.int/csr/sars/coronarecom mendations/en/)

 

 

Figure 1. Immunofluorescence assay (IFA): SARS-CoV-infected Vero cells incubated with patient serum (1:50 dilution) obtained 11 days after the onset of symptoms, showing cytoplasmatic fluorescence. (Source: Source: Institute for Medical Virology, Director: W. Doerr)

http://www.sarsreference.com/archive/verocells_patientserum.jpg

Figure 2. Immunofluorescence assay (IFA): SARS-CoV-infected Vero cells incubated with negative control serum. (Source: Source: Institute for Medical Virology, Director: W. Doerr)

http://www.sarsreference.com/archive/verocells_controlserum.jpg

 

Figure 3. Amplification plot of "TaqMan" (5'nuclease) real-time PCR for the detection of SARS-CoV RNA in clinical specimens. This is a widely used assay, developed by BNI. Primers and fluorescence-labeled probe are located in the polymerase gene of SARS-CoV (Picture source: Institute for Medical Virology, Director: W. Doerr).

 

References

  1. Drosten C, Gunther S, Preiser W, et al. Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome. N Engl J Med 2003; 348:1967-76. Published online Apr 10, 2003. http://SARSReference.com/lit.php?id=12690091
  2. McIntosh K. The SARS coronavirus: rapid diagnostics in the limelight. Clin Chem 2003; 49: 845-6. http://SARSReference.com/lit.php?id=12765977
  3. 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 2003b; 361:1767-72. Published online May 9, 2003. http://image.thelancet.com/extras/03art443 2web.pdf
  4. Poon LL, Wong OK, Luk W, Yuen KY, Peiris JS, Guan Y. Rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (SARS). Clin Chem 2003; 49: 953-5. http://SARSReference.com/lit.php?id=12765993
  5. WHO Update 71. Status of diagnostic tests, training course in China. http://www.who.int/entity/csr/don/2003_06_02 a/en


 

 
 
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