Journal of Clinical Virology 34 (2005) 22–25

First external quality assurance of antibody diagnosticfor SARS-new coronavirus

Matthias Niedriga, ∗, Katrin Leitmeyera, b, Wilina Lim c, Malik Peirisd,John S. Mackenzieb, e, Maria Zambonf

a Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germanyb World Health Organization, Geneva, Switzerland

c Government Virus Unit Hong Kong, Hong Kong, Chinad Department of Microbiology, The University of Hong Kong, Hong Kong, China

e Australian Biosecurity Cooperative Research Centre, Curtin University of Technology, Perth, Australiaf Health Protection Agency, London, UK

Received 23 December 2004; received in revised form 17 January 2005; accepted 19 January 2005

Abstract

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To confirm an infection with the new coronavirus (SARS-CoV) causing the severe acute respiratory syndrome (SARS) diagnosticetection of SARS-CoV specific antibody are necessary. To evaluate the diagnostic performance of laboratories an external qualitEQA) study was performed in 2004. Participating laboratories (9/20) correctly detected anti-SARS antibodies in serum samples wositive results in an immunofluorescence assay. In contrast, only 4/13 laboratories detected most of the anti-SARS antibody posiithout false positive results using enzyme immunoassays (EIA) and/or immunoblot. The overall results clearly demonstrate thatiagnosis of SARS-CoV remains at an early stage of development, with further technical improvements required, particularly with

he use of SARS specific EIAs.2005 Elsevier B.V. All rights reserved.

eywords:SARS; Antibody diagnostic; External quality assurance; EQA

Diagnosis of severe acute respiratory syndrome (SARS)aused by a new coronavirus (SARS-CoV) is of majormportance for assisting the control of any future SARS epi-emic (Drosten et al., 2003; Ksiazek et al., 2003; Peiris et al.,003a,b; Kuiken et al., 2003). The World Health Organiza-

ion (WHO) helps laboratories all over the world to developaboratory capability and a global reference network whichas initiated and coordinated by WHO has assisted in therovision and evaluation of diagnostic tools for detection ofARS-CoV (WHO, 2004a,b). Since the initial epidemic that

∗ Corresponding author. Tel.: +49 30 4547 2370/2321; fax: +49 30 4547321/2390.E-mail addresses:niedrigm@rki.de (M. Niedrig),

eitmeyerk@rki.de (K. Leitmeyer), wllim@pacific.net.hk (W. Lim),alik@hkucc.hku.hk (M. Peiris), j.mackenzie@curtin.edu.au

J.S. Mackenzie), maria.zambon@hpa.org.uk (M. Zambon).

started in November 2002 in south–east China and sglobally in 2003, at least three separate laboratory intion incidents have occurred with some secondary cdemonstrating the need for constant vigilance and reldiagnostic tools (WHO, 2004c; Heymann et al., 2004).

For the acute phase of SARS, RT-PCR methods pioneby several laboratories, are the fastest and most senmethod for detection of the viral genome, and appropfor use in diagnosis in the first few days after illness onSignificant variation is found in the virus load in differepatients and clinical samples, which may relate to the sity of the disease, timing and quality of sampling (Peiris eal., 2003a,b). As a consequence, with current knowleda negative RT-PCR result cannot be interpreted to excSARS-CoV infection. The detection of antibodies agaSARS-CoV is therefore currently a gold standard for cfirmation of SARS infection (Wu et al., 2004). Serologica

386-6532/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.jcv.2005.01.004

M. Niedrig et al. / Journal of Clinical Virology 34 (2005) 22–25 23

assays based on fixed infected cells using immunofluores-cence assays (IFA) or SARS-CoV virus specific enzyme im-munoassays (EIA) have been developed in many differentlaboratories worldwide.

One way to assess preparedness of the diagnosticlaboratories globally is through the conduct of an externalquality assurance (EQA) program providing characterizedserum specimens containing SARS-CoV specific antibody.In collaboration with the WHO, we distributed a panel of 15samples consisting of anti-SARS-CoV positive and negativehuman sera (WHO, 2004a). The serum material obtainedfrom SARS infected patients was provided from variouscountries where SARS-CoV cases occurred. Sera werechecked for SARS-CoV infectivity by inoculation in Verocell cultures (three passages) and by RT-PCR. The sera werediluted in human freshly frozen plasma negative for HIV-1,HBV, HCV and heated to 56◦C for 1 h before lyophilization,for easy distribution and were tested again by two referencelaboratories for specific activity and unspecific binding.

To analyze the sensitivity of different assays, the EQApanel consisted of sequential serum dilutions, resulting insamples with low and high antibody titers against SARS-CoVas well as definite SARS-CoV negative samples. The positivesera were from patients from China, UK and Germany with aclear clinical diagnosis of SARS. All positive sera were con-scientiously analyzed by expert laboratories from China andG inn umanp andw iag-n withk rials

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in positive or negative samples were not considered for eval-uation.

About 52% (13/25) of the laboratories passed theminimum requirements for successful performance(Tables 1 and 2). Three laboratories were not considered forevaluation because of failure to process all EQA samples.The failure of the other 48% of the laboratories to achieveminimum proficiency standards was due to lack of sensi-tivity, and/or to false positive results. From 20 laboratoriesusing IFA, 15 out of 17 (88%) detected the high positive sera(#17, #20) satisfactorily. The performance was worse forlow titer sera (#11, #23, #25) where only 9 out of 17 (53%)detected SARS antibodies. Eight of the 20 laboratories hadgeneral problems regarding specificity, detecting negativesamples as positive in IF assays, possibly due to inadequateinternal positive and negative controls. It is notable that 8/11laboratories with a good performance used a commercialassay, compared with 4/10 with a poor performance.

Six out of 10 laboratories (60%) performing EIA detectedthe high positive sera (#17, #20) satisfactorily, whereas onlyone laboratory (10%) detected the low positive serum (#23),in comparison to IFA where 55% of laboratories detected thissample. One laboratory using an EIA based on SARS specificpeptides showed high reactivity with all samples (Table 2).A similar result was obtained with other peptide based EIAsfor SARS evaluated with these samples. This could be onlya

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ermany for their anti-SARS reactivity by IFA, EIA andeutralization tests. The negative samples consist of hlasma collected before the SARS outbreak in Germanyere not found reactive with any of the SARS specific dostic assays. For analysis of the specificity, two seranown nonspecific reactivity against cell and mitochondtructures were included.

Here we present the results of the first EQA stn SARS-CoV serological diagnosis. The study was

ended as a diagnostic proficiency test, including certiion and publication of anonymized results. Thirty laboories from 18 countries (15 European/Middle Easternustralian/Asian/Oceanian, 3 American, 1 African) partated (see later). The participating institutes included mers of the international WHO SARS reference and veation laboratory network, as well as national and regiARS reference laboratories and three commercial labo

ies (WHO, 2004b).Each participant received a coded panel of 15 sam

istributed by normal mail. Samples were re-suspende00�l distilled water before use. The participants were as

o analyze the material with the diagnostic assays in usARS-CoV diagnosis. Information on the type/format of

est methods used (IFA, EIA, immunoblot, neutralizationay) and whether it was an in-house assay or a commit, was also collected.

The following two criteria were chosen as minimumuirements for overall proficiency: (i) Laboratories hadetect four of the five positive samples correctly (80%);o false positive results were allowed. Indeterminate re

voided with real serum specimens (data not shown).In general the results of the laboratories using more

ne diagnostic assay were better than of the other laboies. Five of the 23 laboratories using IFA assay for SAiagnostic also performed an EIA for confirmation of theults. Four of five laboratories with the best performanceommercial assays or more than one in-house assay.

In conclusion, the results of this first EQA study on SARoV antibody diagnosis indicates the need for perform

mprovement in a significant number of laboratories unaking serological diagnosis of SARS-CoV. Since onlyaboratories have class 3 safety laboratory facilities anxperience for developing and evaluating in-house asommercial assays may be more suitable for SARS seroor laboratories with a low volume of testing. These resultslso likely to be the result of the fact that limited internatioork had been performed or published on the technicaelopment of the assays in use during the time this EQAerformed. A follow up EQA in 12–18 months time woue expected to show substantial improvement as laboraave a chance to develop both the performance of in-hssays and refine testing strategies.

Only a few laboratories were able to develop their oerological assays, partly because of the constraints imy the laboratory handling of SARS-CoV virus, in contr

o the ease with which RT-PCR protocols were disseminnd implemented. Also a variety of different assay forme.g. IFA, EIA, IB) and antigen sources (e.g. purifiirus, recombinant SARS protein, SARS specific peptidontributes to the variability in performance. In this reg

24 M. Niedrig et al. / Journal of Clinical Virology 34 (2005) 22–25

Table 1SARS-CoV external quality assurance—results immunofluorescence assay

Lab.number

Sample number Correct resultsin %#17 pos. #20 pos. #11 pos. #23 pos. #25 pos. #8 neg. #5 neg. #19 neg. #2 neg. #6 neg.a #12 neg.b

11bc + + + + + − − − − − − 10012c + + + + + − − − − − (+/−) 913 + + + + (−) − − − − − − 9120c + + + (+/−) (+/−) − − − − − − 9114c + + + + (−) − − − − − − 915b + + + (−) + − − − − − − 9119 + + + + (−) − − − − − − 9123c + + (−) + + − − − − − − 914bc + + (+/−) (−) + − − − − − − 8630bc + + (−) (−) + − − − − − − 8222c + + (−) + + − − − − − (+) 82

9bc + (−) (−) + (−) − − − − − − 7327c + + (−) (−) (−) − − − − − − 7328 (−) + + (−) + (+) − − − − − 738 + + + + (−) − − − − (+) (+) 737 + + + + + − − (+) − (+) (+) 7325 (+/−) (+/−) (+/−) (+/−) (−) − − − − (+) − 7315 (−) (−) (−) (−) (−) − − − − (+) − 4524 + + + + + (+/−) (+/−) (+) (+/−) (+/−) (+) 4521c (+/−) (+/−) (−) (+/−) (+/−) − (+/−) (+) − (+/−) (+) 3617bc + + n.d. (+/−) + n.d. n.d. n.d. n.d. n.d. n.d.6 n.d. n.d. (−) n.d. n.d. − − n.d. − − −1 n.d. n.d. (−) n.d. n.d. − − n.d. − − −Pos.: serum with antibodies directed against SARS-CoV; neg.: serum without antibodies against SARS-CoV; +: correct positive result;−: negative result;(+/−): indefinite; (+): false positive result; (−): false negative result; n.d.: not done.

a Serum with antibodies reactive with cell core structure.b Serum with antibodies reactive with mitochondria.c Use of commercial assays. Except for 9b and 17b only the IFA from EUROIMMUN was used.

the strategy of testing with peptide based assays as a singletest should be avoided, since it appears from the laboratoriesusing this approach there is a lack of specificity. Diagnosticalgorithms for serological diagnosis of SARS requirerefining.

The approach of sharing protocols and resources forserology testing should be further promoted to guaranteedissemination of high quality assays to ensure the timelyserological confirmation of suspected SARS cases. In themeantime few commercial assays, most of them EIAs are

Table 2SARS-CoV external quality assurance—results EIA

Lab.number

Sample number Correct resultsin %#17 pos. #20 pos. #11 pos. #23 pos. #25 pos. #8 neg. #5 neg. #19 neg. #2 neg. #6 neg.a #12 neg.b

11ac + + (+/−) + + − − − − − − 954ac + + + (−) + − − − − − − 9118d + + + (−) (+/−) − − − − − − 869ac + + (−) (+/−) + − − − − − − 8610c + (+/−) (+/−) (+/−) (+/−) − − − − − − 8230ac + + + (−) + (+/−) − − (+/−) − − 7329d + + + + + (+) (+) (+) (+/−) − (+/−) 7316c (−) + (−) (−) (−) − − − − − − 6426 + + + + + (+) − (+/−) − (+) (+) 555a (−) (−) (−) (−) (−) − − − − − − 5513 + + n.d. (−) + n.d. n.d. − n.d. n.d. n.d.17ac + + n.d. + + n.d. n.d. − n.d. n.d. n.d.2 + + + + + n.d. (+) (+) (+) (+) (+)

Pos.: serum with antibodies directed against SARS-CoV; neg.: serum without antibodies against SARS-CoV; +: correct positive result;−: negative result;(+/−): indefinite; (+): false positive result; (−): false negative result; n.d.: not done.

a Serum with antibodies reactive with cell core structure.b Serum with antibodies reactive with mitochondria.c Use of commercial assays.

d Use of in-house immunoblot assay.

M. Niedrig et al. / Journal of Clinical Virology 34 (2005) 22–25 25

distributed or under development (Chang et al., 2004; Guoet al., 2004; He et al., 2004). The evaluation of these assaysbased on the accessibility of well-characterized SARS-CoVpositive sera is still a major problem for all laboratories andcompanies working on this task.

In the WHO recommended diagnostic strategy, sero-conversion is required to diagnose acute infection. Forestablishing serostatus, while sensitivity is very impor-tant for screening, due to the very low seroprevalenceworldwide, sequential testing using various different anti-gens/methodologies is required to get acceptable positivepredictive value. The present EQA could not take into ac-count this objective of testing or different testing strategies;therefore it is difficult to assess the proficiency of eachparticipant for acute diagnosis or serostatus determination.

When testing for seroconversion, using paired sera, thesensitivity of the assays might be less important than a highspecificity to avoid false positive results. Nevertheless highlysensitive and good specific serology assays will help toimprove the diagnostic performance of the laboratories witha clear benefit for the patient. The EQA performed for SARSantibody diagnostic proved a clear advantage for all par-ticipants, either by demonstrating an adequate performancequality or demonstrate the need for necessary technicalimprovements or the training of the laboratory personal.

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for Communicable Diseases, Beijing, PR China; Queen MaryHospital, Hong Kong, Hong Kong SAR; The Chinese Uni-versity of Hong Kong; Hong Kong SAR.America:Mt. Sinai Hospital, Toronto, Canada; Ministry

of Health and Longterm Care, Etobicoke, Canada; St. Luke’sHospital, Hamilton, Canada.Australia/Oceania:Victorian Infectious Diseases Refer-

ence Laboratory, Melbourne, Australia; Prince of Wales Hos-pital, Randwick NSW, Australia; Canterbury Health Labora-tories, Christchurch, New Zealand.Africa: National Institute of Communicable Diseases

(NICD), Sandringham, Republic of South Africa.

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cknowledgements

The work was partially supported by the EC grant S0002396. The author especially thanks Dr. Angela Mnos, the SARS focal point at WHO, Geneva for provid

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nstitute, Copenhagen, Denmark; Euroimmun, Lubeckermany; Central Public Health Laboratory, LondK; Bernhard Nocht Institut (BNI), Hamburg, GermanioShaf, Tel-Hanan, Israel; HUSLAB, Helsinki, Finlan

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