![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Journal of Clinical Microbiology, September 2001, p. 3390-3392, Vol. 39, No. 9
Department of Virology, University Medical
Center Utrecht, Utrecht, The Netherlands1;
C.N.M., Instituto de Salud Carlos III, Madrid,
Spain3; and King's College London,
London,2 Public Health Laboratory,
Leeds,4 and Manchester Royal
Infirmary, Manchester,5 United Kingdom
Received 1 December 2000/Returned for modification 18 April
2001/Accepted 11 July 2001
A multicenter study of molecular detection of enteroviruses was
conducted using a proficiency panel. Of 70 data sets, 46 (66%) reported correct results for samples containing at least 1 50% infective dose per ml and for negative samples. Variation in
performance between laboratories demonstrates the need for ongoing
quality control.
Nucleic acid amplification
methods are widely used for enterovirus (EV) detection because
they are sensitive, specific, and rapid (1, 3, 6, 10, 12,
17). However, reliable molecular diagnosis requires assay
standardization and continuous monitoring of sensitivity and
specificity. A quality assessment program for EV detection was
therefore established within the framework of the European Union
Concerted Action on Quality Control of Nucleic Acid Amplification in
Diagnostic Virology (QCCA) to assess the proficiency of laboratories
using molecular EV detection methods. Here we describe results of 70 data sets reported by 59 laboratories upon testing an EV proficiency
panel. To our knowledge, this represents the largest such study
reported to date.
The proficiency panel consisted of 12 coded samples, including a
coxsackievirus A9 (CVA9) dilution series, other enterovirus serotypes representing different genetic clusters of human
enteroviruses (5, 11), human parechovirus type 1 (HPEV1;
formerly classified as echovirus 22 but now known to be genetically
distinct from enteroviruses), and negative controls (Table
1). The sources, production, and
characterization of the viruses used are described elsewhere (7,
15). Titration of viral infectivity was performed on the
original virus stocks (50% tissue culture infective doses [TCID50] per milliliter). Virus stocks were inactivated
at 56°C for 30 min, freeze-dried in 1-ml volumes, and stored at
4°C. Freeze drying resulted in an approximately 10-fold reduction in
levels of PCR-detectable viral RNA. Full details of the production and evaluation of the proficiency panel are available on the QCCA website
(www.qcca.org.uk). Prior to release, external quality control
testing was performed by two reference laboratories to assess sample
quality and homogeneity. Four vials of each sample were analyzed in
duplicate using the Amplicor EV-PCR assay (Roche Molecular Systems,
Branchburg, N.J.) and an in-house PCR. A stochastic distribution of
positive results was found in the highly diluted samples close to the
detection limit of the assay in use (data not shown). To assess the
effects of storage and of transportation on sample stability, samples
were tested before and after 1, 2, 4, and 7 days of storage at room
temperature. Identical results were obtained, with the exception of one
borderline sample, which produced a 2.5-fold decrease in optical
density values in the Amplicor EV-PCR assay over a period of 7 days
(data not shown). Storage of the samples for 1 year at 4°C
demonstrated no detectable loss of activity.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3390-3392.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Multicenter Proficiency Testing of Nucleic Acid
Amplification Methods for the Detection of Enteroviruses
and
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
TABLE 1.
Composition of enterovirus proficiency panel and
participant results
The proficiency panel was distributed at ambient temperatures to 63 European laboratories, which were asked to reconstitute each sample with 1 ml of water prior to testing using a molecular assay and to report their results to a neutral office (Manchester, United Kingdom) within 6 weeks. Additionally, a questionnaire was sent to obtain information on the various aspects of amplification procedures. Most participants (83%) received the panel within 3 days. Anonymized data sets were analyzed at the Department of Virology, University Medical Center, Utrecht, The Netherlands.
A total of 71 data sets were reported by 59 laboratories. One laboratory reported a data set with the results of a PCR specific for HPEV1. This was excluded, leaving 70 data sets for final analysis. The response to the questionnaires was high (70 of 71). Results are summarized in Table 1. The detection limits of the various assays as determined using the CVA9 dilution series varied by a factor 103 to 104. Correct dilution series results for the CVA9 samples (i.e., without inconsistent results in the sequence of the dilution series) were reported in 66 data sets. Six and seven of these 66 data sets tested positive for only the lower-dilution CVA9 samples EV-A12 and EV-A06, respectively. In 25 data sets, correct results were reported for the dilution series up to sample EV-A09; the higher-dilution samples EV-A10 and EV-A11 were identified as positive by 20 and 8 data sets, respectively. The participant that failed to detect virus in the sample containing 1% blood also failed to identify the corresponding sample without blood as positive. The five false-positive results (3.6%) were reported by four laboratories (6.8%). Of the three participants that identified sample EV-A04 as positive, only one identified this sample as a parechovirus. The remaining two positive results may represent false-positive results, since there is limited sequence homology between parechoviruses and enteroviruses (4, 14).
To assess proficiency, the test samples were classified as (strongly) positive (>1 TCID50/ml of original virus stock), weakly positive (<1 TCID50/ml), or negative. Sample EV-A04 was excluded from this analysis. A data set reporting correct results for all (strongly) positive and negative samples was considered to indicate adequate proficiency, since this corresponds in sensitivity and specificity to cell-culture based detection. In 46 data sets (66%), this level of proficiency was achieved.
To allow further comparison of performance and its relationship to PCR
methodology in this and future proficiency panel distributions, a
numerical performance score was assigned to each data set as follows:
one point was given for every correct result, and one point was
deducted for every false-positive or false-negative result, with the
exception of negative results on the weakly positive samples. Equivocal
results were not scored. Details of the assays employed were provided
by 68 participating laboratories. Nested PCRs were associated with
better performance than other PCR methods (Table
2). (P = 0.011,
2 analysis, Yates' correction). However, a maximum
score was obtained at least once with each type of assay, indicating
that additional factors, including staff proficiency and laboratory
facilities, are likely to influence performance. This is further
illustrated by the variation in performance observed among participants
using the commercially available Roche Amplicor assay. No other aspects of assay design, such as RNA extraction, primer sequences, cDNA amplification, or PCR product detection, showed any statistical association with performance.
|
Our study has thus shown that the majority of the participating laboratories have succeeded in introducing a reliable EV PCR assay, with several participants demonstrating an increased sensitivity relative to that of viral culture. However, a large group of participants (34%) performed inadequately. Performance was better among nested PCR users, and other aspects of assay design may also influence performance, although such effects could not be verified statistically in this study. Even when using a standardized assay, performance among laboratories varied, as has been observed in other studies of commercially produced PCR assays for enteroviruses (8, 9), human immunodeficiency virus (13, 16), or hepatitis C virus (2). This emphasizes the importance of ongoing quality assessments. For this reason the distribution of EV proficiency panels by the QCCA will continue. This multicenter quality assurance scheme can thus contribute to the optimization and standardization of molecular diagnosis of EV infections.
| |
ACKNOWLEDGMENTS |
|---|
We are grateful to C. W. van Ingen and the Foundation for the Advancement of Public Health and Environmental Protection (SVM) for freeze-drying of the virus samples. We thank L. Sprong for excellent technical assistance.
The European Union Concerted Action Programme was supported with a grant from the EU Biomed 2 program.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: University Medical Center, Department of Virology G04.614, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Phone: 31 (0) 30 2507629. Fax: 31 (0) 30 2505426. E-mail: a.m.vanloon{at}lab.azu.nl.
Member of the European Union Concerted Action on Quality Control of
Nucleic Acid Amplification in Diagnostic Virology.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Casas, I., P. E. Klapper, G. M. Cleator, J. E. Echevarría, A. Tenorio, and J. M. Echevarría. 1995. Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis. J. Med. Virol. 47:378-385[Medline]. |
| 2. | Damen, M., H. T. M. Cuypers, H. L. Zaaijer, H. W. Reesink, W. P. Schaasberg, W. H. Gerlich, H. G. M. Niesters, and P. N. Lelie. 1996. International collaborative study on the second EUROHEP HCV-RNA reference panel. J. Virol. Methods 58:175-185[CrossRef][Medline]. |
| 3. | Glimåker, M., B. Johansson, P. Olcén, A. Ehrnst, and M. Forsgren. 1993. Detection of enteroviral RNA by polymerase chain reaction on cerebrospinal fluid from patients with aseptic meningitis. Scand. J. Infect. Dis. 25:547-557[Medline]. |
| 4. |
Hyypiä, T.,
C. Horsnell,
M. Maaronen,
M. Khan,
N. Kalkkinen,
P. Auvinen,
L. Kinnunen, and G. Stanway.
1992.
A distinct picornavirus group identified by sequence analysis.
Proc. Natl. Acad. Sci. USA
89:8847-8851 |
| 5. | Hyypiä, T., T. Hovi, N. J. Knowles, and G. Stanway. 1997. Classification of enterovirus based on molecular and biological properties. J. Virol. 78:1-11. |
| 6. |
Kämmerer, U.,
B. Kunkel, and K. Korn.
1994.
Nested polymerase chain reaction for specific detection and rapid identification of human picornaviruses.
J. Clin. Microbiol.
32:285-291 |
| 7. | Kapsenberg, J. G. 1988. Picornaviridae: the enteroviruses (polioviruses, coxsacjievirus, echoviruses), p. 693-722. In E. H. Lennette, P. Halonen, and F. A. Murphy (ed.), Laboratory diagnosis of infectious diseases. Principles and practice, vol. 2. Viral, rickettsial, and chlamydial diseases. Springer-Verlag, New York, N.Y. |
| 8. | Lina, B., B. Pozetto, L. Andréoletti, et al. 1996. Multicenter evaluation of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens. J. Clin. Microbiol. 34:3002-3006[Abstract]. |
| 9. |
Muir, P.,
A. Ras,
P. E. Klapper,
G. M. Gleator,
K. Korn,
C. Aepinus,
A. Fomsgaard,
P. Palmer,
A. Samuelsson,
A. Tenorio,
B. Weissbrich, and A. M. van Loon.
1999.
Multicenter quality assessment of PCR methods for detection of enteroviruses.
J. Clin. Microbiol.
37:1409-1414 |
| 10. | Nicholson, F., G. Meetoo, S. Aiyar, J. E. Banatlava, and P. Muir. 1994. Detection of enterovirus RNA in clinical samples by nested polymerase chain reaction for rapid diagnosis of enterovirus infection. J. Virol. Methods 48:155-166[CrossRef][Medline]. |
| 11. |
Pöyry, T.,
L. Kinunen,
T. Hyypiä,
B. Brown,
C. Horsnell,
T. Hovi, and G. Stanway.
1996.
Genetic and phylogenetic clustering of enteroviruses.
J. Gen. Virol.
77:1699-1717 |
| 12. |
Rotbart, H. A.,
H. Sawyer,
S. Fast,
C. Lewinski,
N. Murphy,
E. F. Keyser,
J. Spadoro,
S.-Y. Kao, and M. Loeffelholz.
1994.
Diagnosis of enteroviral meningitis by using PCR with a colorimetric microwell detection assay.
J. Clin. Microbiol.
32:2590-2592 |
| 13. | Schuurman, R., D. Descamps, G. J. Weverling, S. Kaye, J. Tijnagel, I. Williams, R. van Leeuwen, R. Tedder, C. A. B. Boucher, F. Brun-Vezinet, and C. Loveday. 1996. Multicenter comparison of three commercial methods for quantitation of human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 34:3016-3022[Abstract]. |
| 14. |
Stanway, G.,
N. Kalkkinen,
M. Roivainen,
F. Ghazi,
M. Khan,
M. Smyth,
O. Meurman, and T. Hyypiä.
1994.
Molecular and biological characteristics of echovirus 22, a representative of a new picornavirus group.
J. Virol.
68:8232-8238 |
| 15. | Van Loon, A. M., G. C. Cleator, and A. Ras for the European Union Concerted Action on Virus Meningitis and Encephalitis. 1999. External quality assessment of enterovirus detection and typing. Bull. W. H. O. 77:217-220[Medline]. |
| 16. | Yen-Lieberman, B., D. Brambilla, B. Jackson, J. Bremer, R. Coombs, M. Cronin, S. Herman, D. Katzenstein, S. Leung, H. J. Lin, P. Palumbo, S. Rasheed, J. Todd, M. Vahey, and P. Reichelderfer. 1996. Evaluation of a quality assurance program for quantitation of human immunodeficiency virus type 1 RNA in plasma by the AIDS Clinical Trials Group virology laboratories. J. Clin. Microbiol. 34:2695-2701[Abstract]. |
| 17. |
Zoll, G. J.,
W. J. Melchers,
H. Kopecka,
G. Jambroes,
H. J. van der Poel, and J. M. Galama.
1992.
General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections.
J. Clin. Microbiol.
30:160-165 |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. |
|---|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|
