JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p. 3109–3110 0095-1137/07/$08.00⫹0 doi:10.1128/JCM.00697-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 9
Testing of Diagnostic Methods for Detection of Influenza Virus for Optimal Performance in the Context of an Influenza Surveillance Network䌤 Mercedes Pe´rez-Ruiz,1* Ruth Yeste,1 Marı´a-Jose´ Ruiz-Pe´rez,1 Alfonso Ruiz-Bravo,2 Manuel de la Rosa-Fraile,1 and Jose´ Marı´a Navarro-Marı´1 Servicio de Microbiologı´a, Hospital Universitario Virgen de las Nieves, Granada, Spain,1 and Departamento de Microbiologı´a, Universidad de Granada, Granada, Spain2 Received 30 March 2007/Returned for modification 26 April 2007/Accepted 16 July 2007
Influenza surveillance networks must detect early the viruses that will cause the forthcoming annual epidemics and isolate the strains for further characterization. We obtained the highest sensitivity (95.4%) with a diagnostic tool that combined a shell-vial assay and reverse transcription-PCR on cell culture supernatants at 48 h, and indeed, recovered the strain. The clinical specimens were throat and nasal swabs from outpatients (with clinical evidence of acute respiratory infection over a duration of ⱕ72 h and a body temperature of ⱖ38°C) collected by the sentinel physicians. Specimens were sent to the laboratory within the first 6 h following their collection, at 4°C in minimal essential medium supplemented with 1% bovine serum albumin. A 140-l aliquot of the specimen was used for RNA extraction with a QIAmp viral RNA kit (QIAGEN, Hilden, Germany) for use in DD. Other 200-l aliquots were inoculated into MDCK cells (Vircell SL, Granada, Spain) for SV and traditional tube culture (10). The remaining specimen was frozen at ⫺80°C. After a 48-h incubation, cell monolayers from the SV were subjected to DFA with monoclonal antibodies against influenza A and B viruses (Dako Diagnostics Ltd., Cambridgeshire, United Kingdom). The SV supernatant was used for RNA extraction and RTPCR as described above. The traditional tube culture was examined daily during 14 days to observe the appearance of cytopathic effect. Before being discarded as negative, tubes without cytopathic effect were subjected to a hemagglutination test as previously described (9). All positive results by RT-PCR were confirmed by repeating the test with a frozen aliquot of the specimen or cell culture supernatant. The national reference laboratory (National Center for Microbiology, Majadahonda, Madrid, Spain) carried out further characterization of the strains. The data were statistically analyzed with SPSS 13.0.1 software (SPSS Inc., Chicago, IL). Univariate analysis was conducted on the results by the 2 test. A P value of ⬍0.05 was considered significant. A total of 565 specimens were analyzed within the two periods, 299 in the 2004-to-2005 period and 266 in the 2005-to2006 period. Influenza viruses were detected in 152 specimens (26.9%) by any of the three methods. The distribution of the results by method and influenza virus type and subtype is shown in Table 1. The most sensitive method was SV-PCR (95.4%). The two methods that allowed recovery of the virus strain, i.e., SV-DFA and SV-PCR, were compared. Whereas influenza B and H1 were similarly detected by both methods, influenza H3 was detected in 64.7% and 95.3% of samples by
Influenza disease is subjected to surveillance worldwide by national networks that predict the epidemic threshold by reporting clinical and virological data (6). Epidemiological and virological surveillance is carried out by sentinel physicians and virologists, respectively. The clinical criteria for sampling and laboratory testing of influenza viruses vary among different countries. Indeed, a laboratory-confirmed influenza case is defined for each country by the national laboratory network, within the national influenza surveillance network. In Spain, the national laboratory network is currently reporting a laboratory-confirmed influenza case when the virus is isolated in cell culture. Although reverse transcription (RT)-PCR assays are more sensitive than cell culture (5), only those cases detected by virus isolation are being reported as positive for influenza in the surveillance network (7). Many regional laboratories within the national network use the rapid shell-vial assay (SV) (viral culture by SV) because it shortens the time to obtain positive cases. Screening of influenza viruses from the SV is usually done by direct fluorescence assay (DFA), which also allows the differentiation of influenza viruses A and B (10). Subsequently, antigenic characterization by serological or molecular methods is conducted on the isolates. Our laboratory carries out regional influenza surveillance in Andalusia (in the south of Spain) as part of the national influenza laboratory network. During a two-year period, from October 2004 to May 2005 and October 2005 to May 2006, we evaluated several methods for influenza virus detection to determine which was the most reliable and sensitive. For this purpose, we compared three diagnostic methods: multiplex RT-nested-PCR directly on the specimen for the detection and differentiation of influenza A subtypes H1 and H3 and influenza B viruses (DD) (11), SV followed by DFA (SV-DFA), and SV followed by RT-PCR (11) on the cell culture supernatant (SV-PCR).
* Corresponding author. Mailing address: Servicio de Microbiologı´a, Hospital Universitario Virgen de las Nieves, Avda. Fuerzas Armadas, 2, 18014 Granada, Spain. Phone: 34 958 020010. Fax: 34 958 020169. E-mail:
[email protected]. 䌤 Published ahead of print on 25 July 2007. 3109
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TABLE 1. Detection of influenza viruses by the three methods Method
Any method DD SV-DFA SV-PCR
Total no. (%) positive
152 (26.9) 131 (86.2) 119 (78.3) 145 (95.4)
No. (%) positive for influenza virus type and subtype A H1
A H3
B
24 (15.8) 23 (95.8) 24 (100) 23 (95.8)
85 (55.9) 76 (89.4) 55 (64.7) 81 (95.3)
43 (28.3) 32 (74.4) 40 (93) 41 (95.3)
SV-DFA and SV-PCR, respectively (P, 0.016). Positive results were concordant between DD and SV-DFA in 70% of cases, between DD and SV-PCR in 82.8% of cases, and between SV-DFA and SV-PCR in 80.2% of cases (Table 2). By combining both PCR methods, DD and SV-PCR, only one positive sample gave a false-negative result. Direct detection, typing, and/or subtyping can be rapidly carried out by several methods, such as nucleic acid techniques, immunofluorescence assay, or enzyme-linked immunosorbent assay (3, 8, 11). This step can be carried out in each regional laboratory within the national surveillance network. Subsequently, laboratories belonging to the Community Network of Reference Laboratories in Europe must be able to replicate growth in cell lines for isolation of influenza viruses. A selection of the virus isolates is sent to the WHO Collaborating Centre for Reference and Research on Influenza in London for complete characterization (5). Thus, even though multiplex RT-PCR and antigen detection methods can rapidly give a result which eases the management of patients with influenza (1, 5), virus isolation is required in the context of the annual influenza surveillance. In the 2005 to 2006 season, our regional network covered a population of 77,000 individuals, which is 1% of the surveillance coverage (7). We conduct both the detection and isolation, and only the viral isolates are sent to the national reference laboratory. Any delay in sample processing negatively influences virus isolation. Moreover, nasal/throat swabs are not the optimal specimens for influenza virus recovery. Previous reports have used this diagnostic tool, i.e., integrated cell culture-PCR, for improving the recovery of other viruses (2, 4). But few data are available for influenza virus detection in the context of surveillance systems. We have found that the most reliable method for influenza virus detection is SV-PCR. This method recovered 95.4% of the total positive results by any of the three methods at 48 h of sample processing. Only one sample (0.6%) was positive by SV-DFA and negative by SV-PCR. The remaining samples negative by SVPCR were positive only by the DD method. This may be a consequence of delayed sending or processing and/or a suboptimal sampling, which lead to nonviable viruses, in which case only viral RNA can be detected in the specimen. SV-DFA is the method currently recommended for the rapid diagnosis of influenza viruses in different influenza networks. However, our
TABLE 2. Two-by-two concordances of the three methods for influenza virus detection No. with result by: Method
Result
SV-PCR
SV-DFA
Positive
Negative
SV-DFA
Positive Negative
118 28
1 418
DD
Positive Negative
125 20
6 414
Positive
Negative
103 16
28 418
results show that this method may not be the most appropriate, especially when the circulating strain is the H3 subtype. In conclusion, within an influenza surveillance network, the best diagnostic method would be the combination of rapid culture by SV with multiplex RT-PCR on the cell culture supernatant. It reaches the highest sensitivity, recovers the viral strain for further characterization, and is able to simultaneously carry out the detection, typing, and subtyping of influenza viruses. We thank Angeles Rivera and Francisca Garcı´a for their technical assistance. We are indebted to Sean Smith and Lilaj Raz for proofreading the English language. REFERENCES 1. Boivin, G., I. Hardy, and A. Kress. 2001. Evaluation of a rapid optical immunoassay for influenza viruses (FLU OIA test) in comparison with cell culture and reverse transcription-PCR. J. Clin. Microbiol. 39:730–732. 2. Choo, Y. J., and S. J. Kim. 2006. Detection of human adenoviruses and enteroviruses in Korean oysters using cell culture, integrated cell culturePCR, and direct PCR. J. Microbiol. 44:162–170. 3. Dunn, J. J., C. Gordon, C. Kelley, and K. C. Carroll. 2003. Comparison of the Denka-Seiken INFLU A.B-Quick and BD Directigen Flu A⫹B kits with direct fluorescent-antibody staining and shell vial culture methods for rapid detection of influenza viruses. J. Clin. Microbiol. 41:2180–2183. 4. Ebihara, T., R. Endo, X. Ma, N. Ishiguro, and H. Kikuta. 2005. Detection of human metapneumovirus antigens in nasopharyngeal secretions by an immunofluorescent-antibody test. J. Clin. Microbiol. 43:1138–1141. 5. Ellis, J. S., and M. C. Zambon. 2002. Molecular diagnosis of influenza. Rev. Med. Virol. 12:375–389. 6. European Influenza Surveillance Scheme. 2006. Annual report: 2004–2005 influenza season. NIVEL, Netherlands Institute for Health Services Research, Utrecht, the Netherlands. http://www.eiss.org/documents /eiss_annual_report_2004-2005_⫹_cover.pdf. 7. Grupo de Vigilancia de la gripe en Espan ˜a. 2006. Vigilancia de la gripe en Espan ˜a. Sistema centinela. Resumen de la temporada 2005–2006. http://vgripe .isciii.es/gripe/documentos/20052006/InformesAnuales/Gripe0506.pdf. 8. Herrmann, B., C. Larsson, and B. W. Zweygberg. 2001. Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (FLU OIA). J. Clin. Microbiol. 39:134–138. 9. Hsiung, G. D. 1994. Hemagglutination and hemagglutination-inhibition test, p. 69–75. In G. D. Hsiung, C. K. Y. Fong, and M. L. Landry (ed.), Hsiung’s diagnostic virology as illustrated by light and electron microscopy, 4th ed. Yale University Press, New Haven, CT. 10. Navarro-Marı´, J. M., S. Sanbonmatsu-Ga ´mez, M. Pe´rez-Ruiz, and M. de la Rosa-Fraile. 1999. Rapid detection of respiratory viruses by shell vial assay using simultaneous culture of Hep-2, LLC-MK2, and MDCK cells in a single vial. J. Clin. Microbiol. 37:2346–2347. 11. Stockton, J., J. S. Ellis, M. Saville, J. P. Clewley, and M. C. Zambon. 1998. Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses. J. Clin. Microbiol. 36:2990–2995.
