1887

Abstract

The recent recurrence of highly pathogenic avian influenza virus A H5N1 was firstly reported in mid-December 2003 and continued through 2005. This study describes a sensitive and specific real-time RT-PCR method for the detection of influenza A subtype H5 and for monitoring virus loads. Using serial dilutions of influenza A H5N1 cultures, this assay reproducibly determined the lowest detection limit to be approximately 5×10 50 % egg infective doses (EID). In contrast, the minimum detection limit was approximately 3 EID in conventional RT-PCR with WHO primers and 10 EID in antigen-capture ELISA. In tests of serial dilutions of -transcribed influenza A H5 gene RNA, there was linear amplification from 40 copies to 4×10 copies of target RNA per reaction and approximately six copies, and sometimes even as few as three copies, of target RNA tested positive in our assay. Thirty-five throat swabs from ill birds were tested: 33 samples tested positive using this assay. In comparison, 27, 13 and 19 samples tested positive using conventional RT-PCR, antigen-capture ELISA and virus isolation, respectively. To evaluate further the sensitivity of this real-time RT-PCR, a standard panel and 60 H5N1 isolates that contained different clades of influenza virus A/H5N1 were tested and all tested positive. To evaluate the specificity of the assay, 60 throat swabs from patients infected with influenza virus A H1 were tested; all were negative. Thirteen other viruses were also tested and all tested negative.

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2007-05-01
2019-10-13
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References

  1. CDC ( 1997; ). Isolation of avian influenza A (H5N1) viruses from humans – Hong Kong, May–December 1997. MMWR Morb Mortal Wkly Rep 46, 1204–1207.
    [Google Scholar]
  2. CDC ( 2004; ). Cases of influenza A (H5N1) – Thailand, 2004. MMWR Morb Mortal Wkly Rep 53, 100–103.
    [Google Scholar]
  3. Chen, W., Xu, Z., Mu, J., He, B., Yang, L., Lin, L., Meng, S., Mu, F., Gan, H. & other authors ( 2004; ). Real-time quantitative fluorescent reverse transcriptase-PCR for detection of severe acute respiratory syndrome-associated coronavirus RNA. Mol Diagn 8, 231–235.[CrossRef]
    [Google Scholar]
  4. Holland, P. M., Abramson, R. D., Watson, R. & Gelfand, D. H. ( 1991; ). Detection of specific polymerase chain reaction product by utilizing the 5′ to 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88, 7276–7280.[CrossRef]
    [Google Scholar]
  5. Kubar, A., Saygun, I., Ozdemir, A., Yapar, M. & Slots, J. ( 2005; ). Real-time polymerase chain reaction quantification of human cytomegalovirus and Epstein–Barr virus in periodontal pockets and the adjacent gingiva of periodontitis lesions. J Periodontal Res 40, 97–104.[CrossRef]
    [Google Scholar]
  6. Kuypers, J., Wright, N., Ferrenberg, J., Huang, M. L., Cent, A., Corey, L. & Morrow, R. ( 2006; ). Comparison of real-time PCR assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children. J Clin Microbiol 44, 2382–2388.[CrossRef]
    [Google Scholar]
  7. Lee, M. S., Chang, P. C., Shien, J. H., Cheng, M. C. & Shieh, H. K. ( 2001; ). Identification and subtyping of avian influenza viruses by reverse transcription-PCR. J Virol Methods 97, 13–22.[CrossRef]
    [Google Scholar]
  8. Liu, J., Xiao, H., Lei, F., Zhu, Q., Qin, K., Zhang, X. W., Zhang, X. L., Zhao, D., Wang, G. & other authors ( 2005; ). Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309, 1206 [CrossRef]
    [Google Scholar]
  9. Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W. & Deetz, K. ( 1995; ). Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4, 357–362.[CrossRef]
    [Google Scholar]
  10. Munch, M., Nielsen, L. P., Handberg, K. J. & Jorgensen, P. H. ( 2001; ). Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA. Arch Virol 146, 87–97.[CrossRef]
    [Google Scholar]
  11. Ng, E. K. O., Cheng, P. K. C., Ng, A. Y. Y., Hoang, T. L. & Lim, W. W. L. ( 2005; ). Influenza A H5N1 detection. Emerg Infect Dis 11, 1303–1305.[CrossRef]
    [Google Scholar]
  12. Palmer, S., Wiegand, A. P., Maldarelli, F., Bazmi, H., Mican, J. M., Polis, M., Dewar, R. L., Planta, A., Liu, S. & other authors ( 2003; ). New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 41, 4531–4536.[CrossRef]
    [Google Scholar]
  13. Perkins, S. M., Webb, D. L., Torrance, S. A., El Saleeby, C., Harrison, L. M., Aitken, J. A., Patel, A. & DeVincenzo, J. P. ( 2005; ). Comparison of a real-time reverse transcriptase PCR assay and a culture technique for quantitative assessment of viral load in children naturally infected with respiratory syncytial virus. J Clin Microbiol 43, 2356–2362.[CrossRef]
    [Google Scholar]
  14. Starick, E., Romer-Oberdorfer, A. & Werner, O. ( 2000; ). Type- and subtype-specific RT-PCR assays for avian influenza A viruses (AIV). J Vet Med B Infect Dis Vet Public Health 47, 295–301.[CrossRef]
    [Google Scholar]
  15. Tran, T. H., Nguyen, T. L., Nguyen, T. D., Luong, T. S., Pham, P. M., Nguyen, V. C., Pham, T. S., Vo, C. D., Le, T. Q. & other authors ( 2004; ). Avian influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med 350, 1179–1188.[CrossRef]
    [Google Scholar]
  16. Viseshakul, N., Thanawongnuwech, R., Amonsin, A., Suradhat, S., Payungporn, S., Keawchareon, J., Oraveerakul, K., Wongyanin, P., Plitkul, S. & other authors ( 2004; ). The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand. Virology 328, 169–176.[CrossRef]
    [Google Scholar]
  17. Wang, C., Tian, X., Cao, Z., Yu, X., Sun, M., Chen, X., Wang, H. & Tian, K. ( 2004; ). Establishment of H5 avian influenza virus capture ELISA employing biotin labeled monoclonal antibody. Acta Lab Anim Sci Sin 12, 204–207.
    [Google Scholar]
  18. WHO ( 2005; ). Recommended laboratory tests to identify avian influenza A virus in specimens from humans. http://www.who.int/csr/disease/avian_influenza/guidelines/avian_labtests2.pdf
  19. WHO ( 2006; ). Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO. http://www.who.int/csr/disease/avian_influenza/country/cases_table_2006_05_23/en/index.html
  20. Wright, K. E., Wilson, G. A. R., Novosad, D., Dimock, C., Tan, D. & Weber, J. M. ( 1995; ). Typing and subtyping of influenza viruses in clinical samples by PCR. J Clin Microbiol 33, 1180–1184.
    [Google Scholar]
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