Therefore, it is of great importance that sufficient negative controls be included to determine a cutoff value for background hybridization. In addition, we routinely use fold serial dilutions of a titrated influenza A virus stock as input material in our PCR-based assays to provide a semiquantitative estimate of variability between independent assays. Both sets of controls will aid in the determination of a cutoff value for background hybridization and weak positive samples.
By PCR-based assays, diagnosis of influenza A virus infection can be achieved within a single working day, which is significantly faster than the time to diagnosis of infection by classical methods. By virus culture approaches, positive results may be obtained in 24 h or more after inoculation, but a definite negative diagnosis may require culture for up to 2 weeks.
The availability of NA inhibitors for the treatment of influenza virus infection may demand more rapid diagnosis of virus infection in the future.
The benefit of these new drugs appears to depend heavily on the early start of treatment, i. Taken together, our data indicate that the newly designed PCR offers a more sensitive and faster tool for the diagnosis of human influenza A virus infection than virus isolation. Because of the better matching primers, it can be expected that for the detection of animal influenza A viruses this PCR is also more suitable than previous PCR protocols 1 , 3 , 7.
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Fouchier RA 1 ,. Bestebroer TM ,. Herfst S ,. Van Der Kemp L ,. Rimmelzwaan GF ,. Osterhaus AD. Affiliations 1 author 1. A comment on this article appears in " False-positive results obtained by following a commonly used reverse transcription-PCR protocol for detection of influenza A virus. Share this article Share with email Share with twitter Share with linkedin Share with facebook.
Abstract The recently raised awareness of the threat of a new influenza pandemic has stimulated interest in the detection of influenza A viruses in human as well as animal secretions. Free full text.
J Clin Microbiol. PMID: Ron A. Rimmelzwaan , and Albert D. Author information Article notes Copyright and License information Disclaimer. Phone: 31 10 Fax: 31 10 E-mail: ln. This article has been cited by other articles in PMC. RNA isolation. Dot blot hybridization. Virus isolation and propagation. Open in a separate window. Sensitivity and specificity of influenza A virus PCR. Detection of influenza A virus in human throat swab samples. Detection of influenza A virus in bird samples.
Comparison of reverse transcription-PCR with tissue culture and other rapid diagnostic assays for detection of type A influenza virus. Brown T. Analysis of DNA sequences by blotting and hybridization. Current protocols in molecular biology, suppl. New York, N. New clues to the emergence of flu pandemics. Nat Med. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.
Type-specific identification of influenza viruses A, B and C by the polymerase chain reaction. J Virol Methods. Prospective application of reverse transcriptase polymerase chain reaction for diagnosing influenza infections in respiratory samples from a children's hospital. A pandemic warning? Characterisation of the pathogenicity of members of the newly established H9N2 influenza virus lineage in Asia. Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections.
GG Influenza Study Group. N Engl J Med. FDA-cleared rapid molecular assays are available that produce results in approximately minutes. Some of these rapid molecular assays are CLIA-waived for point-of-care use. Some molecular assays are able to detect and discriminate between infections with influenza A and B viruses; other tests can identify specific seasonal influenza A virus subtypes [A H1N1 pdm09, or A H3N2 ]. These assays can yield results in approximately 45 minutes to several hours depending upon the assay.
Notably, the detection of influenza viral RNA or nucleic acids by these assays does not necessarily indicate detection of viable infectious virus or on-going influenza viral replication.
It is important to note that not all assays have been cleared by the FDA for diagnostic use. Some multiplex molecular assays are available that can detect influenza viral nucleic acids and distinguish influenza virus infection from other respiratory pathogens and may also be useful for management of severely immunosuppressed patients, or for use in identifying the cause of an institutional outbreak of respiratory illness. Some tests are CLIA-waived and approved for use in any outpatient setting, whereas others must be used in a moderately complex clinical laboratory.
Detection of influenza virus antigen does not necessarily indicate detection of viable infectious virus or on-going influenza viral replication. None of the rapid influenza diagnostic tests provide any information about influenza A virus subtypes.
The types of specimens acceptable for use i. The specificity and, in particular, the sensitivity of rapid influenza diagnostic tests are lower than for viral culture and RT-PCR and vary by test. Tests with low to moderate sensitivity and high specificity can produce false negative results more commonly than false positive results, especially during peak influenza activity in the community.
In contrast, false-positive RIDT results are less likely, but can occur and are more common during periods of low influenza activity. Therefore, when interpreting results of a rapid influenza diagnostic test, clinicians should consider the of the test in the context of the level of influenza activity in their community See Algorithm to assist in the interpretation of influenza testing results and clinical decision-making during periods when influenza viruses are circulating in the community and Algorithm to assist in the interpretation of influenza testing results and clinical decision-making during periods when influenza viruses are NOT circulating in the community for more information.
Package inserts and the laboratory performing the test should be consulted for more details regarding use of rapid influenza diagnostic tests. Immunofluorescence assays are antigen detection assays that generally require use of a fluorescent microscope to produce results in approximately hours with moderate sensitivity and high specificity. Both direct DFA and indirect fluorescent antibody IFA staining assays are available to detect influenza A and B viral antigens in respiratory tract specimens.
Subtyping or further identification of influenza A viruses is not possible by immunofluorescence assays. One rapid immunofluorescence assay is an RIDT and utilizes an analyzer device to produce results in approximately 15 minutes. PCR amplicons from human, avian, and swine viruses of 15 different subtypes, with between 1. Sequencing of the amplicons showed that the heteroduplex mobility patterns correlated with the sequence divergence between test and reference DNA. The application of the RT-PCR HMA method for rapid screening of samples was assessed with a reference panel of viruses of human, avian, and swine origin.
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