Evaluation of twenty rapid antigen tests for the detection of human influenza A H5N1, H3N2, H1N1, and B viruses

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza‐specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co‐circulating seasonal influenza strains. J. Med. Virol. 81:1918–1922, 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of a rapid test for detection of H5N1 avian influenza virus

The performance of H5 Dot ELISA, a rapid test for detection of avian H5N1 influenza virus, was evaluated using 30 H5N1 strains belonging to 10 major genetic groups of H5N1 influenza virus, 14 strains of non-H5N1 influenza virus and 652 field samples collected from healthy and diseased chickens from markets and poultry farms. The detection limit of the test for all 30 strains of H5N1 virus was < or = 0.1 hemagglutinin (HA) units and the test yielded a negative result when tested against 100 HA units of the non-H5N1 viruses. The test gave a positive result for 87 of the 106 poultry samples from which H5N1 virus was isolated by culture and 3 of 546 culture-negative poultry samples. Compared with virus culture, the overall prediction rate of the test was determined to be 96.6%; the positive prediction rate was 96.7% and negative prediction rate, 96.6%. The false positive rate was 0.5% and false negative rate 17.9%. Considering that the test is also convenient to use, it was concluded that H5 Dot ELISA is suitable for field use in the investigation of H5N1 influenza outbreaks and surveillance in poultry.     

A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1

The neuraminidase of influenza viruses is the target of the inhibitors oseltamivir and zanamivir. Recent reports on influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAI) are a cause for concern. Several amino acid substitutions, each as a consequence of one single nucleotide mutation, are known to confer resistance to NAI. An increase of NAI-resistant viruses appears to be likely as a result of a wider application of NAI for treatment and prophylaxis of seasonal influenza infections. Monitoring the occurrence and spread of resistant viruses is an important task. Therefore, RT-PCR assays were developed with subsequent pyrosequencing analysis (PSQ-PCR). These assays allow a rapid, high-throughput and cost-effective screening of subtype A/H1N1, A/H3N2, and A/H5N1 viruses. Various specimens such as respiratory swabs, allantoic fluid, or cell-propagated viruses can be used and results are available within hours. Several A/H1N1, A/H3N2, and A/H5N1 viruses isolated from human and avian specimens were tested to evaluate the method. Positive controls encoding resistance-associated mutations were created using site-directed mutagenesis. The results obtained with these controls showed that the assay can discriminate clearly the wild-type virus from a mutant virus. The detection limit of minor virus variants within the viral quasispecies amounts to 10%.     

Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2

In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies.     

Utility of the rapid antigen detection BinaxNOW Influenza A&B test for detection of novel influenza A (H1N1) virus

Nasopharyngeal aspirates, collected during outbreaks, of the novel influenza A (H1N1) virus in Barcelona, were used to compare the accuracy of a rapid antigen-based test (Binax) with the real-time RT-PCR assay developed by the CDC. The sensitivity, specificity and positive predictive value of the rapid test are higher in patients less than 18 years old and during the acute stage of the epidemic than in adult patients.     

Performance of rapid-test kits for the detection of the pandemic influenza A/H1N1 virus

The early detection of pandemic influenza strains is a key factor for clinicians in treatment decisions and infection control practices. The aims of this study were to determine the analytical sensitivity and clinical performance of the commercially available influenza rapid tests in Taiwan. Four rapid tests for influenza virus (BinaxNow test, QuickVue test, TRU test, and Formosa Rapid test) were evaluated for their detection limit against four influenza viruses (the 2009 pandemic influenza A virus H1N1, seasonal influenza virus H1N1, H3N2, and influenza B virus) circulating in Taiwan. The viral load of these isolates were quantified by rtRT-PCR and then diluted 2-fold serially for the comparison. The lowest detectable viral load of the pandemic influenza A virus H1N1 by the Formosa Rapid test, QuickVue test, TRU test, and Binax Now test was 5.3 × 10⁴, 1.0 × 10⁵, 1.0 × 10⁵, and 4.2 × 10⁵ copies/μL, respectively. Of these four tests, the two most sensitive tests (the QuickVue test and the Formosa Rapid test) were chosen to evaluate 62 nasopharyngeal specimens from patients who were suspected of infection with pandemic influenza A virus H1N1. The positive rate for the Formosa Rapid test and the QuickVue test were 53.2% (33/62) and 45.2% (28/62) (McNemar's test, P = 0.125), respectively. In conclusion, the Formosa Rapid test was the most sensitive test in the present study for the detection of influenza antigens and its clinical performance was similar to that of the QuickVue test (Kappa = 0.776). This suggests that the Formosa Rapid test could be used to aid clinical decision making in primary health care settings during outbreaks of influenza.     

Evaluation of a new rapid influenza A diagnostic test for detection of pandemic (H1N1) 2009 and seasonal influenza A virus

BackgroundRapid influenza A diagnostic tests (RIDTs) play an important role in the clinical setting, especially in the influenza post-pandemic era with three influenza A viruses in circulation.ObjectivesDetermine the sensitivity and specificity of a new RIDT (FluA Dot) by comparison with BD Directigen EZ FluA+B and CDC rRT-PCR.Study designTwo sets of experiments were conducted to determine the performance of the new test. (1) Serial dilutions of eight pandemic (H1N1) 2009 (pH1N1) isolates, five seasonal H3N2 isolates, five seasonal H1N1 isolates and three recombinant nucleoproteins were tested by FluA Dot assay, Directigen EZ FluA+B test and CDC real-time RT-PCR. (2) Using CDC rRT-PCR as the gold standard, the clinical sensitivity and specificity of the FluA Dot and Directigen EZ FluA+B were evaluated in nasopharyngeal swab (NPS) specimens of 807 patients presenting with influenza-like illness.ResultsThe average analytical sensitivity of FluA Dot (0.06 ng/mL for recombinant nucleoproteins and 2.16 ± 0.85 log 10 TCID₅₀ for viruses) was approximately 10-fold higher than Directigen EZ FluA+B (1–2 ng/mL for recombinant nucleoproteins and 3.54 ± 0.81 log 10 TCID₅₀ for viruses), and was approximately 10-fold lower than the CDC rRT-PCR (1.09 ± 0.69 log 10 TCID₅₀ for viruses). Among 807 NPS specimens tested, the sensitivities and specificities of FluA Dot were 91.1% (95%CI: 86.7–94.4%)/99.7% (95%CI: 98.7–99.9%), and the Directigen EZ FluA+B were 71.9% (95%CI: 65.7–77.6%)/99.8%(95%CI: 99.0–99.9%).ConclusionThe new test (FluA Dot) exhibit higher sensitivity than Directigen EZ FluA+B both in pH1N1 and seasonal influenza A detection. The promising RIDT can play important roles in influenza diagnosis and therapy.     

A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1

A multiplex real-time RT-PCR method for the simultaneous detection of influenza virus types A and B and identification of subtypes H5 and N1 in a single tube is described. The method was developed with four sets of primers and probes which were specific to influenza virus (sub)types A, B, H5, and N1, and evaluated by using a total of 40 influenza virus reference strains, including 17 avian influenza A (12 H5N1, 1 H1N1, 1 H3N2, 1 H4N6, 1 H7N3, and 1 H9N2), 18 human influenza A (11 H3N2, 6 H1N1 and 1 H5N1) and 5 influenza B viruses. The method exhibited a high specificity and sensitivity of approximately 10(1)-10(2)copies/microl for each (sub)type and a high reproducibility with intra- and inter-assay CV from 0.13 to 4.24%. In an analysis of 189 clinical samples from patients during the year 2004 and 2005, the method identified 81 positive samples (42.9%) and identified simultaneously 14 type B samples and 11 subtype N1 samples, in comparison only 46 positive samples (24.3%) identified by the conventional culturing method. The method would be a useful molecular diagnostic tool for large-scale screening of clinical samples for influenza virus.     

On the origin of the human influenza virus subtypes H2N2 and H3N2

The RNA of the human influenza virus Singapore (H2N2) strain has been labeled in vivo by phosphorus-32 and separated by polyacrylamide gel electrophoresis into eight segments, which were correlated to the corresponding gene functions and/or proteins. The base sequence homology between the individual genes (segments) of the H2N2 virus and those of different influenza A strains has been determined by molecular hybridization. Segments 1, 5, 7, and 8 of the Singapore strain exhibit a base sequence homology of almost 100% as compared to the FM1 strain (HlNl), while the homology between the other segments is significantly lower (24–76%). For the Singapore and Hong Kong (H3N2) strains all segments except that coding for the hemagglutinin (HA, 24%) exhibit a homology close to 100%. The ³²P-labeled segment 4 (HA-gene) of the avian influenza A strain duck Ukraine (Hav7Neg2) shows a homology of 92% to Hong Kong, while the homology of at least two other segments is significantly lower. These results are taken as an indication that the H2N2 subtype is derived from the HlN1 subtype by a recombination event retaining four H1N1 segments, while the other four segments are gained from another yet unknown strain. The H3N2 subtype is presumably derived from a H2N2 subtype, retaining seven segments of the H2N2 subtype, while the gene coding for the HA is obtained from the duck Ukraine or another highly related strain.     

rapidSTRIPE H1N1 test for detection of the pandemic swine origin influenza A (H1N1) virus

The rapidSTRIPE H1N1 test, based on a nucleic acid lateral-flow assay, has been developed for diagnosis of a swine-origin influenza A (H1N1) virus. This test is simple and cost-effective and allows specific detection of the S-OIV A (H1N1) virus from swab sampling to final detection on a lateral-flow stripe within 2 to 3 h.     

Differential susceptibility of different cell lines to swine-origin influenza A H1N1, seasonal human influenza A H1N1, and avian influenza A H5N1 viruses

BackgroundThe novel swine-origin influenza A H1N1 virus (S-OIV) causes the current pandemic. Its tissue tropism and replication in different cell lines are not well understood.ObjectiveCompare the growth characteristics of cell lines infected by S-OIV, seasonal influenza A H1N1 (sH1N1) and avian influenza A H5N1 (H5N1) viruses and the effect of temperature on viral replication.Study designCytopathic effect (CPE), antigen expression by immunofluorescence (IF) and viral load profile by quantitative RT-PCR in 17 cell lines infected by S-OIV, sH1N1 and H5N1 were examined. Comparison of their replication efficiency in chick embryo was performed. The effect of temperature on viral replication in Madin–Darby canine kidney (MDCK) cells was determined by TCID₅₀ at 33 °C, 37 °C and 39 °C for 5 consecutive days.ResultsS-OIV replicated in cell lines derived from different tissues or organs and host species with comparable viral load to sH1N1. Among 13 human cell lines tested, Caco-2 has the highest viral load for S-OIV. S-OIV showed a low viral load with no CPE or antigen expression in pig kidney cell PK-15, H5N1 demonstrated the most diverse cell tropism by CPE and antigen expression, and the highest viral replication efficiency in both cell lines and allantoic fluid. All three viruses demonstrated best growth at 37 °C in MDCK cells.ConclusionCell line growth characteristics of S-OIV, sH1N1 and H5N1 appear to correlate clinically and pathologically with involved anatomical sites and severity. Low replication of S-OIV in PK-15 suggests that this virus is more adapted to human than swine.     

Establishment of retroviral pseudotypes with influenza hemagglutinins from H1, H3, and H5 subtypes for sensitive and specific detection of neutralizing antibodies

Pseudotype reporter viruses provide a safe, quantitative, and high-throughput tool for assessing antibody neutralization for many viruses, including high pathogenicity H5 and H7 influenza A strains. However, adapting this system to other influenza subtypes has been hampered by variations in the protease cleavage site of hemagglutinin (HA) that make it less susceptible to the cleavage required for infectivity. In this report several proteases, reporter vectors, and cell substrates were evaluated while optimizing pseudovirus production, and robust methods were established for sensitive and specific neutralization of pseudotypes carrying influenza H1, H3, and H5 subtype HA that correlates well with titers obtained in microneutralization assays involving replicating influenza virus These findings should facilitate broad use of HA-pseudotypes that remove the need for infectious virus in a range of applications, including neutralization assays for serological surveys of viral infection and evaluations of vaccine sera.     

Genetic characterization of H1N1, H1N2 and H3N2 swine influenza virus in Thailand

Swine have been known to be a suitable host for influenza A virus. In Thailand, phylogenetic analysis on swine influenza virus (SIV) has as yet not been attempted. The present report presents molecular and phylogenetic analysis performed on SIV in Thailand. In this study, 12 SIV isolates from the central and eastern part of Thailand were subtyped and the molecular genetics of hemagglutinin and neuraminidase were elucidated. Three subtypes, H1N1, H1N2 and H3N2, are described. Phylogenetic analysis of the SIV hemagglutinin and neuraminidase genes shows individual clusters with swine, human or avian influenza virus at various global locations. Furthermore, amino acid substitutions were detected either at the receptor binding site or the antigenic sites of the hemagglutinin gene.