Semiquantitative one-step RT-PCR for simultaneous identification of human influenza and respiratory syncytial viruses.

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) method was developed for simultaneous detection and typing/subtyping of influenza viruses A/H1, A/H3 or B, and respiratory syncytial viruses A or B, followed by DNA semiquantitation using the Agilent 2100 Bioanalyzer. Such method provides a rapid, specific and sensitive diagnostic tool for detection and semiquantification of respiratory illness specimens.

Superior relative efficacy of live attenuated influenza vaccine compared with inactivated influenza vaccine in young children with recurrent respiratory tract infections.

BACKGROUND: Young children have a high incidence of influenza and influenza-related complications. This study compared the efficacy and safety of cold-adapted influenza vaccine, trivalent (CAIV-T) with trivalent inactivated influenza vaccine (TIV) in young children with a history of recurrent respiratory tract infections (RTIs). METHODS: Children 6 to 71 months of age were randomized to receive 2 doses of CAIV-T (n = 1101) or TIV (n = 1086), 35 +/- 7 days apart before the start of the 2002-2003 influenza season and were followed up for culture-confirmed influenza, effectiveness outcomes, reactogenicity, and adverse events. RESULTS: Overall, 52.7% (95% confidence interval [CI] = 21.6%-72.2%) fewer cases of influenza caused by virus strains antigenically similar to vaccine were observed in CAIV-T than in TIV recipients. Greater relative efficacy for CAIV-T was observed for the antigenically similar A/H1N1 (100.0%; 95% CI = 42.3%-100.0%) and B (68.0%; 95% CI = 37.3%-84.8%) strains but not for the antigenically similar A/H3N2 strains (-97.1%; 95% CI = -540.2% to 31.5%). Relative to TIV, CAIV-T reduced the number of RTI-related healthcare provider visits by 8.9% (90% CI = 1.5%-15.8%) and missed days of school, kindergarten, or day care by 16.2% (90% CI = 10.4%-21.6%). Rhinitis and rhinorrhea, otitis media, and decreased appetite were the only events that were reported more frequently in CAIV-T subjects. There was no difference between groups in the incidence of wheezing after vaccination. CONCLUSIONS: CAIV-T was well tolerated in these children with RTIs and demonstrated superior relative efficacy compared with TIV in preventing influenza illness.

Comparison of the efficacy and safety of live attenuated cold-adapted influenza vaccine, trivalent, with trivalent inactivated influenza virus vaccine in children and adolescents with asthma.

BACKGROUND: Despite their potential for increased morbidity, 75% to 90% of asthmatic children do not receive influenza vaccination. Live attenuated influenza vaccine (LAIV), a cold-adapted, temperature-sensitive, trivalent influenza vaccine, is approved for prevention of influenza in healthy children 5 to 19 years of age. LAIV has been studied in only a small number of children with asthma. METHODS: Children 6 to 17 years of age, with a clinical diagnosis of asthma, received a single dose of either intranasal CAIV-T (an investigational refrigerator-stable formulation of LAIV; n = 1114) or injectable trivalent inactivated influenza vaccine (TIV; n = 1115) in this randomized, open-label study during the 2002-2003 influenza season. Participants were followed up for culture-confirmed influenza illness, respiratory outcome, and safety. RESULTS: The incidence of community-acquired culture-confirmed influenza illness was 4.1% (CAIV-T) versus 6.2% (TIV), demonstrating a significantly greater relative efficacy of CAIV-T versus TIV of 34.7% (90% confidence interval [CI] 9.4%-53.2%; 95% CI = 3.9%-56.0%). There were no significant differences between treatment groups in the incidence of asthma exacerbations, mean peak expiratory flow rate findings, asthma symptom scores, or nighttime awakening scores. The incidence of runny nose/nasal congestion was higher for CAIV-T (66.2%) than TIV (52.5%) recipients. Approximately 70% of TIV recipients reported injection site reactions. CONCLUSIONS: CAIV-T was well tolerated in children and adolescents with asthma. There was no evidence of a significant increase in adverse pulmonary outcomes for CAIV-T compared with TIV. CAIV-T had a significantly greater relative efficacy of 35% compared with TIV in this high-risk population.

Development of a real-time RT-PCR assay for detection and quantitation of parainfluenza virus 3.

A TaqMan-based real-time RT-PCR assay was developed to detect and quantify human parainfluenza virus 3 (PIV3). Two sets of primer-probe pairs were designed based on the nucleotide (nt) sequence of the nucleocapsid (N) gene. The primer-probe pairs were derived from the 3' end of the N gene (set 1) and the 5' region of the gene (set 2), respectively. Using real-time RT-PCR, the sensitivity of set 1 was determined to be about 9 copies of PIV3 genome, while the sensitivity of set 2 was about 93 copies of PIV3 genome. Set 1 was chosen for subsequent experiments. This primer-probe pair detected PIV3, but not any of several other respiratory viruses, indicating that the assay is PIV3 specific. For clinical evaluation, the assay was employed to test 80 nasopharyngeal aspirates from children with respiratory symptoms. The results confirmed the presence of PIV3 in 12 specimens previously identified as positive by culture confirmation, and showed all of which contained more than 100 copies of PIV3 genome. In addition, the method also detected PIV3 genomes in specimens found negative by culture confirmation, indicating the value of this RT-PCR assay. These data thus demonstrate the application of the real-time RT-PCR assay for the detection and quantification of PIV3 in clinical specimens.

Detection of influenza B in clinical specimens: comparison of high throughput RT-PCR and culture confirmation.

Influenza virus is one of the major causes of worldwide respiratory tract infections during the winter season. Here we describe a high throughput (HTP) protocol for rapid diagnosis of influenza B that combines automated viral RNA extraction with detection and quantification by TaqMan-based PCR. Using this methodology, we tested 4176 nasal swabs collected from children enrolled in a European influenza vaccine trial during the winter of 2000 to compare our HTP PCR method to culture confirmation for detection of influenza B. Among these, 37 were positive by culture and 169 were positive by PCR irrespective of virus copy number. However, when specimens with fewer than 20 copies of the viral genome were disregarded, a good correlation between two methods was observed. At this cut-off, 34 specimens were positive and 4106 were negative by both methods. Statistical analysis of the data using culture confirmation as the standard indicated that the sensitivity of HTP RT-PCR for influenza B was 92% (95% CI, 0.83-1), and the specificity was 99% (95% CI, 0.99-1). In summary, HTP RT-PCR was proved to be more rapid and sensitive than culture confirmation, and it correlated significantly with culture confirmation for specimens containing more than 20 copies of the viral genome.

Influenza vaccine concurrently administered with a combination measles, mumps, and rubella vaccine to young children.

Children aged 11 to <24 months received 2 intranasal doses of live attenuated influenza vaccine (LAIV) or placebo, 35+/-7 days apart. Dose 1 was administered concomitantly with a combined measles, mumps, and rubella vaccine (Priorix). Seroresponses to measles and mumps were similar between groups. Compared with placebo, response rates to rubella in LAIV+Priorix recipients were statistically lower at a 15 IU/mL threshold (83.9% vs 78.0%) and the prespecified noninferiority criteria were not met. In a post hoc analysis using an alternate widely accepted threshold of 10 IU/mL, the noninferiority criteria were met (93.4% vs 89.8%). Concomitant administration with Priorix did not affect the overall influenza protection rate of LAIV (78.4% and 63.8% against antigenically similar influenza strains and any strain, respectively).

Molecular evolution of human influenza A/H3N2 virus in Asia and Europe from 2001 to 2003.

Hemagglutinin sequences of 146 human influenza A/H3N2 strains identified in respiratory specimens from Asia and Europe during the 2001-2003 influenza seasons were analyzed by DNA sequencing. Our results suggest that four amino acid substitutions, L25I, H75Q, H155T, and Q156H, led to the antigenic conversion of the previously predominant A/Panama/2007/99-like strains to the more recent A/Fujian/411/2002-like strains.

Detection and characterization of new influenza B virus variants in 2002.

One-hundred five influenza B-positive specimens obtained from southeast Asia in 2002 were categorized on the basis of DNA sequencing of HA1 gene as well as real-time PCR analysis of the NA gene. Phylogenetic analysis of the HA1 gene sequences showed that the majority of the viruses (96.2%) belonged to the B/Victoria/2/87 lineage, while a smaller percentage of the viruses (3.8%) belonged to the B/Yamagata/16/88 lineage. The B/Yamagata/16/88 viruses displayed significant antigenic drift in the deduced amino acid sequences of the HA1 protein, and the B/Victoria/2/87-like viruses consisted of B/Hong Kong/1351/02-like (72.3%) and B/Hong Kong/330/01-like (27.7%) viruses. The B/Hong Kong/1351/02-like viruses were reassortants with the HA gene belonging to the B/Victoria/2/87 lineage and the NA gene belonging to the B/Yamagata/16/88 lineage, whereas both the HA and NA genes of B/Hong Kong/330/01 virus belonged to the B/Victoria/2/87 lineage. In this study, however, all the B/Hong Kong/330/01-like isolates exhibited the B/Yamagata/16/88-like NA gene, which likely resulted from reassortment of B/Hong Kong/330/01 and B/Hong Kong/1351/02 viruses during coinfection. Additional molecular characterization of the six internal genes showed that the M, NS, PA, and PB2 genes of the new variants were B/Hong Kong/1351/02 in origin, whereas the NP and PA genes retained the B/Hong Kong/330/01 origin. Interestingly, these new variants all appeared late in the year 2002. These results support the notion that influenza B viruses continued to evolve through antigenic drift and shift.

Cocirculation and evolution of two lineages of influenza B viruses in europe and Israel in the 2001-2002 season.

Forty-nine influenza B virus isolates collected in Belgium, Finland, Spain, and Israel during the 2001-2002 winter season were categorized into either of two lineages, B/Yamagata/16/88 or B/Victoria/2/87, based on the phylogenetic studies of HA1 sequences. The data trace the geographic spread of B/Victoria/2/87-like viruses and support the emergence of B/Hong Kong/1351/02-like viruses, possibly due to selective advantages of reassortment.

Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR.

Timely diagnosis of respiratory syncytial virus (RSV) infection is critical for appropriate treatment of lower respiratory infection in young children. To facilitate diagnosis, we developed a rapid, specific, and sensitive TaqMan PCR method for detection of RSV A and RSV B. Two sets of primer-probe pairs were selected from the nucleotide sequences encoding the nucleocapsid protein--one targeting RSV A and the other targeting RSV B. The specificity of the TaqMan reverse transcription-PCR assay was evaluated by testing each primer-probe pair against various viruses derived from laboratory virus stocks, as well as clinical respiratory specimens. Fluorescent signals were observed only in the presence of RSV A and/or RSV B. The sensitivity of our quantitative PCR assay was determined on the basis of PFU and virus particle counts. The resulting assay sensitivity was found to be 0.023 PFU, or two copies of viral RNA, for RSV A and 0.018 PFU, or nine copies of viral RNA, for RSV B. This quantitative TaqMan PCR assay was utilized to diagnose 175 nasopharyngeal aspirates obtained from children in Hong Kong with respiratory symptoms during the winter of 2000 and 2001. Among these specimens, TaqMan PCR detected 36 RSV-positive samples, 10 of which were identified as RSV A and 26 of which were identified as RSV B, whereas culture confirmation identified 21 RSV-positive specimens and immunofluorescence identified 32 RSV-positive specimens, all of which were among those identified by PCR. The results confirmed the accuracy of our TaqMan PCR assay and demonstrated its improved sensitivity versus classical methods.