ABSTRACT
BACKGROUND: Although influenza is a major public health concern among adults ≥60 years of age, few large, prospective studies of influenza vaccines have been conducted in this population. The goal of the present study was to directly compare the safety and efficacy of LAIV and TIV in adults ≥60 years of age. MATERIALS AND METHODS: A prospective, randomized, open-label, multicenter trial was conducted in South Africa. In March-April 2002, 3009 community-dwelling ambulatory adults 60-95 years of age were randomized 1:1 to receive a single dose of LAIV or TIV. Surveillance for influenza illness was conducted through November. Serum antibody titers were evaluated in all participants, and interferon-γ enzyme-linked immunosorbent spot assay responses were evaluated in a cohort of subjects. Solicited reactogenicity and adverse events were monitored for days 0-10 postvaccination; serious adverse events were monitored for the entire study. RESULTS: Influenza illness caused by vaccine-matched strains was detected in 0.8% (12/1494) and 0.5% (8/1488) of LAIV and TIV recipients, respectively; the relative efficacy of LAIV vs TIV was -49% (95% CI: -259, 35). As expected, greater serum antibody responses were seen with TIV, and greater cellular responses were seen with LAIV (although not for influenza B). Among subjects with culture-confirmed influenza illness, post hoc analyses revealed trends toward less feverishness (LAIV, 14%; TIV, 46%; P=0.05) and less fever (LAIV, 9%; TIV, 31%; P=0.16) among LAIV recipients. In each treatment group, 38-39% and 24-25% of subjects had baseline hemagglutination inhibition titers of ≤4 for A/H1 and A/H3, but 7 of 8 TIV cases and 7 of 12 LAIV cases of matched-strain influenza occurred among these subjects. Runny nose/nasal congestion (+13%), cough (+5%), sore throat (+5%), lethargy (+3%), and decreased appetite (+2%) were reported by more LAIV vs TIV recipients. Injection site reactions were reported by 27% of TIV recipients. SAEs were reported by a similar proportion of LAIV and TIV recipients (9% vs 8%). CONCLUSIONS: Given the low incidence of influenza in both groups, no conclusions were possible regarding the relative efficacy of LAIV and TIV. There was a trend toward less feverishness/fever among LAIV recipients who developed influenza compared with TIV recipients with influenza, consistent with results from studies comparing the vaccines in children. A disproportionate number of influenza illnesses occurred among baseline seronegative subjects, particularly for those receiving TIV, which suggests that this subgroup has the greatest need for improved influenza vaccination. The safety profiles of LAIV and TIV were consistent with results from previous studies in older adults and no significant safety concerns were identified. clinicaltrials.gov identifier, NCT00192413.
Subject(s)
Influenza Vaccines/immunology , Influenza, Human/prevention & control , Aged , Aged, 80 and over , Antibodies, Viral/blood , Drug Evaluation , Female , Hemagglutination Inhibition Tests , Humans , Immunity, Cellular , Influenza A virus/immunology , Influenza B virus/immunology , Influenza Vaccines/administration & dosage , Influenza Vaccines/adverse effects , Interferon-gamma/blood , Interferon-gamma/metabolism , Male , Middle Aged , Prospective Studies , South Africa , Vaccination/adverse effects , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunology , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/adverse effectsABSTRACT
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).
Subject(s)
Influenza Vaccines/immunology , Measles-Mumps-Rubella Vaccine/immunology , Vaccination/methods , Administration, Intranasal , Antibodies, Viral/blood , Drug Incompatibility , Female , Humans , Infant , Influenza Vaccines/administration & dosage , Influenza, Human/prevention & control , Male , Measles/prevention & control , Measles-Mumps-Rubella Vaccine/administration & dosage , Mumps/prevention & control , Placebos/administration & dosage , Rubella/prevention & control , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vaccines, Combined/administration & dosage , Vaccines, Combined/immunologyABSTRACT
This randomized, double-blind, placebo-controlled study investigated the efficacy, safety, and immunogenicity of LAIV in community-dwelling ambulatory adults > or =60 years of age in South Africa in 2001. Nose and throat swabs were obtained for influenza virus culture based on the symptoms of influenza-like illness. A total of 3242 subjects were enrolled, with a mean age of 69.5 years. The efficacy of LAIV against influenza viruses antigenically similar to the vaccine was 42.3% (95% CI, 21.6-57.8%). Efficacy against A/H3N2 viruses was 52.5% (95% CI, 32.1-67.2%); vaccine efficacy was not observed against antigenically similar B strains. In post hoc analyses, efficacy in subjects 60 to <70 years of age was 41.8% and -22.7% against A/H3N2 and B, respectively and 65.7% and 9.9%, respectively, for subjects > or =70 years. Reactogenicity events were higher among LAIV than placebo recipients during 11 days postvaccination (P=0.042), including runny nose/nasal congestion, cough, sore throat, headache, muscle aches, tiredness, and decreased appetite. Rates of serious adverse events were similar for LAIV and placebo recipients. This was the first demonstration of statistically significant protection by LAIV against culture-confirmed influenza in adults > or =60 years of age. These results suggest that LAIV may provide an additional public health tool in the prevention of influenza in the elderly. (ClinicalTrials.gov identifier, NCT00217230.).
Subject(s)
Influenza Vaccines/immunology , Influenza, Human/prevention & control , Aged , Aged, 80 and over , Antibodies, Viral/blood , Double-Blind Method , Female , Humans , Influenza A Virus, H3N2 Subtype/immunology , Influenza Vaccines/adverse effects , Influenza, Human/epidemiology , Male , Middle Aged , Population Surveillance , South Africa , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunologyABSTRACT
Live attenuated influenza vaccine (LAIV) provides a useful tool to rapidly immunize populations in the developing world to prevent influenza outbreaks. In this noninferiority trial conducted in Asia and South America, where oral poliovirus vaccine (OPV) is still used, 2503 children aged 6 to <36 months with three polio immunizations were randomized to receive LAIV+OPV, placebo+OPV, or LAIV only. Immune responses in children receiving concomitant LAIV+OPV were noninferior to those observed in recipients of either vaccine alone. Response rates for different poliovirus types were similar in recipients of LAIV+OPV and placebo+OPV. Response rates to all influenza strains were similar in LAIV+OPV and LAIV-only recipients. Concomitant OPV and LAIV were safely administered to young children.
Subject(s)
Influenza Vaccines/administration & dosage , Influenza, Human/prevention & control , Poliomyelitis/prevention & control , Poliovirus Vaccine, Oral/administration & dosage , Antibodies, Viral/blood , Child, Preschool , Female , Humans , Immunization Schedule , Infant , Influenza Vaccines/adverse effects , Influenza Vaccines/immunology , Male , Poliovirus Vaccine, Oral/adverse effects , Poliovirus Vaccine, Oral/immunology , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunologyABSTRACT
The highly sensitive gamma interferon (IFN-gamma) enzyme-linked immunosorbent spot (ELISPOT) assay permits the investigation of the role of cell-mediated immunity (CMI) in the protection of young children against influenza. Preliminary studies of young children confirmed that the IFN-gamma ELISPOT assay was a more sensitive measure of influenza memory immune responses than serum antibody and that among seronegative children aged 6 to <36 months, an intranasal dose of 10(7) fluorescent focus units (FFU) of a live attenuated influenza virus vaccine (CAIV-T) elicited substantial CMI responses. A commercial inactivated influenza virus vaccine elicited CMI responses only in children with some previous exposure to related influenza viruses as determined by detectable antibody levels prevaccination. The role of CMI in actual protection against community-acquired, culture-confirmed clinical influenza by CAIV-T was investigated in a large randomized, double-blind, placebo-controlled dose-ranging efficacy trial with 2,172 children aged 6 to <36 months in the Philippines and Thailand. The estimated protection curve indicated that the majority of infants and young children with >or=100 spot-forming cells/10(6) peripheral blood mononuclear cells were protected against clinical influenza, establishing a possible target level of CMI for future influenza vaccine development. The ELISPOT assay for IFN-gamma is a sensitive and reproducible measure of CMI and memory immune responses and contributes to establishing requirements for the future development of vaccines against influenza, especially those used for children.
Subject(s)
Immunity, Cellular , Influenza Vaccines/immunology , Influenza, Human/immunology , Interferon-gamma/blood , Leukocytes, Mononuclear/immunology , Administration, Intranasal , Antibodies, Viral/blood , Antibodies, Viral/immunology , Child, Preschool , Double-Blind Method , Hemagglutination Inhibition Tests , Humans , Infant , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza B virus/immunology , Influenza, Human/prevention & control , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Philippines , Thailand , Vaccination , Vaccines, Attenuated/immunologyABSTRACT
BACKGROUND: This study was designed to evaluate the efficacy and safety of cold-adapted influenza vaccine, trivalent (CAIV-T) against culture-confirmed influenza in children 12 to <36 months of age during 2 consecutive influenza seasons at multiple sites in Asia. METHODS: In year 1, 3174 children 12 to <36 months of age were randomized to receive 2 doses of CAIV-T (n = 1900) or placebo (n = 1274) intranasally > or =28 days apart. In year 2, 2947 subjects were rerandomized to receive 1 dose of CAIV-T or placebo. RESULTS: Mean age at enrollment was 23.5 +/- 7.4 months. In year 1, efficacy of CAIV-T compared with placebo was 72.9% [95% confidence interval (CI): 62.8-80.5%] against antigenically similar influenza subtypes, and 70.1% (95% CI: 60.9-77.3%) against any strain. In year 2, revaccination with CAIV-T demonstrated significant efficacy against antigenically similar (84.3%; 95% CI: 70.1-92.4%) and any (64.2%; 95% CI: 44.2-77.3%) influenza strains. In year 1, fever, runny nose/nasal congestion, decreased activity and appetite, and use of fever medication were more frequent with CAIV-T after dose 1. Runny nose/nasal congestion after dose 2 (year 1) and dose 3 (year 2) and use of fever medication after dose 3 (year 2) were the only other events reported significantly more frequently in CAIV-T recipients. CONCLUSIONS: CAIV-T was well tolerated and effective in preventing culture-confirmed influenza illness over multiple and complex influenza seasons in young children in Asia.
Subject(s)
Adaptation, Physiological , Cold Temperature , Influenza Vaccines/adverse effects , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Orthomyxoviridae/immunology , Vaccines, Attenuated/immunology , Asia/epidemiology , Child, Preschool , Double-Blind Method , Female , Humans , Infant , Influenza, Human/diagnosis , Influenza, Human/epidemiology , Male , Orthomyxoviridae/physiology , Vaccines, Attenuated/adverse effectsABSTRACT
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.
Subject(s)
Influenza A virus/isolation & purification , Influenza B virus/isolation & purification , Respiratory Syncytial Viruses/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Child, Preschool , Humans , Infant , Sensitivity and SpecificityABSTRACT
OBJECTIVE: The goal was to evaluate the safety, tolerability, and efficacy of an investigational, refrigerator-stable formulation of live attenuated influenza vaccine (cold-adapted influenza vaccine-trivalent) against culture-confirmed influenza, acute otitis media, and effectiveness outcomes in young children in day care over 2 consecutive influenza seasons. METHODS: Children 6 to <36 months of age who were attending day care were assigned randomly in year 1 to receive 2 doses of vaccine or placebo intranasally, 35 +/- 7 days apart. In year 2, subjects received 1 dose of the same treatment as in year 1. RESULTS: A total of 1616 subjects (vaccine: 951 subjects; placebo: 665 subjects) in year 1 and 1090 subjects (vaccine: 640 subjects; placebo: 450 subjects) in year 2 were able to be evaluated for efficacy. The mean age at first vaccination was 23.4 +/- 7.9 months. In year 1, the overall efficacy of the vaccine against influenza subtypes similar to the vaccine was 85.4%; efficacy was 91.8% against A/H1N1 and 72.6% against B. In year 2, the overall efficacy was 88.7%; efficacy was 90.0% against H1N1, 90.3% against A/H3N2, and 81.7% against B. Efficacy against all episodes of acute otitis media associated with culture-confirmed influenza was 90.6% in year 1 and 97.0% in year 2. Runny nose or nasal discharge after dose 1 in year 1 was the only reactogenicity event that was significantly more frequent with cold-adapted influenza vaccine-trivalent (82.3%) than placebo (75.4%). CONCLUSIONS: Cold-adapted influenza vaccine-trivalent was well tolerated and effective in preventing culture-confirmed influenza illness in children as young as 6 months of age who attended day care.
Subject(s)
Child Day Care Centers , Influenza Vaccines/therapeutic use , Influenza, Human/prevention & control , Otitis Media/prevention & control , Acute Disease , Child, Preschool , Community-Acquired Infections/prevention & control , Double-Blind Method , Female , Humans , Infant , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza B virus/immunology , Male , Prospective StudiesABSTRACT
BACKGROUND: Live attenuated influenza vaccine (LAIV; FluMist) is a trivalent vaccine containing cold-adapted influenza vaccine viruses that infect and replicate in cells lining the nasopharynx to induce immunity. Recovery of viruses (shedding) is measured by culture of nasal specimens. Shedding of vaccine viruses is not equated with transmission because transmission requires more virus than is detected in many nasal swabs. Previous studies with LAIV did not detect transmission to close contacts. The primary objective of this study was to estimate the probability of transmission to placebo contacts in a day care setting. METHODS: One hundred ninety-seven healthy children aged 9 to 36 months attending day care were randomized to receive vaccine or placebo. Postvaccination viral shedding, safety, genotype and phenotype of shed viruses and probability of transmission were assessed. RESULTS: Eighty percent of 98 vaccine recipients shed at least one vaccine strain. No clinically significant differences in solicited adverse events attributable to vaccine occurred; safety profiles were similar in both groups. Vaccine virus isolates retained their phenotypic characteristics (cold adaptation and temperature sensitivity) and did not revert at nucleotides known to confer an attenuating phenotype. There was one confirmed transmission of a vaccine strain to a single placebo recipient. According to the Reed-Frost model, the calculated probability of transmission to a child after contact with a single vaccinated child was 0.58% (95% confidence interval, 0-1.7%). There was no increased reactogenicity or other safety concerns in the recipient child. CONCLUSIONS: Young children in a day care setting had a high rate of shedding and a low rate of transmission. No clinically significant illness occurred among children who received vaccine or placebo or in the child to whom the vaccine virus was transmitted.
Subject(s)
Influenza Vaccines/therapeutic use , Influenza, Human/prevention & control , Influenza, Human/transmission , Child Day Care Centers , Child, Preschool , Disease Transmission, Infectious , Double-Blind Method , Female , Genotype , Humans , Infant , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza B virus/immunology , Influenza Vaccines/adverse effects , Influenza, Human/immunology , Influenza, Human/virology , Male , Phenotype , Placebos , Prospective Studies , Virus SheddingABSTRACT
Adenovirus (AdV) respiratory infections have usually been associated with species B, C, and E. In this study, we detected 9.4% of AdVs by PCR in 500 nasal swabs from 319 children with influenza-like symptoms. AdV typing by PCR with specific probes showed species C, B, and F as well as coinfection with two species. Coinfection with two AdV species and the presence of species F in respiratory samples are novel findings that should be further investigated.
Subject(s)
Adenovirus Infections, Human/virology , Adenoviruses, Human/classification , Adenoviruses, Human/isolation & purification , Polymerase Chain Reaction/methods , Respiratory Tract Infections/virology , Adenovirus Infections, Human/complications , Adenovirus Infections, Human/epidemiology , Adenoviruses, Human/genetics , Child, Preschool , DNA Probes , Humans , Infant , Respiratory Tract Infections/complications , Respiratory Tract Infections/epidemiology , Species SpecificityABSTRACT
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.
Subject(s)
Amino Acid Substitution , Evolution, Molecular , Influenza A Virus, H3N2 Subtype/genetics , Influenza, Human/epidemiology , Asia/epidemiology , DNA, Viral/analysis , Europe/epidemiology , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza, Human/virology , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNAABSTRACT
BACKGROUND: Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants and young children. Although several experimental RSV vaccines are under investigation, immuno therapy is the only treatment currently available. In assessing the immunogenicity of various vaccine formulations, a plaque reduction neutralization assay for the evaluation of RSV neutralizing antibody has been widely used. The method produces reliable results, but it is tedious and labor intensive as it relies on manual counting by laboratory personnel. To facilitate evaluation of phase II and phase III vaccine clinical trials, a more rapid, reliable and efficient neutralization assay is needed. RESULTS: An improved microneutralization assay for quantifying RSV neutralizing antibodies was developed using an ImmunoSpot Series I Analyzer (Cellular Technology Ltd., Cleveland, OH) for automated plaque counting. The method is an improvement of the established classical microneutralization assay in which immunostained plaques on transparent tissue culture plates are counted manually under a dissecting microscope. Image analyzer technology allows for fully automated counting of plaques distributed throughout an entire well. Adjustments, such as the use of opaque tissue culture plates and the TMB substrate, True Blue (KPL, Gaithersburg, MD), were required to adapt the assay for optimal detection of plaques by the image analyzer. The suitability and the accuracy of the method for counting RSV plaques were determined by comparative testing of a reference serum and two control sera by manual and automated counting methods. The results showed that the two methods were highly correlated (R = 0.9580) and the titers generated by them were within two-fold. CONCLUSION: Our results demonstrate that the semi-automated assay is rapid and reliable. It provides results within two fold to the classical plaque microneutralization assay and is readily applied to the evaluation of neutralizing antibody titers in sera obtained from epidemiology or vaccine clinical trials.
Subject(s)
Automation , Neutralization Tests/methods , Respiratory Syncytial Viruses/isolation & purification , Viral Plaque Assay/methods , Animals , Chlorocebus aethiops , Vero CellsABSTRACT
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.
Subject(s)
Parainfluenza Virus 3, Human/isolation & purification , Respirovirus Infections/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , Child , DNA Primers , Humans , Nasopharynx/virology , Nucleocapsid/genetics , Nucleotides/genetics , Sensitivity and Specificity , Species SpecificityABSTRACT
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.
Subject(s)
Genetic Variation , Influenza B virus/genetics , Influenza B virus/isolation & purification , Influenza, Human/virology , Base Sequence , Child, Preschool , DNA Primers , Humans , Infant , Influenza B virus/classification , Influenza, Human/diagnosis , Influenza, Human/epidemiology , Molecular Sequence Data , New York/epidemiology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Serum antibody titers against the A/Panama/2007/99(H3N2) and A/Fujian/411/2002(H3N2)-like viruses were determined in children 6-35 months of age who received either 1 dose of the inactivated influenza vaccine or the live attenuated influenza vaccine containing the A/Panama strain. Results indicated that the live vaccine induced higher antibody responses than the inactivated vaccine against the A/Panama and A/Fujian-like viruses.
Subject(s)
Antibodies, Viral/immunology , Influenza A Virus, H3N2 Subtype , Influenza A virus/classification , Influenza A virus/immunology , Influenza Vaccines/administration & dosage , Influenza, Human/prevention & control , Vaccination/methods , Age Factors , Antibodies, Viral/analysis , Antibody Formation/immunology , Child, Preschool , Female , Hemagglutination Inhibition Tests , Humans , Immunization Schedule , Incidence , Infant , Influenza Vaccines/immunology , Influenza, Human/epidemiology , Influenza, Human/immunology , Male , Probability , Risk Assessment , Sensitivity and Specificity , Sex Factors , Statistics, Nonparametric , United States/epidemiology , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunologyABSTRACT
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.
Subject(s)
Influenza B virus/isolation & purification , Influenza, Human/diagnosis , Influenza, Human/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Cell Line , Child , Child, Preschool , Dogs , Double-Blind Method , Humans , Influenza B virus/genetics , Influenza, Human/epidemiology , RNA, Viral/analysis , RNA, Viral/isolation & purification , Robotics , Seasons , Sensitivity and Specificity , Taq Polymerase/metabolism , Viral Load , Virus CultivationABSTRACT
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.
Subject(s)
Influenza B virus/classification , Influenza, Human/epidemiology , Phylogeny , Amino Acid Sequence , Base Sequence , DNA Primers , Europe/epidemiology , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza B virus/genetics , Israel/epidemiology , Molecular Sequence Data , Polymerase Chain Reaction/methods , Sequence Alignment , Sequence Homology, Amino Acid , Time FactorsABSTRACT
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.