Live-attenuated influenza vaccine (LAIV) is delivered to vaccine recipients using a nasal spray syringe. LAIV delivered by this method is immunogenic at current doses; however, improvements in nasal delivery might allow for significant dose reduction. We investigated LAIV vaccination in ferrets using a high efficiency nebulizer designed for nasal delivery. LAIV nasal aerosol elicited high levels of serum neutralizing antibodies and protected ferrets from homologous virus challenge at conventional (10(7)TCID(50)) and significantly reduced (10(3)TCID(50)) doses. Aerosol LAIV also provided a significant level of subtype-specific cross-protection. These results demonstrate the dose-sparing potential of nebulizer-based nasal aerosol LAIV delivery.

Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect Nm ctrA, Hi hpd and Sp lytA, and serogroup-specific genes in the cap locus for Nm serogroups A, B, C, W135, X and Y. However, the assay sensitivity for serogroups B, W135 and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays B-synD, W-synG, and Y-synF showed 100% specificity for detecting Nm species, with high sensitivity [B-synD, 99%(75/76); W-synG, 97%(38/39); and Y-synF, 100%(66/66)]. The lower limit of detection (LLD) was 9, 43 and 10 copies/reaction for B-synD, W-synG, and Y-synF assays, respectively, a significant improvement compared to the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of: (i) Nm ctrA, Hi hpd and Sp lytA (NHS); (ii) Nm serogroups A, W135 and X (AWX), and (iii) Nm serogroups B, C and Y (BCY). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for singleplex. Pairwise comparison of real-time PCR between multiplex and singleplex showed that cycle threshold values of the multiplex were similar to those for singleplex. There were no substantial differences in the sensitivity and specificity between these multiplex and singleplex real-time PCR assays.

BACKGROUND: With the introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines, there has been a dramatic reduction of Hib disease in young children and the epidemiological trends of invasive H. influenzae have shifted. METHODS: Data were collected from active surveillance for invasive H. influenzae disease conducted through Active Bacterial Core surveillance sites during 1989-2008. RESULTS: During 1999-2008, the estimated mean annual incidence of H. influenzae infection was 1.62 cases per 100,000 population; 15.3% of cases were fatal. Incidence was higher among adults aged ≥65 years, compared with other age groups. The largest burden of disease among children aged <5 years was in infants aged <1 year; many of these cases occurred during the first month of life in preterm or low-birth weight infants. An estimated 10% of the total burden of disease among children aged <5 years occurred in American Indian and Alaska Native children. During 1989-2008, 7559 cases of H. influenzae disease were reported from Active Bacterial Core surveillance sites. Small increases in the incidence of serotypes a, e, and f were observed during 1989-2008. The largest of these increases was in serotype f and was primarily among adults aged ≥18 years. CONCLUSIONS: Since the introduction of Hib conjugate vaccines, the incidence of invasive disease caused by H. influenzae in the United States has decreased dramatically; however, a considerable burden of non-Hib disease is still present in the oldest and youngest age groups. There is no evidence of substantial replacement disease with non-b serotypes in young children in the United States.

Short-term deferral of the Haemophilus influenzae type b (Hib) vaccine booster dose during a recent U.S. Hib vaccine shortage did not result in widespread Hib carriage in Atlanta, as the Hib carriage rate was found to be 0.3% (1/342). Hi colonization was significantly more common among males and daycare attendees.

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (C(t)) value of 35, with 100% average reaction efficiency and an average R(2) of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average C(t) values from 13.0 to 29.5. The mean sodC C(t) value of these Nm isolates was 17.6+/-2.2 (+/-SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes.

Live-attenuated influenza vaccine (LAIV) delivered by large droplet intranasal spray is efficacious against infection. However, many of the large droplets are trapped in the external nares and do not reach the target nasal airway tissues. Smaller droplets might provide better distribution yielding similar protection with lower doses. We evaluated 20 and 30mum aerosol delivery of influenza virus in mice. A 15s aerosol exposure optimally protected against homologous and heterologous influenza infection and induced a robust immune response. These results demonstrate the feasibility of nasal vaccination using aerosolized particles, providing a strategy to improve vaccine efficacy and delivery.

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.