Amanda J. Leach

From the circumsporozoite protein to the RTS,S/AS candidate vaccine

The RTS,S/AS01E malaria vaccine candidate has recently entered phase 3 testing. Reaching this important milestone is the culmination of more than 20 years of research and development by GlaxoSmithKline and partners and collaborators. The vaccine has been developed to protect young children and infants living in sub-Saharan Africa against clinical and severe disease caused by Plasmodium falciparum infection. Over the past 9 years, RTS,S/AS candidate vaccine has been evaluated in multiple Phase 2 studies. The vaccine was shown to have a favourable safety profile and to be well tolerated in all age groups in which it was tested, including the intended target population of infants and young children in Sub-Saharan Africa. Data obtained so far suggest that RTS,S/AS can be co-administered with other vaccines included in the routine Expanded Program of Immunization (EPI). In Phase 2 testing, the vaccine candidate was shown to confer significant protection against P. falciparum infection and clinical disease, including severe malaria. Furthermore, a trend towards an indirect beneficial effect of the vaccine on non-malarial morbidities has been observed in several trials. In this paper, we will describe the genesis of the RTS,S/AS concept, including the rationale for selecting the circumsporozoite protein (CSP) as the target antigen. Early development history of the vaccine will be briefly described. We will present the most salient results from recent Phase 2 studies conducted in the target pediatric population, which have led to the decision to progress RTS,S/AS to Phase 3 testing. If the Phase 3 results confirm the observations made during Phase 2 testing, the RTS,S/AS vaccine, when broadly implemented and judiciously integrated with other malaria-prevention measures, would have a major public-health impact in Sub-Saharan Africa.

Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: Updated recommendations from the World Health Organization Pneumococcal Carriage Working Group

In 2003 the World Health Organization (WHO) convened a working group and published a set of standard methods for studies measuring nasopharyngeal carriage of Streptococcus pneumoniae (the pneumococcus). The working group recently reconvened under the auspices of the WHO and updated the consensus standard methods. These methods describe the collection, transport and storage of nasopharyngeal samples, as well as provide recommendations for the identification and serotyping of pneumococci using culture and non-culture based approaches. We outline the consensus position of the working group, the evidence supporting this position, areas worthy of future research, and the epidemiological role of carriage studies. Adherence to these methods will reduce variability in the conduct of pneumococcal carriage studies undertaken in the context of pneumococcal vaccine trials, implementation studies, and epidemiology studies more generally so variability in methodology does not confound the interpretation of study findings.

Measuring nasal bacterial load and its association with otitis media

BackgroundNasal colonisation with otitis media (OM) pathogens, particularly Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis, is a precursor to the onset of OM. Many children experience asymptomatic nasal carriage of these pathogens whereas others will progress to otitis media with effusion (OME) or suppurative OM. We observed a disparity in the prevalence of suppurative OM between Aboriginal children living in remote communities and non-Aboriginal children attending child-care centres; up to 60% and <1%, respectively. This could not be explained by the less dramatic difference in rates of carriage of respiratory bacterial pathogens (80% vs 50%, respectively). In this study, we measured nasal bacterial load to help explain the different propensity for suppurative OM in these two populations.MethodsQuantitative measures (colony counts and real-time quantitative PCR) of the respiratory pathogens S. pneumoniae, H. influenzae and M. catarrhalis, and total bacterial load were analysed in nasal swabs from Aboriginal children from remote communities, and non-Aboriginal children attending urban child-care centres.ResultsIn both populations nearly all swabs were positive for at least one of these respiratory pathogens. Using either quantification method, positive correlations between bacterial load and ear state (no OM, OME, or suppurative OM) were observed. This relationship held for single and combined bacterial respiratory pathogens, total bacterial load, and the proportion of respiratory pathogens to total bacterial load. Comparison of Aboriginal and non-Aboriginal children, all with a diagnosis of OME, demonstrated significantly higher loads of S. pneumoniae and M. catarrhalis in the Aboriginal group. The increased bacterial load despite similar clinical condition may predict persistence of middle ear effusions and progression to suppurative OM in the Aboriginal population. Our data also demonstrated the presence of PCR-detectable non-cultivable respiratory pathogens in 36% of nasal swabs. This may have implications for the pathogenesis of OM including persistence of infection despite aggressive therapies.ConclusionNasal bacterial load was significantly higher among Aboriginal children and may explain their increased risk of suppurative OM. It was also positively correlated with ear state. We believe that a reduction in bacterial load in high-risk populations may be required before dramatic reductions in OM can be achieved.

Epidemiology of nasopharyngeal carriage of respiratory bacterial pathogens in children and adults: cross-sectional surveys in a population with high rates of pneumococcal disease

BackgroundTo determine the prevalence of carriage of respiratory bacterial pathogens, and the risk factors for and serotype distribution of pneumococcal carriage in an Australian Aboriginal population.MethodsSurveys of nasopharyngeal carriage of Streptococcus pneumoniae, non-typeable Haemophilus influenzae, and Moraxella catarrhalis were conducted among adults (≥16 years) and children (2 to 15 years) in four rural communities in 2002 and 2004. Infant seven-valent pneumococcal conjugate vaccine (7PCV) with booster 23-valent pneumococcal polysaccharide vaccine was introduced in 2001. Standard microbiological methods were used.ResultsAt the time of the 2002 survey, 94% of eligible children had received catch-up pneumococcal vaccination. 324 adults (538 examinations) and 218 children (350 examinations) were enrolled. Pneumococcal carriage prevalence was 26% (95% CI, 22-30) among adults and 67% (95% CI, 62-72) among children. Carriage of non-typeable H. influenzae among adults and children was 23% (95% CI, 19-27) and 57% (95% CI, 52-63) respectively and for M. catarrhalis, 17% (95% CI, 14-21) and 74% (95% CI, 69-78) respectively. Adult pneumococcal carriage was associated with increasing age (p = 0.0005 test of trend), concurrent carriage of non-typeable H. influenzae (Odds ratio [OR] 6.74; 95% CI, 4.06-11.2) or M. catarrhalis (OR 3.27; 95% CI, 1.97-5.45), male sex (OR 2.21; 95% CI, 1.31-3.73), rhinorrhoea (OR 1.66; 95% CI, 1.05-2.64), and frequent exposure to outside fires (OR 6.89; 95% CI, 1.87-25.4). Among children, pneumococcal carriage was associated with decreasing age (p < 0.0001 test of trend), and carriage of non-typeable H. influenzae (OR 9.34; 95% CI, 4.71-18.5) or M. catarrhalis (OR 2.67; 95% CI, 1.34-5.33). Excluding an outbreak of serotype 1 in children, the percentages of serotypes included in 7, 10, and 13PCV were 23%, 23%, and 29% (adults) and 22%, 24%, and 40% (2-15 years). Dominance of serotype 16F, and persistent 19F and 6B carriage three years after initiation of 7PCV is noteworthy.ConclusionsPopulation-based carriage of S. pneumoniae, non-typeable H. influenzae, and M. catarrhalis was high in this Australian Aboriginal population. Reducing smoke exposure may reduce pneumococcal carriage. The indirect effects of 10 or 13PCV, above those of 7PCV, among adults in this population may be limited.

Pilot trial of a pentavalent pneumococcal polysaccharide/protein conjugate vaccine in Gambian infants

BACKGROUND Invasive pneumococcal disease is a major cause of mortality and morbidity in young children in developing countries. Pneumococcal polysaccharide/protein conjugate vaccines, which are likely to be immunogenic in the very young, offer a potential way for preventing these infections. Therefore a pilot safety and immunogenicity study of a five-valent conjugate vaccine has been undertaken in an area of rural Africa where invasive pneumococcal disease is prevalent. METHODS Thirty Gambian infants were vaccinated with 3 doses of a five-valent pneumococcal conjugate vaccine containing 5 micrograms of type 6B, 14, 18, 19F and 23F polysaccharides conjugated to the diphtheria toxin mutant protein CRM197 at the ages of 2, 3 and 4 months; 30 infants received 2 doses at the ages of 2 and 4 months and 30 infants who received three doses of a Haemophilus influenzae type b vaccine acted as controls. Local and systemic reactions were recorded after vaccination and antibody titers were measured by an enzyme-linked immunosorbent assay. RESULTS No serious local or systemic reactions to vaccination were recorded. Antibody responses to each component of the vaccine were demonstrated. One month after immunization with three doses of vaccine, antibody titers were 3 to 11 times higher than before vaccination (postvaccination titers ranged from 2.49 micrograms/ml for type 19 polysaccharide to 7.59 micrograms/ml for type 14). Elevated titers were well-maintained during the subsequent 4 months. Three doses of vaccine induced higher titers than did two doses. Antibody titers increased 2- to 3-fold over the period of immunization in children who received H. influenzae type b vaccine. CONCLUSIONS A five-valent pneumococcal conjugate vaccine proved safe and immunogenic in Gambian infants. However, a vaccine containing a larger number of serotypes will be necessary to achieve a maximal clinical impact.

Respiratory Bacterial Pathogens in the Nasopharynx and Lower Airways of Australian Indigenous Children with Bronchiectasis

OBJECTIVE To test the hypothesis that bacterial density, strain diversity, and concordance of pathogens between upper and lower airways are higher in children with bronchiectasis than in those with non-bronchiectatic conditions. STUDY DESIGN Nasopharyngeal (NP) swabs and bronchoalveolar lavage (BAL) fluid were cultured from 45 Indigenous children with bronchiectasis and 30 non-Indigenous children with non-bronchiectatic respiratory symptoms. Lower airway infection was defined as >10(4) colony-forming units of respiratory bacteria/mL of BAL fluid. Concordance was determined by phenotype or genotype. RESULTS NP carriage of Streptococcus pneumoniae, nontypable Haemophilus influenzae (NTHi), and Moraxella catarrhalis, and lower airway infection by NTHi (47% vs 3%), were detected significantly more often in the children with bronchiectasis than in those without this condition. BAL specimens from the infected Indigenous children also showed greater strain diversity (71% vs 0%). Strain concordance in NP and BAL cultures was high in both infected subgroups. CONCLUSIONS The high density and diversity of respiratory bacteria, along with strain concordance between upper and lower airways, found in Indigenous children with bronchiectasis suggest a possible pathogenic role of recurrent aspiration of NP secretions.

Effect of health promotion and fluoride varnish on dental caries among Australian Aboriginal children: results from a community-randomized controlled trial

Objectives We tested a dental health program in remote Aboriginal communities of Australias Northern Territory, hypothesizing that it would reduce dental caries in preschool children. Methods In this 2-year, prospective, cluster-randomized, concurrent controlled, open trial of the dental health program compared to no such program, 30 communities were allocated at random to intervention and control groups. All residents aged 18–47 months were invited to participate. Twice per year for 2 years in the 15 intervention communities, fluoride varnish was applied to childrens teeth, water consumption and daily tooth cleaning with toothpaste were advocated, dental health was promoted in community settings, and primary health care workers were trained in preventive dental care. Data from dental examinations at baseline and after 2 years were used to compute net dental caries increment per child (d3mfs). A multi-level statistical model compared d3mfs between intervention and control groups with adjustment for the clustered randomization design; four other models used additional variables for adjustment. Results At baseline, 666 children were examined; 543 of them (82%) were re-examined 2 years later. The adjusted d3mfs increment was significantly lower in the intervention group compared to the control group by an average of 3.0 surfaces per child (95% CI = 1.2, 4.9), a prevented fraction of 31%. Adjustment for additional variables yielded caries reductions ranging from 2.3 to 3.5 surfaces per child and prevented fractions of 24–36%. Conclusions These results corroborate findings from other studies where fluoride varnish was efficacious in preventing dental caries in young children.

Molecular surveillance of true nontypeable Haemophilus influenzae: An evaluation of PCR screening assays

Background Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Methodology/Principal Findings Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Conclusions/Significance Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

Carriage of multiple ribotypes of non-encapsulated Haemophilus influenzae in Aboriginal infants with otitis media

Ribotyping with the restriction enzyme XbaI was used to study the dynamics of Carriage of non-encapsulated Haemophilus influenzae (NCHi) in Aboriginal infants at risk of otitis media. Carriage rates of NCHi in the infants in the community were very high; the median age for detection was 50 days and colonization was virtually 100% by 120 days of age and persisted at a high level throughout the first year of life [1]. Eighteen different ribotypes of NCHi were identified from 34 positive swabs taken from 3 infants over a period of 9 months. The same ribotypes were recovered for up to 3 months from consecutive swabs of individual infants, and 12 of 27 swabs (44.4%) yielded two ribotypes from four colonies typed. Statistical analysis suggested that most swabs would have been positive for two ribotypes if enough colonies had been typed although the second most frequent ribotype was detected on average in only 13% of strains. Early colonization and carriage of multiple ribotypes of NCHi may help to explain the chronicity of carriage and thus the persistence of otitis media in Aboriginal infants.

Crowding and other strong predictors of upper respiratory tract carriage of otitis media-related bacteria in Australian aboriginal and non-aboriginal children

Background: Streptococcus pneumoniae, Moraxella catarrhalis, and nontypeable Haemophilus influenzae is associated with otitis media (OM). Data are limited on risk factors for carriage of these pathogens, particularly for Indigenous populations. We investigated predictors of nasopharyngeal carriage in Australian Aboriginal and non-Aboriginal children. Methods: Nasopharyngeal aspirates were collected up to 7 times before age 2 years from 100 Aboriginal and 180 non-Aboriginal children. Longitudinal modeling estimated effects of environmental factors and concurrent carriage of other bacteria on the probability of bacterial carriage. We present a novel method combining the effects of number of household members and size of house into an overall crowding model. Results: Each additional household member increased the risk of carriage of S. pneumoniae (odds ratio [OR] = 1.45 per additional Aboriginal child in a 4-room house, 95% confidence interval [CI]: 1.15–1.84; OR = 2.34 per additional non-Aboriginal child, 95% CI: 1.76–3.10), with similar effect sizes for M. catarrhalis, and nontypeable Haemophilus influenzae. However, living in a larger house attenuated this effect among Aboriginal children. Daycare attendance predicted carriage of the 3 OM-associated bacteria among non-Aboriginal children. Exclusive breast-feeding at 6 to 8 weeks protected against Streptococcus aureus carriage (OR = 0.42, 95% CI: 0.19–0.90 in Aboriginal children and OR = 0.49, 95% CI: 0.25–0.96 in non-Aboriginal children). OM-associated bacteria were more likely to be present if there was concurrent carriage of the other OM-associated species. Conclusions: This study highlights the importance of household transmission in carriage of OM bacteria, underscoring the need to reduce the crowding in Aboriginal households.