Nasopharyngeal carriage of otitis media pathogens in infants receiving 10-valent non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV10), 13-valent pneumococcal conjugate vaccine (PCV13) or a mixed primary schedule of both vaccines: A randomised controlled trial.

BACKGROUND: Aboriginal children in Northern Australia have a high burden of otitis media, driven by early and persistent nasopharyngeal carriage of otopathogens, including non-typeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae (Spn). In this context, does a combined mixed primary series of Synflorix and Prevenar13 provide better protection against nasopharyngeal carriage of NTHi and Spn serotypes 3, 6A and 19A than either vaccine alone? METHODS: Aboriginal infants (n = 425) were randomised to receive Synflorix™ (S, PHiD-CV10) or Prevenar13™ (P, PCV13) at 2, 4 and 6 months (_SSS or _PPP, respectively), or a 4-dose early mixed primary series of PHiD-CV10 at 1, 2 and 4 months and PCV13 at 6 months of age (SSSP). Nasopharyngeal swabs were collected at 1, 2, 4, 6 and 7 months of age. Swabs of ear discharge were collected from tympanic membrane perforations. FINDINGS: At the primary endpoint at 7 months of age, the proportion of nasopharyngeal (Np) swabs positive for PCV13-only serotypes 3, 6A, or 19A was 0%, 0.8%, and 1.5% in the _PPP, _SSS, and SSSP groups respectively, and NTHi 55%, 52%, and 52% respectively, and no statistically significant vaccine group differences in other otopathogens at any age. The most common serotypes (in order) were 16F, 11A, 10A, 7B, 15A, 6C, 35B, 23B, 13, and 15B, accounting for 65% of carriage. Ear discharge swabs (n = 108) were culture positive for NTHi (52%), S. aureus (32%), and pneumococcus (20%). CONCLUSIONS: Aboriginal infants experience nasopharyngeal colonisation and tympanic membrane perforations associated with NTHi, non-PCV13 pneumococcal serotypes and S. aureus in the first months of life. Nasopharyngeal carriage of pneumococcus or NTHi was not significantly reduced in the early 4-dose combined SSSP group compared to standard _PPP or _SSS schedules at any time point. Current pneumococcal conjugate vaccine formulations do not offer protection from early onset NTHi and pneumococcal colonisation in this high-risk population.

Hospital surveillance predicts community pneumococcal antibiotic resistance in Vietnam.

BACKGROUND: In Vietnam, Streptococcus pneumoniae is a leading cause of disease, including meningitis. Antibiotics are available without physician prescription at community pharmacies and rates of antibiotic non-susceptibility are high. Appropriate treatment and antibiotic stewardship need to be informed by surveillance data. OBJECTIVES: To report community-based pneumococcal antibiotic susceptibility testing data from children enrolled in a pneumococcal conjugate vaccine trial in Ho Chi Minh City [the Vietnam Pneumococcal Project (ViPP)] and compare these with published hospital-based data from the nationwide Survey of Antibiotic Resistance (SOAR) to determine whether hospital surveillance data provide an informative estimate of circulating pneumococcal resistance. METHODS: Pneumococcal isolates from 234 nasopharyngeal swabs collected from ViPP participants at 12 months of age underwent antibiotic susceptibility testing using CLSI methods and the data were compared with SOAR data. RESULTS: Antibiotic susceptibility testing identified penicillin-non-susceptible pneumococci in 93.6% of pneumococcus-positive ViPP swabs (oral, non-meningitis breakpoints). Non-susceptibility to erythromycin, trimethoprim/sulfamethoxazole, clindamycin and tetracycline also exceeded 79%. MDR, defined as non-susceptibility to three or more classes of antibiotic, was common (94.4% of swabs). Low or no resistance was detected for ceftriaxone (non-meningitis breakpoints), ofloxacin and vancomycin. Antibiotic non-susceptibility rates in ViPP and SOAR were similar for several antibiotics tested. CONCLUSIONS: A very high proportion of pneumococci carried in the community are MDR. Despite wide disparities in population demographics between ViPP and SOAR, the non-susceptibility rates for several antibiotics were comparable. Thus, with some qualification, hospital antibiotic susceptibility testing data in Vietnam can inform circulating pneumococcal antibiotic non-susceptibility in young children, the group at highest risk of pneumococcal disease, to guide antibiotic prescribing and support surveillance strategies.

The microbiota in bronchoalveolar lavage from young children with chronic lung disease includes taxa present in both the oropharynx and nasopharynx.

BACKGROUND: Invasive methods requiring general anaesthesia are needed to sample the lung microbiota in young children who do not expectorate. This poses substantial challenges to longitudinal study of paediatric airway microbiota. Non-invasive upper airway sampling is an alternative method for monitoring airway microbiota; however, there are limited data describing the relationship of such results with lung microbiota in young children. In this study, we compared the upper and lower airway microbiota in young children to determine whether non-invasive upper airway sampling procedures provide a reliable measure of either lung microbiota or clinically defined differences. RESULTS: The microbiota in oropharyngeal (OP) swabs, nasopharyngeal (NP) swabs and bronchoalveolar lavage (BAL) from 78 children (median age 2.2 years) with and without lung disease were characterised using 16S rRNA gene sequencing. Permutational multivariate analysis of variance (PERMANOVA) detected significant differences between the microbiota in BAL and those in both OP swabs (p = 0.0001, Pseudo-F = 12.2, df = 1) and NP swabs (p = 0.0001; Pseudo-F = 21.9, df = 1) with the NP and BAL microbiota more different than the OP and BAL, as indicated by a higher Pseudo-F value. The microbiota in combined OP and NP data (upper airways) provided a more comprehensive representation of BAL microbiota, but significant differences between the upper airway and BAL microbiota remained, albeit with a considerably smaller Pseudo-F (PERMANOVA p = 0.0001; Pseudo-F = 4.9, df = 1). Despite this overall difference, paired BAL and upper airway (OP and NP) microbiota were >50 % similar among 69 % of children. Furthermore, canonical analysis of principal coordinates (CAP analysis) detected significant differences between the microbiota from clinically defined groups when analysing either BAL (eigenvalues >0.8; misclassification rate 26.5 %) or the combined OP and NP data (eigenvalues >0.8; misclassification rate 12.2 %). CONCLUSIONS: Upper airway sampling provided an imperfect, but reliable, representation of the BAL microbiota for most children in this study. We recommend inclusion of both OP and NP specimens when non-invasive upper airway sampling is needed to assess airway microbiota in young children who do not expectorate. The results of the CAP analysis suggest lower and upper airway microbiota profiles may differentiate children with chronic suppurative lung disease from those with persistent bacterial bronchitis; however, further research is needed to confirm this observation.

Haemophilus influenzae isolates survive for up to 20 years at -70 °C in skim milk tryptone glucose glycerol broth (STGGB) if thawing is avoided during re-culture.

Haemophilus influenzae remains a major cause of disease worldwide requiring continued study. Recently, isolates of Streptococcus pneumoniae and Moraxella catarrhalis, but not H. influenzae, were reported to survive long-term ultra-freeze storage in STGGB. We show that nontypeable H. influenzae isolates survive for up to 20 years when thawing is avoided.

Nasopharyngeal carriage and macrolide resistance in Indigenous children with bronchiectasis randomized to long-term azithromycin or placebo.

Although long-term azithromycin decreases exacerbation frequency in bronchiectasis, increased macrolide resistance is concerning. We investigated macrolide resistance determinants in a secondary analysis of a multicenter randomized controlled trial. Indigenous Australian children living in remote regions and urban New Zealand Maori and Pacific Islander children with bronchiectasis were randomized to weekly azithromycin (30 mg/kg) or placebo for up to 24 months and followed post-intervention for up to 12 months. Nurses administered and recorded medications given and collected nasopharyngeal swabs 3-6 monthly for culture and antimicrobial susceptibility testing. Nasopharyngeal carriage of Haemophilus influenzae and Moraxella catarrhalis was significantly lower in azithromycin compared to placebo groups, while macrolide-resistant Streptococcus pneumoniae and Staphylococcus aureus carriage was significantly higher. Australian children, compared to New Zealand children, had higher carriage overall, significantly higher carriage of macrolide-resistant bacteria at baseline (16/38 versus 2/40 children) and during the intervention (69/152 versus 22/239 swabs), and lower mean adherence to study medication (63 % versus 92 %). Adherence ≥70 % (versus <70 %) in the Australian azithromycin group was associated with lower carriage of any pathogen [odds ratio (OR) 0.19, 95 % confidence interval (CI) 0.07-0.53] and fewer macrolide-resistant pathogens (OR 0.34, 95 % CI 0.14-0.81). Post-intervention (median 6 months), macrolide resistance in S. pneumoniae declined significantly in the azithromycin group, from 79 % (11/14) to 7 % (1/14) of positive swabs, but S. aureus strains remained 100 % macrolide resistant. Azithromycin treatment, the Australian remote setting, and adherence <70 % were significant independent determinants of macrolide resistance in children with bronchiectasis. Adherence to treatment may limit macrolide resistance by suppressing carriage.

Respiratory bacterial culture from two sequential bronchoalveolar lavages of the same lobe in children with chronic cough.

Identification of bacteria causing lower-airway infections is important to determine appropriate antimicrobial therapy. Flexible bronchoscopy with bronchoalveolar lavage (BAL) is used to obtain lower-airway specimens in young children. The first lavage (lavage-1) is typically used for bacterial culture. However, no studies in children have compared the detection of cultivable bacteria from sequential lavages of the same lobe. BAL fluid was collected from two sequential lavages of the same lobe in 79 children enrolled in our prospective studies of chronic cough. The respiratory bacteria Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Haemophilus parainfluenzae were isolated and identified using standard published methods. H. influenzae was differentiated from Haemophilus haemolyticus using PCR assays. Lower-airway infection was defined as ≥ 104 c.f.u. ml- 1 BAL fluid. We compared cultivable bacteria from lavage-1 with those from the second lavage (lavage-2) using the κ statistic. Lower-airway infections by any pathogen were detected in 46% of first lavages and 39% of second lavages. Detection was similar in both lavages for all pathogens; the κ statistic was 0.7-0.8 for all bacteria except H. parainfluenzae. Of all infections detected in either lavage, 90% were detected in lavage-1 and 78 in lavage-2. However, culture of lavage-2 identified infections that would have been missed in 8% of children, including infections by additional Streptococcus pneumoniae serotypes. Our findings support the continued use of lavage-1 for bacterial culture; however, culture of lavage-2 may yield additional identifications of bacterial pathogens in lower-airway infections.

Ubiquity of putative type III secretion genes among clinical and environmental Burkholderia pseudomallei isolates in Northern Australia.

Type III secretion (TTSI) genes of an HRP (hypersensitivity response and pathogenicity)-like locus were present in all 116 Northern Australian Burkholderia pseudomallei isolates tested but were not detected in other common environmental Burkholderia species. PCR of TTS1 genes may prove valuable as a diagnostic test [corrected].

Modelling of endemic carriage of Haemophilus influenzae in Aboriginal infants in Northern Australia.

Aboriginal infants and children in rural communities in Northern Australia have high rates of nasopharyngeal carriage of nonencapsulated Haemophilus influenzae (NCHi), with positive swab rates of 76%. In this population, the acquisition of NCHi from soon after birth is associated with the onset of otitis media and with muco-purulent nasal discharge, while the long-term persistence of NCHi carriage is associated with the acquisition and turnover of large numbers of antigenically diverse strains. Mathematical models have been fitted to data on the acquisition and loss of encapsulated strains of H. influenzae and 43 different strains of NCHi in 10 children followed from early infancy for up to 2 years. Subject to plausible assumptions, the preferred model estimated the mean time to acquisition of a H. influenzae strain to be 7 days after first becoming exposed after birth. For an infant already carrying H. influenzae, each additional strain was acquired after a mean waiting period of 45 days. On average, 1.50 different strains of H. influenzae were detected in four colonies routinely typed from each positive swab, but it was estimated that another 2.55 strains were 'hidden' behind these more frequent strains. With an average of 4.05 strains per carrier, it was estimated that each strain was carried for an average of 137 days, although detected on only 37% of occasions. Thus we have developed mathematical models that provide estimates for duration of colonisation, time to colonisation, and number of colonising strains in a population in which H. influenzae is highly endemic, characterised by sequential and concurrent carriage of multiple strains in each infant.

Low genetic diversity of Haemophilus influenzae type b compared to nonencapsulated H. influenzae in a population in which H. influenzae is highly endemic.

Immunization with Haemophilus influenzae type b (Hib) conjugate polysaccharide vaccines has dramatically reduced Hib disease worldwide. As in other populations, nasopharyngeal carriage of Hib declined markedly in Aboriginal infants following vaccination, although carriage has not been entirely eliminated. In this study, we describe the genetic characteristics and the carriage dynamics of longitudinal isolates of Hib, characterized by using several typing methods. In addition, carriage rates of nonencapsulated H. influenzae (NCHi) are high, and concurrent colonization with Hib and NCHi is common; we also observed NCHi isolates which were genetically similar to Hib. There is a continuing need to promote Hib immunization and monitor H. influenzae carriage in populations in which the organism is highly endemic, not least because of the possibility of genetic exchange between Hib and NCHi strains in such populations.

Nonencapsulated Haemophilus influenzae in Aboriginal infants with otitis media: prolonged carriage of P2 porin variants and evidence for horizontal P2 gene transfer.

Aboriginal infants in the Northern Territory of Australia experience recurrent otitis media from an early age. Nonencapsulated Haemophilus influenzae (NCHi) colonization of the nasopharynx initially occurs within weeks of birth, persists throughout infancy and most of childhood, and contributes to otitis media. We established previously that the high carriage rates of NCHi in these infants result from concurrent and successive colonization with multiple strains, with sequential elimination of dominant strains. We have now sequenced loops 4, 5, and 6 of the NCHi P2 porin gene and characterized several strains with prolonged carriage times. Furthermore, despite a wide diversity of P2 gene sequences, we have four examples of P2 gene identity for strains with different genetic backgrounds as characterized by PCR ribotyping and randomly amplified polymorphic DNA typing, which leads us to suggest that the P2 gene has been transferred between strains. We also discuss the possibility that the paradoxical observation of cocolonization and prolonged carriage of P2-identical strains is related to immune suppression or tolerance in the host.

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.

Long PCR-ribotyping of nontypeable Haemophilus influenzae.

PCR-ribotyping, a new typing method based on long PCR, has been developed for nontypeable Haemophilus influenzae (NTHi). Ribosomal operons of NTHi were amplified by long PCR and were found to be highly polymorphic for internal HaeIII sites. The technique was applied to 49 isolates previously subjected to conventional ribotyping, and the two methods showed a high level of concordance for serial isolates from individual subjects. PCR-ribotyping provides a powerful new typing tool for strain characterization in epidemiological investigations of NTHi.