Otitis media at 6-monthly assessments of Australian First Nations children between ages 12-36 months: Findings from two randomised controlled trials of combined pneumococcal conjugate vaccines.

OBJECTIVES: In remote communities of northern Australia, First Nations children with hearing loss are disproportionately at risk of poor school readiness and performance compared to their peers with no hearing loss. The aim of this trial is to prevent early childhood persisting otitis media (OM), associated hearing loss and developmental delay. To achieve this, we designed a mixed pneumococcal conjugate vaccine (PCV) schedule that could maximise immunogenicity and thereby prevent bacterial otitis media (OM) and a trajectory of educational and social disadvantage. METHODS: In two sequential parallel, open-label, randomised controlled trials, eligible infants were first allocated 1:1:1 to standard or mixed PCV primary schedules at age 28-38 days, then at age 12 months to a booster dose (1:1) of 13-valent PCV, PCV13 (Prevenar13®, +P), or 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugated vaccine, PHiD-CV10 (Synflorix®, +S). Here we report findings of standardised ear assessments conducted six-monthly from age 12-36 months, by booster dose. RESULTS: From March 2013 to September 2018, 261 children were allocated to booster + P (n = 131) or + S (n = 130). There were no significant differences in prevalence of any OM diagnosis by booster dose or when stratified by primary schedule. We found high, almost identical prevalence of OM in both boost groups at each age (for example 88% of 129 and 91% of 128 children seen, respectively, at primary endpoint age 18 months, difference -3% [95% Confidence Interval -11, 5]). At each age prevalence of bilateral OM was 52%-78%, and tympanic membrane perforation was 10%-18%. CONCLUSION: Despite optimal pneumococcal immunisation, the high prevalence of OM persists throughout early childhood. Novel approaches to OM prevention are needed, along with improved early identification strategies and evaluation of expanded valency PCVs.

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.

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.

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.

Diversity of nontypeable Haemophilus influenzae strains colonizing Australian Aboriginal and non-Aboriginal children.

Nontypeable Haemophilus influenzae (NTHI) strains are responsible for respiratory-related infections which cause a significant burden of disease in Australian children. We previously identified a disparity in NTHI culture-defined carriage rates between Aboriginal and non-Aboriginal children (42% versus 11%). The aim of this study was to use molecular techniques to accurately determine the true NTHI carriage rates (excluding other culture-identical Haemophilus spp.) and assess whether the NTHI strain diversity correlates with the disparity in NTHI carriage rates. NTHI isolates were cultured from 595 nasopharyngeal aspirates collected longitudinally from asymptomatic Aboriginal (n=81) and non-Aboriginal (n=76) children aged 0 to 2 years living in the Kalgoorlie-Boulder region, Western Australia. NTHI-specific 16S rRNA gene PCR and PCR ribotyping were conducted on these isolates. Confirmation of NTHI by 16S rRNA gene PCR corrected the NTHI carriage rates from 42% to 36% in Aboriginal children and from 11% to 9% in non-Aboriginal children. A total of 75 different NTHI ribotypes were identified, with 51% unique to Aboriginal children and 13% unique to non-Aboriginal children (P<0.0001). The strain richness (proportion of different NTHI ribotypes) was similar for Aboriginal (19%, 65/346) and non-Aboriginal children (19%, 37/192) (P=0.909). Persistent carriage of the same ribotype was rare in the two groups, but colonization with multiple NTHI strains was more common in Aboriginal children than in non-Aboriginal children. True NTHI carriage was less than that estimated by culture. The Aboriginal children were more likely to carry unique and multiple NTHI strains, which may contribute to the chronicity of NTHI colonization and subsequent disease.

Impact of recent antibiotics on nasopharyngeal carriage and lower airway infection in Indigenous Australian children with non-cystic fibrosis bronchiectasis.

Indigenous Australian children have increased rates of bronchiectasis. Despite a lack of high-level evidence on effectiveness and antibiotic resistance, these children often receive long-term antibiotics. In this study, we determined the impact of recent macrolide (primarily azithromycin) and ß-lactam antibiotic use on nasopharyngeal colonisation, lower airway infection (>10(4) CFU/mL of bronchoalveolar lavage fluid culture) and antibiotic resistance in non-typeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis isolates from 104 Indigenous children with radiographically confirmed bronchiectasis. Recent antibiotic use was associated with significantly reduced nasopharyngeal carriage, especially of S. pneumoniae in 39 children who received macrolides [odds ratio (OR)=0.22, 95% confidence interval (CI) 0.08-0.63] and 26 children who received ß-lactams (OR=0.07, 95% CI 0.01-0.32), but had no significant effect on lower airway infection involving any of the three pathogens. Children given macrolides were significantly more likely to carry (OR=4.58, 95% CI 1.14-21.7) and be infected by (OR=8.13, 95% CI 1.47-81.3) azithromycin-resistant S. pneumoniae. Children who received ß-lactam antibiotics may be more likely to have lower airway infection with ß-lactamase-positive ampicillin-resistant NTHi (OR=4.40, 95% CI 0.85-23.9). The risk of lower airway infection by antibiotic-resistant pathogens in children receiving antibiotics is of concern. Clinical trials to determine the overall benefit of long-term antibiotic therapy are underway.

Culture and PCR detection of Haemophilus influenzae and Haemophilus haemolyticus in Australian Indigenous children with bronchiectasis.

A PCR for protein D (hpd#3) was used to differentiate nontypeable Haemophilus influenzae (NTHI) from Haemophilus haemolyticus. While 90% of nasopharyngeal specimens and 100% of lower-airway specimens from 84 Indigenous Australian children with bronchiectasis had phenotypic NTHI isolates confirmed as H. influenzae, only 39% of oropharyngeal specimens with phenotypic NTHI had H. influenzae. The nasopharynx is therefore the preferred site for NTHI colonization studies, and NTHI is confirmed as an important lower-airway pathogen.

The nonserotypeable pneumococcus: phenotypic dynamics in the era of anticapsular vaccines.

Nonserotypeable pneumococci (NSP) are commonly carried by Australian Indigenous children in remote communities. The purpose of this study was to characterize carriage isolates of NSP from Indigenous children vaccinated with the seven-valent pneumococcal conjugate vaccine (PCV7) and to use these data to guide decisions on reporting of NSP. A total of 182 NSP were characterized by BOX typing, antibiogram analysis, and multilocus sequence typing (MLST) of common BOX types. NSP positive for the wzg capsule gene were analyzed by a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB-H) targeting capsule genes to determine the serotype. Among 182 NSP, 49 BOX types were identified. MLST of 10 representative isolates found 7 STs, including ST448 (which accounted for 11% of NSP). Non-penicillin susceptibility was evident in 51% of the isolates. Pneumococcal wzg sequences were detected in only 23 (13%) NSP, including 10 that contained an approximately 1.2-kb insert in the region. mPCR/RLB-H identified serotype 14 wzy sequences in all 10 NSP, and 1 also contained a serotype 3-specific wze sequence. Among the remaining 13 wzg-positive NSP, few belonged to the serotypes represented in PCV7. It appears that most NSP identified in Australian Indigenous children are from a true nonencapsulated lineage. Few NSP represented serotypes in PCV7 that suppress capsular expression. High rates of carriage and penicillin resistance and the occasional presence of capsule genes suggest a role for NSP in the maintenance and survival of capsulated pneumococci. To avoid the inflation of pneumococcal carriage and antibiotic resistance rates, in clinical trials, we recommend separate reporting of rates of capsular strains and NSP and the exclusion of data for NSP from primary analyses.

Age-specific cluster of cases of serotype 1 Streptococcus pneumoniae carriage in remote indigenous communities in Australia.

Seven-valent pneumococcal conjugate vaccination commenced in 2001 for Australian indigenous infants. Pneumococcal carriage surveillance detected substantial replacement with nonvaccine serotypes and a cluster of serotype 1 carriage. Our aim was to review Streptococcus pneumoniae serotype 1 carriage and invasive pneumococcal disease (IPD) data for this population and to analyze serotype 1 isolates. Carriage data were collected between 1992 and 2004 in the Darwin region, one of the five regions in the Northern Territory. Carriage data were also collected in 2003 and 2005 from four regions in the Northern Territory. Twenty-six cases of serotype 1 IPD were reported from 1994 to 2007 in the Northern Territory. Forty-four isolates were analyzed by BOX typing and 11 by multilocus sequence typing. In the Darwin region, 26 children were reported carrying serotype 1 (ST227) in 2002 but not during later surveillance. Scattered cases of serotype 1 carriage were noted in two other regions. Cocolonization of serotype 1 with other pneumococcal serotypes was common (34% serotype 1-positive swabs). In conclusion, pneumococcal carriage studies detected intermittent serotype 1 carriage and an ST227 cluster in children in indigenous communities in the Northern Territory of Australia. There was no apparent increase in serotype 1 IPD during this time. The rate of serotype 1 cocolonization with other pneumococcal serotypes suggests that carriage of this serotype may be underestimated.

Molecular characterisation of pneumococcal serotype 16F: Established predominant carriage and otitis media serotype in the 7vPCV era.

Young Australian Aboriginal children in remote communities experience very high rates of pneumococcal carriage and otitis media. Prior to introduction of the 7-valent pneumococcal conjugate vaccine (7vPCV, Prevenar), serotype 16F was an important type found in nasal and ear discharge swabs. Since commencement of pneumococcal immunisation for Aboriginal infants in 2001, 16F has become the predominant established serotype in carriage and otitis media in young Aboriginal children. BOX typing and multi-locus sequence typing revealed a diverse population of serotype 16F strains, and evidence of potential capsule switching from a vaccine serotype 4 to a serotype 16F.

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.