Emerging pneumococcal carriage serotypes in a high-risk population receiving universal 7-valent pneumococcal conjugate vaccine and 23-valent polysaccharide vaccine since 2001

Background In Australia in June 2001, a unique pneumococcal vaccine schedule commenced for Indigenous infants; seven-valent pneumococcal conjugate vaccine (7PCV) given at 2, 4, and 6 months of age and 23-valent pneumococcal polysaccharide vaccine (23PPV) at 18 months of age. This study presents carriage serotypes following this schedule. Methods We conducted cross sectional surveys of pneumococcal carriage in Aboriginal children 0 to 6 years of age living in remote Aboriginal communities (RACs) in 2003 and 2005. Nasal secretions were collected and processed according to published methods. Results 902 children (mean age 25 months) living in 29 communities in 2003 and 818 children (mean age 35 months) in 17 communities in 2005 were enrolled. 87% children in 2003 and 96% in 2005 had received two or more doses of 7PCV. From 2003 to 2005, pneumococcal carriage was reduced from 82% to 76% and reductions were apparent in all age groups; 7PCV-type carriage was reduced from 11% to 8%, and 23PPV-non-7PCV-type carriage from 31% to 25% respectively. Thus non-23PPV-type carriage increased from 57% to 67%. All these changes were statistically significant, as were changes for some specific serotypes. Shifts could not be attributed to vaccination alone. The top 10 of 40 serotypes identified were (in descending order) 16F, 19A, 11A, 6C, 23B, 19F, 6A, 35B, 6B, 10A and 35B. Carriage of penicillin non-susceptible (MIC > = 0.12 μg/mL) strains (15% overall) was detected in serotypes (descending order) 19A, 19F, 6B, 16F, 11A, 9V, 23B, and in 4 additional serotypes. Carriage of azithromycin resistant (MIC > = 2 μg/mL) strains (5% overall), was detected in serotypes (descending order) 23B, 17F, 9N, 6B, 6A, 11A, 23F, and in 10 additional serotypes including 6C. Conclusion Pneumococcal carriage remains high (~80%) in this vaccinated population. Uptake of both pneumococcal vaccines increased, and carriage was reduced between 2003 and 2005. Predominant serotypes in combined years were 16F, 19A, 11A, 6C and 23B. Antimicrobial non-susceptibility was detected in these and 17 additional serotypes. Shifts in serotype-specific carriage suggest a need more research to clarify the association between pneumococcal vaccination and carriage at the serotype level.


Conclusion:
Pneumococcal carriage remains high (~80%) in this vaccinated population. Uptake of both pneumococcal vaccines increased, and carriage was reduced between 2003 and 2005. Predominant serotypes in combined years were 16F, 19A, 11A, 6C and 23B. Antimicrobial nonsusceptibility was detected in these and 17 additional serotypes. Shifts in serotype-specific carriage suggest a need more research to clarify the association between pneumococcal vaccination and carriage at the serotype level.

Background
Australian Aboriginal children in remote communities experience high rates of many infectious diseases [1,2], particularly pneumococcal disease. [3] Concern about antibiotic resistance is used as an argument against greater use of antibiotics. Recent reports describe escalating rates of disease caused by pneumococcal serotypes not included in currently licensed pneumococcal conjugate vaccine (7PCV), [3,4]including an increasing proportion of isolates having antibiotic resistance. [5][6][7] Serotype 19A is of particular interest in Australia [3] and internationally. [8] More recent data suggests 6C as a new and emerging replacement serotype, and that 7PCV is crossprotective for 6A. Prior to 7PCV introduction in 2001, [9] pneumococcal carriage in Indigenous infants was around 80% and approximately 60% were 7PCV serotypes (unpublished data from 3 of the communities also in these surveys). In 2003 and 2005 we conducted wider geographic surveillance of pneumococcal carriage.

Vaccine schedule
From July 2001 7PCV was recommended and funded for Australian Aboriginal and other high-risk infants at 2, 4 and 6 months of age. In the Northern Territory and in South Australia, 23-valent pneumococcal polysaccharide vaccine (23PPV) was recommended as a booster dose for Aboriginal children at 18 months of age. [9] Children less than 2 years of age were offered catch-up 7PCV.

Study setting and participant eligibility criteria
Participating communities were in four regions of South Australia and the Northern Territory. Children were eligible if they were Aboriginal, 0 to 6 years of age, resident in a participating community, and if their parents or caregiver provided written consent.

Swab collection, antibiotic use and vaccination history
Nasal secretions were collected using swabs or nose blowing into a tissue as previously described. [10,11] Pneumo-coccal vaccination dates and antibiotics prescribed in the 5 weeks prior to swab collection were obtained from the NT childhood immunization register and participant medical records.

Microbiological analysis
Nasal secretions were transported, stored and processed as previously described. [10] Pneumococci were identified and one or more colonies (if morphologically distinct) selected for serotyping with antisera from the Statens Serum Institute (Denmark). Antimicrobial susceptibility was determined by disc diffusion [12] and Etest (AB Biodisk, Sweden). We performed PCR for the wciN gene. [13] Statistical analysis Confidence intervals (CI, 95%) and risk differences (RD, with 95% CI) were calculated where appropriate. Stata version 10 was used for all data analysis [14] Ethical Approval and Funding The study was approved by the regional Human Research Ethics Committees and Aboriginal Sub-Committee with right of veto of the Northern Territory Department of Health and Community Services and Menzies School of Health Research. The study was funded by the National Health and Medical Research Council.

Participant characteristics
Nasal secretions were collected from 902 children in 29 communities in 2003 and from 818 children in 17 communities in 2005 (Table 1). Nasal swabs comprised 94% specimens. Mean age was 30 months. 83 were less than 6 months of age.

Antibiotic use in the 5 weeks prior to swab collection
Beta-lactam antibiotics had been recently prescribed (within 5 weeks prior to swabbing) for 14% children in 2003 and for 16% in 2005 (Tables 2 and 3). In general, macrolides were rarely recently prescribed with the exception of 48 of 396 (13%) children the Darwin Rural region (Table 3). Of these 48 children, 45 were from one community and had been prescribed azithromycin for trachoma eradication.

Discussion
Our survey of 1720 young children in remote communities two and four years after commencement of a government funded schedule for Indigenous children of 7PCV at 2, 4 and 6 months followed by a booster 23PPV at 18 months of age, provides a valuable source of pneumococcal serotype and susceptibility data for the region. Two major influences have had the potential to impact on pneumococcal carriage in this population; pneumococcal vaccination and antimicrobial prescribing. In three communities studied extensively in the 1990s, around 50% children carried 7PCV serotypes. Predominant pre-7PCV serotypes were 6B, 19F, 23F, 19A, 16F and 6A (unpublished data from 3 of the communities also in these surveys). Retrospective PCR analysis confirms one of 20 pre-7PCV 6A isolates was 6C. [15] A similar serotype distribution was found in cultures of discharge from acute perforations of the tympanic membrane. [16] In general, this survey found small differences between 2003 and 2005. Vaccine uptake (at least 2 doses of 7PCV) was around 87% in 2003 and increased to 96% in 2005. By 2005, 84% age-eligible children had received 23PPV. Between 2003 and 2005 we detected small but statistically significant reductions in pneumococcal carriage (-6%). This trend was consistent across all age groups. Similarly for 7PCV types (-4%). There was a small reduction in 6A.
Among the group of children not vaccinated, 19A, 16F and 6A and 6C were the predominant serotypes and 7PCV type carriage was less than 10%, indicating a herd effect. Similarly, in very young non-vaccinated infants (mean age 1.9 months) carriage was around 58% and only 8% carried 7PCV vaccine types. Serotypes were diverse and no dominant emerging serotype was evident in this young group.
There is no strong evidence from 23PPV studies to suggest that 23PPV has an impact on NP carriage [17] Our immunogenicity study [18] showed 23PPV to be an effective booster for 7PCV-primed serotypes, and that modest responses to some additional serotypes in 23PPV also occurred. Whether the antibody concentrations achieved would prevent pneumococcal acquisition in the nasopharynx was not assessed. In this survey we did detect Macrolide prescribing was low, but increased significantly between 2003 and 2005, mainly due to recent mass azithromycin use in one community. At the time of our survey in that community there was no statistically significant difference in pneumococcal carriage or carriage of azithromycin resistant strains between children recently prescribed azithromycin and those not (RD = 6% [-5, 18]). Our previous longitudinal study [19] had shown that azithromycin reduces carriage of pneumococci, providing resistant strains with a selective advantage that allows spread within the community. Over time, and in the absence of further azithromycin use or selective pressure, susceptible stains re-populate and the proportion of the population carrying resistant strains declines. [19] The present cross-sectional study does not capture this dynamic process and therefore does not allow any conclusions regarding the relationship between mass azithromycin use and carriage. In Central Australia, recent macrolide prescribing was rare, but carriage of azithromycin resistant strains was clearly higher than other regions. We suggest that this higher background of resistance may be related to a longer period of exposure to azithromycin for trachoma [20] and STI (sexually transmitted infection) [21] eradication programs, in the central Australian region. Data to support this are lacking; the 2006 Trachoma sur-veillance report identified that azithromycin treatment was recommended but was poorly documented in the Northern Territory [20] Whilst carriage prevalence does not fully predict IPD serotypes (particularly outbreak or epidemic serotypes such as serotypes 1 or 5), the majority of annual IPD cases are caused by the serotypes most prevalent in the carriage population. In Australia, rates of IPD caused by serotype 19A in Indigenous children have fluctuated over recent years and serotype 6A became the most common non-vaccine IPD serotype in Indigenous children and the second most common in non-Indigenous children. [3] Hanna [22] compared IPD serotypes in under 5 year old Indigenous children living in far North Queensland preand post-7PCV implementation. They concluded that there was no evidence of non-7PCV serotype replacement. However, the pre-vaccine non-7PCV cases were inflated by a serotype 1 outbreak. Excluding type 1 cases, there was a risk difference in non-7PCV IPD of 55% [95%CI 30,80].
Reporting of pneumococcal epidemiology needs to consider the epidemic and endemic nature of various serotypes and that serotype-specific data are needed to best describe and prepare for shifts over time and in response to vaccines and antibiotic selective pressures.
Otitis media (OM) serotypes closely align with carriage serotype profile, [23] and for Aboriginal children OM is a common and serious infection often resulting in chronic suppurative OM (CSOM). This study showed that the serotypes colonising infants at 2 months of age, and therefore the serotypes most likely causing early OM, [24] are no longer 7PCV-types. Vaccines that are effective in preventing early onset of ear disease (and pneumonia) are urgently needed.

Conclusion
Pneumococcal disease epidemiology is in a transition period in Australia. This surveillance shows that nasal carriage of 7PCV serotypes has been replaced by the serotypes that were the next most common carriage serotypes prior to 7PCV use -namely 19A and 16F. Antibiotic resistance is almost exclusively restricted to these serotypes (and remaining 7PCV serotypes) and requires monitoring at the regional level. The role of 23PPV in limiting carriage and preventing disease in children also needs further investigation. More work is needed to guide design of appropriate surveillance systems that measure serotype shifts and distinguish serotype replacement from secular trends.