Anthony D. Keil

Newly emerging clones of Bordetella pertussis carrying prn2 and ptxP3 alleles implicated in Australian pertussis epidemic in 2008-2010.

Australia is experiencing a prolonged epidemic of pertussis that began in 2008. A total of 194 Bordetella pertussis isolates collected from 2008 through 2010 were typed by single-nucleotide polymorphism (SNP) analysis, by multilocus variable number tandem repeats analysis, and by fim3, prn, and ptxP sequence analyses. Strains with 2 closely related SNP profiles carrying prn2 and ptxP3 from the recently emerged SNP cluster I predominated. The data suggest increasing selection among the B. pertussis population in Australia in favor of strains carrying prn2 and ptxP3 under the pressure of acellular vaccine-induced immunity.

Evidence-based guidelines for use of probiotics in preterm neonates

BackgroundCurrent evidence indicates that probiotic supplementation significantly reduces all-cause mortality and definite necrotising enterocolitis without significant adverse effects in preterm neonates. As the debate about the pros and cons of routine probiotic supplementation continues, many institutions are satisfied with the current evidence and wish to use probiotics routinely. Because of the lack of detail on many practical aspects of probiotic supplementation, clinician-friendly guidelines are urgently needed to optimise use of probiotics in preterm neonates.AimTo develop evidence-based guidelines for probiotic supplementation in preterm neonates.MethodsTo develop core guidelines on use of probiotics, including strain selection, dose and duration of supplementation, we primarily used the data from our recent updated systematic review of randomised controlled trials. For equally important issues including strain identification, monitoring for adverse effects, product format, storage and transport, and regulatory hurdles, a comprehensive literature search, covering the period 1966-2010 without restriction on the study design, was conducted, using the databases PubMed and EMBASE, and the proceedings of scientific conferences; these data were used in our updated systematic review.ResultsIn this review, we present guidelines, including level of evidence, for the practical aspects (for example, strain selection, dose, duration, clinical and laboratory surveillance) of probiotic supplementation, and for dealing with non-clinical but important issues (for example, regulatory requirements, product format). Evidence was inadequate in some areas, and these should be a target for further research.ConclusionWe hope that these evidence-based guidelines will help to optimise the use of probiotics in preterm neonates. Continued research is essential to provide answers to the current gaps in knowledge about probiotics.

Rapid Increase in Pertactin-deficient Bordetella pertussis Isolates, Australia

Acellular vaccines against Bordetella pertussis were introduced in Australia in 1997. By 2000, these vaccines had replaced whole-cell vaccines. During 2008–2012, a large outbreak of pertussis occurred. During this period, 30% (96/320) of B. pertussis isolates did not express the vaccine antigen pertactin (prn). Multiple mechanisms of prn inactivation were documented, including IS481 and IS1002 disruptions, a variation within a homopolymeric tract, and deletion of the prn gene. The mechanism of lack of expression of prn in 16 (17%) isolates could not be determined at the sequence level. These findings suggest that B. pertussis not expressing prn arose independently multiple times since 2008, rather than by expansion of a single prn-negative clone. All but 1 isolate had ptxA1, prn2, and ptxP3, the alleles representative of currently circulating strains in Australia. This pattern is consistent with continuing evolution of B. pertussis in response to vaccine selection pressure.

The Changing Epidemiology of Invasive Pneumococcal Disease in Aboriginal and Non-Aboriginal Western Australians from 1997 through 2007 and Emergence of Nonvaccine Serotypes

BACKGROUND. In 2001, Australia introduced a unique 7-valent pneumococcal conjugate vaccine (7vPCV) 2-, 4-, and 6-month schedule with a 23-valent pneumococcal polysaccharide vaccine (23vPPV) booster for Aboriginal children, and in 2005, 7vPCV alone in a 2-, 4-, and 6-month schedule for non-Aboriginal children. Aboriginal adults are offered 23vPPV but coverage is poor. We investigated trends in invasive pneumococcal disease (IPD) in Western Australia (WA). METHODS. Enhanced IPD surveillance has been ongoing since 1996. We calculated IPD incidence rates for Aboriginal and non-Aboriginal Australians before and after introduction of 7vPCV. RESULTS. A total of 1792 cases occurred during the period 1997-2007; the IPD incidence rate was 47 cases per 100,000 population per year among Aboriginal people and 7 cases per 100,000 population per year in non-Aboriginal people. After introduction of 7vPCV, IPD rates among Aboriginal children decreased by 46% for those <2 years of age and by 40% for those 2-4 years of age; rates decreased by 64% and 51% in equivalent age groups for non-Aboriginal children. IPD rates decreased by >30% in non-Aboriginal people 50 years of age but increased among Aboriginal adults (eg, from 59.1 to 109.6 cases per 100,000 population per year among those 30-49 years of age). Although IPD due to 7vPCV serotypes decreased in all age groups, IPD incidence due to non-7vPCV serotypes increased, and it almost doubled among Aboriginal adults 30-49 years of age (from 48.3 to 97.0 cases per 100,000 population per year). Among non-Aboriginal children, 37% of IPD is now due to serotype 19A. CONCLUSIONS. IPD incidence rates have decreased markedly among children and non-Aboriginal adults with a 3-dose infant 7vPCV schedule. However, IPD due to non-7vPCV serotypes has increased and is of particular concern among young Aboriginal adults, for whom an intensive 23vPPV campaign is needed. An immunization register covering all age groups should be established.

Vaccine Effectiveness Against Laboratory-confirmed Influenza in Healthy Young Children: A Case-Control Study.

Background: The Western Australian Influenza Vaccine Effectiveness study commenced in 2008 to evaluate a new program to provide free influenza vaccine to all children aged 6 to 59 months. We aimed to assess the protective effect of inactivated influenza vaccination in these children. Methods: We conducted a prospective case–control study in general practices and a hospital emergency department, testing all eligible patients for influenza and a range of other common respiratory viruses. Influenza vaccine effectiveness (VE) against laboratory-confirmed influenza was estimated with cases defined as children with an influenza-like illness who tested positive and controls as those with an influenza-like illness who tested negative for influenza virus. We calculated VE using the adjusted odds ratio from multivariate logistic regression. As a surrogate marker for adequate specimen collection, we explored the difference in VE point estimates defining controls as children in whom another respiratory virus was detected. Results: A total of 75 children were enrolled from general practices and 214 through the emergency department, with 12 (27%) and 36 (17%), respectively, having laboratory-confirmed influenza. Using all the influenza-negative controls, the adjusted VE was 58% (95% confidence interval, 9–81). When controls were limited to those with another virus present, the adjusted VE was 68% (95% confidence interval, 26–86). Conclusions: VE estimates were higher when controls included only those children with another respiratory virus detected. Testing for other common respiratory viruses enables the control group to be restricted to those for whom an adequate sample is likely.

Multi-species bacterial biofilm and intracellular infection in otitis media

BackgroundBacteria which are metabolically active yet unable to be cultured and eradicated by antibiotic treatment are present in the middle ear effusion of children with chronic otitis media with effusion (COME) and recurrent acute otitis media (rAOM). These observations are suggestive of biofilm presence or intracellular sequestration of bacteria and may play a role in OM pathogenesis. The aim of this project is to provide evidence for the presence of otopathogenic bacteria intracellularly or within biofilm in the middle ear mucosa of children with COME or rAOM.MethodsMiddle ear mucosal biopsies from 20 children with COME or rAOM were examined for otopathogenic bacteria (either in biofilm or located intracellularly) using transmission electron microscopy (TEM) or species specific fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). One healthy control biopsy from a child undergoing cochlear implant surgery was also examined.ResultsNo bacteria were observed in the healthy control sample. In 2 of the 3 biopsies imaged using TEM, bacteria were observed in mucus containing vacuoles within epithelial cells. Bacterial species within these could not be identified and biofilm was not observed. Using FISH with CLSM, bacteria were seen in 15 of the 17 otitis media mucosal specimens. In this group, 11 (65%) of the 17 middle ear mucosal biopsies showed evidence of bacterial biofilm and 12 demonstrated intracellular bacteria. 52% of biopsies were positive for both biofilm and intracellular bacteria. At least one otopathogen was identified in 13 of the 15 samples where bacteria were present. No differences were observed between biopsies from children with COME and those with rAOM.ConclusionUsing FISH and CLSM, bacterial biofilm and intracellular infection with known otopathogens are demonstrated on/in the middle ear mucosa of children with COME and/or rAOM. While their role in disease pathogenesis remains to be determined, this previously undescribed infection pattern may help explain the ineffectiveness of current treatment strategies at preventing or resolving COME or rAOM.

The role of chronic infection in children with otitis media with effusion: Evidence for intracellular persistence of bacteria

Objective Demonstrate mucosal bacterial infection in children with otitis media with effusion (OME). Study Design and Setting Middle ear mucosal biopsies from 11 children with OME were examined for bacteria utilizing transmission electron microscopy. This was correlated with standard culture and polymerase chain reaction (PCR) of middle ear effusions. Results Gram-positive coccal bacteria were demonstrated in middle ear mucosal epithelial cells of 4 of 11 (36%) children. Morphological appearance of bacteria and detection of pneumolysin DNA by PCR in middle ear fluid suggests a role for persistent intracellular infection with Streptococcus pneumoniae and other gram-positive cocci in some cases of OME. Conclusion Intracellular bacterial infection of middle ear mucosal epithelial cells in children with OME may be an important mechanism for bacterial persistence, and contribute to inflammation and mucus production in the pathogenesis of this condition. Significance Persistent intracellular infection is a novel paradigm for OME pathogenesis in children and may influence antibiotic effectiveness in treatment of this condition.

Effect of Bifidobacterium breve M-16V supplementation on fecal bifidobacteria in preterm neonates - A randomised double blind placebo controlled trial

Background Probiotic supplementation significantly reduces the risk of necrotising enterocolitis (NEC) and all cause mortality in preterm neonates. Independent quality assessment is important before introducing routine probiotic supplementation in this cohort. Aim To assess product quality, and confirm that Bifidobacterium breve (B. breve) M-16V supplementation will increase fecal B. breve counts without adverse effects. Methods and Participants Strain identity (16S rRNA gene sequencing), viability over 2 year shelf-life were confirmed, and microbial contamination of the product was ruled out. In a controlled trial preterm neonates (Gestation <33 weeks) ready to commence or on feeds for <12 hours were randomly allocated to either B. breve M-16V (3×109 cfu/day) or placebo (dextrin) supplementation until the corrected age 37 weeks. Stool samples were collected before (S1) and after 3 weeks of supplementation (S2) for studying fecal B. breve levels using quantitative PCR (Primary outcome). Secondary outcomes included total fecal bifidobacteria and NEC≥Stage II. Categorical and continuous outcomes were analysed using Chi-square and Mann-Whitney tests, and McNemar and Wilcoxon signed-rank tests for paired comparisons. Results A total of 159 neonates (Probiotic: 79, Placebo: 80) were enrolled. Maternal and neonatal demographic characteristics were comparable between the groups. The proportion of neonates with detectable B. breve increased significantly post intervention: Placebo: [S1:2/66 (3%), S2: 25/66 (38%), p<0.001] Probiotic: [S1: 29/74 (40%), S2: 67/74 (91%), p<0.001]. Median S1 B. breve counts in both groups were below detection (<4.7 log cells.g−1), increasing significantly in S2 for the probiotic group (log 8.6) while remaining <4.7 log in the control group (p<0.001). There were no adverse effects including probiotic sepsis and no deaths. NEC≥Stage II occurred in only 1 neonate (placebo group). Conclusion B. breve M-16V is a suitable probiotic strain for routine use in preterm neonates. Trial Registration Australia New Zealand Clinical Trial Registry ACTRN 12609000374268

Effectiveness of Trivalent Flu Vaccine in Healthy Young Children

BACKGROUND: There are few studies evaluating the effectiveness of trivalent influenza vaccination (TIV) in young children, particularly in children <2 years. The Western Australian Influenza Vaccine Effectiveness Study commenced in 2008 to evaluate a program providing TIV to children aged 6 to 59 months. METHODS: An observational study enrolling children with influenza-like illness presenting to a tertiary pediatric hospital was conducted (2008–2012). Vaccination status was determined by parental questionnaire and confirmed via the national immunization register and/or vaccine providers. Respiratory virus polymerase chain reaction and culture were performed on nasopharyngeal samples. The test-negative design was used to estimate vaccine effectiveness (VE) by using 2 control groups: all influenza test-negative subjects and other-virus-detected (OVD) subjects. Adjusted odds ratios were estimated from models with season, month of disease onset, age, gender, indigenous status, prematurity, and comorbidities as covariates. Subjects enrolled in 2009 were excluded from VE calculations. RESULTS: Of 2001 children enrolled, influenza was identified in 389 (20.4%) children. Another respiratory virus was identified in 1134 (59.6%) children. Overall, 295 of 1903 (15.5%) children were fully vaccinated and 161 of 1903 (8.4%) children were partially vaccinated. Vaccine uptake was significantly lower in 2010–2012 after increased febrile adverse events observed in 2010. Using test-negative controls, VE was 64.7% (95% confidence interval [CI]: 33.7%–81.2%). No difference in VE was observed with OVD controls (65.8%; 95% CI: 32.1%–82.8%). The VE for children <2 years was 85.8% (95% CI: 37.9%–96.7%). CONCLUSIONS: This study reveals the effectiveness of TIV in young children over 4 seasons by using test-negative and OVD controls. TIV was effective in children aged <2 years. Despite demonstrated vaccine effectiveness, uptake of TIV remains suboptimal.

Use of data linkage to investigate the aetiology of acute lower respiratory infection hospitalisations in children

Aim:  To document the aetiology of acute lower respiratory infection (ALRI) hospitalisations in Western Australian children by linking population‐based laboratory data with hospital morbidity data.