Robyn L. Marsh

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.

Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data

BackgroundThe rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites.ResultsA workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types.ConclusionsThe application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts.

Three Clinically Distinct Chronic Pediatric Airway Infections Share a Common Core Microbiota

RATIONALE DNA-based microbiological studies are moving beyond studying healthy human microbiota to investigate diverse infectious diseases, including chronic respiratory infections, such as those in the airways of people with cystic fibrosis (CF) and non-CF bronchiectasis. The species identified in the respiratory secretion microbiota from such patients can be classified into those that are common and abundant among similar subjects (core) versus those that are infrequent and rare (satellite). This categorization provides a vital foundation for investigating disease pathogenesis and improving therapy. However, whether the core microbiota of people with different respiratory diseases, which are traditionally associated with specific culturable pathogens, are unique or shared with other chronic infections of the lower airways is not well studied. Little is also known about how these chronic infection microbiota change from childhood to adulthood. OBJECTIVES We sought to compare the core microbiota in respiratory specimens from children and adults with different chronic lung infections. METHODS We used bacterial 16S rRNA gene pyrosequencing, phylogenetic analysis, and ecological statistical tools to compare the core microbiota in respiratory samples from three cohorts of symptomatic children with clinically distinct airway diseases (protracted bacterial bronchitis, bronchiectasis, CF), and from four healthy children. We then compared the core pediatric respiratory microbiota with those in samples from adults with bronchiectasis and CF. MEASUREMENTS AND MAIN RESULTS All three pediatric disease cohorts shared strikingly similar core respiratory microbiota that differed from adult CF and bronchiectasis microbiota. The most common species in pediatric disease cohort samples were also detected in those from healthy children. The adult CF and bronchiectasis microbiota also differed from each other, suggesting common early infection airway microbiota that diverge by adulthood. The shared core pediatric microbiota included both traditional pathogens and many species not routinely identified by standard culture. CONCLUSIONS Our results indicate that these clinically distinct chronic airway infections share common early core microbiota, which are likely shaped by natural aspiration and impaired clearance of the same airway microbes, but that disease-specific characteristics select for divergent microbiota by adulthood. Longitudinal and interventional studies will be required to define the relationships between microbiota, treatments, and disease progression.

Respiratory microbiota: addressing clinical questions, informing clinical practice

Over the last decade, technological advances have revolutionised efforts to understand the role played by microbes in airways disease. With the application of ever more sophisticated techniques, the literature has become increasingly inaccessible to the non-specialist reader, potentially hampering the translation of these gains into improvements in patient care. In this article, we set out the key principles underpinning microbiota research in respiratory contexts and provide practical guidance on how best such studies can be designed, executed and interpreted. We examine how an understanding of the respiratory microbiota both challenges fundamental assumptions and provides novel clinical insights into lung disease, and we set out a number of important targets for ongoing research.

Absence of an important vaccine and diagnostic target in carriage- and disease-related nontypeable Haemophilus influenzae.

ABSTRACT Nontypeable Haemophilus influenzae (NTHi)-associated disease is a major health problem globally. Whole-genome sequence analysis identified the absence of hpd genes encoding Haemophilus protein D in 3 of 16 phylogenetically distinct NTHi isolates. This novel finding is of potential clinical significance, as protein D and hpd represent important NTHi vaccine antigen and diagnostic targets, respectively.

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

ABSTRACT 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 ∼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.

Quantitative PCR of ear discharge from Indigenous Australian children with acute otitis media with perforation supports a role for Alloiococcus otitidis as a secondary pathogen

BackgroundOtitis media is endemic in remote Indigenous communities of Australia’s Northern Territory. Alloiococcus otitidis is an outer ear commensal and putative middle ear pathogen that has not previously been described in acute otitis media (AOM) in this population. The aims of this study were to determine the presence, antibiotic susceptibility and bacterial load of A. otitidis in nasopharyngeal and ear discharge swabs collected from Indigenous Australian children with AOM with perforation.MethodsPaired nasopharyngeal and ear discharge swabs from 27 children with AOM with perforation were tested by A. otitidis quantitative PCR (qPCR). Positive swabs were cultured for 21 days. Total and respiratory pathogen bacterial loads in A. otitidis-positive swabs were determined by qPCR.ResultsA. otitidis was detected by qPCR in 11 ear discharge swabs from 10 of 27 (37%) children, but was not detected in paired nasopharyngeal swabs. A. otitidis was cultured from 5 of 11 qPCR-positive swabs from four children. All A. otitidis isolates had minimum inhibitory concentrations consistent with macrolide resistance. All A. otitidis qPCR-positive swabs were culture-positive for other bacteria. A. otitidis bacterial load ranged from 2.2 × 104-1.1 × 108 cells/swab (median 1.8 × 105 cells/swab). The relative abundance of A. otitidis ranged from 0.01% to 34% of the total bacterial load (median 0.7%). In 6 of 11 qPCR-positive swabs the A. otitidis relative abundance was <1% and in 5 of 11 it was between 2% and 34%. The A. otitidis bacterial load and relative abundance measures were comparable to that of Haemophilus influenzae.ConclusionsA. otitidis can be a dominant species in the bacterial communities present in the ear discharge of Indigenous children with AOM with perforation. The absence of A. otitidis in nasopharyngeal swabs suggests the ear canal as the likely primary reservoir. The significance of A. otitidis at low relative abundance is unclear; however, at higher relative abundance it may be contributing to the associated inflammation. Further studies to better understand A. otitidis as a secondary otopathogen are warranted, particularly in populations at high-risk of progression to chronic suppurative otitis media and where macrolide therapies are being used.

Age-Specific Cluster of Cases of Serotype 1 Streptococcus pneumoniae Carriage in Remote Indigenous Communities in Australia

ABSTRACT 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.

Detection of Biofilm in Bronchoalveolar Lavage from Children With Non-Cystic Fibrosis Bronchiectasis

The presence of Pseudomonas aeruginosa biofilms in lower airway specimens from cystic fibrosis (CF) patients is well established. To date, biofilm has not been demonstrated in bronchoalveolar lavage (BAL) from people with non‐CF bronchiectasis. The aim of this study was to determine (i) if biofilm was present in BAL from children with and without bronchiectasis, and (ii) if biofilm detection differed between sequentially collected BAL.

Toward making inroads in reducing the disparity of lung health in Australian Indigenous and New Zealand Maori ¯ children

Globally, Indigenous populations, which include Aboriginal and Torres Strait islanders in Australia and Māori people in New Zealand (NZ), have poorer health than their non-Indigenous counterparts. Indigenous peoples worldwide face substantial challenges in poverty, education, employment, housing and disconnection from ancestral lands. While addressing social determinants of health is a priority, solving clinical issues is equally important. Indeed, ignoring the latter until social issues improve risks further disparity as this may take generations. A systematic overview of interventions addressing social determinants of health found a striking lack of reliable evaluations.Where evidence was available, health improvement associated with interventions was modest or uncertain. 10 Thus advances in healthcare remain essential and these require the best evidence available in 11 preventing and managing common illnesses, including respiratory illnesses.