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1.  Reduced IL-17A Secretion Is Associated with High Levels of Pneumococcal Nasopharyngeal Carriage in Fijian Children 
PLoS ONE  2015;10(6):e0129199.
Streptococcus pneumonia (the pneumococcus) is the leading vaccine preventable cause of serious infections in infants under 5 years of age. The major correlate of protection for pneumococcal infections is serotype-specific IgG antibody. More recently, antibody-independent mechanisms of protection have also been identified. Preclinical studies have found that IL-17 secreting CD4+ Th17 cells in reducing pneumococcal colonisation. This study assessed IL-17A levels in children from Fiji with high and low pneumococcal carriage density, as measured by quantitative real-time PCR (qPCR). We studied Th17 responses in 54 children who were designated as high density carriers (N=27, >8.21x105 CFU/ml) or low density carriers (N=27, <1.67x105 CFU/ml). Blood samples were collected, and isolated peripheral blood mononuclear cells (PBMCs) were stimulated for 6 days. Supernatants were harvested for cytokine analysis by multiplex bead array and/or ELISA. Th17 cytokines assayed included IL-17A, IL-21, IL-22 as well as TNF-α, IL-10, TGF-β, IL-6, IL-23 and IFNγ. Cytokine levels were significantly lower in children with high density pneumococcal carriage compared with children with low density carriage for IL-17A (p=0.002) and IL-23 (p=0.04). There was a trend towards significance for IL-22 (p=0.057) while no difference was observed for the other cytokines. These data provide further support for the role of Th17-mediated protection in humans and suggest that these cytokines may be important in the defence against pneumococcal carriage.
PMCID: PMC4466549  PMID: 26069966
2.  Single-Plex Quantitative Assays for the Detection and Quantification of Most Pneumococcal Serotypes 
PLoS ONE  2015;10(3):e0121064.
Streptococcus pneumoniae globally kills more children than any other infectious disease every year. A prerequisite for pneumococcal disease and transmission is colonization of the nasopharynx. While the introduction of pneumococcal conjugate vaccines has reduced the burden of pneumococcal disease, understanding the impact of vaccination on nasopharyngeal colonization has been hampered by the lack of sensitive quantitative methods for the detection of >90 known S. pneumoniae serotypes. In this work, we developed 27 new quantitative (q)PCR reactions and optimized 26 for a total of 53 qPCR reactions targeting pneumococcal serotypes or serogroups, including all vaccine types. Reactions proved to be target-specific with a limit of detection of 2 genome equivalents per reaction. Given the number of probes required for these assays and their unknown shelf-life, the stability of cryopreserved reagents was evaluated. Our studies demonstrate that two-year cryopreserved probes had similar limit of detection as freshly-diluted probes. Moreover, efficiency and limit of detection of 1-month cryopreserved, ready-to-use, qPCR reaction mixtures were similar to those of freshly prepared mixtures. Using these reactions, our proof-of-concept studies utilizing nasopharyngeal samples (N=30) collected from young children detected samples containing ≥2 serotypes/serogroups. Samples colonized by multiple serotypes/serogroups always had a serotype that contributes at least 50% of the pneumococcal load. In addition, a molecular approach called S6-q(PCR)2 was developed and proven to individually detect and quantify epidemiologically-important serogroup 6 strains including 6A, 6B, 6C and 6D. This technology will be useful for epidemiological studies, diagnostic platforms and to study the pneumobiome.
PMCID: PMC4370668  PMID: 25798884
4.  Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction 
There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies. The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.
PMCID: PMC4131683  PMID: 24637727
Immunology; Issue 84; Streptococcus pneumoniae; Quellung; serotyping; Neufeld; pneumococcus
5.  Silica Desiccant Packets for Storage and Transport of Streptococcus pneumoniae and Other Clinically Relevant Species 
PLoS ONE  2013;8(8):e72353.
Bacterial isolates are often transported between laboratories for research and diagnostic purposes. Silica desiccant packets (SDPs), which are inexpensive and do not require freezing, were evaluated for storage and recovery of bacterial isolates. Conditions such as inoculum size, swab type and temperature of storage were investigated using ten Streptococcus pneumoniae isolates. The optimized protocol was then tested using 49 additional S. pneumoniae isolates representing 40 serogroups. Overall, S. pneumoniae growth was considered satisfactory (>100 colony forming units) for 98/109 (89.9%) and 20/20 (100%) swabs after 14 days at room temperature or 28 days at 4° C, respectively. Storage in SDPs did not impact on the ability of S. pneumoniae isolates to be subsequently serotyped. When the survival of nine other clinically relevant bacterial species was tested, seven were viable after 28 days at room temperature, the exceptions being Neisseria gonorrhoeae and Haemophilus influenzae. SDPs are suitable for transport and short-term storage of bacterial species including S. pneumoniae.
PMCID: PMC3737130  PMID: 23940811
6.  Detection of group a streptococcal pharyngitis by quantitative PCR 
BMC Infectious Diseases  2013;13:312.
Group A streptococcus (GAS) is the most common bacterial cause of sore throat. School-age children bear the highest burden of GAS pharyngitis. Accurate diagnosis is difficult: the majority of sore throats are viral in origin, culture-based identification of GAS requires 24–48 hours, and up to 15% of children are asymptomatic throat carriers of GAS. The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for detecting GAS pharyngitis and assess its suitability for clinical diagnosis.
Pharyngeal swabs were collected from children aged 3–18 years (n = 91) and adults (n = 36) located in the Melbourne area who presented with sore throat. Six candidate PCR assays were screened using a panel of reference isolates, and two of these assays, targeting speB and spy1258, were developed into qPCR assays. The qPCR assays were compared to standard culture-based methods for their ability to detect GAS pharyngitis. GAS isolates from culture positive swabs underwent emm-typing. Clinical data were used to calculate McIsaac scores as an indicator of disease severity.
Twenty-four of the 127 samples (18.9%) were culture-positive for GAS, and all were in children (26%). The speB qPCR had 100% sensitivity and 100% specificity compared with gold-standard culture, whereas the spy1258 qPCR had 87% sensitivity and 100% specificity. Nine different emm types were found, of which emm 89, 3, and 28 were most common. Bacterial load as measured by qPCR correlated with culture load. There were no associations between symptom severity as indicated by McIsaac scores and GAS bacterial load.
The speB qPCR displayed high sensitivity and specificity and may be a useful tool for GAS pharyngitis diagnosis and research.
PMCID: PMC3711935  PMID: 23844865
7.  In vitro growth of pneumococcal isolates representing 23 different serotypes 
BMC Research Notes  2013;6:208.
Although there are established methodologies for pneumococcal carriage studies, recent studies have extended these by conducting a culture amplification step prior to pneumococcal identification or serotyping. However, few data are available comparing the growth of different serotypes.
We compared the growth of individual isolates representing 23 serotypes in serum broth, and Todd-Hewitt broth from four different manufacturers. Following overnight incubation of low inocula, there were differences in the final growth densities of individual isolates. These can be minimised with the use of optimal media.
These data caution against using broth culture amplification of nasopharyngeal samples for some applications.
PMCID: PMC3664610  PMID: 23701729
Serum broth; Todd-Hewitt broth; Streptococcus pneumoniae
8.  Inhibition of Streptococcus pneumoniae adherence to human epithelial cells in vitro by the probiotic Lactobacillus rhamnosus GG 
BMC Research Notes  2013;6:135.
Colonization of the nasopharynx by Streptococcus pneumoniae is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. Probiotic bacteria can influence disease outcomes through various mechanisms, including inhibition of pathogen colonization. Here, we examine the effect of the probiotic Lactobacillus rhamnosus GG (LGG) on S. pneumoniae colonization of human epithelial cells using an in vitro model. We investigated the effects of LGG administered before, at the same time as, or after the addition of S. pneumoniae on the adherence of four pneumococcal isolates.
LGG significantly inhibited the adherence of all the pneumococcal isolates tested. The magnitude of inhibition varied with LGG dose, time of administration, and the pneumococcal isolate used. Inhibition was most effective when a higher dose of LGG was administered prior to establishment of pneumococcal colonization. Mechanistic studies showed that LGG binds to epithelial cells but does not affect pneumococcal growth or viability. Administration of LGG did not lead to any significant changes in host cytokine responses.
These findings demonstrate that LGG can inhibit pneumococcal colonization of human epithelial cells in vitro and suggest that probiotics could be used clinically to prevent the establishment of pneumococcal carriage.
PMCID: PMC3641997  PMID: 23561014
Probiotic; LGG; Pneumococci; Colonization; in vitro model
9.  Production of latex agglutination reagents for pneumococcal serotyping 
BMC Research Notes  2013;6:49.
The current ‘gold standard’ for serotyping pneumococci is the Quellung test. This technique is laborious and requires a certain level of training to correctly perform. Commercial pneumococcal latex agglutination serotyping reagents are available, but these are expensive. In-house production of latex agglutination reagents can be a cost-effective alternative to using commercially available reagents. This paper describes a method for the production and quality control (QC) of latex reagents, including problem solving recommendations, for pneumococcal serotyping.
Here we describe a method for the production of latex agglutination reagents based on the passive adsorption of antibodies to latex particles. Sixty-five latex agglutination reagents were made using the PneuCarriage Project (PCP) method, of which 35 passed QC. The other 30 reagents failed QC due to auto-agglutination (n=2), no reactivity with target serotypes (n=8) or cross-reactivity with non-target serotypes (n=20). Dilution of antisera resulted in a further 27 reagents passing QC. The remaining three reagents passed QC when prepared without centrifugation and wash steps. Protein estimates indicated that latex reagents that failed QC when prepared using the PCP method passed when made with antiserum containing ≤ 500 μg/ml of protein. Sixty-one nasopharyngeal isolates were serotyped with our in-house latex agglutination reagents, with the results showing complete concordance with the Quellung reaction.
The method described here to produce latex agglutination reagents allows simple and efficient serotyping of pneumococci and may be applicable to latex agglutination reagents for typing or identification of other microorganisms. We recommend diluting antisera or removing centrifugation and wash steps for any latex reagents that fail QC. Our latex reagents are cost-effective, technically undemanding to prepare and remain stable for long periods of time, making them ideal for use in low-income countries.
PMCID: PMC3570367  PMID: 23379961
Latex agglutination; Serotyping; Streptococcus pneumoniae
10.  Effect of Pneumococcal Vaccination on Nasopharyngeal Carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian Children 
Journal of Clinical Microbiology  2012;50(3):1034-1038.
The 7-valent pneumococcal conjugate vaccine (PCV7) reduces carriage of vaccine type Streptococcus pneumoniae but leads to replacement by nonvaccine serotypes and may affect carriage of other respiratory pathogens. We investigated nasopharyngeal carriage of S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian infants participating in a pneumococcal vaccine trial using quantitative PCR. Vaccination did not affect pathogen carriage rates or densities, whereas significant differences between the two major ethnic groups were observed.
PMCID: PMC3295152  PMID: 22170924
12.  Multilocus Sequence Typing of Streptococcus pneumoniae by Use of Mass Spectrometry ▿  
Journal of Clinical Microbiology  2011;49(11):3756-3760.
Multilocus sequence typing (MLST) is an important tool for the global surveillance of bacterial pathogens that is performed by comparing the sequences of designated housekeeping genes. We developed and tested a novel mass spectrometry-based method for MLST of Streptococcus pneumoniae. PCR amplicons were subjected to in vitro transcription and base-specific cleavage, followed by analysis of the resultant fragments by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Comparison of the cleavage fragment peak patterns to a reference sequence set permitted automated identification of alleles. Validation experiments using 29 isolates of S. pneumoniae revealed that the results of MALDI-TOF MS MLST matched those obtained by traditional sequence-based MLST for 99% of alleles and that the MALDI-TOF MS method accurately identified two single-nucleotide variations. The MADLI-TOF MS method was then used for MLST analysis of 43 S. pneumoniae isolates from Papua New Guinean children. The majority of the isolates present in this population were not clonal and contained seven new alleles and 30 previously unreported sequence types.
PMCID: PMC3209096  PMID: 21880964
13.  Molecular Epidemiology of Streptococcus pneumoniae Serogroup 6 Isolates from Fijian Children, Including Newly Identified Serotypes 6C and 6D▿  
Journal of Clinical Microbiology  2010;48(11):4298-4300.
Multilocus sequence typing (MLST) was applied to all unique serotype 6C and 6D isolates and a random selection of serotype 6B and 6A isolates from nasopharyngeal swabs from Fijian children enrolled in a recent vaccine trial. The results suggest that Fijian serotype 6D has arisen independently from both serotypes 6A/C and 6B.
PMCID: PMC3020807  PMID: 20810769
14.  Comparison of Citrated Human Blood, Citrated Sheep Blood, and Defibrinated Sheep Blood Mueller-Hinton Agar Preparations for Antimicrobial Susceptibility Testing of Streptococcus pneumoniae Isolates ▿  
Journal of Clinical Microbiology  2010;48(10):3770-3772.
The use of Mueller-Hinton agar supplemented with citrated human or citrated sheep blood was compared with the use of routinely used Mueller-Hinton agar supplemented with defibrinated sheep blood for antimicrobial susceptibility testing of Streptococcus pneumoniae. The alternate supplements were found to be unsatisfactory, particularly for testing resistant isolates, and therefore are not recommended.
PMCID: PMC2953122  PMID: 20668133
15.  Identification of Newly Described Streptococcus pneumoniae Serotype 6D by Use of the Quellung Reaction and PCR ▿  
Journal of Clinical Microbiology  2010;48(9):3378-3379.
We tested 121 pneumococcal serogroup 6 isolates (including 30 serotype 6C and 24 serotype 6D isolates) by serotype-specific PCR and the Quellung reaction, using “old” and “new” pool B, factor 6b, and new factor 6d antisera. In combination with group B and other factor antisera, factor 6d antiserum can reliably identify the newly described serotype 6D.
PMCID: PMC2937698  PMID: 20610680
16.  Treating infant colic with the probiotic Lactobacillus reuteri: double blind, placebo controlled randomised trial 
Objective To determine whether the probiotic Lactobacillus reuteri DSM 17938 reduces crying or fussing in a broad community based sample of breastfed infants and formula fed infants with colic aged less than 3 months.
Design Double blind, placebo controlled randomised trial.
Setting Community based sample (primary and secondary level care centres) in Melbourne, Australia.
Participants 167 breastfed infants or formula fed infants aged less than 3 months meeting Wessel’s criteria for crying or fussing: 85 were randomised to receive probiotic and 82 to receive placebo.
Interventions Oral daily L reuteri (1×108 colony forming units) versus placebo for one month.
Main outcomes measures The primary outcome was daily duration of cry or fuss at 1 month. Secondary outcomes were duration of cry or fuss; number of cry or fuss episodes; sleep duration of infant at 7, 14, and 21 days, and 1 and 6 months; maternal mental health (Edinburgh postnatal depression subscale); family functioning (paediatric quality of life inventory), parent quality adjusted life years (assessment of quality of life) at 1 and 6 months; infant functioning (paediatric quality of life inventory) at 6 months; infant faecal microbiota (microbial diversity, colonisation with Escherichia coli), and calprotectin levels at 1 month. In intention to treat analyses the two groups were compared using regression models adjusted for potential confounders.
Results Of 167 infants randomised from August 2011 to August 2012, 127 (76%) were retained to primary outcome; of these, a subset was analysed for faecal microbial diversity, E coli colonisation, and calprotectin levels. Adherence was high. Mean daily cry or fuss time fell steadily in both groups. At 1 month, the probiotic group cried or fussed 49 minutes more than the placebo group (95% confidence interval 8 to 90 minutes, P=0.02); this mainly reflected more fussing, especially for formula fed infants. The groups were similar on all secondary outcomes. No study related adverse events occurred.
Conclusions L reuteri DSM 17938 did not benefit a community sample of breastfed infants and formula fed infants with colic. These findings differ from previous smaller trials of selected populations and do not support a general recommendation for the use of probiotics to treat colic in infants.
Trial registration Current Controlled Trials ISRCTN95287767.
PMCID: PMC3972414  PMID: 24690625

Results 1-16 (16)