Updated emm-typing protocol for Streptococcus pyogenes.

OBJECTIVES: PCR-based typing of the emm gene Streptococcus pyogenes often results in the amplification of multiple bands. This has resulted in the misclassification of strains into types based on non-emm gene sequences. We aimed to improve the specificity of the emm typing PCR reaction using a primer called CDC3, the sequence for which has been previously used to identify emm genes in silico. METHODS: The proposed primer CDC3 was validated in silico from a global database of 1688 GAS genomes and in vitro with 32 isolates. PCR reactions were performed on genomic DNA from each isolate, using the published CDC1 forward primer with the CDC2 reverse primer or the new CDC3 reverse primer. The products were examined by gel electrophoresis, and representative PCR products were sequenced. RESULTS: In 1688 S. pyogenes genomes, the previous CDC2 reverse primer annealed in silico in 1671 emm genes and also in 2109 non emm genes in close proximity, whereas the new CDC3 primer annealed in 1669 emm genes only. The remaining 19 genes without a CDC3 binding site were chimeric emm genes. The PCR pair CDC1+CDC3 produced a single band at appropriate molecular weight in all 32 isolates tested, while the CDC1+CDC2 pair produced more than one band in 13 of 32 isolates (40%). CONCLUSIONS: The new CDC3 primer is more specific for emm genes than the previous CDC2 primer and represents a simple solution to reduce the potential for mistyping S. pyogenes strains.

Validation of an automated colony counting system for group A Streptococcus.

BACKGROUND: The practice of counting bacterial colony forming units on agar plates has long been used as a method to estimate the concentration of live bacteria in culture. However, due to the laborious and potentially error prone nature of this measurement technique, an alternative method is desirable. Recent technologic advancements have facilitated the development of automated colony counting systems, which reduce errors introduced during the manual counting process and recording of information. An additional benefit is the significant reduction in time taken to analyse colony counting data. Whilst automated counting procedures have been validated for a number of microorganisms, the process has not been successful for all bacteria due to the requirement for a relatively high contrast between bacterial colonies and growth medium. The purpose of this study was to validate an automated counting system for use with group A Streptococcus (GAS). METHODS: Twenty-one different GAS strains, representative of major emm-types, were selected for assessment. In order to introduce the required contrast for automated counting, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) dye was added to Todd-Hewitt broth with yeast extract (THY) agar. Growth on THY agar with TTC was compared with growth on blood agar and THY agar to ensure the dye was not detrimental to bacterial growth. Automated colony counts using a ProtoCOL 3 instrument were compared with manual counting to confirm accuracy over the stages of the growth cycle (latent, mid-log and stationary phases) and in a number of different assays. The average percentage differences between plating and counting methods were analysed using the Bland-Altman method. RESULTS: A percentage difference of ±10 % was determined as the cut-off for a critical difference between plating and counting methods. All strains measured had an average difference of less than 10 % when plated on THY agar with TTC. This consistency was also observed over all phases of the growth cycle and when plated in blood following bactericidal assays. Agreement between these methods suggest the use of an automated colony counting technique for GAS will significantly reduce time spent counting bacteria to enable a more efficient and accurate measurement of bacteria concentration in culture.

Group A streptococcal strains isolated in Lao People's Democratic Republic from 2004 to 2013.

Epidemiological data regarding group A streptococcal (GAS) infections in South East Asia are scarce with no information from Laos. We characterized emm types, emm clusters and the antibiotic resistance profile of 124 GAS isolates recovered in Laos during 2004-2013. Most strains were recovered from skin and invasive infections (76% and 19%, respectively). Thirty-four emm types were identified as belonging to 12 emm clusters and no novel emm types were identified. No significant differences were observed in the distribution of emm types or emm clusters according to age or site of recovery (skin or invasive infections). There was moderate strain diversity in this country but considerable differences in emm-type distribution between Laos, Thailand and Cambodia. Vaccine coverage was high for the J8 vaccine candidate. The theoretical coverage for the 30-valent vaccine candidate needs further investigation. Antibiotic resistance was moderate to erythromycin and chloramphenicol (8% and 7%, respectively) and low to ofloxacin (<1%).

ASO titer or not? When to use streptococcal serology: a guide for clinicians.

Clinicians frequently request serologic tests to provide evidence of prior infection by Streptococcus pyogenes, especially when suspecting a diagnosis of acute rheumatic fever or post-streptococcal glomerulonephritis. However, the interpretation of these tests is difficult and should take account of the clinical features, epidemiological setting, and pre-test probability, as well as the specific aspects of the assay. This review details the characteristics of streptococcal serologic assays and provides recommendations for their use and interpretation.

Updated model of group A Streptococcus M proteins based on a comprehensive worldwide study.

Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.

Differences in nasopharyngeal bacterial carriage in preschool children from different socio-economic origins.

A prospective cohort study of preschool healthy children (3-6 years old) from two distinct socio-economic settings in the Brussels area, Belgium, was conducted during the years 2006-2008. The objectives were to evaluate nasopharyngeal colonization by Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis and Haemophilus influenzae at the time of PCV7 vaccine introduction and to assess the socio-economic level impact on flora composition and antibiotic resistance. Three hundred and thirty-three children were included and a total of 830 nasopharyngeal samples were collected together with epidemiological data. Pneumococcal serotypes and antibiotic resistance profiles were determined. Risk factors for carriage and bacterial associations were analysed by multivariate logistic regression. Carriage rates were high for all pathogens. Fifty per cent of the children were colonized at least once with S. aureus, 69% with S. pneumoniae, 67% with M. catarrhalis and 83% with H. influenzae. PCV7 uptake was higher among children from a higher socio-economic setting and S. pneumoniae serotypes varied accordingly. Children from lower socio-economic schools were more likely to carry M. catarrhalis, S. aureus and antibiotic-resistant S. pneumoniae, including a high proportion of non-typeable pneumococcal strains. Positive associations between S. pneumoniae and H. Influenza, between H. influenzae and M. catarrhalis and between H. influenzae and S. aureus were detected. Our study indicates that nasopharynx flora composition is influenced not only by age but also by socio-economic settings. A child's nasopharynx might represent a unique dynamic environment modulated by intricate interactions between bacterial species, host immune system and PCV7 immunization.

[The pain of the newborn: between reality and perception]. / La douleur des nouveau-nés: entre réalité et perception.

UNLABELLED: A newborn hospitalized in neonatology suffers a lot of painful and fully perceived procedures. However this pain is not enough taken into consideration. There are various reasons for this failure. The objective of our study was to analyze the perception of 3 groups of participants (parents, nurses and doctors) about newborns'pain. We wanted to compare these perceptions with pain scales (EDIN and BBdol scale) and to study their connection with newborn illness severity and mortality risk scores (SNAP and CRIB). POPULATION AND METHOD: We have led a prospective study involving 80 newborns. Questionnaires assessing, with the help of a visual analogic scale, the pains' perception and the efficiency of the treatment of this pain were given to the 3 groups of participants. RESULTS: Parents assessed that their newborn feels an important pain (median: 5/10), that was not correlated with pain scales. Nurses and doctors assessed a lower level of pain (median: 2/10), greatly correlated with the pain scales. Parents assessed that the treatment of pain was better when the newborn was severely ill. The nurses, and even more the doctors, assessed the opposite effect. The nurses appeared to hold an intermediate position between parents and doctors. Nurses underlined moreover some lack of communication of the doctors about the newborns' pain. This communication problem is a major hindrance to the adequate treatment of pain.