Accurate identification of Streptococcus pseudopneumoniae and other mitis group Streptococci identified as atypical Streptococcus pneumoniae in Tunisian pediatric population.

Accurate identification of Mitis group streptococci especially Streptococcus pneumoniae and Streptococcus pseudopneumoniae seems difficult due to the lack of specific and sensitive tests. We performed an approach for the identification of atypical pneumococci in pediatric Tunisian population. In this study, 49 streptococcal isolates that were considered as atypical S. pneumoniae were analyzed by: optochin susceptibility in ambient and 5% CO2 atmosphere, oxgall disk sensitivity, PCR targeting several genes and antimicrobial susceptibility.The combined results of biochemical and molecular methods showed the presence of 23 S. pneumoniae, 7 S. pseudopneumoniae, and 19 other mitis group. Among S. pseudopneumoniae, all isolates were collected from respiratory tract samples and showed a high level of resistance to ß-lactams with a MIC90 of 32 mg L-1. Two isolates of S. pseudopneumoniae showed the typical phenotype of optochin resistance described in the literature. All isolates could be identified only by molecular tests. Among Streptococcus pneumonaie, all strains harbored the lytA gene and the Spn9802 fragment. But only 14 strains were encapsulated.This study describes several assays for the identification of atypical pneumococci in order to gain insights on the nature of isolate and raise alert about the presence of these strains in the pediatric Tunisian community.

Phylogeography and phylogeny of Rhinoviruses collected from Severe Acute Respiratory Infection (SARI) cases over successive epidemic periods in Tunisia.

Rhinoviruses (RV) are a major cause of Severe Acute Respiratory Infection (SARI) in children, with high genotypic diversity in different regions. However, RV type diversity remains unknown in several regions of the world. In this study, the genetic variability of the frequently circulating RV types in Northern Tunisia was investigated, using phylogenetic and phylogeographic analyses with a specific focus on the most frequent RV types: RV-A101 and RV-C45. This study concerned 13 RV types frequently circulating in Northern Tunisia. They were obtained from respiratory samples collected in 271 pediatric SARI cases, between September 2015 and November 2017. A total of 37 RV VP4-VP2 sequences, selected among a total of 49 generated sequences, was compared to 359 sequences from different regions of the world. Evolutionary analysis of RV-A101 and RV-C45 showed high genetic relationship between different Tunisian strains and Malaysian strains. RV-A101 and C45 progenitor viruses' dates were estimated in 1981 and 1995, respectively. Since the early 2000s, the two types had a wide spread throughout the world. Phylogenetic analyses of other frequently circulating strains showed significant homology of Tunisian strains from the same epidemic period, in contrast with earlier strains. The genetic relatedness of RV-A101 and RV-C45 might result from an introduction of viruses from different clades followed by local dissemination rather than a local persistence of an endemic clades along seasons. International traffic may play a key role in the spread of RV-A101, RV-C45, and other RVs.

Epidemiological study of bacterial meningitis in Tunisian children, beyond neonatal age, using molecular methods: 2014-2017.

BACKGROUND: Since the 1990s, the epidemiology of bacterial meningitis worldwide has changed thanks to vaccination. In Tunisia, the main causative pathogens were Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae serotype b (Hib). Only Hib vaccination was available during our study period. OBJECTIVES: We performed a laboratory case report based-study of suspected bacterial meningitis in Northern Tunisia from January 2014 to June 2017. METHODS: CSF samples obtained from children beyond neonatal age with suspicion of meningitis were tested by two real time PCRs, targeting pneumococcus, meningococcus and Hib, and conventional methods. RESULTS: Using real-time PCR, 63 were positive including ten supplementary cases compared to conventional methods. A general decrease of bacterial meningitis cases was demonstrated comparing to previous data. Pneumococcus was predominant (69.84%) followed by meningococcus (28.57%) and Hib (1.59%). The main serotypes were 14, 19F, 6B and 23F for pneumococcus and serogroup B for meningococcus. Most cases occurred during cold season and children under one year were the most affected by bacterial meningitis. CONCLUSION: Our study suggests the predominance of pneumococcal cases. It may provide valuable data on meningitis epidemiology before the introduction of pneumococcal vaccine, which may be useful for future evaluation.

Molecular diagnosis of bacterial meningitis by multiplex real time PCR in Tunisian children.

INTRODUCTION: Bacterial meningitis is a medical emergency requiring a fast and reliable diagnosis. Molecular methods such as real-time PCR (rt-PCR) offer an attractive alternative. Thus, this study aims to establish multiplex rt-PCRs detecting N. meningitidis, S. pneumoniae and H. influenzae b from cerebrospinal fluid in Tunisian children beyond neonatal age. METHODOLOGY: Using bioinformatic tools and experimentation, we validated the specificity and optimal criteria of PCRs for primers and probes of plyA (S. pneumoniae), ctrA and sodC (N. meningitidis) and bexA genes (H. influenzae b). We performed one multiplex RT-PCR for detection of S. pneumoniae and N. meningitidis targeting plyA and ctrA, sodC genes respectively, simultaneously with a singleplex RT-PCR for H. influenzae b. The sensitivity and specificity of our methods were assessed. Then, we tested our methods for 122 CSF samples collected from suspected meningitis cases between 2014 and 2016 in Bechir Hamza Children's Hospital of Tunis. RESULTS: Our results have shown the sensitivity of the designed PCRs was up to 10-4 DNA dilution and the specificity was 100%. PCR evaluation has shown 51 positive samples: 38 of pneumococcal cases, 12 meningococcal cases, 1 case of H. influenzae b with 8.57% and 50% of supplementary positive cases rates respectively. CONCLUSIONS: Our assay proved to be very sensitive, specific and rapid for bacterial meningitis diagnosis. In the recent context of Hib vaccination, the possibility of detecting S. pneumoniae and N. meningitidis separately constitute an attractive opportunity. Nevertheless, simultaneous detection of Hib remains relevant in specific clinical context and for epidemiologic study.

Molecular Characteristics of Erythromycin-Resistant Streptococcus pyogenes Strains Isolated from Children Patients in Tunis, Tunisia.

AIM: The aims of our study were to characterize phenotypically and genotypically erythromycin-resistant Streptococcus pyogenes or group A streptococci (ERGAS) isolates, to evaluate macrolide resistance and to analyze the association between emm types and virulence factors. Included in this study were all ERGAS strains isolated from 2000 to 2013 at the Children's hospital of Tunis. Antimicrobial susceptibility was performed according to the CA-SFM guidelines. Macrolide resistance genes were revealed by polymerase chain reaction (PCR) method. Virulence factor genes (pyrogenic exotoxin genes and superantigen gene) were detected by PCR, and the emm types were defined by the sequencing of the variable 5' end of the emm gene. RESULTS: Among the 289 GAS isolates collected, 15 (5.2%) were resistant to erythromycin; 7 of the strains were assigned to the cMLSB phenotype (46.6%); 5 harbored ermB gene alone (33.3%); and 2 strains coharbored ermB and mefA (13.3%). The remaining (53.4%) were assigned to the M phenotype and harbored the mefA gene. The frequency of detection of each toxin gene among ERGAS was 13.4% for speA (2 strains), 53.4% for speC (8 strains), and 13.4% for ssa (2 strains). Emm types 1, 58, 11, and 78 were the most frequent among ERGAS strains. The distribution of the cMLSB and M phenotypes changed over the period of investigation with a decrement of cMLSB phenotype and ermB gene that predominated between 2000 and 2006 and an increase of M phenotype and mefA gene between 2007 and 2013, but this difference was nonstatistically significant because of the low number of resistant strains. Emm types 1, 58, and 4 were only present among strains assigned to the M phenotype. However strains assigned to the cMLSB phenotype were associated to emm11, emm22, emm28, emm78, or emm76. There was diversity in emm distribution in ERGAS between the two study periods. There was diversity in emm distribution among ERGAS particularly in 2000-2006. Indeed, from 2000 to 2006, the 6 ERGAS belonged to 5 different emm types (22, 28, 76, 11, and 4), while between 2007 and 2013, seven among the nine ERGAS belonged to only 2 emm types 58 and 1. The speA gene was present only among emm1 isolates, and the ssa gene was associated with emm4 and emm78 types. All emm78, emm28, and emm11 strains harbored speC gene. CONCLUSIONS: Our study revealed a low frequency of ERGAS and few emm types were associated with these strains.