Serotype distribution and antimicrobial sensitivity profile of Streptococcus pneumoniae carried in healthy toddlers before PCV13 introduction in Niamey, Niger

Background To mitigate the burden of pneumococcal infections in Niger, a 13-valent pneumococcal vaccine, PCV13, was introduced for routine child vaccination in July 2014. In order to provide pre-vaccine baseline data and allow appreciation of changes on carriage due to vaccination, we analyzed retrospectively pneumococcal isolates obtained from healthy, 0 to 2 year old children prior to the vaccine introduction. Methods From June 5, 2007, to May 26, 2008,1200 nasopharyngeal swabs were collected from healthy 0 to 2 year old children and analyzed by standard microbiological methods. Serotyping was done by SM-PCR and the data were analyzed with R version 2.15.0 (2012-03-30). Results Streptococcus pneumoniae was detected in 654/1200 children (54.5%) among whom 339 (51.8%) were males. The ages of the study subjects varied from few days to 26 months (mean = 7.1, median = 6, 95% CI [6.8-7.4]). Out of 654 frozen isolates, 377 (54.8%) were able to be re-grown and analyzed. In total, 32 different serogroups/serotypes were detected of which, the most prevalent were 6/(6A/6B/6C/6D) (15.6%), 23F (10.6%), 19F (9.3%), 14 (9%), 19A (5.6%), 23B (4.0%), 25F/38 (3.7%), 18/(18A/18B/18C/18F) (2.9%) and PCR non-typeable (16.4%). Eleven serogroups/serotypes accounting for 57.3% (216/377) were of PCV13 types. Of the 211/377 (56%) isolates tested for drug sensitivity, 23/211 (10.9%), 24/211 (11.4%), 9/211(4.3%) and 148/210 (70.5%) were respectively resistance to oxacillin, chloramphenicol, erythromycin and tetracycline. Thirteen of the oxacillin resistant isolates were additionally multidrug-resistant. No resistance was however detected to gentamy-cin500μg and to fluoroquinolones (ø Norfloxacin5μg <7mm). Age > 3 months and presence in family of more than one sibling aged < 6 years were significant risk factors for carriage. Conclusion A global rate of 54.5% pneumococcal carriage was detected in this study. The introduced PCV13 vaccine should cover 57.3% (216/377) of circulating serogroups/serotypes, among which were those resistant to antibiotics. Age > 3 months and presence in family of children aged < 6 years were significant factors for pneumococcal carriage. The present data should help understanding post vaccine introduction changes in pneumococcal carriage and infections for better action. The study subjects were enrolled in the maternal and infant protection health center, PMI Yantala Haut, a public, primary health care center delivering care services for all but mainly for mothers and children. In 2007, the center covered a population of about 53.224 inhabitants in a catchment area of 0–5 km within commune I of Niamey, the capital city of Niger. PMI Yantala is a close neighbor of Centre de Recherche Médicale et Sanitaire, CERMES, a public but autonomous health research institute where the this study was conducted. CERMES was created to carry out research on meningitis and schistosomiasis since 1980 and had undergone important structural and administrative changes in 2000 to include activities in public health, research and training of health personnel. The center had been first an associate member of International Network of Pasteur Institutes (RIIP) from 2003 to 2007 before becoming a full member of the network in 2007. The study was conducted from June 5, 2007, to May 26, 2008 and enrolled healthy children taken to PMI Yantala Haut health center for routine systematic vaccination. The criteria for a child to be enrolled were being aged between 0 to 2 year old, being healthy without any history of infectious disease in the last 3 months to 7 days before the enrollment date and obtaining signed consent of parents, next of kin or of a person having witnessed the parent’s or next of kin’s oral consent. A questionnaire form was then addressed to mothers or next of kin for the collection of socio-demographic information. Nasopharyngeal samples were subsequently collected once for each participant according to WHO recommendations [12] by the study physician using a flexible cotton swab (Calgiswab, COPAN Strasbourg). The inclusion was done once in a given day of each week, throughout the study period. The study protocol, the consent and questionnaire forms were all submitted to and approved by the Niger National Consultative Committee of Ethic through deliberation N° 02/2007/CCNE of February1, 2007. Collected swabs were immediately inoculated on plates of Colombia agar medium containing 15 mg/l Nalidixic acid (Bio-Rad Ref. 60534) and supplemented with 7–10% sheep blood. Plates were then incubated at 36±0.5°C under approximate 5% CO2 atmosphere (GENbag BioMérieux®. Ref. 45533) for18 ± 2 hours. Suspected colonies were further selected and purified on new blood agar plates on which an optochin disc (OPT-F BioMérieux®. Ref.55912) was applied and incubated under the same conditions as stated above. SPN were identified as a Gram-positive diplococci, α-hemolytic, oxydase and catalase negative colonies with an optochin inhibition zone ≥14 mm (Ref. SFM-CA edition 2008) and further confirmed by latex agglutination using Slidex® Pneumo-Kit (BioMérieux Ref. 58821). All confirmed SPN strains were kept at -80°C in Trypto-Casein-Soya (TCS) broth containing 0.5% glucose, 2% skimmed milk and 10% glycerol until use. Serotyping was performed by Sequential Multiplex PCR (SM-PCR) as described by Collard et al [7] with little change in that samples consist of pneumococcal isolates rather than CSF and PCR run in 12 reactions rather than 8. DNA extracts were obtained by suspending frozen isolates in 500μl PBS buffer and heating to 100°C for 10 minutes. Antimicrobial sensitivity testing was performed using the Kirby–Bauer disc diffusion method, based on recommendations of the French Committee for Antibiotic sensitivity testing (CA-SFM, 2013). Inhibition zones were read with OSIRIS (Expert version 3.2, BIO-RAD) with manual adjustment where necessary. Minimum Inhibitory Concentrations (MIC) were measured using BioMérieux E-tests (BioMérieux). In this study, an isolate was considered less susceptible to penicillin when an inhibition zone of less than 26 mm was observed around oxacillin5μg disc and resistant when in addition the measured MIC of cefotaxim was > 0.5μg (CA-SFM, 2013 edition). All data collected have been confidentially recorded into a MySQL database and extracted to Excel for analysis with open source R v.2.15.0 (2012-03-30); The R Foundation for Statistical Computing, ISBN 3-900051-07 0. X2 test was used to compare frequencies or means. Risk factors for SPN carriage were first analyzed individually by univariate (single variable) analysis then examined together in multivariable (multiple variables) analysis by logistic regression model. The study was conducted in accordance with the then last Helsinki Declaration and started after approval obtained from the Niger National Consultative Committee of Ethic through deliberation N° 02/2007/CCNE of February1, 2007. Detailed information about the study aim, the sampling method and its related risk, the use and confidentiality of data were all made clear to parents or to next of kin before obtaining and signing their consent. Parents or next of kin were free to refuse the participation of their child, to ask question or to have access to the laboratory result when they wished. Sample collection, analysis and waste management were carried out according to WHO recommendations[12]