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Prevalence of Kingella kingae oropharyngeal carriage and predominance of type a and type b polysaccharide capsules among French young children

2018; Elsevier BV; Volume: 25; Issue: 1 Linguagem: Inglês

10.1016/j.cmi.2018.07.033

1469-0691

Romain Basmaci, K. Deschamps, Yaël Levy, Vincent Mathy, François Corrard, Franck Thollot, Stéphane Béchet, Elsa Sobral, Philippe Bidet, Robert Cohen, Stéphane Bonacorsi,

Streptococcal Infections and Treatments

Kingella kingae is the leading pathogen of osteoarticular infection in children under 4 years in different countries [[1]Yagupsky P. Kingella kingae: carriage, transmission, and disease.Clin Microbiol Rev. 2015; 28: 54-79Crossref PubMed Scopus (127) Google Scholar]. However, only a few studies have described the healthy carriage of K. kingae. In Israel and Switzerland, the highest colonization rate was around 10% in the children aged 12–24 months [1Yagupsky P. Kingella kingae: carriage, transmission, and disease.Clin Microbiol Rev. 2015; 28: 54-79Crossref PubMed Scopus (127) Google Scholar, 2Anderson de la Llana R. Dubois-Ferriere V. Maggio A. Cherkaoui A. Manzano S. Renzi G. et al.Oropharyngeal Kingella kingae carriage in children: characteristics and correlation with osteoarticular infections.Pediatr Res. 2015; 78: 574-579Crossref PubMed Scopus (18) Google Scholar]. To our knowledge, no epidemiological data on the healthy carriage is available in France. We aimed to determine the rate of and the factors associated with healthy carriage of K. kingae in young children in France, as well as the capsular serotype of these strains, known to be associated either with invasive or carriage strains [[3]Porsch E.A. Starr K.F. Yagupsky P. St Geme 3rd, J.W. The Type a and Type b polysaccharide capsules predominate in an International Collection of Invasive Kingella kingae isolates.mSphere. 2017; 2 (pii:e00060-17)Crossref PubMed Scopus (17) Google Scholar]. Between May 2015 and June 2016, 217 healthy children aged from 6 to 36 months were prospectively enrolled. Throat samples were collected, as previously described [[4]Basmaci R. Ilharreborde B. Bidet P. Doit C. Lorrot M. Mazda K. et al.Isolation of Kingella kingae in the oropharynx during K. kingae arthritis in children.Clin Microbiol Infect. 2012; 18: E134-E136Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar], by nine paediatricians from five different French departments (from Parisian Region: Paris, Seine-et-Marne, Seine-Saint-Denis, Val-De-Marne; and from Meurthe-et-Moselle, located in the Grand Est region), after recording the written consent of at least one of their parents. Children who had received antibiotics during the last 7 days were not eligible for inclusion. The protocol was approved by the Saint-Germain-en-Laye Ethics Committee (Comité de Protection des Personnes Ile-de-France XI). Continuous variables were compared by Mann–Whitney U-test. Categorical variables were compared by Fisher exact test or chi-squared test. All analyses were performed with R statistical package 3.3.2 (R Foundation for Statistical Computing, Vienna, Austria). A p value < 0.05 was considered statistically significant. Demographic and clinical features of the children included in the study are described in Table S2. In all, 171 children (78.8%) were included by two paediatricians ('two-paediatrician group') and 46 (21.2%) by the seven others ('seven-paediatrician group'). In the seven-paediatrician group, children were more frequently cared for out-of-home than in the two-paediatrician group (p < 0.001) (Table S2). To reliably define a K. kingae carriage, we first identified the positive rtxA samples, and then we needed a negative groEL PCR (highly specific of Kingella negevensis) [[5]El Houmami N. Bzdrenga J. Durand G.A. Minodier P. Seligmann H. Prudent E. et al.Molecular tests that target the RTX locus do not distinguish between Kingella kingae and the recently described Kingella negevensis species.J Clin Microbiol. 2017; 55: 3113-3122Crossref PubMed Scopus (23) Google Scholar], to discriminate K. kingae (rtxA+/groEL–) from K. negevensis (rtxA+/groEL+). No rtxA+/groEL + samples were found, which excluded the possibility of a mixed colonization. To consolidate the identification of K. kingae, we also performed cpn60 real-time PCR [[6]Ilharreborde B. Bidet P. Lorrot M. Even J. Mariani-Kurkdjian P. Liguori S. et al.New Real-Time PCR-Based Method for Kingella kingae DNA Detection: Application to Samples Collected from 89 Children with Acute Arthritis.J Clin Microbiol. 2009; 47: 1837-1841Crossref PubMed Scopus (154) Google Scholar]. All the rtxA-positive samples were cpn60-positive, but some rtxA-negative samples were cpn60-positive. To explore this discrepancy we attempted to sequence the cpn60 allele [[7]Basmaci R. Yagupsky P. Ilharreborde B. Guyot K. Porat N. Chomton M. et al.Multilocus sequence typing and rtxA toxin gene sequencing analysis of Kingella kingae isolates demonstrates genetic diversity and international clones.PLoS One. 2012; 7e38078Crossref PubMed Scopus (35) Google Scholar]. When the sequences could be confidently read, we observed that the rtxA-positive samples exhibited a known K. kingae cpn60 allele, whereas the rtxA-negative samples exhibited an allele closely related to a different species (Simonsiella muelleri). Kingella kingae was detected by PCR in 11 (5.1%; 95% CI 2.6%–8.9%) out of the 217 children. No K. kingae strain could be isolated by culture on a selective medium, as previously described [[4]Basmaci R. Ilharreborde B. Bidet P. Doit C. Lorrot M. Mazda K. et al.Isolation of Kingella kingae in the oropharynx during K. kingae arthritis in children.Clin Microbiol Infect. 2012; 18: E134-E136Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. The peak of prevalence of healthy carriage appeared in children aged 18–23 months (11.4%; 95% CI 3.2%–26.7%) (see Supplementary material, Fig. S1). For capsular typing, we performed a multiplex PCR allowing four types to be distinguished: 'a', 'b', 'c' and 'd', using a modified protocol (see Supplementary material, Table S1). To identify the capsule type based on molecular weight after gel electrophoresis, we used eight K. kingae strains with known capsule type as positive controls (two per capsule type) (see Supplementary material, Fig. S2a). Among the 11 K. kingae-positive throat samples, the capsule type was successfully determined in nine cases (see Supplementary material, Fig. S2b). Capsules a and b were identified in four samples each, and simultaneous capsules a and c were identified in one sample, and no amplification product was visualized for the two remaining samples. Whether a lack of PCR sensitivity was observed in an oropharyngeal sample or capsules other than those already known could be elaborated by the species remains to be determined. Demographic and clinical characteristics of the 11 K. kingae healthy carriers, compared with their 206 non-carrier counterparts, are described in Table S2. Healthy carriers were more frequently cared for out-of-home than non-carriers (63.6% versus 21.4%, respectively; p 0.004), especially in day-care centre (63.6% versus 17.0%, respectively; p 0.002). Hence, the prevalence of carriage was higher in children cared for out-of-home than in children cared for at home (13.7% versus 2.4%, respectively; p 0.004). Of interest, although not significant, six out of seven (85.7%) carriers who were cared for out-of-home attended at least 4 days per week (Table S2), leading to a prevalence of carriage of 15.8% (6/38; 95% CI 6.0%–31.3%) among children attending a day-care centre at least 4 days per week. Those results appeared similar to those previously described in other countries [2Anderson de la Llana R. Dubois-Ferriere V. Maggio A. Cherkaoui A. Manzano S. Renzi G. et al.Oropharyngeal Kingella kingae carriage in children: characteristics and correlation with osteoarticular infections.Pediatr Res. 2015; 78: 574-579Crossref PubMed Scopus (18) Google Scholar, 8Amit U. Dagan R. Yagupsky P. Prevalence of pharyngeal carriage of Kingella kingae in young children and risk factors for colonization.Pediatr Infect Dis J. 2013; 32: 191-193Crossref PubMed Scopus (29) Google Scholar]. Although not significant, we observed a higher carriage rate during spring (8/117; 6.8%) and autumn (2/32; 6.3%) than during winter (1/68; 1.5%) (p 0.23). A high sensitivity of the oropharyngeal culture method has been observed in children with K. kingae septic arthritis [[4]Basmaci R. Ilharreborde B. Bidet P. Doit C. Lorrot M. Mazda K. et al.Isolation of Kingella kingae in the oropharynx during K. kingae arthritis in children.Clin Microbiol Infect. 2012; 18: E134-E136Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar], but no K. kingae strain could be isolated from the oropharynx of the healthy children in the current study. Although Ceroni et al. [[9]Ceroni D. Llana R.A. Kherad O. Dubois-Ferriere V. Lascombes P. Renzi G. et al.Comparing the oropharyngeal colonization density of Kingella kingae between asymptomatic carriers and children with invasive osteoarticular infections.Pediatr Infect Dis J. 2013; 32: 212-214Crossref Scopus (18) Google Scholar] have demonstrated that the colonization density of K. kingae, based on real-time rtxA PCR results, among healthy paediatric carriers is not inferior to that observed in children with skeletal system infections, the lack of specificity of this PCR cannot rule out that ill children present a higher bacterial load than asymptomatic children. Several limitations could be identified in our study. First, the low number of carriers identified may lead to a decrease in the representativeness of our results. Second, the rate of children who were cared for out-of-home in the two-paediatrician group (78.8% of the study population) was lower than that in the seven-paediatrician group, the latter being close to that observed in France [[10]Angoulvant F. Cohen R. Doit C. Elbez A. Werner A. Bechet S. et al.Trends in antibiotic resistance of Streptococcus pneumoniae and Haemophilus influenzae isolated from nasopharyngeal flora in children with acute otitis media in France before and after 13 valent pneumococcal conjugate vaccine introduction.BMC Infect Dis. 2015; 15: 236Crossref PubMed Scopus (37) Google Scholar] (17.0%, 47.8% and 45%, respectively). This may have led to an underestimate of the carriage rate in our study. The first study on the K. kingae healthy carriage in France revealed a similar carriage rate compared with other countries; and day-care centre attendance appeared as an important associated factor. Capsule types a and b, associated with invasive infection, were commonly observed in our healthy population. Further studies are required to describe the genotypes and the antibiotic susceptibility patterns of the K. kingae carriage strains. C. Levy reports grants, personal fees and non-financial support from Pfizer, and grants from GSK, Sanofi and Merck, outside the submitted work; S. Béchet reports grants from Pfizer, GSK, Sanofi and Merck, outside the submitted work; and R. Cohen reports grants, personal fees and non-financial support from Pfizer, and grants and personal fees from GSK, Merck and Sanofi, outside the submitted work. The other authors have nothing to disclose. This work was supported by the Association Clinique et Thérapeutique Infantile du Val de Marne. The funder played no role in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication.