Nasal carriage of Streptococcus pneumoniae in healthy children and adults in northern Taiwan

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Diagnostic Microbiology and Infectious Disease 59 (2007) 265 – 269 www.elsevier.com/locate/diagmicrobio

Nasal carriage of Streptococcus pneumoniae in healthy children and adults in northern Taiwan Chih-Jung Chen a,c , Yhu-Chering Huang a,⁎, Lin-Hui Su b , Tzou-Yien Lin a a

Division of Pediatric Infectious Diseases, Department of Pediatrics, Chang Gung ChildrenTs Hospital, Kweishan 333, Taoyuan, Taiwan b Department of Clinical Pathology, Chang Gung Memorial Hospital, Kweishan 333, Taoyuan, Taiwan c Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Kweishan 333, Taoyuan, Taiwan Received 20 March 2007; accepted 22 May 2007

Abstract A total of 427 subjects were surveyed for the carriage of Streptococcus pneumoniae before the introduction of pneumococcal conjugate vaccine in northern Taiwan. Positive results were identified in 27% of 94 children attending 6 classes in a child care center (CCC), 14% of 122 pupils attending 4 classes in 2 elementary schools, and 1.4% of 74 adolescents attending 3 classes in a junior middle school. None of 137 health care workers working in neonatal intensive care units of a university-affiliated hospital was colonized with S. pneumoniae. Three (12%) of 25 isolates from the children attending the CCC were susceptible to penicillin (MIC b0.1 μg/mL), whereas 8 (44%) of 18 isolates from the pupils and adolescents were susceptible (P = 0.0312). Of the 43 isolates, 11 serotypes were identified and serotypes/serogroups 6 (13 isolates), 23 (10 isolates), and 19 (7 isolates) were the 3 most common serotypes/serogroups. The genotypes of the 43 isolates were diverse, but it was not infrequently seen that several isolates from the subjects in a single class shared a common genotype. Conclusively, the carriage rate of S. pneumoniae is inversely correlated with the age of the subjects. The isolates from the children attending the CCC had a significantly higher penicillin-nonsusceptible rate than those from the pupils and adolescents. Transmission of the same clone in a single class may have frequently occurred. © 2007 Elsevier Inc. All rights reserved. Keywords: Streptococcus pneumoniae; Nasal carriage; Taiwan

1. Introduction Colonization of Streptococcus pneumoniae in the nasopharynx is believed to be an important reservoir for community-wide spread of this pathogen and a key factor preceding pneumococcal diseases (Gray et al., 1980). The carriage rate in children is affected by the environmental and socioeconomic factors including number of siblings, income, exposure to antibiotics, parental smoking, and day-care center attendance (Bogaert et al., 2004a; Ghaffar et al., 1999). In individual host, the colonization mainly depends on age. Several studies have shown that age younger than 2 years is associated with the highest carriage rate (Sung et al., 1995; Ussery et al., 1996; Yagupsky et al., 1998). The rate declines thereafter to a stable and low prevalence in

⁎ Corresponding author. Tel.: +886-3-3281200; fax: +886-3-3288957. E-mail address: [email protected] (Y.-C. Huang). 0732-8893/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2007.05.012

adolescents (Bogaert et al., 2004b). In contrast to abundant information in the children, there were few data focused on adulthood pneumococcal carriage, and even fewer studies investigated the carriage in both pediatric and adult population simultaneously (Regev-Yochay et al., 2004). In Taiwan, 60% to 84% of clinical isolates of S. pneumoniae were nonsusceptible to penicillin (Hsueh and Luh, 2002). The isolates from the children had a significantly higher rate of resistances to β-lactams and other non–β-lactam antibiotics than those from the adults (Chen et al., 2006; Chiou et al., 1998; Siu et al., 2002). There has been no report regarding the carriage of S. pneumoniae among adult population in Taiwan. During 2001 and 2002, we conducted 2 studies to investigate the extent of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in the community of northern Taiwan (Huang et al., 2005; Huang et al., 2004). The carriage rates of S. pneumoniae among these healthy children and adults were also assessed before the introduction of heptavalent

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pneumococcal conjugate vaccine (PCV-7). The microbiologic characteristics of these isolates were determined and compared. 2. Materials and methods Between November 2001 and June 2002, a survey for nasal carriage of S. pneumoniae and S. aureus was conducted among 290 school children after written informed consents were obtained from their guardians. The subjects included 94 of 121 children aged 3 to 4 years attending 6 classes (class A–F, Table 1) at a child care center (CCC) affiliated to a tertiary care hospital (Chang Gung Memorial Hospital, Taiwan); 122 of 134 pupils attending 2 classes (classes G and H) of grade 2, 1 class (class I) of grade 5, and 1 class (class J) of grade 6 in 2 municipal elementary schools; and 74 of 115 adolescents attending 3 classes of grade 8 at a municipal junior middle school. All 4 schools are in northern Taiwan. A total of 137 health care workers (HCWs) working in the neonatal intensive care units of Chang Gung Children's Hospital, including 20 physicians, 87 nurses, 27 respiratory therapists, and 3 clerks, were also sampled during October 2003 for a surveillance culture survey. A culture of both sides of the anterior nares was obtained from each subject. For school children, all cultures were taken on a single visit to each school. Specimen for culture were obtained with a cotton swab, placed in the transport medium (Venturi Transystem; Copan Innovation, Limmerick, Ireland), and then transported to and processed in our microbiologic laboratory within 4 h. Isolates of S. aureus and S. pneumoniae were identified according to the standard

methods (Clinical and Laboratory Standard Institute, 2006). S. aureus was identified by coagulase testing. Identification of S. pneumoniae isolates was confirmed by optochin sensitivity and bile solubility test. All the colonizing isolates were frozen at −70 °C until use. The MICs of penicillin, cefuroxime, ceftriaxone, and teicoplanin to S. pneumoniae isolates were determined by Etest (AB Biodisk, Solna, Sweden) according to the manufacture's instructions. The Clinical and Laboratory Standards Institute (CLSI) (2006) breakpoints for meningitis were used to interpret the E-test result, and the susceptibility to erythromycin and vancomycin was determined by the disc diffusion method according to CLSI guideline. Pulsed-field gel electrophoresis (PFGE) was used to fingerprint all S. pneumoniae isolates according to the procedure described previously (Hsieh et al., 2004). Briefly, the DNA was digested with SmaI. Bands were stained with ethidium bromide and visualized with ultraviolet light. PFGE patterns with a difference of N3 bands were considered to be distinct. The PFGE types were designated with Arabic numbers consecutively. The S. pneumoniae isolates were tested at the Statens Seruminstitut (Copenhagen, Denmark) using duplicate slide agglutination assays with capsular group-specific antisera to determine the serotype/serogroup. The categoric variables were compared by χ2 or Fisher exact test where appropriate. The trends of prevalence rate of S. pneumoniae and S. aureus nasal carriage in children from the 3 levels of schools and in HCWs were tested by the Mantel–Haenszel χ2 method using SAS 8.1 (Stokes et al., 2000). Statistical significance was determined at P b 0.05.

3. Results Table 1 The nasal carriage rate of S. pneumoniae and S. aureus in previously healthy children and health care workers in northern Taiwan Category

Subject no.

S. pneumoniae carriage no. (%)

Children care center Class A Class B Class C Class D Class E Class F Elementary schools a Class G (grade 2) Class H (grade 2) Class I (grade 5) Class J (grade 6) Junior middle school Class K (grade 8) Class L (grade 8) Class M (grade 8) HCWs b Total

94 15 20 16 15 11 17 122 32 31 28 31 74 15 28 31 137 427

25 (26.6) 0 9 (45) 3 (18.8) 4 (26.7) 3 (27.2) 6 (35.3) 17 (13.9) 5 (15.6) 6 (19.4) 3 (10.7) 3 (9.7) 1 (1.35) 1 (6.7) 0 0 0 43 (10.0)

S. aureus carriage no. (%) 20 (21.3)

57 (46.7)

32 (43.2)

38 (27.7) 147 (34.4)

a Class G, H, and J belonged to elementary school I; class I belonged to elementary school II. b HCWs in the neonatal intensive care units.

Of the 427 subjects enrolled, 43 (10.0%) were colonized with S. pneumoniae and 147 (34.4%) were colonized with S. aureus. The detailed carriage rates of S. aureus and MRSA among the subjects were shown elsewhere (Huang et al., 2005; Huang et al., 2004), and for contrast, the carriage rates of S. aureus among the subjects are presented in Table 1 partly. Table 1 also illustrates the detailed carriage rates of S. pneumoniae among the subjects. The carriage rate of S. pneumoniae was inversely correlated with the age of the subjects, and the carriage rate was highest (26.6%) in the young children attending the CCC, declined in pupils (17.5% in grade 2 and 10.2% in grades 5 and 6), and reached an extremely low rate in adolescents (1.35%) and the HCWs (0%). The trend of the carriage rate decline with age was highly significant (P b 0.0001). For S. aureus, there was also an age-related distribution, with a higher incidence in the pupils and adolescents compared with the young children and the HCWs (P b 0.0001). All of the 43 S. pneumoniae isolates were susceptible to vancomycin and teicoplanin but resistant to erythromycin (Table 2). Only 11 (25.6%) isolates were susceptible to penicillin. The resistance rates were also high for cefuroxime

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Table 2 Antimicrobial susceptibility of 43 colonized S. pneumoniae isolates from healthy Taiwanese school children Drug

No. of isolates (%) All isolates (n = 43)

Penicillin Vaccine serotypes b Nonvaccine serotypes Cefuroxime Ceftriaxone Erythromycin Teicoplanin Vancomycin

CCC isolates (n = 25)

Pupil and adolescent isolates (n = 18)

S

I

R

S

I

R

S

I

R

11 (25.6) 2 (18.2) 9 (81.8) 18 (41.9) 24 (55.8) 0 43 (100) 43 (100)

20 (46.5) 11 (55.0) 9 (45.0) 1 (2.3) 19 (44.2) 1 (2.3) 0 0

12 (27.9) 11 (91.7) 1 (8.3) 24 (55.8) 0 42 (97.7) 0 0

3 (12.0) a 0 3 (100) 6 (24.0) a 10 (40.0) a 0 25 (100) 25 (100)

12 (48.0) 8 (66.7) 4 (33.3) 0 15 (60.0) 0 0 0

10 (40.0) 9 (90.0) 1 (10.0) 19 (76.0) 0 25 (100) 0 0

8 (44.4) a 2 (25.0) 6 (75.0) 12 (66.7) a 14 (77.8) a 0 18 (100) 18 (100)

8 (44.4) 3 (37.5) 5 (62.5) 1 (5.6) 4 (22.2) 1 (5.6) 0 0

2 (11.1) 2 (100) 0 5 (27.8) 0 17 (94.4) 0 0

S = sensitive; I = intermediate resistant; R = resistant. a Isolates from pupils and adolescents were more frequently susceptible to penicillin (P = 0.0312), cefuroxime (P = 0.0051), and ceftriaxone (P = 0.00281) than isolates from children attending child care center. b Strains with serotypes covered by the 7-valent (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) pneumococcal conjugate vaccine.

(58.1%) and ceftriaxone (44.2%). Of the 43 isolates, 26 (60.5%) were resistant to 3 or 4 antibiotics (Table 3). When compared with the isolates from the children attending CCC, the isolates from pupils and adolescents were more frequently susceptible to penicillin (44.4% versus 12.0%, P = 0.0312), cefuroxime (66.7% versus 24.0%, P = 0.0051), and ceftriaxone (77.8% versus 40.0%, P = 0.0281) (Table 2). Multidrug (≥3 antibiotics) resistance was more commonly seen in the isolates from CCC than those from pupils and adolescents (80% versus 33.3%, P = 0.004, Table 3). The detailed PFGE pattern and serotype of each isolate are displayed in Fig. 1. A total of 11 serotypes and 18 genotypes were identified among the 43 S. pneumoniae isolates. Twenty-four (55.8%) isolates expressed the serotypes covered by the PCV-7. The carriage of vaccine-type strains declined from 17 (68%) of 25 isolates from the children attending CCC to 7 (39%) of 18 isolates from the pupils and adolescents with borderline significance (P = 0.0579). Serogroups 6 (14 isolates), 23 (10 isolates), and 19 (7 isolates) were the 3 most common serogroups identified. Among the 43 isolates, the PFGE patterns were diverse. The isolates with the same PFGE pattern were with the same serotype, whereas the isolates with the same serotype may be with different genotypes. However, it was not infrequently seen that several isolates from a single class shared a common PFGE pattern. Five of 6 isolates with serotype 6B from class B were found to be PFGE type 6, and 2 strains with serotype 6B from class D also shared a common PFGE type (type 35). The genetically similar strains were also identified in classes E and F in the CCC and classes G, H, and I in both elementary schools.

previous reports (Bogaert et al., 2004b; Gunnarsson et al., 1998). The extremely low carriage rate in the adolescents (1.4%) and even noncarriage identified in the adults, however, were unexpected. Transnasal route of nasopharyngeal sample has been the preferable sampling method for pneumococcal carriage in children (Bogaert et al., 2004a). Using the nasal swab samples may be considered inadequate, and the carriage rate could be therefore underestimated. However, the carriage rates of 13.9% and 26.6% in the pupils and the young children, respectively, were in agreement with 2 previous reports from Taiwan using nasopharyngeal samples (Chiou et al., 1998; Lo et al., 2003). In addition, parallel to the decline in pneumococcal colonization with age, we found a simultaneous increase in S. aureus colonization rate in children. The results were consistent with the observations in a study regarding the nasopharyngeal colonization of S. aureus and S. pneumoniae among healthy children in Netherlands (Bogaert et al., 2004b). Based on these findings, we believed that the carriage rate of S. pneumoniae presented here should reflect the true condition in Taiwan and not be affected substantially by the sampling method. Table 3 Distribution of 43 colonizing S. pneumoniae isolates according to antibioticresistant profiles Resistant profile

No. of isolates (%) All isolates (n = 43)

CCC isolates (n = 25)

Pupil and adolescent isolates (n = 18)

18 7 1 6 11

14 (56.0) 5 (20.0) 1 (4.0) 2 (8.0) 3 (12.0)

4 2 0 4 8

4. Discussion

ERY–PEN–CXM–CRO ERY–PEN–CXM ERY–PEN–CRO ERY–PEN ERY

(41.9) (16.3) (2.3) (14.0) (25.8)

(22.2) (11.1) (0) (22.2) (44.4)

Results from this study disclosed that an inverse correlation existed between the carriage rate of S. pneumoniae and the age of subjects, which were compatible with

Multidrug (≥3 antibiotics) resistance was more commonly identified in the isolates from young children than in the isolates from pupils and adolescents (20/25 versus 6/18, P = 0.004). ERY = erythromycin; PEN = penicillin; CXM = cefuroxime; CRO = ceftriaxone.

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Rodriguez and Fresnadillo Martinez, 2002; Ghaffar et al., 1999). We detected a high prevalence rate (74.4%) of penicillinnonsusceptible S. pneumoniae isolates among the colonization isolates from Taiwanese children. The highly penicillinresistant strains (MIC ≥2 μg/mL) were not uncommon and accounted for 28% of the colonization strains. The rates of resistance to erythromycin (100%) and ceftriaxone (44.2%) were even more noteworthy. The subsidiary analysis disclosed that the majority (69%) of the resistant strains (and highly resistant strains) belonged to the serotypes covered by PCV-7. On the contrary, 82% of the isolates with nonvaccine serotypes were susceptible to penicillin (Table 2). CCCs attendance is a significant risk factor associated with pneumococcal colonization and carriage of resistant strains (Finkelstein et al., 2003). By serotyping and genotyping, the transmission of the same clone may have occurred in 14 of 25 pneumococcal isolates from young children attending 4 of the 6 classes in the CCC. Furthermore, the presumed intraclass transmission occurred primarily in drug-resistant vaccine-serotype strains including serotype 6B, 19F, and 23F (Table 3). Active immunization with currently licensed PCV-7 incorporating these serotypes seemed promising in reducing the horizontal spread of the resistant strains in young children and further decreasing the incidence of pneumococcal diseases. PCV-7 has been shown to reduce the acquisition and carriage of vaccine serotypes S. pneumoniae in the children (Dagan et al., 1996). The herd immunity of the PCV via interruption of the transmission of the vaccine-serotypes was also demonstrated by several large-scale surveillance studies of invasive pneumococcal diseases in the United States after universal vaccination (Kyaw et al., 2006; Poehling et al., 2006). The introduction and mass vaccination of PCV-7 in Taiwan may stop the clonal spread of S. pneumoniae-resistant strains and decrease the burden of antibiotic resistance. Fig. 1. Phylogenetic tree of 43 colonizing S. pneumoniae isolates. PFGE patterns were digitalized and analyzed with BioNumerics Fingerprint types and Cluster Analysis software (Applied Maths, Austin, TX). The scale above the dendrogram shows the percentage of similarity among different strains. The first letter of each strain designation indicates the class from which the strain was isolated (Table 1 for reference). The squares represent presumed intraclass transmission of 20 isolates.

Acknowledgment This study was supported by a grant from Chang Gung Memorial Hospital, Taiwan (CMRPG33128). References

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