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Serotype distribution of Streptococcus pneumoniae strains in the nasopharynx of healthy Turkish children.

Background & objectives: There is scanty information available on the pharyngeal carriage of Streptococcus pneumoniae and serotype distribution in healthy Turkish children. We therefore undertook this study to determine the serotype prevalence of pharyngeal S. pneumoniae Isolates of healthy school children in Turkey.

Methods: Pharyngeal swabs were collected from 1440 healthy children (Ages 6-13) between April 2003 and March 2004. S. pneumoniae was identified by standard microbiological culture methods. The serotyping was performed and penicillin minimal inhibitory concentration was detected by the E test.

Results: S. pneumoniae carrier rate was 13.9 per cent (n=201) and penicillin resistance 12.9 per cent (n=26). Twenty four of 201 (11.9%) isolates were found intermediately resistant and 2 of 201 (1%) highly resistant by E test. A total of 169 of 201 (84%) pneumoeoccal Isolates were typable with the available antisera. The six most frequent serotypes were 6, 19, 1, 23, 20 and 17. The majority of penicillin-resistant isolates were serotypes 20, 23, 14, 6 and 19.

Interpretation & conclusion: The serotype distribution of the isolates showed diversity and that some common circulating serotypes are invasive. A majority of invasive serotypes are covered by pneumococcal conjugate vaccine formulations in at risk groups of children suggesting that vaccine use could reduce the incidence of invasive pneumococcal disease.

Key words Penicillin resistance--pharyngeal carriage--serotyping--Streptococcus pneumoniae--Turkey

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The nasopharyngeal carriage of Streptococcus pneumoniae is an important risk factor for pneumococcal diseases. Pharyngeal S. pneumoniae carriage and serotype distribution in healthy Turkish children have been established in a few studies (1-3). Nasopharyngeal carriage rate was 8.5 (2) and 15.8 per cent (1), carriage rates for penicillin resistant Pneumococcus 2.7 (2), 2.7 (3), 7 per cent (1). Serotypes 6, 23, 19 and 9 were found most frequent (2).

It is known that serotype distribution of strains causing invasive disease, nasopharyngeal colonization and antibiotic resistance are linked to age, geography, and socio-economic conditions of that population. Analysis of the leading serotypes in a particular area is important to evaluate the efficacy of vaccines (4-6). Therefore, serotyping of S. pneumoniae isolates has become more important to monitor the serotypes prevalent in the general population and to track resistance patterns.

Two vaccines for pneumococcal infections are currently available: the older 23-valent polysaccharide vaccine [PPV; Pnu-Imune 23 (1, 2, 3, 4, 5, 8, 9, 12, 14, 17, 19, 20, 22, 23, 26, 34, 43, 51, 54, 57, 68 and 70)] and the 7-valent pneumococcal conjugate vaccine [PCV; Prevnar (14, 6B, 19F, 18C, 23F, 4 and 9V)], was approved for use in 2000. 9-valent vaccine contains additional serotypes 1 and 5, and 11 valent vaccine in addition has serotypes 3, 7 and 6(7).

We carried out this study to assess the prevalence of S. pneumoniae serotypes in the nasopharynx of healthy school children in Mersin, Turkey.

Material & Methods

This cross-sectional study was undertaken during April 2003 and March 2004. Healthy children (ages 6-13) from centre of Mersin city were enrolled for the isolation of pharyngeal carriage of S. pneumoniae. 1440 healthy children out of a total of 2300 children from three primary schools were randomly selected in the centre of Mersin city. Permission was given for these 3 primary schools out of a total of 173 schools in the city. All children underwent a physical examination regarding upper respiratory tract infection. Children free from underlying chronic illness (immunologic diseases; neoplastic disorders; renal, cardiac, hepatic, or haematological diseases; bronchodysplasia; Down's syndrome; chronic otitis media with effusion) or even a mild acute upper or lower respiratory tract infection at the time of enrolment were considered eligible.

The study protocol was approved by institutional ethics committee of the Mersin University. It was also approved by the administrations of each school and written informed consent was obtained from the parents or supervisor of each child. The study group included 1440 school children without any respiratory infections (including rhinitis, tonsillitis, laryngitis, acute otitis media, sinusitis, acute bronchitis, and pneumonia) and no antibiotic treatment was given during the preceding three months.

Pharyngeal specimens were collected with throat swabs into anterior pillars of the oropharynx without touching the uvula or the tongue and tonsils, kept in Stuart transport medium (Oxoid Limited, Hampshire, England) which were sent to the microbiology laboratory of Department of Microbiology and Medical Microbiology, Mersin University within 1- 3 h and immediately processed.

Swabs were inoculated immediately onto trypticase soy agar plus (Oxoid Limited, Hampshire, England) 5 per cent sheep blood plates containing gentamicin (5 [micro]g/ml) and incubated at 35-37[degrees]C in 5-10 per cent C[O.sub.2]. After overnight incubation, colonies that were morphologically identical with S. pneumoniae (small, gray, and showing mucoid surrounded by a greenish zone of haemolysis) were subcultured onto the same media. Before incubation, a 6 mm 5 [micro]g optochin disk (Becton Dickinson Microbiology System, Cockeysville, MD, US) was placed aseptically, on the centre of the inoculum. Then the plates were incubated overnight in an atmosphere with 5 per cent C[O.sub.2]. Alpha haemolytic strains with a growth-inhibition zone of >14 mm in diameter, (susceptible to optochin) were considered as S. pneumoniae.

One or two colonies of presumptive pneumococci were selected and stored at -70[degrees]C in brain heart infusion (BHI) broth-glycerol stock solution [9.9% (wl/v) glycerol, 3.33% (w/v) BHI broth] (ARUP Laboratories Reagent Lab, USA) while the antibiotic susceptibility screening and serotype determination were carried out. Frozen isolates were subcultured onto Columbia agar with 5 per cent sheep blood (BD BBL, Sparks, MD, USA), and incubation for 24 h at 35[degrees]C in 5 per cent C[O.sub.2], when needed.

S. pneumoniae ATCC 49619 was used as control strain. Antimicrobial susceptibility of oxacillin (1 [micro]g) was determined by using of the Kirby-Bauer disk diffusion method according to The National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Penicillin resistant and intermediate strains were determined by the NCCLS guidelines (8), and were subjected to the E test (AB Biodisk, Sonla, Sweden) for the determination of minimum inhibitory concentration (MICs of the drug). The E tests were performed on Mueller-Hinton agar (Oxoid, Basingstoke, and Hampshire, United Kingdom) supplemented with 5 per cent defibrinated sheep blood. Inoculates were prepared in Mueller-Hinton broth by direct suspension of pneumococci colonies grown overnight on sheep blood agar to a density that matched a 0.5 McFarland opacity standard tube. Based on the results, isolates were interpreted as susceptible (<0.06 [micro]g/ml), intermediate (0.1-1 [micro]g/ml), or completely nonsusceptible (> 2 [micro]g/ml), according to the NCCLS protocol (8).

After isolates had been subcultured from the frozen BHI-glycerol stock solution onto Columbia agar with 5 per cent sheep blood, the working culture was used to perform slide agglutination, by using S. pneumoniae antisera and following the recommended procedure (supplied by Denka Seiken Co. Ltd., Japan). These isolates were tested one at a time, first with the polyvalent antisera and then with the respective monovalent antisera included in the polyvalent serotype that showed agglutination. The typing schema for the slide agglutination method consists of 8 polyvalent and 15 monovalent serotype antisera. (Table I).

To check for spontaneous agglutination of the bacterial cells, phosphate-buffered saline was tested with each isolate. A glass slide, divided into partitions was used for testing. One drop of each of the eight polyvalent antisera and a drop of phosphate buffered saline were placed on a partition of the slide. A 10 [micro]l loopful of bacterial cells was then placed into each partition and mixed with a loop using a new sterile loop with each respective antiserum. The slide was rotated by hand for 30 secs. Coarse aggregation of the bacterial cells with a clear background was considered a positive result for the antiserum showing agglutination. A homogenous emulsion of bacterial cells was interpreted as a negative result. The serotype obtained at least two of the three times was used for the analysis, and final results were recorded as matching, discordant, or unable to type (10).

Statistical analysis: Differences between groups were examined by [chi square] and Fisher's exact test. P < 0.05 was considered as significant.

Results & Discussion

Median age (range) of participants at elementary schools was 8.8 yr (6-13 yr). S. pneumoniae carriage Was detected in 201 (13.9%) children. Of the 201 S. pneumoniae isolates, 26 (12.9%) were resistant to penicillin. The rate of penicillin resistance was low (12.9%); the prevalence of high level resistance to penicillin was low (1.0%) (n=2), and the intermediate level penicillin resistance was 11.9 per cent (n=24).

Of the 201 isolates, 169 (84%) were serotyped with the available (1,2,3,4,5,6,7,8,14,17,19,20,22, 23,34) antisera. Serotypes 6 (n=19), 19 (n=18) and 23 (n=17), were found frequently (Table II), followed by serotypes 1 (n=16), 20 (n=15) and 17 (n=14). No statistically significant difference was found for the serotypes and age and sex.

Thirty two isolates (16%) could not be serotyped due to cross reactions with more than one antisera and non availability of the antisera for some. The majority of penicillin-resistant isolates were serotypes 20, 23, 14, 6 and 19. Among high level resistance to penicillin, the most predominant serotype was 19.

S. pneumoniae pharyngeal carriage was seen in 12.9 per cent of the healthy children in the present study to earlier reports (1,2,11). The serotypes most frequently colonizing in healthy population (3, 19F, 23F, 19A, 6B and 14) were those commonly involved in invasive pneumococcal diseases, highlighting the importance of nasopharyngeal colonization in the development of serious community infections (12).

Previous studies on among healthy Turkish infants and children have found 6, 23, 19 and 9 as the most common nasopharyngeal serotypes. In our study, most frequent serotypes were 1, 6, 19, 23, 20 and 17 in healthy school children. These serotypes are commonly involved in invasive pneumococcal diseases, highlighting the importance of nasopharyngeal colonization in the development of serious community infections.

In Hungary, the dominant serotype among penicillin-resistant strains has been reported to be 1913. In France, Maugein et a1 (14) found serotypes 14 and 23 as the most common penicillin-resistant serotypes. Syrjanen et a1 (15) reported 6B, 6A, 11, 19F and 23F, as the most common nasopharyngeal serotypes in children. Serotypes 6 (28%), 23 (28%), 19 (17%) and 9 (12%) were found as dominant penicillin-resistant S. pneumoniae among healthy Turkish infants and children (2).

The serotypes included in the conjugate vaccine are involved in about 80 per cent of invasive pneumococcal infections in children younger than 6 yr (16).

All serotypes are not completely covered by vaccine formulations (the 23 valent polysaccharide vaccine, 7-valent, 9-valent and 11-valent). However, penicillin-resistant and invasive serotypes are covered by the same formulations underlying the importance of serotyping of pathogenic strains in our region.

In conclusion, the present study suggests that the serotype distribution of the isolates showed diversity and most of the S. pneumoniae isolates circulating in the population were invasive. A majority of invasive serotypes are covered by pneumococcal conjugate vaccine formulations in risk groups of children suggesting that vaccine use could reduce the incidence of invasive pneumococcal disease.

Acknowledgment

Authors acknowledge the financial support received from the Research Foundation of Mersin University, Mersin, Turkey (No. BAP-TF-TTB-GA-2006-I).

Received November 29, 2005

References

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(7.) Zimmerman RK. Pneumococcal conjugate vaccine for young children. Am Fam Physician 2001; 63 : 1991-8.

(8.) National Committee for Clinical Laboratory Standards, Performance standards for antimicrobial disk susceptibility test-sixth edition: Approved standard. NCCLS document M2-A6. N. Pennsylvania. 2004.

(9.) Jorgensen JH, Howell AW, Maher LA. Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae by using the E-test. J Clin Microbiol 1991; 29 : 109-14.

(10.) Shutt CK, Samore M, Carroll KC. Comparison of the Denka Seiken slide agglutination method to the quellung test for serotyping of Streptococcus pneumoniae isolates. J Clin Microbiol 2004; 42 : 1274-6.

(11.) Bayer A. Antibiotic resistance of streptococcus pneumoniae strains isolated from nasopharynx in healthy school children in Mersin district. Department of Microbiology and Medical Microbiology, Mersin University Faculty of Medicine, Mersin. Doctor of Medicine Thesis, 2004.

(12.) Marchisio P, Esposito S, Schito GC, Marchese A, Cavagna R, Principi N. Hercules Project Collaborative Group. Nasopharyngeal carriage of Streptococcus pneumoniae in healthy children: implications for the use of heptavalent pneumococcal conjugate vaccine. Emerg Infect Dis 2002; 8 : 479-84.

(13.) Dobay O, Rozgonyi F, Hajdu E, Nagy E, Knausz M, Amyes SG. Antibiotic susceptibility and serotypes of Streptococcus pneumoniae isolates from Hungary. J Antimicrob Chemother 2003; 51 : 887-93.

(14.) Maugein J, Guillemot D, Dupont MJ, Fosse T, Laurans G, Roussel-Delvallez M, et al. Clinical and microbiological epidemiology of Streptococcus pneumoniae bacteremia in eight French counties. Clin Microbiol Infect 2003; 9 : 280-8.

(15.) Syrjanen RK, Kilpi TM, Kaijalainen TH, Herva EE, Takala AK. Nasopharyngeai carriage of Streptococcus pneumoniae in Finnish children younger than 2 years old. J Infect Dis 2001; 184: 451-9.

(16.) Laval CB, de Andrade AL, Pimenta FC, de Andrade JG, de Oliveira RM, Silva SA, et al. Serotypes of carriage and invasive isolates of Streptococcus pneumoniae in Brazilian children in the era of pneumococcal vaccines. Clin Microbiol Infect 2006; 12 : 50-5.

Reprint requests: Dr. Gonul Asian, Mersin Universitesi Tip Fakuitesi, Mikrobiyoloji ve Klinik Mikrobiyologi Anabilim, Dali 33169 Mersin, Turkey e-mail: dragaslan@gmail.com

Gonul Aslan, Gurol Emekdas, Mujgan Bayer, Mehmet Sami Serin, Necdet Kuyucu & Arzu Kanik

Department of Microbiology & Medical Microbiology, Mersin University Faculty of Medicine Mersin, Turkiye
Table I. Denka Seiken slide agglutination polyvalent
and monovalent serotype scheme

Polyvalent serogroup Monovalent serotype

1 1,2,3,4,5
2 6,8,9,10
3 11,12,14,15,16
4 17,18,21,22
5 20,29,31,33,34,35,47
6 23,25,28,41,46
7 27,32,36,38,39
8 7,19,24,40

Table II. Distribution of serotypes of Streptococcus
pneumoniae strains of Mersin city

 Number of
 strains

Serotype n %

1 16 9.46
2 7 4.14
3 9 5.32
4 6 3.55
5 8 4.73
6 19 11.24
7 3 1.77
8 5 2.95
14 8 4.73
17 14 8.28
19 18 10.65
20 15 8.82
22 9 5.32
23 17 10.05
34 15 8.87
Total 169
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Author:Aslan, Gonul; Emekdas, Gurol; Bayer, Mujgan; Serin, Mehmet Sami; Kuyucu, Necdet; Kanik, Arzu
Publication:Indian Journal of Medical Research
Geographic Code:7TURK
Date:Apr 1, 2007
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