Eur J Pediatr (2015) 174:373–381 DOI 10.1007/s00431-014-2408-1
ORIGINAL ARTICLE
Radical serotype rearrangement of carried pneumococci in the first 3 years after intensive vaccination started in Hungary Adrienn Tóthpál & Szilvia Kardos & Krisztina Laub & Károly Nagy & Tamás Tirczka & Mark van der Linden & Orsolya Dobay
Received: 16 June 2014 / Revised: 6 August 2014 / Accepted: 19 August 2014 / Published online: 2 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Streptococcus pneumoniae is responsible for a significant amount of morbidity and mortality worldwide. Healthy carriers, mainly young children, are the most important sources of infections. In the current study, we aimed to determine the changes that have occurred since the introduction of PCV-7 in Hungary. Nasal specimens were collected from 1,022 healthy children aged 3–6 years attending daycare centres. After thorough identification, pneumococcal isolates were serotyped, and their antibiotic sensitivity was determined. The carriage rate was found to be 34.9 %. A huge serotype rearrangement was detected compared to earlier results, with the previously leading serotype 14 having completely disappeared. Serotypes 11A, 35F, 19A, 6B, 15B, Communicated by Peter de Winter A. Tóthpál : S. Kardos : K. Laub : K. Nagy : O. Dobay (*) Institute of Medical Microbiology, Semmelweis University, BudapestNagyvárad tér 4, 1089, Hungary e-mail: [email protected]
3 and 38 were most prevalent, and 29 different types were identified in total. The PCV-7 types were responsible for 16.5 % of all serotypes, and 36.0 % are not covered by any pneumococcal vaccines. The isolates were sensitive to most tested antibiotics, except erythromycin (resistance was 21.6 %). Only one penicillin-resistant strain was found. The newly and rapidly emerging non-vaccine serotypes are much more sensitive, except serotype 19A. Conclusion: Due to PCV vaccination, a complete serotype arrangement occurred also in Hungary. The old “paediatric” serotypes were replaced by serotypes 11A, 35F, 19A, 6B, 15B, 3 and 38. Keywords Pneumococcus . PCV-7 vaccination . Carriage . Serotype changing . Day-care centres
K. Nagy e-mail: [email protected]
Abbreviations CDC Centers for Disease Control and Prevention DCC Day-care centre EUCAST The European Committee on Antomicrobial Susceptibility Testing GNRCS German National Reference Centre for Streptococci NVT Non-vaccine type PCV-7 7-valent pneumococcal conjugate vaccine PCV-13 13-valent pneumococcal conjugate vaccine VT Vaccine type
T. Tirczka Pneumococcal Reference Laboratory, National Center for Epidemiology, Budapest, Hungary e-mail: [email protected]
Introduction
M. van der Linden German National Reference Center for Streptococci; Department of Medical Microbiology, University Hospital RWTH Aachen, Aachen, Germany e-mail: [email protected]
Streptococcus pneumoniae is one of the most important bacterial pathogens worldwide, causing a wide range of illnesses from milder infections to life-threatening invasive diseases and is responsible for the death of nearly 1 million
A. Tóthpál e-mail: [email protected] S. Kardos e-mail: [email protected] K. Laub e-mail: [email protected]
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children per year, especially due to severe pneumonia [30]. Pneumococci often colonise the nasopharynx; it is estimated that all healthy individuals become carriers for shorter or longer periods during life. Under certain circumstances, pneumococci can spread to the mucosa causing respiratory tract infections (sinusitis, otitis media) or enter the normally sterile body fluids causing sepsis and/or meningitis. Additionally, the bacterium can be transmitted to other susceptible persons in the community (e.g. older family members); therefore, carriers represent the most important natural reservoir for pneumococcal infections [2, 8, 28]. Carriage peaks in early childhood, and children attending day-care centres (DCCs) are shown to have significantly higher colonisation rates [8, 10]. A study from the Netherlands demonstrated a 1.6 times higher rate in DCC attendees compared to those staying at home [1]. To prevent pneumococcal infections, several attempts were made to develop a vaccine, based on the major protective antigen of the bacterium, the polysaccharide capsule. Currently, two types of vaccines are available. The 23-valent polysaccharide vaccine (Pneumovax or PPV, Merck) has been in use since 1982, containing 23 of the >90 currently known capsular polysaccharides. As children younger than 2–2.5 years are not able to develop a T cell-dependent immune response against polysaccharides, a conjugate vaccine (PCV-7) was developed for them, which was licenced in 2000 in the USA and later in Europe. In recent years, higher-valent vaccines have followed (PCV-10 by GSK and PCV-13 by Pfizer). Conjugate vaccines have been proven to have an impact not only on pneumococcal diseases but also on nasopharyngeal carriage [15], hence, cutting the infection chain in the beginning, providing an indirect population-wide effect [27]. In Hungary, PCV-7 was introduced in 2005, but in the first 3 years, only very few children obtained vaccination due to a high price and a low awareness. In October 2008, PCV-7 was made freely available for children under the age of 2 years in the framework of a pneumococcus surveillance programme, and in April 2009, it was included in the national immunisation programme as a recommended but not obligatory vaccine in the 2+1 scheme. The vaccination rate increased to >80 % within a year, and it has reached nearly 100 % by now [18, 21]. In September 2010, PCV-13 replaced PCV-7 in Hungary, and PCV-13 was fully integrated in the national immunisation programme on 1 July 2014. In a previous project, we had surveyed nasal carriage of S. pneumoniae in healthy children attending day-care centres, just before widespread vaccine uptake started [36]. The aim of the current study was to make a comparison of the serotype distribution and vaccine coverage between the pre- and postvaccination eras in Hungary.
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Materials and methods Study population The study involved 1,022 healthy children aged between 3 and 6 years, attending 18 DCCs, located in different regions of Hungary, between February 2010 and February 2012. Informed consent obtained from the parents was a condition for enrolment. Besides informing the parents about the purpose of the study, data were asked about the children’s vaccination status, siblings, history of recurrent otitis media or other severe infections and smoking in the family. The number of the ethical permit for the study is TUKEB 4-3/2009, issued by the Regional and Institutional Committee of Science and Research Ethics of Semmelweis University. For statistical analysis, as appropriate, the chisquare test was used. Specimen collection Nasal samples were collected from both nostrils of the children with sterile cotton swabs. The samples were transported to the laboratory on active charcoalcontaining transport media (Transwab, Medical Wire & Equipment, Corsham, UK) and were immediately inoculated onto Müller-Hinton blood agar plates. Samples were incubated overnight at 37 °C in 5 % CO2 atmosphere. Identification of pneumococci Suspected colonies showing typical colony morphology of S. pneumoniae (α-haemolysis and flat colonies collapsed in the middle) were subcultured and further tested for optochin sensitivity (5 μg discs, Mast Diagnostica, Bootle, UK). The identity of the strains was confirmed by detecting the lytA (autolysin) gene by PCR in every case [17], and confirmed strains were stored at −80 °C on cryobeads (Mast Diagnostica). Antibiotic susceptibility testing The antibiotic sensitivity of the strains to penicillin, cefotaxime, imipenem, erythromycin, telithromycin, clindamycin, levofloxacin, moxifloxacin and vancomycin was determined by the agar dilution method using an A400 multipoint inoculator (AQS Manufacturing Ltd., Southwater, UK) and by E-test (Liofilchem, Roseto, Italy), on Müller-Hinton blood agar plates. Incubation was done at 37 °C in 5 % CO2, and ATCC 49619 was used as control strain. The susceptibility and resistance rates were determined using the breakpoints recommended by the EUCAST guidelines [33]. As our collection consisted of carried strains (i.e. non-meningitis cases), we used the ≤0.06/>2 breakpoints for penicillin. Macrolide resistant strains were tested for the presence of the erm(B) and mef genes [11, 32], and the distinction between mefA and mefE was carried out by BamHI digestion, which generates two fragments in mefA, but none in mefE, as described before [11, 26]. Serotyping Determination of the serogroups was done by the Pneumotest-Latex kit (Statens Serum Institut, Copenhagen,
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Denmark) using antisera, and the factor determination was done by PCR using primers described by the CDC [6], or others [3]. Several strains were sent to the German National Reference Centre for Streptococci (GNRCS; Aachen) or to the Hungarian National Center for Epidemiology for further serotyping.
Results Carriage rate and vaccination status Among the 1,022 children, 357 were colonised with pneumococcus; this means a carriage rate of 34.9 %. Although the ratio of genders was nearly equalised (51.4 % males), the carriage rate was slightly higher among males: 36.8 versus 33.0 % in females (statistically not significant, p = 0.207). In one occasion, we detected double carriage: in a boy previously vaccinated with Pneumovax, one strain each of serotypes 3 and 34 was present; therefore, the number of pneumococcal isolates is 358. The degree of colonisation varied widely from pure pneumococcal culture to just a few colonies among the normal flora. In the examined age group of 3–6 years, carriage peaked at 3 years with 43.3 % (Fig. 1). As the isolates of this study were collected between January 2010 and February 2012, from children aged 3 years or older, those who were vaccinated received PCV-7; hence, we could test the effect only of PCV-7. Out of all children, 393 (38.5 %) were vaccinated. However, it must be noted that we based our calculations on the parents’ positive answers, and often, they were not aware whether their children had received pneumococcal vaccination or not. Therefore, in this question, we only evaluated the sure-positive (n=393) or sure-negative (n=388) answers. Among the 393 PCV-7-vaccinated children, 156 (39.7 %) were carriers; this is a higher rate than that observed among the non-vaccinated children (128/388, 33.0 %), but the difference is not statistically significant (p=0.051). The coverage of the conjugate vaccines is much higher in the non-
Fig. 1 Carriage rates at different ages
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vaccinated group, (calculated with the Broome method [4], the PCV-7 efficacy would be 1–11/22.7=52 %), but this figure equalises for the Pneumovax coverage (Table 1). This phenomenon can easily be explained by the different serotype distribution: the vaccinated children characteristically carry non-vaccine serotypes (with the exception of serotypes 6B and 19A) (Table 2). Among the PCV-13 minus PCV-7 serotypes, 19A was represented in higher rate (10.3 %). Notably, out of the 27 serotype 6B isolates (which is a PCV-7 type), 14 were found in previously vaccinated (PCV-7 or Pneumovax) children. Based on the answers given by the parents in the questionnaire, it was demonstrated that those children who had otitis media and/or pneumonia/meningitis (possibly of pneumococcal origin) in the past were more frequently carriers (Table 3). Regarding the family-related factors, having siblings surprisingly showed a negative association with carriage, indicating that fellow children are more important sources of pneumococcal colonisation than older (school-age) or younger (before attending any community) siblings in the household. Furthermore, there was an only non-significant difference in the exposure to passive smoking, although this is usually an identifiable risk factor for carriage. High percentage (35.2 %) of all children were exposed to passive smoking (34.4 % of the carriers and 35.6 % of the non-carriers). Nonetheless, none of these correlations were statistically significant. Antibiotic susceptibility Only one single isolate (serotype 19A) was resistant to penicillin, but only at the lowest level (MIC=4 mg/L), while 78.4 % of all strains were fully susceptible (Table 4). No resistance was observed to cefotaxime, imipenem, vancomycin and moxifloxacin. On the other hand, 21.6 % of the isolates were resistant to erythromycin. Ten strains out of these showed the M phenotype (low-level resistance to erythromycin—MIC=4–16 mg/ L—and sensitivity to clindamycin); these all had the mefE gene, and five of these were serotype 6A. The majority, however, showed the macrolide-lincosamide-streptogramin B (MLSB) type (high-level resistance to both), and serotype 19A was dominant (54.0 %) in this group; all serotype 19A strains were ermB positive. Seven isolates possessed ermB and mefE together (four strains serotype 19A, three strains serotype 19F). In the case of telithromycin, the strains could be divided clearly into two distinct groups. Those that had a telithromycin MIC ≤0.03 mg/L were all macrolide sensitive, and none of them were serotype 19A. On the other hand, those with an MIC ≥0.06 mg/L contained all the macrolide resistant isolates, M types and MLSB types as well. Out of the 12 levofloxacin-resistant strains (MIC=4 mg/L), eight were serotype 11A.
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Table 1 Carriage rate and vaccine coverage of the vaccinated and non-vaccinated children Non-vaccinated n=388a
PCV-7 vaccinated n=393
Pneumovax vaccinated n=37b
All children n=1,022
PCV-7 coverage PCV-10 coverage
33.0 % (n=128) 22.7 % 23.4 %
39.7 % (n=156) 10.9 % 10.9 %
37.8 % (n=14) 4/15c 4/15
34.9 % (n=357) 16.5 % 16.8 %
PCV-13 coverage Pneumovax coverage Non-vaccine types
45.3 % 61.7 % 32.8 %
25.8 % 58.3 % 41.7 %
8/15 12/15 3/15
36.3 % 59.8 % 36.0 %
Carriage rate
a
only the surely negative answers were included in the calculation
b
out of the 37 children, 19 received both Pneumovax and PCV-7
c
one child carried 2 pneumococci
Serotypes and vaccine coverage The prevalence of the more frequent serotypes in ranking order was the following: 11A (n=49), 35F (n=33), 19A (n=31), 6B (n=27), 15B (n=27), 3 (n=24) and 38 (n=22), but 29 different serotypes were identified. The complete serotype distribution is shown in Fig. 2. All NT strains are truly non-typable as determined by the GNRCS. The calculated vaccine coverage is 16.5 % for PCV-7, 16.8 % for PCV-10 (the very small difference is due to the fact that we had no serotype 5 or 7F isolates, and only one single isolate of serotype 1—these are the additional serotypes belonging to PCV-10) and 36.3 % for PCV-13. Pneumovax would cover an additional 27.7 %, but it does not include serotype 6A, so the coverage for Pneumovax is 59.8 %. This means that 36.0 % of these carried strains are not covered by any of the currently available vaccines.
Table 2 Serotype distribution in the vaccinated and nonvaccinated carriers
a
only the surely negative answers were included in the calculation
b
one child carried 2 pneumococci
The isolates belonging to the leading serotype 11A were very sensitive to penicillin (most of them had MIC = 0.015 mg/L) and also to macrolides, but they had the highest levofloxacin MICs (4 mg/L). The serotype 19A isolates were (with a very few exceptions) macrolide resistant, and most of them had a penicillin MIC≥0.25 mg/L. Many of the serotype 6B strains had high-level macrolide resistance, while the 6A strains were mostly sensitive, or had the M phenotype, with a resulting low-level macrolide resistance. Isolates with elevated MICs to penicillin (intermediate category) had serotypes 19A, 19F, 6A, 6B, 23A, 23F and 35B. Serotypes 3, 15A, 15B, 23F, 35F and most of the “minor” types as well were fully sensitive to all tested antibiotics, with especially low MIC values.
Serotype
Non-vaccinated n=128a
PCV-7 vaccinated n=156
Pneumovax vaccinated n=14b
6B 18C 19F 23F 1 3 6A 19A 8 10A 11A
10 5 6 8 1 12 7 9 1 3 12
11 – 4 2 – 4 3 16 – 3 29
3 – 1 – – 2 1 1 – – 2
15B 22F 33F NVTs
10 2 – 42 (32.8 %)
14 5 3 55 (35.3 %)
1 1 – 3
PCV-7
PCV-13−PCV-7
Pneumovax−PCV-13
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Table 3 Differences in the possible risk factors usually associated with pneumococcal colonisation, among the carriers and non-carriers. The p value was calculated with the chi-square test. Carriers (n=357)
Non-carriers (n=665)
Otitis media in the past Yes 86 (38.6 %) 137 (61.4 %) No 272 (34.0 %) 527 (66.0 %) Pneumonia or meningitis in the past Yes 22 (40.0 %) 33 (60.0 %) No 336 (34.7 %) 631 (65.3 %) Having siblings Yes 251 (33.6 %) 495 (66.4 %) No 107 (38.8 %) 169 (61.2 %) Gender Male 193 (36.8 %) Female 164 (33.0 %) Passive exposure to smokinga Yes No
74 (34.7 %) 141 (35.8 %)
p value
0.211 (NS)
0.427 (NS)
0.128 (NS)
332 (63.2 %) 333 (67.0 %)
0.207 (NS)
140 (65.4 %) 253 (64.2 %)
0.766 (NS)
a
In this question, only 608 answers were collected (215 carriers and 393 non-carriers)
Discussion Carriage rate Compared to our previous study carried out a few years ago, just before conjugate vaccination started in Hungary [36], the carriage rate has decreased slightly from 37.7 to 34.9 %. This suggests that the conjugate vaccines have a non-significant reductive effect on the carriage rate; they rather influence the distribution of the carried serotypes. Other studies have also observed that despite conjugate vaccination, the overall carriage rate remained virtually unchanged [7, 25]. Comparing vaccinated and non-vaccinated children, we could in fact observe a higher carriage rate among the vaccinated ones. However, there was a significant difference in the distribution of serotypes, with much more NVTs in the
Table 4 Antibiotic susceptibility results of the 358 carried isolates (MIC in mg/L) Penicillin Cefotaxime Imipenem Erythromycin Clindamycin Telithromycin Levofloxacin Moxifloxacin Vancomycin
vaccinated children. Among the VTs, serotypes 6B and 19A were represented in higher rates. We detected double carriage only in one occasion. This low co-colonisation rate might be the result of the culture-base technique we applied. Several papers suggest that co-colonisation is often underestimated, and to reveal the real rates, more sophisticated methods such as multiplex PCR or microarray should be used [12, 37]. We found a slightly higher colonisation rate in males; this was also found by others, e.g. among Brazilian adolescents [5]. As these authors have also suggested, a higher pneumococcal carriage rate may be one of the reasons leading to the observations that male gender is a definitive risk factor for pneumonia [14]. The highest level of carriage was observed at 3 years of age (43.3 %), which declined to 18.4 % [5] by 6 years of age. This is in accordance with the results of a large-scale review, where the peak incidence was identified also at 3 years [2]. After that age, colonisation steadily declines to below 3 % by the age of 19 years [5]. The 20–40-year-old adults are least effected by pneumococcal infections, which start increasing above 50 years, but the source is not any more the self carriage, rather small children in the close environment. Antibiotic susceptibility The isolates in this study were generally sensitive to the drugs tested, except for the macrolides. Only one isolate (0.35 %) was resistant to penicillin (MIC= 4 mg/L), and 21.6 % were resistant to erythromycin. These are much lower figures compared to those of disease-causing strains in Hungary. According to the nationwide reporting system data maintained by the National Center for Epidemiology (NCE), penicillin resistance of clinical isolates has been varying between 1.5 and 5 % in the last 10 years (based on approximately 4,000 specimens annually), and the higher rates are observed in hospitalised patients and invasive specimens [24]. Regarding macrolide resistance of clinical isolates, although it shows a decreasing tendency over the last few years (from a previously decade-long stable ~40 %, it gradually fell to ~28 % between 2008 and 2012), it is still higher than that detected by us for the carried strains.
MIC range
MIC50
MIC90
S (%)
I (%)
R (%)
≤0.016–4 0.004–1 0.004–5 0.03–>512 0.016–>256 0.004–2 0.06–4 0.06–0.5 0.06–2
0.015 0.015 0.008 0.125 0.125 0.03 1 0.25 0.25
0.25 0.25 0.06 >256 >128 0.125 2 0.5 1
78.4 97.7 100 77.7 82.7 96.4 95.6 100 100
21.3 2.3 – 0.7 – 2.8 – – –
0.3 0 0 21.6 17.3 0.8 4.4 0 0
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Fig. 2 Distribution of serotypes of the 358 carried isolates in this study. Yellow columns, PCV-7 serotypes; green columns, additional PCV-13 serotypes; purple columns, additional Pneumovax serotypes; blue columns, non-vaccine serotypes. It is worth to note that 6A is not included in Pneumovax
Furthermore, the sensitivity data found in this study are slightly better even compared to those previously observed by us for carried strains [36]. All these findings are most likely related to the drastic rearrangement of serotypes occurring everywhere where vaccination was introduced, the newly emerging types being—luckily—much more sensitive to antibiotics than the previously worldwide dominating “paediatric” clones. Serotypes and vaccine coverage If we compare the current results to those obtained in our previous study [36] conducted Table 5 Comparison of serotype prevalence and vaccine coverage between the pre- and postvaccination periods (n=135 and 358, respectively)
in the very last year of the pre-vaccination era, we find striking differences in serotype distribution (Table 5). While in the prevaccination era, the well-known paediatric serotypes were still dominant (14, 19F, 23F, 6A, 6B in ranking order); these were rapidly replaced by others. For example, although serotype 14 has been the leading type before, it fully disappeared as soon as children became vaccinated. The frequency of 19F, 23F and 6A also decreased to a great extent. On the other hand, the previously absent serotypes 19A and 35F emerged rapidly, competing for the second position. Serotype 19A is especially of great concern, as it was shown to have high invasive disease
Serotype
Pre-vaccination March 2009–February 2010 No. of strains (%)
Post-vaccination February 2010–February 2012 No. of strains (%)
14 19F 23F 6A 6B 15B 11A 18C
21 (15.6) 18 (13.3) 17 (12.6) 15 (11.1) 15 (11.1) 14 (10.4) 8 (5.9) 4 (3.0)
0 (0) 11 (3.1) 14 (3.9) 15 (4.2) 27 (7.5) 27 (7.5) 49 (13.7) 5 (1.4)
3 35F 19A Vaccine coverage (%) PCV-7 PCV-10 PCV-13 Pneumovax
3 (2.2) 0 (0) 0 (0)
24 (6.7) 33 (9.2) 31 (8.7)
55.6 56.3 69.6 86.7
16.5 16.8 36.3 59.8
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potential and antibiotic resistance capacity and has often been reported worldwide as an emerging serotype after the implementation of PCV-7 [31, 35, 38, 39, 41]. For instance, van Gils et al. reported that nasopharyngeal acquisition of serotype 19A was significantly higher in children who completed the 2+1 vaccination schedule with PCV-7, compared to the unvaccinated ones [39]. Interestingly, although serotype 6B is part of PCV-7, its prevalence did not decrease that much throughout the years. Similar findings were published by Dagan et al., where lower immunogenicity of serotype 6B was found in the case of 2+1 versus 3+1 dosing schedule [9]. In the UK, lower initial effectiveness was observed in the case of serotype 6B, compared to overall PCV-7 serotypes (65 vs 87 %), not long after the introduction of PCV-7 [13]. In Hungary, the 2+1 dosing schedule was applied, and it will be interesting to follow the changes in the frequency of serotype 6B a few years after PCV-7/PCV-13 use. Serotype 3 became three times more frequent, and, many, previously rare serotypes also emerged. As a consequence of this robust serotype rearrangement, the vaccine coverage rates declined sharply, as seen in Table 5. By now, 36.0 % of the carried strains would not be covered by any of the vaccines. Among the carried strains in Hungary, the three additional serotypes belonging to PCV-10 (1, 5, 7F) do not seem to be prevalent, unlike in many other European countries such as Germany, UK or Belgium, where serotypes 1 and 7F rank among the three most frequent types causing invasive pneumococcal infections in children [41] or are involved in carriage [29]. van Hoek et al. have investigated the disease potential of the different serotypes based on more than 23,000 invasive pneumococcal disease (IPD) isolates from England [40]. They found significant differences regarding disease focus and mortality rates. For example, serotypes 6A and 22F were responsible for the highest quality-adjusted life year (QALY) loss in children 30 years) died [19, 20, 22, 23]. In summary, we can state that PCV-7 had a striking effect also in Hungary, observable very soon after widespread vaccination started. Apparently serotype 14 responds most sensitively to the vaccination and can be eliminated easily. Vaccination coverage of >80 % of the target population provides a sufficient herd protection [41], and in Hungary PCV coverage is close to 100 %. According to the estimations and economic models based on European epidemiological data [16, 41], the highervalency PCVs will have better public health benefits by including the most important emerging non-PCV-7 serotypes. The question arises whether the newly emerging serotypes retain their antibiotic sensitivity or become more resistant and virulent. Acknowledgments The results were presented in part at the 22nd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), London, UK (31 March–3 April 2012, poster number P1034). This study was financially supported by the Hungarian National Scientific Research Fund (OTKA K108631). Conflict of interest ML has been a member of advisory boards for and has received research grants and speakers honorary fees from Pfizer, GSK, Merck and SanofiPasteurMSD.
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23. National Center for Epidemiology (2013) “Reportable infectious diseases in Hungary in 2012” Epinfo 20: 281–295 [in Hungarian] 24. National Center for Epidemiology, National Bacteriological Surveillance Management Team (2003–2012) NBS annual reports. Available via www.oek.hu Accessed June 2014 25. Oikawa J, Ishiwada N, Takahashi Y, Hishiki H, Nagasawa K, Takahashi S, Watanabe M, Chang B, Kohno Y (2014) Changes in nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis among healthy children attending a day-care centre before and after official financial support for the 7-valent pneumococcal conjugate vaccine and H. influenzae type b vaccine in Japan. J Infect Chemother 20:146–149 26. Oster P, Zanchi A, Cresti S, Lattanzi M, Montagnani F, Cellesi C, Rossolini GM (1999) Patterns of macrolide resistance determinants among community-acquired Streptococcus pneumoniae isolates over a 5-year period of decreased macrolide susceptibility rates. Antimicrob Agents Chemother 43:2510–2512 27. Pletz MW, Maus U, Krug N, Welte T, Lode H (2008) Pneumococcal vaccines: mechanism of action, impact on epidemiology and adaption of the species. Int J Antimicrob Agents 32:199–206 28. Sá-Leão R, Tomasz A, Sanches IS, Nunes S, Alves CR, Avo AB, Saldanha J, Kristinsson KG, de Lencastre H (2000) Genetic diversity and clonal patterns among antibioticsusceptible and -resistant Streptococcus pneumoniae colonizing children: day care centers as autonomous epidemiological units. J Clin Microbiol 38:4137–4144 29. Sá-Leão R, Nunes S, Brito-Avô A, Frazão N, Simões AS, Crisóstomo MI, Paulo AC, Saldanha J, Santos-Sanches I, de Lencastre H (2009) Changes in pneumococcal serotypes and antibiotypes carried by vaccinated and unvaccinated day-care centre attendees in Portugal, a country with widespread use of the seven-valent pneumococcal conjugate vaccine. Clin Microbiol Infect 15:1002–1007 30. Scott JA (2007) The preventable burden of pneumococcal disease in the developing world. Vaccine 25:2398–2405 31. Sharma D, Baughman W, Holst A, Thomas S, Jackson D, da Gloria CM, Beall B, Satola S, Jerris R, Jain S, Farley MM, Nuorti JP (2013) Pneumococcal carriage and invasive disease in children before introduction of the 13-valent conjugate vaccine: comparison with the era before 7-valent conjugate vaccine. Pediatr Infect Dis J 32:45–53 32. Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L (1996) Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 40:2562–2566 33. The European Committee on Antimicrobial Susceptibility Testing. EUCAST guidelines. Available via http://www.eucast.org/clinical_ breakpoints/ Accessed June 2014 34. Tirczka T, Berta B (2012) Results of the Streptococcus pneumoniae serotyping and antibiotic resistance surveillance, between 01.01.2008–31.08, 2011. Microbiol Circ 3:3–17 35. Tocheva AS, Jefferies JM, Christodoulides M, Faust SN, Clarke SC (2013) Distribution of carried pneumococcal clones in UK children following the introduction of the 7-valent pneumococcal conjugate vaccine: a 3-year cross-sectional population based analysis. Vaccine 31:3187–3190 36. Tóthpál A, Kardos S, Hajdú E, Nagy K, Linden M, Dobay O (2012) Nasal carriage of Streptococcus pneumoniae among Hungarian children before the wide use of the conjugate vaccine. Acta Microbiol Immunol Hung 59:107–118 37. Turner P, Hinds J, Turner C, Jankhot A, Gould K, Bentley SD, Nosten F, Goldblatt D (2011) Improved detection of nasopharyngeal cocolonization by multiple pneumococcal serotypes by use of latex agglutination or molecular serotyping by microarray. J Clin Microbiol 49:1784–1789
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381 40. van Hoek AJ, Andrews N, Waight PA, George R, Miller E (2012) Effect of serotype on focus and mortality of invasive pneumococcal disease: coverage of different vaccines and insight into non-vaccine serotypes. PLoS ONE 7:e39150 41. Weil-Olivier C, van der Linden M, de Schutter I, Dagan R, Mantovani L (2012) Prevention of pneumococcal diseases in the post-seven valent vaccine era: a European perspective. BMC Infect Dis 12:207
ORIGINAL ARTICLE
Radical serotype rearrangement of carried pneumococci in the first 3 years after intensive vaccination started in Hungary Adrienn Tóthpál & Szilvia Kardos & Krisztina Laub & Károly Nagy & Tamás Tirczka & Mark van der Linden & Orsolya Dobay
Received: 16 June 2014 / Revised: 6 August 2014 / Accepted: 19 August 2014 / Published online: 2 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Streptococcus pneumoniae is responsible for a significant amount of morbidity and mortality worldwide. Healthy carriers, mainly young children, are the most important sources of infections. In the current study, we aimed to determine the changes that have occurred since the introduction of PCV-7 in Hungary. Nasal specimens were collected from 1,022 healthy children aged 3–6 years attending daycare centres. After thorough identification, pneumococcal isolates were serotyped, and their antibiotic sensitivity was determined. The carriage rate was found to be 34.9 %. A huge serotype rearrangement was detected compared to earlier results, with the previously leading serotype 14 having completely disappeared. Serotypes 11A, 35F, 19A, 6B, 15B, Communicated by Peter de Winter A. Tóthpál : S. Kardos : K. Laub : K. Nagy : O. Dobay (*) Institute of Medical Microbiology, Semmelweis University, BudapestNagyvárad tér 4, 1089, Hungary e-mail: [email protected]
3 and 38 were most prevalent, and 29 different types were identified in total. The PCV-7 types were responsible for 16.5 % of all serotypes, and 36.0 % are not covered by any pneumococcal vaccines. The isolates were sensitive to most tested antibiotics, except erythromycin (resistance was 21.6 %). Only one penicillin-resistant strain was found. The newly and rapidly emerging non-vaccine serotypes are much more sensitive, except serotype 19A. Conclusion: Due to PCV vaccination, a complete serotype arrangement occurred also in Hungary. The old “paediatric” serotypes were replaced by serotypes 11A, 35F, 19A, 6B, 15B, 3 and 38. Keywords Pneumococcus . PCV-7 vaccination . Carriage . Serotype changing . Day-care centres
K. Nagy e-mail: [email protected]
Abbreviations CDC Centers for Disease Control and Prevention DCC Day-care centre EUCAST The European Committee on Antomicrobial Susceptibility Testing GNRCS German National Reference Centre for Streptococci NVT Non-vaccine type PCV-7 7-valent pneumococcal conjugate vaccine PCV-13 13-valent pneumococcal conjugate vaccine VT Vaccine type
T. Tirczka Pneumococcal Reference Laboratory, National Center for Epidemiology, Budapest, Hungary e-mail: [email protected]
Introduction
M. van der Linden German National Reference Center for Streptococci; Department of Medical Microbiology, University Hospital RWTH Aachen, Aachen, Germany e-mail: [email protected]
Streptococcus pneumoniae is one of the most important bacterial pathogens worldwide, causing a wide range of illnesses from milder infections to life-threatening invasive diseases and is responsible for the death of nearly 1 million
A. Tóthpál e-mail: [email protected] S. Kardos e-mail: [email protected] K. Laub e-mail: [email protected]
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children per year, especially due to severe pneumonia [30]. Pneumococci often colonise the nasopharynx; it is estimated that all healthy individuals become carriers for shorter or longer periods during life. Under certain circumstances, pneumococci can spread to the mucosa causing respiratory tract infections (sinusitis, otitis media) or enter the normally sterile body fluids causing sepsis and/or meningitis. Additionally, the bacterium can be transmitted to other susceptible persons in the community (e.g. older family members); therefore, carriers represent the most important natural reservoir for pneumococcal infections [2, 8, 28]. Carriage peaks in early childhood, and children attending day-care centres (DCCs) are shown to have significantly higher colonisation rates [8, 10]. A study from the Netherlands demonstrated a 1.6 times higher rate in DCC attendees compared to those staying at home [1]. To prevent pneumococcal infections, several attempts were made to develop a vaccine, based on the major protective antigen of the bacterium, the polysaccharide capsule. Currently, two types of vaccines are available. The 23-valent polysaccharide vaccine (Pneumovax or PPV, Merck) has been in use since 1982, containing 23 of the >90 currently known capsular polysaccharides. As children younger than 2–2.5 years are not able to develop a T cell-dependent immune response against polysaccharides, a conjugate vaccine (PCV-7) was developed for them, which was licenced in 2000 in the USA and later in Europe. In recent years, higher-valent vaccines have followed (PCV-10 by GSK and PCV-13 by Pfizer). Conjugate vaccines have been proven to have an impact not only on pneumococcal diseases but also on nasopharyngeal carriage [15], hence, cutting the infection chain in the beginning, providing an indirect population-wide effect [27]. In Hungary, PCV-7 was introduced in 2005, but in the first 3 years, only very few children obtained vaccination due to a high price and a low awareness. In October 2008, PCV-7 was made freely available for children under the age of 2 years in the framework of a pneumococcus surveillance programme, and in April 2009, it was included in the national immunisation programme as a recommended but not obligatory vaccine in the 2+1 scheme. The vaccination rate increased to >80 % within a year, and it has reached nearly 100 % by now [18, 21]. In September 2010, PCV-13 replaced PCV-7 in Hungary, and PCV-13 was fully integrated in the national immunisation programme on 1 July 2014. In a previous project, we had surveyed nasal carriage of S. pneumoniae in healthy children attending day-care centres, just before widespread vaccine uptake started [36]. The aim of the current study was to make a comparison of the serotype distribution and vaccine coverage between the pre- and postvaccination eras in Hungary.
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Materials and methods Study population The study involved 1,022 healthy children aged between 3 and 6 years, attending 18 DCCs, located in different regions of Hungary, between February 2010 and February 2012. Informed consent obtained from the parents was a condition for enrolment. Besides informing the parents about the purpose of the study, data were asked about the children’s vaccination status, siblings, history of recurrent otitis media or other severe infections and smoking in the family. The number of the ethical permit for the study is TUKEB 4-3/2009, issued by the Regional and Institutional Committee of Science and Research Ethics of Semmelweis University. For statistical analysis, as appropriate, the chisquare test was used. Specimen collection Nasal samples were collected from both nostrils of the children with sterile cotton swabs. The samples were transported to the laboratory on active charcoalcontaining transport media (Transwab, Medical Wire & Equipment, Corsham, UK) and were immediately inoculated onto Müller-Hinton blood agar plates. Samples were incubated overnight at 37 °C in 5 % CO2 atmosphere. Identification of pneumococci Suspected colonies showing typical colony morphology of S. pneumoniae (α-haemolysis and flat colonies collapsed in the middle) were subcultured and further tested for optochin sensitivity (5 μg discs, Mast Diagnostica, Bootle, UK). The identity of the strains was confirmed by detecting the lytA (autolysin) gene by PCR in every case [17], and confirmed strains were stored at −80 °C on cryobeads (Mast Diagnostica). Antibiotic susceptibility testing The antibiotic sensitivity of the strains to penicillin, cefotaxime, imipenem, erythromycin, telithromycin, clindamycin, levofloxacin, moxifloxacin and vancomycin was determined by the agar dilution method using an A400 multipoint inoculator (AQS Manufacturing Ltd., Southwater, UK) and by E-test (Liofilchem, Roseto, Italy), on Müller-Hinton blood agar plates. Incubation was done at 37 °C in 5 % CO2, and ATCC 49619 was used as control strain. The susceptibility and resistance rates were determined using the breakpoints recommended by the EUCAST guidelines [33]. As our collection consisted of carried strains (i.e. non-meningitis cases), we used the ≤0.06/>2 breakpoints for penicillin. Macrolide resistant strains were tested for the presence of the erm(B) and mef genes [11, 32], and the distinction between mefA and mefE was carried out by BamHI digestion, which generates two fragments in mefA, but none in mefE, as described before [11, 26]. Serotyping Determination of the serogroups was done by the Pneumotest-Latex kit (Statens Serum Institut, Copenhagen,
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Denmark) using antisera, and the factor determination was done by PCR using primers described by the CDC [6], or others [3]. Several strains were sent to the German National Reference Centre for Streptococci (GNRCS; Aachen) or to the Hungarian National Center for Epidemiology for further serotyping.
Results Carriage rate and vaccination status Among the 1,022 children, 357 were colonised with pneumococcus; this means a carriage rate of 34.9 %. Although the ratio of genders was nearly equalised (51.4 % males), the carriage rate was slightly higher among males: 36.8 versus 33.0 % in females (statistically not significant, p = 0.207). In one occasion, we detected double carriage: in a boy previously vaccinated with Pneumovax, one strain each of serotypes 3 and 34 was present; therefore, the number of pneumococcal isolates is 358. The degree of colonisation varied widely from pure pneumococcal culture to just a few colonies among the normal flora. In the examined age group of 3–6 years, carriage peaked at 3 years with 43.3 % (Fig. 1). As the isolates of this study were collected between January 2010 and February 2012, from children aged 3 years or older, those who were vaccinated received PCV-7; hence, we could test the effect only of PCV-7. Out of all children, 393 (38.5 %) were vaccinated. However, it must be noted that we based our calculations on the parents’ positive answers, and often, they were not aware whether their children had received pneumococcal vaccination or not. Therefore, in this question, we only evaluated the sure-positive (n=393) or sure-negative (n=388) answers. Among the 393 PCV-7-vaccinated children, 156 (39.7 %) were carriers; this is a higher rate than that observed among the non-vaccinated children (128/388, 33.0 %), but the difference is not statistically significant (p=0.051). The coverage of the conjugate vaccines is much higher in the non-
Fig. 1 Carriage rates at different ages
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vaccinated group, (calculated with the Broome method [4], the PCV-7 efficacy would be 1–11/22.7=52 %), but this figure equalises for the Pneumovax coverage (Table 1). This phenomenon can easily be explained by the different serotype distribution: the vaccinated children characteristically carry non-vaccine serotypes (with the exception of serotypes 6B and 19A) (Table 2). Among the PCV-13 minus PCV-7 serotypes, 19A was represented in higher rate (10.3 %). Notably, out of the 27 serotype 6B isolates (which is a PCV-7 type), 14 were found in previously vaccinated (PCV-7 or Pneumovax) children. Based on the answers given by the parents in the questionnaire, it was demonstrated that those children who had otitis media and/or pneumonia/meningitis (possibly of pneumococcal origin) in the past were more frequently carriers (Table 3). Regarding the family-related factors, having siblings surprisingly showed a negative association with carriage, indicating that fellow children are more important sources of pneumococcal colonisation than older (school-age) or younger (before attending any community) siblings in the household. Furthermore, there was an only non-significant difference in the exposure to passive smoking, although this is usually an identifiable risk factor for carriage. High percentage (35.2 %) of all children were exposed to passive smoking (34.4 % of the carriers and 35.6 % of the non-carriers). Nonetheless, none of these correlations were statistically significant. Antibiotic susceptibility Only one single isolate (serotype 19A) was resistant to penicillin, but only at the lowest level (MIC=4 mg/L), while 78.4 % of all strains were fully susceptible (Table 4). No resistance was observed to cefotaxime, imipenem, vancomycin and moxifloxacin. On the other hand, 21.6 % of the isolates were resistant to erythromycin. Ten strains out of these showed the M phenotype (low-level resistance to erythromycin—MIC=4–16 mg/ L—and sensitivity to clindamycin); these all had the mefE gene, and five of these were serotype 6A. The majority, however, showed the macrolide-lincosamide-streptogramin B (MLSB) type (high-level resistance to both), and serotype 19A was dominant (54.0 %) in this group; all serotype 19A strains were ermB positive. Seven isolates possessed ermB and mefE together (four strains serotype 19A, three strains serotype 19F). In the case of telithromycin, the strains could be divided clearly into two distinct groups. Those that had a telithromycin MIC ≤0.03 mg/L were all macrolide sensitive, and none of them were serotype 19A. On the other hand, those with an MIC ≥0.06 mg/L contained all the macrolide resistant isolates, M types and MLSB types as well. Out of the 12 levofloxacin-resistant strains (MIC=4 mg/L), eight were serotype 11A.
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Table 1 Carriage rate and vaccine coverage of the vaccinated and non-vaccinated children Non-vaccinated n=388a
PCV-7 vaccinated n=393
Pneumovax vaccinated n=37b
All children n=1,022
PCV-7 coverage PCV-10 coverage
33.0 % (n=128) 22.7 % 23.4 %
39.7 % (n=156) 10.9 % 10.9 %
37.8 % (n=14) 4/15c 4/15
34.9 % (n=357) 16.5 % 16.8 %
PCV-13 coverage Pneumovax coverage Non-vaccine types
45.3 % 61.7 % 32.8 %
25.8 % 58.3 % 41.7 %
8/15 12/15 3/15
36.3 % 59.8 % 36.0 %
Carriage rate
a
only the surely negative answers were included in the calculation
b
out of the 37 children, 19 received both Pneumovax and PCV-7
c
one child carried 2 pneumococci
Serotypes and vaccine coverage The prevalence of the more frequent serotypes in ranking order was the following: 11A (n=49), 35F (n=33), 19A (n=31), 6B (n=27), 15B (n=27), 3 (n=24) and 38 (n=22), but 29 different serotypes were identified. The complete serotype distribution is shown in Fig. 2. All NT strains are truly non-typable as determined by the GNRCS. The calculated vaccine coverage is 16.5 % for PCV-7, 16.8 % for PCV-10 (the very small difference is due to the fact that we had no serotype 5 or 7F isolates, and only one single isolate of serotype 1—these are the additional serotypes belonging to PCV-10) and 36.3 % for PCV-13. Pneumovax would cover an additional 27.7 %, but it does not include serotype 6A, so the coverage for Pneumovax is 59.8 %. This means that 36.0 % of these carried strains are not covered by any of the currently available vaccines.
Table 2 Serotype distribution in the vaccinated and nonvaccinated carriers
a
only the surely negative answers were included in the calculation
b
one child carried 2 pneumococci
The isolates belonging to the leading serotype 11A were very sensitive to penicillin (most of them had MIC = 0.015 mg/L) and also to macrolides, but they had the highest levofloxacin MICs (4 mg/L). The serotype 19A isolates were (with a very few exceptions) macrolide resistant, and most of them had a penicillin MIC≥0.25 mg/L. Many of the serotype 6B strains had high-level macrolide resistance, while the 6A strains were mostly sensitive, or had the M phenotype, with a resulting low-level macrolide resistance. Isolates with elevated MICs to penicillin (intermediate category) had serotypes 19A, 19F, 6A, 6B, 23A, 23F and 35B. Serotypes 3, 15A, 15B, 23F, 35F and most of the “minor” types as well were fully sensitive to all tested antibiotics, with especially low MIC values.
Serotype
Non-vaccinated n=128a
PCV-7 vaccinated n=156
Pneumovax vaccinated n=14b
6B 18C 19F 23F 1 3 6A 19A 8 10A 11A
10 5 6 8 1 12 7 9 1 3 12
11 – 4 2 – 4 3 16 – 3 29
3 – 1 – – 2 1 1 – – 2
15B 22F 33F NVTs
10 2 – 42 (32.8 %)
14 5 3 55 (35.3 %)
1 1 – 3
PCV-7
PCV-13−PCV-7
Pneumovax−PCV-13
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Table 3 Differences in the possible risk factors usually associated with pneumococcal colonisation, among the carriers and non-carriers. The p value was calculated with the chi-square test. Carriers (n=357)
Non-carriers (n=665)
Otitis media in the past Yes 86 (38.6 %) 137 (61.4 %) No 272 (34.0 %) 527 (66.0 %) Pneumonia or meningitis in the past Yes 22 (40.0 %) 33 (60.0 %) No 336 (34.7 %) 631 (65.3 %) Having siblings Yes 251 (33.6 %) 495 (66.4 %) No 107 (38.8 %) 169 (61.2 %) Gender Male 193 (36.8 %) Female 164 (33.0 %) Passive exposure to smokinga Yes No
74 (34.7 %) 141 (35.8 %)
p value
0.211 (NS)
0.427 (NS)
0.128 (NS)
332 (63.2 %) 333 (67.0 %)
0.207 (NS)
140 (65.4 %) 253 (64.2 %)
0.766 (NS)
a
In this question, only 608 answers were collected (215 carriers and 393 non-carriers)
Discussion Carriage rate Compared to our previous study carried out a few years ago, just before conjugate vaccination started in Hungary [36], the carriage rate has decreased slightly from 37.7 to 34.9 %. This suggests that the conjugate vaccines have a non-significant reductive effect on the carriage rate; they rather influence the distribution of the carried serotypes. Other studies have also observed that despite conjugate vaccination, the overall carriage rate remained virtually unchanged [7, 25]. Comparing vaccinated and non-vaccinated children, we could in fact observe a higher carriage rate among the vaccinated ones. However, there was a significant difference in the distribution of serotypes, with much more NVTs in the
Table 4 Antibiotic susceptibility results of the 358 carried isolates (MIC in mg/L) Penicillin Cefotaxime Imipenem Erythromycin Clindamycin Telithromycin Levofloxacin Moxifloxacin Vancomycin
vaccinated children. Among the VTs, serotypes 6B and 19A were represented in higher rates. We detected double carriage only in one occasion. This low co-colonisation rate might be the result of the culture-base technique we applied. Several papers suggest that co-colonisation is often underestimated, and to reveal the real rates, more sophisticated methods such as multiplex PCR or microarray should be used [12, 37]. We found a slightly higher colonisation rate in males; this was also found by others, e.g. among Brazilian adolescents [5]. As these authors have also suggested, a higher pneumococcal carriage rate may be one of the reasons leading to the observations that male gender is a definitive risk factor for pneumonia [14]. The highest level of carriage was observed at 3 years of age (43.3 %), which declined to 18.4 % [5] by 6 years of age. This is in accordance with the results of a large-scale review, where the peak incidence was identified also at 3 years [2]. After that age, colonisation steadily declines to below 3 % by the age of 19 years [5]. The 20–40-year-old adults are least effected by pneumococcal infections, which start increasing above 50 years, but the source is not any more the self carriage, rather small children in the close environment. Antibiotic susceptibility The isolates in this study were generally sensitive to the drugs tested, except for the macrolides. Only one isolate (0.35 %) was resistant to penicillin (MIC= 4 mg/L), and 21.6 % were resistant to erythromycin. These are much lower figures compared to those of disease-causing strains in Hungary. According to the nationwide reporting system data maintained by the National Center for Epidemiology (NCE), penicillin resistance of clinical isolates has been varying between 1.5 and 5 % in the last 10 years (based on approximately 4,000 specimens annually), and the higher rates are observed in hospitalised patients and invasive specimens [24]. Regarding macrolide resistance of clinical isolates, although it shows a decreasing tendency over the last few years (from a previously decade-long stable ~40 %, it gradually fell to ~28 % between 2008 and 2012), it is still higher than that detected by us for the carried strains.
MIC range
MIC50
MIC90
S (%)
I (%)
R (%)
≤0.016–4 0.004–1 0.004–5 0.03–>512 0.016–>256 0.004–2 0.06–4 0.06–0.5 0.06–2
0.015 0.015 0.008 0.125 0.125 0.03 1 0.25 0.25
0.25 0.25 0.06 >256 >128 0.125 2 0.5 1
78.4 97.7 100 77.7 82.7 96.4 95.6 100 100
21.3 2.3 – 0.7 – 2.8 – – –
0.3 0 0 21.6 17.3 0.8 4.4 0 0
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Fig. 2 Distribution of serotypes of the 358 carried isolates in this study. Yellow columns, PCV-7 serotypes; green columns, additional PCV-13 serotypes; purple columns, additional Pneumovax serotypes; blue columns, non-vaccine serotypes. It is worth to note that 6A is not included in Pneumovax
Furthermore, the sensitivity data found in this study are slightly better even compared to those previously observed by us for carried strains [36]. All these findings are most likely related to the drastic rearrangement of serotypes occurring everywhere where vaccination was introduced, the newly emerging types being—luckily—much more sensitive to antibiotics than the previously worldwide dominating “paediatric” clones. Serotypes and vaccine coverage If we compare the current results to those obtained in our previous study [36] conducted Table 5 Comparison of serotype prevalence and vaccine coverage between the pre- and postvaccination periods (n=135 and 358, respectively)
in the very last year of the pre-vaccination era, we find striking differences in serotype distribution (Table 5). While in the prevaccination era, the well-known paediatric serotypes were still dominant (14, 19F, 23F, 6A, 6B in ranking order); these were rapidly replaced by others. For example, although serotype 14 has been the leading type before, it fully disappeared as soon as children became vaccinated. The frequency of 19F, 23F and 6A also decreased to a great extent. On the other hand, the previously absent serotypes 19A and 35F emerged rapidly, competing for the second position. Serotype 19A is especially of great concern, as it was shown to have high invasive disease
Serotype
Pre-vaccination March 2009–February 2010 No. of strains (%)
Post-vaccination February 2010–February 2012 No. of strains (%)
14 19F 23F 6A 6B 15B 11A 18C
21 (15.6) 18 (13.3) 17 (12.6) 15 (11.1) 15 (11.1) 14 (10.4) 8 (5.9) 4 (3.0)
0 (0) 11 (3.1) 14 (3.9) 15 (4.2) 27 (7.5) 27 (7.5) 49 (13.7) 5 (1.4)
3 35F 19A Vaccine coverage (%) PCV-7 PCV-10 PCV-13 Pneumovax
3 (2.2) 0 (0) 0 (0)
24 (6.7) 33 (9.2) 31 (8.7)
55.6 56.3 69.6 86.7
16.5 16.8 36.3 59.8
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potential and antibiotic resistance capacity and has often been reported worldwide as an emerging serotype after the implementation of PCV-7 [31, 35, 38, 39, 41]. For instance, van Gils et al. reported that nasopharyngeal acquisition of serotype 19A was significantly higher in children who completed the 2+1 vaccination schedule with PCV-7, compared to the unvaccinated ones [39]. Interestingly, although serotype 6B is part of PCV-7, its prevalence did not decrease that much throughout the years. Similar findings were published by Dagan et al., where lower immunogenicity of serotype 6B was found in the case of 2+1 versus 3+1 dosing schedule [9]. In the UK, lower initial effectiveness was observed in the case of serotype 6B, compared to overall PCV-7 serotypes (65 vs 87 %), not long after the introduction of PCV-7 [13]. In Hungary, the 2+1 dosing schedule was applied, and it will be interesting to follow the changes in the frequency of serotype 6B a few years after PCV-7/PCV-13 use. Serotype 3 became three times more frequent, and, many, previously rare serotypes also emerged. As a consequence of this robust serotype rearrangement, the vaccine coverage rates declined sharply, as seen in Table 5. By now, 36.0 % of the carried strains would not be covered by any of the vaccines. Among the carried strains in Hungary, the three additional serotypes belonging to PCV-10 (1, 5, 7F) do not seem to be prevalent, unlike in many other European countries such as Germany, UK or Belgium, where serotypes 1 and 7F rank among the three most frequent types causing invasive pneumococcal infections in children [41] or are involved in carriage [29]. van Hoek et al. have investigated the disease potential of the different serotypes based on more than 23,000 invasive pneumococcal disease (IPD) isolates from England [40]. They found significant differences regarding disease focus and mortality rates. For example, serotypes 6A and 22F were responsible for the highest quality-adjusted life year (QALY) loss in children 30 years) died [19, 20, 22, 23]. In summary, we can state that PCV-7 had a striking effect also in Hungary, observable very soon after widespread vaccination started. Apparently serotype 14 responds most sensitively to the vaccination and can be eliminated easily. Vaccination coverage of >80 % of the target population provides a sufficient herd protection [41], and in Hungary PCV coverage is close to 100 %. According to the estimations and economic models based on European epidemiological data [16, 41], the highervalency PCVs will have better public health benefits by including the most important emerging non-PCV-7 serotypes. The question arises whether the newly emerging serotypes retain their antibiotic sensitivity or become more resistant and virulent. Acknowledgments The results were presented in part at the 22nd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), London, UK (31 March–3 April 2012, poster number P1034). This study was financially supported by the Hungarian National Scientific Research Fund (OTKA K108631). Conflict of interest ML has been a member of advisory boards for and has received research grants and speakers honorary fees from Pfizer, GSK, Merck and SanofiPasteurMSD.
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