The authors have declared that no competing interests exist.
Conceived and designed the experiments: AP MLG VS MPL MGP ET. Performed the experiments: LD FC DL FDA MDG RC. Analyzed the data: MFV RC. Wrote the manuscript: RC AP. Discussed the results and commented the manuscript: AP MLG MPL ET MGP VS RC LD FC DL MDG MFV.
Current address: Unit of Clinical Microbiology, Hub Laboratory of the “Greater Romagna Area”, Pievestina, Cesena, Italy
In mid 2010, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent conjugate vaccine (PCV13) for childhood immunization in Italy. Our objective in this study was to obtain a snapshot of pneumococcal carriage frequency, colonizing serotypes, and antibiotic resistance in healthy children in two Italian cities one year after PCV13 was introduced.
Nasopharyngeal swabs were obtained from 571 children aged 0-5 years from November 2011-April 2012. Pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. Penicillin and/or erythromycin non-susceptible isolates were analyzed by Multi Locus Sequence Typing (MLST).
Among the children examined, 81.2% had received at least one dose of PCV7 or PCV13 and 74.9% had completed the recommended vaccination schedule for their age. Among the latter, 57.3% of children had received PCV7, 27.1% PCV13, and 15.6% a combination of the two vaccines. The overall carriage rate was 32.9%, with children aged 6-35 months the most prone to pneumococcal colonization (6-23 months OR: 3.75; 95% CI: 2.19-6.43 and 24-35 months OR: 3.15, 95%CI: 2.36-4.22). A total of 184 pneumococcal isolates were serotyped and divided into PCV7 (5.4%), PCV13 (18.0%), and non-PCV13 (82.0%) serotypes. Serotypes 6C, 24F, and 19A were the most prevalent (10.3%, 8.6%, and 8.1%, respectively). The proportion of penicillin non-susceptible (MIC >0.6 mg/L) isolates was 30.9%, while 42.3% were erythromycin resistant. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively.
Our results revealed low rates of PCV7 and PCV13 serotypes in Italian children, potentially due to the effects of vaccination. As the use of PCV13 continues, its potential impact on vaccine serotypes such as 19A and cross-reactive serotypes such as 6C will be assessed, with this study providing a baseline for further analysis of surveillance isolates.
The ecological niche of
In Italy, PCV7 was licensed in 2001 but its use was gradually implemented in Italian regions due to different local immunization strategies [
A thorough evaluation of the impact of PCV7 on the incidence of IPD in Italy was not possible since the nationwide surveillance program for invasive bacterial diseases began in 2007 (
However, during the implementation of PCV7, a change in the relative rates of the most common serotypes was observed, due to a reduction of PCV7 serotypes and a concurrent increase in non-PCV7 serotypes, most of which were included in PCV13, such as 1, 7F, and 19A (
Since nasopharyngeal colonization is necessary for invasive disease, it is critical to understand the effects of vaccines on pneumococcal ecology. Nasopharyngeal isolates reflect strains currently circulating in the community; therefore, changes in the distribution of colonizing serotypes following immunization could predict the prevalent serotypes causing IPD. Thus, surveillance of colonization has become an important component of the vaccination monitoring process. Recently, Simell et al [
Few studies regarding pneumococcal carriage have been conducted in Italy, and most were performed prior to the introduction of PCV7 [
In the present study, we examined the nasopharyngeal carriage in children aged 0-5 years in two large Italian cities, Bologna and Milan, at the beginning of PCV13 use. These two cities are located in two regions of North Italy (Emilia-Romagna and Lombardy, respectively), each with a different policy regarding PCV7 implementation [
To evaluate pneumococcal carriage in Italian children, a cross-sectional study was conducted between November 2011 and April 2012 in children aged 0-5 years in two Italian cities, Bologna (Emilia-Romagna region) and Milan (Lombardy region), located in regions with different pneumococcal vaccination policies. The Emilia-Romagna region actively provided pneumococcal immunization to all infants since 2006; while, the Lombardy region initially offered vaccination only to disease-specific risk groups of children. Since 2009, Lombardy has offered vaccination to all infants upon the parents’ or pediatricians’ request [
A structured questionnaire was filled in by the pediatrician and was used to collect the children’s demographic data (age, gender, number of family members, presence of young siblings under 6 years, daycare attendance, and presence of at least one smoking parent) and their PCV7/PCV13 vaccination status. Children who had received all the recommended doses for their age, according to the Italian schedule (2+1 doses at 3, 5, and 11/13 months), or who had received a catch-up dose at >1 year of age, were considered completely vaccinated. Children who had not completed the recommended vaccination schedule were classified as partially vaccinated.
The study was approved by the Ethics Committee of S. Orsola-Malpighi University Hospital in Bologna (reference number: 1946/2011) and the Ethics Committee of Hospital Maggiore Policlinico in Milan, according to the Italian legislation. Parents or guardians of the participating children provided written informed consent. The procedures followed were in accordance with the European Statements for Good Clinical Practice and the Declaration of Helsinki of the World Medical Association.
Nasopharyngeal specimens were obtained using a nylon flocked swab (eSwab, Copan, Brescia, Italy), according to the WHO pneumococcal colonization detection protocol [
Pneumococcal isolates were detected by colony morphology and identification was confirmed by optochin susceptibility and bile solubility testing. Individual isolates were serotyped by latex agglutination and the Quellung reaction using commercially available antisera (Statens Serum Institut, Copenhagen, Denmark).
Susceptibilities to penicillin, ceftriaxone, erythromycin, clindamycin, tetracycline, and chloramphenicol were determined by the antimicrobial gradient strip diffusion method (Etest, bioMérieux, Durham, USA). The breakpoints proposed by the EUCAST (
Data were checked for outliers, duplicate records, and distribution of the variables. Age, computed by date of examination minus date of birth, was analyzed as a categorical (0-5; 6-23; 24-35; and 36-71 months) variable. For each variable, missing values were tabulated by age, sex, categories of outcome (colonization status), exposure (vaccination status), and recruitment center. We examined the association between colonization status and vaccination exposure (unvaccinated was the reference category). Potential risk factors for nasopharyngeal carriage of
A total of 571 children, 270 from Bologna and 301 from Milan, aged 0-5 years, were enrolled in the study. The mean age was 35 months (SD 17.15) and 316 (55.3%) participants were male. Overall, 78.9% (451/571) of children attended day care centers, and this percentage was higher (97%) in the 24-71 month old group. Only 30.8% (176/571) of children had siblings aged <6 years and 37.1% (212/571) had at least one smoking parent. The only difference observed between the children populations of the two cities with regard to demographic characteristics was a higher presence of young siblings in Milan, which was borderline significant (p= 0.051).
The vaccination status was known for 531 children: 81.2% (431/531) had received at least one dose of PCV7 or PCV13 and 74.9% (398/531) had completed the recommended vaccination schedule for their age. Specifically, 57.3% (228/398) received PCV7, 27.1% (108/398) PCV13, and 15.6% (62/398) a combination of the two vaccines. According to the Italian schedule [16], children vaccinated with a combination of vaccines receive 2 or 3 doses of PCV7 followed by 1 dose of PCV13 (48/62 children, 77.4%) or 1 or 2 doses of PCV7 followed by 2 doses of PCV13 (14/62 children, 22.6%). By age-adjusted analysis, a statistically significant difference was found in the proportion of vaccinated children between Bologna and Milan. In Bologna, 98.8% of children had received at least one vaccine dose and 97.8% were completely vaccinated compared with 68.1% and 57.5%, respectively, in Milan (p < 0.001).
The overall pneumococcal carriage rate was 32.9% (188/571) and there was no significant difference between Bologna and Milan (34.4% and 31.5%, respectively, p= 0.5). The colonization rate increased from 7.1% in the younger age group (0-5 months) to 33.3% (6-23 months), 33.0% (24-35 months), and 35.1% (36-71 months) in the older aged groups (
Total children (N=571) | Univariate |
Multivariate |
||||||
---|---|---|---|---|---|---|---|---|
N (%) | N (%) | OR | (95%CI) | P | OR | (95%CI) | P | |
0 to 5 months | 28 (4.90) | 2 (7.14) | 1 | 1 | ||||
6 to 23 months | 132 (23.11) | 44 (33.33) | 6.50 | (5.44; 7.77) | <0.001 | 3.75 | (2.19; 6.43) | <0.001 |
24 to 35 months | 124 (21.71) | 41 (33.06) | 6.42 | (4.70; 8.77) | <0.001 | 3.15 | (2.36; 4.22) | <0.001 |
36 to 71 months | 287 (50.26) | 101 (35.19) | 7.06 | (5.85; 8.52) | <0.001 | 3.03 | (1.00; 9.21) | 0.051 |
no | 120 (21.01) | 19 (15.83) | 1 | 1 | ||||
yes | 451 (78.98) | 169 (37.47) | 3.55 | (2.77; 4.56) | <0.001 | 2.31 | (0.89; 5.98) | 0.084 |
0 | 392 (68.65) | 122 (31.12) | 1 | 1 | ||||
≥1 | 176 (30.82) | 65 (36.93) | 1.25 | (1.21; 1.29) | <0.001 | 1.20 | (0.91; 1.57) | 0.193 |
no | 356 (62.34) | 127 (35.67) | 1 | 1 | ||||
yes | 212 (37.12) | 60 (28.30) | 0.71 | (0.58; 0.87) | 0.01 | 0.70 | (0.61; 0.80) | <0.001 |
unvaccinated | 100 (17.51) | 29 (29.0) | 1 | 1 | ||||
PCV7 | 228 (39.93) | 85 (45.21) | 1.45 | (1.42; 1.49) | <0.001 | 1.34 | (0.97; 1.87) | 0.073 |
PCV7/13 + PCV13 | 170 (29.77) | 50 (37.28) | 1.02 | (0.87; 1.19) | 0.250 | 1.04 | (0.74; 1.43) | 0.839 |
partially vaccinated | 33 (5.77) | 10 (30.30) | 1.06 | (0.92; 1.22) | 0.870 | 0.86 | (0.72; 1.03) | 0.121 |
a Data for 3 children are missing; b data for 40 children are missing
In univariate analysis, colonization was associated with age, day-care center attendance, presence of young siblings in the family, and prior vaccination with PCV7 (
Overall, 189 pneumococcal isolates were obtained from 188 children, as 2 different isolates were obtained in one child. Five isolates were not viable upon storage and could not be serotyped. Of the 184 isolates, 31 different serotypes were identified, with 6C (19/184, 10.3%), 24F (16/184, 8.6%), and 19A (15/184, 8.1%) being the most common; in addition, 5.4% of the isolates (10/184) were non-typeable (NT) (
Within each serotype, the orange portion indicates isolates from Bologna and the blue portion isolates from Milan. The arrowheads indicate PCV13 serotypes.
Total children | PCV7 (N = 10) |
PCV13 (N = 33) |
non-PCV13 (N = 151) |
||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
N | N | OR | (95%CI) | P | N | OR | (95%CI) | P | N | OR | (95%CI) | P | |||
unvaccinated | 100 | 5 | 1 | 9 | 1 | 18 | 1 | ||||||||
PCV7 | 228 | 1 | 0.13 | (0.02; 0.86) | 0.034 | 5 | 0.34 | (0.06; 2.07) | 0.242 | 43 | 1.59 | (0.96; 2.65) | 0.073 | ||
PCV7/13+PCV13 | 170 | 4 | 0.30 | (0.25; 0.37) | <0.001 | 16 | 0.69 | (0.66; 0.72) | <0.001 | 69 | 1.90 | (1.40; 2.57) | <0.001 | ||
partially vaccinated | 33 | 0 | - | 0 | - | 10 | 1.99 | (1.97; 2.01) | <0.001 | ||||||
unknown | 40 | 3 | 11 |
The ranking order of serotypes was slightly shifted between the two populations (
Ranking order | No. isolates | % | Cumulative % | Ranking order | No. isolates | % | Ranking order | No. isolates | % | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|
6C | 1 | 19 | 10.3 | 10.3 | 1 | 11 | 11.8 | 1 | 8 | 8.7 | ||
24F | 2 | 16 | 8.6 | 19.0 | 2 | 10 | 10.7 | 3 | 6 | 6.5 | ||
19A | 3 | 15 | 8.1 | 27.1 | 3 | 9 | 9.6 | 3 | 6 | 6.5 | ||
11A | 4 | 13 | 7.0 | 34.2 | 5 | 6 | 6.4 | 2 | 7 | 7.6 | ||
15B | 4 | 13 | 7.0 | 41.3 | 5 | 6 | 6.4 | 2 | 7 | 7.6 | ||
23B | 5 | 11 | 5.9 | 47.2 | 8 | 3 | 3.2 | 1 | 8 | 8.7 | ||
23A | 6 | 10 | 5.4 | 52.7 | 6 | 5 | 5.3 | 4 | 5 | 5.4 | ||
NT | 6 | 10 | 5.4 | 58.1 | 6 | 5 | 5.3 | 4 | 5 | 5.4 | ||
10A | 7 | 8 | 4.3 | 62.5 | 4 | 8 | 8.6 | - | 0 | 0 | ||
35B | 8 | 7 | 3.8 | 66.3 | 7 | 4 | 4.3 | 5 | 3 | 3.2 | ||
15A | 9 | 6 | 3.2 | 69.5 | 8 | 3 | 3.2 | 5 | 3 | 3.2 | ||
19F | 9 | 6 | 3.2 | 72.8 | 10 | 1 | 1.0 | 4 | 5 | 5.4 | ||
35F | 9 | 6 | 3.2 | 76.0 | 10 | 1 | 1.0 | 4 | 5 | 5.4 | ||
15C | 10 | 5 | 2.7 | 78.8 | 9 | 2 | 2.1 | 5 | 3 | 3.2 | ||
21 | 10 | 5 | 2.7 | 81.5 | 9 | 2 | 2.1 | 5 | 3 | 3.2 | ||
38 | 10 | 5 | 2.7 | 84.2 | 8 | 3 | 3.2 | 6 | 2 | 2.1 | ||
3 | 11 | 3 | 1.6 | 85.8 | 10 | 1 | 1.0 | 6 | 2 | 2.1 | ||
22F | 11 | 3 | 1.6 | 87.5 | 10 | 1 | 1.0 | 6 | 2 | 2.1 | ||
31 | 11 | 3 | 1.6 | 89.1 | 9 | 2 | 2.1 | 7 | 1 | 1.0 | ||
6A | 12 | 2 | 1.0 | 90.2 | 10 | 1 | 1.0 | 7 | 1 | 1.0 | ||
7F | 12 | 2 | 1.0 | 91.3 | 9 | 2 | 2.1 | - | 0 | 0 | ||
8 | 12 | 2 | 1.0 | 92.3 | 9 | 2 | 2.1 | - | 0 | 0 | ||
14 | 12 | 2 | 1.0 | 93.4 | 10 | 1 | 1.0 | 7 | 1 | 1.0 | ||
16 | 12 | 2 | 1.0 | 94.5 | 10 | 1 | 1.0 | 7 | 1 | 1.0 | ||
29 | 12 | 2 | 1.0 | 95.6 | - | 0 | 0 | 6 | 2 | 2.1 | ||
33F | 12 | 2 | 1.0 | 96.7 | 10 | 1 | 1.0 | 7 | 1 | 1.0 | ||
1 | 13 | 1 | 0.5 | 97.2 | - | 0 | 0 | 7 | 1 | 1.0 | ||
6B | 13 | 1 | 0.5 | 97.8 | - | 0 | 0 | 7 | 1 | 1.0 | ||
12F | 13 | 1 | 0.5 | 98.3 | 10 | 1 | 1.0 | - | 0 | 0 | ||
18C | 13 | 1 | 0.5 | 98.9 | - | 0 | 0 | 7 | 1 | 1.0 | ||
20 | 13 | 1 | 0.5 | 99.4 | 10 | 1 | 1.0 | - | 0 | 0 | ||
34 | 13 | 1 | 0.5 | 100 | - | 0 | 0 | 7 | 1 | 1.0 | ||
All | 184 | 93 | 91 |
Using the EUCAST meningitis breakpoints for penicillin (MIC > 0.06 mg/L), 57 isolates out of 184 (30.9%) were penicillin non-susceptible. Of these, 11 (6.0%) showed high penicillin resistance (MIC> 1 mg/L). For the non-meningitis breakpoints, 30.4% (56/184) were intermediate to penicillin (MIC 0.12-2 mg/L), and only 1 isolate (MIC=3 mg/L) was fully resistant (MIC > 2 mg/L). Nine isolates out of 184 (4.8%) were non-susceptible to ceftriaxone (MIC >0.5 mg/L), 8 were intermediate, and only 1 isolate was fully resistant (MIC=3 mg/L). The rates of resistance to the other antibiotics were: erythromycin, 42.3% (78/184); clindamycin, 36.4% (67/184); tetracycline, 36.9% (68/184); and chloramphenicol, 1.6% (3/184). In addition, 23.9% (44/184) were non-susceptible to both penicillin and erythromycin. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively. The most prevalent serotypes (6C, 24F, and 19A) included mainly penicillin and/or erythromycin non-susceptible isolates, which accounted for 89.4% of 6C, 75.0% of 24F, and 80.0% of 19A isolates. A high rate of antibiotic non-susceptibility was also present in serotypes 23B, 15A, 19F, 3, 14, 33F, and in NT isolates (
Within each serotype, pneumococcal isolates were divided into 4 categories: susceptible to penicillin and erythromycin (green, PEN-S + ERY-S), non-susceptible to penicillin only (red, PEN-R), non-susceptible to erythromycin only (yellow, ERY-R), and non-susceptible to penicillin and erythromycin (striped, PEN-R + ERY-R).
The 93 isolates that were non-susceptible to penicillin and/or erythromycin were genotyped. A complete MLST profile was obtained for 54 isolates, while a partial MLST profile was used for 39 isolates to infer the ST [
CCa (No. isolates) | STsb (No. isolates) | Serotype (No. isolates) | Resistance patternsc (No. isolates) |
---|---|---|---|
156 (16) | 2372 (4), 7121 (3) | 23B (7) | P (7) |
8511 (1) | 19F (1) | PT (1) | |
162 (4) | 24F (4) | PECT (1), ECT (2), EC (1) | |
143 (2) | 14 (2) | PCxECT (1), PCxEC (1) | |
138 (1) | 6C (1) | ECT (1) | |
469 (1) | 6B (1) | ECT (1) | |
315 (13) | 386 (13) | 6C (13) | PECT (11), ECT (2) |
230 (12) | 230 (8) | 24F (8) | PECT (8) |
276 (1), 2013 (1), 2674 (1), 9037 (1) | 19A (4) | PCxECT (2), PT (2) | |
344 (8) | 344 (5), 3097 (1), 4149 (1), 8448 (1) | NT (8) | PCxECT (1), PECT (5), PET (1), PCx (1) |
199 (6) | 416 (5), 2343 (1) | 19A (6) | ECT (5), EC (1) |
63 (6) | 63 (1), 374 (1), 2613 (1), 8512 (1), 8872 (1) | 15A (5) | PECT (1), PEC (1), PET (1), ECT (1), ET (1) |
2543 (1) | 10A (1) | ECT (1) | |
193 (6) | 179 (4) | 19F (4) | PECT (4) |
193 (1) | 15B (1) | ECT (1) | |
1877 (1) | 21 (1) | ET (1) | |
439 (3) | 42 (1), 2404 (1), 8449 (1) | 23A (3) | ECT (1), EC (1), P (1) |
320 (3) | 320 (2) | 19A (2) | PCxECT (2)d |
236 (1) | 19F (1) | PCxET (1) | |
180 (3) | 180 (3) | 3 (3) | ECTCh (2), ECT (1) |
Se (3) | 1577 (3) | 15B (3) | ECT (3) |
62 (2) | 62 (2) | 11A (2) | ET (1), E (1) |
717 (2) | 717 (2) | 33F (2) | ECT (1), EC (1) |
473 (2) | 473 (1) | 6A (1) | PE (1) |
6759 (1) | 6C (1) | ECT (1) | |
395 (1) | 1692 (1) | 6C (1) | EC (1) |
Se (1) | 8447 (1) | 6C (1) | PET (1) |
393 (1) | 3098 (1) | 38 (1) | P (1) |
460 (1) | 446 (1) | 35F (1) | P (1) |
2991 (1) | 2991 (1) | 35F (1) | ECT (1) |
198 (1) | 4346 (1) | 35B (1) | P (1) |
1262 (1) | 8450 (1) | 15C (1) | PET (1) |
3548 (1) | 1766 (1) | 31 (1) | E (1) |
a Clonal complex (CC) and b Sequence type (ST) as defined using information from the MLST website (
Surveillance of pneumococcal carriage is a good method for monitoring the impact of pneumococcal conjugate vaccines at the population level [
In studies conducted in other countries, the pneumococcal carriage rate in young children ranged from 2% to more than 80%, depending on the geographical areas, the socio-economic conditions, and the methods used in the carriage study, including the sampling site (nasopharynx versus oropharynx) [
In our study, the pneumococcal carriage rate was 32.9%, which is consistent with the rates reported in recent studies using cultural methods [
The proportion of children completing the recommended vaccinations for their age with PCV7, PCV13, or a combination of the two was 74.9%. There was a difference in the immunization rates between Bologna and Milan (97.8% versus 57.5%), which likely reflects the different vaccination strategies implemented in the two regions [
A total of 31 different serotypes were found, with 7 (6C, 24F, 19A, 11A, 15B, 23B, and 23A) representing the majority of those detected; in addition, 5.4% and 17.9% were PCV7 and PCV13 serotypes, respectively. The low rate of PCV13 serotypes was expected since some (1, 3, 5, and 7F) are rarely found in the nasopharynx, irrespective of vaccination, likely because they colonize only for a short period of time and at a low density [
This study was a cross-sectional analysis of nasopharyngeal carriage in the Italian pediatric population; therefore, we could not evaluate the changes in serotypes distribution following vaccination. However, variations could be inferred from carriage studies performed in Italy before PCV7 implementation, when more than 50% of the serotypes detected in children were PCV7 ones [
PCV7 and PCV13 appeared to be protective against the collective PCV7 and PCV13 serotypes, however protection against individual vaccine serotypes could not be evaluated due to the low number of isolates. In time, the impact of PCV13 on the pneumococcal reservoir will become more evident, and will likely lead to a decline in serotype 19A, which was still prominent in our study. Future studies can also monitor the effect of PCV13 on serotype 6C, which was most common in our study. Cross-reaction between serotype 6A and 6C has been demonstrated in vitro [
Serotype 6C was circulating in the pre-vaccine era in many parts of the world [
The high prevalence of serotype 24F among Italian children is not surprising since this serotype was circulating in our country and a cause of IPD prior to PCV7 [
Overall, half of the colonizing pneumococci were antibiotic non-susceptible. With the exception of serotype 19A, most of these isolates had non-PCV13 serotypes, such as 6C, 24F, and 15A. Although only one isolate had a penicillin MIC >2 mg/L and was considered fully resistant to penicillin, the rate of penicillin non-susceptibility was significantly higher than that reported for IPD isolates in Italy (31% versus 14%) [
There are some limitations of this study. This was a cross-sectional study of nasopharyngeal carriage; therefore, it is not possible to evaluate changing trends in serotypes or antibiotic resistance, but only to obtain a snapshot of the current situation. In addition, colonization of multiple pneumococcal isolates was not investigated; therefore, some serotypes may have been underestimated. Multiple isolates have been reported in 1.5-50% of carriers [
In conclusion, this study analyzed the pneumococci colonizing healthy children one year after PCV13 was introduced in Italy. It is important to continue to monitor vaccine impact and coverage, using the baseline established here for future comparisons of pneumococcal serotypes.
We thank Dr. Mauro Carlo Benozzi, Dr. Fernando Specchia and Prof. Andrea Pession for recruitment of children. We also thank all participating children and their families, without whom this study would not have been possible. We are indebted to Dr. Alba Finarelli for information about the vaccination status of children from Bologna and to Dr. Stefania Iannazzo for encouragement. We acknowledge the use of the pneumococcal MLST database which is located at Imperial College London and is funded by the Wellcome Trust.