Surveillance and outbreak reports

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1 Surveillnce nd outbrek reports Decresing incidence nd chnges in serotype distribution of invsive pneumococcl disese in persons ged under 18 yers since introduction of 1-vlent nd 13-vlent conjugte vccines in Portugl, July 28 to June 212 S I Aguir 1, M J Brito 2, A N Horácio 1, J P Lopes 1, M Rmirez (rmirez@fm.ul.pt) 1, J Melo-Cristino 1, on behlf of the Portuguese Group for the Study of Streptococcl Infections nd the Portuguese Study Group of Invsive Pneumococcl Disese of the Peditric Infectious Disese Society 3 1. Instituto de Microbiologi, Instituto de Medicin Moleculr, Fculdde de Medicin, Universidde de Lisbo, Lisbon, Portugl 2. Centro Hospitlr de Lisbo Centrl, Lisbon, Portugl 3. Members of the groups re listed t the end of the rticle Cittion style for this rticle: Aguir SI, Brito MJ, Horácio AN, Lopes JP, Rmirez M, Melo-Cristino J, on behlf of the Portuguese Group for the Study of Streptococcl Infections nd the Portuguese Study Group of Invsive Pneumococcl Disese of the Peditric Infectious Disese Society. Decresing incidence nd chnges in serotype distribution of invsive pneumococcl disese in persons ged under 18 yers since introduction of 1-vlent nd 13-vlent conjugte vccines in Portugl, July 28 to June 212. Euro Surveill. 214;19(12):pii=275. Avilble online: Article submitted on 28 August 213 / published on 27 Mrch 214 The 1-vlent pneumococcl conjugte vccine (PCV1) becme vilble in Portugl in mid-29 nd the 13-vlent vccine (PCV13) in erly 21. The incidence of invsive pneumococcl disese (IPD) in ptients ged under 18 yers decresed from 8.19 cses per 1, in 28 9 to 4.52/1, in However, IPD incidence due to the serotypes included in the 7-vlent conjugte vccine (PCV7) in children ged under two yers remined constnt. This fll resulted from significnt decreses in the number of cses due to: (i) the dditionl serotypes included in PCV1 nd PCV13 (1, 5, 7F; from 37.6% to 2.6%), prticulrly serotype 1 in older children; nd (ii) the dditionl serotypes included in PCV13 (3, 6A, 19A; from 31.6% to 16.2%), prticulrly serotype 19A in younger children. The decrese in serotype 19A before vccintion indictes tht it ws not triggered by PCV13 dministrtion. The decrese of serotype 1 in ll groups, concomitnt with the introduction of PCV1, is lso unlikely to hve been triggered by vccintion, lthough PCVs my hve intensified nd supported these trends. PCV13 serotypes remin mjor cuses of IPD, ccounting for 63.2% of isoltes recovered in Portugl in , highlighting the potentil role of enhnced vccintion in reducing peditric IPD in Portugl. Introduction Introduction of the 7-vlent pneumococcl conjugte vccine (PCV7) led to chnges in the circulting serotypes of Streptococcus pneumonie nd lso often to decreses in the incidence of invsive pneumococcl disese (IPD) worldwide [1-3]. Two new pneumococcl conjugte vccine (PCV) formultions re now commercilly vilble nd re used for children [4]. A 1-vlent formultion (PCV1) including, in ddition to the PCV7 serotypes, serotypes 1, 5 nd 7F nd 13-vlent conjugte vccine (PCV13), including ll PCV1 serotypes plus serotypes 3, 6A nd 19A. The few vilble erly reports point to the effectiveness of these expnded vlency vccines ginst the serotypes included in their formultions [4-8]. In Portugl, PCVs re not included in the ntionl immunistion pln. Hd they been, they would hve been offered free of chrge. Nevertheless, since 21, when the vccine becme vilble, there hs been stedy increse in PCV7 uptke bought privtely, without ny reimbursement, reching 75% of children ged 2 yers or under in 28 [9]. PCV1 becme vilble for childhood vccintion in mid-29 nd PCV13 in erly 21. Soon fter PCV13 becme vilble, ccording to sles dt, this vccine ws mostly used (dt not shown). In previous studies, we showed tht significnt chnges in the serotypes cusing IPD in children followed PCV7 vilbility in Portugl [9,1] nd tht there ws evidence for herd effect in the dult popultion [9,11,12]. Serotypes 1 nd 7F, both included in PCV1 nd PCV13, nd serotype 19A, included in PCV13, emerged s mjor cuses of peditric (persons ged under 18 yers) IPD in the post-pcv7 period in Portugl [1]. Given the limited informtion on the efficcy of PCV1 nd PCV13, this study imed t documenting the potentil effects of vccintion on serotype distribution, ntimicrobil resistnce nd incidence of peditric IPD in Portugl from July (week 26) 28 nd June (week 25)

2 Tble 1 Popultion under 18 yers during the study period, Portugl, Clendr yer Number of persons per ge group 11 months months 2 4 yers 5 17 yers Totl 28 13,746 11, ,186 1,454,385 1,977, ,759 13,11 311,641 1,444,426 1,957, ,492 96,995 32,67 1,429,777 1,929, ,73 99, ,727 1,412,241 1,92, ,651 97, ,215 1,45,356 1,89,813 Methods Bcteril isoltes Since 27, the Portuguese Group for the Study of Streptococcl Infections nd the Portuguese Study Group of Invsive Pneumococcl Disese of the Peditric Infectious Disese Society hve monitored pneumococcl invsive infections in Portugl. During the study period, this involved the microbiology lbortories nd peditric deprtments of 61 hospitls throughout Portugl. The network includes centres covering the entire country, including ll referrl hospitls nd most centres where microbiologicl dignostic services re vilble. All centres included in the study reported during the entire period. A cse of IPD ws defined s person from whom n isolte of S. pneumonie ws recovered from normlly sterile body site (not including middle er fluid) or from whom pneumococcl DNA ws detected in cerebrospinl fluid (CSF) or pleurl fluid. Isoltes recovered up to 28 were previously chrcterised [9,1,13]. Isoltes recovered from ptients ged under 18 yers between July 28 nd June 212 were included in the present study. Epidemiologicl yers were defined s spnning from week 26 of one yer to week 25 of the following yer. Only one isolte from ech ptient ws considered. All strins were identified s S. pneumonie by colony morphology nd hemolysis on blood gr pltes, optochin susceptibility nd bile solubility. The lyta gene ws used to identify pneumococci in CSF or pleurl fluid. Incidences were clculted bsed on the entire Portuguese popultion of the relevnt ge groups using dt vilble from the Instituto Ncionl de Esttístic [14] (Tble 1), using the popultion dt of the first clendr yer of ech epidemiologicl yer. The clcultion ssumes tht ll IPD cses were treted t the 61 hospitls in our network. Four ge groups were considered: infnts ged less thn 12 months, children ged months, children ged from two to four yers nd children nd dolescents ged from five yers to less thn 18 yers. Serotyping nd ntimicrobil susceptibility testing Serotyping ws performed by the stndrd cpsulr rection test using the chessbord system [15] nd specific ser (Sttens Serum Institut, Copenhgen, Denmrk). Serotypes were clssified into vccine serotypes, i.e. those included in PCV7 (serotypes 4, 6B, 9V, 14, 18C, 19F, 23F), the dditionl three found in PCV1 (ddpcv1: 1, 5, 7F), the dditionl three found Tble 2 Cses of invsive pneumococcl disese nd vilble Streptococcus pneumonie isoltes from ptients ged under 18 yers, Portugl, July 28 June 212 Epidemiologicl Number of cses b /vilble isoltes, by ge group yers 11 months months 2 4 yers 5 17 yers Totl /49 32/26 36/25 4/33 162/ /35 27/23 33/21 38/35 136/ /2 21/19 16/13 27/25 87/ /17 16/16 22/19 24/16 86/68 Totl 139/121 96/84 17/78 129/19 471/392 From week 26 of one yer to week 25 of the following yer. b Cse numbers comprise ptients from whom pneumococci were isolted from normlly sterile site or pneumococcl DNA ws detected in cerebrospinl fluid or pleurl fluid. 2

3 Figure 1 Incidence of invsive pneumococcl disese in persons ged under 18 yers, Portugl, July 28 June months months yers 5 17 yers The brs represent 95% confidence intervls for the incidence estimtes. Epidemiologicl yers: from week 26 of one yer to week 25 of the following yer. b Number of cses per 1, in specified ge group. in PCV13 (ddpcv13: 3, 6A, 19A) nd non-vccine serotypes (NVT). Etest strips (AB Biodisk, Soln, Sweden) were used to determine the miniml inhibitory concentrtions (MICs) for penicillin, cefotxime, ceftrixone nd levofloxcin. We opted to use the Clinicl nd Lbortory Stndrds Institute (CLSI) guidelines becuse these were the stndrds in use in Portugl during the study period. In 28, the CLSI chnged the recommended brekpoints to those currently used to interpret MIC vlues [16]. Unless otherwise stted, we hve used the CLSIrecommended brekpoints before 28 [17] s epidemiologicl brekpoints tht llow comprison with previous studies. Isoltes were further chrcterised by determining their susceptibility to erythromycin, clindmycin, vncomycin, linezolid, tetrcycline, trimethoprim-sulfmethoxzole nd chlormphenicol by the Kirby-Buer disk diffusion technique, ccording to the CLSI recommendtions nd interprettive criteri [16]. Mcrolide resistnce phenotypes were identified using double disc test with erythromycin nd clindmycin. Simultneous resistnce to erythromycin nd clindmycin defines de MLS B phenotype (resistnce to mcrolides, lincosmides nd streptogrmin B) while non-susceptibility only to erythromycin defines the M phenotype. Sttisticl nlysis Simpson s index of diversity (SID) nd respective 95% confidence intervls (CIs) ws used to mesure the serotype diversity [18]. The SID mesures the probbility tht two isoltes smpled t rndom will not shre the sme serotype. The Cochrn Armitge test ( modifiction of the Person chi-squred test to incorporte suspected ordering in the effects of the ctegories of the second vrible) ws used for trends with the flse discovery rte (FDR) correction for multiple testing [19]. A p vlue of less thn.5 ws considered significnt for ll tests. Results Isolte collection Between July 28 nd June 212, totl of 471 cses of IPD were reported in Portugl. Their distribution by ge nd epidemiologicl yer is shown (Tble 2) nd the nnul incidence of IPD by ge group is presented (Figure 1). Although strting from very disprte vlues, IPD incidence decresed significntly in ll ge groups when compring 28 9 with ( 11 months, p<.1; months nd 2 4 yers, p=.2; nd 5 17 yers, p=.3; ll robust fter FDR) (Tble 3). For the mjority of IPD cses (n=43), pneumococci were isolted from normlly sterile site. Only 41 cses involved solely the identifiction of pneumococcl DNA in CSF or pleurl fluid: for these cses, no cpsulr serotype informtion is vilble. Of the 43 IPD cses from whom S. pneumonie bcteri were recovered, 392 isoltes were vilble for further chrcteristion. The remining 38 isoltes were lost before reching the centrl lbortory for chrcteristion. Avilble isoltes were recovered from blood (n=335, 85.5%), CSF (n=39, 9.9%), pleurl fluid (n=15, 3.8%) nd peritonel fluid (n=3,.8%). Serotype distribution We detected 39 different cpsulr types s well s non-typble isoltes mong the 392 vilble isoltes (Figure 2). The most frequent, which ccounted for 57% (n=225) of isoltes from ll nlysed IPD cses in the study period, were serotypes 1 (n=74, 18.9%), 19A (n=72, 18.4%), 7F (n=43, 11.%), nd 14 (n=36, 9.2%). The proportion of IPD cses cused by PCV7 serotypes remined reltively constnt, t round 21%. However, the frction of IPD cses tht could hve been potentilly prevented by PCV1 nd PCV13 decresed significntly during the study period, from 59.4% to 47.1% (p=.8) nd from 91.% to 63.% (p=.24), respectively. This ws ccompnied by n increse in serotype diversity when compring 28 9 (SID=.846, CI95%:.81 to.882) with (SID=.957, 95% CI: ). In order to estimte the incidence of IPD due to individul serotypes, the cses for which no isolte ws vilble (n=79) were ssumed to hve the sme serotype distribution s tht found mong isoltes from 3

4 Tble 3 Incidence of invsive pneumococcl infections cused by Streptococcus pneumonie serotypes included in conjugte vccine formultions by ge group, Portugl, July 28 June 212 Age group 17 yers 5 17 yers 2 4 yers months 11 months Serotype group (95% confidence intervls) PCV ( ) 1.41 ( ).82 ( ) 1.1 ( ) PCV ( ) 3.66 ( ) 2.1 ( ) 2.17 ( ) PCV ( ) 5.27 ( ) 3.1 ( ) 2.87 ( ) NVT.69 ( ) 1.68 ( ) 1.41 ( ) 1.65 ( ) All serotypes 8.19 ( ) 6.95 ( ) 4.51 ( ) 4.52 ( ) PCV7.33 (.14.79).38 (.17.85) (.27) (.27) PCV ( ) 1.65 ( ).83 ( ) 1.17 ( ) PCV ( ) 2.1 ( ) 1.51 ( ) 1.17 ( ) NVT.8 (.2.41).53 ( ).38 (.17.86).53 ( ) All serotypes 2.75 ( ) 2.63 ( ) 1.89 ( ) 1.7 ( ) PCV ( ) 2.2 ( ).41 (.8 2.) 2.36 ( ) PCV1 9.5 ( ) 8.7 ( ) 3.67 ( ) 3.14 ( ) PCV ( ) 9.58 ( ) 4.7 ( ) 5.11 ( ) NVT ( 1.21) 1.1 ( ) 1.22 ( ) 2.36 ( ) All serotypes ( ) 1.59 ( ) 5.3 ( ) 7.46 ( ) PCV ( ) 7.98 ( ) 6.84 ( ) 7.3 ( ) PCV ( ) 11.4 ( ) 6.84 ( ) 7.3 ( ) PCV ( ) 2.51 ( ) ( ) 9.4 ( ) NVT 3.64 ( ) 5.7 ( ) 7.98 ( ) 7.3 ( ) All serotypes ( ) ( ) ( ) 16.8 ( ) PCV ( ) 7.7 ( ) 8.1 ( ) 7.38 ( ) PCV ( ) 1.99 ( ) 9.15 ( ) 8.85 ( ) PCV ( ) ( ) ( ) ( ) NVT 8.5 ( ) ( ) 1.3 ( ) 1.32 ( ) All serotypes 52.5 ( ) ( ) ( ) 25.8 ( ) NVT: non-vccine serotypes; PCV: pneumococcl conjugte vccine. Epidemiologicl yers: from week 26 of one yer to week 25 of the following yer. b Number of cses per 1, in specified ge group. the sme epidemiologicl yer nd ge group. The overll incidence of IPD in ll ge groups due to PCV7, ddpcv1 nd ddpcv13 serotypes decresed during the study period (p=.26, p<.1 nd p<.1 respectively, robust fter FDR) while tht of cses with NVT incresed (p=.27, lso robust fter FDR) (Figure 3A, Tble 3). While IPD incidence due to the serotypes included in the conjugte vccines decresed, these were still significnt cuse of IPD in ll ge groups, ccounting for 62.3% (n=43) of the isoltes in When considering individul serotypes (for which t lest five isoltes were detected), the incidence of IPD due to serotypes 1 (p<.1), 19A (p<.1), 7F (p=.24) nd 14 (p=.15) decresed nd increses in serotype 1A (p=.1) were noted. However, only the decrese in serotypes 1 nd 19A incidence nd the increse in 1A incidence were supported fter djustment by FDR. In contrst to the continul decreses in the number of isoltes of serotype 1 nd 19A during the study period, serotype 7F decresed only in fter the introduction of PCV1 (Tble 4). These resulted in decreses in the proportion of isoltes of ddpcv1 from 37.6% in 28 9 to 2.6% in nd of ddpcv13 from 31.6% in 28 9 to 16.2% in The overll chnges in IPD incidence re the result of very different dynmics in the vrious ge groups (Figure 3 nd Tble 3). In the youngest ge group ( 11 months), IPD incidence due to the dditionl serotypes included in the expnded vlency conjugte vccines decresed (ddpcv1, p=.6; ddpcv13, p<.1, both robust fter FDR) while the incidence due to PCV7 serotypes nd NVTs did not chnge significntly. A similr sitution occurred in those ged months, but here only the ddpcv13 (p=.4) ws robust fter FDR. 4

5 Figure 2 Streptococcus pneumonie isoltes expressing serotypes present in conjugte vccines cusing invsive pneumococcl infections, Portugl, July 28 June 212 (n=392) Susceptible (to both penicillin nd erythromycin) ERP PNSP EPNSP 11 months (n=121) months (n=84) 2 4 yers (n=78) 5 17 yers (n=19) Number of isoltes B 9V 14 18C 19F 23F 1 5 7F 3 6A 19A Other (NVT) PCV7 (21.4%) PCV1 (51.8%) PCV13 (77.%) EPNSP: isoltes presenting both erythromycin resistnce nd penicillin non-susceptibility; ERSP: erythromycin-resistnt isoltes; NVT: nonvccine serotypes; PNSP: penicillin non-susceptible isoltes. The number of isoltes expressing ech serotype in ech of the ge groups considered is shown. The serotypes included in ech of the conjugte vccines re indicted by the rrows nd the percentge of the totl number of isoltes expressing the serotypes included in ech of the vccines is indicted. A totl of 27 NVT were detected, representing 9 isoltes s follows: 1A, 15A nd 23B (n=7 ech); 22F (n=6); 8, 15B, 15C, 21 nd 24F (n=5 ech); 25A (n=4); 9N, 11A, 2, 24A, 33A nd non-typble (n=3 ech); 6C, 16F, 29, 33F nd 35F (n=2 ech); 7C, 12B, 18B, 28A, 34 nd 45 (n=1 ech). In ptients ged 2 4 yers, there ws no significnt chnge in IPD incidence due to ddpcv13, but there ws substntil decrese in IPD incidence due to the ddpcv1 serotypes (p<.1) ccompnied by n increse in IPD incidence due to NVTs (p=.7), both supported fter djustment by FDR. In the older ge group (5 17 yers), in which no individuls were vccinted with PCV1 or PCV13, the decreses in IPD incidence due to ddpcv1 nd ddpcv13 serotypes were not sttisticlly significnt: the only significnt chnge ws the decrese in IPD incidence due to PCV7 serotypes (p=.6, robust fter FDR). Antimicrobil susceptibility Resistnce to the tested ntimicrobils is summrised in Figure 2 nd Tble 5. Overll, 11/392 isoltes (28.1%) were non-susceptible to penicillin (PNSP): 82 (2.9%) expressed low-level resistnce nd 28 (7.1%) high-level resistnce [17]. Considering the current CLSI brekpoints for prenterl penicillin [16], 16/39 isoltes from CSF would hve been considered resistnt nd three isoltes (.8%) from 353 non-meningitis cses would hve been considered non-susceptible, ll intermeditely resistnt. Resistnce to erythromycin (ERP) ws found in 13/392 isoltes (26.%), of which 78 isoltes (75.7%) expressed the MLS B phenotype nd 25 (24.3%) the M phenotype. All isoltes were susceptible to levofloxcin, vncomycin nd linezolid. The simultneous expression of erythromycin resistnce nd penicillin non-susceptibility (EPNSP) ws found in 64/392 (16.3%) of the isoltes. Within the study period, there ws modest decrese in ERP (when compring 36/133 (27.1%) in 28 9 with 16/68 (23.5%) in ) nd more noticeble decrese in PNSP (when compring 45/133 (33.8%) in 28 9 with 14/68 (2.6%) in ). 5

6 Figure 3 Incidence of invsive pneumococcl disese due to vccine serotypes in ptients ged under 18 yers, Portugl, July 28 June A. Under 18 yers PCV7 4 1,5,7F ,6A,19A NVT B. 11 months C months D. 2 4 yers E yers NVT: non-vccine serotypes; PCV7: 7-vlent pneumococcl conjugte vccine. Epidemiologicl yers: from week 26 of one yer to week 25 of the following yer. b Number of cses per 1, in specified ge group. 6

7 Tble 4 Serotypes of the isoltes responsible for invsive pneumococcl disese in ptients ged under 18 yers, Portugl, July 28 June 212 (n=392) Serotype Number of isoltes A B F V C A F F NVT Totl Tble 5 Antimicrobil resistnce of Streptococcus pneumonie isoltes responsible for invsive pneumococcl disese in ptients ged under 18 yers, Portugl, July 28 June 212 (n=392) Antibiotic 11 months (n=121) Number of resistnt isoltes (%) b months (n=84) 2 4 yers (n=78) [5 17 yers (n=19) PEN c 43 (35.5) 38 (45.2) 15 (19.2) 14 (12.8) MIC MIC CTX 4 (3.3) 7 (8.3) 1 (1.3) 2 (1.8) MIC MIC CRO 6 (5.) 4 (4.8) (.) 2 (1.8) MIC MIC ERY 46 (38.) 29 (34.5) 15 (19.2) 13 (11.9) CLI 34 (28.1) 23 (27.4) 13 (16.7) 8 (7.3) CHL 6 (5.) 4 (4.8) (.) 4 (3.7) SXT 27 (22.3) 21 (25.) 16 (2.5) 15 (13.8) TET 29 (24.) 21 (25.) 11 (14.1) 13 (11.9) NVT: non-vccine type. Epidemiologicl yers: from week 26 of one yer to week 25 of the following yer. CHL: chlormphenicol; CLI: clindmycin; CRO: ceftrixone; CTX: cefotxime; ERY: erythromycin; LEV: levofloxcin; MIC: miniml inhibitory concentrtion; PEN: penicillin; SXT: trimethoprimsulfmethoxzole; TET: tetrcycline. Epidemiologicl yers: from week 26 of one yer to week 25 of the following yer. b Unless otherwise specified. c Number of isoltes nd percentge of penicillin non-susceptible isoltes is indicted. Together, serotypes 19A nd 14 contributed gretly to ERP (62/13, 6.2%) nd PNSP (64/11, 58.2%) (Figure 2). Serotypes included in PCV1 represented 33/46 (71.7%), 21/39 (53.8%) nd 17/64 (26.6%) of PNSP, ERP nd EPNSP, respectively, while serotypes included in PCV13 constituted 41/46 (89.1%), 32/39 (82.1%) nd 49/64 (76.6%), respectively. Discussion The most importnt finding of our study is the decrese in incidence of IPD in ll ge groups nlysed. Moreover, the chnges in distribution of the serotypes of pneumococci cusing IPD in Portugl tht ccompnied the introduction of PCV7 [9,1] hve continued with the introduction of PCV1 nd PCV13. Overll, IPD incidence due to the serotypes included in the PCVs decresed (Tble 3 nd Figure 3A): this ffected prticulrly the dditionl serotypes included in PCV1 nd PCV13 nd to lesser extent the serotypes included in PCV7. The incidence of IPD mens tht the ctul number of isoltes per serotype in ech epidemiologicl yer is smll (Tble 4). This is even more mrked when strtifying by ge group nd in the lst yers of the study, due to the decrese in IPD incidence. The resons behind the persistence of PCV7 serotypes s cuses of IPD my be multifctoril [12,2]. Chief mong those could be the slower uptke nd lower vccintion coverge in Portugl when compred with countries where PCV7 ws introduced in the ntionl immunistion pln [1,2]. As with serotype 19A (see below), the high proportion of resistnt isoltes expressing PCV7 serotypes, prticulrly to penicillin nd mcrolides, could be n importnt fctor in their persistence. Serotype 14, which is known to represent more virulent clones [21], remins the most importnt PCV7 serotype in IPD. In contrst to our dt, recent study in Portugl found the virtul elimintion of PCV7 serotypes from nsophryngel crrige, with the exception of serotype 19F, serotype lredy ssocited with crrige before the introduction of PCVs [22]. The serotype dynmics underpinning the chnges in IPD incidence were different in the vrious ge groups. In the youngest children (under 2 yers), including those vccinted with either PCV1 or PCV13, there were decreses in IPD cused by the serotypes included in the PCVs (Figure 3, B nd C). While reduction in 7

8 incidence due to ddpcv1 could be expected in 29 1, since the PCV1 vccine ws vilble from the outset of the epidemiologicl yer, the reduction in incidence due to ddpcv13 occurred in spite of PCV13 becoming vilble only in erly 21. IPD incidence due to PCV7 serotypes decresed slightly, but not significntly: these serotypes remin importnt cuses of IPD in spite of more thn decde of PCV7 use. The 2 4 yers ge group strted including children potentilly vccinted with t lest one dose of PCV1 or PCV13 in In this ge group, there ws strong decrese in the IPD incidence due to ddpcv1 serotypes lredy in (Figure 3D), wheres no significnt chnge ws seen in incidence due to ddpcv13. In the older ge group, (5 17 yers), the decreses in IPD incidence cused by ddpcv1 occurring between 28 9 nd nd those due to ddpcv13 serotypes in were not sttisticlly significnt. Tken together, our dt suggest tht there my be different resons for the decreses in the incidence due to the vrious serotypes included in both PCV1 nd PCV13 nd those included only in PCV13. Limited dt vilble from observtionl studies in different geogrphicl regions hve documented decreses in IPD incidence due to PCV13 serotypes following PCV13 introduction nd of serotypes 1 nd 19A in prticulr [6,7]. A field study showed cler effectiveness of PCV13 ginst serotype 19A IPD [4], while nother study demonstrted reduced nsophryngel cquisition of both serotypes 1 nd 19A [8]. Decreses in the proportion of isoltes expressing serotypes 1 nd 5, included in both PCV1 nd PCV13, nd of serotype 6A, included only in PCV13, were noted in the dult ( 18 yers) popultion in Portugl between 29 nd 211 [12]. In contrst, the proportion of isoltes expressing serotype 19A, lso included only in PCV13, did not chnge significntly s cuse of IPD in the dult popultion in the country [12]. The herd effect tht is known to occur due to PCV7, nd now lso expected for PCV1 nd PCV13, is predicted to be delyed reltive to the effects on the vccinted children [2,9,23]. It is therefore unlikely tht the chnges seen in dults, nd now in older unvccinted peditric ge groups, cn be ttributed to PCV use. These dt emphsise the importnce of unexplined temporl trends in the distribution of pneumococcl serotypes [24] s potentil confounders in observtionl studies such s ours. Similr to our observtions, vccine tril in Alsk sw decreses in the incidence of PCV13 serotype IPD even before direct effect of vccintion would be expected [2]. Although mrked fluctutions in the incidence of serotype 1 IPD hve been known to occur, no such chnges hve been documented in IPD due to serotype 19A, one of the most importnt serotypes tht emerged in the post-pcv7 er nd serotype tht remins stble cuse of IPD in dults in Portugl [1,12,25]. Another importnt difference between isoltes expressing these two serotypes is their ntimicrobil resistnce. While serotype 1 isoltes re mostly susceptible to the ntimicrobils most frequently used to tret pneumococcl infections, serotype 19A isoltes re frequently resistnt to penicillin nd the mcrolides [1,24,25], chrcteristic tht hs been mintined throughout the study period (Figure 2). Our dt rgue tht fctors other thn vccintion, such s unrelted temporl trends, triggered the decrese in the incidence of the two serotypes tht hd become the leding cuses of peditric IPD in the post-pcv7 yers: serotypes 1 nd 19A (Tble 4) [1]. The reson for these chnges remins unknown nd my be different for these two serotypes. However, even if the observed serotype chnges were not triggered by vccintion, PCV use might hve reinforced them. Although the decreses in incidence of PCV13 serotypes were prtly offset by n increse in incidence of NVT IPD, the overll result ws substntil reduction in the incidence of peditric IPD. An increse in NVT IPD incidence ws noted in most ge groups, lthough only in the 2 4 yer-olds ws this increse sttisticlly significnt. The decrese in the incidence of cses due to PCV13 serotypes resulted in multiple serotypes becoming more prominent cuses of IPD, leding to incresed serotype diversity of IPD cses. Serotype 1A ws the only NVT serotype rising significntly in n overll nlysis. However, this serotype ws not mong the most frequent NVT serotypes detected in crrige study [22] nor ws it found to be prticulrly invsive [21], rising the possibility tht this will not be sustined increse. The decrese in overll PNSP during the study period reflected primrily the decrese of serotype 19A. When compring with previous dt [1], resistnce to penicillin nd cefotxime remined unchnged in ll ge groups nd there ws modest decline in ceftrixone resistnce. On the other hnd, resistnce to erythromycin nd clindmycin rose in the youngest children (ged <2 yers) but remined pproximtely constnt in the older ge groups. Serotype 19A nd the PCV7 serotypes, prticulrly serotype 14, remined the most importnt serotypes in terms of resistnce, suggesting potentil influence of PCVs in the dynmics of ntimicrobil resistnce. Since the criterion for identifiction of n IPD cse is the isoltion of pneumococci from normlly sterile body site or the identifiction of pneumococcl DNA in cerebrospinl fluid (CSF) or pleurl fluid, nd this is lmost exclusively done in hospitl lbortories, we believe tht few cses of lbortory-confirmed IPD would hve been dignosed outside of our network. The ctive nture of the surveillnce nd the involvement of lrge number of hospitls covering the entire country offer further ressurnce tht our surveillnce system identified most cses. We cnnot gurntee tht 8

9 the serotype distribution of the cses where isoltes were unvilble followed the serotype distribution of vilble isoltes, but we consider tht the pproch dopted minimises potentil bis nd describes the ctul sitution. However, the smll proportion of cses with unknown serotype informtion ensures tht our extrpoltion does not ffect the results. In spite of substntil decreses in PCV13 IPD incidence in ll ge groups (Tble 3), these serotypes remin the most importnt cuses of IPD, being responsible for 63.2% of IPD cses in ptients under 18 yers in (Tble 4). The persistence of these serotypes, including the PCV7 serotypes tht hve been subject to vccine pressure for more thn decde, suggests tht this could be due to the reltively modest vccintion coverge in Portugl. Coverge peked round 28 t 75% but declined to 63% in 212 (dt not shown), lower coverge thn in countries where PCVs re in the ntionl immunistion pln nd highlighting the potentil benefits of incresing vccintion coverge. Although the study period is probbly too close to vccine introduction to expect herd effect in older unvccinted children, the reltively lower vccintion coverge my lso compromise the extent of this effect. This my reduce the overll benefits of vccintion, including the potentil protection of infnts younger thn 9 dys [26] who re not currently protected by direct vccintion. Continued surveillnce will monitor the extent of PCV13 success by evluting the cpcity of PCV13 serotypes to persist in spite of vccintion nd in documenting the chnges in ntimicrobil resistnce tht ccompny the chnging serotypes nd the emergence of ny NVT replcement serotypes. Members of the Portuguese Group for the Study of Streptococcl Infections Teres Vz, Mríli Gião, Rui Ferreir (Centro Hospitlr do Brlvento Algrvio), An Buschy Fonsec (Hospitl de Cscis), Henrique Oliveir (Centro Hospitlr de Coimbr), An Cristin Silv, Hermíni Cost (Centro Hospitlr de Entre Douro e Voug), Mrgrid Pinto, Odete Chntre, João Mrques, Isbel Peres, Isbel Dniel, Em Cns, Teres Ferreir, Cristin Mrcelo (Centro Hospitlr de Lisbo Centrl), Lurdes Monteiro, Luís Mrques Lito (Centro Hospitlr Lisbo Norte), Teres Mrques, Cristin Toscno, Filomen Mrtins, Mri An Pessnh, Els Gonçlves, Teres Moris (Centro Hospitlr de Lisbo Ocidentl), Pulo Lopes, Luís Felício, Angelin Lmeirão (Centro Hospitlr de Vil Nov de Gi / Espinho), An Pul Mot Vieir, Mrgrid Tomz (Centro Hospitlr do Alto Ave), Ros Bento (Centro Hospitlr do Bixo Alentejo), Mri Helen Rmos, An Pul Cstro (Centro Hospitlr do Porto), Fernndo Fonsec (Centro Hospitlr d Póvo do Vrzim / Vil do Conde), An Pul Cstro, (Centro Hospitlr de Trás os Montes e Alto Douro), Grç Ribeiro, Rui Tomé, Celeste Pontes, Luís Boventur (Hospitis d Universidde de Coimbr), Nuno Cnhoto, Teres Afonso (Centro Hospitlr do Funchl), Teres Pin, Helen Peres (Hospitl Curry Cbrl, Lisbo), Ilse Fontes, Pulo Mrtinho (Hospitl de Snt Luzi, Elvs), An Domingos, Gin Mrrão, José Grossinho (Hospitl de Snto André, Leiri), Mnuel Ribeiro (Hospitl de São João, Porto), Albert Fustino, Adelide Alves (Hospitl de Brg), Mri Pul Pinheiro, R. Semedo (Hospitl Dr. José Mri Grnde, Portlegre), Adrin Coutinho (Hospitl do Espírito Snto, Évor), Luís Cbrl, Olg Neto (Hospitl dos SAMS, Lisbo), Luís Sncho (Hospitl Dr. Fernndo d Fonsec, Amdor / Sintr), José Diogo, An Rodrigues, Isbel Nscimento (Hospitl Grci de Ort, Almd), Elmno Rmlheir, Fernnd Bess (Hospitl Infnte D. Pedro, Aveiro), I. Mrques, José Miguel Ribeiro (Hospitl de São Teotónio, Viseu), Mri Antóni Red, Vlquíri Alves (Hospitl Pedro Hispno, Mtosinhos), Engráci Rposo, Mri Lurdes Mglhães, Helen Rochs, Anbel Silv (Instituto Ncionl de Súde Ricrdo Jorge, Porto), Mrgrid Rodrigues (Hospitl Reynldo dos Sntos, Vil Frnc de Xir), Mri Fvil Meneses, José Germno de Sous (Hospitl CUF Descoberts), Mrin Bettencourt Vin, Isur Terr (Centro Hospitlr do Tâmeg e Sous), Vitóri Rodrigues, Ptríci Pereir (Hospitl Betriz Ângelo, Loures), Jesuín Durte (Centro Hospitlr de Setúbl), Pul Pinto (Hospitl Distritl de Sntrém), Ezequiel Moreir (Centro Hospitlr do Médio Ave), João Atíde Ferreir (Hospitl de Fro), Adíli Vicente (Centro Hospitlr do Oeste Norte), Pulo Pixão (Hospitl d Luz), Ntáli Novis (Hospitl d Figueir d Foz). Members of the Portuguese Study Group of Invsive Pneumococcl Disese of the Peditric Infectious Disese Society Sóni Aires (Centro Hospitlr de Entre Douro e Voug), Cristin Ferreir (Centro Hospitlr do Alto Ave), Eurico Gspr (Centro Hospitlr de Trás os Montes e Alto Douro), Mnuel Ferreir, Fernnd Pereir (Centro Hospitlr do Nordeste), Mri José Dinis (Centro Hospitlr d Póvo do Vrzim / Vil do Conde), Álvro Sous, Pulo Teixeir (Centro Hospitlr do Médio Ave), José Amorim (Centro Hospitlr do Alto Minho), Cláudi Monteiro (Centro Hospitlr do Tâmeg e Sous), Isbel Crvlho (Centro Hospitlr de Vil Nov de Gi / Espinho), Sofi Aros (Hospitl Pedro Hispno, Mtosinhos), Mrgrid Guedes, Lur Mrques, An Brg (Centro Hospitlr do Porto), Mrgrid Tvres (Hospitl de São João, Porto), Isbel Cunh (Hospitl de Brg), Lurdes Vicente (Hospitl Amto Lusitno, Cstelo Brnco), Mri Mnuel Zrcos (Hospitl de Snto André, Leiri), Helen Almeid (Centro Hospitlr do Oeste Norte), Silvi Almeid (Hospitl Infnte D. Pedro, Aveiro), Fernnd Rodrigues, Cristin Resende (Centro Hospitlr de Coimbr), Euláli Afonso (Hospitis d Universidde de Coimbr), Luís Mendes (Hospitl d Figueir d Foz), Cristin Fri (Hospitl de São Teotónio, Viseu), An Luís Teixeir (Centro Hospitlr d Cov d Beir, Covilhã), António Mendes (Hospitl Sous Mrtins, Gurd), Teres Tomé, Mónic Rebelo, Mri João Brito (Centro Hospitlr de Lisbo Centrl), Filomen Pereir (IPO, Lisbo), Gustvo Rodrigues (Hospitl Lusíds, Lisbo), Alexndr Cost, An Teixeir (Centro Hospitlr de Lisbo Ocidentl), Sofi Lim (Hospitl Betriz Ângelo, Loures), Éric Lim (Hospitl d Luz), Mri An S. Nunes (Hospitl Cruz Vermelh, Lisbo), Filip Prt (Centro Hospitlr Lisbo Norte), Pedro Flores (Hospitl CUF Descoberts), Mnuel Brndão (Hospitl dos SAMS, Lisbo), João Cldo Nunes (Hospitl Distritl de Sntrém), Rosário Mss (Centro Hospitlr do Médio Tejo, Abrntes), Florbel Cunh (Hospitl Reynldo dos Sntos, Vil Frnc de Xir), Pul Correi (Hospitl Dr. Fernndo d Fonsec, Amdor / Sintr), Anbel Brito (Hospitl de Cscis), João Frnco (Hospitl Grci de Ort, Almd), Cristin Didelet (Centro Hospitlr do Brreiro Montijo), Estel Veig (Centro Hospitlr de Setúbl), Crl Cruz (Hospitl do Espírito Snto, Évor), Grç Seves (Centro Hospitlr do Bixo Alentejo), Céu Novis (Hospitl Dr. José Mri Grnde, Portlegre), Mri João Virtuoso (Hospitl de Fro), Nncy Guerreiro (Centro Hospitlr do Brlvento Algrvio), Améli Cvco (Centro Hospitlr do Funchl), Frncisco Gomes (Hospitl de Snto Espírito, Angr do Heroísmo), 9

10 Dor Gomes (Hospitl d Hort), Isbel Monteiro (Hospitl do Divino Espírito Snto, Pont Delgd). Acknowledgements S.I. Aguir nd A.N. Horácio were supported by grnts SFRH/ BPD/78376/211nd SFRH/BD/8125/211, respectively, from Fundção pr Ciênci e Tecnologi, Portugl. This work ws prtilly supported by Fundção pr Ciênci e Tecnologi, Portugl (PTDC/DTP-EPI/1759/212) nd unrestricted reserch grnts from Pfizer nd GlxoSmithKline. Conflict of interest JMC reserch grnts dministered through his university nd honorri for serving on spekers bureus of Pfizer, Giled nd Novrtis. MR honorri for serving on the spekers bureu of Pfizer nd for consulting for GlxoSmithKline. Authors contributions JMC, MJB nd MR developed the design for the study; MJB, JMC nd the Portuguese Group for the Study of Streptococcl Infections nd the Portuguese Study Group of Invsive Pneumococcl Disese of the Peditric Infectious Disese Society coordinted the collection of the surveillnce dt; SIA, ANH nd JPL chrcterised the isoltes; JMC, SIA nd MR nlysed the dt; JMC nd MR drfted the mnuscript; ll co-uthors reviewed nd contributed to the finl version of the mnuscript. References 1. Pilishvili T, Lexu C, Frley MM, Hdler J, Hrrison LH, Bennett NM, et l. Sustined reductions in invsive pneumococcl disese in the er of conjugte vccine. J Infect Dis. 21;21(1): Miller E, Andrews NJ, Wight PA, Slck MP, George RC. Herd immunity nd serotype replcement 4 yers fter seven-vlent pneumococcl conjugte vccintion in Englnd nd Wles: n observtionl cohort study. Lncet Infect Dis. 211;11(1): Rodenburg GD, de Greeff SC, Jnsen AG, de Melker HE, Schouls LM, Hk E, et l. 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