Dominance of vaccine serotypes in pediatric invasive pneumococcal infections in Portugal (2012-2015)

1 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x www.na u e.com/scien i ic epo s Dominance o accine se o ypes in pedia ic in asi e pneumococcal in ec ions in Po ugal (2012–2015) Ca a ina Sil a-Cos a1, Ma ia J. B i o2, Sand a I. Aguia 1, Joana P. Lopes1, Má io Rami ez 1, José Melo-C is ino1, Po uguese G oup o he S udy o S ep ococcal In ec ions* & he Po uguese S udy G oup o In asi e Pneumococcal Disease o he Pedia ic In ec ious Disease Socie y* We e alua ed he impac o con inued 13- alen pneumococcal conjuga e accine (PCV13) use in he p i a e ma ke (up ake o 61%) in pedia ic in asi e pneumococcal disease (pIPD) in Po ugal (2012–2015). The mos equen ly de ec ed se o ypes we e: 3 (n = 32, 13.8%), 14 (n = 23, 9.9%), 1 (n = 23, 9.9%), 7F (n = 15, 6.4%), 19A (n = 13, 5.6%), 6B and 15B/C (bo h n = 12, 5.2%), and 24F, 10A and 12B (all wi h n = 10, 4.3%). Taken oge he , non-PCV13 se o ypes we e esponsible o 42.2% o pIPD wi h a known se o ype. The use o PCR o de ec and se o ype pneumococci in bo h pleu al and ce eb ospinal luid samples con ibu ed o 18.1% (n = 47) o all pIPD. Se o ype 3 was mos ly de ec ed by PCR (n = 21/32, 65.6%) and esul ed om a ele an numbe o accine ailu es. The incidence o pIPD a ied in he di e en age g oups bu wi hou a clea end. The e we e no ob ious declines o he incidence o pIPD due o se o ypes included in any o he PCVs, and PCV13 se o ypes s ill accoun ed o he majo i y o pIPD (57.8%). Ou s udy indica es ha a highe accina ion up ake may be necessa y o ealize he ull bene i s o PCVs, e en a e 15 yea s o mode a e use, and highligh s he impo ance o using molecula me hods in pIPD su eillance, since hese can lead o subs an ially inc eased case asce ainmen and iden i ica ion o pa icula se o ypes as causes o pIPD. The in oduc ion o pneumococcal conjuga e accines (PCVs) in o ou ine in an immuniza ion p og ams wo ld- wide has led o signi ican dec eases in o e all incidence o in asi e pneumococcal disease (IPD), bo h in child en and in adul s1–5. Mo eo e , massi e changes in he dis ibu ion o S ep ococcus pneumoniae se o ypes we e also no ed, wi h majo dec eases in he incidence o IPD caused by accine se o ypes (VTs), i.e. hose included in PCV o mula ions1,2,6,7. Howe e , his was accompanied in some cases by an inc ease in he incidence o IPD due o non- accine se o ypes (NVTs)5,8,9. Addi ionally, an imic obial suscep ibili y may also be a ec ed by he use o PCVs since hese a ge se o ypes equen ly associa ed wi h an imic obial esis ance10,11. In Po ugal, al hough a ailable only in he p i a e ma ke , he in oduc ion o he 7- alen pneumococcal con- juga e accine (PCV7) in 2001 led o signi ican changes in he se o ypes esponsible o pedia ic IPD (pIPD)2. Among he mos ele an changes was he ise o non-PCV7 se o ypes 1, 7F and 19A as majo causes o pIPD12. Du ing his pe iod up ake peaked a 75% a ound 20081. In mid-2009, PCV10 became a ailable, including PCV7 se o ypes and addi ionally se o ypes 1, 5 and 7F. Soon a e his, in ea ly 2010, PCV13 was in oduced in Po ugal, u he including se o ypes 3, 6A and 19A, and became he leading accine used in Po ugal. Despi e a decline in PCV up ake o 62% in 2012, which emained s able un il 2014 a 61%13, he in oduc ion o highe alency accines was accompanied by declines o pIPD incidence, wi h a dec ease om 8.19 cases pe 100,000 inhabi an s in 2008–2009 o 4.52 pe 100,000 inhabi an s in 2011–20121. Despi e PCV7 use since 2001, his dec ease was mainly due o dec eases in he numbe o cases due o se o ypes included in he newes ac- cines, pa icula ly se o ype 1 and 19A in olde and younge child en, espec i ely, and no o u he educ ions in he numbe o cases due o PCV7 se o ypes, including in child en unde 2 yea s o age. Tha s udy also sugges ed 1Ins i u o de Mic obiologia, Ins i u o de Medicina Molecula , Faculdade de Medicina, Uni e sidade de Lisboa, Lisboa, Po ugal. 2Cen o Hospi ala de Lisboa Cen al, Lisboa, Po ugal. *A comp ehensi e lis o conso ium membe s appea s a he end o he pape . Co espondence and eques s o ma e ials should be add essed o M.R. (email: [email p o ec ed]) Recei ed: 30 Ap il 2018 Accep ed: 27 No embe 2018 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x ha some o he se o ype changes may no ha e been igge ed by accina ion1, emphasizing he impo ance o he na u al luc ua ions o se o ypes and he need o pe o m con inuous epidemiological su eillance. In July 2015, PCV13 was in oduced in he Na ional Immuniza ion Plan (NIP), o child en bo n a e Janua y 2015, wi h doses gi en a 2, 4 and 12 mon hs o age. The aims o his wo k we e o de e mine he incidence o pIPD in Po ugal, as well as se o ype dis ibu ion and suscep ibili y pa e ns among he pneumococcal popula- ion be ween July 2012 and June 2015, jus p io o he in oduc ion o PCV13 in he NIP. Resul s Isola e collec ion. Be ween July 2012 and June 2015, a o al o 259 cases o pIPD we e epo ed. Table1 summa izes hei dis ibu ion by age and epidemiological yea while Fig.1 p esen s he annual incidence o pIPD by age g oup. We ound no empo al and geog aphic clus e ing o cases which could sugges he exis ence o ou - b eaks. Al hough he e we e a ia ions in he incidence o pIPD in all age g oups hese did no show clea ends (TableS1). Fo 27 cases (10.4%) nei he he isola e no a pa ien sample was sen o he cen al labo a o y o cha ac e iza ion. A o al o 185 isola es (79.7%) and 47 samples (20.2%) o LCR (n = 6) o pleu al luid (n = 41) posi i e o S. pneumoniae we e a ailable o u he cha ac e iza ion. A ailable isola es we e eco e ed om blood (n = 149, 80.6%), CSF (n = 25, 13.4%), pleu al luid (n = 7, 3.8%), and syno ial luid (n = 4, 2.2%). Se o ype dis ibu ion. Among he 232 isola es and pa ien samples 39 di e en capsula ypes we e de ec ed, including samples o which he se o ype could no be unambiguously de e mined– 7F/7A (n = 3), 25A/38 (n = 2), se og oup 6 (n = 1), 33F/33A/37 (n = 1), 29/35B (n = 1), as well as non- ypable isola es (Figs2 and 3). This ansla ed in o a high se o ype di e si y (SID = 0.943; CI95% 0.933 o 0.954). The mos equen se o ypes, which accoun ed o 69% o all pIPD in he s udy pe iod, we e se o ypes 3 (n = 32, 13.8%), 14 (n = 23, 9.9%), 1 (n = 23, 9.9%), 7F (n = 15, 6.4%), 19A (n = 13, 5.6%), 6B and 15B/C (bo h n = 12, 5.2%), and 24F, 10A and 12B (all wi h n = 10, 4.3%). O e all pIPD due o he se o ypes included in PCV13 emained signi ican , accoun ing o 57.8% (21.6% PCV7) o he isola es and pa ien samples. When conside ing indi idual se o ypes (n > 5) in all age g oups, we no ed signi ican dec eases in he numbe o cases om 2012–2013 o 2014–2015 o se o ypes 1 and 15B/C, and an inc ease o se o ype 14, bu none o hese changes was s a is ically suppo ed (Table2). To es ima e he incidence o pIPD due o indi idual se o ypes, he cases o which no isola e was a ailable we e assumed o ha e he same se o ype dis ibu ion as ha ound among isola es om he same epidemiolog- ical yea and age g oup. Since he molecula se o yping echnique used on cul u e nega i e samples comp ised all he se o ypes included in PCV13, when a se o ype could no be de e mined by his me hod, he case was Epidemiological yea sa Cases/numbe o missing samples, by age g oup 0–11 mon hs 12–23 mon hs 2–4 yea s 5–17 yea s To al 2012–2013 21/2 15/2 30/2 19/3 85/9 2013–2014 33/5 9/1 23/0 19/1 84/7 2014–2015 20/3 22/2 23/2 25/4 90/11 To al 74/10 46/5 76/4 63/8 259/27 Table 1. Cases and missing samples om in asi e pneumococcal disease om pa ien s <18 y s, Po ugal, July 2012–June 2015. aF om week 26 o one yea o week 25 o he ollowing yea . Figu e 1. Incidence o in asi e pneumococcal disease in child en and adolescen s in Po ugal (2012–2013 o 2014–2015). The 95% con idence in e als o he incidence es ima es a e indica ed. www.na u e.com/scien i ic epo s/ 3 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x conside ed caused by an NVT se o ype. Cases whose se o ype emained unde e mined (7F/7A, 25A/38, se o- g oup 6, 33F/33A/37, 29/35B) and non- ypable isola es we e conside ed NVTs. As wi h o e all pIPD incidence, in some age g oups he e we e a ia ions in he incidence o pIPD due o he se o ypes included in each o he conjuga e accine o mula ions, bu hese showed no clea end (Fig.4). An imic obial suscep ibili y. Suscep ibili y o an imic obials was es ed among he 185 a ailable isola es and is summa ized in Figs2 and 3 and Table3. O e all 43 isola es (23.2%) we e nonsuscep ible o penicillin (PNSP) – 34 (18.4%) exp essed low-le el esis - ance and 9 (4.8%) high-le el esis ance. Conside ing he cu en CLSI b eakpoin s o pa en e al penicillin14, 6 isola es (24%) om he CSF would ha e been conside ed esis an and 6 isola es (3.8%) om non-meningi is Figu e 2. Numbe o samples ep esen ing se o ypes p esen in conjuga e accines causing in asi e in ec ions in Po ugal (2012–2013 o 2014–2015). The numbe o samples ep esen ing each se o ype in each o he age g oups conside ed is indica ed. Isola es p esen ing bo h e y h omycin esis ance and penicillin non- suscep ibili y (EPNSP) a e ep esen ed by ed ba s. Penicillin non-suscep ible isola es (PNSP) a e indica ed by o ange ba s. E y h omycin esis an isola es (ERSP) a e indica ed by yellow ba s. Isola es suscep ible o bo h penicillin and e y h omycin a e ep esen ed by g een ba s. Cases whe e no sample was a ailable o whe e pneumococci we e de ec ed exclusi ely by PCR and o which suscep ibili y is unknown, a e indica ed by whi e ba s. The se o ypes included in each o he conjuga e accines a e indica ed by he a ows. NVT – non- accine se o ypes, i.e., se o ypes no included in any o he cu en ly a ailable conjuga e accines (PCV7, PCV10 and PCV13). The alues indica ed below he a ows a e he p opo ion o each g oup in he o e all cases (n = 259). Figu e 3. Numbe o samples ep esen ing se o ypes no p esen in conjuga e accines causing in asi e in ec ions in Po ugal (2012–2013 o 2014–2015). See he legend o Fig.2. NT – Non- ypeable. Some se o ypes could no be unambiguously de e mined and a e indica es as such (see ex ). Among he 12 isola es iden i ied as 15B/C, 8 we e o iginally yped as 15B and 4 isola es we e yped as 15C. www.na u e.com/scien i ic epo s/ 4 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x cases would ha e been conside ed nonsuscep ible, all in e media ely esis an . Resis ance o e y h omycin (ERP) was ound in 42 isola es (22.7%), o which 35 isola es (83.3%) exp essed he cons i u i e MLSB pheno ype and 7 (16.7%) he M pheno ype. All isola es we e suscep ible o le o loxacin, ancomycin and linezolid. The simul ane- ous exp ession o e y h omycin esis ance and penicillin non-suscep ibili y (EPNSP) was ound in 13.5% o he isola es. The e we e no signi ican ends in esis ance wi hin he s udy pe iod. Se o ype Numbe o isola esa 2012–2013 2013–2014 2014–2015 1 12 5 6 3 12 10 10 6B 5 4 3 7Fb536 10 A 352 12B 0 8 2 14 5 6 12 15B/C 7 3 2 19 A 517 24 F 523 Table 2. Se o ypes esponsible o ≥10 IPD cases wi h a known se o ype. aEpidemiological yea s: om week 26 o one yea o week 25 o he ollowing yea . bThe h ee cases in which he de e mined se o ype was 7 F/7A a e no included. Figu e 4. Incidence o in asi e pneumococcal disease in child en and adolescen s in Po ugal (2011–2012 o 2014–2015). NVT – non- accine se o ypes. www.na u e.com/scien i ic epo s/ 5 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x Se o ype 14 con ibu ed g ea ly o e y h omycin esis ance (31.0%) and o penicillin nonsuscep ibili y (46.5%) (Fig.2). Se o ypes included in PCV7 ep esen ed 77.8%, 41.1% and 56.0% o PNSP, ERSP and EPNSP, espec- i ely, while se o ypes included in PCV13 cons i u ed 77.8%, 52.9% and 68.0%, espec i ely. Discussion pIPD incidence a ied only modes ly and wi hou a clea end in 2012–2015, in con as o he signi ican declines in pIPD incidence seen in 2008–2012, pa icula ly among he younge g oups1. As was seen p e iously, PCV7 se o ypes emain impo an causes o pIPD (wi h incidences a ying be ween 0.94–1.12 cases/100,000 in 2012–2015), wi h he slowe up ake and lowe accina ion co e age eached in Po ugal, when compa ed o coun ies whe e PCV7 was in oduced in he NIP, and he high esis ance o isola es exp essing PCV7 se o ypes, pa icula ly o penicillin and he mac olides (77% o all PNSP and 41% o all ERP), possibly being impo an ac o s o hei pe sis ence1. The se o ype dynamics we e di e en in he a ious age g oups, bu he dominan ea u e o he 2012–2015 pe iod was se o ype ins abili y, wi h he incidence o he se o ypes included in he di e en PCVs and NVTs luc ua ing wi hou a de ini e end in he a ious age g oups. This may be due o he use o PCVs ou side o he NIP and he exis ence o a conside able numbe o un accina ed child en ha emain suscep ible o VT disease. The a iable yea ly dissemina ion o PCV se o ypes in he la e g oup could accoun o some o he luc ua ions seen. In neighbo ing Spain, he swi ch in Mad id om adminis e ing PCV13 wi hin he NIP o he p i a e sec o , wi h a concomi an decline in up ake om 95% o 67–73% (simila o he one in Po ugal), led o a s agna ion in he decline o PCV13 pIPD15, sugges ing ha a high accina ion up ake is needed o eap he ull bene i s o PCV use seen elsewhe e16,17. Al hough he numbe o pIPD cases iden i ied solely by molecula me hods was no e y di e en om ha in 2008–20121, he p opo ion was highe in 2012–2015 (n = 41/471 = 8.6% in 2008–2012 e sus n = 47/259 = 18.1% in 2012–2015). In con as o ou las s udy, in his pe iod we se o yped by molecula me hods samples om which no isola e was eco e ed. This esul ed in a signi ican imp o emen in he iden i ica ion o he se o ypes causing complica ed pneumonias, in which pneumococci we e equen ly iden i ied exclusi ely by molecula me hods in pleu al luid, and which cons i u ed 20.2% o all samples wi h se o ype in o ma ion. Se o ype 3, he mos equen ly de ec ed se o ype in 2012–2015 (Fig.2 and Table2) was p edominan ly de ec ed in pleu al luid samples by molecula me hods (n = 21/32, 65.6%) and, as epo ed and discussed elsewhe e13, was equen ly associa ed wi h accine ailu es. The pe sis ence o se o ype 3 despi e accina ion and i s associa ion wi h compli- ca ed pneumonia, oge he wi h he decline o o he accine se o ypes associa ed wi h o he disease p esen a ions and posi i e cul u es, is po en ially con ibu ing o he inc ease in he ela i e impo ance o molecula me hods in iden i ying pIPD cases. Se o ype 3 was no among he mos p e alen se o ypes in p e ious yea s1,2,12, bu his would ha e also been he case in his s udy i molecula me hods had no been used o se o yping di ec ly om cul u e nega i e pa ien samples. A ecen s udy om England and Wales looking a IPD in all age g oups, epo ed ha PCR-con i med cases we e a mino i y o all IPD (<4%) and we e he e o e excluded om he anal- ysis pe o med by he au ho s17. Howe e , in ou con ex molecula me hods did con ibu e o he iden i ica ion An ibio ica Numbe o esis an isola es (%)b 0–11 mon hs (n = 59) 12–23 mon hs (n = 36) 2–4 yea s (n = 49) 5–17 yea s (n = 41) PENc17 (28.8) 12 (33.3) 8 (16.3) 6 (14.6) MIC90 1.5 1.5 1 0.094 MIC50 0.016 0.016 0.016 0.016 CROc4 (6.8) 1 (2.8) 0 (0) 0 (0) MIC90 1 1 0.75 0.064 MIC50 0.02 0.023 0.016 0.016 CTXc2 (3.4) 2 (5.6) 0 (0) 0 (0) MIC90 0.75 0.75 0.5 0.094 MIC50 0.02 0.02 0.02 0.02 ERY 20 (33.9) 13 (36.1) 4 (8.2) 5 (12.2) CLI 16 (27.1) 11 (30.6) 3 (6.1) 5 (12.2) CHL 2 (3.4) 1 (2.8) 2 (4.1) 0 (0) SXT 16 (27.1) 5 (13.9) 8 (16.3) 7 (17.1) TET 12 (20.3) 5 (13.9) 4 (8.2) 3 (7.3) VAN 0 (0) 0 (0) 0 (0) 0 (0) LVX 0 (0) 0 (0) 0 (0) 0 (0) LZD 0 (0) 0 (0) 0 (0) 0 (0) Table 3. An imic obial esis ance o S ep ococcus pneumoniae isola es esponsible o in asi e disease in pa ien s <18 y s, Po ugal, July 2012–June 2015 (n = 185). aCHL: chlo amphenicol; CLI: clindamycin; CRO: ce iaxone; CTX: ce o axime; ERY: e y h omycin; LVX: le o loxacin; LZD: linezolid; PEN: penicillin; SXT: ime hop im-sul ame hoxazole; TET: e acycline; VAN: ancomycin. bUnless o he wise speci ied. cNumbe and pe cen age o non-suscep ible isola es is indica ed. www.na u e.com/scien i ic epo s/ 6 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x o a signi ican ac ion o all pIPD cases in child en and he dominance o pa icula se o ypes in complica ed pneumonia cases can ha e impo an consequences o he o e all se o ype dis ibu ion o IPD in child en, as seen he e. The mos impo an NVTs we e 15B/C, 10A, 12B and 24F. Mos o hese se o ypes (15B/C, 10A and 24F) we e also among he mos p e alen causes o pos -PCV13 IPD in se e al o he coun ies16 and we e associa ed wi h cases o pIPD in Po ugal in 2008–20121. In con as , se o ype 12B was no ound among he p e alen se o ypes elsewhe e and was de ec ed only once in pIPD in Po ugal in 2008–20121, sugges ing a possible ecen eme gence o a pa icula ly i ulen lineage. The PCV7 se o ypes, pa icula ly se o ype 14, emained he mos impo an se o ypes esponsible o an imi- c obial esis ance, sugges ing ha his may be a ac o d i ing hei pe sis ence. Rela i e o he p e ious pe iod1 esis ance dec eased only modes ly, despi e he sha p dec ease o se o ype 19A, a PCV13 se o ype which was associa ed wi h esis ance. Asymp oma ic ca iage wi h PCV13 se o ypes was common in Po ugal be o e he use o PCV13, al hough i was subjec o empo al luc ua ions18. Howe e , he e icacy o accines in e adica ing col- oniza ion, and he e o e in educing exposu e, is known o a y be ween accine se o ypes. Fo ins ance, despi e being a ge ed by all PCVs a ailable o da e, se o ype 19F emained common in nasopha yngeal ca iage in child en in Po ugal in he la e pos -PCV7 pe iod19 and in he pos -PCV13 pe iod20, including among accina ed child en. Such di e ences may mean ha he o e all exposu e o child en o ce ain se o ypes is no signi ican ly changed, po en ially playing an impo an ole in hei pe sis ence as causes o pIPD among bo h accina ed and un accina ed child en. Besides hei con inued ci cula ion in ca iage, ano he impo an ac o o conside is he di e en in asi e disease po en ial o dis inc se o ypes, measu ing hei p opensi y o cause pIPD. In he case o se o ypes 19A and 14, only he la e was shown o ha e an enhanced in asi e disease po en ial when aken as a whole, al hough lineages wi h a iable in asi e disease po en ial we e ound wi hin bo h se o ypes21. Ou s udy has se e al limi a ions. The s udy was no designed o collec in o ma ion impo an o assess he se e i y o he in ec ions caused by he di e en se o ypes (e.g. hospi aliza ion, ICU admission, 30-day mo ali y) no he accina ion s a us o he cases, which would be impo an o iden i y accine ailu es. We belie e ha ew cases o con i med pIPD we e diagnosed ou side o ou ne wo k since he c i e ia o he iden i ica ion o a pIPD case a e he isola ion o pneumococci o he iden i ica ion o pneumococcal DNA in no mally s e ile luids, es s which a e almos exclusi ely pe o med in hospi al labo a o ies. The s abili y o ou su eillance ne wo k, he ac i e na u e o he su eillance and he in ol emen o bo h pedia ic and mic obiology depa men s o many hospi als co e ing he en i e coun y, u he subs an ia es he iden i ica ion o mos cases by ou su eillance. We canno gua an ee ha he se o ype dis ibu ion o he cases whe e isola es we e una ailable ollowed he se o ype dis ibu ion o a ailable isola es. Howe e , he small p opo ion o cases wi h unknown se o ype in o ma ion ensu es ha ou ex apola ion will no g ea ly a ec he esul s. Following he subs an ial declines in PCV13 pIPD seen p e iously1, hese se o ypes ha e no declined u he and emain he mos impo an causes o pIPD (Fig.2). The pe sis ence o hese se o ypes, including he PCV7 se o ypes ha ha e been subjec o accine p essu e o mo e han a decade, sugges s ha hei pe sis ence could be due in pa o he ela i ely mode a e accina ion up ake in Po ugal. Up ake eached 75% a ound 20081 bu declined o 61% in ecen yea s13, a signi ican ly lowe up ake han in coun ies whe e PCVs a e in he NIP and highligh ing he po en ial bene i s o inc easing accina ion up ake. Con inued su eillance will moni o he ex en o VT esilience and will cla i y i he accine ailu es seen wi h se o ype 3 and i s ise o mos impo an pIPD se o ype will be main ained ollowing he in oduc ion o PCV13 in he NIP, when a high accine up ake (≥95%) is expec ed. Ma e ials and Me hods Bac e ial isola es. Since 2007, he Po uguese G oup o he S udy o S ep ococcal In ec ions and he Po uguese S udy G oup o In asi e Pneumococcal Disease o he Pedia ic In ec ious Disease Socie y ha e mon- i o ed pneumococcal in asi e in ec ions in Po ugal. The s udy was app o ed by he Ins i u ional Re iew Boa d o he Cen o Académico de Medicina de Lisboa. These we e conside ed su eillance ac i i ies and we e exemp om in o med consen . All me hods we e pe o med in acco dance wi h he ele an guidelines and egula ions. The da a and isola es we e de-iden i ied so ha hese we e i e ie ably unlinked o an iden i iable pe son. Du ing he su eillance pe iod his in ol ed he mic obiology labo a o ies and pedia ic depa men s o 55 hospi als h oughou Po ugal. All cen e s epo ed du ing he en i e pe iod. A case o IPD was de ined as a pe son om whom an isola e o S. pneumoniae was eco e ed om a no mally s e ile body si e (no including middle ea luid) o om whom pneumococcal DNA was de ec ed in ce eb ospinal luid (CSF) o pleu al luid. Isola es eco e ed up o June 2012 we e p e iously cha ac e ized1,2,12,22. Only isola es eco e ed om pedia ic pa ien s (<18 yea s) and eco e ed be ween July 2012 (week 26) and June 2015 (week 25) we e included in he p esen s udy. Da a on pleu al luid samples was epo ed p e iously13. Epidemiological yea s we e de ined as spanning om week 26 o week 25 o he ollowing yea . Only one isola e om each pa ien in a 90-day in e al was included. All s ains we e iden i ied as S. pneumoniae by colony mo phology and hemolysis on blood aga pla es, op ochin suscep i- bili y and bile solubili y. In he case o body luids whe e he de ec ion o pneumococci was pe o med by molec- ula me hods, wo S. pneumoniae genes (ly A and wzg) we e used o bac e ial iden i ica ion13. Incidences we e calcula ed based on he en i e Po uguese popula ion o he ele an age g oups using da a a ailable om he i s calenda yea o each epidemiological yea om “Ins i u o Nacional de Es a ís ica” (www.ine.p ). This calcula ion assumes ha all pIPD cases a e ea ed wi hin he 55 hospi als in ou ne wo k. Se o yping and an imic obial suscep ibili y es ing. Se o yping was pe o med by he s anda d cap- sula eac ion es using he chessboa d sys em and speci ic se a (S a ens Se um Ins i u , Copenhagen, Denma k). In cases whe e he diagnosis was done by molecula me hods, he se o ypes we e also de e mined by PCR wi h a eac ion a ge ing 21 se o ypes13. Se o ypes we e classi ied in o accine se o ypes (VT), i.e., hose included in www.na u e.com/scien i ic epo s/ 7 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x PCV7 (se o ypes 4, 6B, 9V, 14, 18C, 19F, 23F), he addi ional h ee ound in PCV10 ela i e o PCV7 (addPCV10: 1, 5, 7F), he addi ional h ee ound in PCV13 ela i e o PCV10 (addPCV13: 3, 6A, 19A), and non- accine se o- ypes (NVT). Since he PCR eac ion includes all PCV13 se o ypes, when a se o ype could no be iden i ied his was g ouped in o he NVT g oup. E es s ips (AB Biodisk, Solna, Sweden) we e used o de e mine he minimal inhibi o y concen a ions (MICs) o penicillin, ce o axime, ce iaxone and le o loxacin. In 2008, he Clinical and Labo a o y S anda ds Ins i u e (CLSI) changed he ecommended b eakpoin s o hose cu en ly used o in e p e MIC alues14. Unless o he wise s a ed, we used he CLSI- ecommended b eakpoin s p io o 200823 as epidemiological b eakpoin s, allowing he compa ison wi h p e ious s udies. Isola es we e u he cha ac e ized by de e mining hei suscep ibili y o e y h omycin, clindamycin, anco- mycin, linezolid, e acycline, ime h op im-sul ame hoxazole and chlo amphenicol by he Ki by-Baue disk di usion echnique, acco ding o he CLSI ecommenda ions and in e p e a i e c i e ia14. Mac olide esis ance pheno ypes we e iden i ied using a double disc es wi h e y h omycin and clindamycin. Simul aneous esis ance o e y h omycin and clindamycin de ines he MLSB pheno ype ( esis ance o mac olides, lincosamides and s ep- og amin B) while non-suscep ibili y only o e y h omycin indica es he M pheno ype. S a is ical analysis. Simpson’s index o di e si y (SID) and espec i e 95% con idence in e als (CI95%) was used o measu e se o ype di e si y24. The Coch an-A mi age es was used o ends wi h he alse disco e y a e (FDR) co ec ion o mul iple es ing25. A p < 0.05 was conside ed signi ican o all es s. Da a A ailabili y The da ase s gene a ed du ing he cu en s udy a e a ailable om he co esponding au ho on easonable e- ques . Re e ences 1. Aguia , S. I. e al. Dec easing incidence and changes in se o ype dis ibu ion o in asi e pneumococcal disease in pe sons aged unde 18 yea s since in oduc ion o 10- alen and 13- alen conjuga e accines in Po ugal, July 2008 o June 2012. Eu o Su eill. 19, h ps://doi.o g/10.2807/1560-7917.ES2014.19.12.20750 (2014). 2. Aguia , S. I., Se ano, I., Pin o, F. R., Melo-C is ino, J. & Rami ez, M. Changes in S ep ococcus pneumoniae se o ypes causing in asi e disease wi h non-uni e sal accina ion co e age o he se en- alen conjuga e accine. Clin. Mic obiol. In ec . 14, 835–843 (2008). 3. Ho ácio, A. N. e al. The majo i y o adul pneumococcal in asi e in ec ions in Po ugal a e s ill po en ially accine p e en able in spi e o signi ican declines o se o ypes 1 and 5. PLoS ONE 8, h ps://doi.o g/10.1371/jou nal.pone.0073704 (2013). 4. Esposi o, S. & P incipi, N., ESCMID Vaccine S udy G oup. Di ec and indi ec e ec s o he 13- alen pneumococcal conjuga e accine adminis e ed o in an s and young child en. Fu u e Mic obiol. 10, 1599–1607 (2015). 5. Waigh , P. A. e al. E ec o he 13- alen pneumococcal conjuga e accine on in asi e pneumococcal disease in England and Wales 4 yea s a e i s in oduc ion: an obse a ional coho s udy. Lance In ec . Dis. 15, 535–543 (2015). 6. Balsells, E., Guillo , L., Nai , H. & Kyaw, M. H. Se o ype dis ibu ion o S ep ococcus pneumoniae causing in asi e disease in child en in he pos -PCV e a: A sys ema ic e iew and me a-analysis. PloS One 12, h ps://doi.o g/10.1371/jou nal.pone.0177113 (2017). 7. Ga i ia-Agudelo, C. L., Jo dan-Villegas, A., Ga cia, C. & McC acken, G. H. The e ec o 13- alen pneumococcal conjuga e accine on he se o ype dis ibu ion and an ibio ic esis ance p o iles in child en wi h in asi e pneumococcal disease. J. Pedia . In ec . Dis. Soc. 6, 253–259 (2017). 8. Weinbe ge , D. M., Malley, R. & Lipsi ch, M. Se o ype eplacemen in disease a e pneumococcal accina ion. Lance 378, 1962–1973 (2011). 9. Camilli, R. e al. Impac o pneumococcal conjuga e accine (PCV7 and PCV13) on pneumococcal in asi e diseases in I alian child en and insigh in o e olu ion o pneumococcal popula ion s uc u e. Vaccine 35, 4587–4593 (2017). 10. Tomczyk, S. e al. P e en ion o an ibio ic-nonsuscep ible in asi e pneumococcal disease wi h he 13- alen pneumococcal conjuga e accine. Clin. In ec . Dis. 62, 1119–1125 (2016). 11. Dagan, R. & Klugman, K. P. Impac o conjuga e pneumococcal accines on an ibio ic esis ance. Lance In ec . Dis. 8, 785–795 (2008). 12. Aguia , S. I., B i o, M. J., Gonçalo-Ma ques, J., Melo-C is ino, J. & Rami ez, M. Se o ypes 1, 7F and 19A became he leading causes o pedia ic in asi e pneumococcal in ec ions in Po ugal a e 7 yea s o hep a alen conjuga e accine use. Vaccine 28, 5167–5173 (2010). 13. Sil a-Cos a, C. e al. Pedia ic complica ed pneumonia caused by S ep ococcus pneumoniae se o ype 3 in 13- alen pneumococcal conjuga e accinees, Po ugal, 2010–2015. Eme g. In ec . Dis. 24, 1307–1314 (2018). 14. Clinical and Labo a o y S anda ds Ins i u e. Pe o mance s anda ds o an imic obial suscep ibili y es ing - wen y- i h in o ma ional supplemen . CLSI documen M100–S25 (Clinical and Labo a o y S anda ds Ins i u e, 2015). 15. Picazo, J. e al. E ec o he di e en 13- alen pneumococcal conjuga e accina ion up akes on he in asi e pneumococcal disease in child en: Analysis o a hospi al-based and popula ion-based su eillance s udy in Mad id, Spain, 2007–2015. PloS One 12, h ps:// doi.o g/10.1371/jou nal.pone.0172222 (2017). 16. Cohen, R., Cohen, J. F., Chalumeau, M. & Le y, C. Impac o pneumococcal conjuga e accines o child en in high- and non-high- income coun ies. Expe Re . Vaccines 16, 625–640 (2017). 17. Ladhani, S. N. e al. Rapid inc ease in non- accine se o ypes causing in asi e pneumococcal disease in England and Wales, 2000-17: a p ospec i e na ional obse a ional coho s udy. Lance In ec . Dis. 18, 441–451 (2018). 18. Paulo, A. C. & Sá-Leão, R. Pe iodic cycles o pneumococcal se o ypes ca ied by child en be o e and a e 7- alen pneumococcal conjuga e accine. PloS One 12, h ps://doi.o g/10.1371/jou nal.pone.0176723 (2017). 19. Rod igues, F. e al. Resu gence o se o ype 19F ca iage in p eschool child en in Po ugal in he con ex o con inuing mode a e conjuga e pneumococcal accine up ake. Clin. In ec . Dis. 57, 473–474 (2013). 20. Va len e, C. e al. Impac o he 13- alen pneumococcal conjuga e accine on S ep ococcus pneumoniae mul iple se o ype ca iage. Vaccine 34, 4072–4078 (2016). 21. Sá-Leão, R. e al. Analysis o in asi eness o pneumococcal se o ypes and clones ci cula ing in Po ugal be o e widesp ead use o conjuga e accines e eals he e ogeneous beha io o clones exp essing he same se o ype. J. Clin. Mic obiol. 49, 1369–1375 (2011). 22. Se ano, I., Rami ez, M. & Melo-C is ino, J., Po uguese Su eillance G oup o he S udy o Respi a o y Pa hogens. In asi e S ep ococcus pneumoniae om Po ugal: implica ions o accina ion and an imic obial he apy. Clin. Mic obiol. In ec . 10, 652–656 (2004). 23. Clinical and Labo a o y S anda ds Ins i u e. Pe o mance s anda ds o an imic obial suscep ibili y es ing - se en een h in o ma ional supplemen . CLSI documen M100–S17 (Clinical and Labo a o y S anda ds Ins i u e, 2007). 24. Ca iço, J. A. e al. Illus a ion o a common amewo k o ela ing mul iple yping me hods by applica ion o mac olide- esis an S ep ococcus pyogenes. J. Clin. Mic obiol. 44, 2524–2532 (2006). 25. Benjamini, Y. & Hochbe g, Y. Con olling he alse disco e y a e - a p ac ical and powe ul app oach o mul iple es ing. J R S a Soc Se B S a . Me hodol. 57, 289–300 (1995). www.na u e.com/scien i ic epo s/ 8 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x Acknowledgemen s This wo k was pa ly suppo ed by Fundação pa a a Ciência e a Tecnologia, Po ugal (PTDC/DTP- EPI/1555/2014), LISBOA-01-0145-FEDER-007391, p ojec co unded by FEDER, h ough POR Lisboa 2020 - P og ama Ope acional Regional de Lisboa, PORTUGAL 2020 and Fundação pa a a Ciência e a Tecnologia, and an un es ic ed In es iga o ini ia ed p ojec om P ize . The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Au ho Con ibu ions J.M.C., M.J.B. and M.R. concei ed and designed he expe imen s. P.G.S.S.I. and P.G.I.P.D.P.I.D.S. collec ed da a. C.S.C., S.I.A., J.P.L. pe o med he expe imen s. C.S.C., J.M.C. and M.R. analyzed he da a. C.S.C., J.M.C. and M.R. con ibu ed o he w i ing o he manusc ip and all au ho s e ised and app o ed he e sion o be submi ed. Addi ional In o ma ion Supplemen a y in o ma ion accompanies his pape a h ps://doi.o g/10.1038/s41598-018-36799-x. Compe ing In e es s: J.M.C. has ecei ed esea ch g an s adminis e ed h ough his uni e si y and ecei ed hono a ia o se ing on he speake s bu eaus o P ize and Me ck Sha p and Dohme. M.R. has ecei ed hono a ia o se ing on he speake s bu eau o P ize and o consul ing o GlaxoSmi hKline and Me ck Sha p and Dohme. The o he au ho s decla e no con lic o in e es . No company o inancing body had any in e e ence in he decision o publish. Publishe ’s no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C e- a i e Commons license, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons license and you in ended use is no pe - mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his license, isi h p://c ea i ecommons.o g/licenses/by/4.0/. © The Au ho (s) 2019 www.na u e.com/scien i ic epo s/ 9 SCIENTIFIC RePoR S | (2019) 9:6 | DOI:10.1038/s41598-018-36799-x Conso ia Po uguese G oup o he S udy o S ep ococcal In ec ions Te esa Vaz3, Ma ília Gião3, Rui Fe ei a3, Ana B uschy Fonseca4, Hen ique Oli ei a5, Ana C is ina Sil a6, He mínia Cos a6, Ma ia Fá ima Sil a6, Ma ia Amélia A onso6, Ma ga ida Pin o2, Ode e Chan e2, João Ma ques2, Isabel Pe es2, Isabel Daniel2, Ema Canas2, Te esa Fe ei a2, C is ina Ma celo2, Lu des Mon ei o8, Luís Ma ques Li o8, Filomena Ma ins9, Ma ia Ana Pessanha9, Elsa Gonçal es9, Te esa Mo ais9, Te esa Ma ques9, C is ina Toscano9, Paulo Lopes10, Luísa Felício10, Angelina Lamei ão10, Ana Paula Mo a Viei a11, Ma ga ida Tomaz11, Rosa Ben o12, Ma ia Helena Ramos13, Ana Paula Cas o13, Fe nando Fonseca14, Ana Paula Cas o15, G aça Ribei o16, Rui Tomé Ribei o16, Celes e Pon es16, Luísa Boa en u a16, Ca a ina Cha es16, Te esa Reis16, Nuno Canho o17, Te esa A onso17, Te esa Pina18, Helena Pe es18, Ilse Fon es19, Paulo Ma inho19, Ana Domingos20, Gina Ma ão20, José G ossinho20, Manuela Ribei o21, Helena Gonçal es21, Albe a Faus ino22, Adelaide Al es22, Ma ia Cá men Iglesias22, Ma ia Paula Pinhei o23, R. Semedo23, Ad iana Cou inho24, Luísa Cab al25, Olga Ne o25, Luísa Sancho26, José Diogo27, Ana Rod igues27, Isabel Nascimen o27, Elmano Ramalhei a28, Fe nanda Bessa28, Raquel Diaz28, Isabel Vale29, Ana Ca alho29, José Miguel Ribei o29, Ma ia An ónia Read30, Valquí ia Al es30, Ma ga ida Mon ei o30, Eng ácia Raposo31, Ma ia Lu des Magalhães31, Helena Rochas31, Anabela Sil a31, Ma ga ida Rod igues32, José Mo a F ei as33, Sand a Viei a33, Ma ia Fa ila Meneses34, José Ge mano de Sousa34, Ma iana Be encou Viana35, Isau a Te a35, Vi ó ia Rod igues36, Pa ícia Pe ei a36, Jesuína Dua e37, Paula Pin o38, Ezequiel Mo ei a39, João A aíde Fe ei a40, Adília Vicen e41, Paulo Paixão42 & Na ália No ais43 Po uguese S udy G oup o In asi e Pneumococcal Disease o he Pedia ic In ec ious Disease Socie y Sónia Ai es6, C is ina Fe ei a11, Eu ico Gaspa 15, Manuela Fe ei a1, Fe nanda Pe ei a44, Ma ia José Dinis14, Ál a o Sousa13, Paulo Teixei a39, José Amo im33, Cláudia Mon ei o35, Isabel Ca alho10, So ia A osa30, Ma ga ida Guedes13, Lau a Ma ques13, Ana B aga13, Ma ga ida Ta a es21, Isabel Cunha22, Lu des Vicen e45, Ma ia Manuel Za cos20, Helena Almeida41, Sil ia Almeida28, Fe nanda Rod igues5, C is ina Resende5, Eulália A onso16, Luísa Mendes7, C is ina Fa ia29, Ana Luísa Teixei a46, An ónio Mendes47, Te esa Tomé2, Mónica Rebelo2, Filomena Pe ei a48, Gus a o Rod igues49, Alexand a Cos a10, Ana Teixei a10, So ia Lima36, É ica Laima42, Ma ia Ana S. Nunes50, Filipa P a a8, Ped o Flo es34, Manuela B andão25, João Calado Nunes38, Rosá io Massa51, Flo bela Cunha32, Paula Co eia26, Anabela B i o4, João F anco27, C is ina Didele 52, Es ela Veiga37, Ca la C uz24, G aça Se es12, Céu No ais23, Ma ia João Vi uoso40, Nancy Gue ei o3, Amélia Ca aco17, F ancisco Gomes53, Do a Gomes54 & Isabel Mon ei o55 3Cen o Hospi ala do Ba la en o Alga io, Po imão, Po ugal. 4Hospi al de Cascais, Cascais, Po ugal. 5Cen o Hospi ala de Coimb a, Coimb a, Po ugal. 6Cen o Hospi ala de En e Dou o e Vouga, San a Ma ia da Fei a, Po ugal. 7Hospi al da Figuei a da Foz, Figuei a da Foz, Po ugal. 8Cen o Hospi ala Lisboa No e, Lisboa, Po ugal. 9Cen o Hospi ala Lisboa Ociden al, Lisboa, Po ugal. 10Cen o Hospi ala de Vila No a de Gaia/Espinho, Vila No a de Gaia e Espinho, Po ugal. 11Cen o Hospi ala do Al o A e, Guima ães, Po ugal. 12Cen o Hospi ala do Baixo Alen ejo, Beja, Po ugal. 13Cen o Hospi ala do Po o, Po o, Po ugal. 14Cen o Hospi ala da Pó oa do Va zim/Vila do Conde, Pó oa do Va zim e Vila do Conde, Po ugal. 15Cen o Hospi ala de T ás os Mon es e Al o Dou o, Vila Real e Peso da Régua e Cha es, Cha es, Po ugal. 16Hospi ais da Uni e sidade de Coimb a, Coimb a, Po ugal. 17Hospi al Cen al do Funchal, Funchal, Po ugal. 18Hospi al Cu y Cab al, Lisboa, Po ugal. 19Hospi al de San a Luzia, El as, Po ugal. 20Hospi al de San o And é, Lei ia, Po ugal. 21Hospi al de São João, Po o, Po ugal. 22Hospi al de B aga, B aga, Po ugal. 23Hospi al D . José Ma ia G ande, Po aleg e, Po ugal. 24Hospi al do Espí i o San o, É o a, Po ugal. 25Hospi al dos SAMS, Lisboa, Po ugal. 26Hospi al D . Fe nando da Fonseca, Amado a, Po ugal. 27Hospi al Ga cia de O a, Almada, Po ugal. 28Hospi al In an e D. Ped o, A ei o, Po ugal. 29Hospi al de São Teo ónio, Viseu, Po ugal. 30Hospi al Ped o Hispano, Ma osinhos, Po ugal. 31Ins i u o Nacional de Saúde Rica do Jo ge, Po o, Po ugal. 32Hospi al Reynaldo dos San os, Vila F anca de Xi a, Po ugal. 33Cen o Hospi ala do Al o Minho, Pon e de Lima e Viana do Cas elo, Po ugal. 34Hospi al CUF Descobe as, Lisboa, Po ugal. 35Cen o Hospi ala do Tâmega e Sousa, Ama an e e Guilhu e, Po ugal. 36Hospi al Bea iz Ângelo, Lou es, Po ugal. 37Cen o Hospi ala de Se úbal, Se úbal, Po ugal. 38Hospi al Dis i al de San a ém, San a ém, Po ugal. 39Cen o Hospi ala do Médio A e, San o Ti so e Vila No a de Famalicão, Famalicão, Po ugal. 40Hospi al de Fa o, Fa o, Po ugal. 41Cen o Hospi ala do Oes e No e, Caldas da Rainha, Po ugal. 42Hospi al da Luz, Lisboa, Po ugal. 43Hospi al da Figuei a da Foz, Figuei a da Foz, Po ugal. 44Cen o Hospi ala do No des e, B agança, Macedo de Ca alei os e Mi andela, Po ugal. 45Hospi al Ama o Lusi ano, Cas elo B anco, Po ugal. 46Cen o Hospi ala da Co a da Bei a, Co ilhã, Po ugal. 47Hospi al Sousa Ma ins, Gua da, Po ugal. 48IPO, Lisboa, Po ugal. 49Hospi al Lusíadas, Lisboa, Po ugal. 50Hospi al C uz Ve melha, Lisboa, Po ugal. 51Cen o Hospi ala do Médio Tejo, Ab an es, Po ugal. 52Cen o Hospi ala do Ba ei o Mon ijo, Ba ei o Mon ijo, Po ugal. 53Hospi al de San o Espí i o, Ang a do He oísmo, Po ugal. 54Hospi al da Ho a, Ho a, Po ugal. 55Hospi al do Di ino Espí i o San o, Pon a Delgada, Po ugal.