Serotype and molecular epidemiology of pneumococci before and after pneumococcal vaccination

The pneumococcus is a major human pathogen that causes morbidity and mortality worldwide. Pneumococci are normally carried asymptomatically in the nasopharynx of healthy pre-school children. Pneumococcal infections can range from acute otitis media (AOM) and pneumonia to invasive pneumococcal disease (IPD). Vaccination with conjugate pneumococcal vaccines (PCVs) leads to a reduction of serotypes targeted by the vaccines (vaccine-type, VT) but can also lead to replacement by serotypes not targeted by PCVs (nonvaccine-type, NVT). Iceland introduced the 10-valent PCV (PHiD-CV, GSK) into the national immunization program in 2011. The aim of the study was to assess the impact of PHiD-CV on pneumococci isolated from samples from the nasopharynx of healthy children attending day-care centres (DCCs), middle ear (ME) from children with AOM and lower respiratory tract (LRT) from adults with suspected pneumonia. Serotype distribution, genetic lineages and antimicrobial resistance was analysed by comparing the period before vaccine introduction (PreVac; 2009-2011) to the period post vaccine introduction (PostVac; 2012-2017). Nasopharyngeal swabs were collected annually (March, 2009-2017), from children <7 years old attending 15 DCCs in the greater Reykjavik area. All pneumococci isolated from ME samples from children <7 years old submitted to Landspitali University Hospital from 2009-2017 were included. ME isolates were analysed as the mean number of isolates detected each year (avg/yr) to correct for the difference in the number of years between the two study periods. All pneumococci from LRT samples from adults ≥18 years old were included in the study. The LRT isolates were analysed as the number of detected isolates according to the population size for the referral area. Two PostVac periods were defined for LRT: PostVac-I; 2012-2014 and PostVac-II; 2015-2017. All isolates were serotyped using latex agglutination and/or PCR and/or sequence based serotyping. Antimicrobial susceptibility testing was according to the methods and criteria of EUCAST. Whole genome sequencing (WGS) was done on selected isolates. Multilocus sequence types (STs) were extracted from the WGS data and STs were assigned to clonal complexes (CCs). Phylogenetic trees were constructed and annotated with serotypes and CCs. A two-sided Fisher´s exact test was used to calculate p-values and the level of significance was set ≤0.05. A total of 5,570 pneumococcal isolates were analysed: 3,020 from carriage, 1,330 from ME, 1,047 from LRT and 173 from IPD. Carriage rates remained unchanged between the study periods (67.3% vs. 61.5%; p=0.090). The total number of ME samples and pneumococci isolated from ME and LRT samples decreased PostVac. Overall, VTs decreased PostVac in nasopharyngeal, ME and LRT samples (p<0.001, all sample groups), with a concurrent increase in NVTs in nasopharyngeal and ME samples (p<0.001, both) and LRT samples from adults 18-64 years old (p=0.008). Serotype 23B replaced serotype 23F as the most prevalent serotype in carriage PostVac (from 0.7/1,000 samples PreVac to 49.3 PostVac; p<0.001). Serotype 19F was the most prevalent serotype in both study periods in ME but decreased PostVac: ME from 80.7 isolates avg/yr PreVac to 11.2 PostVac (p<0.001). Serotype 19F was the most prevalent serotype PreVac in LRT samples but decreased from 58.2/100,000 adults PreVac to 10.7 PostVac-II (p<0.001). The majority of serotype 19F isolates were penicillin non-susceptible pneumococci (PNSP) and multidrug resistant (MDR). All PNSP/MDR serotype 19F isolates were members of CC236/271/32019F, variants of the international MDR Taiwan19F-14 lineage. Serotype 23B had a predilection for carriage and serotype 19F for the middle ear. Serotype 6C increased overall between the periods in carriage (from 6.5/1,000 samples to 49.0; p<0.001), ME (from 0.3 isolates avg/yr to 6.8; p<0.001) and LRT samples (from 0.6/100,000 adults to 7.5; p=0.021). A large number of serotype 6C were PNSP and MDR. NVT serotypes that increased significantly PostVac in nasopharyngeal and ME samples from children in different age strata were: 15A, 15B/C, 21, 22F, 23A, 35F and 35B. Non-encapsulated isolates were the most prevalent pneumococci overall in LRT samples PostVac and increased in adults 18-64 years of age (from 4.2/100,000 adults 18-64 years PreVac to 16.2 PostVac-II; p=0.028). The majority were PNSP and MDR. The study revealed that carriage rates were similar in both study periods. The annual number of ME samples and pneumococcal-positive cultures in ME and LRT samples decreased following PHiD-CV implementation. Significant shifts in serotype distribution followed vaccination where VTs decreased and were replaced by NVTs. Herd effect was seen within the older unvaccinated population within four years of vaccine introduction. Isolates of the MDR CC236/271/32019F have nearly been eliminated after vaccine introduction. Whether this lineage will be replaced by other MDR lineages, such as CC3156C, remains to be seen.Pneumókokkar eru mikilvægir sýkingavaldar í mönnum. Heilbrigð börn á leikskólaaldri geta borið pneumókokka í nefkoki án nokkurra einkenna en pneumókokkar geta leitt til miðeyrnasýkingar, lungnabólgu og ífarandi sýkinga. Bólusetning með próteintengdum bóluefnum leiðir til fækkunar stofna af hjúpgerðum sem bóluefnið veitir vörn gegn (bóluefnis hjúpgerð, e. vaccine type: VT) en getur auk þess leitt til fjölgunar stofna af hjúpgerðum sem bóluefnið veitir ekki vörn gegn (ekki bóluefnis hjúpgerð, e. non-vaccine type: NVT). Tíu-gilda próteintengda pneumókokka bóluefnið (PHiD-CV, GSK) var innleitt í ungbarnabólusetningar á Íslandi árið 2011. Markmið rannsóknarinnar var að meta áhrif PHiD-CV á pneumókokka sem ræktaðir voru úr nefkoki heilbrigðra leikskólabarna, miðeyrum (ME) barna með miðeyrnarbólgu og neðri öndunarvegum (LRT) fullorðinna einstaklinga með grun um lungnabólgu. Í rannsókninni var dreifing hjúpgerða, raðgerða (e. sequence types, ST) og sýklalyfjanæmi pneumókokkastofna fundið. Gögn fyrir bólusetningu (2009-2011) voru borin saman við gögn eftir bólusetningu (2012-2017). Nefkoksstrokum var safnað árlega (í mars, 2009-2017) frá börnum <7 ára frá 15 mismunandi leikskólum á stór-höfuðborgarsvæðinu. Allir pneumókokkastofnar sem ræktuðust frá ME barna <7 ára á Landspítala Háskólasjúkrahúsi árin 2009-2017 voru greindir. Til að leiðrétta fyrir mismunandi fjölda ára fyrir og eftir bólusetningu var meðalfjöldi ME stofna á ári skoðaðir á hverju tímabili fyrir sig. Öll sýni frá LRT fullorðinna ≥18 ára voru með í rannsókninni. Pneumókokkastofnum frá LRT eftir bólusetningu var skipt í tvö tímabil: eftir-I (2012-2014) og eftir-II (2015-2017). Allir stofnar voru hjúpgreindir með latex kekkjun og/eða PCR og/eða hjúpgerðin fengin úr raðgreiningargögnum. Sýklalyfjanæmi stofna var mælt og túlkað samkvæmt EUCAST staðli. Annar hver stofn frá árunum 2009-2014 var heilraðgreindur. ST voru fengnar úr raðgreiningargögnunum og þeim raðað í klónal komplexa (CC). Skyldleikatré voru gerð og lituð með tilliti til hjúpgerða og CC. Tveggjaþátta Fisher´s exact próf var notað til að reikna p-gildi og marktækni var miðuð við p ≤0,05. Alls voru 5570 stofnar rannsakaðir yfir allt tímabilið: 3020 frá nefkoki, 1330 frá ME, 1047 frá LRT og 173 úr ífarandi sýkingum. Berahlutfall leikskólabarna var óbreytt milli tímabilanna tveggja (fyrir 67,3% og eftir 61,5%; p=0,090). Sýnum frá ME fækkaði eftir bólusetningu og jafnframt fjöldi pneumókokkaræktanna frá ME og LRT. Pneumókokkastofnum af VT hjúpgerðum fækkaði í öllum sýnaflokkum milli tímabilanna (p<0,001, allir sýnaflokkar) með samhliða fjölgun á pneumókokkastofnum af NVT hjúpgerðum í sýnum frá berum, ME (p<0,001, báðir sýnaflokkar) og LRT frá fullorðnum 18-64 ára (p=0,008). Hjúpgerð 23B tók við af hjúpgerð 23F sem algengasta hjúpgerðin í berasýnum eftir bólusetningu (úr 0,7/1000 sýni fyrir bólusetningu í 49,3 eftir; p<0,001). Stofnar af hjúpgerð 19F voru algengastir bæði tímabilin í ME en fækkaði úr 80,7 á ári fyrir bólusetningu í 11,2 eftir (p<0,001). Stofnar af hjúpgerð 19F voru algengastir fyrir bólusetningu í LRT en fækkaði úr 58,2/100000 fullorðnir fyrir bólusetningu í 10,7 eftir-II (p<0,001). Meirihluti stofna af hjúpgerð 19F var með minnkað næmi fyrir penisillíni (PNSP) og voru fjölónæmir (MDR). Allir PNSP/MDR stofnar af hjúpgerð 19F tilheyrðu CC236/271/32019F, afbrigði af alþjóðlega MDR Taiwan19F -14 klóninum. Hjúpgerð 23B var oftar í nefkoki heilbrigðra leikskólabarna en hjúpgerð 19F sýkti oftar miðeyru barna. Stofnum af hjúpgerð 6C fjölgaði í heildina milli tímabilanna í sýnum frá berum (úr 6,5/1000 sýni fyrir bólusetningu í 49,0 eftir; p<0,001), ME (úr 0,3 á ári fyrir í 6,8 eftir; p