Pneumococcal serotypes and risk factors in adult community-acquired pneumonia 2018-20; a multicentre UK cohort study.

Background: Higher-valency pneumococcal vaccines are anticipated. We aimed to describe serotype distribution and risk factors for vaccine-serotype community-acquired pneumonia (CAP) in the two years pre-SARS-CoV-2 pandemic. Methods: We conducted a prospective cohort study of adults hospitalised with CAP at three UK sites between 2018 and 2020. Pneumococcal serotypes were identified using a 24-valent urinary-antigen assay and blood cultures. Risk factors associated with vaccine-type pneumonia caused by serotypes in the 13-, 15- and 20-valent pneumococcal conjugate vaccines (PCV13, PCV15, PCV20) and 23-valent pneumococcal polysaccharide vaccine (PPV23) were determined from multivariable analysis. Findings: Of 1921 adults hospitalised with CAP, 781 (40.7%, 95% confidence intervals (CI) 38.5-42.9%) had pneumococcal pneumonia. A single PCV13-serotype was detected in 242 (31.0%, 95% CI 27.8-34.3%) pneumococcal CAP patients, mostly serotype 3 (171/242, 70.7%, 95% CI 64.5-76.0%). The additional two PCV15-serotypes were detected in 31 patients (4%, 95% CI 2.8-5.6%), and PCV20-non13-serotypes in 192 (24.6%), with serotype 8 most prevalent (123/192, 64.1%, 95% CI 57.1-70.5%). Compared to PCV13-serotype CAP, people with PCV20-non13 CAP were younger (median age 62 versus 72 years, p < 0.001) and less likely to be male (44% versus 61%, p = 0.01). PPV23-non13-serotypes were found in 252 (32.3%, 95% CI 29.1-35.6%) pneumococcal CAP patients. Interpretation: Despite mature infant pneumococcal programmes, the burden of PCV13-serotype pneumonia remains high in older adults, mainly due to serotype 3. PCV20-non13-serotype pneumonia is more likely in younger people with fewer pneumococcal risk factors. Funding: Unrestricted investigator-initiated research grant from Pfizer, United Kingdom; support from National Institute for Health Research (NIHR) Biomedical Research Centre, Nottingham.

Pneumococcal carriage following PCV13 delivered as one primary and one booster dose (1 + 1) compared to two primary doses and a booster (2 + 1) in UK infants.

In January 2020 the UK changed from a 2 + 1 schedule for 13-valent pneumococcal conjugate vaccine (PCV13) to a 1 + 1 schedule (doses at 3 and 12 months) based on a randomized immunogenicity trial comparing the two schedules. Carriage prevalence measured at the time of booster and 6 months later in 191 of the 213 study infants was 57 % (109/191) and 60 % (114/190) respectively. There were eight episodes of vaccine-type (VT) or vaccine-related 6C carriage in the 2 + 1 and six in the 1 + 1 group; ≥4-fold rises in serotype-specific IgG in 71 children with paired post-booster and follow up blood samples at 21-33 months of age were found in 20 % (7/35) of the 2 + 1 and 15 % (6/41) of the 1 + 1 group. VTs identified in carriage and inferred from serology were similar comprising 3, 19A and 19F. Dropping a priming dose from the 2 + 1 PCV 13 schedule did not increase VT carriage in the study cohort. Ongoing population level carriage studies will be important to confirm this.

Increased Incidence of Invasive Pneumococcal Disease among Children after COVID-19 Pandemic, England.

During July-December 2021, after COVID-19 restrictions were removed in England, invasive pneumococcal disease incidence in children <15 years of age was higher (1.96/100,000 children) than during the same period in 2020 (0.7/100,000 children) and in prepandemic years 2017-2019 (1.43/100,000 children). Childhood vaccine coverage should be maintained to protect the population.

PneumoKITy: A fast, flexible, specific, and sensitive tool for Streptococcus pneumoniae serotype screening and mixed serotype detection from genome sequence data.

Determination of serotypes of Streptococcus pneumoniae is essential for monitoring current vaccine programmes. Since October 2017, pneumococcal serotypes in England have been derived from whole genome sequencing (WGS) data using our bioinformatic tool PneumoCaT. That tool was designed for serotype determination from pure cultures in a reference laboratory. To help determine multiple serotypes in pneumococcal carriage samples, we developed a new software tool named PneumoKITy (Pneumococcal K-mer Integrated Typing) that uses the powerful Mash k-mer screening method for pneumococcal serotyping. Mash k-mer screening is more sequence specific and much faster than the mapping method used in PneumoCaT and can determine 54 (58.1  %) of the 93 serotypes in the SSI Diagnostica phenotypical serotyping scheme to type level with the remainder called to serogroup or subgroup level (e.g., 11A/D). PneumoKITy can be run on both FastQ and assembly input, requiring up to 11× less memory and running up to 29× faster than the current version of PneumoCaT (1.2.1) on FastQ files. PneumoKITy can be used as a rapid, flexible serotype screening method which adds sensitive detection of mixed serotypes, e.g., for nasopharyngeal carriage studies where the presence of multiple serotypes is common. PneumoKITy's ability to function from assembly file, for pure culture serotype detection, increases its speed. This speed potentially enables the software to be run using low infrastructure overhead via web-based platforms. PneumoKITy could be used as a fast initial screening method with other tools used for those serotypes that could not be fully determined to type level if necessary. PneumoKITy was found to be highly accurate and sensitive when run on a panel of FastQ files derived from mixed cultures with all serotypes in 47/51 (92.2  %) of samples being accurately detected. PneumoKITy was also able to accurately estimate the relative abundance of serotypes in the same sample. Estimates being within a mean relative abundance of 1.5 % of the expected abundance in mixtures with known concentrations. PneumoKITy was able to detect minor serotypes with expected abundance of 1 % in the known mixture serotypes. PneumoKITy is a rapid, flexible tool with wide-ranging applications outside of the pure-culture, reference laboratory serotyping remit of PneumoCaT.

Structural, Genetic, and Serological Elucidation of Streptococcus pneumoniae Serogroup 24 Serotypes: Discovery of a New Serotype, 24C, with a Variable Capsule Structure.

Pneumococcal capsules are important in pneumococcal pathogenesis and vaccine development. Although conjugate vaccines have brought about a significant reduction in invasive pneumococcal disease (IPD) caused by vaccine serotypes, the relative serotype prevalence has shifted with the dramatic emergence of serotype 24F in some countries. Here, we describe 14 isolates (13 IPD and 1 non-IPD) expressing a new capsule type, 24C, which resembles 24F but has a novel serological profile. We also describe the antigenic, biochemical, and genetic basis of 24F and 24C and the related serotypes 24A and 24B. Structural studies show that 24B, 24C, and 24F have identical polysaccharide backbones [ß-Ribf-(1→4)-α-Rhap-(1→3)-ß-GlcpNAc-(1→4)-ß-Rhap-(1→4)-ß-Glcp] but with different side chains, as follows: 24F has arabinitol-phosphate and 24B has ribitol-phosphate. 24C has a mixture of 24F and 24B repeating units, with the ratio of ribitol to arabinitol being strain dependent. In contrast, the 24A capsule has a backbone without ß-Ribf but with arabinitol-phosphate and phosphocholine side chains. These structures indicate that factor-sera 24d and 24e recognize arabinitol and ribitol, respectively, which explains the serology of serogroup 24, including those of 24C. The structures can be genetically described by the bispecificity of wcxG, which is capable of transferring arabinitol or ribitol when arabinitol is limiting. Arabinitol is likely not produced in 24B but is produced in reduced amounts in 24C due to various mutations in abpA or abpB genes. Our findings demonstrate how pneumococci modulate their capsule structure and immunologic properties with small genetic changes, thereby evading host immune responses. Our findings also suggest a potential for new capsule types within serogroup 24.

Invasive pneumococcal disease due to 22F and 33F in England: A tail of two serotypes.

BACKGROUND: A 15-valent pneumococcal conjugate vaccine (PCV15) aims to protect against serotype 22F and 33F in addition to the serotypes within the 13-valent PCV (PCV13) which was introduced to the UK childhood immunisation programme in April 2010. Little is known about the specific epidemiology, clinical features or outcomes of invasive pneumococcal disease (IPD) due to these two serotypes. METHODS: Public Health England (PHE) conducts enhanced IPD surveillance in England. Hospital laboratories routinely submit invasive pneumococcal isolates to PHE for serotyping and enhanced clinical information is collected through questionnaires sent to general practitioners. IPD due to serotypes 22F and 33F diagnosed during 2014/15-2018/19 were compared with IPD due to PCV13 serotypes and remaining serotypes. RESULTS: In total, 25,415 isolates (93.4%) were serotyped and questionnaires were completed for 22,097 (86.9%) cases. Serotype 22F was responsible for 1,788 (7.0%) and serotype 33F for 893 (3.5%) cases compared to 19.9% (n = 5,047) for PCV13 and 69.6% (n = 17,687) for the remaining serotypes. IPD incidence increased for both serotypes since 2005/06, especially in older adults, but plateaued after PCV13 introduction. Comorbidity prevalence was 68.7% (n = 1,037) for serotype 22F and 67.2% (n = 505) for serotype 33F, with invasive pneumonia being the most common clinical presentation 1,067/1,482; 72.0%, and 514/755; 68.1%, respectively. There were 3,617 deaths within 30 days of disease onset, including 236 (CFR, 15.4%) among 22F, 128 (CFR, 16.5%) among 33F and 21.3% (925/4,350) among PCV13-type IPD cases. When compared with PCV13-type IPD, serotype 22F (aOR 0.58, 95%CI 0.49-0.68, p < 0.001) and 33F (aOR 0.73, 95%CI 0.59-0.91, p = 0.004) were independently associated with lower odds of death. The major circulating sequence types (STs) in 22F (ST 433, ST698) and 33F (ST717, ST100, ST673) were not associated with an increased risk of death compared to the other STs. CONCLUSIONS: Serotype 22F and 33F-type IPD are associated with a lower risk of death compared to PCV13-type, with those presenting with septicaemia more likely to have a fatal outcome compared to pneumonia. PCV15 has the potential to prevent up to an additional 10% of IPD cases in England.

Impact of the Coronavirus Disease 2019 (COVID-19) Pandemic on Invasive Pneumococcal Disease and Risk of Pneumococcal Coinfection With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): Prospective National Cohort Study, England.

BACKGROUND: Streptococcus pneumoniae coinfection with influenza results in synergistic lethality, but there are limited data on pneumococcal coinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Public Health England conducts invasive pneumococcal disease (IPD) and SARS-CoV-2 surveillance in England. IPD trends during 2000/2001-2019/2020 epidemiological years were analyzed and cases during February-June 2020 linked with laboratory-confirmed SARS-CoV-2 infections. Multivariable logistic regression was used to assess risk factors for death. RESULTS: IPD incidence in 2019/2020 (7.6/100 000; n = 3964) was 30% (IRR, .70; 95% CI, .18-2.67) lower compared with 2018/2019 (10.9/100 000; n = 5666), with large reductions observed across all age groups during March-June 2020. There were 160 886 SARS-CoV-2 and 1137 IPD cases during February-June 2020, including 40 IPD/coronavirus disease 2019 (COVID-19) co-infections (.025% [95% CI, .018-.034] of SARS-CoV-2 infections; 3.5% [2.5-4.8] of IPD cases), 21 with COVID-19 diagnosed 3-27 days after IPD, and 27 who developed COVID-19 ≥28 days after IPD. Case-fatality rates (CFRs) were 62.5 (25/40), 47.6% (10/21), and 33.3% (9/27), respectively (P < .001). In addition to an independent association with increasing age and serotype group, CFR was 7.8-fold (95% CI, 3.8-15.8) higher in those with IPD/COVID-19 coinfection and 3.9-fold (95% CI, 1.4-10.7) higher in patients who developed COVID-19 3-27 days after IPD compared with patients with IPD only. CONCLUSIONS: Large declines in IPD were observed following COVID-19 lockdown. IPD/COVID-19 coinfections were rare but associated with high CFR, mainly in older adults. The rarity, age and serotype distribution of IPD/COVID-19 coinfections do not support wider extension of pneumococcal vaccination.

Re-validation and update of an extended-specificity multiplex assay for detection of Streptococcus pneumoniae capsular serotype/serogroup-specific antigen and cell-wall polysaccharide in urine specimens.

National surveillance of pneumococcal disease at the serotype level is essential to assess the effectiveness of vaccination programmes. We previously developed a highly sensitive extended-specificity multiplex immunoassay for detection of Streptococcus pneumoniae serotype-specific antigen in urine in the absence of isolates. The assay uses human mAbs that detect the 24 pneumococcal serotype/groups targeted by the pneumococcal conjugate vaccines (PCVs) and pneumococcal polysaccharide vaccine (PPV-23) plus some cross-reactive types and the pneumococcal cell-wall polysaccharide. However, the previous assay had some limitations, namely the reduced specificity of the serotype 7F, 20 and 22F assays, for which non-specific binding in urine samples was observed. Here we report on the further development and re-validation of a new version of the assay (version 2.1), which offers improved sensitivity towards serotypes 7F, 18C and 19F and increased specificity for serotypes 7F, 20 and 22F by replacement of some of the antibody clones with new clones. Using a panel of urine specimens from patients diagnosed with community-acquired pneumonia or pneumococcal disease, the overall clinical sensitivity of this version of the assay based on isolation of S. pneumoniae from a normally sterile site is 94.3 % and the clinical specificity is 93.6 %, in comparison with clinical sensitivity and specificity values of 96.2 % and 89.9 % in the previous assay.

Evolution of Streptococcus pneumoniae Serotype 3 in England and Wales: A Major Vaccine Evader.

Despite its inclusion in pneumococcal conjugate vaccine 13 (PCV13), Streptococcus pneumoniae serotype 3 remains a major cause of invasive pneumococcal disease in England and Wales. Previous studies have indicated that there are distinct lineages within serotype 3 clonal complex 180 and the clade distributions have shifted in recent years with the emergence of clade II. We undertook whole genome sequencing and genomic analysis of 616 serotype 3 isolates from England and Wales between 2003 and 2018, including invasive and carriage isolates. Our investigations showed that clade II has expanded since 2014 and now represents 50% of serotype 3 invasive pneumococcal disease (IPD) isolates in England and Wales. Genomic analysis of antibiotic resistance and protein antigen genes showed that distinct profiles are present within the clades which could account for the recent emergence of this clade. This investigation highlights the importance and utility of routine whole genome sequencing and its ability to identify new and emerging variation at the single nucleotide level which informs surveillance and will impact future vaccine development.

The Genomics of Streptococcus Pneumoniae Carriage Isolates from UK Children and Their Household Contacts, Pre-PCV7 to Post-PCV13.

We used whole genome sequencing (WGS) analysis to investigate the population structure of 877 Streptococcus pneumoniae isolates from five carriage studies from 2002 (N = 346), 2010 (N = 127), 2013 (N = 153), 2016 (N = 187) and 2018 (N = 64) in UK households which covers the period pre-PCV7 to post-PCV13 implementation. The genomic lineages seen in the population were determined using multi-locus sequence typing (MLST) and PopPUNK (Population Partitioning Using Nucleotide K-mers) which was used for local and global comparisons. A Roary core genome alignment of all the carriage genomes was used to investigate phylogenetic relationships between the lineages. The results showed an influx of previously undetected sequence types after vaccination associated with non-vaccine serotypes. A small number of lineages persisted throughout, associated with both non-vaccine and vaccine types (such as ST199), or that could be an example of serotype switching from vaccine to non-vaccine types (ST177). Serotype 3 persisted throughout the study years, represented by ST180 and Global Pneumococcal Sequencing Cluster (GPSC) 12; the local PopPUNK analysis and core genome maximum likelihood phylogeny separated them into two clades, one of which is only seen in later study years. The genomic data showed that serotype replacement in the carriage studies was mostly due to a change in genotype as well as serotype, but that some important genetic lineages, previously associated with vaccine types, persisted.

Effect of Pneumococcal Conjugate Vaccines on Pneumococcal Meningitis, England and Wales, July 1, 2000-June 30, 2016.

We describe the effects of the 7-valent (PCV7) and 13-valent (PCV13) pneumococcal conjugate vaccines on pneumococcal meningitis in England and Wales during July 1, 2000-June 30, 2016. Overall, 84,473 laboratory-confirmed invasive pneumococcal disease cases, including 4,160 (4.9%) cases with meningitis, occurred. PCV7 implementation in 2006 did not lower overall pneumococcal meningitis incidence because of replacement with non-PCV7-type meningitis incidence. Replacement with PCV13 in 2010, however, led to a 48% reduction in pneumococcal meningitis incidence by 2015-16. The overall case-fatality rate was 17.5%: 10.7% among patients <5 years of age, 17.3% among patients 5-64 years of age, and 31.9% among patients >65 years of age. Serotype 8 was associated with increased odds of death (adjusted odds ratio 2.9, 95% CI 1.8-4.7). In England and Wales, an effect on pneumococcal meningitis was observed only after PCV13 implementation. Further studies are needed to assess pneumococcal meningitis caused by the replacing serotypes.

Invasive Pneumococcal Disease in UK Children <1 Year of Age in the Post-13-Valent Pneumococcal Conjugate Vaccine Era: What Are the Risks Now?

BACKGROUND: Invasive pneumococcal disease (IPD) has declined significantly since the introduction of pneumococcal conjugate vaccines (PCVs). It is not known whether certain infant populations remain at higher risk of IPD in countries with established 13-valent PCV (PCV13) programs. We aimed to describe the epidemiology, clinical characteristics, serotype distribution, and outcomes of IPD in infants, and to estimate the relative risk of PCV13-type, non-PCV13-type, and overall IPD in premature infants compared to term infants during a 4-year period after the PCV13 program was established. METHODS: This was a prospective, enhanced national surveillance of laboratory-confirmed IPD in England in infants aged <1 year diagnosed during 2013-2016. RESULTS: There were 517 cases of IPD (incidence: 19/100000 infants). Incidence was significantly higher in premature infants compared with those born at term (49/100000 vs 17/100000; incidence rate ratio [IRR], 2.87; P < .001), with infants born before 28 weeks' gestation having the highest incidence (150/100000; IRR, 8.8; P < .001). Of the 454 IPD cases with serotyped isolates, most were caused by non-PCV13 serotypes (369 cases, 71.4%), with 85 cases (16.4%) due to PCV13 serotypes. There were 31 deaths (case fatality rate [CFR], 6.2% [95% confidence interval, 4.3%-8.6%]). Premature infants did not have a higher CFR than term infants (P = .62). CONCLUSIONS: IPD incidence in infants remains lower than rates reported prior to PCV7 introduction in England. The risk of IPD remains significantly higher in premature infants compared to infants born at term, for both PCV13 and non-PCV13 serotypes. Any changes to the infant PCV13 immunization schedule may disproportionally affect premature infants.

Pneumococcal carriage in children and their household contacts six years after introduction of the 13-valent pneumococcal conjugate vaccine in England.

BACKGROUND: In April 2010, 13-valent pneumococcal conjugate vaccine (PCV13) replaced PCV7 in the infant immunisation schedule in England and Wales. Despite limited serotype replacement in invasive pneumococcal disease (IPD) during the first four post-PCV13 years, non-vaccine type (NVT) IPD increased substantially in 2014/15. We undertook a carriage study in 2015/16 to help understand the reasons for this increase. METHODS AND FINDINGS: Families with a child aged <5 years attending a participating general practice in Gloucestershire or Hertfordshire were invited to provide nasopharyngeal swabs from all consenting members. Swabs from 650 individuals (293 under five, 73 five to twenty and 284 >twenty years) were cultured and serotyped for Streptococcus pneumoniae. Results were compared with those from three previous household studies conducted in the same populations between 2001 to 2013, and with the serotypes causing IPD to estimate case-carrier ratios (CCRs). Overall carriage prevalence did not differ between the four carriage studies with reductions in vaccine-type carriage offset by increases in NVT carriage. While no individual NVT serotype showed an increase in CCR from 2012/13, the composition of the serotypes comprising the NVT group differed such that the overall CCR of the NVT group had significantly increased since 2012/13. Carriage of two PCV13 serotypes, 3 and 19A, was found in 2015/16 (3/650 = 0.5% and 2/650 = 0.3% respectively) with no overall reduction in carriage prevalence of PCV13-7 serotypes since 2012/13, though 6C prevalence, a vaccine-related serotype, had reduced from 1.8% in 2012/13 to 2/648 (0.3%) in 2015/16, p = 0.013. CONCLUSIONS: There was continuing evolution in carried NVTs six years after PCV13 introduction which, in addition to being vaccine-driven, could also reflect natural secular changes in certain NVTs. This poses challenges in predicting future trends in IPD. Elimination of carriage and disease due to serotypes 3 and 19A may not be achieved by PCV13.

Discovery and description of a new serogroup 7 Streptococcus pneumoniae serotype, 7D, and structural analysis of 7C and 7D.

Streptococcus pneumoniae is characterised into 92 serotypes based on antigenic reactions of commercial rabbit sera to the capsular polysaccharides. During development of a bioinformatic serotyping tool (PneumoCaT), an isolate exhibited a novel codon at residue 385 of the glycosyltransferase gene wcwK encoding a distinct amino acid, which differentiates genogroup 7. Investigation by repeat serotyping and Quellung reaction revealed a novel pattern of factor sera with the isolate reacting very strongly with 7f, but also with 7e factor sera. The structure of the capsular polysaccharide was determined by NMR spectroscopy to be an approximately 5:1 combination of the structures of 7C and 7B, respectively, and the structure of 7C was also elucidated. All data from whole genome sequencing, NMR spectroscopy, production of antisera and serotyping of the novel 7 strain shows that it is a new serotype, which will be named in the Danish nomenclature as 7D.

Rapid increase in non-vaccine serotypes causing invasive pneumococcal disease in England and Wales, 2000-17: a prospective national observational cohort study.

BACKGROUND: Pneumococcal conjugate vaccines (PCVs) have substantially reduced the incidence of invasive pneumococcal disease caused by vaccine serotypes; however, replacement disease with non-PCV serotypes remains a concern. We describe the population effect of the seven-valent and 13-valent PCVs (PCV7 and PCV13) on invasive pneumococcal disease in England and Wales. METHODS: Using national invasive pneumococcal disease surveillance data for 2016/17, we compared incidence rate ratios (IRRs) against pre-PCV13 (2008/09-2009/10) and pre-PCV7 (2000/01-2005/06) baselines. We also estimated the number of invasive pneumococcal disease cases prevented since the introduction of PCVs. FINDINGS: In 2016/17, overall invasive pneumococcal disease incidence (9·87 cases per 100 000; 5450 cases) across all age groups was 37% lower (IRR 0·63, 95% CI 0·60-0·65) than pre-PCV7 incidence (14·79 per 100 000; 8167 cases) and 7% lower (0·93; 0·89-0·97) than pre-PCV13 incidence (10·13 per 100 000; 5595 cases). By 2016/17, PCV7-type invasive pneumococcal disease incidence across all age groups had decreased by 97% (0·24 per 100 000; 0·03, 0·02-0·04) compared with the pre-PCV7 period, whereas additional PCV13-type invasive pneumococcal disease decreased by 64% (1·66 per 100 000; 0·36, 0·32-0·40) since the introduction of PCV13. Invasive pneumococcal disease incidence due to non-PCV13 serotypes doubled (7·97 per 100 000; 1·97, 1·86-2·09) since the introduction of PCV7, and accelerated since 2013/14-especially serotypes 8, 12F, and 9N, which were responsible for more than 40% of invasive pneumococcal disease cases by 2016/17. Invasive pneumococcal disease incidence in children younger than 5 years remained stable since 2013/14, with nearly all replacement disease occurring in adults. We estimated 38 366 invasive pneumococcal disease cases were prevented in the 11 years since the introduction of PCV7. INTERPRETATION: Both PCV7 and PCV13 have had a major effect in reducing the burden of invasive pneumococcal disease in England and Wales; however, rapid increases in some non-PCV13 serotypes are compromising the benefits of the programme. FUNDING: Public Health England.

Development of an Extended-Specificity Multiplex Immunoassay for Detection of Streptococcus pneumoniae Serotype-Specific Antigen in Urine by Use of Human Monoclonal Antibodies.

Current pneumococcal vaccines cover the 10 to 23 most common serotypes of the 92 presently described. However, with the increased usage of pneumococcal-serotype-based vaccines, the risk of serotype replacement and an increase in disease caused by nonvaccine serotypes remains. Serotype surveillance of pneumococcal infections relies heavily on culture techniques, which are known to be insensitive, particularly in cases of noninvasive disease. Pneumococcal-serotype-specific urine assays offer an alternative method of serotyping for both invasive and noninvasive disease. However, the assays described previously cover mainly conjugate vaccine serotypes, give little information about circulating nonvaccine serotypes, and are currently available only in one or two specialist laboratories. Our laboratory has developed a Luminex-based extended-range antigen capture assay to detect pneumococcal-serotype-specific antigens in urine samples. The assay targets 24 distinct serotypes/serogroups plus the cell wall polysaccharide (CWP) and some cross-reactive serotypes. We report that the assay is capable of detecting all the targeted serotypes and the CWP at 0.1 ng/ml, while some serotypes are detected at concentrations as low as 0.3 pg/ml. The analytical serotype specificity was determined to be 98.4% using a panel of polysaccharide-negative urine specimens spiked with nonpneumococcal bacterial antigens. We also report clinical sensitivities of 96.2% and specificities of 89.9% established using a panel of urine specimens from patients diagnosed with community-acquired pneumonia or pneumococcal disease. This assay can be extended for testing other clinical samples and has the potential to greatly improve serotype-specific surveillance in the many cases of pneumococcal disease in which a culture is never obtained.

External Quality Assurance for Laboratory Identification and Capsular Typing of Streptococcus pneumoniae.

An external quality assessment (EQA) scheme for pneumococcal serotype identification has been performed over a period of 11 years, by a network of European pneumococcal reference laboratories. We report the results from the EQA, and present an assessment of the acceptability and utility of the EQA scheme. Reports from 22 EQA panels distributed in 2005-2016 were analysed. Each EQA panel consisted of seven isolates. A questionnaire including seven questions related to the acceptability and utility of the EQA scheme was distributed to all participating laboratories. Altogether, 154 pneumococcal isolates were tested. Of the 92 serologically distinct serotypes currently defined, 49 serotypes were included in the rounds. Discrepant results were observed in eight EQA rounds, involving 11 isolates (7.1%, 95% CI: 4% to 12%). All participating laboratories reported that the EQA scheme was useful for quality assurance purposes. Our results show that comparable serotyping data can be obtained in different laboratories. The EQA participation helps to keep the typing procedures at a high standard and provides data for accreditation purposes. The EQA is helpful when new technologies are introduced, and reveal limitations of both genotypic and phenotypic methods. Continuation of the presented EQA scheme is planned.

Pneumococcal 23B Molecular Subtype Identified Using Whole Genome Sequencing.

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae and the target of all currently licensed pneumococcal vaccines. At present, there are 92 serologically distinct pneumococcal serotypes. Structural and antigenic variation of capsular types is the result of genetic variation within the capsular polysaccharide synthesis (CPS) locus; however, genetic variation may not always result in phenotypic differences which produce novel serotypes. With the introduction of high throughput whole genome sequencing, discovery of novel genotypic variants is not unexpected and this study describes a novel variant of the serotype 23B CPS operon. This novel variant was characterized as a novel genotypic subtype (23B1) with ∼70% homology to the published 23B CPS sequence. High sequence variability was determined in eight cps genes involved in sugar biosynthesis. However, there was no distinction between the classic 23B serotype and 23B1 serologically or in terms of polysaccharide structure. Phylogenetic and eBURST analysis revealed a distinct lineage for 23B1 with multiple clones (UK, Thailand, and USA) that arose at different points during pneumococcal evolution. Analysis of the UK S. pneumoniae isolates (n = 121) revealed an upsurge of 23B1 ST2372 in 2011, after which this previously unseen ST increased to reach 50% proportion of the 23B sequenced isolates from 2013 and remained prevalent within our sequenced isolates from later years. Therefore, although the 23B1 variant appears to have no phenotypic impact and cannot be considered as novel serotype, it appears to have led to a genetic restructuring of the UK serotype 23B population.

Clinical streptococcal isolates, distinct from Streptococcus pneumoniae, but containing the ß-glucosyltransferase tts gene and expressing serotype 37 capsular polysaccharide.

The major virulence factor of the pneumococcus, and target for conjugate vaccines, is the polysaccharide capsule, which is usually encoded by the highly variable cps locus. Serotype 37 is an unusual pneumococcal type in which the single ß-glucosyltransferase gene responsible for serotype capsule production (tts) is located outside of the capsular operon region. Using a previously described automated whole genome sequence (WGS)-based serotyping bioinformatics tool, PneumoCaT, we identified and investigated seven clinical isolates (three from blood cultures) of non-pneumococcal streptococci containing a highly homologous tts and included them in a study panel of 20 isolates which included a 11 further clinical isolates of S. pneumoniae serotype 37, a reference strain of serotype 37 and the S. pseudopneumoniae type strain BAA 960T. The seven non-pneumococcal isolates generated novel alleles at all pneumococcal MLST loci and gave low percentage similarity (<45%) to S. pneumoniae or S. pseudopneumoniae species by comparison of short sequence patterns in genomic data (k-mer analysis). The S. pseudopneumoniae BAA-960T isolate generated two novel alleles in the MLST and gave a high similarity (>99%) to the reference sequence for BAA-960T. Twelve isolates gave high similarity (>77%) to the Streptococcus pneumoniae 5652-06 serotype 19A reference genome sequence and had previously reported MLST alleles. Each of the seven clinical non-pneumococcal strains and all of the 12 S. pneumoniae possessed a ß-glycosyltransferase gene (tts) with >95% similarity to the pneumococcal tts reference DNA sequence with 20-22 non-synonymous SNPs. All but two strains in which the tts gene was detected gave positive reactions for serotype 37 in slide agglutination tests with serotype 37 typing sera. Phylogenetic analysis using both SNP and MLST data showed distinct clades corresponding to strains identified as pneumococcus or non-pneumococcus by kmer WGS analysis. Extended k-mer database analysis and ribosomal MLST placed the non-pneumococcal isolates within the S. mitis group. Biochemical and bile solubility assays showed differences between the unusual isolates and S. pneumoniae. All isolates had detectable pneumolysin (ply) genes, but only those that identified as pneumococcus contained the genes for autolysin (lytA) or the ABC transporter lipoprotein A (piaA) with >80% coverage and >95% similarity. Here we report the existence of a novel group of strains distinct from S. pneumoniae, but which can express a pneumococcal serotype 37 capsular polysaccharide which can be associated with clinical disease.