The changing epidemiology of diphtheria in the United Kingdom, 2009 to 2017.

BackgroundDiphtheria is a potentially fatal disease caused by toxigenic strains of Corynebacterium diphtheriae, C. ulcerans or C. pseudotuberculosis.AimOur objective was to review the epidemiology of diphtheria in the United Kingdom (UK) and the impact of recent changes in public health management and surveillance.MethodsPutative human toxigenic diphtheria isolates in the UK are sent for species confirmation and toxigenicity testing to the National Reference Laboratory. Clinical, epidemiological and microbiological information for toxigenic cases between 2009 and 2017 are described in this population-based prospective surveillance study.ResultsThere were 33 toxigenic cases of diphtheria aged 4 to 82 years. Causative species were C. diphtheriae (n = 18) and C. ulcerans (n = 15). Most C. diphtheriae cases were cutaneous (14/18) while more than half of C. ulcerans cases had respiratory presentations (8/15). Two thirds (23/33) of cases were inadequately immunised. Two cases with C. ulcerans infections died, both inadequately immunised. The major risk factor for C. diphtheriae aquisition was travel to an endemic area and for C. ulcerans, contact with a companion animal. Most confirmed C. diphtheriae or C. ulcerans isolates (441/507; 87%) submitted for toxigenicity testing were non-toxigenic, however, toxin positivity rates were higher (15/23) for C. ulcerans than C. diphtheriae (18/469). Ten non-toxigenic toxin gene-bearing (NTTB) C. diphtheriae were also detected.ConclusionDiphtheria is a rare disease in the UK. In the last decade, milder cutaneous C. diphtheriae cases have become more frequent. Incomplete vaccination status was strongly associated with the risk of hospitalisation and death.

Neonatal invasive Haemophilus influenzae disease in England and Wales: epidemiology, clinical characteristics, and outcome.

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) frequently causes noninvasive upper respiratory tract infections in children but can cause invasive disease, mainly in older adults. An increased burden of invasive NTHi disease in the perinatal period has been reported by a number of studies. Here we describe the epidemiology, clinical characteristics, and outcome of neonatal invasive H. influenzae disease in England and Wales over a 5-year period. METHODS: Public Health England conducts enhanced national surveillance of invasive H. influenzae disease in England and Wales. Detailed clinical information was obtained for all laboratory-confirmed cases in infants aged ≤31 days during 2009-2013. RESULTS: Overall, 118 live-born neonates had laboratory-confirmed invasive H. influenzae disease: 115 (97%) were NTHi, 2 were serotype f, and 1 was serotype b. NTHi was isolated within 48 hours of birth (early-onset) in 110 of 115 (96%) cases, and 70 of 110 (64%) presented with septicemia. Only 17 mothers (15%) had suspected bacterial infection requiring antibiotics during labor. Few (8/110 [7%]) neonates had comorbidities. The incidence of early-onset NTHi increased exponentially with prematurity, from 0.9 per 100 000 (95% confidence interval [CI], .6-1.4) in term neonates to 342 per 100 000 (95% CI, 233.9-482.7) in neonates born at <28 weeks' gestation (incidence rate ratio, 365 [95% CI, 205-659]; P < .001). Case fatality for early-onset NTHi was 19% (21/110); each additional gestational week reduced the odds of dying by 21% (odds ratio, 0.79 [95% CI, .69-.90]; P < .01). A quarter of neonates who survived experienced long-term complications. CONCLUSIONS: Early-onset neonatal NTHi disease is strongly associated with premature birth and causes significant morbidity and mortality.

Invasive pneumococcal disease 3 years after introduction of a reduced 1 + 1 infant 13-valent pneumococcal conjugate vaccine immunisation schedule in England: a prospective national observational surveillance study.

BACKGROUND: The UK transition from a 2 + 1 to a 1 + 1 infant immunisation schedule with the 13-valent pneumococcal conjugate vaccine (PCV13) on Jan 1, 2020, coincided with the start of the COVID-19 pandemic. We describe the epidemiology of invasive pneumococcal disease (IPD) in England over 6 financial years (April 1 to March 31) between 2017-18 and 2022-23. METHODS: We used prospective national surveillance data, including serotyping and whole-genome sequencing of invasive isolates, to analyse IPD trends in England by age and financial year. We compared breakthrough infections and vaccine failure rates in 2022-23 among children eligible for the 1 + 1 schedule with rates in cohorts of children eligible for the 2 + 1 schedule between 2017-18 and 2019-20. We assessed genomic changes over time by comparing Global Pneumococcal Sequencing Clusters and multilocus sequence types among PCV13 serotypes causing IPD. FINDINGS: There were 4598 laboratory-confirmed IPD cases in 2022-23, 3025 in 2021-22, 1240 in 2020-21, and 5316 in 2019-20. IPD incidence in 2022-23 was 14% lower than in 2019-20 (incidence rate ratio [IRR] 0·86, 95% CI 0·81-0·91; p<0·001). IPD incidence in 2022-23 compared with 2019-20 was 34% higher in children (aged <15 years) (378 cases vs 292 cases; IRR 1·34, 95% CI 1·08-1·68; p=0·009) and 17% lower in adults (aged 15 years and older; 4220 vs 5024; 0·83, 0·78-0·88; p<0·001). The proportion of PCV13-type IPD increased from 19·4% (95% CI 18·2-20·4; 957 of 4947) in 2019-20 to 29·7% (28·3-31·0; 1283 of 4326) in 2022-23, mainly due to serotype 3, but also serotypes 19F, 19A, and 4, alongside a decrease in non-PCV13 serotypes 8, 12F, and 9N. The increase in IPD incidence due to serotypes 3, 19A, and 19F was driven by clonal expansion of previously circulating strains, whereas serotype 4 expansion was driven by newer strains (ie, sequence types 801 and 15603). Breakthrough infections and vaccine failure rates were similar in children eligible for the 1 + 1 (1·08 per 100 000 person-years) and 2 + 1 (0·76 per 100 000 person-years; IRR 1·42, 95% CI 0·78-2·49; p=0·20) PCV13 schedules. INTERPRETATION: Overall, IPD incidence in England was lower in 2022-23, 2 years after removal of pandemic restrictions, than in 2019-20. Breakthrough and vaccine failure rates were not significantly different between children who received the 1 + 1 compared with the 2 + 1 PCV13 immunisation schedule. The post-pandemic increase in childhood IPD incidence and especially PCV13-type IPD will require close monitoring. FUNDING: None.

Trends in invasive Haemophilus influenzae serotype a disease in England from 2008-09 to 2021-22: a prospective national surveillance study.

BACKGROUND: Invasive Haemophilus influenzae serotype a (Hia) disease is rare, with most cases reported among Indigenous populations in North America. In England, national surveillance was enhanced following an increase in laboratory-confirmed invasive Hia disease since the 2016-17 epidemiological year. This study aimed to describe the epidemiological trends, clinical characteristics of cases, and assess potential genomic drivers. METHODS: Hospital laboratories in England routinely submit invasive H influenzae isolates to the UK Health Security Agency for confirmation and serotyping. In this prospective national surveillance study we contacted the general practitioners and clinicians of all patients with laboratory-confirmed invasive Hia from the 2008-09 to the 2021-22 epidemiological year to complete a clinical questionnaire on demographics, underlying conditions, clinical presentation, complications, outcomes, and travel history of the patient. All Hia invasive isolates from residents in England were included in the study; non-invasive isolates were excluded. Multilocus sequence typing (MLST), whole genome single-nucleotide polymorphism, and k-mer-based analysis of bacterial isolates were performed following Illumina whole-genome sequencing (WGS). Outcomes included epidemiological trends, clinical characteristics of confirmed Hia cases, and genomic analyses. FINDINGS: From the 2008-09 to the 2021-22 epidemiological years, there were 52 cases of invasive infection with H influenzae serotype a in England (25 [48%] in female patients and 27 [52%] in male patients). There were zero to two annual Hia cases (accounting for <0·5% of serotyped H influenzae isolates) until 2015-16, after which cases increased across England to 19 cases in 2021-22 (incidence 0·03 cases per 100 000), when Hia accounted for 19 (4%) of 484 serotyped H influenzae isolates, 19 (19%) of 100 capsulated cases, and 37% (19 of 52) of all H influenzae cases between 2008-09 and 2021-22. Most of the recent increase in cases occurred among individuals aged 65 years and older (17 [33%] of 52), who typically presented with bacteraemic pneumonia (13 [76%] of 17), and infants younger than 1 year, who had the highest incidence and were more likely to present with meningitis (five [50%] of ten). Overall case fatality rate was 7·7% (95% CI 2·1-19·7; four of 52 patients). WGS found that closely related MLST sequence types ST1511 (20 [39%] of 51), ST23 (13 [25%] of 51), and ST56 (seven [14%] of 51) accounted for most cases, with no evidence of serotype b strains switching capsule to Hia. Duplication of the capsule operon, associated with more severe disease, was present in 32 (80%) of 40 of these sequence types. Analysis of the core and accessory genome content grouped most isolates into a single strain. INTERPRETATION: The persistent increase in invasive Hia cases across England and across all age groups suggests widespread transmission, consistent with reports from other European countries, and will require close monitoring. FUNDING: UK Health Security Agency.

Household transmission of non-toxigenic diphtheria toxin gene-bearing Corynebacterium diphtheriae following a cluster of cutaneous cases in a specialist outpatient setting.

Introduction. Combination of PCR and Elek testing to identify toxigenic corynebacteria has revealed organisms described as non-toxigenic toxin-gene bearing (NTTB) Corynebacterium diphtheriae or C. ulcerans (i.e. PCR tox positive; Elek negative). These organisms carry part or all of tox, but are unable to express diphtheria toxin (DT) and present a challenge to clinical and public health case management.Gap analysis/Hypothesis. There are few data on the theoretical risk of NTTB reversion to toxigenicity. This unique cluster and subsequent epidemiologically linked isolates allowed the opportunity to determine any change in DT expression status.Aim. To characterize a cluster of infections due to NTTB in a skin clinic and subsequent cases in two household contacts.Methodology. Epidemiological and microbiological investigations were carried out according to existing national guidance at the time. Susceptibility testing used gradient strips. The tox operon analysis and multi-locus sequence typing (MLST) was derived from whole-genome sequencing. Alignment of the tox operon and phylogenetic analyses were performed using clustalW, mega, the public core-genome MLST (cgMLST) scheme and an in-house bioinformatic single nucleotide polymorphism (SNP) typing pipeline.Results. Isolates of NTTB C. diphtheriae were recovered from four cases (cases 1 to 4) with epidermolysis bullosa attending the clinic. Two further isolates were subsequently recovered from case 4, >18 months later, and from two household contacts (cases 5 and 6) after a further 18 months and 3.5 years, respectively. All eight strains were NTTB C. diphtheriae biovar mitis, belonged to the same sequence type (ST-336) with the same deletion in tox. Phylogenetic analysis showed relatively high diversity between the eight strains with 7-199 SNP and 3-109 cgMLST loci differences between them. The number of SNPs between the three isolates from case 4 and two household contacts (cases 5 and 6) was 44-70 with 28-38 cgMLST loci differences.Conclusions. We report a cluster of NTTB C. diphtheriae cases in a skin clinic and evidence of onward household transmission. We conclude the deletion in the tox was responsible for the non-expression of DT. There was no evidence of reversion to DT expression over the 6.5 year period studied. These data informed revision to guidance in the management of NTTB cases and their contacts in the UK.

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.

Invasive Haemophilus influenzae serotype e and f disease, England and Wales.

Haemophilus influenzae infection causes serious invasive disease, but incidence of the most virulent serotype, Hib, has dropped since introduction of routine Hib vaccination. In England and Wales, the incidence of 2 other serotypes, Hie and Hif, is increasing; during 2001-2010, there was an 11.0% year-on-year increase in Hif and a 7.4% increase in Hie. In 2009-2010, Hif incidence was 0.090/100,000 persons and Hie incidence 0.030/100,000, with higher rates among infants and older adults. Hie had a more severe clinical course; although outcome at 6 months was comparable for the 2 serotypes, case-fatality rate within 7 days of diagnosis was higher for Hie, even after adjustment for age and comorbidities. Multilocus sequence typing revealed a single major circulating clone for both Hif (sequence type 124; 89/99 isolates, 90%) and Hie (sequence type 18; 21/33, 64%), but no association between type and clinical disease or outcome was found.

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.

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.

Childhood bacterial meningitis in Ulaanbaatar, Mongolia, 2002-2004.

BACKGROUND: Childhood bacterial meningitis is severe and largely preventable by vaccination. Few data on childhood bacterial meningitis in Northeast and Central Asia exist. Our aim was to determine the incidence and etiology of childhood bacterial meningitis in Ulaanbaatar, Mongolia. METHODS: We conducted prospective, population-based, active hospital surveillance for clinical meningitis in children 2 months to 5 years of age. Clinical data, blood, and cerebrospinal fluid were collected according to a standard protocol. Laboratory testing was performed at 2 reference laboratories in Ulaanbaatar. RESULTS: From February 2002 to January 2005, 201 suspected meningitis cases were identified in residents of Ulaanbaatar. The average annual incidence rate for confirmed and probable bacterial meningitis (cases with culture-negative, purulent cerebrospinal fluid) was 68 cases per 100,000 children aged 2 months to 5 years. The average annual incidence rate of confirmed cases was 28 cases per 100,000 children for Haemophilus influenzae type b meningitis, 11 cases per 100,000 children for pneumococcal meningitis, and 13 cases per 100,000 children for meningococcal meningitis. Adjusting for cases without complete cerebrospinal fluid information and culture-negative, probable bacterial cases, the estimated incidence rate was 40 cases per 100,000 children for H. influenzae type b meningitis, 15 cases per 100,000 children for pneumococcal meningitis, and 17 cases per 100,000 children for meningococcal meningitis. CONCLUSION: H. influenzae type b is the leading cause of childhood bacterial meningitis in Ulaanbaatar, and the incidence rate is higher than that reported from other Asian countries. These data supported the recent introduction of H. influenzae type b conjugate vaccine in Mongolia. Ongoing surveillance will monitor the impact of the vaccine.

Changes in genetic diversity of the Bordetella pertussis population in the United Kingdom between 1920 and 2006 reflect vaccination coverage and emergence of a single dominant clonal type.

Pertussis (whooping cough) is a potentially fatal respiratory disease caused by the bacterium Bordetella pertussis. Despite effective vaccination programs, there has been concern in some developed countries that pertussis cases are on the increase. We characterized 703 clinical B. pertussis isolates collected in the United Kingdom between 1920 and 2006 using multilocus variable-number tandem repeat analysis (MLVA), pertactin (prnA) and pertussis toxin (ptxA) genotyping, and serotyping. The results showed that the genetic diversity of the bacterial population decreased during periods of high vaccine coverage. However, it was elevated between 1977 and 1986, when vaccine coverage in the United Kingdom was low and epidemics occurred. A high proportion of MLVA types during this epidemic period were novel, and the prnA(2) and prnA(3) alleles were seen for the first time in the United Kingdom. MLVA-27 appeared in 1982, was codominant during the 1998-to-2001 period, and comprised approximately 70% of isolates during both the 2002-to-2004 and the 2005-to-2006 periods. The United Kingdom is dominated currently by an MLVA-27 prnA(2) ptxA(1) serotype Fim3 clonal type. Even during recent periods dominated by MLVA-27, many novel types were found at low frequencies, suggesting that either there are a large number of uncommon MLVA types circulating at low frequencies or new types are constantly arising. This supports a hypothesis that MLVA-27 is under some form of positive selection conferring increased survival in a highly vaccinated population. There has been no significant change to the bacterial population in the first 2 years since the United Kingdom switched from a whole-cell to an acellular vaccine.

Role of PCR in the diagnosis of pertussis infection in infants: 5 years' experience of provision of a same-day real-time PCR service in England and Wales from 2002 to 2007.

As part of an enhanced surveillance programme for pertussis in England and Wales, a real-time PCR service for the detection of Bordetella pertussis was introduced for infants aged

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.

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.

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.

Detection of anti-pertussis toxin IgG in oral fluids for use in diagnosis and surveillance of Bordetella pertussis infection in children and young adults.

Bordetella pertussis infection is being increasingly recognized as a cause of prolonged, distressing cough (without whooping symptoms) in children and young adults. Diagnosis of infection in this population is important for treatment and surveillance purposes, and may also prove useful in reducing transmission to unvaccinated babies, for whom disease can be fatal. Serum IgG titres against pertussis toxin (PT) are routinely used as a marker of recent or persisting B. pertussis infection. However, collection of serum from young children is difficult, and compliance amongst these subjects to give samples is low. To circumvent these problems, an IgG-capture ELISA capable of detecting anti-PT IgG in oral fluid was devised. The assay was evaluated by comparison to a serum ELISA, using 187 matched serum and oral fluid samples from children (aged 5-16 years) with a history of prolonged coughing, whose serum anti-PT titre had already been determined (69 seropositive, 118 seronegative). The results showed that, using a cutoff of 70 arbitrary units (AU), the oral fluid assay detected seropositive subjects with a sensitivity of 79.7% [95% confidence interval (CI) 68.3-88.4] and a specificity of 96.6% (95% CI 91.5-99.1). Thus, oral fluid titres of >or=70 AU would possess a positive predictive value of 76.2-93.2% for pertussis amongst children with chronic coughs when used as a surrogate for the serum ELISA (assuming disease prevalence of 12-37%). This oral fluid ELISA will greatly assist in the convenience of B. pertussis disease diagnosis and surveillance.

Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets for serogroup and serotype prediction using an automated pipeline.

Streptococcus pneumoniae typically express one of 92 serologically distinct capsule polysaccharide (cps) types (serotypes). Some of these serotypes are closely related to each other; using the commercially available typing antisera, these are assigned to common serogroups containing types that show cross-reactivity. In this serotyping scheme, factor antisera are used to allocate serotypes within a serogroup, based on patterns of reactions. This serotyping method is technically demanding, requires considerable experience and the reading of the results can be subjective. This study describes the analysis of the S. pneumoniae capsular operon genetic sequence to determine serotype distinguishing features and the development, evaluation and verification of an automated whole genome sequence (WGS)-based serotyping bioinformatics tool, PneumoCaT (Pneumococcal Capsule Typing). Initially, WGS data from 871 S. pneumoniae isolates were mapped to reference cps locus sequences for the 92 serotypes. Thirty-two of 92 serotypes could be unambiguously identified based on sequence similarities within the cps operon. The remaining 60 were allocated to one of 20 'genogroups' that broadly correspond to the immunologically defined serogroups. By comparing the cps reference sequences for each genogroup, unique molecular differences were determined for serotypes within 18 of the 20 genogroups and verified using the set of 871 isolates. This information was used to design a decision-tree style algorithm within the PneumoCaT bioinformatics tool to predict to serotype level for 89/94 (92 + 2 molecular types/subtypes) from WGS data and to serogroup level for serogroups 24 and 32, which currently comprise 2.1% of UK referred, invasive isolates submitted to the National Reference Laboratory (NRL), Public Health England (June 2014-July 2015). PneumoCaT was evaluated with an internal validation set of 2065 UK isolates covering 72/92 serotypes, including 19 non-typeable isolates and an external validation set of 2964 isolates from Thailand (n = 2,531), USA (n = 181) and Iceland (n = 252). PneumoCaT was able to predict serotype in 99.1% of the typeable UK isolates and in 99.0% of the non-UK isolates. Concordance was evaluated in UK isolates where further investigation was possible; in 91.5% of the cases the predicted capsular type was concordant with the serologically derived serotype. Following retesting, concordance increased to 99.3% and in most resolved cases (97.8%; 135/138) discordance was shown to be caused by errors in original serotyping. Replicate testing demonstrated that PneumoCaT gave 100% reproducibility of the predicted serotype result. In summary, we have developed a WGS-based serotyping method that can predict capsular type to serotype level for 89/94 serotypes and to serogroup level for the remaining four. This approach could be integrated into routine typing workflows in reference laboratories, reducing the need for phenotypic immunological testing.

Clinical and Molecular Epidemiology of Childhood Invasive Nontypeable Haemophilus influenzae Disease in England and Wales.

INTRODUCTION: In countries with established Haemophilus influenzae type b (Hib) immunization programs, nontypeable H. influenzae (NTHi) is now responsible for nearly all invasive H. influenzae cases across all age groups. METHODS: Public Health England (PHE) conducts enhanced national surveillance of invasive H. influenzae disease in England and Wales. Invasive NTHi isolates submitted to Public Health England from children of ages 1 month to 10 years during 2003-2010 were characterized by multilocus sequence typing (MLST). Detailed clinical information was obtained for all laboratory-confirmed cases of invasive NTHi disease in children during 2009-2013. RESULTS: In England and Wales, there were 7797 cases of invasive H. influenzae disease diagnosed during 2000-2013 and 1585 (20%) occurred in children aged 1 month to 10 years, where NTHi was responsible for 31-51 cases (incidence, 0.53-0.92/100,000) annually. Detailed clinical follow-up of 214 confirmed NTHi cases diagnosed in this age-group during 2009-2013 revealed that 52% (n = 111) occurred in <2-year-old and 52% (n=110) had comorbidity. Bacteremic pneumonia was the most common clinical presentation (n = 99, 46%), 16% (n = 34) required intensive care and 11% (n = 23) died. Characterization by biotyping and MLST of 316 NTHi strains from children with invasive disease during 2003-2010 revealed a genetically heterogeneous population (155 MLSTs) with diverse biotypes and no association with comorbidity status, clinical disease or outcome. CONCLUSIONS: The high level of genetic diversity in invasive NTHi strains highlights the difficulties in developing an effective vaccine against this pathogen.

Modelling anti-pertussis toxin IgG antibody decay following primary and preschool vaccination with an acellular pertussis vaccine in UK subjects using a modified oral fluid assay.

Recent vaccination with pertussis vaccine can confound serological and oral fluid (OF) assays targeting anti-pertussis toxin (anti-PT) IgG antibodies as a marker of recent infection. This study sought to establish the minimum potentially confounding time period based on experimental data to assist interpretation from such samples submitted from UK subjects for pertussis diagnosis. Anti-PT IgG antibody response and decay were measured post-vaccination using a modified OF IgG antibody-capture ELISA (GACELISA). Data were obtained from 72 infants after the third acellular pertussis vaccine dose in the primary schedule (4 months of age) and from 119 children after the single dose at preschool age (3 years 4 months to 5 years 8 months of age). Specimens were taken at approximately 1 month intervals for 9 months post-primary immunization (third dose) and 13 months post-preschool booster (PSB). The modified GACELISA demonstrated a sensitivity of 52/56 (92.9 %: 95 % CI 82.7-98.0) and a specificity of 120/128 (93.8 %: 95 % CI 88.0-97.3) and showed good agreement with the National Reference Laboratory standard anti-PT IgG serum ELISA (rank correlation = 0.80) and the original OF assay (rank correlation = 0.79). Modelling of the decline in antibody titres showed a reduction of 54 % and 34 % for each doubling of time after day 14 for the post-third primary dose and post-PSB subjects, respectively. These data suggest that the minimum confounding time period is approximately 300 days for samples obtained post-primary immunization and at least 3 years for samples submitted from UK children following immunization with the PSB. These data will greatly assist the interpretation of single high diagnostic anti-PT IgG titres by allowing an estimate of the positive predictive value, when the number of days post-immunization and prevalence are known or assumed.