Neonatal invasive disease caused by Streptococcus agalactiae in Europe: the DEVANI multi-center study.

PURPOSE: Group B streptococcus (GBS) remains a leading cause of invasive disease, mainly sepsis and meningitis, in infants < 3 months of age and of mortality among neonates. This study, a major component of the European DEVANI project (Design of a Vaccine Against Neonatal Infections) describes clinical and important microbiological characteristics of neonatal GBS diseases. It quantifies the rate of antenatal screening and intrapartum antibiotic prophylaxis among cases and identifies risk factors associated with an adverse outcome. METHODS: Clinical and microbiological data from 153 invasive neonatal cases (82 early-onset [EOD], 71 late-onset disease [LOD] cases) were collected in eight European countries from mid-2008 to end-2010. RESULTS: Respiratory distress was the most frequent clinical sign at onset of EOD, while meningitis is found in > 30% of LOD. The study revealed that 59% of mothers of EOD cases had not received antenatal screening, whilst GBS was detected in 48.5% of screened cases. Meningitis was associated with an adverse outcome in LOD cases, while prematurity and the presence of cardiocirculatory symptoms were associated with an adverse outcome in EOD cases. Capsular-polysaccharide type III was the most frequent in both EOD and LOD cases with regional differences in the clonal complex distribution. CONCLUSIONS: Standardizing recommendations related to neonatal GBS disease and increasing compliance might improve clinical care and the prevention of GBS EOD. But even full adherence to antenatal screening would miss a relevant number of EOD cases, thus, the most promising prophylactic approach against GBS EOD and LOD would be a vaccine for maternal immunization.

Diphtheria in the WHO European Region, 2010 to 2019.

BackgroundDiphtheria is uncommon in the World Health Organization (WHO) European Region. Nevertheless, sporadic cases, sometimes fatal, continue to be reported.AimTo report on diphtheria cases and coverage with first and third doses of diphtheria, tetanus and pertussis vaccines (DTP1 and DTP3, respectively) for 2010-19 in the Region with a focus on 2019.MethodsData on diphtheria cases were obtained from WHO/United Nations International Children's Emergency Fund (UNICEF) Joint Reporting Forms submitted annually by the Region's Member States. WHO/UNICEF Estimates of National Immunization Coverage for DTP1 and DTP3 were summarised for 2010-19. For 2019, we analysed data on age, and vaccination status and present data by country on DTP1 and DTP3 coverage and the percentage of districts with ≥ 90% and < 80% DTP3 coverage.ResultsFor 2010-19, 451 diphtheria cases were reported in the Region. DTP1 and DTP3 coverage was 92-96% and 95-97%, respectively. For 2019, 52 cases were reported by 11 of 48 countries that submitted reports (including zero reporting). Thirty-nine countries submitted data on percentage of their districts with ≥ 90% and < 80% DTP3 coverage; 26 had ≥ 90% districts with ≥ 90% coverage while 11 had 1-40% districts with < 80% coverage.ConclusionLong-standing high DTP3 coverage at Regional level probably explains the relatively few diphtheria cases reported in the Region. Suboptimal surveillance systems and inadequate laboratory diagnostic capacity may also be contributing factors. Still, the observed cases are of concern. Attaining high DTP3 coverage in all districts and implementing recommended booster doses are necessary to control diphtheria and prevent outbreaks.

Uncovering Infant Group B Streptococcal (GBS) Disease Clusters in the United Kingdom and Ireland Through Genomic Analysis: A Population-based Epidemiological Study.

BACKGROUND: The true frequency of hospital outbreaks of invasive group B streptococcal (iGBS; Streptococcus agalactiae) disease in infants is unknown. We used whole genome sequencing (WGS) of iGBS isolates collected during a period of enhanced surveillance of infant iGBS disease in the UK and Ireland to determine the number of clustered cases. METHODS: Potentially linked iGBS cases from infants with early (<7 days of life) or late-onset (7-89 days) disease were identified from WGS data (HiSeq 2500 platform, Illumina) from clinical sterile site isolates collected between 04/2014 and 04/2015. We assessed time and place of cases to determine a single-nucleotide polymorphism (SNP) difference threshold for clustered cases. Case details were augmented through linkage to national hospital admission data and hospital record review by local microbiologists. RESULTS: Analysis of sequences indicated a cutoff of ≤5 SNP differences to define iGBS clusters. Among 410 infant iGBS isolates, we identified 7 clusters (4 genetically identical pairs with 0 SNP differences, 1 pair with 3 SNP differences, 1 cluster of 4 cases with ≤1 SNP differences) of which 4 clusters were uncovered for the first time. The clusters comprised 16 cases, of which 15 were late-onset (of 192 late-onset cases with sequenced isolates) and 1 an early-onset index case. Serial intervals between cases ranged from 0 to 59 (median 12) days. CONCLUSIONS: Approximately 1 in 12 late-onset infant iGBS cases were part of a hospital cluster. Over half of the clusters were previously undetected, emphasizing the importance of routine submission of iGBS isolates to reference laboratories for cluster identification and genomic confirmation.

SpeS: A Novel Superantigen and Its Potential as a Vaccine Adjuvant against Strangles.

Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of proinflammatory cytokines. Streptococcus equi (S. equi) and Streptococcus zooepidemicus (S. zooepidemicus) produce sAgs that play an important role in their ability to cause disease. Strangles, caused by S. equi, is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of S. zooepidemicus, SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) class II molecules. The fusion of SpeS and SpeSY39I to six S. equi surface proteins using two different peptide linkers was conducted to determine if MHC class II-binding properties were maintained. Proliferation assays, qPCR and flow cytometry analysis showed that SpeSY39I and its fusion proteins induced less mitogenic activity and interferon gamma expression when compared to SpeS, whilst retaining Antigen-Presenting Cell (APC)-binding properties. Our data suggest that SpeSY39I-surface protein fusions could be used to direct vaccine antigens towards antigen-presenting cells in vivo with the potential to enhance antigen presentation and improve immune responses.

European Multicentre Tics in Children Studies (EMTICS): protocol for two cohort studies to assess risk factors for tic onset and exacerbation in children and adolescents.

Genetic predisposition, autoimmunity and environmental factors [e.g. pre- and perinatal difficulties, Group A Streptococcal (GAS) and other infections, stress-inducing events] might interact to create a neurobiological vulnerability to the development of tics and associated behaviours. However, the existing evidence for this relies primarily on small prospective or larger retrospective population-based studies, and is therefore still inconclusive. This article describes the design and methodology of the EMTICS study, a longitudinal observational European multicentre study involving 16 clinical centres, with the following objectives: (1) to investigate the association of environmental factors (GAS exposure and psychosocial stress, primarily) with the onset and course of tics and/or obsessive-compulsive symptoms through the prospective observation of at-risk individuals (ONSET cohort: 260 children aged 3-10 years who are tic-free at study entry and have a first-degree relative with a chronic tic disorder) and affected individuals (COURSE cohort: 715 youth aged 3-16 years with a tic disorder); (2) to characterise the immune response to microbial antigens and the host's immune response regulation in association with onset and exacerbations of tics; (3) to increase knowledge of the human gene pathways influencing the pathogenesis of tic disorders; and (4) to develop prediction models for the risk of onset and exacerbations of tic disorders. The EMTICS study is, to our knowledge, the largest prospective cohort assessment of the contribution of different genetic and environmental factors to the risk of developing tics in putatively predisposed individuals and to the risk of exacerbating tics in young individuals with chronic tic disorders.

Comparison of molecular serotyping approaches of Streptococcus agalactiae from genomic sequences.

BACKGROUND: Group B streptococcus (GBS) capsular polysaccharide is one of the major virulence factors underlying invasive GBS disease and a component of forthcoming vaccines. Serotype classification of GBS is based on the capsule polysaccharide of which ten variants are known to exist (Ia, Ib, II-IX). Current methods for GBS serotype assignment rely on latex agglutination or PCR while more recently a whole genome sequencing method was reported. In this study, three distinct algorithms for serotype assignment from genomic data were assessed using a panel of 790 clinical isolates. METHODS: The first approach utilised the entire capsular locus coupled with a mapping methodology. The second approach continues from the first and utilised a SNP-based methodology across the conserved cpsD-G region to differentiate serotypes Ia-VII and IX. Finally the third approach used the variable cpsG -K region coupled with a mapping methodology. All three approaches were assessed for typeability (percentage of isolates assigned a serotype) and concordance to the latex agglutination methodology. RESULTS: Following comparisons, the third approach using the variable cpsG-K region demonstrated the best performance with 99.9% typeability and 86.7% concordance. Overall, of the 105 discordant isolates, 71 were resolved following retesting of latex agglutination and whole genome sequencing, 20 failed to assign a serotype using latex agglutination and only 14 were found to be truly discordant on re-testing. Comparison of this final approach with the previously described assembly-based approach returned identical results. CONCLUSIONS: These results demonstrated that molecular capsular typing using whole genome sequencing and a mapping-based approach is a viable alternative to the traditional, latex agglutination-based serotyping method and can be implemented in a public health microbiology setting.

Scarlet Fever Upsurge in England and Molecular-Genetic Analysis in North-West London, 2014.

Scarlet fever notifications surged across the United Kingdom in spring 2014. Molecular epidemiologic investigation of Streptococcus pyogenes infections in North-West London highlighted increased emm4 and emm3 infections coincident with the upsurge. Unlike outbreaks in other countries, antimicrobial resistance was uncommon, highlighting an urgent need to better understand the drivers of scarlet fever activity.

Invasive Group A Streptococcus Infection among Children, Rural Kenya.

To determine the extent of group A Streptococcus (GAS) infections in sub-Saharan Africa and the serotypes that cause disease, we analyzed surveillance data for 64,741 hospital admissions in Kilifi, Kenya, during 1998-2011. We evaluated incidence, clinical presentations, and emm types that cause invasive GAS infection. We detected 370 cases; of the 369 for which we had data, most were skin and soft tissue infections (70%), severe pneumonia (23%), and primary bacteremia (14%). Overall case-fatality risk was 12%. Incidence of invasive GAS infection was 0.6 cases/1,000 live births among neonates, 101/100,000 person-years among children <1 year of age, and 35/100,000 among children <5 years of age. Genome sequencing identified 88 emm types. GAS causes serious disease in children in rural Kenya, especially neonates, and the causative organisms have considerable genotypic diversity. Benefit from the most advanced GAS type-specific vaccines may be limited, and efforts must be directed to protect against disease in regions of high incidence.

Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data.

Group B streptococcus (GBS) capsular serotypes are major determinants of virulence and affect potential vaccine coverage. Here we report a whole-genome-sequencing-based method for GBS serotype assignment. This method shows strong agreement (kappa of 0.92) with conventional methods and increased serotype assignment (100%) to all 10 capsular types.

Molecular and epidemiological review of toxigenic diphtheria infections in England between 2007 and 2013.

Human infections caused by toxigenic corynebacteria occur sporadically across Europe. In this report, we undertook the epidemiological and molecular characterization of all toxigenic corynebacterium strains isolated in England between January 2007 and December 2013. Epidemiological aspects include case demographics, risk factors, clinical presentation, treatment, and outcome. Molecular characterization was performed using multilocus sequence typing (MLST) alongside traditional phenotypic methods. In total, there were 20 cases of toxigenic corynebacteria; 12 (60.0%) were caused by Corynebacterium ulcerans, where animal contact was the predominant risk factor. The remaining eight (40.0%) were caused by Corynebacterium diphtheriae strains; six were biovar mitis, which were associated with recent travel abroad. Adults 45 years and older were particularly affected (55.0%; 11/20), and typical symptoms included sore throat and fever. Respiratory diphtheria with the absence of a pharyngeal membrane was the most common presentation (50.0%; 10/20). None of the eight C. diphtheriae cases were fully immunized. Diphtheria antitoxin was issued in two (9.5%) cases; both survived. Two (9.5%) cases died, one due to a C. diphtheriae infection and one due to C. ulcerans. MLST demonstrated that the majority (87.5%; 7/8) of C. diphtheriae strains represented new sequence types (STs). By adapting several primer sequences, the MLST genes in C. ulcerans were also amplified, thereby providing the basis for extension of the MLST scheme, which is currently restricted to C. diphtheriae. Despite high population immunity, occasional toxigenic corynebacterium strains are identified in England and continued surveillance is required.

External quality assessments for microbiologic diagnosis of diphtheria in Europe.

The European Diphtheria Surveillance Network (EDSN) ensures the reliable epidemiological and microbiologic assessment of disease prevalence in the European Union. Here, we describe a survey of current diagnostic techniques for diphtheria surveillance conducted across the European Union and report the results from three external quality assessment (EQA) schemes performed between 2010 and 2014.

Emerging trends in the epidemiology of invasive group B streptococcal disease in England and Wales, 1991-2010.

BACKGROUND: Few cross-population studies examining the epidemiology of invasive group B streptococcal (GBS) disease have been undertaken. To identify longitudinal trends in the burden and characteristics of infections, national surveillance data on diagnoses in England and Wales from 1991 to 2010 were analyzed. METHODS: A parallel review of laboratory-confirmed invasive GBS infection surveillance reports and isolates submitted to the national reference laboratory was undertaken. Cases were defined as GBS isolated from a normally sterile site. RESULTS: A total of 21 386 reports of invasive GBS infection were made between 1991 and 2010. The annual rate of reports doubled over the 20 years from 1.48 to 2.99 per 100 000 population. Significant increases were seen in all age groups but most pronounced in adults. Rates of early-onset (0-6 days) infant disease fluctuated but showed a general rise between 2000 and 2010 from 0.28 to 0.41 per 1000 live births. Rates of late-onset (7-90 days) disease increased steadily between 1991 and 2010 from 0.11 to 0.29 per 1000 live births. Resistance to erythromycin increased markedly from 2.5% in 1991 to 15% in 2010. The distribution of serotypes varied according to patient age and over time with type III increasing among early-onset cases and decreasing in adults. CONCLUSIONS: Although risk of invasive GBS infection remains highest within the first few days of life, the relative burden of disease is shifting toward adults. The rise in incidence and antibiotic resistance makes development of an effective and safe vaccine all the more pressing.

Molecular analysis of an outbreak of lethal postpartum sepsis caused by Streptococcus pyogenes.

Sepsis is now the leading direct cause of maternal death in the United Kingdom, and Streptococcus pyogenes is the leading pathogen. We combined conventional and genomic analyses to define the duration and scale of a lethal outbreak. Two postpartum deaths caused by S. pyogenes occurred within 24 h; one was characterized by bacteremia and shock and the other by hemorrhagic pneumonia. The women gave birth within minutes of each other in the same maternity unit 2 days earlier. Seven additional infections in health care and household contacts were subsequently detected and treated. All cluster-associated S. pyogenes isolates were genotype emm1 and were initially indistinguishable from other United Kingdom emm1 isolates. Sequencing of the virulence gene sic revealed that all outbreak isolates had the same unique sic type. Genome sequencing confirmed that the cluster was caused by a unique S. pyogenes clone. Transmission between patients occurred on a single day and was associated with casual contact only. A single isolate from one patient demonstrated a sequence change in sic consistent with longer infection duration. Transmission to health care workers was traced to single clinical contacts with index cases. The last case was detected 18 days after the first case. Following enhanced surveillance, the outbreak isolate was not detected again. Mutations in bacterial regulatory genes played no detectable role in this outbreak, illustrating the intrinsic ability of emm1 S. pyogenes to spread while retaining virulence. This fast-moving outbreak highlights the potential of S. pyogenes to cause a range of diseases in the puerperium with rapid transmission, underlining the importance of immediate recognition and response by clinical infection and occupational health teams.

A three year descriptive study of early onset neonatal sepsis in a refugee population on the Thailand Myanmar border.

BACKGROUND: Each year an estimated four million neonates die, the majority in the first week of life. One of the major causes of death is sepsis. Proving the incidence and aetiology of neonatal sepsis is difficult, particularly in resource poor settings where the majority of the deaths occur. METHODS: We conducted a three year observational study of clinically diagnosed early onset (<7 days of age) neonatal sepsis (EONS) in infants born to mothers following antenatal care at the Shoklo Malaria Research Unit clinic in Maela camp for displaced persons on the Thailand-Myanmar border. Episodes of EONS were identified using a clinical case definition. Conventional and molecular microbiological techniques were employed in order to determine underlying aetiology. RESULTS: From April 2009 until April 2012, 187 infants had clinical signs of EONS, giving an incidence rate of 44.8 per 1000 live births (95% CI 38.7-51.5). One blood culture was positive for Escherichia coli, E. coli was detected in the cerebrospinal fluid specimen in this infant, and in an additional two infants, by PCR. Therefore, the incidence of bacteriologically proven EONS was 0.7 per 1000 live births (95% CI 0.1-2.1). No infants enrolled in study died as a direct result of EONS. CONCLUSION: A low incidence of bacteriologically proven EONS was seen in this study, despite a high incidence of clinically diagnosed EONS. The use of molecular diagnostics and nonspecific markers of infection need to be studied in resource poor settings to improve the diagnosis of EONS and rationalise antibiotic use.

Polymorphisms in regulator of protease B (RopB) alter disease phenotype and strain virulence of serotype M3 group A Streptococcus.

Whole-genome sequencing of serotype M3 group A streptococci (GAS) from oropharyngeal and invasive infections in Ontario recently showed that the gene encoding regulator of protease B (RopB) is highly polymorphic in this population. To test the hypothesis that ropB is under diversifying selective pressure among all serotype M3 GAS strains, we sequenced this gene in 1178 strains collected from different infection types, geographic regions, and time periods. The results confirmed our hypothesis and discovered a significant association between mutant ropB alleles, decreased activity of its major regulatory target SpeB, and pharyngitis. Additionally, isoallelic strains with ropB polymorphisms were significantly less virulent in a mouse model of necrotizing fasciitis. These studies provide a model strategy for applying whole-genome sequencing followed by deep single-gene sequencing to generate new insight to the rapid evolution and virulence regulation of human pathogens.

Population genetics of group B Streptococcus from maternal carriage in an ethnically diverse community in London.

Introduction: Maternal immunization against Group B Streptococcus (GBS) has the potential to significantly reduce the burden of neonatal GBS infections. Population genetics of GBS from maternal carriage can offer key insights into vaccine target distribution. Methods: In this study we characterized the population structure of GBS isolates from maternal carriage (n = 535) in an ethnically diverse community in London, using whole genome sequencing. Results: The isolates clustered into nine clonal complexes (CCs) but the majority (95%) belonged to five lineages: CC1 (26%), CC19 (26%), CC23 (20%), CC17 (13%) and CC8/10 (10%). Nine serotypes were identified, the most common were serotypes III (26%), V (21%), II (19%) and Ia (19%). Other serotypes (Ib, IV, VI, VII, IX) represented less than 10% of all isolates each. Intra-lineage serotype diversity was observed in all major CCs but was highest in CC1, which revealed nine serotypes. Nearly all isolates (99%) carried at least one of the four alpha family protein genes (alpha, alp1, alp23, and rib). All isolates were susceptible to penicillin. We found 21% and 13% of isolates to be resistant to clarithromycin and clindamycin, respectively. Prevalence of macrolide-lincosamide-streptogramin B (MLSB) resistance genes was 22% and they were most common in CC19 (37%) and CC1 (28%), and isolates with serotypes V (38%) and IV (32%). We identified some associations between maternal ethnicity and GBS population structure. Serotype Ib was significantly less common among the South Asian compared to Black women (S. Asian: 3/142, Black: 15/135, p = 0.03). There was also a significantly lower proportion of CC1 isolates among the White other (24/142) in comparison to Black (43/135) and S. Asian (44/142) women (p = 0.04). We found a significantly higher proportion of CC17 isolates among the White other compared to S. Asian women (White other: 32/142, S. Asian: 10/142, p = 0.004). Conclusion: Our study showed high prevalence of GBS vaccine targets among isolates from pregnant women in London. However, the observed serotype diversity in CC1 and high prevalence of MLSB resistance genes in CC19 demonstrates presence of high risk lineages, which might act as a reservoir of non-vaccine strains and antimicrobial resistance determinants.

Evaluation and validation of a commercial ELISA versus the in vitro toxin neutralization assay for determination of diphtheria anti-toxin in human serum.

Introduction. Diphtheria is a potentially life-threatening infection and remains endemic in many low- and middle-income countries (LMICs). A reliable, low-cost method for serosurveys in LMICs is warranted to estimate the accurate population immunity to control diphtheria.Hypothesis/Gap Statement. The correlation between the ELISA results against diphtheria toxoid and the gold standard diphtheria toxin neutralization test (TNT) values is poor when ELISA values are <0.1 IU ml-1, which results in inaccurate estimates of susceptibility in populations when ELISA is used for measuring antibody levels.Aim. To explore methods to accurately predict population immunity and TNT-derived anti-toxin titres from ELISA anti-toxoid results.Methodology. A total of 96 paired serum and dried blood spot (DBS) samples collected in Vietnam were used for comparison of TNT and ELISA. The diagnostic accuracy of ELISA measurement with reference to TNT was assessed by area under the receiver operating characteristic (ROC) curve (AUC) and other parameters. Optimal ELISA cut-off values corresponding to TNT cut-off values of 0.01 and 0.1 IU ml-1 were identified by ROC analysis. A method based on the multiple imputation approach was also applied to estimate TNT measurements in a dataset that only included ELISA results. These two approaches were then applied to ELISA results previously generated from 510 subjects in a serosurvey in Vietnam.Results. The ELISA results on DBS samples showed a good diagnostic performance compared to TNT. The cut-off values for ELISA measurement corresponding to the TNT cut-off values of 0.01 IU ml-1 were 0.060 IU ml-1 in serum samples, and 0.044 IU ml-1 in DBS samples. When a cut-off value of 0.06 IU ml-1 was applied to the 510 subject serosurvey data, 54 % of the population were considered susceptible (<0.01 IU ml-1). The multiple imputation-based approach estimated that 35 % of the population were susceptible. These proportions were much larger than the susceptible proportion estimated by the original ELISA measurements.Conclusion. Testing a subset of sera by TNT combined with ROC analysis or a multiple imputation approach helps to adjust ELISA thresholds or values to assess population susceptibility more accurately. DBS is an effective low-cost alternative to serum for future serological studies for diphtheria.

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.

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia.

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.

Diphtheria in the postepidemic period, Europe, 2000-2009.

Diphtheria incidence has decreased in Europe since its resurgence in the 1990s, but circulation continues in some countries in eastern Europe, and sporadic cases have been reported elsewhere. Surveillance data from Diphtheria Surveillance Network countries and the World Health Organization European Region for 2000-2009 were analyzed. Latvia reported the highest annual incidence in Europe each year, but the Russian Federation and Ukraine accounted for 83% of all cases. Over the past 10 years, diphtheria incidence has decreased by >95% across the region. Although most deaths occurred in disease-endemic countries, case-fatality rates were highest in countries to which diphtheria is not endemic, where unfamiliarity can lead to delays in diagnosis and treatment. In western Europe, toxigenic Corynebacterium ulcerans has increasingly been identified as the etiologic agent. Reduction in diphtheria incidence over the past 10 years is encouraging, but maintaining high vaccination coverage is essential to prevent indigenous C. ulcerans and reemergence of C. diphtheriae.