Analysis of Bordetella pertussis clinical isolates circulating in European countries during the period 1998-2012.

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998-2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.

Antibody-mediated inhibition of Bordetella pertussis adenylate cyclase-haemolysin-induced macrophage cytotoxicity is influenced by variations in the bacterial population.

Whooping cough is a vaccine-preventable disease presenting with epidemic cycles linked to natural and/or vaccine-driven evolution of the aetiological agent of the disease, Bordetella pertussis. Adenylate cyclase-haemolysin (AC-Hly) is a major toxin produced by this pathogen, which mediates macrophage apoptosis in vitro and in vivo. While current acellular pertussis vaccine (APV) formulations do not include AC-Hly, they all contain pertussis toxin and can comprise filamentous haemagglutinin (FHA), which interacts with AC-Hly, and pertactin (PRN), which has been hypothesized also to interact with AC-Hly. We aimed to study the capacity of specific antibodies to inhibit the in vitro B. pertussis AC-Hly-mediated cytotoxicity of J774A.1 murine macrophages in a background of a changing bacterial population. We demonstrate that: (i) clinical isolates of different types or PRN phenotype are all cytotoxic and lethal in the mouse model of respiratory infection; (ii) lack of PRN production does not impact AC-Hly-related phenotypes; (iii) anti-AC-Hly antibodies inhibit cell lysis whatever the phenotype of the isolate, while anti-PRN antibodies significantly inhibit cell lysis provided the isolate produces this antigen, which might be relevant in vivo for APV-induced immunity; and (iv) anti-FHA antibodies only inhibit lysis induced by isolates collected in 2012, maybe indicating specific characteristics of epidemic lineages of B. pertussis.

The utility of the PCR melting profile technique for typing Corynebacterium diphtheriae isolates.

UNLABELLED: Selection of appropriate typing method depends on a number of factors, including the scale of the investigation, the rapidity required of the results and the financial and technical resources available. Several typing methods have been applied to Corynebacterium diphtheriae genotyping, but most are laborious and time-consuming or require expensive equipment. We report an evaluation of the utility of the PCR melting profile technique for simple and easy-to-perform genotyping of C. diphtheriae. We compared the method with ribotyping-the 'gold standard' for C. diphtheriae typing-and PFGE, MLST, AFLP, RAPD and spoligotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: Occurrence of Corynebacterium diphtheriae infections-in the form of diphtheria in endemic countries and in the form of invasive infections in countries with high antidiphtheria vaccination coverage-indicates the need for maintenance of ability to genotype this pathogen by laboratories. Application of an appropriate typing method is essential not only in outbreak investigations for understanding and predicting epidemics but also in monitoring of the evolution and spread of epidemic clones of C. diphtheriae. The PCR melting profile method presented in the study is a good alternative for C. diphtheriae typing.

Toxigenic Corynebacterium ulcerans in a fatal human case and her feline contacts, France, March 2014.

In March 2014, a person in their eighties who was diagnosed with extensive cellulitis due to toxigenic Corynebacterium ulcerans died from multiple organ failure. Environmental investigation also isolated C. ulcerans in biological samples from two stray cats in contact with the case. This finding provides further evidence that pets can carry toxigenic C. ulcerans and may be a source of the infection in humans.

Pertussis vaccination in adults in France: Overview and suggestions for improvement.

In France, the goal of the pertussis vaccination program is to protect newborns. All infants are vaccinated under the program and then given booster shots up to the age of 25 years. Pregnant women are likewise vaccinated, with the cocooning strategy reserved for infants born to unvaccinated mothers. Real-world data shows (i) inadequate coverage among adolescents and adults under 25; (ii) improper use of the tetanus, diphtheria, and polio (Td/IPV) vaccine in children under six years, adolescents, and young adults; and (iii) underdiagnosis of pertussis in adults. Older patients or those with specific chronic medical conditions are at risk of developing severe disease. Improving the diagnosis and surveillance of pertussis in adults and seniors would be one of the first steps in the right direction. Aligning pertussis vaccination in adults with the Td/IPV program (boosters at 45, 65 years of age, and then every 10 years) would make the vaccination schedule simpler, easier to understand, and easier to implement. Large-scale awareness campaigns targeting this population would increase coverage, thereby boosting the effectiveness of the other measures.

Insertion sequences shared by Bordetella species and implications for the biological diagnosis of pertussis syndrome.

The molecular diagnosis of pertussis and parapertussis syndromes is based on the detection of insertion sequences (IS) 481 and 1001, respectively. However, these IS are also detected in the genomes of various Bordetella species, such that they are not specific for either B. pertussis or B. parapertussis. Therefore, we screened the genome of recently circulating isolates of Bordetella species to compare the prevalence of IS481, IS1001 and, also IS1002 with previously published data and to sequence all IS detected. We also investigated whether the numbers of IS481 and IS1001 copies vary in recently circulating isolates of the different Bordetella species. We used the polymerase chain reaction (PCR) method for screening the genome of circulating isolates and to prepare the fragments for sequencing. We used Southern blotting and quantitative real-time PCR for quantification of the numbers of IS. We found no significant diversity in the sequences of the IS harboured in the genomes of the Bordetella isolates screened, except for a 71-nucleotide deletion from IS1002 in B. bronchiseptica. The IS copy numbers in the genome of recently circulating isolates were similar to those in reference strains. Our results confirm that biological diagnosis targeting the IS481 and IS1001 elements are not specific and detect the species B. pertussis, B. holmesii and B. bronchiseptica (IS481), and B. parapertussis and B. bronchiseptica (IS1001).

Pediatric ambulatory pertussis epidemiology in France, recent updates.

OBJECTIVES: Following various changes in the vaccine strategy in 2013 and the mandatory vaccination in 2018, we aimed to analyze the vaccination status, age, and source of contamination of pertussis and parapertussis cases in outpatient surveillance. PATIENTS AND METHODS: Confirmed pertussis and parapertussis cases were enrolled by 35 pediatricians. RESULTS: From 2014 to 2022, 73 confirmed cases of pertussis (n = 65) and parapertussis (n = 8) were reported. For children below 6 years of age, the number of cases with a 2 + 1 schedule (n = 22) was higher than that of those with a 3 + 1 schedule (n = 7). The age of cases with a 3 + 1 or a 2 + 1 schedule was not significantly different (3.8y ± 1.4 vs 4.2y ± 1.5). The main source of contamination was either adults or adolescents. CONCLUSION: Vaccination status and source of contamination are crucial to study the impact of vaccination recommendations.

Characterization and comparison of invasive Corynebacterium diphtheriae isolates from France and Poland.

Corynebacterium diphtheriae, the agent of diphtheria, is rarely responsible for bacteremia. However, high numbers of bacteremia have been reported in countries with extensive immunization coverage. Here, we used molecular and phenotypic tools to characterize and compare 42 invasive isolates collected in France (including New Caledonia) and Poland over a 23-year period.

Evaluation of the Andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing Gram-positive bacilli.

Matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Dual infection with Bordetella pertussis and Mycoplasma pneumoniae in three infants: case reports.

Studying pertussis-like respiratory infections, we report the cases of three infants with evidence of both Bordetella pertussis and Mycoplasma pneumoniae. Bordetella infection was identified by the real-time polymerase chain reaction (RT-PCR) of nasopharyngeal specimens. Neither B. pertussis nor B. parapertussis were recovered on the culture of nasopharyngeal aspirates (NPAs) from any subjects. M. pneumoniae etiology was diagnosed by culture and RT-PCR. The evolution was fatal for all of the subjects. We conclude that, among patients with Bordetella infection, co-infection with another respiratory pathogen is often probable, and these mixed infections might cause a more severe form of illness, sometimes leading to death.

Multilocus sequence types of invasive Corynebacterium diphtheriae isolated in the Rio de Janeiro urban area, Brazil.

Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types.

What to do and what not to do in serological diagnosis of pertussis: recommendations from EU reference laboratories.

Bordetella pertussis-specific antibodies can be detected by enzyme-linked immunosorbent assays (ELISAs) or multiplex immunoassays. Assays use purified or mixed antigens, and only pertussis toxin (PT) is specific for B. pertussis. The interpretation of results can be based on dual-sample or single-sample serology using one or two cut-offs. The EU Pertstrain group recommends that: (i) ELISAs and multiplex immunoassays should use purified non-detoxified PT as an antigen, that they should have a broad linear range and that they should express results quantitatively in International Units per millilitre (IU/ml); (ii) a single or dual diagnostic cut-off for single-serum serology using IgG-anti-PT between 50 and 120 IU/ml should be used, and diagnostic serology cannot be validly interpreted for one year after vaccination with acellular pertussis (aP) vaccines; (iii) IgA-anti-PT should only be used with indeterminate IgG-anti-PT levels or when a second sample cannot be obtained. This group discourages using: (i) other antigens in routine diagnostics, as they are not specific; (ii) micro-agglutination, due to its lack of sensitivity; (iii) immunoblots for pertussis serodiagnosis, as results cannot be quantified; (iv) other methods, such as complement fixation or indirect immunofluorescence, due to their low sensitivity and/or specificity.

Pertussis incidence among adolescents and adults surveyed in general practices in the Paris area, France, May 2008 to March 2009.

Since the introduction in 1998 of an adolescent pertussis vaccine booster (for persons aged 11-13 years) in France, the incidence of pertussis in adolescents and adults has been unknown. We therefore undertook a study to estimate the incidence of pertussis in these population groups and to evaluate the feasibility of a real-time electronic surveillance system for pertussis in general practices in France. The general practitioners selected for the study were located in Paris and the surrounding area. Polymerase chain reaction (PCR) or measurement of anti-pertussis toxin IgG levels by enzyme-linked immunosorbent assay (ELISA) was used to confirm the infection. Among the 204 patients enrolled in the study, 46 (23%) were diagnosed as having pertussis: 21 were confirmed cases, 24 were clinical cases and one was an epidemiological case. The median age of the 204 patients was 44 years and 134 (66%) were female. The median duration of the patients' cough at enrolment was 24 days. No clinical difference was observed between those with and without a pertussis diagnosis. The incidence of pertussis was estimated to be 145 (95% confidence interval: 121-168) per 100,000 population based on the results from the 10-month study period (calculated for 12 months). Problems in sample collection were identified: pertussis sentinel surveillance cannot be developed without training the staff of medical laboratories who take the biological samples. French health authorities were alerted and training procedures were developed.

Diphtheria in the south of France, March 2011.

In March 2011, a 40 year-old French man was diagnosed with diphtheria caused by toxigenic Corynebacterium diphtheriae. Fifty-three close contacts were identified from whom throat samples were analysed. C. diphtheriae was found only in the asymptomatic partner of the index case. The two cases had travelled in Spain during the incubation period of the index case. Investigation around the second case identified 13 new close contacts.None of them was found to be infected.

The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18).

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor required for the early phases of lung colonization. It can invade eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes the formation of unregulated cAMP levels. CyaA intoxication leads to evident toxic effects on macrophages and neutrophils. Here, we demonstrate that CyaA uses the alpha(M)beta(2) integrin (CD11b/CD18) as a cell receptor. Indeed, the saturable binding of CyaA to the surface of various hematopoietic cell lines correlated with the presence of the alpha(M)beta(2) integrin on these cells. Moreover, binding of CyaA to various murine cell lines and human neutrophils was specifically blocked by anti-CD11b monoclonal antibodies. The increase of intracellular cAMP level and cell death triggered by CyaA intoxication was also specifically blocked by anti-CD11b monoclonal antibodies. In addition, CyaA bound efficiently and triggered intracellular cAMP increase and cell death in Chinese hamster ovary cells transfected with alpha(M)beta(2) (CD11b/CD18) but not in cells transfected with the vector alone or with the alpha(X)beta(2) (CD11c/CD18) integrin. Thus, the cellular distribution of CD11b, mostly on neutrophils, macrophages, and dendritic and natural killer cells, supports a role for CyaA in disrupting the early, innate antibacterial immune response.

Comparison of whole-cell versus acellular pertussis vaccine effectiveness in school clusters of pertussis, France, 2013.

OBJECTIVES AND METHOD: We conducted a prospective study in 2013 to compare the whole-cell versus acellular pertussis vaccines effectiveness and duration of protection, following the occurrence of pertussis clusters. RESULTS: During seven school outbreaks, we identified 102 clinical pertussis cases, including 10 cases biologically confirmed by Bordetella pertussis specific PCR, among a cohort of 305 children in 2nd to 6th grade. The risk of pertussis when vaccinated with an acellular vaccine alone was 1.6 (RR=1.6; 95% CI=1.1-2.5) times higher than when vaccinated with a whole-cell vaccine or using a combined schedule. CONCLUSIONS: The limited duration of protection conferred by the acellular vaccine reinforces the 2013 introduction of the pertussis booster at six years old.

[Pertussis: data collection and vaccinal strategy]. / La coqueluche : collecte de données et choix des stratégies vaccinales.

UNLABELLED: Renacoq is a pediatric hospital-based surveillance network in France, set up in April 1996 to monitor the trend of pertussis among children and the impact of vaccination strategies. METHOD: The authors studied the link between data collection and public health policy. Microbiologists from 43 hospitals notify diagnosis of pertussis among children less than 16 years of age. Pediatricians complete a questionnaire for infants less than 6 months of age fulfilling the case definitions. Positive cultures are sent to the National reference laboratory to validate biological results. Data collected from 1996 to 2007 was analyzed, as well as its interaction with changes in pertussis vaccine policy. RESULTS: The introduction of adolescent and adult boosters was largely supported by Renacoq data but this was not the case for interruption of whole cell vaccine use. The impact of adolescent booster is moderate because of a limited vaccine coverage. There was no observed impact of the adult booster but the coverage is very weak. The introduction and then the sole use of acellular vaccine did not have any impact on Renacoq data. DISCUSSION: The study illustrates the burden of the disease among infants and the link between surveillance data collection and public health decision. It highlights the difficulty to implement new vaccine strategies and the importance of data collection, stressing the need for a better consideration of hospital practitioners involved in public healthcare surveillance.

[Awareness and adherence to Pertussis vaccination guidelines by occupational medicine physicians in Paris healthcare institution]. / Connaissance et application des recommandations vaccinales concernant la coqueluche par la médecine du travail des établissements de santé de Paris.

OBJECTIVE: A questionnaire was used on 44 public and private hospital physicians in Paris to evaluate their knowledge of and adherence to Vaccination Guidelines, three years after their introduction. RESULTS: Eighty per cent of the physicians answered and 92.5% were aware of the vaccination guidelines but only 2 out of 4 respected the targeted vaccination in young adults even when the vaccine was available. A policy of pertussis vaccination was applied only in 12 institutions, but even in these, the rate of vaccinated healthcare workers remained low or was not documented. CONCLUSION: Pertussis is a potential risk to newborns not or partially vaccinated in France. Even if the vaccine is available, adherence to pertussis vaccination guidelines must be improved. Efforts should be made to better publicize and apply pertussis vaccination guidelines.

Incidence of pertussis in subjects aged 50years and older in France in 2013-2014.

OBJECTIVE: To assess the incidence of pertussis (whooping cough) in subjects aged 50years and older in France. METHODS: Participating family physicians (FPs) using the patient record management software AxiSanté® included patients aged 50years and older, who had signed an informed consent form, presenting with persistent cough for 7 to 21days. Bordetella genetic material was detected by polymerase chain reaction (PCR) on nasopharyngeal samples collected at the FP's discretion. RESULTS: A total of 42 FPs included 129 patients from June 2013 to August 2014 (large cities: 38; medium-sized cities: 57; rural areas: 34); 106 samples were analyzed. Overall, 30 pertussis cases were diagnosed: 10 cases confirmed by PCR, 18 purely clinical cases, and two direct epidemiological cases. The crude incidence rate per 100,000 patients aged≥50years was 103.6 (95% CI: 69.9-47.9): 77.1 in large cities, 103.1 in medium-sized cities, and 143.9 in rural areas. The extrapolated incidence rate per 100,000 persons aged≥50years was 187.1 (95% CI: 126.2-67.1): 131.1 in large cities, 256.1 in medium-sized cities, and 242.2 in rural areas. CONCLUSION: The population aged 50years and older can serve as a reservoir. Its role in Bordetella pertussis circulation should be taken into account for pertussis booster vaccination programs.