Susceptibility of clinical isolates of Bordetella pertussis to chemicals.

THE AIM OF STUDY: To test clinical isolates of Bordetella pertussis from the National Reference Laboratory for Pertussis and Diphtheria for susceptibility to commonly available disinfectants. Another aim was to determine the concentration and exposure time for each chemical under real conditions of use and possibly to detect the emergence of resistance to disinfectants among 34 strains of B. pertussis referred to the National Reference Laboratory for Pertussis and Diphtheria in 2014 and 2015. MATERIAL AND METHODS: A total of 34 clinical isolates of Bordetella pertussis were tested for susceptibility to chemical disinfectants by three different methods. The microsuspension method was used for the primary screening, and the tests were carried out without protein contamination. Further testing was conducted in accordance with standard EN 14885, where the test procedure consists of several steps. Step 1 involves quantitative suspension methods (Phase 2, Step 1), and step 2 uses methods designed for practice (Phase 2, Step 2). The quantitative suspension method modified according to EN 13727+A2 was used in step 1 to confirm bactericidal activity of the test products under the dirty conditions. In step 2, clinical isolates were tested using a quantitative carrier test method under the dirty conditions modified according to EN 14561. Based on this standard, the real conditions of product use are simulated. Four disinfectants differing in composition and intended use were tested. RESULTS: Disinfectant No. 1 showed bactericidal activity at a concentration of 0.5% after 2 min of exposure in the case of immersion or at a concentration of 5% after 2 min of exposure when treated by wiping. Disinfectant No. 2 was active at a concentration of 0.1% after 2 min of exposure or at a concentration of 1% after 2 min of exposure, respectively. Disinfectant No. 3 did not show bactericidal activity even at a concentration of 100% after 5 min of exposure. Disinfectant No. 4 showed bactericidal activity at a concentration of 10% after 5 min of exposure or at a concentration of 30% after 2 min of exposure. CONCLUSIONS: None of the strains tested was resistant. Using the methods that simulate the real conditions of use of disinfectants Nos. 1 and 2, it was possible to determine the concentration and exposure time needed to achieve disinfection of surfaces under the dirty conditions. Disinfectants Nos. 3 and 4 are not primarily intended for the treatment of surfaces but for the treatment of the skin and mucous membranes. The results obtained with the latter two products are interesting but inconclusive as the real conditions of their use were not simulated accurately. KEYWORDS Bordetella pertussis - susceptibility - disinfectants.

Seroepidemiology of whooping cough in the Czech Republic: estimates of incidence of infection in adults.

OBJECTIVES: Despite widespread vaccination programmes, pertussis remains a significant health burden in many countries. Low awareness of the disease, the high rate of asymptomatic cases in adults and difficulties with diagnosis could explain the under-reporting of pertussis. The lack of data on actual incidence constitutes an obstacle for public health authorities for the implementation of a vaccination strategy against pertussis in adults. The aim of this study was to evaluate the seroprevalence of antibodies against Bordetella pertussis infection in adults and to estimate the actual incidence of the disease compared with the reported incidence. STUDY DESIGN: Prospective, multicentre seroprevalence study. METHODS: The study was conducted in 2000 adult subjects aged ≥18 years who had not received pertussis vaccination within the last 5 years. All enrolled subjects provided a blood sample for serum testing of IgG antibodies against pertussis toxin, performed by enzyme-linked immunosorbent assay, to indicate if they had an acute infection or if they had been infected with pertussis within the last 12 months or earlier. Results were validated in accordance with the guidelines of the European Sero-epidemiology Network 2 and were expressed in ESEN units/ml. RESULTS: A positive concentration of anti-pertussis toxin antibodies, indicating previous pertussis infection, was demonstrated in 39.9% (n = 799) of all study subjects, and 0.40% (n = 8) of subjects had a concentration suggestive of a recent infection (within the last 12 months). The highest antibody seroprevalence was observed in subjects aged 18-29 years (1.46%). No cases of acute infection were detected. CONCLUSIONS: During the study period, the reported incidence of pertussis in the adult population was 0.84/100,000 inhabitants. Based on the seroprevalence results from this study, it is estimated that the actual incidence of pertussis could be as much as 699/100,000 inhabitants. The actual incidence of pertussis in adults in the Czech Republic could therefore be at least 200-fold higher than the reported incidence. These findings support the need for pertussis vaccination in the adult population.

[Multiple-locus variable number tandem repeat analysis of Bordetella pertussis strains collected in the Czech Republic in 1967-2015: spread of a variant adapted to the population with a high vaccination coverage]. / Multiple-locus variable number tandem repeat analýza souboru kmenu Bordetella pertussis v CR z období 1967-2015: rozsírení varianty adaptované na proockovanou populaci.

AIM: To perform multiple-locus variable number tandem repeat analysis (MLVA) of B. pertussis strains from the collection of the National Reference Laboratory for Diphtheria and Pertussis (NRL/DIPE), National Institute of Public Health (NIPH), Prague. The study strains were isolated from clinical specimens collected mostly in the Czech Republic over a nearly 50-year period from 1967 to 2015 (June). The isolates from three periods characterized by different vaccination strategies and trends in pertussis are compared for genetic diversity and distribution of MLVA types (MT). Based on the results obtained, the suitability for use of MLVA in the analysis of epidemic outbreaks of B. pertussis in the Czech Republic is considered. MATERIAL AND METHODS: DNA samples extracted from B. pertussis strains included in the present study were examined by MLVA using the standard protocol. Data were processed by means of the eBURST algorithm and the calculation of the Simpson diversity index (DI) was used for the statistical analysis. Data were analyzed as a whole and also separately for strains from the three periods: 1967-1980, 1990-2007, and 2008-2015 (June). RESULTS: Fourteen different MT were detected in the study strains, with three of them not being reported before. The most common MTs were MT27 and MT29. MT29 was predominant in 1967-1980 while MT27 was the most prevalent in 1990-2007 and 2008-2015 (June). The DI was the lowest (0.49) in 2008-2015 (June), and comparably higher DIs were calculated for the two previous periods (i.e. 0.667 for 1967-1980 and 0.654 for 1990-2007). CONCLUSION: MLVA revealed a decrease in genetic diversity and shifts in MT distribution of B. pertussis strains isolated from clinical specimens in the Czech Republic from 1967 to 2015 (June). These shifts in the Czech Republic can be characterized as a progressive increase in global MTs at the expense of the locally unique ones. The most common MT, similarly to other geographical areas with long-term high vaccination coverage, is MT27. The results of MLVA of 136 B. pertussis strains can provide a background for using this method in molecular epidemiological analysis of smaller groups of strains.

[Antigenic variability of Bordetella pertussis strains isolated in 1967-2010 in the Czech Republic--possible explanation for the rise in cases of pertussis?]. / Antigenní variabilita kmenu Bordetella pertussis izolovaných v letech 1967-2010 v Ceské republice--mozné vysvetlení vzestupu nemocnosti pertusí?

OBJECTIVE: Comparison of antigenic structures of Bordetella pertussis (B. pertussis) strains isolated from 1967 to 2010 in the Czech Republic. MATERIAL AND METHODS: Seventy strains of B. pertussis were referred to the National Reference Laboratory (NRL) for Pertussis and Diphtheria within the surveillance of pertussis from all over the Czech Republic (CR) between 1967 and 2010. To study the strains, the analysis was performed of the genome sequences encoding the surface immunogenic structures--the pertussis toxin S1 subunit gene (ptxA), pertactin gene region 1 (prnA), type 3 fimbriae gene (fim3)--and pertussis toxin promoter (ptxP) responsible for the regulation of the production of pertussis toxin. RESULTS: For the study set of B. pertussis strains, the sequencing analysis revealed changes in all genomic regions studied. The isolates from three periods differ in the allelic profile. In period I (19671978) with the use of whole cell pertussis vaccine (wP), the following two profiles were the most common: ptxP(1), ptxA(2), prnA(1), fim3(1) and ptxP(1), ptxA(1), prnA(3), fim3(1). In period 2 (19902007) with the switch to acellular pertussis vaccine (aP), the most common profile was: ptxP(3), ptxA(1), prnA(2), fim3(2). Period 3 (20082010) with the use of aP was characterized by the predominance of the following two profiles which had never been found in period 1: ptxP(3), ptxA(1), prnA(2), fim3(2) and ptxP(3) ptxA(1), prnA(2), fim3(1). CONCLUSIONS: Sequencing of the genomic regions ptxP, ptxA, prnA, and fim3 of B. pertussis strains isolated in the CR between 1967 and 2010 confirmed changes in the allelic variants of these regions. The incidence of strains carrying the new allelic variants was increasing after 1995 at the expense of those carrying the original variants. The study results can be interpreted as a partial genetic escape of pathogenic strains of B. pertussis beyond the reach of the pertussis vaccines.

Minimum inhibitory concentrations of erythromycin and other antibiotics for Czech strains of Bordetella pertussis.

AIM OF THE STUDY: To test the susceptibility to first-line and alternative antibiotics of 70 Bordetella pertussis (B. pertussis) strains recovered from patients with whooping cough through national pertussis surveillance in the Czech Republic (CR) in 1967-2010. MATERIAL AND METHODS: The minimum inhibitory concentrations (MICs) of erythromycin, clarithromycin, azithromycin, ciprofloxacin, and co-trimoxazole were tested by the reference agar dilution method on Bordet-Gengou agar with 15 % defibrinated sheep blood. RESULTS: Each of the 70 study strains was inhibited by two concentrations of erythromycin and azithromycin (0.06 and 0.12 mg/l) and by three concentrations of clarithromycin (0.03, 0.06, and 0.12 mg/l), with the highest concentration of the MIC range being 0.12 mg/l for all these similar antibiotics. Tested in a 2-fold geometric dilution series, the concentration of erythromycin required to inhibit 90 % of the study strains (MIC90) was one dilution step lower (0.06 mg/l) than those of clarithromycin and azithromycin (0.12 mg/l). All study strains were inhibited by a single concentration of ciprofloxacin (0.06 mg/l) and two concentrations of co-trimoxazole (0.12 and 0.25 mg/l). CONCLUSION: The panel of 70 Czech strains of B. pertussis appears to be homogeneous in terms of the MICs of the antibiotics tested, with two to three low concentrations being effective against all strains. To be inhibited, no strain required a higher concentration of erythromycin, clarithromycin, azithromycin, ciprofloxacin, or co-trimoxazole.

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.

[Pertussis trend in children under one year of age in the Czech Republic in 1997-2013]. / Trend pertuse u detí do jednoho roku zivota v Ceské republice v letech 1997-2013.

STUDY OBJECTIVE: To characterize the epidemiological situation of pertussis in children under one year of age in the Czech Republic in 1997-2013. MATERIAL AND METHODS: The study cohort consisted of children under one year of age with laboratory confirmed pertussis reported to the communicable disease system from 1997 to 2013. A total of 265 pertussis cases were reported in children under one year of age over the study period. Selected demographic data, need for hospitalization, and vaccination history were evaluated in the study cohort. RESULTS: Children under one year of age have shown a steady upward trend in reported cases of pertussis since the 1990s. The reported incidence of pertussis in this age group was the lowest in 1998 (1.1/100,000 population) and the highest in 2013 (31.3/100,000). In 1997-2013, 265 pertussis cases were reported in children under one year of age, 128 females and 137 males, to the communicable disease system in the Czech Republic. Most of these children, nearly 77%, developed pertussis within the first four months of life. Of the 265 children, 79% were not vaccinated before the onset of the disease and 21% were immunized with at least one dose of pertussis vaccine before developing the disease. As many as 75% of the children with pertussis needed hospitalization. Most of them, nearly 81%, were hospitalized with pertussis in the first four months of life and 90% of them in the first six months of life. CONCLUSIONS: In 1997-2013, an upward trend was observed in pertussis cases in children under one year of age. Most children developed the disease within the first four months of life while not vaccinated against pertussis. This fact unambiguously supports the "cocoon" strategy, i.e. vaccination of the closest contacts of the child, and a booster dose at 25 years of age. At the same time, a question arises whether to provide vaccination to pregnant women.