Alex-Mikael Barkoff

Appearance of Bordetella pertussis strains not expressing the vaccine antigen pertactin in Finland.

Despite extensive vaccinations, resurgence of pertussis has been reported in many countries ([9][1]). Recently, emergence of Bordetella pertussis isolates not expressing the vaccine antigen pertactin (Prn) or pertussis toxin was described in France, where surveillance of the clinical isolates has

Nasopharyngeal bacterial colonization and gene polymorphisms of mannose-binding lectin and toll-like receptors 2 and 4 in infants.

Background Human nasopharynx is often colonized by potentially pathogenic bacteria. Gene polymorphisms in mannose-binding lectin (MBL), toll-like receptor (TLR) 2 and TLR4 have been reported. The present study aimed to investigate possible association between nasopharyngeal bacterial colonization and gene polymorphisms of MBL, TLR2 and TLR4 in healthy infants. Methodology/Principal Findings From August 2008 to June 2010, 489 nasopharyngeal swabs and 412 blood samples were taken from 3-month-old healthy Finnish infants. Semi-quantitative culture was performed and pyrosequencing was used for detection of polymorphisms in MBL structural gene at codons 52, 54, and 57, TLR2 Arg753Gln and TLR4 Asp299Gly. Fifty-nine percent of subjects were culture positive for at least one of the four species: 11% for Streptococcus pneumoniae, 23% for Moraxella catarrhalis, 1% for Haemophilus influenzae and 25% for Staphylococcus aureus. Thirty-two percent of subjects had variant types in MBL, 5% had polymorphism of TLR2, and 18% had polymorphism of TLR4. Colonization rates of S. pneumoniae and S. aureus were significantly higher in infants with variant types of MBL than those with wild type (p = .011 and p = .024). Colonization rates of S. aureus and M. catarrhalis were significantly higher in infants with polymorphisms of TLR2 and of TLR4 than those without (p = .027 and p = .002). Conclusions Our study suggests that there is an association between nasopharyngeal bacterial colonization and genetic variation of MBL, TLR2 and TLR4 in young infants. This finding supports a role for these genetic variations in susceptibility of children to respiratory infections.

Differences in avidity of IgG antibodies to pertussis toxin after acellular pertussis booster vaccination and natural infection.

BACKGROUND Pertussis toxin (PT) is a specific virulence factor of Bordetella pertussis and it is included in all acellular pertussis vaccines (aP). Although immunity after infection seems to persist longer than that after vaccination, the exact mechanism(s) is not known. Primary aim of this study was to develop an ELISA method for measuring avidity index (AI) of IgG-anti-PT antibodies and to compare antibody responses after booster vaccination and infection. Secondary aim was to evaluate if the AI-ELISA has potential in the serodiagnosis of pertussis. MATERIAL Serum samples from a total of 409 subjects were included in the study. Paired sera were taken from 97 adolescents who received booster vaccine ten years ago (dTpa-004) and from 80 young adults who received a second booster dose ten years after the previous booster vaccine (dTpa-040). Thirty-two paired sera from culture-confirmed pertussis patients, 161 single sera from serologically diagnosed patients and 39 single sera from healthy controls were included. AI of IgG-anti-PT antibodies were determined with newly developed ELISA using diethylamine (DEA) as a bond breaking agent. The IgG-anti-PT antibodies were measured by standardized ELISA. RESULTS A significant increase was found in antibody concentrations and AI between PRE and one month POST vaccination ten years ago [GMC for antibody: 7.9 IU/ml vs. 98.3 IU/ml (p=0.0001); for AI: 40.4% vs. 56.1% (p=0.0001)]. Similar result was observed after the second booster dose [GMC for antibody: 9.2 IU/ml vs. 92.4 IU/ml (p=0.0001); for AI: 36.1% vs. 59.5% (p=0.0001)] and between the first and second sera of culture-confirmed patients [GMC for antibody: 6.9 IU/ml vs. 285.1 IU/ml (p=0.0001); for AI: 40.5% vs. 68.4% (p=0.0001)]. Healthy controls showed lower levels of both antibodies and AI. CONCLUSIONS Our results suggest that there may be difference in quality and quantity of antibodies to PT after vaccination and after infection. Furthermore, AI could be a help for vaccine studies.

Gene Polymorphism in Toll-like Receptor 4: Effect on Antibody Production and Persistence After Acellular Pertussis Vaccination During Adolescence

BACKGROUND Toll-like receptors play an important role in the regulation of adaptive immunity. This study aimed to investigate whether Toll-like receptor 4 (TLR4) polymorphisms influence the production and persistence of antibodies after acellular pertussis booster vaccination during adolescence. METHODS Seventy-five subjects received a single dose of diphtheria and tetanus toxoids and acellular pertussis vaccine 10 years ago, during adolescence. The same cohort was followed up at 3, 5, and 10 years after this booster vaccination. Pyrosequencing was used for detecting polymorphism in TLR4. Concentrations of anti-pertussis vaccine antibodies were measured by standardized enzyme-linked immunosorbant assay and published elsewhere. RESULTS The fold increase in antibodies to pertussis toxin after original vaccination 10 years ago was significantly lower in subjects with TLR4 polymorphism than in those without (55% vs 86%; P = .028). At the 3-year follow-up evaluation, geometric mean concentrations of anti-pertussis vaccine antibodies were significantly lower in subjects with TLR4 polymorphism, compared with those without the polymorphism (for pertussis toxin, P = .028; for filamentous hemagglutinin, P = .047; and for pertactin, P = .046). CONCLUSIONS This study suggests that TLR4 Asp299Gly polymorphism might influence production and persistence of antibodies after pertussis booster vaccination in adolescents. However, the results should be interpreted with caution as the number of subjects included in this study was limited.

A rapid ELISA-based method for screening Bordetella pertussis strain production of antigens included in current acellular pertussis vaccines.

INTRODUCTION Despite extensive vaccinations, there have been pertussis epidemics in many countries including the Netherlands, the UK, Australia and the USA. During these epidemics Bordetella pertussis strains not producing the vaccine antigen pertactin (Prn) are emerging and increasing in numbers. However, methods for confirming PRN production of B. pertussis isolates are combined PCR or PCR-based sequencing tests and western blotting. Furthermore, data about production of pertussis toxin (PT) and filamentous hemagglutinin (FHA) of these isolates are scarce. Fimbriae (Fim) production is usually determined by agglutination and reported as serotype. In this study we developed an easy, accurate and rapid method for screening PT and FHA production. Methods for Prn and Fim production have been published earlier. METHODS We analyzed altogether 109 B. pertussis strains, including 103 Finnish B. pertussis strains collected during 2006-2013, international strain Tohama I, French strains FR3496 (PT-negative), FR3693 (Prn-negative) and FR4624 (FHA-negative) and Fim-serotype reference strains S1 (producing only Fim2) and S3 (producing only Fim3). An indirect ELISA with whole bacterial cells as coating antigen was developed and used for rapid screening of the B. pertussis strains. Production of different antigens (PT, FHA, Prn, Fim2 and Fim3) was detected with specific monoclonal antibodies (mAbs). RESULTS From the 103 Finnish B. pertussis strains tested, all were positive for PT, FHA and Fim. Four were found negative for Prn, and they were isolated during 2011-2013. CONCLUSIONS The newly developed method proved to be useful and simple for rapid screening of different antigen production of B. pertussis isolates.

Surveillance of Circulating Bordetella pertussis Strains in Europe during 1998 to 2015

ABSTRACT One reason for increased pertussis incidence is the adaptation of Bordetella pertussis to vaccine-induced immunity by modulating its genomic structure. This study, EUpert IV, includes 265 isolates collected from nine European countries during 2012 to 2015 (n = 265) and compares the results to previous EUpert I to III studies (1998 to 2009). The analyses included genotyping, serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat analysis (MLVA). Genotyping results showed only small variations among the common virulence genes of B. pertussis. The frequencies of serotypes Fim2 and Fim3 varied among the four collections. Genomic analyses showed that MLVA type 27 increased to 80% between the periods of 1998 to 2001 and 2012 to 2015. Two PFGE profiles, BpSR3 (29.4%) and BpSR10 (27.2%), constituted more than 50% of the circulating isolates in the present collection. Our study indicates that the European B. pertussis population is changing and became more homogenous after the introduction of acellular pertussis vaccines.

A rapid lateral flow immunoassay for serological diagnosis of pertussis

Current serological diagnosis of pertussis is usually done by ELISA. However, the ELISAs are often central-laboratory based, require trained staff and have long turnaround times. A rapid point-of-care (POC) assay for pertussis serology would aid in both diagnosis and surveillance of the disease. While lateral flow immunoassays (LFIA) are simple to use and ideal for point-of-care diagnostics, they were limited to qualitative assays until recently. In this study, we developed a quantitative LFIA with fluorescent Eu-nanoparticle reporters for the detection of anti-pertussis toxin (PT) IgG. The assay was evaluated by testing 198 serum samples with varying anti-PT IgG levels and the result was compared to those obtained with standardized anti-PT IgG ELISA. At the diagnostic cutoff of 100 IU/mL in ELISA, the LFIA had a concordance of 92% with the ELISA, with a specificity of 96% [95% confidence interval (CI): 89-99%] and a sensitivity of 88% [CI: 77-94%]. The developed LFIA has a turnaround time of one hour and requires only a simple manipulation by the user and an instrument for the quantitative detection of the signal. We conclude that the LFIA is specific and sensitive for serological diagnosis of pertussis and is suitable for a POC test.

Antimicrobial susceptibility testing of Finnish Bordetella pertussis isolates collected during 2006–2017

OBJECTIVES Macrolides, such as azithromycin and erythromycin, are first-line drugs for the (prophylactic) treatment of pertussis. This study aimed to screen for macrolide-, quinolone- or trimethoprim/sulfamethoxazole (SXT)-resistant strains among Finnish Bordetella pertussis isolates. METHODS Antimicrobial susceptibility testing was performed on 148 B. pertussis strains isolated during 2006-2017. Isolates were analysed by allele-specific PCR for detection of the macrolide resistance-associated mutation A2047G in the 23S rRNA gene. The gyrA gene was sequenced for detection of the A260G mutation associated with quinolone resistance. For phenotyping, a random selection was made by selecting every third isolate (n=50) to determine the minimum inhibitory concentrations (MICs) for erythromycin and azithromycin by Etest and the inhibition zone size for nalidixic acid (NAL) and SXT by single disk diffusion assay. RESULTS Neither the macrolide resistance-associated mutation A2047G nor the quinolone resistance-associated mutation A260G was detected in any of the B. pertussis isolates. MICs of azithromycin and erythromycin ranged between 0.016-0.19μg/mL and 0.016-0.25μg/mL, respectively. The size of the inhibition zone surrounding the NAL disk ranged between 22-27mm in diameter. The inhibition zone surrounding the SXT disk ranged between 24-37mm in diameter. No isolates resistant to any of the tested antimicrobials were identified. CONCLUSIONS The allele-specific PCR is a simple and useful tool for screening B. pertussis resistance to macrolides. All Finnish isolates tested were susceptible to macrolides, quinolones and SXT.

O-180 Avidity Of Anti-pertussis Toxin (pt) Igg-antibodies After Primary And Booster Pertussis Vaccination And Their Association With Il-17a Gene Polymorphism

Background and aims High levels of antibodies and avidity indicates good protection after vaccination. However, there are only a few studies measuring avidity of PT-antibodies in children. Recent studies suggest that Th17 specific immunity acquired from Diphtheria-Tetanus-acellular-Pertussis (DTaP) vaccination may provide efficient protection against pertussis. In this study, we aimed to investigate concentration and avidity of anti-PT-IgG antibodies (PT-Abs) after primary and booster vaccination and their association with gene polymorphism of IL-17A. Methods Altogether, 325 serum samples were included. From these, 72 were collected from unvaccinated infants at 2.6 months of age, 203 from primary vaccinated 13-month-old children, and 50 from DTaP vaccinated adults. Concentration and avidity of PT-Abs were measured by ELISA. SNP detection of IL-17A was performed using Sequenom iPlex Gold system. Results Quantity of PT-Abs showed significant increase after primary vaccination in infants. When primary and booster vaccinations were compared, significantly higher levels of PT-Abs were observed after booster vaccination, whereas higher levels of avidity were found after primary vaccination. Frequencies of three IL-17A genotypes identified was 33% (G/G), 47% (G/A) and 20% (A/A) in 203 infants. Subjects with IL-17A G/G genotype had significantly lower avidity of PT-Abs than those with the other two genotypes. However, there was no significant difference in levels of PT-antibodies between these genotypes. Conclusions Our results indicate that avidity of PT-Abs is higher after primary vaccination than after booster vaccination. This study also suggests that gene polymorphism of IL-17A may influence quality of PT-Abs after primary vaccination in infants.