Macrolide Resistance in Bordetella pertussis: Current Situation and Future Challenges

Pertussis is a highly contagious respiratory infection caused by Bordetella pertussis bacterium. The mainstay of treatment is macrolide antibiotics that reduce transmissibility, shorten the duration of symptoms and decrease mortality in infants. Recently, the macrolide resistance of B. pertussis has been reported globally but is especially widespread in mainland China. In this review, we aim to summarise the current understanding of the epidemiology, resistance mechanisms and clinical implications of B. pertussis macrolide resistance. Since the first appearance of macrolide-resistant B. pertussis in Arizona, USA, in 1994, only sporadic cases have been reported outside China. In certain parts of China, on the other hand, up to 70-100% of the recent clinical isolates have been found to be macrolide resistant. Reasons for macrolide resistance being centred upon China during the last decade can only be speculated on, but the dominant B. pertussis lineage is different between China and most of the high-income countries. It seems evident that efforts to increase awareness, guide molecular epidemiological surveillance and carry out systematic screening of B. pertussis positive samples for macrolide resistance should be implemented globally. In addition, practices to improve the clinical care of infants with pertussis caused by resistant strains should be studied vigorously

Evaluation of Anti-PT Antibody Response after Pertussis Vaccination and Infection: The Importance of Both Quantity and Quality

Pertussis toxin (PT) is considered the main virulence factor causing whooping cough or pertussis. The protein is widely studied and its composition was revealed and sequenced already during the 1980s. The human immune system creates a good response against PT when measured in quantity. However, the serum anti-PT antibodies wane rapidly, and only a small amount of these antibodies are found a few years after vaccination/infection. Therefore, multiple approaches to study the functionality (quality) of these antibodies, e.g., avidity, neutralizing capacity, and epitope specificity, have been investigated. In addition, the long-term B cell memory (Bmem) to PT is crucial for good protection throughout life. In this review, we summarize the findings from functional PT antibody and Bmem studies. These results are discussed in line with the quantity of serum anti-PT antibodies. PT neutralizing antibodies and anti-PT antibodies with proper avidity are crucial for good protection against the disease, and certain epitopes have been identified to have multiple functions in the protection. Although PT-specific Bmem responses are detectable at least five years after vaccination, long-term surveillance is lacking. Variation of the natural boosting of circulating Bordetella pertussis in communities is an important confounding factor in these memory studie</p

Whole blood based point-of-care assay for the detection of anti-pertussis toxin IgG antibodies

Current serological diagnosis of pertussis is usually done by ELISA to determine serum specific anti-pertussis toxin (PT) IgG antibodies. 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. In this study, a quantitative lateral flow assay (LFA) with fluorescent Eu-nanoparticle reporters was used for the detection of anti-PT antibodies from whole blood. The assay was eval-uated by testing overall 141 samples including 25 before and 116 one month after acellular pertussis booster vaccination. LFA results were compared to those obtained with standardized anti-PT IgG ELISAs with paired serum samples. Correlation between the assays was high (Pearson R = 0.832), and the achieved analytical sensitivity of the LFA was 29 IU/mL, which would be sufficient for clinically relevant cutoffs for determining recent infections. The paired samples, collected pre-and post-booster, demonstrated a significant increase in anti-PT IgG antibodies similar to that detected by ELISA. The developed LFA opens up several alternatives for a suitable POC test also in middle-and low-income countries

Pertussis toxin neutralizing antibody response after an acellular booster vaccination in Dutch and Finnish participants of different age groups

Pertussis incidence has increased in many countries and the disease occurs among all age groups, suggesting the need for booster immunizations through life. In addition to determining the concentration of anti-pertussis toxin (PT) antibodies, the ability of PT neutralizing antibodies (PTNAs) could be used to assess vaccine responses.Altogether 258 participants [7–10-year-old (N = 73), 11–15-year-old (N = 85), 20–35-year-old (N = 50) and 60–70-year-old (N = 50)] were included. Sera were collected before, one month, and one year after a single dose of a three pertussis component containing acellular pertussis vaccine. The adolescents were primed in childhood either by acellular or whole-cell vaccination. PTNA titres were determined by a Chinese hamster ovary cell assay and anti-PT IgG/IgA antibody concentrations by multiplex immunoassay.In all age groups, a significant increase in levels of PTNAs and anti-PT IgG was observed one month after vaccination and remained at least two-fold higher one year post-booster, in comparison to pre-booster. Young adults had the lowest response. The strongest increase in PTNAs was observed in participants who had ≥10 IU/mL concentration of anti-PT IgG antibodies pre-booster. At pre-booster, whole-cell-primed adolescents had higher PTNAs than acellular-primed peers (p = 0.047). One year post-booster, the Finnish whole-cell-primed adolescents had a higher level of PTNAs than acellular-primed adolescents (p = 0.049), however, this was not observed in Dutch adolescents. In conclusion, PTNAs increased after vaccination in all age groups, and the strongest increase was related to the presence of high pre-booster antibodies.</p

Differences in epitope-specific antibodies to pertussis toxin after infection and acellular vaccinations

Objectives: Pertussis toxin (PT) is a component of all acellular pertussis vaccines. PT must be detoxified to be included in acellular vaccines, which results in conformational changes in the functional epitopes of PTs. Therefore, induced epitope-specific antibodies to PT may vary after vaccinations or natural infections, and this information could reveal biomarkers implicated for protection and successful immunisation. Methods: Pertussis toxin epitope-specific antibodies in sera from 152 vaccinated children and 72 serologically confirmed patients were tested with a blocking ELISA, based on monoclonal antibodies that target protective PT epitopes. Results: All study groups induced considerable antibody titres to subunit 1 (S1). Of interest, S3 7E10-specific antibodies were present in patients, but not after vaccinations (P < 0.001). The impact of glutaraldehyde treatment of PT was visible on epitope 1D7 (S1), whereas epitopes 1B7 (S1) and 10D (S1) were more preserved. Antibodies to these epitopes were higher after three primary vaccine doses than after a single booster dose. Conclusion: The high amount of 7E10-specific antibodies in patients suggests this epitope might be functionally relevant in protection. The overall characteristics of epitope-specific antibodies are influenced by infection or vaccination background, by the used detoxification method of PT and by the amount of the toxin used in immunisation. </div

High heterogeneity in methods used for the laboratory confirmation of pertussis diagnosis among European countries, 2010 : integration of epidemiological and laboratory surveillance must include standardisation of methodologies and quality assurance

Despite extensive childhood immunisation, pertussis remains one of the world's leading causes of vaccinepreventable deaths. The current methods used for laboratory diagnosis of pertussis include bacterial culture, polymerase chain reaction (PCR) and enzymelinked immunosorbent assay (ELISA) serology. We conducted a questionnaire survey to identify variations in the laboratory methods and protocols used among participating countries included in the European surveillance network for vaccine-preventable diseases (EUVAC.NET). In February 2010, we performed the survey using a web-based questionnaire and sent it to the country experts of 25 European Union countries, and two European Economic Area (EEA) countries, Norway and Iceland. The questionnaire consisted of 37 questions which covered both general information on surveillance methods and detailed laboratory methods used. A descriptive analysis was performed. Questionnaires were answered by all 27 contacted countries. Nineteen countries had pertussis reference laboratories at the national level; their functions varied from performing diagnosis to providing technical advice for routine microbiology laboratories. Culture, PCR and serology were used in 17, 18 and 20 countries, respectively. For PCR, nine laboratories used insertion sequence IS481 as the target gene, which is present in multiple copies in the Bordetella pertussis genome and thus has a greater sensitivity over single copy targets, but has been proved not to be specific for B. pertussis. Antibodies directed against pertussis toxin (PT) are specific for B. pertussis infections. For ELISA serology, only 13 countries' laboratories used purified PT as coating antigen and 10 included World Health Organization (WHO) or Food and Drug Administration (FDA) reference sera in their tests. This present survey shows that methods used for laboratory confirmation of pertussis differ widely among European countries and that there is a great heterogeneity of the reference laboratories and functions. To evaluate the effects of different pertussis immunisation programmes in Europe, standardisation and harmonisation of the laboratory methods are needed.peer-reviewe

Circulation of pertussis and poor protection against diphtheria among middle-aged adults in 18 European countries

Reported incidence of pertussis in the European Union (EU) and the European Economic Area (EEA) varies and may not reflect the real situation, while vaccine-induced protection against diphtheria and tetanus seems sufficient. We aimed to determine the seroprevalence of DTP antibodies in EU/EEA countries within the age groups of 40-49 and 50-59 years. Eighteen countries collected around 500 samples between 2015 and 2018 (N = 10,302) which were analysed for IgG-DTP specific antibodies. The proportion of sera with pertussis toxin antibody levels ≥100 IU/mL, indicative of recent exposure to pertussis was comparable for 13/18 countries, ranging between 2.7-5.8%. For diphtheria the proportion of sera lacking the protective level (</p

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

Memory B Cell Activation Induced by Pertussis Booster Vaccination in Four Age Groups of Three Countries

Background: Immunogenicity of acellular pertussis (aP) vaccines is conventionally assessed by measuring antibody responses but antibody concentrations wane quickly after vaccination. Memory B cells, however, are critical in sustaining long-term protection and therefore may be an important factor when assessing pertussis immunity after vaccination. Aim: We studied pertussis specific memory B cell (re)activation induced by an aP booster vaccination in four different age groups within three countries. Materials and methods: From a phase IV longitudinal interventional study, 268 participants across Finland, the Netherlands and the United Kingdom were included and received a 3-component pertussis booster vaccine: children (7-10y, n=53), adolescents (11-15y, n=66), young adults (20-34y, n=74), and older adults (60-70y, n=75). Memory B cells at baseline, day 28, and 1 year post-vaccination were measured by a pertussis toxin (Ptx), filamentous haemagglutinin (FHA), and pertactin (Prn) specific ELISpot assay. Antibody results measured previously were available for comparison. Furthermore, study participants were distributed into groups based on their baseline memory B cell frequencies, vaccine responses were monitored between these groups. Results: Geometric mean (GM) memory B cell frequencies for pertussis antigens at baseline were low. At 28 days post-vaccination, these frequencies increased within each age group and were still elevated one year post-booster compared to baseline. Highest frequencies at day 28 were found within adolescents (GM: 5, 21, and 13, for Ptx, FHA and Prn, respectively) and lowest within older adults (GM: 2, 9, and 3, respectively). Moderate to strong correlations between memory B cell frequencies at day 28 and antibody concentrations at day 28 and 1 year were observed for Prn. Memory B cell frequencies > 1 per 100,000 PBMCs at baseline were associated with significantly higher memory responses after 28 days and 1 year. Conclusions: An aP booster vaccine (re)activated memory B cells in all age groups. Still elevated memory B cell frequencies after one year indicates enhanced immunological memory. However, antigen specific memory B cell activation seems weaker in older adults, which might reflect immunosenescence. Furthermore, the presence of circulating memory B cells at baseline positively affects memory B cell responses. This study was registered at www.clinicaltrialsregister.eu: No. 2016-003678-42.</p

A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens.

BACKGROUND: Bordetella pertussis continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination. MATERIAL AND METHODS: Longitudinal WB samples were collected from a small set of subjects (n = 38) aged 7-70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated B. pertussis lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay. RESULTS: The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles. CONCLUSIONS: The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future