High prevalence of currently circulating Bordetella pertussis isolates not producing vaccine antigen pertactin in Slovenia
Authors: T. Kastrin, A.-M. Barkoff, M. Paragi, M.G. Vitek, J. Mertsola, Q. He
Journal: Clinical Microbiology and Infection (2018)

In Slovenia, whole-cell pertussis vaccine (WCV) was introduced in 1959 and was replaced by acellular pertussis vaccine (ACV) in 1999. The primary vaccination schedule contains three doses of ACV during the first year of life and one booster dose in the second year. Three-, four- or five-valent vaccine (infanrix/IPV + Hib; pertactin (PRN) included) were used for primary vaccination in the period 1999–2007. It was followed by ACV Pentaxim (PRN not included) vaccine, which was used during 2008–2011. Five-valent infanrix/IPV + Hib was reintroduced for 2012–2016, and from 2017 onwards, Pentaxim has been again used. Additional booster (Boostrix, PRN included) was introduced for 8-year-olds in 2009. Vaccination coverage for the primary vaccination (three first doses) has been high since 2002 (>90%). We studied vaccine antigen production of Slovenian Bordetella pertussis isolates in the ACV era. In Slovenia, no ethical approval is required for retrospective studies as long as no clear identification is permitted between the patient (name, social security number, date of birth) and the study sample (local procedure). In the current study, no data were included that could identify study subjects. Therefore, no ethical approval was needed for this study. We included all isolated strains in the country (n = 27), which were collected from four out of nine Slovenian health regions during 2006–2017 (eight in 2006–2011 and 19 in 2014–2017). Vaccination status, clinical information and age of patients were collected (Table 1). We measured production of pertussis toxin, PRN, filamentous haemagglutinin (FHA), and fimbriae 2 and 3 (Fim 2/3) with monoclonal antibody–based enzyme-linked immunosorbent assay [[1]Barkoff A.M. Guiso N. Guillot S. Xing D. Markey K. Berbers G. et al.A rapid ELISA- based method for screening Bordetella pertussis strain production of antigens included in current acellular pertussis vaccines.J Immunol Methods. 2014; 408: 142-148Crossref PubMed Scopus (7) Google Scholar]. PRN-deficient isolates were further studied by sequencing of the prn gene. Genotyping of prn, ptxP and ptxA genes of all isolates was performed as previously described [[2]Barkoff A.M. Mertsola J. Pierard D. Dalby T. Vermedal Hoegh S. Guillot S. et al.Surveillance of circulating Bordetella pertussis strains in Europe during 1998–2015.J Clin Microbiol. 2018; (56e01998-17)Crossref PubMed Scopus (21) Google Scholar].Table 1Strain characteristics, production of vaccine antigens and molecular typing resultsStrain IDAge of patient (years)Year of collectionPlace of originVaccination statusNo. of dosesDate of last dosePRN productionMechanism PRN−FHA productionPT productionSerotype (Fim 2 or 3)prnptxAptxP7162017RadovljicaCompletely412.03.2009Yes—YesYes221311182017RadovljicaCompletely431.07.2001NoInsertion of IS481 in 1613YesYes221314182017PodnartCompletely412.01.2000Yes—YesYes221316262017Dolenje BrdoCompletely4No dataNoInsertion of IS481 in 1613YesYes221336172017LesceCompletely4No dataNoInsertion of IS481 in 1613YesYes221377172017RadovljicaCompletely430.5.2001NoInsertion of IS481 in 1613YesYes2213105202017KranjCompletely427.01.1999NoInsertion of IS481 in 1613YesYes2213112132017IdrijaCompletely514.06.2012NoInsertion of IS481 in 1613YesYes2213123202017KranjCompletely428.10.1998NoInsertion of IS481 in 1613YesYes2213146162017LesceCompletely509.12.2009NoDeletion in the prom+ geneYesYes2NA13150462017IdrijaCompletelyNo dataNo dataNoInsertion of IS481 in 1613YesYes221316162017IdrijaCompletely409.10.2012NoInsertion of IS481 in 1613YesYes2213171172017LjubljanaCompletely410.01.2002NoInsertion of IS481 in 1613YesYes2213191132017DomžaleCompletely507.09.2012NoInsertion of IS481 in 1613YesYes2213211142017DomžaleCompletely409.11.2004NoInsertion of IS481 in 1613YesYes221325552017DomžaleCompletely406.11.2013NoInsertion of IS481 in 1614YesYes2213109152016SelcaNot vaccinated——NoInsertion of IS481 in 1613YesYes2213137172016PodnartCompletely419.04.2001NoInsertion of IS481 in 1613YesYes2213CE 2014782014CeljeNo dataNo dataNo dataNoInsertion of IS481 in 1613YesYes221321142011LjubljanaNo dataNo dataNo dataYes—YesYes3213LJ 2010482010LjubljanaCompletelyNo dataNo dataYes—YesYes22131002 months2010CerkvenjakNo dataNo dataNo dataYes—YesYes321376132010LjubljanaCompletely45.2.1999Yes—YesYes222, 3 or 419582009CeljeCompletely428.09.2001Yes—NoYes32131267NA2006CeljeNo dataNo dataNo dataYes—YesYes32132563NA2006CeljeNo dataNo dataNo dataYes—YesYes32131985NA2006CeljeNo dataNo dataNo dataYes—YesYes3213All patients had had cough when culture was made. FHA, filamentous haemagglutinin; Fim, fimbriae; NA, not available; PRN, pertactin; PT, pertussis toxin. Open table in a new tab All patients had had cough when culture was made. FHA, filamentous haemagglutinin; Fim, fimbriae; NA, not available; PRN, pertactin; PT, pertussis toxin. Of 27 isolates, 17 did not produce PRN and one did not produce FHA. All eight isolates collected in 2006–2011 produced PRN, whereas 17 (89.5%) of 19 strains isolated in 2014–2017 did not produce PRN. Increase of PRN deficient strains between the two time periods was statistically significant (p < 0.0001, Fisher's exact test). Of the 17 PRN-deficient isolates, 16 (94.1%) had a reverse insertion of an IS481 element at position 1613 of the prn gene, and one had a deletion in the prn promoter and coding region at position −297 to 1325 (partly replaced by a fragment of IS1663 element). Interestingly, one isolate collected in 2009 did not produce FHA. All isolates produced pertussis toxin and Fim 2 or Fim 3. Almost all isolates (26/27) had different allele of ptxA (ptxA1), ptxP (ptxP3) and prn (prn 2) compared to B. pertussis strains, which are used for production of ACVs (ptxA2 or ptxA4, ptxP1 and prn 1 or prn 7, respectively) [[2]Barkoff A.M. Mertsola J. Pierard D. Dalby T. Vermedal Hoegh S. Guillot S. et al.Surveillance of circulating Bordetella pertussis strains in Europe during 1998–2015.J Clin Microbiol. 2018; (56e01998-17)Crossref PubMed Scopus (21) Google Scholar]. Of 17 subjects from whom PRN-deficient strains were isolated, 15 were completely vaccinated (Table 1). These 15 subjects were from nine different communities of the country. All subjects had coughing symptoms when the culture sample was taken, and some also had typical paroxysmal cough. In this study, we showed that PRN-deficient B. pertussis isolates are currently dominant in Slovenia. The first PRN-deficient isolate was detected in the community of Celje in 2014 and the most recent in the community of Radovljica. Moreover, of three isolates collected in the community of Radovljica in 2017, two were PRN deficient and one was PRN positive, suggesting that a mixture of PRN-positive and -negative strains are still cocirculating in parts of the country. In addition, the serotype of the strains also changed, although the ACVs used in this country do not contain Fim antigens. This change may be partly due to the immunity induced during the nationwide epidemic of 2006–2007, when Fim 3 strains dominated. In Slovenia, surveillance data revealed that pertussis remains endemic and epidemic [[3]European Center for Disease Prevention and Control (ECDC) ECDC surveillance atlas. ECDC, Stockholm, Sweden2018Google Scholar]. However, there could be several other factors affecting the recent switch from Fim3 to Fim 2 strains in Slovenia. In this study, the frequency of PRN-deficient strains significantly increased between the periods of 2006–2011 (0) and 2014–2017 (89.5%) after the epidemic period of 2006–2007. Nevertheless, ACV containing PRN was introduced in 1999 and no PRN-deficient isolates were found before 2014, 15 years after the introduction of ACV. However, this finding is based on a very small sample of isolates (eight isolates over the 2006–2011 period). During the WCV era, one PRN-deficient strain was found in Italy in the mid-1990s [[4]Stefanelli P. Fazio C. Fedele G. Spensieri F. Ausiello C.M. Mastrantonio P. A natural pertactin deficient strain of Bordetella pertussis shows improved entry in human monocyte-derived dendritic cells.New Microbiol. 2009; 32: 159-166PubMed Google Scholar]. A recent study also shows a similar increase in many European countries using ACVs [[2]Barkoff A.M. Mertsola J. Pierard D. Dalby T. Vermedal Hoegh S. Guillot S. et al.Surveillance of circulating Bordetella pertussis strains in Europe during 1998–2015.J Clin Microbiol. 2018; (56e01998-17)Crossref PubMed Scopus (21) Google Scholar]. Interestingly, a decrease in frequency of PRN-deficient strains was reported in Japan, where the proportion of ACVs without PRN in use was increased in 2012 [[5]Hiramatsu Y. Miyaji Y. Otsuka N. Arakawa Y. Shibayama K. Kamachi K. Significant decrease in pertactin-deficient Bordetella pertussis isolates, Japan.Emerg Infect Dis. 2017; 23: 699-701Crossref PubMed Scopus (19) Google Scholar]. In Slovenia, an ACV without PRN was reintroduced in 2017, so it remains to be seen whether there will also be a decrease of PRN-deficient strains in this country in the future. Most of the PRN-deficient isolates (88.2%) were collected from vaccinated subjects, thus confirming that PRN-deficient B. pertussis is capable of circulating and transmitting among fully immunized populations. In addition, PRN-deficient strains are capable of causing the disease with common pertussis symptoms. However, we need to keep in mind that the duration of protection after ACV and WCV vaccination is not lifelong. In this study, many subjects' previous vaccination had occurred more than 10 years before, thus indicating waning immunity. In summary, this is the first report to show a high frequency of PRN-deficient isolates in Slovenia. Almost 90% of the cultured isolates were PRN deficient in 2014–2017. Appearance of the PRN-deficient isolates occurred 15 years after the introduction of ACVs containing PRN. Our results emphasize that continuous monitoring of B. pertussis strains is needed because vaccine antigen–deficient isolates are spreading. All authors report no conflicts of interest relevant to this letter. We thank E. Laakso, University of Turku for help with laboratory work.