Journal of Infection (2013) xx, 1e6

www.elsevierhealth.com/journals/jinf

Bordetella pertussis: Why is it stillcirculating?

Nicole Guiso a,b,*

a Institut Pasteur, Molecular Prevention and Therapy of Human Diseases Unit, Paris, FrancebCentre National de la Recherche Scientifique (CNRS), URA3012, France

Accepted 20 September 2013Available online - - -

KEYWORDSBordetella pertussis;Whooping cough;Vaccine;Diagnostic

* Institut Pasteur, PTMMH, 25 rue duE-mail address: nicole.guiso@paste

0163-4453/$36 ª 2013 The British Infehttp://dx.doi.org/10.1016/j.jinf.2013

Please cite this article in press as: Gj.jinf.2013.09.022

Summary Bordetella pertussis is the causal agent of whooping cough, a highly contagiousrespiratory disease that is life-threatening in infants under the age of three months and mayalso be very severe in pregnant women and seniors. This disease can be prevented by vaccina-tion but it remains a public health problem in many developed and developing countries.1 So,why is B. pertussis still circulating?

We need to consider several aspects of this vaccine-preventable disease when answering thisquestion: (i) the history of the disease and the historical context in which the vaccine wasdeveloped; (ii) the type of vaccine used; (iii) the vaccination strategy and coverage; (iv) thedisease surveillance after the introduction of generalized vaccination and (v) the surveillancefor the causal agent of the disease.ª 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

1. History of whooping cough and its causalagents

Whooping cough was characterized much more recentlythan many other infectious diseases. Nils Rosen von Rose-nstein has suggested that this illness first occurred inFrance in 1414, but the first description of the diseasewas published by Guillaume de Baillou, after an epidemic inParis, France, in 1578.2 During the 16th and the 17th cen-turies, a large number of descriptions were published, sug-gesting an expansion of the disease.2

Dr Roux, 75724 Paris cedex 15, Fur.fr

ction Association. Published by E.09.022

uiso N, Bordetella pertussis: Why

In 1900, Jules Bordet identified the causal agent of thedisease in the expectoration of a five-month-old child withwhooping syndrome but initially was unable to isolate themicrobe, due to its instability. It was not until 1906 that hewas able to isolate the causal bacterium, after developing,with Octave Gengou, a medium containing potato extractand blood, which is now known as Bordet Gengou medium.3

These difficulties of isolation reflect the fragility of thebacterium. The agent, a Gram-negative bacterium, wascalled Bordetella pertussis in tribute to Bordet. A secondagent was isolated by G. Eldering in the 1930s,4 from pa-tients with pertussis-like symptoms. The disease caused

rance. Tel.: þ33 1 45 68 83 34; fax: þ33 1 40 61 35 33.

lsevier Ltd. All rights reserved.

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2 N. Guiso

by this bacterium is clinically indistinguishable from thatcaused by B. pertussis but is of shorter duration. This bac-terium was named Bordetella parapertussis.

These two microbes are difficult to isolate and identify.Indeed, only a few biochemical characters, such as oxidase,nitrate reductase, urease and citrate activities, can beused for this purpose. The identification of the two speciescan be confirmed by using specific antisera.

The principal evidence in favor of the microbe whichgrows in Bordet Gengou medium being the causal agent ofwhooping cough was (i) its agglutination by serum from aconvalescent child and (ii) the absence of agglutinationobserved with serum from an uninfected child.3 However,not all microbes are equally strongly agglutinated and thenotion of intraspecies polymorphism had already been putforward as early as 1907 by Bordet and Gengou.5

The typical disease observed in non-immune individualshas four stages: (i) a few days without symptoms aftercontact with an infected individual; (ii) a “catarrhal phase”with nonspecific rhinorrhea lasting about one week (iii) a“paroxysmal phase” of several weeks in which the patientdisplays a typical cough followed by whoops and (iv) severalweeks of less severe coughing referred to as the “conva-lescence phase”. Various complications may be associatedwith B. pertussis infection in non immune subjects,including pneumonia, broken ribs and incontinence. Beforethe advent of vaccination, the disease was considered to bea pediatric disease, because mortality, morbidity and inci-dence were highest in infants and children. Most affectedchildren caught the disease through contact with other chil-dren, at school, subsequently infecting their younger sib-lings at home. Mortality rates in children were very high,but whooping cough was rarely observed in adults, whoseimmunity was naturally boosted by contact with infectedchildren throughout their lives.

2. Development and evolution of pertussisvaccines

Two types of vaccine have been developed since thediscovery of the causal agents of the disease: a pertussiswhole-cell vaccine composed of heat-killed bacteria (Pw)and a pertussis subunit or acellular vaccine (Pa) composedof purified and detoxified components of the bacterium.6

No specific vaccine against B. parapertussis has beendeveloped.

The Pw vaccine was the first to be introduced. It was notpossible to identify, purify and study the toxins produced bythis bacterium, as had been done for the tetanus anddiphtheria toxins, due to the difficulties encountered in theculture of the bacterium. The Pw vaccine was thusdeveloped and used, and the bacteria included in thevaccine were regularly changed until the introduction offermentors, after which the strain composition of individualvaccines remained constant. The final strains chosen werethose circulating during the development of Pw vaccines.The protective efficacy of effective Pw vaccines can be ashigh as 88e90%.1 However, there are two major problemswith Pw vaccines: (i) they are difficult to produce in areproducible manner, due to the difficulties involved in B.pertussis culture: in trials carried out between 1990 and

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1995, the efficacy of some Pw vaccines was found to beas low as 30%6; (ii) they frequently induce secondary reac-tions, including local reactions, fever, febrile seizures andhypotonic episodes. These major problems led to consider-able efforts to identify and characterize the virulence de-terminants of B. pertussis and to identify the pertussisantigens responsible for inducing protective immunity.The virulence determinants of this bacterium are classifiedas adhesins and toxins. The major adhesins produced arefilamentous hemagglutinin (FHA), pertactin (PRN) and thefimbrial proteins FIM2 and FIM3. The major toxins producedare (i) the pertussis toxin (PT), an “ADP-ribosylating toxin”;(ii) the adenylate cyclase-hemolysin (AC-Hly), a “Repeats intoxin” (RTX) toxin and (iii) the tracheal cytotoxin (TCT), afragment of the bacterial peptidoglycan.7 The character-ization of these molecules led to the replacement of thewhole bacterium in the vaccine with a small number of pu-rified and detoxified antigens. These Pa vaccines are saferthan Pw vaccines. All Pa vaccines contain purified anddetoxified PT, either alone or together with FHA, FHAplus PRN, or FHA plus PRN and FIM. The efficacy of Pa vac-cines with at least two components (e.g. PT þ FHA) in pla-cebo randomized controlled studies in infants is about75e85%.6 Vaccines for children (Pa vaccines) include alarger amount of antigens than those for adolescents andadults (pa vaccines). All Pa and pa vaccines are combinedwith tetanus and diphtheria anatoxoids and, sometimes,also with poliomyelitis, Haemophilus influenzae type band hepatitis B vaccines. These Pa and pa vaccines can beused for primary vaccination but, unlike Pw vaccineswhich are reactogenic, they can also be used for boostervaccinations. However, the epidemiology, transmissionand characterization of the disease have changed sincethe introduction of generalized vaccination.

3. Epidemiology and vaccination strategy

After the development of the Pw vaccine, the vaccinationstrategy recommended in many developed countries wasprimary vaccination at three, four and five months of age,with a booster injection at two years of age. This strategyled to a considerable decrease in mortality and morbidity inregions in which vaccination coverage was high.7 However,this proved to be a honeymoon period. The Pw vaccinesused were produced by different manufacturers anddiffered considerably in efficacy (both the efficiency andeffectiveness of vaccines should be taken into accountwhen considering efficacy). Furthermore, Pw vaccinesused induced secondary effects of various severities someof which were exaggerated. Some countries, such as Swe-den decided to stop vaccination entirely. In others, vacci-nation was still recommended but the coverage decreasedsuch as in the United Kingdom, Germany and Italy and ina third group of countries, including France, the UnitedStates and Canada, vaccination coverage remained veryhigh.

A few years later, the incidence of the disease increasedsubstantially in the countries that had stopped theirvaccination programs or in which coverage had fallen,resulting in high levels of morbidity and mortality. Severalstudies were carried out, particularly in the United

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Why is Bordetella pertussis still circulating? 3

Kingdom. These studies showed that although the Pwvaccine had secondary effects, these were reversible.This and a landmark legal ruling by Lord Justice Stuart-Smith against those claiming compensation from the vac-cine manufacturer, led to a progressive return to highcoverage rates in the United Kingdom, and a decrease inthe incidence of the disease.8,9 The consequences of thesehistorical differences in vaccination coverage between re-gions are differences between their current pertussisepidemiology.10

Another epidemiological change occurred in regions withhigh vaccination coverage using effective Pw vaccines. Asudden change in disease transmission was observed 30years after the introduction of vaccination. An increase inthe number of infants admitted to hospital due to contam-ination by a sibling or their parents was observed, mostlydue to waning immunity in the immunized population.Thus, child-to-child transmission was replaced byadolescent/adult-to-infant transmission.10 A resurgence ofwhooping cough was observed following an initial decreasein morbidity and mortality in infants. Several studiesshowed that neither vaccine-induced nor natural immunityis life-long,11,12 and that whooping cough can affect individ-uals of all ages. This disease is thus not an exclusively pedi-atric problem. This pattern of transmission was notobserved before the introduction of vaccination becausemost individuals got the disease by the age of about fiveto seven years, and their immunity was subsequently regu-larly boosted by contact with infected children. Nowadays,individuals are vaccinated when they are young, but theirimmunity subsequently wanes due to the absence of con-tact with infected children. There is evidence to suggestthat the immunity induced by Pw and Pa vaccines is strongfor five to eight years, gradually declining thereafter.11

There was therefore a need to reinforce herd immunity,but with which vaccine? It was not possible to use Pw vac-cines, due to their secondary effects. Booster vaccinesonly became available with the development and releaseonto the market of Pa vaccines. In France, adolescentboosters were first introduced in 1998, the cocooning strat-egy (giving boosters to close contacts of newborn infants) in2004 and adult boosters in 2008,13 and several other Euro-pean and North American countries have now introducedadolescent and adult boosters.10,14 So what impact hasthe introduction of adolescent boosters had in France? Sur-veillance in France is hospital-based and was established in1996, for monitoring trends in whooping cough in infantsand the impact of vaccination strategies. The surveillancenetwork includes 42 pediatric hospitals and the NationalReference Center and is coordinated by the Ministry ofHealth http://www.invs.sante.fr/Dossiers-thematiques/Maladies-infectieuses/Maladies-a-prevention-vaccinale.This surveillance system has revealed that most affectedunvaccinated infants under the age of two months of ageare infected by adults. This trend was confirmed by a com-parison of the results of two studies performed 10 yearsapart, just before and 10 years after the introduction ofadolescent and adult boosters, by a network of generalpractitioners in the Parisian area. The incidence of whoop-ing cough in 2010 was 145/100,000,15 much lower than thatrecorded 10 years previously for the same area.16 Theaverage age of the contaminating contact has increased

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over the last 15 years, from 21.6 years in 1996e1998 to28.7 in 2007e2010.17 It is therefore now important to in-crease vaccination coverage in adults.

However, there is little awareness of this problem amongeither healthcare workers or the public. Training andcommunication are required, to increase vaccinationcoverage in adolescents and adults. Whooping cough is stillthought of as a childhood disease, even though it may alsoaffect adolescents and adults, who can then transmit thedisease to newborns too young to be vaccinated. Publichealth authorities also need to understand that the pro-tection of these infants involves not only vaccination of themother and the father, but also of healthcare workers,daycare workers and grandparents coming into contact withthe infant, who may also transmit the disease.

4. Whooping cough diagnosis

Whooping cough is a bacterial disease of the respiratorytract. As such, its diagnosis might be expected to be simple,but this is not the case. The major diagnostic problems are:(i) A lack of awareness of the disease. Healthcare workersand members of the public still think of whooping cough asa disease affecting only young children. It is thereforelargely under-diagnosed in adults, because this diagnosis isnot considered in older subjects. (ii) The heterogeneity ofclinical expression. The clinical signs of whooping coughdiffer to some extent between infants, older children,adolescents, and adults.18 Indeed, clinical expression of thedisease depends strongly on the level of immunity of the in-dividual concerned. It is no longer possible to diagnosewhooping cough on the basis of clinical evidence alone inregions of high vaccination coverage. Biological diagnosisis required. (iii) A lack of availability of biological diagnosisreagents and (iv) The lack of a standardized method for bio-logical diagnosis.

Biological diagnosis may be either direct, by culture,with isolation of the causal agent of the disease or bydetection of genetic material from the bacterium respon-sible by real-time PCR. Alternatively, it may be indirect, asin the measurement of anti-PT antibody levels in the serumof the patient with suspected B. pertussis infection.

If the patient has been coughing for less than threeweeks, direct diagnostic methods can be used. Culture isthe conventional gold standard method, but real-timePCR is both faster and more sensitive, and can be very use-ful for rapid diagnosis of the disease in adolescents andadults. Such diagnosis requires a nasopharyngeal aspirateor swab (Institut Pasteur video on taking nasopharyngealswabs [French] http://www.pasteur.fr/pasteur/film_cnr/prelev.swf. Accessed June 21, 2012). The use of real-timePCR has become universal and this biological diagnosismethod is increasingly replacing culture methods. Howev-er, the use of insertion sequences as multicopy targets,despite being the most sensitive diagnostic method forrapid detection of the disease, appears to be nonspecific,resulting in a high frequency of false positive results,mostly in adolescents and adults, leading to unnecessarytreatment with macrolides or assertions of vaccine fail-ure.19,20 Should sensitivity be favored over specificity? Weurgently need to address this issue.

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If the patient has been coughing for three weeks ormore, it is not usually possible to isolate the bacteria orto detect bacterial genetic material. In such cases, indirectmethods can be used. This is particularly valuable for ado-lescents and adults, who tend to consult their general prac-titioners late. ELISA with purified PT as the antigen is therecommended technique and a single high titeris taken asindicative of recent infection if the patient was not vacci-nated recently (less than one year).21,22

Nevertheless it is important to diagnose the disease asrapidly as possible, so that the patient can be treated,which should prevent transmission as the disease is highlycontagious in its early stages. B. pertussis is sensitive tomany antibiotics, but macrolides, such as clarithromycinand azithromycin, are recommended for treatment. How-ever, although antibiotic treatment may prevent transmis-sion of the bacterium to other patients, it does notmodify the course of the disease.

5. Surveillance for the causal agents ofwhooping cough

Whatever the surveillance method used, it is important toremember that the agent of the disease targeted by thevaccine must also be taken into account. Eubacteria evolveand adapt rapidly to new ecosystems and have been doingso for longer time than human beings.

As discussed above, PCR is the fastest and most sensitivemethod for whooping cough diagnosis. However, theroutine use of PCR for biological diagnosis should not stopreference laboratories from isolating the bacterium andanalyzing its evolution, for two reasons: (i) It will always beimportant to monitor antibiotic resistance. In 2012, weisolated a macrolide-resistant B. pertussis for the first timein Europe.23 Such evolution cannot easily be monitored iflaboratories do not continue to isolate bacteria. (ii) B.pertussis has always been considered a homogeneous spe-cies. However, the use of new molecular techniques andthe generalization of vaccination have revealed polymor-phism within this species.24

Bordet and Gengou first reported the polymorphism ofagglutinogen expression on the surface of the bacteriummore than a century ago.5 In 1993e1994, we demonstratedthat the isolates circulating in France were different fromthe vaccine strains.25 However, data from a national trans-mission study performed in France over the same period26

showed that vaccine efficacy was still as high as 94%. In2000, the EUPERtstrain network (Finland, France, Germany,Sweden and The Netherlands, with the addition ofDenmark, Italy, Norway, Poland and the United Kingdom afew years later) was established in Europe to analyze thepolymorphism of B. pertussis by various typing techniques.These studies showed that the level of polymorphism waslow although an analysis of clinical isolates showed that iso-lates similar to the vaccine strains were no longer circu-lating in regions of high Pw vaccination coverage.24 ThePw vaccine induced immunity that successfully controlledvaccine strain types, but not all B. pertussis types. The in-fluence of vaccine-induced immunity was confirmed by theanalysis of isolates circulating in Niakkhar, Senegal, a re-gion with low vaccination coverage, in 1991e1995.27 The

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isolates circulating in this region were different from thosecollected in France over the same period, but were similarto those circulating in France before the introduction of thevaccine.28

Since 2000, use of Pa vaccines has predominated inFrance, and vaccination coverage is increasing in adoles-cent and adult populations. Herd immunity is thereforeincreasing but is induced by Pa vaccines, which targetcertain virulence determinants of the bacterium but notthe whole bacterium. We have hypothesized that this mayfavor the circulation of isolates not expressing the vaccineantigens.29 Isolates not expressing vaccine antigens havebeen collected sporadically before and since the introduc-tion of vaccination30,31 but they do not seem to havebecome more common over time. However, seven years af-ter the introduction of Pa vaccines in France we observedthe circulation of B. pertussis isolates not expressing PRNand the prevalence of these isolates has increased since2005.30,32 Similar observations have recently been reportedin Finland and Japan.33,34

All these observations suggest that vaccine-inducedimmunity is again influencing the B. pertussis population.Furthermore, we have observed that, as reported for B.pertussis, B. parapertussis isolates currently circulating inFrance do not express PRN.30,35 This observation suggeststhat Pa vaccine-induced immunity has also affected B. par-apertussis because, in the St. Petersburg region in Russia,where a Pw vaccine is still used, the circulating isolatesof B. parapertussis still express PRN.36

6. Why is B. pertussis still circulating?

Based on current scientific knowledge, it appears that B.pertussis is still circulating because our vaccination strat-egy is no longer appropriate. This vaccine strategy hasgreatly decreased morbidity and mortality in young chil-dren, the only group within the population routinely vacci-nated, but it has recently become clear that the immunityinduced by vaccination or by natural infection is not life-long. Individuals may suffer from whooping cough severaltimes during their lives.

It is therefore important to both highlight the need forhigh coverage and timely administration of each vaccinedose in infants as recommended by WHO1 and to strengthenherd immunity by introducing pre-school, adolescent andadult booster vaccinations with Pa vaccines.

In 2011 the US Advisory Committee on ImmunizationPractices recommended that pregnant women be vacci-nated during the third trimester. The UK authorities did thesame in October 2012 faced by a rapid rise in pertussis casesand associated infant deaths that year. The transfer ofmaternally anti-PT antibodies may help to protect theinfant during the first weeks of life. However, the bluntingof infant immune responses in the presence of maternalantibodies may be of concern. Furthermore, acceptance ofimmunization of pregnant women is likely to be difficult inmany countries and for many individuals.

The cocooning strategy of (re-)immunization of closecontacts of newborns is theoretically the best recommen-dation but in practice very difficult to implement at highrates.

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Why is Bordetella pertussis still circulating? 5

7. Conclusions and new perspectives

The history of Bordetella species and pertussis vaccineshighlight the importance of ongoing surveillance of boththe disease and the microbial population for diseases thatcan be prevented by vaccination. Vaccination strategiesmay need to be adjusted regularly on the basis of epidemi-ological data and vaccine manufacture may need to bemodified in line with new scientific knowledge. There isalso a need for continuous communication with publichealth authorities, healthcare workers and the public, toprovide information about the reasons for changes in thetype of vaccine or vaccination strategy used and about ac-curate biological diagnosis and other issues.

We still have a long way to go before the circulation of B.pertussis can be considered to be under control. However,in the short term, a key priority is increasing herd immunitythrough the introduction of booster vaccinations for adoles-cents and adults. Pa and pa vaccines are safe and boostersmay be administered at any time in a subject’s life,37,38 asfor tetanus and diphtheria vaccines.

It is particularly important to increase herd immunity inseniors and to establish surveillance of the carriage of Bor-detella bronchiseptica, a known ancestor of B. pertussisand B. parapertussis.39 B. bronchiseptica can infect awide range of mammals, including humans, mostly immuno-deficient elderly individuals.40 Pa vaccines can induce aprotective immune response to B. bronchiseptica (Guiso,unpublished data).

Disease surveillance, with standardized, biological diag-nostic methods, is required. Such diagnosis must be bothsensitive and specific. The recent discovery that Bordetellaholmesii can also be detected in the respiratory tracts ofadults and adolescents, teaches us an important lesson.20

Surveillance systems should be comparable worldwide,with a uniform case definition, but this is not currently thecase.

Further studies are required to identify ways of increasingthe duration of vaccine-induced immunity. A change in theadjuvant used could be oneway forward. New antigens couldalso be incorporated into the vaccines. PT has always beenconsidered as themajor toxin produced by B. pertussis and isincluded in all Pa and pa vaccines currently on the market.However, AC-Hly has been shown to be as important as PTfor the virulence of B. pertussis41e43 and should thereforebe among the first antigens considered for the improvementof vaccine formulations.

Conflict of interest

The author has no conflict of interest to report concerningthis article. The author has participated in the past insymposia organized by Sanofi pasteur and GlaxoSmithKlineand has undertaken scientific activities with Sanofi pasteurand GlaxoSmithKline.

References

1. World Health Organisation. Pertussis vaccine: who position pa-per. Wkly Epidemiol Rec 2010;40:385e400.

Please cite this article in press as: Guiso N, Bordetella pertussis: Whyj.jinf.2013.09.022

2. Lapin JH. Whooping cough. 1 ed. Springfield, IL: Charles C.Thomas; 1943.

3. Bordet J, Gengou O. Le microbe de la coqueluche. Ann InstPasteur 1906;20:731e41.

4. Eldering G, Kendrick P. Bacillus parapertussis: a species resem-bling both Bacillus pertussis and Bacillus bronchiseptica, butidentical with neither. J Bacteriol 1938;35:561e72.

5. Bordet J, Gengou O. Note complementaire sur le microbe de lacoqueluche. Ann Inst Pasteur 1907;21:720e6.

6. Edwards KM, Decker MD. Pertussis vaccine. In: Plotkin S,Orenstein WA, Offit PA, editors. Vaccines. 4th ed. Philadelphia:Saunders; 2004. p. 471e528.

7. Mattoo S, Cherry JD. Molecular pathogenesis, epidemiology,and clinical manifestations of respiratory infections due to Bor-detella pertussis and other Bordetella subspecies. Clin Micro-biol Rev 2005;18:326e82.

8. Campbell H, Amirthalingam G, Andrews N, Fry NK, George RC,Harrison TG, et al. Accelerating control of pertussis in Englandand Wales. Emerg Infect Dis 2012;18:38e47.

9. Grimprel E, Baron S, Levy-Bruhl D, Garnier JM, Njamkepo E,Guiso N, et al. Influence of vaccination coverage on pertussistransmission in France. Lancet 1999;354:1699e700.

10. Zepp F, Heininger U, Mertsola J, Bernatowska E, Guiso N,Roord J, et al. Rationale for pertussis booster vaccinationthroughout life in Europe. Lancet Infect Dis 2011;11:557e70.

11. Wendelboe AM, Van Rie A, Salmaso S, Englund JA. Duration ofimmunity against pertussis after natural infection or vaccina-tion. Pediatr Infect Dis J 2005;24:S58e61.

12. Grimprel E, Begue P, Anjak I, Njamkepo E, Francois P, Guiso N.Long-term human serum antibody responses after immuniza-tion with whole-cell pertussis vaccine in France. Clin DiagnLab Immunol 1996;3:93e7.

13. Anonymous. Calendrier vaccinal 2008-Avis du Haut conseil dela sante publique. Bull Epidemiol Heb 2008;16e17:1e20.

14. Advisory Committee on Immunization Practices (ACIP). Updatedrecommendations for use of tetanus toxoid, reduced diphtheriatoxoid and acellular pertussis vaccine (Tdap) in pregnantwomen and persons who have or anticipate having close contactwith an infant aged <12 months. MMWR 2011;60:1424e6.

15. Lasserre A, Laurent E, Turbelin C, Hanslik T, Blanchon T,Guiso N. Pertussis incidence among adolescents and adults sur-veyed in general practices in the Paris area, France, May 2008to March 2009. Euro Surveill 2011;16 piiZ19783.

16. Gilberg S, Njamkepo E, Du Chatelet IP, Partouche H,Gueirard P, Ghasarossian C, et al. Evidence of Bordetellapertussis infection in adults presenting with persistent coughin a French area with very high whole-cell vaccine coverage.J Infect Dis 2002;186:415e8.

17. Belchior E, Bonmarin I, Guillot S, Savitch Y, Levy-Bruhl D,Guiso N, et al. Whooping cough surveillance in French hospitalRenacoq net: data from 1996 to 2010. In: 30th annual meetingof the European Society for paediatric infectious diseases.Tessaloniki, Greece 2012 [Poster session].

18. Cherry JD, Tan T, Wirsing von Konig CH, Forsyth KD,Thisyakorn U, Greenberg D, et al. Clinical definitions ofpertussis: summary of a global pertussis initiative roundtablemeeting, February 2011. Clin Infect Dis 2012;54:1756e64.

19. Miranda C, Porte L, Garcia P. Bordetella holmesii in nasopha-ryngeal samples from Chilean patients with suspected Borde-tella pertussis infection. J Clin Microbiol 2012;50:1505author reply 6.

20. Njamkepo E, Bonacorsi S, Debruyne M, Gibaud SA, Guillot S,Guiso N. Reply to Bordetella holmesii in nasopharyngeal sam-ples from Chilean patients with suspected Bordetella pertussisinfection. J Clin Microbiol 2012;50:1506.

21. Guiso N. What to do and what not to do in serological diagnosisof pertussis: recommendations from EU reference laboratories.Eur J Clin Microbiol Infect Dis 2011;30:307e12.

is it still circulating?, J Infect (2013), http://dx.doi.org/10.1016/

6 N. Guiso

22. Riffelmann M, Thiel K, Schmetz J, Wirsing von Koenig CH. Per-formance of commercial enzyme-linked immunosorbent assaysfor detection of antibodies to Bordetella pertussis. J Clin Mi-crobiol 2010;48:4459e63.

23. Guillot S, Descours G, Gillet Y, Etienne J, Floret D, Guiso N.Macrolide-resistant Bordetella pertussis infection in newborngirl, France. Emerg Infect Dis 2012;18:966e8.

24. Mooi FR, He Q, Guiso N. Phylogeny, evolution, and epidemiologyof Bordetellae. In: Locht, INSERM U629 and Institut Pasteur deLille F Camille, editor. Bordetella: molecular microbiology.Norfolk, United Kingdom: Horizon Bioscience; 2007. p. 17e45.

25. Guiso N. Isolation, identification and characterization of Bor-detella pertussis. Basel: Karger; 1997.

26. Baron S, Njamkepo E, Grimprel E, Begue P, Desenclos JC,Drucker J, et al. Epidemiology of pertussis in French hospitalsin 1993 and 1994: thirty years after a routine use of vaccina-tion. Pediatr Inf Dis J 1998;17:412e8.

27. Njamkepo E, Cantinelli T, Guigon G, Guiso N. Genomic analysisand comparison of Bordetella pertussis isolates circulating inlow and high vaccine coverage areas. Microbes Infect 2008;10:1582e6.

28. Weber C, Boursaux-Eude C, Coralie G, Caro V, Guiso N. Poly-morphism of Bordetella pertussis isolates circulating the lastten years in France, where a single effective whole-cell vac-cine has been used for more than thirty years. J Clin Microbiol2001;39:4396e403.

29. Guiso N. Bordetella pertussis and pertussis vaccines. ClinInfect Dis 2009;49:1565e9.

30. Hegerle N, Paris AS, Brun D, Dore G, Njamkepo E, Guillot S,et al. Evolution of French Bordetella pertussis and Bordetellaparapertussis isolates: increase of Bordetellae not expressingpertactin. Clin Microbiol Infect 2012;18:E340e6.

31. Stefanelli P, Fazio C, Fedele G, Spensieri F, Ausiello CM,Mastrantonio P. A natural pertactin deficient strain of Borde-tella pertussis shows improved entry in human monocyte-derived dendritic cells. New Microbiol 2009;32:159e66.

32. Bouchez V, Brun D, Cantinelli T, Dore G, Njamkepo E, Guiso N.First report and detailed characterization of B. pertussis iso-lates not expressing Pertussis Toxin or Pertactin. Vaccine2009;27:6034e41.

Please cite this article in press as: Guiso N, Bordetella pertussis: Whyj.jinf.2013.09.022

33. Barkoff AM, Mertsola J, Guillot S, Guiso N, Berbers G, He Q.Appearance of Bordetella pertussis strains not expressing vac-cine antigen pertactin in Finland. Clin Vaccine Immunol 2012;19:1703e4.

34. Otsuka N, Han HJ, Toyoizumi-Ajisaka H, Nakamura Y,Arakawa Y, Shibayama K, et al. Prevalence and genetic charac-terization of pertactin-deficient Bordetella pertussis in Japan.PLoS One 2012;7:e31985.

35. Bouchez V, Brun D, Dore G, Njamkepo E, Guiso N. Bordetellaparapertussis isolates not expressing pertactin circulating inFrance. Clin Microbiol Infect 2011;17:675e82.

36. Kurova N, Njamkepo E, Brun D, Tseneva G, Guiso N. Monitoringof Bordetella isolates circulating in Saint Petersburg, Russia be-tween 2001 and 2009. Res Microbiol 2010;161:810e5.

37. Beytout J, Launay O, Guiso N, Fiquet A, Baudin M, Richard P,et al. Safety of Tdap-IPV given one month after Td-IPV boosterin healthy young adults: a placebo- controlled trial. Hum Vac-cine 2009;5:315e21.

38. Halperin SA, Scheifele D, De Serres G, Noya F, Meekison W,Zickler P, et al. Immune responses in adults to revaccinationwith a tetanus toxoid, reduced diphtheria toxoid, and acellularpertussis vaccine 10 years after a previous dose. Vaccine 2012;30:974e82.

39. Diavatopoulos DA, Cummings CA, Schouls LM, Brinig MM,Relman DA, Mooi FR. Bordetella pertussis, the causative agentof whooping cough, evolved from a distinct, human-associatedlineage of B. bronchiseptica. PLoS Pathog 2005;1:373e83.

40. Gueirard P, Weber C, Le Coustumier A, Guiso N. Human Borde-tella bronchiseptica infection related to contact with infectedanimals: persistence of bacteria in host. J Clin Microbiol 1995;33:2002e6.

41. Betsou F, Sebo P, Guiso N. The C-terminal domain is essentialfor protective activity of the Bordetella pertussis adenylatecyclase-hemolysin. Infect Immun 1995;63:3309e15.

42. Carbonetti NH. Pertussis toxin and adenylate cyclase toxin: keyvirulence factors of Bordetella pertussis and cell biology tools.Future Microbiol 2010;5:455e69.

43. Khelef N, Zychlinsky A, Guiso N. Bordetella pertussis inducesapoptosis in macrophages: role of adenylate cyclase-hemo-lysin. Infect Immun 1993;61:4064e71.

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