Molecular epidemiology of invasive Haemophilus influenzae disease in Portugal: an update of the post-vaccine period, 2011–2018


Haemophilus influenzae reference laboratory from Portugal characterized the entire collection of 260 H. influenzae invasive isolates received between 2011 and 2018, with the purpose of updating the last published data (2002–2010). Capsular serotypes and antimicrobial susceptibility patterns were determined. The ftsI gene encoding the transpeptidase domain of PBP3 was sequenced for β-lactamase-negative ampicillin-resistant (BLNAR) isolates. Multilocus sequence typing (MLST) was performed to examine genetic relatedness among isolates. The majority of H. influenzae invasive isolates are nonencapsulated (NTHi-79.2%). Among encapsulated isolates (20.8%), the most characterized serotype was serotype b (13.5%), followed by serotype f (3.1%), serotype a (2.7%), and serotype e (1.5%). In contrast to NTHi that mainly affected the elderly (64.0%; ≥ 65 years old), most encapsulated isolates were characterized in preschool children (55.6%). Comparing the two periods, β-lactamase production increased from 10.4 to 13.5% (p = 0.032) and low-BLNAR (MIC ≥ 1 mg/L) isolates from 7.7 to 10.5% (p = 0.017). NTHi showed high genetic diversity (60.7%), in opposition to encapsulated isolates that were clonal within each serotype. Interestingly, ST103 and ST57 were the predominant STs among NTHi, with ST103 being associated with β-lactamase-producers and ST57 with non-β-lactamase-producers. In Portugal, susceptible and genetically diverse NTHi H. influenzae continues to be responsible for invasive disease, mainly in the elderly. Nevertheless, we are now concerned with Hib circulating in children we believe to have been vaccinated. Our data reiterates the need for continued surveillance, which will be useful in the development of public health prevention strategies.

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  1. 1.

    Watt JP, Wolfson LJ, O'Brien KL, Henkle E, Deloria-Knoll M, McCall N, Lee E, Levine OS, Hajjeh R, Mulholland K, Cherian T (2009) Burden of disease caused by Haemophilus influenzae type b in children younger than 5 years: global estimates. Lancet 374(9693):903–911

    PubMed  Google Scholar 

  2. 2.

    Ulanova M, Tsang RS (2009) Invasive Haemophilus influenzae disease: changing epidemiology and host-parasite interactions in the 21st century. Infect Genet Evol 9(4):594–605

    PubMed  Google Scholar 

  3. 3.

    Rubach MP, Bender JM, Mottice S, Hanson K, Weng HY, Korgenski K, Daly JA, Pavia AT (2011) Increasing incidence of invasive Haemophilus influenzae disease in adults, Utah, USA. Emerg Infect Dis 17(9):1645–1650

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Pittman M (1931) Variation and type specificity in the bacterial species Haemophilus influenzae. J Exp Med. 53(4):471–492

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Peltola H (2000) Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates. Clin Microbiol Rev 13(2):302–317

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    van Alphen L, Bijlmer HA (1990) Molecular epidemiology of Haemophilus influenzae type b. Pediatrics 85(4):636–642

    PubMed  Google Scholar 

  7. 7.

    McIntyre PB, O'Brien KL, Greenwood B, van de Beek D (2012) Effect of vaccines on bacterial meningitis worldwide. Lancet 380(9854):1703–1711.

    Article  PubMed  Google Scholar 

  8. 8.

    WHO-Immunization, Vaccines and Biologicals (2019) Haemophilus influenzae type b. Accessed 18 November 2019

  9. 9.

    Campos J, Hernando M, Roman F, Perez-Vazquez M, Aracil B, Oteo J, Lazaro E, de Abajo F (2004) Analysis of invasive Haemophilus influenzae infections after extensive vaccination against H. influenzae type b. J Clin Microbiol 42(2):524–529

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Dworkin MS, Park L, Borchardt SM (2007) The changing epidemiology of invasive Haemophilus influenzae disease, especially in persons ≥65 years old. Clin Infect Dis 44:810–816

    PubMed  Google Scholar 

  11. 11.

    Ladhani SN, Collins S, Vickers A, Litt DJ, Crawford C, Ramsay ME, Slack MP (2012) Invasive Haemophilus influenzae serotype e and f disease, England and Wales. Emerg Infect Dis 18(5):725–732

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Puig C, Grau I, Marti S, Tubau F, Calatayud L, Pallares R, Liñares J, Ardanuy C (2014) Clinical and molecular epidemiology of Haemophilus influenzae causing invasive disease in adult patients. PLoS One 9(11):e11271.

    CAS  Article  Google Scholar 

  13. 13.

    Butler DF, Myers AL (2018) Changing epidemiology of Haemophilus influenzae in children. Infect Dis Clin North Am 32(1):119–128

    PubMed  Google Scholar 

  14. 14.

    Giufrè M, Fabiani M, Cardines R, Riccardo F, Caporali MG, D'Ancona F, Pezzotti P, Cerquetti M (2018) Increasing trend in invasive non-typeable Haemophilus influenzae disease and molecular characterization of the isolates, Italy, 2012–2016. Vaccine 36(45):6615–6622

    PubMed  Google Scholar 

  15. 15.

    Ladhani S, Slack MP, Heath PT, von Gottberg A, Chandra M, Ramsay ME, European Union Invasive Bacterial Infection Surveillance Participants (2010) Invasive Haemophilus influenzae disease, Europe, 1996–2006. Emerg Infect Dis 16:455–463

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    ECDC- European Centre for Disease Prevention and Control (2019) Haemophilus influenzae. In: ECDC. Annual epidemiological report for 2017. ECDC, Stockholm

    Google Scholar 

  17. 17.

    Whittaker R, Economopoulou A, Dias JG, Bancroft E, Ramliden M, Celentano LP, European Centre for Disease Prevention and Control Country Experts for Invasive Haemophilus influenzae Disease (2017) Epidemiology of invasive Haemophilus influenzae disease, Europe, 2007–2014. Emerg Infect Dis 23(3):396–404

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Bajanca P, Caniça M, Multicenter Study Group (2004) Emergence of nonencapsulated and encapsulated non-b-type invasive Haemophilus influenzae isolates in Portugal (1989–2001). J Clin Microbiol 42(2):807–810

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Bajanca-Lavado MP, Simões AS, Betencourt CR, Sá-Leão R, Portuguese Group for Study of Haemophilus influenzae invasive infection (2014) Characteristics of Haemophilus influenzae invasive isolates from Portugal following routine childhood vaccination against H. influenzae serotype b (2002–2010). Eur J Clin Microbiol Infect Dis 33:603–610

    CAS  PubMed  Google Scholar 

  20. 20.

    Ubukata K, Shibasaki Y, Yamamoto K, Chiba N, Hasegawa K, Takeuchi Y, Sunakawa K, Inoue M, Konno M (2001) Association of amino acid substitutions in penicillin-binding protein 3 with beta-lactam resistance in beta-lactamase-negative ampicillin-resistant Haemophilus influenzae. Antimicrob Agents Chemother 45(6):1693–1699

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    The European Committee on Antimicrobial Susceptibility Testing. (Version 9.0, 2019) Breakpoint tables for interpretation of MICs and zone diameters: 50–54. Accessed 18 November 2019

  22. 22.

    Shuel ML, Tsang RSW (2009) Canadian β-lactamase-negative Haemophilus influenzae isolates showing decreased susceptibility toward ampicillin have significant penicillin binding protein 3 mutations. Diagn Microbiol and Infect Dis 63:379–383

    CAS  Google Scholar 

  23. 23.

    Barbosa AR, Giufrè M, Cerquetti M, Bajanca-Lavado MP (2011) Polymorphism in ftsI gene and β-lactam susceptibility in Portuguese Haemophilus influenzae strains: clonal dissemination of β-lactamase-positive isolates with decreased susceptibility to amoxicillin/clavulanic acid. J Antimicrob Chemother 66(4):788–796

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Lam TT, Claus H, Elias J, Frosch M, Vogel U (2015) Ampicillin resistance of invasive Haemophilus influenzae isolates in Germany 2009–2012. Int J Med Microbiol 305(7):748–755

    CAS  PubMed  Google Scholar 

  25. 25.

    Heinz E (2018) The return of Pfeiffer's bacillus: Rising incidence of ampicillin resistance in Haemophilus influenzae. Microb Genom 4(9).

  26. 26.

    Meats E, Feil EJ, Stringer S, Cody AJ, Goldstein R, Kroll JS, Popovic T, Spratt BG (2003) Characterization of encapsulated and noncapsulated Haemophilus influenzae and determination of phylogenetic relationships by multilocus sequence typing. J Clin Microbiol 41(4):1623–1636

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Erwin AL, Smith AL (2007) Nontypeable Haemophilus influenzae: understanding virulence and commensal behavior. Trends Microbiol 15:355–362

    CAS  PubMed  Google Scholar 

  28. 28.

    LaCross NC, Marrs CF, Gilsdorf JR (2013) Population structure in nontypeable Haemophilus influenzae. Infect Genet Evol 14:125–136

    PubMed  Google Scholar 

  29. 29.

    De Chiara M, Hood D, Muzzi A, Pickard DJ, Perkins T, Pizza M, Dougan G, Rappuoli R, Moxon ER, Soriani M, Donati C (2014) Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure. Proc Natl Acad Sci USA 111:5439–5444

    PubMed  Google Scholar 

  30. 30.

    Pinto M, González-Díaz A, Machado MP, Duarte S, Vieira L, Carriço JA, Marti S, Bajanca-Lavado MP, Gomes JP (2019) Insights into the population structure and pan-genome of Haemophilus influenzae. Infect Genet Evol 67:126–135.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Falla TJ, Crook DW, Brophy LN, Maskell D, Kroll JS, Moxon ER (1994) PCR for capsular typing of Haemophilus influenzae. J Clin Microbiol 32(10):2382–2386

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Cerquetti M, Giufrè M, Cardines R, Mastrantonio P (2007) First characterization of heterogeneous resistance to imipenem in invasive nontypeable Haemophilus influenzae isolates. Antimicrob Agents Chemother 51:3155–3161

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Kumar S, Stecher G, Tamura K (2016) Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Francisco AP, Vaz C, Monteiro PT, Melo-Cristino J, Ramirez M, Carriço JA (2012) PHYLOViZ: phylogenetic inference and data visualization for sequence based typing methods. BMC bioinformatics 13(1):87–97

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Tristram S, Jacobs MR, Appelbaum PC (2007) Antimicrobial resistance in Haemophilus influenzae. Clin Microbiol Rev 20(2):368–389

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    García-Cobos S, Campos J, Lázaro E, Román F, Cercenado E, García-Rey C, Pérez-Vázquez M, Oteo J, de Abajo F (2007) Ampicillin-resistant non-β-lactamase-producing Haemophilus influenzae in Spain: recent emergence of clonal isolates with increased resistance to cefotaxime and cefixime. Antimicrob Agents Chemother 51:2564–2573

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Dabernat H, Delmas C, Seguy M, Pelissier R, Faucon G, Bennamani S, Pasquier C (2002) Diversity of beta-lactam resistance-conferring amino acid substitutions in penicillin-binding protein 3 of Haemophilus influenzae. Antimicrob Agents Chemother 46(7):2208–2218

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Agrawal A, Murphy TF (2011) Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era. J Clin Microbiol 49(11):3728–3732

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Van Eldere J, Slack MP, Ladhani S, Cripps AW (2014) Non-typeable Haemophilus influenzae, an under-recognised pathogen. Lancet Infect Dis 14(12):1281–1292

    PubMed  Google Scholar 

  40. 40.

    Soeters HM, Blain A, Pondo T, Doman B, Farley MM, Harrison LH, Lynfield R, Miller L, Petit S, Reingold A, Schaffner W, Thomas A, Zansky SM, Wang X, Briere EC (2018) Current epidemiology and trends in invasive Haemophilus influenzae disease-United States, 2009–2015. Clin Infect Dis 67(6):881–889

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Garmendia J, Viadas C, Calatayud L, Mell JC, Martí-Lliteras P, Euba B, Llobet E, Gil C, Bengoechea JA, Redfield RJ, Liñares J (2014) Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection. PLoS ONE 9:e97020.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Charania NA, Moghadas SM (2017) Modelling the effects of booster dose vaccination schedules and recommendations for public health immunization programs: the case of Haemophilus influenzae serotype b. BMC Public Health 17(1):705.

    Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Garcia S, Lagos R, Muñoz A, Picón T, Rosa R, Alfonso A, Abriata G, Gentile A, Romanin V, Regueira M, Chiavetta L, Agudelo CI, Castañeda E, De la Hoz F, Higuera AB, Arce P, Cohen AL, Verani J, Zuber P, Gabastou JM, Pastor D, Flannery B, Andrus J (2012) Impact of vaccination against Haemophilus influenzae type b with and without a booster dose on meningitis in four South American countries. Vaccine 30(2):486–492

    PubMed  Google Scholar 

  44. 44.

    Wang S, Tafalla M, Hanssens L, Dolhain J (2017) A review of Haemophilus influenzae disease in Europe from 2000–2014: challenges, successes and the contribution of hexavalent combination vaccines. Expert Rev Vaccines 16(11):1095–1105

    CAS  PubMed  Google Scholar 

  45. 45.

    Giufrè M, Cardines R, Accogli M, Pardini M, Cerquetti M (2013) Identification of Haemophilus influenzae clones associated with invasive disease a decade after introduction of H. influenzae serotype b vaccination in Italy. Clin Vaccine Immunol 20(8):1223–1229

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    Tsang RS, Ulanova M (2017) The changing epidemiology of invasive Haemophilus influenzae disease: emergence and global presence of serotype a strains that may require a new vaccine for control. Vaccine 35(33):4270–4275

    PubMed  Google Scholar 

  47. 47.

    Bruce MG, DeByle TZ, Singleton Z, Hurlburt R, Bruden D, Wenger JD (2013) Haemophilus influenzae serotype a invasive disease, Alaska, USA, 1983–2011. Emerg Infect Dis 19(6):932–937

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Resman F, Ristovski M, Ah J, Forsgren A, Gilsdorf JR, Jasir A, Kaijser B, Kronvall G, Riesbeck K (2012) Invasive disease caused by Haemophilus influenzae in Sweden 1997–2009; evidence of increasing incidence and clinical burden of non-type b strains. Clin Microbiol Infect 17(11):1638–1645

    Google Scholar 

  49. 49.

    Giufrè M, Cardines R, Brigante G, Orecchioni F, Cerquetti M (2017) Emergence of invasive Haemophilus influenzae type a disease in Italy. Clin Infect Dis 64(11):1626–1628

    PubMed  Google Scholar 

  50. 50.

    Garcia-Cobos S, Campos J, Roman F, Carrera C, Perez-Vazquez M, Aracil B, Oteo J (2008) Low beta-lactamase-negative ampicillin-resistant Haemophilus influenzae strains are best detected by testing amoxicillin susceptibility by the broth microdilution method. Antimicrob Agents Chemother 52(7):2407–2414

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Tsang RSW, Shuel M, Whyte K, Hoang L, Tyrrell G, Horsman G, Wylie J, Jamieson J, Lefebvre B, Haldane D, Gad RR, German GJ, Needle R (2017) Antibiotic susceptibility and molecular analysis of invasive Haemophilus influenzae in Canada, 2007 to 2014. J Antimicrob Chemother 72(5):1314–1319

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Clinical and Laboratory Standards Institute (CLSI) (2011) Performance standards for antimicrobial susceptibility testing; Twenty-first informational supplement. CLSI document M100-S21. CLSI, Wayne, PA, USA

  53. 53.

    Erwin AL, Sandstedt SA, Bonthuis PJ, Geelhood JL, Nelson KL, Unrath WC, Diggle MA, Theodore MJ, Pleatman CR, Mothershed EA, Sacchi CT, Mayer LW, Gilsdorf JR, Smith AL (2008) Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing. J Bacteriol 190(4):1473–1483

    CAS  PubMed  Google Scholar 

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Members of the Portuguese Group for the Study of Haemophilus influenzae invasive infection are as follows: Ana Maria Jesus and Luísa Teixeira (Centro Hospitalar Barreiro Montijo); José Melo-Cristino, Luís Lito, Maria Dinah Carvalho, and José Gonçalo Marques (Centro Hospitalar Universitário Lisboa Norte); Margarida Pinto, Tiago Silva, and João Farela Neves (Centro Hospitalar Universitário de Lisboa Central); Filomena Martins, Maria Pessanha, and Cristina Toscano (Centro Hospitalar de Lisboa Ocidental); Henrique Oliveira and Fernanda Rodrigues (Centro Hospitalar e Universitário de Coimbra); Gina Marrão and Maria Manuel Zarcos (Centro Hospitalar de Leiria); Angélica Ramos, Manuela Ribeiro, and Artur Bonito Vitor (Centro Hospitalar de S. João, Porto); Mariana Viana and Cláudia Monteiro (Centro Hospitalar Tâmega e Sousa); Gabriela Abreu and Diana Moreira (Centro Hospitalar Vila Nova de Gaia/Espinho); Ana Paula Castro and Joana Carvalho (Centro Hospitalar de Trás-os-Montes); Hermínia Costa (Centro Hospitalar de Entre Douro e Vouga); Maria João Virtuoso (Centro Hospitalar Universitário do Algarve); Teresa Afonso, Nuno Canhoto, and Cristina Freitas (Hospital Dr. Nélio Mendonça, Funchal); Adriana Coutinho and Carla Cruz (Hospital Espírito Santo Évora); José Diogo and Filipa Nunes (Hospital Garcia de Orta, Almada); Elmano Ramalheira and Maria Manuel Flores (Hospital Infante D. Pedro, Aveiro); Luísa Sancho, Teresa Sardinha, and Paula Correia (Hospital Professor Doutor Fernando da Fonseca, Amadora); Isabel Brito and Diana Almeida (Hospital Distrital da Figueira da Foz); Alberta Faustino and Manuela Alves (Hospital de Braga); Margarida Rodrigues and Florbela Cunha (Hospital Reynaldo dos Santos, Vila Franca de Xira); Sandra Vieira and Idalina Maciel (Hospital de Santa Luzia, Viana do Castelo); Rosa Bento and Graças Seves (Hospital José Joaquim Fernandes, Beja).


This work was supported by National Institute of Health, Lisbon, Portugal.

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Correspondence to Maria Paula Bajanca-Lavado.

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Not applicable to the present study. In Portugal, invasive Haemophilus influenzae disease is of mandatory notification, where isolates depleted of personal data are sent to the Reference Laboratory for H. influenzae at the National Institute of Health for molecular characterization.

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Not applicable to the present study. This is a population-based study to define epidemiological trends, where no personal or clinical data is available.

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Heliodoro, C.I.M., Bettencourt, C.R., Bajanca-Lavado, M.P. et al. Molecular epidemiology of invasive Haemophilus influenzae disease in Portugal: an update of the post-vaccine period, 2011–2018. Eur J Clin Microbiol Infect Dis 39, 1471–1480 (2020).

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