We aimed to characterize Haemophilus influenzae invasive isolates recovered in Portugal over a 9-year period (2002-2010) following the inclusion of H. influenzae serotype b (Hib) conjugate vaccination in the National Immunization Program (NIP) in the year 2000 and compare the results with those obtained in a similar study from the pre-vaccination era (1989-2001) previously described by us. As part of a laboratory-based passive surveillance system, 144 invasive isolates obtained in 28 Portuguese hospitals were received at the National Reference Laboratory for Bacterial Respiratory Infections and were characterized. Capsular types and antibiotic susceptibility patterns were determined. The ftsI gene encoding PBP3 was sequenced for β-lactamase-negative ampicillin-resistant (BLNAR) isolates. Genetic relatedness among isolates was examined by multilocus sequencing typing (MLST). Most isolates (77.1%) were non-capsulated, a significant increase compared to the pre-vaccination era (19.0%, p < 0.001). Serotype b strains decreased significantly (from 81.0 to 13.2%, p < 0.001) and serotype f increased significantly (from 0.8 to 6.9%, p = 0.03). Ten percent of the isolates were β-lactamase producers, a value lower than that previously observed (26.9%, p = 0.005). Eight percent of all isolates were BLNAR. A high genetic diversity among non-capsulated isolates was found. By contrast, capsulated isolates were clonal. The implementation of Hib vaccination has resulted in a significant decline in the proportion of serotype b H. influenzae invasive disease isolates. Most episodes of invasive disease occurring in Portugal are now due to fully susceptible, highly diverse, non-capsulated strains. Given the evolving dynamics of this pathogen and the increase in non-type b capsulated isolates, continuous surveillance is needed.

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Background: Haemophilus influenzae (Hi) is responsible for a number of human diseases ranging from chronic infections to meningitis. Most of the isolates are nonencapsulated (NC), although it is known until now six serotypes (a-f). Hi serotype b (Hib) vaccine was adopted in Portugal since 1994 and obligated since the year 2000 to children less than five years old. Our aim is to analyse the serotypes of Hi invasive strains in parallel with possible changes in antimicrobial susceptibility, before and after the introduction of vaccine.Methods: We collected 104 invasive Hi isolates (50 CSF, 52 blood cultures and 2 pleural fluids) from 1989 to 2000, and 111 (22 CSF, 81 blood cultures and 8 pleural fluids) from 2001 to 2008, from several Hospital Laboratories in Portugal. Fifty two strains (50%) were isolated from children (<18 years old) in the first period and 34 (32.7%) after the vaccination period. Identification of Hi was confirmed by standard procedures. Susceptibility testing was performed by a microdilution assay, according to the CLSI guidelines. $-lactamase production was determined with nitrocefin. Serotyping was performed by PCR.Results: In relation to ampicillin resistance, $-lactamase producing was detected in 28.8% of the strains in the period 1989-2000, and 12.6% between 2001-2008. Five strains (5.2%), all isolated in the pos vaccine period, had MIC values to ampicillin and clavulanic acid of 2 mg/L, which indicates BLNAR possible. In relation to serotype characterization, in the pre-vaccine period, 68.3% of the strains were Hib and 31.7% were NC; after the introduction of the vaccine, 12.6% were Hib, 79,3% NC, and 8.1% non-b-type (2.7% a and 5.4% f). Conclusion:Considering the two periods 1989-2000 and 2001-2008 we observe a rise in NC strains and a decline in serotype b, in invasive disease in Portugal. Non-b-type strains were characterized only after the introduction of the vaccine. In this way, we document changes in invasive infections following the introduction of the vaccine in Portugal, with an emergence of NC and an increase of serotypes other than b. We also expect a new rule in the virulence mechanisms of these strains.