| Summary: | Background: Haemophilus influenzae is an important cause of serious childhood invasive disease despite the use of the vaccine against serotype b strains (Hib). Six capsular types, a-f, have been identified to date, although most of strains are non-capsulated (NC). Multilocus Sequencing Typing (MLST) is a powerful method that allows a precise and unambiguous characterization of H. influenzae genotypes. A partnership between the National Institute of Health and the Society for Paediatric Infectious Diseases aimed to characterize invasive childhood infection in Portugal. The objective of this study was to genotype isolates by MLST to follow the epidemiology of disease. Material / Methods: This study was conducted between 1 January 2010 and 31 December 2015. During this period 41 H. influenzae strains were analysed, mostly isolated from blood (88%). Antibiotic resistance was assessed by the microdilution assay and β-lactamase production was determined with nitrocefin. Capsular status was characterized by polymerase chain reaction using primers and conditions described in the literature. MLST was performed by amplifying and sequencing internal fragments of the 7 housekeeping genes (adk, atpG, frdB, fucK, mdh, pgi and recA). Sequences were analyzed and submitted to the MLST website http://pubmlst.org/hinfluenzae/ for assignment of the sequence type (ST). To display the allelic distances between the obtained STs, we applied the goeBURST algorithm implemented in the PHYLOViZ platform. Results: Antimicrobial susceptibility testing showed that most strains were susceptible to all beta-lactams studied, with only three strains being ampicillin resistant due to beta-lactamase production. Most of invasive disease was due to the presence of NC strains (27/41; 66%), while 14 isolates (34%) were capsulated and characterized as follow: two serotype a (5%), 10 b (24%) and two f (5%). As expected, MLST typing revealed high genetic variability among 27 NC isolates, which had 24 (89%) different sequence types (STs), with four new STs represented by previously unidentified allele combinations. In opposition, capsulated isolates were very clonal: all 10 Hib were assigned to CC6 (eight strains ST6, one ST 1149, one ST 190), the two Hia strains were assigned to CC 23 (ST 23) and the two Hif belonged to CC124 (ST 124 and ST 1188) (Figure 1). Conclusions: Our data indicate that invasive disease among Portuguese children is now due to highly genetically diverse, fully susceptible NC strains, suggesting that no particular virulent clone is responsible for epidemiological change of disease, after vaccine implementation in the year 2000. Nevertheless, we are concerned about Hib disease (24% of the isolates) despite the higher vaccine coverage. MLST typing continues to show a high genetic diversity among NC strains and clonal relationships among capsulated isolates. In conclusion, in order to monitor the evolving dynamics of this pathogen and the epidemiology of invasive disease, ongoing surveillance is needed to monitor the true magnitude of this problem.
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