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- Epidemiology and molecular characterization of invasive disease in children twenty years after the implementation of Haemophilus influenzae serotype b vaccine in Portuguese Immunization ProgrammePublication . Bajanca-Lavado, Maria Paula; Bettencout, Célia; Cunha, Florbela; Gonçalo-Marques, José; Study Group of invasive Haemophilus influenzae disease in of the Pediatric Infection Disease SocietyBackground: Haemophilus influenzae is an important human pathogen responsible for severe childhood invasive disease, despite the implementation of the vaccine against serotype b isolates (Hib), in our National Immunization Programme (NIP), in June 2000. The use of the vaccine lead to a reduction in Hib invasive disease, together with the emergence of non-encapsulated (NTHi), and capsulated non-b-type isolates. This study aims to characterize H. influenzae invasive disease in children, twenty years after the introduction of the Hib vaccine in NIP. Methods Hundred-twenty invasive H. influenzae isolates collected from children in 33 Hospitals, between January 2010 and December 2020, were characterized at the National Reference Laboratory for Haemophilus influenzae. Antibiotic susceptibility was assessed by a microdilution assay. Capsular status was identified by PCR as previously described. MLST was performed as described in the literature. Sequences were analysed and submitted to the MLST website (https://pubmlst.org/hinfluenzae/) for assignment of the sequence type (ST). goeBURST analysis was performed using the PHYLOViZ platform. Results Childhood invasive disease was mainly due to NTHi (55.8%; 67/120), although Hib still in circulation (29.2%; 35/120). Twenty-two cases of vaccine failures were responsible for 62.9% of Hib disease, with 59% of cases occurring in last four years. Non-b capsular types isolates were distributed as follow: 9.2% serotype a (11/120), 1.6% serotype e (2/120) and 4.2% serotype f (5/120). Most isolates were susceptible to all antibiotics studied, with 8.3% (10/120) being ampicillin resistant by β-lactamase producing. MLST revealed, as expected, high genetic variability (77.1%), with 37 different STs among 48 NTHi isolates. In opposition, encapsulated isolates were clonal with Hia assigned to CC23 (ST23-n=6; ST1511-n=1), Hib to CC6 (ST6-n=27, ST190, ST1149 and ST1231 with one isolate each), Hie to CC18 (ST18-n=2) and Hif to CC124 (ST124-n=2, ST1188-n=1). Conclusions Our data suggests that after vaccine implementation, invasive disease among Portuguese children is mainly due to highly genetically diverse, susceptible NTHi isolates. Nevertheless, we are concerned about Hib disease (~30%) despite the higher vaccine coverage observed in our country. Ongoing surveillance should be continued, in order to monitor the burden of the disease, especially Hib, and develop additional public health prevention strategies.
- Characterization of Haemophilus influenzae non-invasive disease in children, in Portugal: 2015-2022Publication . Duarte, Sara; Bajanca Lavado, Maria Paula; Portuguese Group for the Study of Haemophilus influenzae Invasive InfectionBackground: Haemophilus influenzae is a Gram-negative bacterium that colonizes the human upper respiratory tract, where it can remain asymptomatic. It can also progress from colonizer to pathogen and cause acute mucosal infections, such as otitis and conjunctivitis, particularly in children. These infections are frequently associated to NTHi H. influenzae. Empirical treatment with antibiotics is of concern due to possible emergence and dissemina-tion of resistant strains. Aims / Methods: We aim to characterize H. influenzae isolates from two epidemiologically relevant non-invasive diseases, otitis media and conjunctivitis, in Portugal, from 2015 to 2022 and compare this data with results from invasive disease. From January 2015 to December 2022, 134 H. influenzae isolates (78-ear-swab; 56-eye-swab) were collected in the National Reference Laboratory for Haemophilus influenzae, based at the NIH, in Lisbon. Most isolates were from children (99.5%; 132/134). Capsular status was characterized by conventional PCR. Beta-lactamase producers were identified with nitrocefin. Antimicrobial susceptibility was determined by microdilution, according to EUCAST guidelines, for several antibiotics of interest. Genetic diversity was studied by MLST and ST assigned in PubMLST (https://pubmlst.org/organisms/haemophilus-influenzae/). Results: Among 134 H. influenzae isolates, 99.3% were NTHi (133/134), whereas only one encapsulated isolate was found, and characterized as Hia (0.8%, 1/134). Beta- lactamase producers accounted for 6.7% (9/134). Antibiotic susceptibility results (n=113) showed that most isolates were susceptible to the antibiotics tested, with the exception of 34.5% resistance to trimethoprim-sulfamethoxazole (39/113). In the course of this study, we highlight the characterization of a beta-lactamase negative, NTHi isolate, resistant to ampicillin, cefotaxime, cefuroxime, amoxicillin-clavulanic acid, cefepime, and trimethoprim/sulfamethoxazole. This is the first time that we characterized resistance to cefotaxime in H. influenzae, in our country. The isolate, from a 67 years old man with multi-microorganisms corneal ulcer, was characterized as BLNAR group III-like, ST3 (confirmed by WGS). High genetic diversity was observed among NTHi, as expected, with 22 different STs assigned for 31 isolates (71% 22/31), although ST12 and ST34 included three isolates each. When comparing the MLSTs results of isolates from both invasive and non-invasive diseases, we observed that 41% (9/22) of the STs were shared among both diseases: ST-12, ST-34, ST-142, ST-160, ST-262, ST-367, ST-396, ST-1034, and ST-1411.
- Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp.Publication . Sierra, Y.; González-Díaz, A.; Tubau, F.; Carrera-Salinas, A.; Moleres, J.; Bajanca-Lavado, Maria Paula; Garmendia, J.; Domínguez, M.; Ardanuy, C.; Marti, S.Background: Several discrepancies were found in clinical routine regarding trimethoprim-sulfamethoxazole (SXT) susceptibility determination depending on antimicrobial susceptibility (AST) method used and growth media. We aimed to compare the determinants of SXT resistance with established susceptibility values for fastidious Haemophilus spp., in order to provide recommendations for optimal SXT measurement. Materials/methods: We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. SXT susceptibility was tested by microdilution, E-test, and disc diffusion using both Mueller-Hinton Fastidious (MH-F) and Haemophilus Test Medium (HTM) following EUCAST and CLSI criteria respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome sequenced isolates. Results: Strains presented generally higher rates of SXT resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related mechanisms were as follows: I95L and F154S/V in FolA; 3 and 15 base pair insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. Conclusions: Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorised as susceptible to SXT despite having resistance-related mechanisms. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the gold-standard method of microdilution.
- Characterization of Haemophilus influenzae from healthy children attending day-care centers in the Lisbon región, 2015-2019Publication . Bajanca Lavado, Maria Paula; Cavaco, Luís Filipe; Fernandes, Mariana; Touret, Tiago; Candeias, CatarinaHaemophilus influenzae colonizes the human upper respiratory tract, where it can remain asymptomatically. It can also progress from colonizer to pathogen and cause mucosal or invasive infections. The aim of this study was to unravel epidemiological aspects of H. influenzae nasopharyngeal colonization in healthy children attending day-care centers in Portugal. Methods Between 2015 and 2019, 1518 nasopharyngeal samples were collected from children up to six years old, attending day-care centers in Lisbon region. Samples were cultured in chocolate agar with bacitracin and isovitalex and screened for the presence of H. influenzae based on colony morphology. Pure cultures were obtained and capsular serotype was determined by PCR. β-lactamase production was assessed with nitrocefin. Antibiotic susceptibility was determined for all β-lactamase producer isolates and all encapsulated isolates by a microdilution assay. Genetic characterization based on whole genome sequencing (WGS) analysis was performed for encapsulated isolates. Results H. influenzae was presumptively identified in 1280 samples indicating a high carriage rate (84.3%). Of these, most isolates (96.7%) were non-encapsulated (NT). The 42 encapsulated isolates belonged to serotypes a (n=4), b (n=1), e (n=14), and f (n=23). A total of 7.5% (n=96) of the isolates were β-lactamase producers although a higher percentage (11.9%) was observed in encapsulated isolates. Most isolates were susceptible to all antimicrobials tested. MLST revealed low genetic variability among encapsulated isolates: Hia-ST23, Hib-ST6, Hie-ST18 and ST122; and Hif-ST124, ST973 and ST2346. Analysis of presence/absence of 105 virulence genes showed that this is associated with the serotype, with serotype b isolates having the highest number of virulence genes. Virulence genes associated with specific structures or functions, such as iron acquisition, adherence, biofilm development or immune evasion, were present in all isolates. Conclusion Our results show that, in children attending day-care centers in the Lisbon region, the proportion of H. influenzae carriers is high and that circulation of encapsulated isolates is rare. Characterization of circulating isolates is important for community surveillance as these isolates may progress to cause severe invasive disease.
- Haemophilus influenzae invasive disease in Portugal, 2017-2018 - what have been changing after Hib vaccine implementation?Publication . Bettencourt, Célia; Bajanca Lavado, Maria Paula; The Portuguese Group for the “Study of Haemophilus influenzae invasive infection”Introduction and Aims: Haemophilus influenzae is a frequent colonizer of the upper airways that can easily spread through respiratory tract leading to invasive infections in both children and adults. H. influenzae serotype b (Hib) has been prevented by vaccination, in Portugal, since 2000, but the emergence of non-typeable isolates (NTHi), as well as non-b serotypes responsible for invasive disease have been documented.1,2 We aim to characterize H. influenzae invasive isolates recovered in Portugal, during the last two years and compare results from previous studies. 1,2 Materials and Methods: As part of a laboratory-based surveillance system, 108 invasive H. influenzae were received at the National Reference Laboratory for Haemophilus influenzae for characterization. Capsular status was identified by PCR with primers and conditions described in the literature.3 Antibiotic susceptibility was determined by microdilution, according to EUCAST guidelines. 4 β-lactamase production was assessed with nitrocefin. Genetic relatedness among the isolates was examined by MLST as previously described.5 Sequences were analysed and submitted to the MLST website (https://pubmlst.org/hinfluenzae/) for assignment of the sequence type (ST). goeBURST analysis was performed using the PHYLOViZ platform.6 Results: Among 108 H. influenzae isolates, 90% were from blood, and 66% were from adults. Serotyping revealed that 74% were NTHi (80/108) and 26% (28/108) were capsulated isolates. Among capsulated isolates, 10.7% were Hia, 60.7% Hib, 10.7% Hie and 7.1% Hif. Most of the isolates were susceptible to the antibiotics studied, with the exception of 12.9% (14/108) ampicillin resistant by β-lactamase producing and 9.6% (9/94) that correlates with BLNAR phenotype (MIC≥1 to ampicillin and co-amoxiclave). MLST profiles revealed, as expected, high genetic variability (74.1%), with 23 different STs among 31 NTHi isolates tested. In opposition, encapsulated isolates were clonal with all Hia assigned to CC23, Hib assigned to CC6, (ST6 and ST1231), Hie to CC18, and Hif to CC124. Conclusion: After vaccine implementation and the great decline observed in Hib invasive disease (from 81% in 1989-2001 to 13% in 2002-2010),1,2 we are now concerned about Hib isolates (16%) that still in circulation in our country, affecting mostly children (88%), and accounting for 47% of vaccine failures. In addition, Hia increased, from 2% in the last period of five years (2012-2016) to 3% in this two years period (2017-2018), with all isolates from infants, up to two years old. It is important to continue the surveillance of H. influenzae invasive disease to monitor the true magnitude of these problems and develop public health prevention strategies.
- Haemophilus influenzae Serotype b Vaccines: Public Health Impact in Prevention of Invasive DiseasePublication . Bajanca Lavado, Maria PaulaWorldwide vaccination introduction in the National Immunization Programs have contributed to control and prevent a considerable number of diseases. This is one of most relevant Public Health resources in the area of prevention and control of diseases and epidemics. The human-restricted bacterium Haemophilus influenzae is responsible for respiratory infections in both children and adults. While colonization begins in the upper airways, it can spread throughout the respiratory tract potentially leading to severe invasive infections. H. influenzae is divided into two major groups: non-encapsulated (NCHi) and encapsulated isolates, being the last group further characterized into six antigenically distinct serotypes (a through f), which differ in chemical and antigenic composition of the polysaccharide capsule, the major virulence factor. H. influenzae serotype b (Hib) has been a major cause of morbidity and mortality, being responsible for more than 95% of invasive disease. Prevention of Hib disease started in the early 1980s, with the license of the “first-generation Hib vaccine” that contained the pure polysaccharide of the Hib capsule. This vaccine was poorly immunogenic in children younger than 18 months. In the mid-1980s and in 1990s, Hib conjugate vaccines (PRP-OMP and PRP-CRM) were evaluated and considered immunogenic in children younger than 2 years of age. These vaccines were licensed and routinely used to prevent Hib disease that dropped dramatically soon after its introduction. In 2006, the World Health Organization (WHO) universally recommended the implementation of Hib vaccination in all infant immunization programs worldwide, which is currently in use in 192 countries, including all EU/EEA member states. Recently, other formulations of vaccines, including the aggregation of several antigens in the same injection, lead to an ultimate formula of a hexavalent vaccine (Hepatitis B, Hib, Diphtheria, Tetanus, Pertussis and Polio) that have showed good results in contributing to decrease Hib disease, with increase of coverage and maintaining effectiveness of previous vaccine combinations. With decline of Hib disease, the emergence of infections by other serotypes as well as by NCHi have been observed, highlighting the need for the development of new vaccines such as a vaccine against NCHi disease. Several studies have been conducted, but the development of an effective vaccine against NCHi is still an ongoing subject of research.
- Spatial and temporal genomic homogeneity among Haemophilus influenzae serotype fPublication . Gonzalez-Diaz, Aida; Pinto, Miguel; Cubero, Meritxell; Langereis, Jeroen D.; van der Ende, Arie; Bajanca-Lavado, Maria Paula; Ardanuy, Carmen; Marti, SaraBackground: Haemophilus influenzae is an opportunistic pathogen highly adapted to the human respiratory tract which is often reported as the etiologic agent of infectious diseases. After the introduction of serotype b vaccine, non-typeable H. influenzae (NTHi) has become the most frequent cause of respiratory infection, followed in frequency by serotype f strains (Hif). The aim of this study was to analyze the genomic diversity among invasive and colonizing Hif isolates by whole genome sequencing (WGS).
- Rare serotype c Haemophilus influenzae invasive isolate: characterization of the first case in PortugalPublication . Bajanca Lavado, Maria Paula; Pinto, Miguel; Carvalho, Maria Dinah; Jantarada Domingos, Gonçalo; Melo-Cristino, JoséWe report for the first time in Portugal a serotype c Haemophilus influenzae isolated from an adult, with HIV-1 infection. Whole-genome sequencing characterized the isolate as clonal complex ST-7, albeit with a novel MLST (ST2754) due to a unique atpG profile. Integration of this genome with other available H. influenzae serotype c genomes from PubMLST revealed its overall genetic distinctiveness, with the closest related isolate being identified in France in 2020. This surveillance study, involving collaboration among hospitals and reference laboratory, successfully contributed to the identification and characterization of this rare serotype.