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- Doença invasiva por Haemophilus Influenzae na criança: estudo multicêntrico nacional 2010-2014Publication . Gonçalo-Marques, José; Cunha, Florbela; Bettencourt, Célia; Lavado, Paula; Grupo de Estudo da Doença Invasiva a Haemophilus influenzae na criançaIntrodução: Uma parceria Sociedade de Infecciologia Pediátrica / Instituto Nacional de Saúde Dr. Ricardo Jorge propôs-se avaliar a epidemiologia, fatores de risco, clínica, serotipos e suscetibilidade aos antibióticos da doença invasiva a Haemphilus influenzae nas crianças, em Portugal. Métodos: Estudo prospetivo descritivo multicêntrico, entre 1 de janeiro de 2010 e 30 de junho de 2014 (54 meses). Cada estirpe foi enviada ao Instituto Nacional de Saúde Dr. Ricardo Jorge acompanhada de inquérito clínico. Foi pesquisada produção de β-lactamase, resistência antibiótica, cápsula e serotipo. Resultados: Foram analisadas 38 estirpes (18 hospitais) isoladas em hemocultura (34), liquor (três) e líquido articular (uma). A incidência global foi 0,45/100000. Identificaram-se 25 (65,7%) Haemophilus influenzae não-capsulados, nove (23,7%) serotipo b (seis falências vacinais), duas (5,3%) serotipo a e duas (5,3%) serotipo f. As idades variaram entre 1 mês e 15 anos (lactentes 44,7%; 5 anos ou mais 26,3%); 23,7% tinham patologia prévia. As apresentações clínicas foram pneumonia (13), meningite (cinco), bacteriemia (cinco), infeção respiratória alta (quatro), bronquiolite (três) sépsis sem foco (três), artrite (duas), celulite periorbitária (uma), celulite (uma), epiglotite (uma). A meningite foi manifestação de 33,3% das infeções por serotipo b e de 4% das devidas a Haemophilus influenzae não-capsulados (p < 0,05). As infeções respiratórias altas bacteriémicas predominaram acima dos 5 anos (30% vs 3,6%; p < 0,05). Registaram-se sequelas neurológicas num caso (2,6%) e um óbito (2,6%). Verificou-se produção de β-lactamase em 7,9% e resistência à cefuroxima em 18,4%. Sem resistências a amoxicilina / clavulanato, cefotaxima, rifampicina. Discussão: A doença invasiva por Haemophilus influenzae atingiu diferentes grupos etários, predominando nos lactentes. As estirpes mais prevalentes foram de Haemophilus influenzae não-capsulados, causando maioritariamente bacteriemia e manifestações respiratórias. O serotipo b manteve-se em circulação sendo responsável por dois casos / ano.
- 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.
- Haemophilus influenzae invasive disease in children – preliminary results from the Portuguese Study GroupPublication . Bajanca-Lavado, Maria Paula; Betencourt, Célia; Cunha, Florbela; Gonçalo-Marques, José; Portuguese Study Group of Invasive Haemophilus influenzae Disease of The Pediatric Infectious Disease SocietyIntroduction: Haemophilus influenzae (H. influenzae) can cause life-threatening infections especially in children. Although six capsular serotypes (a-f) have been identified to date, H. influenzae serotype b (Hib) has long been a major cause of morbidity and mortality. The Hib conjugate vaccine was introduced in the Portuguese Immunization Program in June 2000 and lead to a dramatically decrease of invasive disease. The National Reference Laboratory for Bacterial Respiratory Infections, based at the National Institute of Health in Lisbon, is the reference laboratory for H. influenzae. In the beginning of 2010, the Pediatric Infectious Disease Society and our Laboratory started a surveillance study on invasive H. influenzae infections in paediatric age, with the participation of 30 Hospitals all over Portugal. Material and Methods: From January 2010 to December 2012 we received 28 strains from patients under 18 years old. Twenty-four strains were isolated from blood, three from cerebrospinal fluid, and one from a net joint fluid. Twenty two isolates (78.6%) were from pre-school children (≤5 years old). Males accounted for 78.6% of the cases. β-lactamase production was determined with nitrocefin. Minimum inhibitory concentrations (MIC) was determined for 13 antibiotics by a microdilution assay, according to CLSI guidelines. Serotyping was performed by PCR. MLST was performed for strains isolated after 2011. Results and Discussion: Serotype characterization showed that the majority of the cases (75%) were due to non-capsulated strains (NC). Of the 7 capsulated strains, five were serotype b (two vaccine failures) and two serotypes a and f respectively. Two strains were β-lactamase producers (7.1%). All other strains were susceptible to all antibiotics tested, except for trimethoprim-sulfamethoxazole with 22.2% of resistance. According to other studies MLST also revealed a great diversity among NC strains: 8 different STs in 11 strains. Comparing the results of this surveillance with our first studies, in pre-vaccine era, we are facing a change in the epidemiology of H. influenzae invasive disease with NC, fully susceptible strains, being responsible for invasive disease in Portugal.
- Haemophilus influenzae serotype b vaccine failure in Portugal: a new threat?Publication . Bajanca Lavado, Maria Paula; Bettencourt, Célia; Cunha, Florbela; Marques, José GonçaloIntroduction and Aims: Invasive disease due to Haemophilus influenzae type-b (Hib) suffered a dramatic reduction in countries that introduced routine immunization of infants with the conjugate vaccine. However, along with the relative increase of H. influenzae non-typeable invasive strains (NTHI), the emergence of non-b serotypes as well as Hib disease due to vaccine failure (VF) have been described.1- 4 The aim of our study is to identify and characterize Hib VF in children living in Portugal. Materials and Methods: From January 2010 to December 2018, 94 invasive H. influenzae strains isolated from paediatric patients in 25 Hospitals were characterized. Serotype was identified by PCR with primers and conditions described in the literature.5 Antibiotic susceptibility was determined by microdilution. Genetic relatedness was examined by MLST as previously described.6 Sequences were analysed and submitted to the MLST website (https://pubmlst.org/hinfluenzae/) for assignment of the sequence type (ST). A case of VF was considered if invasive Hib disease occurred ≥2 weeks after one Hib vaccine dose, given after the first birthday, or ≥1 week after ≥2 doses, given at <1 year of age.1 Results: Among 94 invasive H. influenzae isolates, 29 (30.8%) were Hib, with half of the cases occurring in the last two years and 72% among pre-school children. Eighteen (62%) cases were considered VF: three infants, seven between 13 and 47 months old and eight ≥4 years old. A risk factor for VF was identified only in one case. The main diagnosis were pneumonia (6), meningitis (5), epiglottitis (3) bacteremia (2), sepsis (1), and arthritis (1). One patient died. All isolates from VF cases were characterized in CC6 (ST6, ST190, ST1231) according to the expected results for Hib. In addition, WGS published data from our laboratory showed that five Hib isolates from VF, segregated together with Hib isolates (37) from both pre and pos-vaccination periods.7 All VF isolates were susceptible to ampicillin. Conclusion: Although the numbers are small, an alert is made by this study, as Hib VF seems to be increasing in previously healthy children with a clinical course that may be as severe as the observed in unvaccinated children. Also 44% of the VF occurred at an age (≥4 years-old) where invasive Hib disease was unusual before Hib-conjugate vaccination. Further analysis should be made considering vaccine formulations used, as no difference in vaccine schedule was made in the NIP, possible decline of protective antibody titers and a correlation with Hib carriage in our population.
- Multilocus sequencing typing as a tool to investigate childhood Haemophilus influenzae invasive disease in PortugalPublication . Bettencourt, Célia; Borges, Vitor; Gonçalo-Marques, José; Cunha, Florbela; Bajanca-Lavado, Maria Paula; on behalf of Portuguese Study Group of Invasive Haemophilus influenzae Disease of the Pediatric Infectious Disease SocietyBackground: 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.