Browsing by Author "Bajanca-Lavado, Maria Paula"
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- Alteração à epidemiologia da Infeção a Haemophilus influenzae, após a introdução da vacina para o H. influenzae serotipo b. Análise de estudos realizados no INSA, 1989-2012Publication . Bajanca-Lavado, Maria PaulaResumo: O Haemophilus influenzae (H. influenzae) é um microrganismo Gram negativo cujo nicho ecológico é o trato respiratório humano. Para além de colonizar a nasofaringe de pessoas saudáveis, o H. influenzae é normalmente responsável por infeções respiratórias e ainda infeções invasivas graves como a meningite e a septicemia, principalmente nas crianças (Tristram, 2007). As estirpes capsuladas, nomeadamente as de serotipo b (Hib) eram, até à introdução da vacina conjugada, responsáveis pela maior parte dos casos de infeção invasiva (Peltola, 2000). A vacina para o Hib foi licenciada em Portugal em 1994 e incluída no Programa Nacional de Vacinação (PNV) no ano 2000, para crianças até aos 5 anos de idade. Como consequência, as infeções invasivas de serotipo b foram praticamente eliminadas nas populações onde a vacina foi implementada (Wenger, 1998). No entanto, esta vacina não protege contra a infeção invasiva por estirpes não capsuladas (NC) ou de serotipos não b sendo, de momento, as estirpes NC as responsáveis pela maior parte destas infeções (Ladhani et al., 2010; Ulanova & Tsang, 2009). Desde 1989 que, primeiro no Laboratório de Resistência aos Antibióticos e, mais recentemente, no Laboratório Nacional de Referência de Infeções Respiratórias a agentes bacterianos, se têm realizado estudos em infeções por Haemophilus influenzae, nas várias vertentes, de vigilância epidemiológica, referência e investigação. Até ao momento contamos com uma coleção de isolados clínicos de mais de 12000 estirpes, isoladas de todas as infeções e de todas as idades, de vários Hospitais geograficamente distribuídos em Portugal. Colaboramos com a Sociedade Portuguesa de Pediatria, no que se refere à infeção invasiva na criança. Relativamente a projetos de investigação temos colaborado com alguns laboratórios de investigação, em Portugal e no Estrangeiro. É diversa a metodologia utilizada para estudar este microrganismo, recorrendo-se a diferentes técnicas para cada tipo de estudo, que vão desde técnicas fenotípicas para determinar a susceptibilidade aos antibióticos até técnicas de biologia molecular, como PCR e sequenciação para estudos genotípicos. Para além destas, e com o objetivo de estudar a clonalidade e disseminação das estirpes, utilizamos ainda duas técnicas de tipagem molecular: Pulsed-Field-Gel-Electrophoresis (PFGE) (Campos et al., 2004) e Multilocus Sequencing Type (MLST) (Meats et al, 2003). Serão apresentados resultados de alguns estudos, tanto de vigilância epidemiológica, como de investigação, realizados no nosso laboratório, e que demonstram a alteração na epidemiologia da infeção a H. influenzae após a introdução da vacina, em Portugal (Bajanca et al, 2004; Barbosa et al, 2011; Calado et al, 2011) Referências Bibliográficas: • Bajanca P, Caniça M, & the Multicenter Study Group. J Clin Microbiol 2004; 42:807-10. • Barbosa R, Giufrè M, Cerquetti M, & Bajanca-Lavado, P. J Antimicrob Chemother 2011; 66:788-96. • Calado R, Betencourt C, Gonçalves H, Cristino N, Calhau P & Bajanca-Lavado, P. Diagn Microbiol Infect Dis 2011; 69:111-13. • Campos J, Hernando M, Román F, Pérez-Vazquez M, Aracil B, Oteo J et al, J Clin Microbiol 2004; 42:524-9. • Ladhani S, Slack M, Heath P, von Gottberg A, Chandra M. Ramsay M & EUIBIS participants. Emerg Infect Dis 2010; 16:455-63. • Peltola H. Clin Microbiol Rev 2000; 13:302-17. • Tristam S, Jacobs M & Appelbaum P. Clin Microbiol Rev 2007; 20:368-89. • Ulanova M, Tsang R. Infect Genet Evol 2009; 9:594-605. • Wenger D. Pediatrr Infect Dis J 1998; 17 (Suppl.9):S132-36. • Meats E, Feil EJ, Stringer S, Cody AJ, Goldstein R, Kroll JS, et al, J Clin Microbiol 2003; 41: 1623-1636.
- Ampicillin Resistance Mechanisms in Clinical Haemophilus influenzae: What is Happening in Portugal?Publication . Bajanca-Lavado, Maria PaulaHaemophilus influenzae (Hi) remains a key etiological agent of upper and lower respiratory tract infections. Two major mechanisms are involved in ampicillin (AMP) resistance: β-lactam hydrolysis due to β-lactamase production (TEM-1 or ROB-1) and decreased affinity of penicillin-binding protein 3 (PBP3) for β-lactam antibiotics as a result of ftsI gene mutations encoding PBP3. Isolates exhibiting this latter resistant mechanism are termed β-lactamase-non-producing ampicillin-resistant (BLNAR), while isolates with both resistant mechanisms are defined as β-lactamase-positive amoxicillin-clavulanic acid-resistant (BLPACR). A variety of amino acid (AA) substitutions within the transpeptidase domain of PBP3 are mainly responsible for resistance. According to specific substitutions, these isolates have been classified in one of three mutational groups: I-III. Group II was further divided into subgroups IIa-IId. More recently, a new group was described, “III-like”, with additionally AA substitutions to the ones described in group III. Decreased ampicillin susceptibility have been associated to group I and II, while group III is normally associated with high resistance levels to ampicillin. Isolates with the non enzymatic resistance mechanisms have been described and emerging worldwide. In this context, we aimed to characterize ampicillin resistance mechanisms in clinical Hi strains isolated in Portugal. Amplification and sequencing of ftsI gene was performed in 568 clinical Hi isolates. Analysis of mutations characterized 61% of isolates as gBLNAR or gBLPACR. Most of the strains were included in group II (85%), with predominance of IIb (61%). Rare isolates were of group I and no isolate was classified in group III, although few strains were of group III-like. Our results are indicative of wide dissemination of a non-enzymatic resistance mechanism to β-lactams among H. influenzae isolates circulating in our country, probably due to inappropriate use of oral antibiotics, which is a matter of concern. A better understanding of this issue may help to establish adequate therapeutic and preventive measures to avoid selection or dissemination of such strains.
- 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.
- Caracterização da Infeção A Haemophilus Influenzae em Crianças após a Introdução da Vacina para o Haemophilus Influenzae Serótipo B no Programa Nacional de VacinaçãoPublication . Bajanca-Lavado, Maria Paula; Betencourt, Célia; Cristóvão, PaulaINTRODUÇÃO: O Haemophilus influenzae (Hi) é um microrganismo Gram negativo cujo nicho ecológico é o trato respiratório humano. É responsável por infeções respiratórias e infeções invasivas graves como a meningite e septicemia, principalmente nas crianças. O Hi apresenta seis serotipos capsulares distintos: a, b, c, d, e, f. Até ao início do seculo XXI o serótipo b (Hib) era responsável pela maior parte dos casos de infeção invasiva. Assim, no ano 2000, foi introduzida a vacina para o Hib no Programa Nacional de Vacinação, para crianças até aos 5 anos de idade. O OBJECTIVO deste estudo é caracterizar as estirpes de Hi isoladas entre 2001 e 2010, de crianças em idade pré-escolar (≤ 5 anos), dando destaque ao serotipo capsular e à resistência aos antibióticos. MATERIAL E MÉTODOS: Caracterizaram-se 1550 estirpes de Hi isoladas no período pós vacinal, de 26 Laboratórios Hospitalares em Portugal. A produção de β-lactamase foi pesquisada com nitrocefin. A determinação da concentração inibitória mínima (CIM, mg/L) foi realizada pelo método de microdiluição em placa; os “breakpoints” utilizados foram os estabelecidos pelo CLSI. Foi efetuada a pesquisa de cápsula e caracterizado o serotipo capsular por “Polymerase Chain Reaction”. RESULTADOS: As infeções mais comuns foram: infeção respiratória: 51%; conjuntivite: 29,9%; otite: 11,6% e infeção invasiva: 3,4%; A resistência à ampicilina por produção de beta-lactamase foi caracterizada em 12,6% da amostra. Entre as estirpes não produtoras de β-lactamase detetaram-se 2,9% com diminuição da susceptibilidade à ampicilina (CIM ≥2 mg/L). Estas estirpes designam-se por BLNAR (β-lactamase negativas e resistentes à ampicilina). Trimetoprim/Sulfametoxasol (SXT) foi o antibiótico que apresentou resistência (I+R) mais elevada, com 29% das estirpes. Em relação ao serotipo capsular, 98,5% das estirpes não apresentavam cápsula, sendo caracterizadas como não capsuladas (NC). CONCLUSÃO: Os nossos resultados revelam uma alteração na epidemiologia da infeção a Hi após a introdução da vacina, com um aumento de estirpes NC e a caracterização de outros serotipos, como a, d, e, f. Na resistência aos antibióticos há a salientar um aumento das estirpes BLNAR. Os resultados apresentados demonstram a importância de dar continuidade a esta monitorização através de mais e melhores estudos de vigilância.
- Caracterização da infeção a Haemophilus influenzae em crianças após a introdução da vacina para o Haemophilus influenzae serótipo b no Programa Nacional de VacinaçãoPublication . Bajanca-Lavado, Maria Paula; Betencourt, Célia; Cristóvão, Paula; Portuguese Laboratory Network for the Surveillance of Haemophilus influenzae InfectionsIntrodução: O Haemophilus influenzae (Hi) é um microrganismo Gram negativo cujo nicho ecológico é o trato respiratório humano. É responsável por infeções respiratórias e infeções invasivas graves como a meningite e septicemia, principalmente nas crianças. O Hi apresenta seis serotipos capsulares distintos: a, b, c, d, e, f. Até ao início do seculo XXI o serótipo b (Hib) era responsável pela maior parte dos casos de infeção invasiva. Assim, no ano 2000, foi introduzida a vacina para o Hib no Programa Nacional de Vacinação, para crianças até aos 5 anos de idade. O objectivo deste estudo é caracterizar as estirpes de Hi isoladas entre 2001 e 2010, de crianças em idade pré-escolar (≤ 5 anos), dando destaque ao serotipo capsular e à resistência aos antibióticos. Material e Métodos: Caracterizaram-se 1550 estirpes de Hi isoladas no período pós vacinal, de 26 Laboratórios Hospitalares em Portugal. A produção de β-lactamase foi pesquisada com nitrocefin. A determinação da concentração inibitória mínima (CIM, mg/L) foi realizada pelo método de microdiluição em placa; os “breakpoints” utilizados foram os estabelecidos pelo CLSI. Foi efetuada a pesquisa de cápsula e caracterizado o serotipo capsular por “Polymerase Chain Reaction”. Resultados: As infeções mais comuns foram: infeção respiratória: 51%; conjuntivite: 29,9%; otite: 11,6% e infeção invasiva: 3,4%; A resistência à ampicilina por produção de beta-lactamase foi caracterizada em 12,6% da amostra. Entre as estirpes não produtoras de β-lactamase detetaram-se 2,9% com diminuição da susceptibilidade à ampicilina (CIM ≥2 mg/L). Estas estirpes designam-se por BLNAR (β-lactamase negativas e resistentes à ampicilina). Trimetoprim/Sulfametoxasol (SXT) foi o antibiótico que apresentou resistência (I+R) mais elevada, com 29% das estirpes. Em relação ao serotipo capsular, 98,5% das estirpes não apresentavam cápsula, sendo caracterizadas como não capsuladas (NC). Conclusão: Os nossos resultados revelam uma alteração na epidemiologia da infeção a Hi após a introdução da vacina, com um aumento de estirpes NC e a caracterização de outros serotipos, como a, d, e, f. Na resistência aos antibióticos há a salientar um aumento das estirpes BLNAR. Os resultados apresentados demonstram a importância de dar continuidade a esta monitorização através de mais e melhores estudos de vigilância.
- Characteristics of Haemophilus influenzae invasive isolates from Portugal following routine childhood vaccination against H. influenzae serotype b (2002-2010)Publication . Bajanca-Lavado, Maria Paula; Simões, Alexandra; Betencourt, Célia; Sá-Leão, Raquel; Portuguese Group for the Study of Haemophilus influenzae invasive infectionWe 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.
- Characterization of ampicillin resistance mechanisms in clinical Haemophilus influenzae strains isolated in Portugal between 2009 and 2012Publication . Guilherme, Elsa; Bajanca-Lavado, Maria PaulaIntroduction: Haemophilus influenzae (Hi) is mainly responsible for respiratory infections and empirical therapy is used most of times. Ampicillin resistance is a problem of concern since some strains have diminished susceptibility to β-lactams through a non-enzymatic mechanism that involves decreased affinity of β-lactams for altered penicillin-binding proteins (PBPs). Strains exhibiting this resistance mechanism are referred as β-lactamase-negative ampicillin resistant (BLNAR). The aim of this study is to characterize ampicillin resistance mechanisms in clinical isolates of Hi in Portugal. Material and Methods: Two hundred and thirty-five isolates chosen according to their ampicillin MICs: 139 BLNAR (MIC≥1mg/L), 33 susceptible strains (BLNAS; MIC<1mg/L) and 63 β-lactamase producers (BLPAR) were analyzed. The ftsI gene encoding PBP3 was amplified and sequenced. MIC was determined for 13 antibiotics by a microdilution assay, according to CLSI guidelines. Results and Discussion: Of the 235 Hi isolates 199 had mutations in the ftsI transpeptidase domain as follow: 136 gBLNAR out of 139 BLNAR strains (98%) and 44 gBLPACR out of 63 BPLAR strains (70%). Of note, 19 out of 33 BLNAS (58%) presented mutations being designated as gBLNAR. Among gBLNAR and gBLPACR strains there were 43 different mutation patterns, that were included in the six previously described groups and subgroups (I, IIa, IIb, IIc, IId, III-like). The most common amino acid substitutions were located near KTG motif: N526K (160/199, 80.4%), V547I (140/199, 70.4%) and N569S (131/199, 65.8%). Strains with mutations were less susceptible to the β-lactam antibiotics studied. Comparing these results with previously ones, performed in our laboratory (between 2001 and 2008) we are assisting to an increase of susceptible strains (ampicillin MIC≤2mg/L) as well as resistant strains (beta-lactamase producers) with mutations in the ftsI gene, being so called gBLNAR and gBLPACR. CLSI breakpoints alone can’t characterize these strains as susceptible or resistant in the susceptibility tests performed routinely in the laboratory. In this way, a continuous research on breakpoints and methodologies to better define strains of this kind is of crucial importance. In conclusion, we emphasize the importance of continuing surveillance studies of this nature as essential tools to define trends in the antibiotic resistance of Hi.
- 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.
- Epidemiology of Haemophilus influenzae invasive disease in Portugal, 2011-2016Publication . Heliodoro, Catarina; Bettencourt, Célia; Bajanca-Lavado, Maria Paula; The Portuguese Group for the Study of Haemophilus influenzae invasive infectionIntroduction: Haemophilus influenzae, despite being a common commensal of the upper respiratory tract, is also an important pathogen, capable of causing severe invasive disease, in both children and adults. The epidemiology of invasive disease has changed since the introduction of Hib conjugate vaccines in 1990s, with a shift in the predominant serotype from Hib to non-capsulated H. influenzae (NTHi) and non-b serotypes. Aims: We aim to characterize H. influenzae invasive isolates recovered in Portugal, over a 6-year period (2011-2016), and compare results with previous studies. Materials and Methods: As part of a laboratory-based passive surveillance system, 174 invasive isolates, originated from 36 different Portuguese hospitals, were received at the National Reference Laboratory for Haemophilus influenzae. Capsular status was identified by PCR amplification of bexA gene and capsular type was determined by amplification of capsule-specific genes (a-f), as previously described.6 β-lactamase production was assessed with nitrocefin. Antibiotic susceptibility was determined by the microdilution assay, according to EUCAST guidelines. Genetic relatedness among the isolates was examined by MLST, by amplifying and sequencing internal fragments of the 7 housekeeping genes (adk, atpG, frdB, fucK, mdh, pgi, and recA), as previously described.7 Sequences were analysed and submitted to the MLST website (https://pubmlst.org/hinfluenzae/) for assignment of the sequence type (ST). To display the allelic distances between the obtained STs, goeBURST analysis was performed using the PHYLOViZ platform. Results: Invasive disease was mainly due to NTHi strains (143/174; 82.2%). Encapsulated strains accounted to 17.8% of the isolates (31/174) and were characterized as follows: 12.9% serotype a (4/31), 67.7% serotype b (21/31), 6.5% serotype e (2/31), and 12.9% serotype f (4/31). Most strains were susceptible to the studied antibiotics, with 12.1% (21/174) of the isolates being β-lactamase producers. MLST profiles revealed high genetic variability (71.3%), with 57 different STs among 80 NTHi isolates. In contrast, encapsulated strains were clonal; serotype b was assigned to CC6, (ST6 and ST190), serotype a to ST23, serotype e to ST18, and serotype f to ST124. Conclusion: Invasive disease in Portugal is predominantly due to susceptible, highly genetically diverse NTHi strains, although non b type strains emerged after introduction of the Hib vaccine (5.7%). We are concerned with Hib strains (12.1%) which are still circulating in our country, especially in children (57.1%). Due to its evolving dynamics, ongoing surveillance is needed, in order to monitor the burden of the disease, and develop public health prevention strategies.
- Haemophilus influenzae Carriage among Healthy Children in Portugal, 2015-2019Publication . Bajanca-Lavado, Maria Paula; Cavaco, Luís; Fernandes, Mariana; Touret, Tiago; Candeias, Catarina; Simões, Alexandra S.; Sá-Leão, RaquelHaemophilus influenzae is an important cause of mucosal and invasive infections and a common colonizer of the upper respiratory tract. As there are no recent data on H. influenzae carriage in Portugal, we aimed to characterize carriage samples and investigate possible parallelisms with disease isolates. Between 2016–2019, 1524 nasopharyngeal samples were obtained from children (0–6 years) attending day-care. H. influenzae were serotyped and screened for β-lactamase production. Strains producing β-lactamase and/or those that were encapsulated were further characterized by antibiotype; encapsulated strains were also investigated for MLST and the presence of antimicrobial resistance and virulence genes (extracted from whole genome sequencing). The overall carriage rate was 84.1%. Most isolates (96.7%) were nonencapsulated. Encapsulated strains were of serotypes f (1.8%), e (1.1%), a (0.3%), and b (0.1%). MLST showed clonality within serotypes. Although the lineages were the same as those that were described among disease isolates, colonization isolates had fewer virulence determinants. Overall, 7.5% of the isolates were β-lactamase positive; one isolate had blaTEM-82, which has not been previously described in H. influenzae. A single isolate, which was identified as H. parainfluenzae, had an incomplete f-like cap locus. In conclusion, circulation of serotype b is residual. The few encapsulated strains are genetically related to disease-causing isolates. Thus, surveillance of H. influenzae carriage should be maintained.
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