Vaccine 29 (2011) Contents lists available at ScienceDirect. Vaccine. journal homepage:

Transcription

1 Vaccine 29 (2011) Contents lists available at ScienceDirect Vaccine journal homepage: Carriage of Haemophilus influenzae among Brazilian children attending day care centers in the era of widespread Hib vaccination Camila Xavier de Carvalho a, André Kipnis a, Licia Thörn b, João Guimarães de Andrade c, Fabiana Pimenta d, Maria Cristina de Cunto Brandileone e, Rosemeire Cobo Zanella e, Brendan Flannery f, Sabrina Sgambatti g, Ana Lucia Andrade h, a Laboratory of Molecular Bacteriology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, Brazil b Division of Epidemiology, Secretary of Health of Goiânia, Brazil c Department of Tropical Medicine and Dermatology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiania, Brazil d Respiratory Diseases Branch, Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States e Bacteriology Branch, Instituto Adolfo Lutz, Secretary of Health of the State of São Paulo, Brazil f Pan American Health Organization, Brasília, Brazil g Department of Medicine, Catolica University of Goiás, Goiânia, Brazil h Department of Community Health, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, Brazil article info abstract Article history: Received 5 August 2010 Received in revised form 24 November 2010 Accepted 13 December 2010 Available online 1 January 2011 Keywords: H. influenzae carriage Hib vaccination Nontypeable H. influenzae Haemophilus influenzae type b vaccine was introduced into the Immunization Program of Brazil in 1999 and no study has evaluated the impact of Hib vaccination in H. influenzae carriage so far. In June 2010, Brazil introduced the 10-valent pneumococcal nontypeable H. influenzae (NTHi) conjugate vaccine (PHiD- CV). We investigated the prevalence of encapsulated H. influenzae and NTHi isolates in nasopharyngeal samples of 1192 children attending day-care centers in Goiânia, central Brazil. H. influenzae carriage rate was 32.1% and 38.4% of them carried -lactamase TEM-1 gene. Serotype f (4.6%) was the most frequent encapsulated isolate, type b was recovered in only 0.7% and carriage rate of NTHi was 23.3%. Recurrent acute otitis media and NTHi were independently associated with colonization by -lactamase producing H. influenzae. Changes in frequency of H. influenzae carriage isolates should be carefully monitored to assess the impact of the PHiD-CV on NTHi carriage in young children Elsevier Ltd. All rights reserved. 1. Introduction Prior to the introduction of Haemophilus influenzae type b (Hib) conjugate vaccines for infants, Hib was the leading bacterial cause of meningitis among children younger than five years of age worldwide [1]. Widespread use of Hib conjugate vaccines rapidly reduced the burden of Hib meningitis and invasive Hib infections [1 3]. In many countries with routine infant immunization against Hib disease, serotypes other than type b and non-typeable (or unencapsulated) H. influenzae (NTHi) have overtaken Hib among invasive Corresponding author at: Departamento de Saúde Coletiva, Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás. Rua 235, esq. com 1 a. Avenida, Setor Leste Universitário, CEP: , Goiânia, Goiás, Brazil. Tel.: ; fax: addresses: camilamicrobiol@hotmail.com (C.X. de Carvalho), akipnis@iptsp.ufg.br (A. Kipnis), liciakamila@gmail.com (L. Thörn), jguima0@hotmail.com (J.G. de Andrade), gzy7@cdc.gov (F. Pimenta), brandi@ial.sp.gov.br (M.C.d.C. Brandileone), cobo@ial.sp.gov.br (R.C. Zanella), flanneryb@bra.ops-oms.org (B. Flannery), sasgambatti@gmail.com (S. Sgambatti), ana@iptsp.ufg.br, ana@pq.cnpq.br (A.L. Andrade). isolates [4 6]. Hib vaccination was introduced into the Brazilian Immunization Program at 2, 4, and 6 months of age, without booster dose, since July A sustained high coverage of vaccination (around 95%) has been maintained and invasive diseases due to type b sharply fell in the vaccine target population soon after the Hib vaccination [7,8]. The capsule of H. influenzae is a virulence factor that plays a role in invasiveness [9]. Unencapsulated NTHi isolates more commonly colonize the human nasopharynx and are associated with noninvasive diseases including acute otitis media (AOM) and pneumonia, although NTHi are increasingly isolated from patients with blood stream infections [5,10,11]. Colonization of the nasopharynx is likely the initial event that can lead to AOM, pneumonia and invasive infection [12]. Few studies have examined nasopharyngeal colonization with H. influenzae in the Hib vaccine era [13,14]. In 2010, Brazil became the first country to introduce into its national immunization program a novel pneumococcal conjugate vaccine containing 10 serotype-specific polysaccharides, eight of which are conjugated to protein D, a conserved membrane protein from H. influenzae (PHiD-CV) [15]. A similar vaccine containing 11 pneumococcal polysaccharides and the protein D carrier reduced X/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi: /j.vaccine

2 C.X. de Carvalho et al. / Vaccine 29 (2011) carriage of H. influenzae in 42.6% and also NTHi colonization and NTHi-associated otitis media in clinical trials [16,17]. As part of a study of nasopharyngeal colonization with Streptococcus pneumoniae conducted among children attending day-care centers (DCCs) in Goiânia, Brazil [18], we measured the prevalence of colonization with encapsulated and NTHi prior to the introduction of PHiD-CV as part of the national program. Additionally, the study examined variables associated with carriage of -lactamase positive H. influenzae. This data will provide a baseline to assess the impact of the novel 10-valent pneumococcal conjugate vaccine on carriage of H. influenzae among young children in Brazil. 2. Materials and methods 2.1. Study population Between August and December 2005, demographic data and nasopharyngeal (NP) swabs were collected from children aged 2 59 months in 62 of the 70 municipal DCCs in the municipality of Goiania (population 1,201,007), capital of the central Brazilian state of Goiás. All day-care attendees had received three doses of Hib vaccine. The number of children sampled per DCC was proportional to the number of children attending the center. We calculated that 1100 children would be necessary to estimate prevalence of H. influenzae colonization with 95% confidence, assuming 20% of NTHi carriage [14,19], with a design effect = 1.5. Socio-demographic characteristics (age of child, sex, household size, number of siblings, and participant siblings) were obtained by parent interview. Information on history of fever or hospitalization in the previous 3 months and history of AOM were also collected as proxies for antibiotic use and consequently potential risk factors for carriage of non-susceptible H. influenzae. The study protocol was approved by the Regional Ethical Committee of the Federal University of Goiás and written informed consent was obtained from each child s parents or legal representative Laboratory methods NP specimens were collected and placed into Stuart transport medium tubes (Transwabs, Medical Wire, Corsham, UK). Swabs were transferred to the Laboratory of Bacteriology of the Federal University of Goiás and processed immediately. Bacteria isolation and identification was performed according to the WHO recommendations [20]. Methods for the characterization of nasopharyngeal carriage of S. pneumoniae have been previously described [21]. For identification of H. influenzae, nasopharyngeal specimens were inoculated on chocolate agar supplemented with bacitracin (300 g/ml) and incubated in 5 7% CO 2 at 37 C for h. Gram staining was performed for those colonies with suggestive morphology of H. influenzae. Identification of H. influenzae was confirmed by the satellitism test with Staphylococcus aureus (ATCC 23922) co-culture as well as by biochemical tests (fermentation of glucose, xylose, lactose, mannose, sucrose; ornithine decarboxylase, indole production and urease test). DNA was extracted by the boiling method [22] from one colony per plate of confirmed H. influenzae cultures. Molecular identification of H. influenzae was done by amplification of the P6 gene [23], while the presence of capsule was confirmed by amplification of the bexa gene. Capsulated H. influenzae typing was performed by multiplex PCR [22]. Screening of capsule-deficient type b strains (b phenotype) was performed by PCR of the b capsule gene for all H. influenzae strains negative for the amplification of the bexa gene. This phenotype is genetically similar to type b, however b isolates do not produce PRP capsule [24]. Screening for - lactamase genes was carried out with TEM-1 and ROB-1 primers to amplify the blatem-1 and blarob-1 genes, respectively [25]. These genes are the most frequent and clinically relevant in b- lactamase production by Haemophilus [26]. Standard strains used as positive controls in PCR reactions were Hia ATCC9006, Hib ATCC33533, Hic ATCC9007, Hid ATCC9332, Hie ATCC8142 and Hif ATCC Data analysis Age was stratified into <24 months and 24 months groups. History of three episodes of AOM in 6 months or more than three episodes in 12 months was defined as recurrent AOM. Values of 95% confidence interval (95% CI) of the prevalence of H. influenzae and NHTi carriage were adjusted by DCCs. Logistic regression was used to analyze risk factors associated with carriage of - lactamase positive H. influenzae. Results were presented as odds ratios (OR) with 95% CI. All variables with p-values less than 0.10 in univariate analyses were included in a multivariate model to identify factors independently associated with carriage of H. influenzae containing the -lactamase TEM-1 gene. Significant variables were selected based on likelihood ratio tests [27]. A probability level of 0.05 (two-tailed) was used to determine statistical significance. 3. Results A total of 1276 children between 2 and 59 months of age attending 62 out of 70 public DCCs in Goiânia were recruited for swab collection. Eighty-four children were excluded from the analysis because they had taken an antibiotic in the previous 7 days. Therefore, 1192 children were available for the study. The median number of children sampled per DCC was 16.5 (range 7 50). The median age of the participants was 39 months (range 4 59), and 645 (54.1%) were male. A total of 129 children were siblings of other study participants. Overall, 80.5% of the children had taken antibiotics (76% amoxicillin) at least twice in the last six months. History of antibiotic use in the previous 3 months was more frequent among children 24 months compared to those <24 months (p = 0.003). A total of 137 (11.5%) children had attended other DCCs and 10.3% had been admitted to hospital in the previous six months. A significant association was found between fever or hospitalization in the previous three months and antibiotic use in the previous three months (p < 0.01). The annual median household income was U$2643 which is below the Brazilian poverty line Colonization with H. influenzae The prevalence of H. influenzae nasopharyngeal colonization among the 1192 day-care attendees was 32.1% (95% CI ). Colonization by DCC ranged from 20.0% to 62.5%. Encapsulated and NTHi isolates accounted for 8.8% (95% CI ) and 23.3% (95% CI ), respectively. No significant association was found between carriage rates of NTHi (p = 0.068), beta-lactamase production (p > 0.05), and different nurseries (p > 0.05) among siblings. Among encapsulated organisms, the most frequent serotype was f (4.6%), which predominated in older children. Serotype a (2.0%) was more frequent in young children, aged between 4 and 11 months. Type b was only recovered from 8 children (0.7%) (Fig. 1). Among NTHi, 17 (1.4%) of 278 were type b mutants (b ). The age distribution of capsular types showed that for all H. influenzae types the number of isolates increased with the age-group. However, isolates of type f and NTHi predominated in older children ( 36 months of age).

3 1440 C.X. de Carvalho et al. / Vaccine 29 (2011) Table 1 Clinical and demographic characteristics associated with -lactamase gene Haemophilus influenzae colonization in children attending 62 day care centers in Goiania, Brazil. Variables -Lactamase gene p value Yes = 147 a N (%) No = 236 a N (%) Fig. 1. Distribution of capsular types of Haemophilus influenzae carriage by age of children in day-care centers Factors associated with presence of ˇ-lactamase gene Of the 383 children colonized with H. influenzae, 47 (38.4%) were colonized with organisms possessing the TEM-1 -lactamase gene. The prevalence of TEM-1 was significantly higher in NTHi (43.2%) compared to the typeable strains (25.7%). No H. influenzae isolate carried the ROB-1 gene. -Lactamase positive isolates predominated in children above 11 months of age (Fig. 2). Colonization with NTHi (p = 0.001) and recurrent AOM (p = 0.029) were associated with colonization of -lactamase producing H. influenzae (Table 1). After adjustment in the multivariate model, colonization with NTHi (OR, 1.84; 95% CI, ; p = 0.002) and recurrent AOM (OR, 3.14; 95% CI ; p = 0.021) remained independently associated with the risk of -lactamase positive H. influenzae carriage. There was no interaction between recurrent AOM and NTHi (p = 0.06) Colonization with Haemophilus influenzae and Streptococcus pneumoniae Of the 383 children colonized with H. influenzae, 235 (61.4%) were co-colonized with S. pneumoniae, while 451 (78.6%) of 574 children not colonized with H. influenzae carried S. pneumoniae in their nasopharynx. Carriage of H. influenzae was negatively associated with carriage of S. pneumoniae (OR, 0.43; 95% CI ; p < 0.001). 4. Discussion In this cross-sectional study of children attending DCCs in a large Brazilian city, the overall prevalence of H. influenzae carriage was similar to or lower than that reported among children attending DCCs, living in orphanages and pre-schools students from studies conducted in Europe and North America after the introduction of Hib conjugate vaccines [14,28]. Also, the prevalence of NTHi colonization in the current study (23%) was lower than that observed in children in USA (64%) and France (40%) after Hib vaccination [14,28]. Frequent antibiotic use (mainly amoxicillin) may have reduced the overall prevalence of H. influenzae colonization, % H. influenzae organisms 100% 80% 60% 40% 20% 0% n=11 n=58 n=108 n= >=36 Child's age in months TEM-1 NEG Fig. 2. Distribution of Haemophilus influenzae carriage -lactams gene (TEM-1) according to the age of the attendees. Age <24 months 23 (15.6) 46 (19.5) Sex Male 84 (57.1) 125 (53.0) Capsular type capsulated 27 (18.4) 78 (33.1) Having recurrent AOM b 12 (8.2) 7 (3.0) Co-colonization with S. 85 (58.0) 150 (64.0) pneumoniae Previous hospitalization c 8 (5.4) 17 (7.2) Previous antibiotic c 60 (43.8) 108 (46.6) Fever c 42 (28.6) 66 (28.0) Previous day-care 18 (12.2) 21 (8.9) attendance Pneumonia c 145 (98.6) 231 (97.9) History of asthma c 8 (5.5) 10 (4.2) Previous flu-like c 60 (40.8) 91 (38.6) Mother s schooling Illiterate 0 (0.0) 2 (0.9) d Elementary 102 (69.9) 141 (61.0) High school 37 (25.3) 76 (32.9) College 7 (4.8) 12 (5.2) NTHi, nontypeable H. influenzae; DCC, day-care center; AOM, acute otitis media. a Missing values for any variable were excluded from the analysis, so the total does not always equal to 147 or 236. b Three episodes of AOM in 6 months or more than three episodes in 12 months. c In the previous 3 months. d Chi square, 3 df = 4.80 (p > 0.05). despite exclusion of 7% of the original sample of children who had received antibiotics in the previous 7 days. Seasonal differences in H. influenzae carriage may also play a role; sampling in Goiânia was conducted from August to December (corresponding to winter and spring time in the southern hemisphere), while H. influenzae and S. pneumoniae colonization in Brazil is reportedly higher during May to July, following the rainy season [29]. We could not rule out the possibility of niche replacement by non-typeable H. influenzae as a result of pneumococcal seven-valent conjugate vaccination to explain the higher rates of NTHi in countries where this vaccine has been extensively used. Another point is that carriage studies have used different methodologies. We characterized only a single H. influenzae colony recovered from each child and used PCR to identify capsular type, which may be more accurate than slide agglutination methods [30]. Carriage studies using similar methodologies are needed to compare prevalence of H. influenzae in different settings and populations. Few carriage studies have examined factors associated with reduced susceptibility of H. influenzae to -lactams. In the current study, reduced susceptibility to -lactam antibiotics was not measured directly. However, presence of the TEM-1 gene is highly predictive of -lactam resistance, with positive predictive values ranging from 94% to 100% [26,28]. Frequent exposure to antibiotics exerts selective pressure favoring -lactamase-producing H. influenzae strains. We found that history of antibiotic use was more frequent among older children. NTHi strains being more common colonizers of the nasopharynx have more antibiotic exposure and were more likely than encapsulated organisms to possess the TEM-1 gene. Plasmid-mediated production of TEM-1 or ROB- 1 -lactamase is the most common mechanism of H. influenzae resistance [31]. The global distribution of these enzymes is highly variable with ROB-1 being found almost exclusively in North America Canada, USA, Mexico [26]. In our study all H. influenzae strains that produced -lactamase were TEM-type. The clinical relevance of -lactamase-producing H. influenzae is not well known, although it was found to be associated with

4 C.X. de Carvalho et al. / Vaccine 29 (2011) recurrent acute otitis media in our study. NTHi is one of the most common causes of recurrent AOM in childhood [32 34]. The relative proportion of AOM cases caused by NTHi has increased since the introduction of the 7-valent pneumococcal conjugate vaccine in developed countries [32,35,36]. AOM is the main reason for ambulatory and emergency room visits by children in both developing and developed countries, contributing to high rates of antibiotic prescription [37,38]. -lactams, especially ampicillin, are the most frequently prescribed antibiotics for children treated as outpatients in Goiânia, where the study was conducted [33]. Therefore, - lactamase-producing NTHi would complicate the management of AOM, an association already reported in children failing first-line AOM therapy in olther studies [32,35,36]. The low rate of Hib carriage in this survey (<1%) is not a surprise, even though day-care attendees are at increased risk for H. influenzae colonization. In Brazil, Hib carriage rates before the routine vaccination ranged from 4.8% to 7.3% [39,40]. Hib vaccination coverage in Goiânia is maintained close to 100% [41]. In England, the prevalence of Hib carriage was lower than 2% in children after vaccination [42]. In USA, studies in attendees four years after vaccination detected a 46% decrease in type b Hi colonization in children who had received the conjugate vaccine [43]. Hib conjugate vaccines induce high levels of mucosal anti-hib antibody, which reduce Hib carriage in the nasopharynx as well as transmission of Hib in the community [9,44,45]. Although anti-hib antibodies are serotypespecific, reduction of Hib carriage as a result of vaccination could create an ecological niche for other H. influenzae or other bacteria [2]. However, the prevalence of nasopharyngeal colonization with encapsulated H. influenzae other than serotype b in this study was low, similar to reports from Spain, France and USA [14,19,28]. Serotype f was the most commonly identified H. influenzae type, from just 4.6% of the children attending day care. Serotype f has been associated with increased invasive infections in United States after Hib immunization [4,10,46]. Finally, we may have underestimated the prevalence of colonization with encapsulated H. influenzae by characterizing only one single colony from each child s sample culture [11]. In conclusion, this survey of H. influenzae carriage among children attending public day-care centers in a major Brazilian city provides a baseline to evaluate the effect of the PHiD-CV containing protein D from H. influenzae on NTHi carriage. Clinical trials are underway to evaluate the effectiveness of this vaccine on AOM. In the Pneumococcal Otitis Efficacy Trial (POET) with the 11-valent pneumococcal vaccine, a 35.6% protection against AOM by NTHi was observed [16]. In addition, a reduction of 41.4% on NTHi nasopharyngeal colonization in the vaccine recipients was found compared with the control group [17]. With the introduction of this novel pneumococcal vaccine into the routine childhood immunization schedule in Brazil, changes in frequency not only in pneumococcal but also in NTHi carriage and disease should be monitored. Whether the PHiD-CV will protect against NTHi carriage and disease is an exciting issue; if so, it would be an additional bonus to vaccination, since the vaccine was primarily designed to prevent pneumococcal disease. Acknowledgements This investigation was sponsored by The National Council for Scientific and Technological Development/CNPq (Research Grants no /2004-3, /2007-1) and the Secretariat of Health of Goiânia Municipality. The investigation was supported by the National Institute of Science and Technology for Health Technology Assessment/IATS (CNPq). AL Andrade (grant no /2007-8), A. Kipnis (grant no /2009-8), MC Brandileone (grant no /2007-2) and R Zanella (grant no /2007-4) are fellowships of CNPq. Transparency declaration: In the last 24 months, AL Andrade has received a research grant from Pfizer to conduct the LEAP Study (Latin American Epidemiologic Assessment of Pneumococcus), and has served as an advisor/consultant for Pfizer. References [1] Peltola H. Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates. Clin Microbiol Rev 2000;13(2): [2] Bisgard KM, Kao A, Leake J, Strebel PM, Perkins BA, Wharton M. Haemophilus influenzae invasive disease in the United States, : near disappearance of a vaccine preventable childhood disease. Emerg Infect Dis 1998;4(2): [3] Nizet V, Colina KF, Almquist JR, Rubens CE, Smith AL. A virulent nonencapsulated Haemophilus influenzae. J Infect Dis 1996;173(1): [4] Adam HJ, Richardson SE, Jamieson FB, Rawte P, Fisman DN. Changing epidemiology of invasive Haemophilus influenzae in Ontario, Canada: Evidence for herd effects and strain replacement due to Hib vaccination. Vaccine 2010;28(24): [5] Ladhani S, Slack MPE, Heath PT, von Gottberg A, Chandra M, Ramsay ME, et al. Invasive Haemophilus influenzae Disease, Europe, Emerg Infec Dis 2010;16(3): [6] Morris SK, Moss JW, Halsey N. Haemophilus influenzae type b conjugate vaccine use and effectiveness. Lancet Infec Dis 2008;8(7): [7] Simões LL, Andrade AL, Laval CA, Oliveira RM, Silva SA, Martelli CM, et al. Impact of Haemophilus influenzae b (Hib) vaccination on meningitis in Central Brazil. Rev Saude Publica 2004;38(5): [8] Miranzi Sde S, de Moraes SA, de Freitas IC. Impact of the Haemophilus influenzae type b vaccination program on HIB meningitis in Brazil. Cad Saude Publica 2007;23(7): [9] Barbour M, Phil D. Conjugate vaccines and the carriage of Haemophilus influenzae type b. Emerg Infect Dis 1996;2(3): [10] Dworkin MS, Park L, Borchardt SM. The changing epidemiology of invasive Haemophilus influenzae disease, especially in persons > or =65 years old. Clin Infect Dis 2007;44(6): [11] Faden H, Duffy L, Williams A, Krystofik DA, Wolf J. Epidemiology of nasopharyngeal colonization with nontypeable Haemophilus influenzae in the first 2 years of life. J Infect Dis 1995;172(1): [12] Harabuchi Y, Faden H, Yamanaka N, Duffy L, Wolf J, Krystofik D. Nasopharyngeal colonization with nontypeable Haemophilus influenzae and recurrent otitis media. Tonawanda/Williamsville Pediatrics. J Infect Dis 1994;170(4): [13] Peerbooms PG, Engelen MN, Stokman DA, van Benthem BH, van Weert ML, Bruisten SM, et al. Nasopharyngeal carriage of potential bacterial pathogens related to day care attendance, with special reference to the molecular epidemiology of Haemophilus influenzae. J Clin Microbiol 2002;40(8): [14] Dabernat H, Plisson-Sauné MA, Delmas C, Séguy M, Faucon G, Pélissier R, et al. Haemophilus influenzae carriage in children attending French day care centers: a molecular epidemiological study. J Clin Microbiol 2003;41(4): [15] Dagan R, Frasch C. Clinical characteristics of a novel 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine candidate (PHiD-CV). Introduction. Pediatr Infect Dis J 2009;28(4 Suppl.):S63 5. [16] Prymula R, Peeters P, Chrobok V, Kriz P, Novakova E, Kaliskova E, et al. Pneumococcal capsular polysaccharides conjugated to protein D for prevention of acute otitis media caused by both Streptococcus pneumoniae and non-typable Haemophilus influenzae: a randomised double-blind efficacy study. Lancet 2006;367(9512): [17] Prymula R, Kriz P, Kaliskova E, Pascal T, Poolman J, Schuerman L. Effect of vaccination with pneumococcal capsular polysaccharides conjugated to Haemophilus influenzae-derived protein D on nasopharyngeal carriage of Streptococcus pneumoniae and H. influenzae in children under 2 years of age. Vaccine 2009;28(1):71 8. [18] Franco CM, Andrade AL, Andrade JG, Almeida e Silva S, Oliveira RM, Pimenta FC, et al. Survey of nonsusceptible nasopharyngeal Streptococcus pneumoniae isolates in children attending day-care centers in Brazil. Pediatr Infect Dis J 2010;29(1):77 9. [19] Bou R, Dominguez A, Fontanals D, Sanfeliu I, Pons I, Renau J, et al. Prevalence of Haemophilus influenzae pharyngeal carriers in the school population of Catalonia. Working Group on invasive disease caused by Haemophilus influenzae. Eur J Epidemiol 2000;16(6): [20] World Health Organization & Centers for Disease Control and Prevention. Laboratory manual for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. Available from: pdf [accessed June 10, 2010]. [21] Ministério da Saúde do Brasil. DATASUS. Informações de Saúde. Imunizações Doses Aplicadas; Available at: exe?pni/cnv/dpnigo.def [accessed July 5, 2010]. [22] van Ketel RJ, de Wever B, van Alphen L. Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification. J Med Microbiol 1990;33(4):271 6.

5 1442 C.X. de Carvalho et al. / Vaccine 29 (2011) [23] Falla TJ, Crook DWM, Brophy LN, Maskell D, Kroll JS, Moxon ER. PCR for capsular typing of Haemophilus influenzae. J Clin Microbiol 1994;32(10): [24] St Geme 3rd JW, Falkow S. Loss of capsule expression by Haemophilus influenzae type b results in enhanced adherence to and invasion of human cells. Infect Immun 1991;59(4): [25] Tenover FC, Mohammed MJ, Gorton TS, Dembek ZF. Detection and reporting of organisms producing extended-spectrum beta-lactamases: survey of laboratories in Connecticut. J Clin Microbiol 1999;37(12): [26] Farrell DJ, Morrissey I, Bakker S, Buckridge S, Felmingham DGR. Global distribution of TEM-1 and ROB-1 b-lactamases in Haemophilus influenzae. J Antimicrob Chemother 2005;56(4): [27] Hosmer DW, Lemeshow S. Applied logistic regression. 2nd ed. Wiley, John & Sons; 2000 [incorporated, 672 pp.]. [28] Barbosa-Cesnik C, Farjo R, Patel M, Gilsdorf J, McCoy SI, Pettigrew M, et al. Predictors for Haemophilus influenzae colonization, antibiotic resistance and for sharing an identical isolate among children attending 16 licensed day-care centers in Michigan. Pediatr Infect Dis J 2006;25(3): [29] Laval CB, Andrade AL, Pimenta FC, Andrade JG, Oliveira RM, Silva SA, et al. Serotypes of carriage and invasive isolates of Streptococcus pneumoniae in Brazilian children in the era of pneumococcal vaccines. Clin Microbiol Infect 2006;12(1):50 5. [30] Bokermann S, Zanella RC, Lemos AP, de Andrade AL, Brandileone MC. Evaluation of methodology for serotyping invasive and nasopharyngeal isolates of Haemophilus influenzae in the ongoing surveillance in Brazil. J Clin Microbiol 2003;41(12): [31] Scriver SR, Walmsley SL, Kau CL, Hoban DJ, Brunton J, McGeer A, et al. Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their b-lactamases. Canadian Haemophilus Study Group. Antimicrob Agents Chemother 1994;38(7): [32] Block SL, Hedrick J, Harrison CJ, Tyler R, Smith A, Findlay R, et al. Community-wide vaccination with the heptavalent pneumococcal conjugate significantly alters the microbiology of acute otitis media. Pediatr Infect Dis J 2004;23(9): [33] Dagan R, Leibovitz E. Bacterial eradication in the treatment of otitis media. Lancet Infect Dis 2002;2(10): [34] McEllistrem MC, Adams JM, Patel K, Mendelsohn AB, Kaplan SL, Bradley JS, et al. Acute otitis media due to penicillin nonsusceptible Streptococcus pneumoniae before and after the introduction of the pneumococcal conjugate vaccine. Clin Infect Dis 2005;40(12): [35] Casey JR, Pichichero ME. Changes in frequency and pathogens causing acute otitis media in Pediatr Infect Dis J 2004;23(9): [36] Pichichero ME. Evolving shifts in otitis media pathogens: relevance to a managed care organization. Am J Manag Care 2005;11:S [37] McCaig LF, Besser RE, Hughes JM. Trends in antimicrobial prescribing rates for children and adolescents. JAMA 2002;287(23): [38] Grijalva CG, Nuorti JP, Griffin MR. Antibiotic prescription rates for acute respiratory tract infections in US ambulatory settings. JAMA 2009;302(7): [39] Forleo-Neto E, Oliveira CF, Maluf EMCP, Bataglin C, Araújo JMR, Kunz LF, et al. Decreased point prevalence of Haemophilus influenzae type b (Hib) oropharyngeal colonization by mass immunization of Brazilian children less than 5 years old with Hib polyribosylribitol phosphate polysaccharide tetanus toxoid conjugate vaccine in combination with diphtheria-tetanus toxoids pertussis vaccine. J Infect Dis 1999;180(4): [40] Bricks LF, Mendes CMF, Lucarevschi BR, Oplustil CP, Zanella RC, Bori A, et al. Oropharyngeal colonization by Haemophilus influenzae in healthy children from Taubaté (São Paulo), prior to the Haemophilus influenzae type b vaccination program in Brazil. Rev Hosp Clin Fac Med Sao Paulo 2004;59(5): [41] Ministério da Saúde do Brasil. DATASUS. Informações de Saúde. Imunizações Doses Aplicadas; Available at: exe?pni/cnv/dpnigo.def [accessed in July 5, 2010]. [42] Heath PT, Booy R, Azzopardi HJ, Slack MP, Fogarty J, Moloney AC, et al. Non-type b Haemophilus influenzae disease: clinical and epidemiologic characteristics in the Haemophilus influenzae type b vaccine era. Pediatr Infect Dis J 2001;20(3): [43] Murphy TV, Pastor P, Medley F, Osterholm MT, Granoff DM. Decreased Haemophilus colonization in children vaccinated with Haemophilus influenzae type b conjugate vaccine. J Pediatr 1993;122(4): [44] Adegbola RA, Mulholland EK, Secka O, Jaffar S, Greenwood BM. Vaccination with a Haemophilus influenzae type b conjugate vaccine reduces oropharyngeal carriage of H. influenzae type b among Gambian children. J Infect Dis 1998;177(6): [45] Rushdy A, Ramsay M, Heath PT, Azzopardi HJ, Slack MP. Infant Hib vaccination and herd immunity. J Pediatr 1999;134(2): [46] Urwin G, Krohn JA, Deaver-Robinson K, Wenger JD, Farley MM. Invasive disease due to Haemophilus influenzae serotype f: clinical and epidemiologic characteristics in the H. influenzae serotype b vaccine era. The Haemophilus influenzae Study Group. Clin Infect Dis 1996;22(6):