Abstract

Since the introduction of Haemophilus influenzae type b (Hib) conjugate vaccines, meningitis caused by serotypes other than Hib has gained in importance. We conducted active hospital-based surveillance for meningitis over an 11-year period in Salvador, Brazil. H. influenzae isolates were serotyped and analyzed by polymerase chain reaction, pulsed-field gel electrophoresis, and DNA sequencing to identify strains with a specific deletion (IS1016) in the bexA gene (IS1016-bexA). We identified 43 meningitis cases caused by non-type b H. influenzae: 28 (65%) were caused by type a (Hia), 9 (21%) were caused by noncapsulated strains, and 3 (7%) each were caused by types e and f. Hia isolates clustered in 2 clonal groups; clonal group A strains (n = 9) had the IS1016-bexA deletion. Among children <5 years of age, meningitis caused by Hia from clonal group A had higher case-fatality than meningitis caused by clonal group B. Despite small numbers, these results indicate that the presence of the IS1016-bexA deletion is associated with enhanced virulence in non-type b H. influenzae.

Introduction of Haemophilus influenzae type b (Hib) conjugate vaccines into childhood immunization programs has dramatically reduced the incidence of Hib meningitis in countries using Hib vaccines [1–3]. Hib conjugate vaccines are highly efficacious against invasive Hib disease [4], decrease Hib carriage among vaccinated children, and reduce transmission and invasive disease among nonimmunized children [5].

Hib conjugate vaccines do not prevent H. influenzae disease caused by other serotypes, raising the potential for the emergence of H. influenzae disease due to virulent organisms with non–type b capsules [6–11]. Detection of meningitis due to non-type b H. influenzae has increased following widespread use of Hib conjugate vaccines as a result of improved surveillance and use of molecular techniques, which have reduced the serotyping errors associated with slide agglutination [12–14]. Molecular methods have also been used to identify genetic elements in invasive non-type b H. influenzae isolates, including presence of a partial deletion of IS1016 in the bexA gene commonly found in Hib isolates. The IS1016-bexA deletion is a putative virulence factor that has been identified in invasive Hia isolates from patients with severe disease in The Gambia and the United States [9, 10, 15], but not in all areas where Hia strains have been isolated [11, 16]. Acquisition of virulence factors from Hib strains could possibly lead to the emergence of non-type b H. influenzae disease.

Previously, we reported a transient increase in meningitis due to H. influenzae serotype a after introduction of Hib conjugate vaccine in Salvador, the third largest urban center in Brazil [17, 18]. Clinical outcomes of meningitis cases due to non-type b H. influenzae were similar to those of cases due to Hib [17, 18]. To investigate the role of the IS1016-bexA deletion in clinical outcomes of meningitis cases due to non-type b H. influenzae, we analyzed data from 11 years of active, hospital-based meningitis surveillance in Salvador, Brazil.

Methods

Surveillance. Meningitis is a nationally notifiable disease in Brazil, with mandatory reporting of all suspect meningitis cases to public health authorities. We conducted active surveillance for meningitis among patients admitted to Couto Maia Hospital in Salvador, Brazil. According to state health guidelines, all suspected cases of meningitis in the region are referred to Couto Maia Hospital for diagnostic procedures, including lumbar puncture and examination of cerebrospinal fluid. Couto Maia Hospital accounted for 98% of reported meningitis cases among persons from the metropolitan area of Salvador during the study period [19].

We analyzed data for H. influenzae meningitis cases identified from 9 March 1996 through 8 September 2007. A case of H. influenzae meningitis was defined as a patient who had: (1) clinical presentation of meningitis, characterized by fever, meningismus, and altered mental status; (2) abnormal cerebrospinal fluid examination; and (3) cerebrospinal fluid or blood culture positive for H. influenzae. The study team reviewed laboratory records 5 days a week to identify new culture isolations of H. influenzae. Patients were enrolled in the study according to informed consent procedures approved by the Institutional Review Boards of the Oswaldo Cruz Foundation, Brazilian Ministry of Health, and the New York–Presbyterian Hospital (New York, New York). We used a standardized data entry form to collect information on demographic characteristics, clinical presentation, laboratory results, and outcome from the patient's medical records. Number of doses of Hib conjugate vaccine received prior to hospitalization and dates of vaccination were obtained from patient immunization records.

Strain identification and serotyping. H. influenzae was identified by Gram stain morphology and growth requirement for hemin and nicotinamide adenine dinucleotide. Commercial antiserum (Becton Dickinson) was used to determine capsular serotype. Each isolate was tested for slide agglutination with the complete panel of type a-specific to type f-specific antisera (Becton Dickinson) and a saline control. A semi-nested polymerase chain reaction (PCR) method was used to amplify serotype- specific and nonspecific DNA sequences from the H. influenzae capsular loci [20]. Isolates were defined as noncapsulated if agglutination was not observed with the 6 type-specific antisera and if PCR capsular loci sequences conserved among serotypes were not detectable by PCR [20].

Pulsed-field gel electrophoresis characterization (PFGE). H. influenzae non-type b clinical isolates were examined by PFGE after digestion of bacterial DNA with Sma I (New England Biolabs), as previously described [21, 22]. The Sma I fingerprints were analyzed using GelCompar II software (Applied Maths). A 1.5%-band position tolerance was used for gel comparisons. Cluster analysis was performed using the un-weighted-pair-group method, and the relatedness between isolates was interpreted according to the criteria of Tenover [23].

Identification of the IS1016-bexA partial deletion and sequencing.H. influenzae non-type b isolates and a random sample of 20 Hib isolates were evaluated by PCR for identification of a partial deletion of the bexA gene, using the IS1016 and bexA primers as previously described [15]. For DNA sequencing, PCR products were purified with the QIAquick PCR purification kit (Qiagen) and subjected to sequence analysis. The DNA sequences from both strands were edited, assembled, and aligned using MEGA4 and BioEdit software. The sequences were compared with those of the Hib strains AF549213 [24], S62752 [25], and the type a strain DQ086152 [10], available in the NCBI Gene bank.

Multilocus sequence typing (MLST). MLST was performed for 2 H. influenzae type a isolates that were randomly selected among the isolates which had and did not have the IS1016- bexA deletion. Chromosomal DNA was extracted using a Qiagen genomic Kit (Qiagen). PCR was used to amplify 450-base pair (bp) internal fragments of 7 housekeeping genes (adk, atpG, frdB, fucK, mdh, pgi, and recA), according to previously described methods [26]. Sequences were submitted to the online MLST database (http://www.mlst.net), which in turn assigned alleles at each locus and a sequence type.

Statistical analysis. Data were entered and analyzed using EpiInfo software, version 3.3.2 (Centers for Disease Control and Prevention). Fisher's exact test and the Wilcoxon rank-sum test were used for comparison of proportions and continuous data, respectively. A significant difference was defined by a 2-tailed P-value < .05.

Mean annual incidence of H. influenzae meningitis was compared for the period prior to introduction of Hib vaccination (March 1996 through July 1999) and after Hib vaccine introduction (August 1999 through September 2007). Incidence was calculated for the metropolitan area of Salvador by dividing the number of cases among residents of metropolitan Salvador by the estimated population from the 2000 national census [27].

Results

During the study period, we identified 615 cases of H. influenzae meningitis. Among the 573 cases (93%) for which an isolate was serotyped, 43 episodes (8%) were caused by H. influenzae non-type b strains (Table 1). The majority of H. influenzae non-type b isolates were type a (28 isolates; 65%), followed by noncapsulated (9 isolates; 21%), type e (3 isolates; 7%), and type f (3 isolates; 7%). The proportion of H. influenzae meningitis cases caused by a non-type b isolate increased from 2% (8 of 424) to 23% (35 of 149) after the introduction of routine Hib immunization (P < .001). This increase was largely explained by the 91% reduction in the incidence of Hib meningitis between the pre- and post-vaccine periods (from 2.45 to 0.24 cases per 100,000 population; P < .001). The incidence of meningitis due to non-type b H. influenzae increased after the introduction of the Hib conjugate vaccine, mainly because of an increase in disease due to Hia. Meningitis cases due to Hia did not cluster spatially with respect to the neighborhood of residence during pre- and postvaccine periods.

Table 1

Cases and Incidences of Haemophilus influenzae Meningitis in Salvador, Brazil, according to Period of Identification and Serotype

Table 1

Cases and Incidences of Haemophilus influenzae Meningitis in Salvador, Brazil, according to Period of Identification and Serotype

Hia and Hib meningitis occurred mainly among children <5 years of age, whereas meningitis due to H. influenzae types e, f, and noncapsulated strains occurred in older age groups (Table 2). Case-fatality was also higher for Hia and Hib meningitis cases than it was for meningitis cases due to other serotypes (Table 2). The age group distribution and case fatality rate for H. influenzae type a cases did not differ between the pre- and postvaccine period.

Table 2

Characteristics of Haemophilus influenzae Meningitis Cases from Salvador, Brazil, according to Serotype

Table 2

Characteristics of Haemophilus influenzae Meningitis Cases from Salvador, Brazil, according to Serotype

We were able to obtain information on immunization status for 26 (74%) of the 35 meningitis cases due to non-type b H. influenzae identified in the postvaccine period. Although 75% (13 of 17) of the patients with cases due to H. influenzae type a isolates had received 2 or 3 Hib vaccine doses, only 11% (1 of 9) of the cases due to H. influenzae type e, f, and noncapsulated isolates received the same number of Hib vaccine doses (P < .01).

PFGE analysis for the 43 H. influenzae non-type b isolates discriminated 15 distinct patterns (Figure 1). The 28 H. influenzae type a isolates had 2 different patterns, cluster A (9 isolates), and cluster B (19 isolates), whereas H. influenzae types e, f, and noncapsulated strains were heterogeneous (Figure 1). MLST analysis determined that PFGE clusters A and B corresponded to sequence type (ST) 4 and 23, respectively. PCR analysis identified the 339-bp IS1016-bexA partial deletion product in 9 of the 43 H. influenzae non-type b isolates. All of the 9 H. influenzae isolates containing the IS1016-bexA deletion were serotype a and belonged to PFGE cluster A (ST4). Among the 28 Hia isolates, 5 and 23 were isolated during the pre- and postvaccine periods, respectively. The proportion of Hia isolates with the IS1016-bexA deletion was 40% (2 of 5) and 30% (7 of 23) in the pre- and postvaccine periods, respectively, and this difference was not statistically significant (P = .65).

Figure 1

Dendrogram showing the genetic relationships among the 43 non-type b Haemophilus influenzae (Hib) isolates obtained from meningitis cases in Salvador, Brazil, as determined by pulsed-field gel electrophoresis (PFGE). The columns, from left to right, show the isolate identification number, serotype, period of isolation in relation to introduction of Hib conjugate vaccine, the PFGE pattern designation, and presence of the IS1016bexA partial deletion. NC, noncapsulated.

Figure 1

Dendrogram showing the genetic relationships among the 43 non-type b Haemophilus influenzae (Hib) isolates obtained from meningitis cases in Salvador, Brazil, as determined by pulsed-field gel electrophoresis (PFGE). The columns, from left to right, show the isolate identification number, serotype, period of isolation in relation to introduction of Hib conjugate vaccine, the PFGE pattern designation, and presence of the IS1016bexA partial deletion. NC, noncapsulated.

Patients with meningitis cases caused by Hia strains belonging to cluster A or B were similar with respect to sex, age, and characteristics of cerebrospinal fluid (Table 3). However, the case-fatality rate for patients with meningitis caused by Hia strains that had the IS1016-bexA deletion was 33% (3 of 9 patients died), compared with 5% (1 of 19) for patients with cases caused by Hia strains with complete IS1016-bexA (P = .06) (Table 3). Among children <5 years of age with H. influenzae type a meningitis, 38% (3 of 8) of individuals with isolates that contained the IS1016-bexA deletion died, whereas none of the 16 patients with isolates that did not contain the IS1016-bexA deletion died (P = .03) (Table 3).

Table 3

Characteristics for the Haemophilus influenzae Type a Meningitis Cases Identified through Active Surveillance in Salvador, Brazil, according to the Presence on the Isolate of the IS1016-bexA Partial Gene Deletion

Table 3

Characteristics for the Haemophilus influenzae Type a Meningitis Cases Identified through Active Surveillance in Salvador, Brazil, according to the Presence on the Isolate of the IS1016-bexA Partial Gene Deletion

Sequencing of the PCR products confirmed the presence of an IS1016-bexA deletion in the 9 H. influenzae type a ST4 isolates. The size and location of the deletion, as well as the flanking region sequences, was identical to that previously reported for an invasive serotype a strain that was isolated in Georgia in 2005, except for 1 nucleotide in position 98 (GenBank accession number DQ086152) (Figure 2). However, the sequence of the regions flanking the IS1016-bexA deletion for the ST4 isolates differed at 4 nucleotide sites from corresponding sequences for 2 previously reported Hib strains (GenBank accession numbers AF549213 [HI 1007-Georgia] and S62752 [RM 7004-Gambia]) and 3 of 4 Hib stains isolated during surveillance in Salvador. One Hib strain isolated from an individual in Salvador had a flanking region sequence that differed at only 1 nucleotide from the corresponding sequence in serotype a ST4 isolates (Figure 2).

Figure 2

Nucleotide sequence of the IS1016-bexA deletion region of Haemophilus influenzae type b and type a strains. The arrowhead identifies the site of the deletion (base pair 66) with the sequence of the bexA gene and IS1016 denoted in capital and lower case letter, respectively. The consensus sequence was obtained from the H. influenzae type b isolates (GenBank AF549213) from the United States and The Gambia (GenBank S62752) and compared with an H. influenzae type a isolate from the United States (GenBank DQ086152) and 4 H. influenzae type b (H503, H636, H637, and H492) and 9 H. influenzae type a (H493, H532, H235, H417, H481, H607, H614, H628, and H638) isolates from Salvador, Brazil. Dots indicate identical nucleotides to the consensus sequence. The box denotes nucleotides specific to H. influenzae type a.

Figure 2

Nucleotide sequence of the IS1016-bexA deletion region of Haemophilus influenzae type b and type a strains. The arrowhead identifies the site of the deletion (base pair 66) with the sequence of the bexA gene and IS1016 denoted in capital and lower case letter, respectively. The consensus sequence was obtained from the H. influenzae type b isolates (GenBank AF549213) from the United States and The Gambia (GenBank S62752) and compared with an H. influenzae type a isolate from the United States (GenBank DQ086152) and 4 H. influenzae type b (H503, H636, H637, and H492) and 9 H. influenzae type a (H493, H532, H235, H417, H481, H607, H614, H628, and H638) isolates from Salvador, Brazil. Dots indicate identical nucleotides to the consensus sequence. The box denotes nucleotides specific to H. influenzae type a.

Discussion

Widespread use of Hib conjugate vaccines has substantially reduced the incidence of Hib meningitis [1–3, 28, 29], resulting in increased awareness of meningitis due to other H. influenzae serotypes [7, 8, 11]. Because Hib conjugate vaccines are effective in reducing Hib nasopharyngeal carriage [30, 31], it was hypothesized that non-type b strains could potentially occupy the niche left by Hib and consequently increase the risk of invasive disease caused by non-type b strains. To date, however, there has been little evidence of a substantial replacement of Hib disease by disease caused by other serotypes, a phenomenon known as serotype replacement [17, 32].

Among the capsulated H. influenzae strains that are not type b, type a has the capsular polysaccharides most closely related to those of type b. In animal challenge studies, reports have found that Hia is the most virulent capsulated H. influenzae after Hib [33]. The H. influenzae type a meningitis cases from this study occurred among similar age groups and had case-fatality rates similar to those for Hib meningitis. In contrast, cases due to serotype e, serotype f, and noncapsulated serotypes occurred at older ages and tended to have a better prognosis. These findings are consistent with prior clinical and epidemiological characterizations of invasive disease due to H. influenzae non-type b and support the hypothesis that type a isolates are the most virulent capsulated H. influenzae serotype after type b [33]. Although H. influenzae type a invasive infections typically occur in healthy children [9, 10, 12, 16, 34], infections due to serotype e, serotype f, and noncapsulated serotypes mostly occur among adults with underlying conditions, such as cancer [8, 35, 36].

In this study, we found that patients with H. influenzae type a meningitis had an increased risk of death when the IS1016-bexA partial deletion was present in the clinical isolate. The association did not appear to be confounded by other prognostic factors, such as patients’ age and disease duration prior to hospitalization. This finding is both plausible and analogous with what is known about virulence factors for Hib, for which the IS1016-bexA deletion stabilizes duplicated loci and leads to increased production of capsular polysaccharide [25, 33]. Hib capsular loci amplification has been found to inhibit complement-mediated bacteriolysis and opsonization [37]. Capsular amplification and the IS1016-bexA deletion have been identified in Hia invasive isolates [9, 10, 12, 15]. However, this study provides the first evidence, to our knowledge, for the significant association between the IS1016-bexA deletion and poor clinical outcome from Hia invasive disease.

However, the IS1016-bexA partial deletion was present in a minority of the H. influenzae type a isolates (9 of 28 isolates). Other investigations have also identified isolates of H. influenzae type a causing invasive infections resembling Hib invasive disease in the absence of the IS1016-bexA partial deletion [11, 16]. Additional studies in other geographical settings and with larger sample sizes are warranted to confirm the role of the IS1016-bexA deletion as a virulence factor in H. influenzae type a invasive disease. Furthermore, we did not evaluate whether the presence of the IS1016-bexA deletion was associated with neurological sequelae, hearing impairment, or other markers of disease severity. Finally, further studies are needed to determine whether clinical isolates with the IS1016-bexA deletion exhibit enhanced virulence in animal models for Hia infection.

Results of this study suggest that Hia strains causing meningitis in Salvador have been stable over time. Sequence type 23 has been isolated in Malaysia, Canada, and New Guinea [11, 26], which suggests worldwide spread of these clones. Interestingly, sequence type 4, previously isolated in Kenya and The Gambia [26], was the first non-type b strain identified as having the IS1016-bexA partial deletion [15]. In addition, Sill et al [38] described a Canadian case of H. influenzae type a invasive disease due to an ST4 strain containing the IS1016-bexA partial deletion [38]. This isolate was closely related on the basis of PFGE analysis to 2 Hia strains possessing the IS10116-bexA deletion that were isolated from patients with invasive disease in Georgia [10]. Future studies are needed to investigate whether the ST4 clone is entirely responsible for the global spread of H. influenzae type a strains containing the IS1016-bexA partial deletion. These findings highlight the need to continue surveillance for H. influenzae invasive disease to monitor for the potential emergence of non-type b H. influenzae virulent clones.

Acknowledgments

We thank the study patients and their families; the clinical, laboratory and administrative staff of Hospital Couto Maia, especially Ana MariaMaia and Neide Oliveira Silvap; Ricardo Martinez and Tatiana Lobo, for their participation in data collection and processing; Neci Ivo Ramos, Nilda Lúcia Nunes Ivo, Maria Auxiliadora Macedo de Lima Machado, and Helena Macedo, for providing information on the Hib immunization programand meningitis case notifications; Hermes P. da Silva Filho, for support on the sequence analysis; and Brendan Flannery, for manuscript review.

References

1
Adegbola
RA
Secka
O
Lahai
G
, et al.  . 
Elimination of Haemophilus influenzae type b (Hib) disease from The Gambia after the introduction of routine immunisation with a Hib conjugate vaccine: a prospective study
Lancet
 , 
2005
, vol. 
366
 (pg. 
144
-
150
)
2
Farhoudi
D
Lofdahl
M
Giesecke
J
Invasive Haemophilus influenzae type b disease in Sweden 1997–2003: epidemiological trends and patterns in the post-vaccine era
Scand J Infect Dis
 , 
2005
, vol. 
37
 (pg. 
717
-
722
)
3
WCenters for Disease Control and Prevention
Progress toward elimination of Haemophilus influenzae type b invasive disease among infants and children--United States 1998–2000
MMWR Morb Mortal Wkly Rep
 , 
2002
, vol. 
51
 (pg. 
234
-
237
)
4
Eskola
J
Kayhty
H
Takala
AK
, et al.  . 
A randomized, prospective field trial of a conjugate vaccine in the protection of infants and young children against invasive Haemophilus influenzae type b disease
N Engl J Med
 , 
1990
, vol. 
323
 (pg. 
1381
-
1387
)
5
Hviid
A
Melbye
M
Impact of routine vaccination with a conjugate Haemophilus influenzae type b vaccine
Vaccine
 , 
2004
, vol. 
22
 (pg. 
378
-
382
)
6
Perdue
DG
Bulkow
LR
Gellin
BG
, et al.  . 
Invasive Haemophilus influenzae disease in Alaskan residents aged 10 years and older before and after infant vaccination programs
JAMA
 , 
2000
, vol. 
283
 (pg. 
3089
-
3094
)
7
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
, vol. 
22
 (pg. 
1069
-
1076
)
8
Cerquetti
M
Ciofi degli Atti
ML
Cardines
R
Salmaso
S
Renna
G
Mastrantonio
P
Invasive type e Haemophilus influenzae disease in Italy
Emerg Infect Dis
 , 
2003
, vol. 
9
 (pg. 
258
-
261
)
9
Adderson
EE
Byington
CL
Spencer
L
, et al.  . 
Invasive serotype a Haemophilus influenzae infections with a virulence genotype resembling Haemophilus influenzae type b: emerging pathogen in the vaccine era?
Pediatrics
 , 
2001
, vol. 
108
 pg. 
E18
 
10
Kapogiannis
BG
Satola
S
Keyserling
HL
Farley
MM
Invasive infections with Haemophilus influenzae serotype a containing an IS1016-bexA partial deletion: possible association with virulence
Clin Infect Dis
 , 
2005
, vol. 
41
 (pg. 
e97
-
103
)
11
Tsang
RS
Mubareka
S
Sill
ML
Wylie
J
Skinner
S
Law
DK
Invasive Haemophilus influenzae in Manitoba, Canada, in the postvaccination era
J Clin Microbiol
 , 
2006
, vol. 
44
 (pg. 
1530
-
1535
)
12
Ogilvie
C
Omikunle
A
Wang
Y
St Geme
IJ
3rd
Rodriguez
CA
Adderson
EE
Capsulation loci of non-serotype b encapsulated Haemophilus influenzae
J Infect Dis
 , 
2001
, vol. 
184
 (pg. 
144
-
149
)
13
Satola
SW
Collins
JT
Napier
R
Farley
MM
Capsule gene analysis of invasive Haemophilus influenzae: accuracy of serotyping and prevalence of IS1016 among nontypeable isolates
J Clin Microbiol
 , 
2007
, vol. 
45
 (pg. 
3230
-
3238
)
14
LaClaire
LL
Tondella
ML
Beall
DS
, et al.  . 
Identification of Haemophilus influenzae serotypes by standard slide agglutination serotyping and PCR-based capsule typing
J Clin Microbiol
 , 
2003
, vol. 
41
 (pg. 
393
-
396
)
15
Kroll
JS
Moxon
ER
Loynds
BM
Natural genetic transfer of a putative virulence-enhancing mutation to Haemophilus influenzae type a
J Infect Dis
 , 
1994
, vol. 
169
 (pg. 
676
-
679
)
16
Hammitt
LL
Block
S
Hennessy
TW
, et al.  . 
Outbreak of invasive Haemophilus influenzae serotype a disease
Pediatr Infect Dis J
 , 
2005
, vol. 
24
 (pg. 
453
-
456
)
17
Ribeiro
GS
Reis
JN
Cordeiro
SM
, et al.  . 
Prevention of Haemophilus influenzae type b (Hib) meningitis and emergence of serotype replacement with type a strains after introduction of Hib immunization in Brazil
J Infect Dis
 , 
2003
, vol. 
187
 (pg. 
109
-
116
)
18
Ribeiro
GS
Lima
JB
Reis
JN
, et al.  . 
Haemophilus influenzae meningitis 5 years after introduction of the Haemophilus influenzae type b conjugate vaccine in Brazil
Vaccine
 , 
2007
, vol. 
25
 (pg. 
4420
-
4428
)
19
Case notification records
2005
Salvador, Brazil
Secretary of Health for the State of Bahia
20
Falla
TJ
Crook
DW
Brophy
LN
Maskell
D
Kroll
JS
Moxon
ER
PCR for capsular typing of Haemophilus influenzae
J Clin Microbiol
 , 
1994
, vol. 
32
 (pg. 
2382
-
2386
)
21
Curran
R
Hardie
KR
Towner
KJ
Analysis by pulsed-field gel electrophoresis of insertion mutations in the transferrin-binding system of Haemophilus influenzae type b
J Med Microbiol
 , 
1994
, vol. 
41
 (pg. 
120
-
126
)
22
Saito
M
Umeda
A
Yoshida
S
Subtyping of Haemophilus influenzae strains by pulsed-field gel electrophoresis
J Clin Microbiol
 , 
1999
, vol. 
37
 (pg. 
2142
-
2147
)
23
Tenover
FC
Arbeit
RD
Goering
RV
, et al.  . 
Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing
J Clin Microbiol
 , 
1995
, vol. 
33
 (pg. 
2233
-
2239
)
24
Satola
SW
Schirmer
PL
Farley
MM
Complete sequence of the cap locus of Haemophilus influenzae serotype b and nonencapsulated b capsule-negative variants
Infect Immun
 , 
2003
, vol. 
71
 (pg. 
3639
-
3644
)
25
Kroll
JS
Moxon
ER
Loynds
BM
An ancestral mutation enhancing the fitness and increasing the virulence of Haemophilus influenzae type b
J Infect Dis
 , 
1993
, vol. 
168
 (pg. 
172
-
176
)
26
Meats
E
Feil
EJ
Stringer
S
, et al.  . 
Characterization of encapsulated and noncapsulated Haemophilus influenzae and determination of phylogenetic relationships by multilocus sequence typing
J Clin Microbiol
 , 
2003
, vol. 
41
 (pg. 
1623
-
1636
)
27
Instituto Brasileiro de Geografia e Estatística
Anuário Estatístico do Brasil
1996
, vol. 
56
 
Rio de Janeiro, Brazil
Instituto Brasileiro de Geografia e Estatística
28
Dagan
R
Fraser
D
Roitman
M
, et al.  . 
Effectiveness of a nationwide infant immunization program against Haemophilus influenzaeb. The Israeli Pediatric Bacteremia and Meningitis Group
Vaccine
 , 
1999
, vol. 
17
 (pg. 
134
-
141
)
29
Peltola
H
Aavitsland
P
Hansen
KG
Jonsdottir
KE
Nokleby
H
Romanus
V
Perspective: a five-country analysis of the impact of four different Haemophilus influenzae type b conjugates and vaccination strategies in Scandinavia
J Infect Dis
 , 
1999
, vol. 
179
 (pg. 
223
-
229
)
30
Forleo-Neto
E
de Oliveira
CF
Maluf
EM
, 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
, vol. 
180
 (pg. 
1153
-
1158
)
31
Millar
EV
O'Brien
KL
Levine
OS
Kvamme
S
Reid
R
Santosham
M
Toward elimination of Haemophilus influenzae type B carriage and disease among high-risk American Indian children
Am J Public Health
 , 
2000
, vol. 
90
 (pg. 
1550
-
1554
)
32
Bajanca
P
Canica
M
Emergence of nonencapsulated and encapsulated non-b-type invasive Haemophilus influenzae isolates in Portugal (1989–2001)
J Clin Microbiol
 , 
2004
, vol. 
42
 (pg. 
807
-
810
)
33
Jin
Z
Romero-Steiner
S
Carlone
GM
Robbins
JB
Schneerson
R
Haemophilus influenzae type a infection and its prevention
Infect Immun
 , 
2007
, vol. 
75
 (pg. 
2650
-
2654
)
34
Millar
EV
O'Brien
KL
Watt
JP
, et al.  . 
Epidemiology of invasive Haemophilus influenzae type A disease among Navajo and WhiteMountain Apache children, 1988–2003
Clin Infect Dis
 , 
2005
, vol. 
40
 (pg. 
823
-
830
)
35
Bruun
B
Gahrn-Hansen
B
Westh
H
Kilian
M
Clonal relationship of recent invasive Haemophilus influenzae serotype f isolates from Denmark and the United States
J Med Microbiol
 , 
2004
, vol. 
53
 (pg. 
1161
-
1165
)
36
Campos
J
Hernando
M
Roman
F
, et al.  . 
Analysis of invasive Haemophilus influenzae infections after extensive vaccination against H. influenzae type b
J Clin Microbiol
 , 
2004
, vol. 
42
 (pg. 
524
-
529
)
37
Noel
GJ
Brittingham
A
Granato
AA
Mosser
DM
Effect of amplification of the Cap b locus on complement-mediated bacteriolysis and opsonization of type b Haemophilus influenzae
Infect Immun
 , 
1996
, vol. 
64
 (pg. 
4769
-
4775
)
38
Sill
ML
Zhou
J
Law
DK
, et al.  . 
Molecular characterization of four Haemophilus influenzae serotype a strains isolated from patients in Quebec, Canada
Can J Microbiol
 , 
2007
, vol. 
53
 (pg. 
1191
-
1194
)
Financial support: Brazilian National Research Council (491345/2005-4), Research Support Foundation for the State of Bahia (FAPESB- 1431040054051), and National Institute of Health, USA (D43 TW00919 and R01 TW007303).
Potential conflicts of interest: none reported.

Author notes

a
J.B.T.L. and G.S.R. contributed equally to this work.