Haemophilus influenzae serotype b and a capsule

Journal of Medical Microbiology (2013), 62, 655–657
Case Report
DOI 10.1099/jmm.0.052357-0
Haemophilus influenzae serotype b and a capsuledeficient type mutant (b”) invasive disease in a
partially vaccinated child in Brazil
Nathalia G. S. Caldeira,1 Ivano de Filippis,1 Tânia Catão Arruda,2
Maria Eulália Côrte Real,3 Alice Batalha de Jesus1
and Antonio Eugenio C. C. de Almeida1
Correspondence
1
Antonio Eugenio C.C. de Almeida
2
Instituto Nacional de Controle de Qualidade em Saúde, INCQS/FIOCRUZ, Rio de Janeiro, Brazil
[email protected]
Laboratório Central/LACEN-PE, Recife, Pernambuco, Brazil
3
Hospital C. Picanço, Recife, Pernambuco, Brazil
Received 14 September 2012
Accepted 21 December 2012
We report a rare case of infection by two different types of Haemophilus influenzae strains in a
child who received only one dose of the H. influenzae serotype b (Hib) conjugate vaccine
(DTwP+Hib). The strains were recovered from blood and cerebrospinal fluid (CSF) and were
phenotypically identified as Hib and non-typable H. influenzae, respectively, after serological
tests. The two strains were characterized by PCR capsular typing, multilocus sequence typing
and PFGE. Our results suggest that the infection was caused by the bloodstream invasion by a
single Hib strain, followed by the diffusion of the bacteria across the blood–brain barrier and into
the CSF. The strain recovered from the CSF, however, was identified as a capsule-deficient type
mutant (b”) strain. Despite the high efficacy of the Hib conjugate vaccine, the increase in the
numbers of strains able to escape the immune system of the vaccinated population advocates
continued surveillance.
Introduction
Haemophilus influenzae serotype b (Hib) has been an
important cause of paediatric morbidity and mortality. The
routine use of the conjugate Hib vaccine has produced a
dramatic decline in the incidence of invasive Hib disease
over a very short period of time (Slack et al., 1998; Peltola,
2000; Heath et al., 2001; Kriz et al., 2005). As a consequence,
non-b and non-typable H. influenzae (NTHi) isolates are
nowadays the leading cause of invasive H. influenzae disease
in countries with ongoing Hib vaccination programmes
(Sarangi et al., 2000; Bajanca et al., 2004; Ito et al., 2011).
In encapsulated H. influenzae strains, the genes for the
production of the polysaccharide capsule are organized in a
capsulation (cap) locus, formed by three different functional regions. Regions I (bexABCD) and III are common
to all capsular types and contain the genes necessary for
transport and processing of the capsular structures, while
region II contains serotype-specific biosynthesis genes
(Kroll et al., 1991; Van Eldere et al., 1995).
Abbreviations: CSF, cerebrospinal fluid; Hib, H. influenzae serotype b;
MLST, multilocus sequence typing; NTHi, non-typable H. influenzae;
SAT, slide agglutination test.
The GenBank/EMBL/DDBJ accession numbers for partial bcs3 gene
sequences of H. influenzae strains are JN411086 and JN411087.
052357 G 2013 SGM
Since the implementation of routine immunization against
Hib just over a decade ago, non-encapsulated strains have
taken on greater importance as a cause of bacteraemia,
meningitis and other forms of invasive disease (Ito et al.,
2011). Non-encapsulated strains are now commonly found
to cause sepsis in invasive H. influenzae infection in the
elderly. Nonetheless, strain replacement of Hib with
serotype f and non-typable strains in children under
5 years has been reported (Adam et al., 2010).
This study describes a rare case of septicaemia and
meningitis due to a Hib strain and a capsule-deficient
type mutant (b2) strain isolated simultaneously from
blood and cerebrospinal fluid (CSF) in a child.
Case report
A 5-month-old male was admitted to the Municipal
Hospital of the Recife, Pernambuco State, in the northeastern region of Brazil after falling from a swing 2 days
earlier. He had symptoms and signs of meningitis such as
lethargy, convulsion, bulged fontanelle, neck rigidity and a
39 uC fever. Meningitis infection was considered as a
possible cause and a spinal tap was performed. The child’s
past medical history was notable for frequent upper
respiratory tract infections but there was no evidence
of any immunocompromising condition. The patient had
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655
N. G. S. Caldeira and others
received only one dose of the Hib conjugate vaccine
(DTwP+Hib). Two weeks before admission the child had
whooping cough. Samples of CSF and blood were collected
and submitted for laboratory analysis. Blood tests revealed
leukocytosis and the CSF results showed abundant white
cells (1024 ml21) and a protein level of 40 mg dl21, with
90 % neutrophils. The glucose level was 10 mg dl21. A
Gram stain revealed Gram-negative bacilli. Bacterial
meningitis was diagnosed based on clinical and laboratory
findings, and empirical treatment with ceftriaxone sodium
hydrate was promptly initiated (120 mg kg21 day21, every
12 h). After culture confirmation, the antibiotic therapy
with ceftriaxone was continued for 7 days with 100 mg
kg21. Blood culture for aerobic and anaerobic microorganisms was carried out by inoculating 1 ml blood
sample into 20 ml trypticase soy broth containing sodium
polyanthol sulfonate (Becton Dickinson). Blood culture
was positive after incubation at 35 uC for 5 days. CSF was
cultured on brain heart infusion chocolate agar (Difco)
enriched with 10 % defibrinated horse blood at 37 uC for
24 h, and bacterial growth was detected after 24 h. Gram
staining of bacterial colonies recovered from blood and
CSF samples was performed, showing abundant pleomorphic Gram-negative rods, suggesting Haemophilus
species. The suspected cultures were sent to the National
Institute for Quality Control in Health (INCQS) for
additional identification and serotypes tests, such as the
slide agglutination test (SAT), biotyping, antimicrobial
sensitivity and PCR capsular typing tests, as previously
described by Falla et al. (1994) and Ledeboer & Doern
(2011). The serotyping tests were repeated and five
individual colonies of the blood culture and CSF isolates
were tested and were identified by the SAT as Hib and
NTHi, respectively. The results of the biotyping test
indicated that both isolates were biotype II. After antibiotic
therapy, the patient showed a complete resolution of
symptoms, return of normal function and normalization of
blood parameters.
The negative serology result combined with the negative
PCR result for the bexABCD (region I), but positive result
for type b polysaccharide capsule gene bcs3 (region II),
confirms that strain P3271 isolated from the CSF was
actually a type b capsule-deficient mutant (b2) lacking the
bexABCD or bexA gene (region I) – a strain with the
capsulation locus but with a 1.2 kb deletion within the
bexA gene that is necessary for polysaccharide export (Falla
et al., 1994; Van Eldere et al., 1995). On the other hand,
strain P3271/A isolated from the blood showed a positive
serology result for type b and positive PCR result for
capsule determination and capsular type, which confirms
that this isolate was a regular Hib strain. MLST analysis was
performed as previously described by Meats et al. (2003)
and typing based on the bacterial genomic DNA fingerprinting pattern obtained by PFGE has been reported to
determine whether the two isolates were the same strain
(Smith & Cantor, 1987; Tenover et al., 1995; Saito et al.,
1999; Qin et al., 2012). Both strains were classified as ST-6
656
by MLST analysis and PFGE analysis confirmes the MLST
result, since the two strains showed identical fingerprinting
patterns (Fig. 1), suggesting that they could be the same
isolate. Partial sequences of the bcs3 gene were deposited in
GenBank under accession numbers JN411086 and
JN411087.
Discussion
Trends in the changing epidemiology of invasive H.
influenzae disease should alert health authorities to increase
the surveillance of this important bacterial disease. In the
past, surveillance of invasive H. influenzae disease had been
restricted to monitoring only Hib cases, but with the
success of the Hib immunization programme, most
countries have focused their attention on the invasive
non-type b type disease, caused most commonly by NTHi
strains (van Belkum et al., 1994; Ito et al., 2011).
The results of our study show clearly that a b2 strain
caused meningitis in a vaccinated child. It is likely that a
Hib strain invaded the patient’s bloodstream, rapidly
reaching the meninges and causing meningitis. We cannot
confirm whether part of the Hib strain population lost or
modified part of the cap genetic machinery, changing to
Hib2 while in the blood or after crossing the blood–brain
barrier, but it is likely that the immune system played an
important role in this event as the strain was circulating in
Fig. 1. PFGE (agarose). Lanes: M, lambda DNA PFGE ladder;
Hib”, isolate from CSF; Hib, isolate from blood.
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Journal of Medical Microbiology 62
H. influenzae b capsule-deficient mutant in Brazil
the bloodstream. Considering that the child had one dose
of vaccine, we could even speculate about a possible
positive selection caused by the immune barrier, allowing
the escape of the Hib2 strain, considering that the child
showed only meningeal signs. This hypothesis would be of
high concern since it could occur in vaccinated children. In
any case, these findings suggest that Hib2 strains may have
the same pathogenic potential as Hib strains. (Rahman
et al., 2008; Ito et al., 2011).
Considering that serotyping by SAT is less accurate, being
unable to detect Hib2, and invasive H. influenzae infections
have become rare, we recommend a combination of culture
and PCR for the detection of H. influenzae infections. The
PCR capsule typing is a rapid, sensitive and specific
diagnostic test for confirming negative serotype results.
Because of the low numbers of invasive H. influenzae
isolates, mistyping might have an important influence on
surveillance.
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Ledeboer, N. A. & Doern, G. V. (2011). Haemophilus. In Manual of
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Acknowledgements
This study was supported by CNPq/INCQS/FIOCRUZ/MS.
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