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Department of Epidemiology and Clinical Microbiology, National Institute of Public Health
Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Warsaw, Poland
ABSTRACT
Among 1,272 H. influenzae isolates obtained from patients with lower respiratory tract infections, 5.6% were found to be encapsulated. The most common were serotypes b (40.3%) and e (38.9%) followed by serotypes f (16.7%) and d (4.1%). All encapsulated isolates within a particular serotype showed high genetic homogeneity and did not produce -lactamases.
TEXT
Haemophilus influenzae is responsible for respiratory tract infections as well as severe invasive infections such as meningitis, bacteremia-sepsis, epiglottitis, and septic arthritis. Although the overwhelming majority of invasive Haemophilus sp. infections are caused by H. influenzae serotype b, other serotypes as well as nontypeable isolates (NTI) have also been associated with invasive diseases (2, 3, 6, 12, 13, 17, 18, 24). The aim of this study was to investigate the prevalence and serotype distribution of encapsulated H. influenzae isolated from patients with lower respiratory tract infections (LRTIs).
From 1996 through 2001, 1,272 H. influenzae isolates, representing the whole country, were collected during a surveillance study in 25 centers in Poland. The study included outpatients and hospitalized patients (sampled within 48 h of admission) with clinically proven community-acquired LRTIs (exacerbations of chronic obstructive pulmonary disease or pneumonia) from whom samples of bronchoalveolar lavage, blood, or sputum (evaluated with respect to the numbers of white blood cells and epithelial cells and the presence of the mucus threads) were taken (1). Although in local laboratories all isolates were identified by standard procedures, 4.07% of isolates were misidentified. PCRs were run to confirm species identification and to detect capsule- and serotype-specific genes. Supernatants obtained from boiled colonies of H. influenzae were used for duplex PCR for species identification and capsule gene detection (4, 5). For isolates for which the specific products for capsule genes were obtained, the PCRs for serotypes a to f determination were run with primers a1-a2, b1-b2, c1-c2, d1-d2, e1-e2, f1-f2, respectively (4). Quality control strains of H. influenzae of serotypes a to f used for PCR were provided by Oxford Public Health Laboratory (John Radcliffe Hospital). The H. parainfluenzae strain used as the negative control (strain no. 2259/96) was derived from our collection. The production of -lactamases was determined by a nitrocefin assay according to the instructions of the manufacturer (bioMerieux, Marcy l'Etoile, France). The relatedness among encapsulated isolates was evaluated by restriction fragment length polymorphism of SmaI-digested chromosomal DNA analysis, using pulsed-field gel electrophoresis (PFGE), and interpreted as previously described (21, 22).
A total of 72 (5.6%) H. influenzae isolates were found to be encapsulated. The most common were H. influenzae serotypes b (40.3%; n = 29) and e (38.9%; n = 28) followed by serotypes f (16.7%; n = 12) and d (4.1%; n = 3). The results of comparisons of the frequencies of recovery of different serotypes and NTI sorted according to specimen type and patient age are presented in Table 1. Among NTI, 4.4% (n = 56) were found to be producers of -lactamases whereas none of the encapsulated isolates produced such enzymes. All but one of H. influenzae serotype b isolates were classified as one PFGE type with seven subtypes (G1 to G7); G1, the most common subtype, was characteristic of 48.3% (n = 14) of these isolates. All but one of the H. influenzae serotype e isolates belonged to one PFGE type with 6 subtypes (A1 to A6). Of the 12 H. influenzae serotype f isolates, 6 had identical PFGE patterns (type C1) and an additional 5 (with type D1 and D2 patterns) were possibly related to PFGE type C1. Identical PFGE patterns were characteristic of all three H. influenzae serotype d isolates. The PFGE patterns of 72 encapsulated H. influenzae isolates are presented in Fig. 1.
In this study, as would be expected for a country with no program of mass vaccination against H. influenzae serotype b, the most prevalent serotype among the encapsulated H. influenzae isolates was H. influenzae serotype b, but, astonishingly, almost the same percentage of isolates represented H. influenzae serotype e. Although the results of carriage studies have mainly presented H. influenzae serotype e isolates as colonizers, few authors have described H. influenzae serotype e as an etiologic agent of serious, as well as fatal, infections (2, 3, 20, 24). Despite the reports that in the populations with mass vaccination against H. influenzae serotype b the rates of colonization and infection by other serotypes may increase, the high proportion of H. influenzae serotype e found in our study is probably connected with serotype detection by PCR, because in some isolates, in particular those representing H. influenzae serotype e, the expression of capsular polysaccharide is not sufficient to be detected by slide agglutination (2, 3, 11, 16, 17, 24). Therefore, until recently the prevalence of this serotype could have been underestimated, because most studies had used slide agglutination as a standard method. Moreover, the study conducted by LaClaire et al. revealed not only the wrong serotype identification but also that 68% of the isolates recognized as H. influenzae serotype b by slide agglutination did not possess the necessary capsular genes when identified by PCR (11). Therefore, the real number of H. influenzae serotype b cases, including vaccine failures, may be considerably lower (11). The third most common serotype in this study was f, which of the non-H. influenzae serotype b serotypes is known to be mostly associated with invasive disease and a very high mortality rate of up to 30% (12, 19, 23).
The rate of -lactamase producers among NTI, although consistent with previous Polish data, is low in comparison with the results of the SENTRY and the PROTEKT projects for other countries, which showed on average 28.6 and 16.6% of the H. influenzae as producers of -lactamases, respectively (8-10). Although resistance is generally more frequent among H. influenzae serotype b isolates than among NTI, it is variable depending on the source of isolation; i.e., it is more prevalent among isolates responsible for meningitis than among those responsible for otitis media (7, 21). In this study none of the encapsulated isolates produced -lactamases. This is in contrast to the results seen with a group of H. influenzae meningitis isolates in Poland, of which 97% were identified as H. influenzae serotype b, with 14% of them producing -lactamases (M. Kadubowski, A. Skoczyska, A. Klarowicz, and W. Hryniewicz, Abstr. 6th Inter. Meet. Microb. Epidemiol. Markers, abstr. 168, 2003).
In this study, all the encapsulated isolates within a particular serotype showed high genetic homogeneity. It is well known and especially well documented for H. influenzae serotype b isolates that the genetic structure of encapsulated isolates, in contrast to those of NTI, is clonal and that chromosomal recombination is an infrequent event in encapsulated isolates (12, 14, 15, 21). The most common PFGE subtype (G1) among H. influenzae serotype b isolates tested was also the most frequent among H. influenzae serotype b strains responsible for meningitis in Poland (91%), Italy, and the Czech Republic (data not shown) (21; M. Kadubowski et al., Abstr. 6th Inter. Meet. Microb. Epidemiol. Markers).
A detailed comparison of our results with results from other studies is difficult, because almost all the papers on this subject are focused on invasive diseases (3, 17, 19, 24). There is a need for further studies, using PCR as the "gold standard," concentrated on other infections caused by encapsulated non-H. influenzae serotype b isolates as well as on potentially invasive isolates from carriers. This knowledge is particularly important, since H. influenzae serotype b vaccine dramatically decreases not only the incidence of invasive H. influenzae serotype b disease but also the rate of H. influenzae serotype b carriage, which may influence the epidemiology, with respect to both carriage and invasiveness, of other encapsulated non-H. influenzae serotype b H. influenzae isolates. At present, because of PCR-based methods, we have the possibility to assess more correctly the role of encapsulated non-H. influenzae serotype b isolates in the epidemiology of infectious diseases caused by this species. Data from our study strongly indicate an increasing role of non-H. influenzae serotype b encapsulated isolates and thus a need for careful monitoring of the H. influenzae serotypes responsible for both invasive and noninvasive infections.
ACKNOWLEDGMENTS
The work was done in the Sera & Vaccines Central Research Laboratory in the Department of Epidemiology and Clinical Microbiology (presently the National Institute of Public Health). During the period in which the work was done, M.L. was an undergraduate student (Division of Biology, University of Warsaw).
Present address: Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Warsaw, Poland.
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