Evaluation of sampling sites for detection of upper respiratory tract carriage of Streptococcus pneumoniae and Haemophilus influenzae among healthy Filipino infants.

Two sampling techniques, nasal swabbing and oropharyngeal swabbing, for detection of the upper respiratory tract carriage of Streptococcus pneumoniae and Haemophilus influenzae were studied prospectively with 296 healthy Filipino infants at various ages: 6 to 8, 10 to 12, 14 to 17, 18 to 22, 32 to 39, and 46 to 65 weeks. In all age groups S. pneumoniae was isolated significantly more often (P < 0.0001) from the nasal site than from the oropharyngeal site. H. influenzae was found equally often at both sites.     

Risk factors for the nasopharyngeal carriage of respiratory pathogens by Portuguese children: phenotype and antimicrobial susceptibility of Haemophilus influenzae and Streptococcus pneumoniae

Between 1997 and 2000 nasopharyngeal specimens were obtained from 466 children < or = 12 years old attending the Pediatric Emergency Department at S. Francisco Xavier Hospital, Lisbon, to evaluate risk factors for nasopharyngeal carriage of Haemophilus influenzae and Streptococcus pneumoniae and to characterize their phenotype and antimicrobial susceptibility. The attending pediatrician completed written questionnaires about the children's demographic and clinical histories. Over half the children (52.8%) carried H. influenzae and/or S. pneumoniae. Forty-one percent of these children had H. influenzae, 22.8% had S. pneumoniae and 36.2% had both. Risk factors identified for carriage of respiratory pathogens were: age below 3 years (p < 0.05), black race (p < 0.01), attending a daycare center (p < 0.05), and having a lower respiratory infection (p < 0.05). Asthmatic children were less likely to be carriers (p = 0.004). About two-thirds of H. influenzae isolates were susceptible to all antibiotics tested, 7.9% were beta-lactamase producers, 16.4% were nonsusceptible to trimethoprim, and 6.9% were intermediately resistant to clarithromycin. Over half (57.1%) of S. pneumoniae isolates were susceptible to all antibiotics tested, 21.1% were multiresistant, 23.3% were nonsusceptible to penicillin, and about 20% were resistant to macrolides. Low-level resistance to third-generation cephalosporins was detected in 2.3%. The data reflect the controversy surrounding risk factors of nasopharyngeal colonization. These may have significant implications on clinical practice and on antimicrobial strategies to prevent the appearance of further resistant strains. Our findings highlight the importance to investigate the relationship between asthma and carriage.     

Effect of pneumococcal vaccination on nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian children

The 7-valent pneumococcal conjugate vaccine (PCV7) reduces carriage of vaccine type Streptococcus pneumoniae but leads to replacement by nonvaccine serotypes and may affect carriage of other respiratory pathogens. We investigated nasopharyngeal carriage of S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Fijian infants participating in a pneumococcal vaccine trial using quantitative PCR. Vaccination did not affect pathogen carriage rates or densities, whereas significant differences between the two major ethnic groups were observed.     

Immunoglobulin A1 protease production by Haemophilus influenzae and Streptococcus pneumoniae.

Bacterial strains of Haemophilus species and Streptococcus pneumoniae were examined for synthesis of the enzyme immunoglobulin A1 (IgA1) protease. Of 36 H. influenzae strains examined, 35 produced IgA1 protease; strains included all six capsular types, unencapsulated variants of types b and d, and untypable H. influenzae. Eight Haemophilus strains (non-H. influenzae) were studied, and two produced IgA1 protease. All 10 strains of S. pneumoniae produced IgA1 protease; these strains included 9 different capsular polysaccharide types and 1 untypable strain. Both IgA1 proteases cleaved myeloma IgA1 and secretory IgA but not myeloma IgA2, IgM, or IgG as determined by immunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes cleaved IgA1 myeloma sera, but not IgA2, into two fragments. The apparent molecular weight of the cleaved fragments was dependent both on the apparent molecular weight of the cleaved fragments was dependent both on the specific IgA1 protease assayed and the specific IgA1 substrate utilized. It is postulated that both carbohydrate variation between the IgA1 substrates studied and the ability of S. pneumoniae glycosidases to cleave carbohydrates from glycoprotein offer an explanation for the different fragment sizes observed.     

Comparison of methods for serotyping isolates of Haemophilus influenzae.

This study compared four methods for serotyping isolates of Haemophilus influenzae. Slide agglutination with commercial antisera (Difco Laboratories and Wellcome Diagnostics), coagglutination (Phadebact Haemophilus Test [Pharmacia Fine Chemicals]), latex agglutination with affinity-purified anticapsular antibody, and counterimmunoelectrophoresis with multiple antisera were used to serotype 80 isolates of H. influenzae. Coagglutination and counterimmunoelectrophoresis correctly identified all 80 isolates as either type b or not type b. Slide agglutination and latex agglutination each successfully identified 76 of the 80 isolates; however, each of these two methods failed to type four isolates because of agglutination of controls. We recommend slide agglutination or coagglutination as the serotyping methods of choice in most laboratories because they are simple, accurate, and rapid. Slide agglutination with Difco antiserum can be performed at the lowest cost.     

Nasopharyngeal Haemophilus influenzae carriage in Japanese children attending day-care centers

We conducted a prospective bacteriological survey to investigate antibiotic resistance-related genetic characteristics and the turnover of nasopharyngeal Haemophilus influenzae carriage in healthy children in day-care centers (DCCs). A total of 363 nasopharyngeal mucus samples were collected from children aged 0 to 6 years attending two DCCs in the summer of 2004 (n = 184) and the following winter (n = 179). We obtained 172 H. influenzae isolates and analyzed them by antimicrobial susceptibility testing, PCR for bla_TEM-1 and the penicillin-binding protein (PBP) gene, and pulsed-field gel electrophoresis (PFGE). The overall carriage rate was 47.4% (172/363), and 37.2% of the isolates (64/172) were ampicillin (AMP) resistant. All the resistant isolates had a PBP mutation(s), while only three isolates had TEM-1. The carriage rate was significantly higher in the winter than in the summer (56.4% and 38.6%, respectively), owing to the increase in the numbers of AMP-susceptible H. influenzae isolates in the winter. Children aged ≤3 years showed a higher rate of carriage of H. influenzae isolates with an AMP resistance gene(s) than those aged ≥4 years (21.9% and 12.6%, respectively). Forty-two strains with different PFGE patterns were obtained from among the 172 isolates. Only five strains were observed in both seasons. None of the strains isolated in the summer was isolated from the same carrier in the winter. Twenty-seven strains (64.3%) were isolated from two or more children, and 25 of these were each isolated from children belonging to the same DCC. These results indicate the spread of H. influenzae, particularly those with a PBP mutation(s), and the highly vigorous genetic turnover and substantial horizontal transmission of this pathogen in healthy children attending DCCs in Japan.     

Factors affecting pharyngeal Haemophilus influenzae type b colonization rates in children.

Over 1,300 children were studied in an analysis of factors that might affect pharyngeal colonization with Haemophilus influenzae type b. Our semiquantitative methods for the culture of H. influenzae type b, consisting of inoculation of 0.001 ml of throat swab fluid on antiserum agar plates and division of the results into three grades of intensity, showed agreement as to intensity of colonization in over 80% of repeat throat cultures. Our data also suggest that throat swabs are more efficient than nasopharyngeal swabs for detecting colonization, particularly for older children. All 17 H. influenzae type b carriers found with either method were detected with throat swabs, but six had negative nasopharyngeal cultures; four of these six were lightly colonized older children. Furthermore, colony counts were apt to be higher on plates inoculated with throat swab fluids. The frequency of pharyngeal H. influenzae type b colonization in children visiting health department clinics and pediatricians' offices was low during the first 6 months of life (0.7%) but averaged 3 to 5% throughout the rest of childhood. Approximately two-thirds of the carriers were colonized at an intensity too low to be detected by standard laboratory techniques. No influence on colonization rates was found for sex, race, season, economic status, or common childhood infectious diseases such as coryza or otitis media.     

Anti-adhesive activity of human casein against Streptococcus pneumoniae and Haemophilus influenzae

The casein fraction of human milk was found to inhibit the attachment of Streptococcus pneumoniae and Haemophilus influenzae human respiratory tract epithelial cells. The inhibitory activity for S. pneumoniae remained after heat and trypsin treatment of the casein and was found in oligosaccharides released from casein. kappa-Casein, which is the most highly glycosylated casein component, inhibited pneumococcal attachment at concentrations similar to the whole casein fraction. The results are consistent with the known recognition of GlcNAc beta 1-3Gal by S. pneumoniae, since human milk and bovine colostrum, which contain GlcNAc, inhibited attachment, but mature bovine milk lacking GlcNAc did not. The effect on H. influenzae was similar to that on S. pneumoniae in that the attachment was inhibited by human casein and bovine colostrum, but not by either mature bovine milk or by the bovine casein fraction. The kappa-casein component of human milk was a less efficient inhibitor of H. influenzae attachment than the whole casein fraction and the free oligosaccharides were inactive. This anti-microbial effect of human casein represents a new mechanism for the protection by breast-milk against respiratory tract infection.     

Detection of Streptococcus pneumoniae and Haemophilus influenzae type b antigens in acute nonbacteremic pneumonia.

Commercially available latex agglutination and coagglutination reagents were evaluated for their ability to detect bacterial antigens in the sera of 165 patients to determine their suitability for rapid diagnosis of pneumonia. These reagents were used to detect the polysaccharide capsular antigens of Haemophilus influenzae type b and Streptococcus pneumoniae in nonbacteremic patients known to be respiratory culture positive for these organisms. The reagents were unable to detect the polysaccharide antigens in sera from nonbacteremic patients. Patients with a clinical diagnosis of pneumonia who had respiratory or extrarespiratory infections with a variety of organisms were also tested. No evidence of cross-reactivity or of false-positive reactions was observed with either reagent. Because a negative agglutination test may occur during the course of a nonbacteremic infection, these reagents should not be used alone, and if used, they should be used only in conjunction with standard bacteriological tests.     

Invasive infections caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae among children in St Petersburg, Russia.

This study investigated the causes of invasive bacterial infections in children aged <15 years in St Petersburg, Russia, during 2001-2003, using culture and antigen detection methods (rapid antigen latex agglutination (RAL)) for normally sterile body fluids. A pathogen was detected in 90 cases (culture 50, RAL 40). Neisseria meningitidis was the most common pathogen (66%), followed by Haemophilus influenzae (19%) and Streptococcus pneumoniae (16%). Meningitis was the main clinical diagnosis (68/90, 76%), with N. meningitidis serogroup B, H. influenzae type b (Hib), and S. pneumoniae serogroup 1 being the most common isolates. Hib was less prevalent in St Petersburg than it was in industrialised countries before the introduction of Hib vaccinations.     

Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae by using the E-test.

The E-test (PDM Epsilometer; AB Biodisk, Solna, Sweden) is an antimicrobial agent gradient-coated plastic test strip which allows MIC determinations on agar media. The test is performed in a manner similar to the agar disk diffusion procedure. A collection of Haemophilus influenzae and Streptococcus pneumoniae strains possessing various resistance mechanisms was used to evaluate the E-test method. H. influenzae strains were tested with both Haemophilus test medium (HTM) and PDM ASM II chocolate agar, while the S. pneumoniae strains were tested on Mueller-Hinton sheep blood agar. E-test MICs for a total of 10 antimicrobial agents were compared with broth microdilution MICs determined according to National Committee for Clinical Laboratory Standards methods. In general, E-test MICs for both species were quickly and easily interpreted and agreed within one log2 MIC increment in 89.8% of tests with H. influenzae and in 80.4% of pneumococcal tests. The majority of disagreements between the E-test and conventional MICs occurred with trimethoprim-sulfamethoxazole because of trailing and diffuse E-test MIC endpoints with both species. Ampicillin MICs for beta-lactamase-producing H. influenzae determined by the E-test differed at times from those determined by conventional testing because of the vagaries of interpreting colonies growing within the E-test inhibition ellipses. E-test penicillin MICs for pneumococci tended to be 1 to 2 log2 dilutions lower than those determined by using Mueller-Hinton broth supplemented with lysed horse blood. Nevertheless, strains of both species with documented resistance to the study drugs were detected by E-tests, i.e., 0.7% of the tests had very major errors with H. influenzae and 0.8% had very major errors with S. pneumoniae. Thus, the E-test represents a potential alternative method for antimicrobial susceptibility testing of these two fastidious bacterial species.     

Haemophilus influenzae and Streptococcus pneumoniae induce different intracerebral mRNA cytokine patterns during the course of experimental bacterial meningitis

Using in situ hybridization with radiolabelled oligonucleotide probes, we studied the mRNA expression of IL-1β, IL-4, IL-6, IL-10, IL-12, tumour necrosis factor-alpha (TNF-α), TNF-β, interferon-gamma (IFN-γ), and transforming growth factor-beta (TGF-β) in the brain during the lethal course of experimental meningitis in a rat model inoculated intracisternally with Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae and in uninfected control rats inoculated with the same volume of PBS. The production of IL-1β, IL-4, IL-6 and IFN-γ was also evaluated by immunohistochemistry. In the brain of Hib-inoculated rats, there was marked mRNA expression of IL-1β, IL-6, TNF-α, IL-12 and IFN-γ. IL-1β, IL-6 and TNF-α were up-regulated throughout the observation period at 2, 8 and 18 h post-inoculation (p.i.), with similar patterns of induction. The Th1 cytokines IFN-γ and TNF-β were up-regulated within 8 h p.i. IL-10 and TGF-β were down-regulated at 18 h p.i., while IL-4 was not detected. In contrast, the brain of S. pneumoniae-inoculated rats showed lower levels of IL-1β, IL-6 and TNF-α, but higher levels of TNF-β and detectable mRNA expression of IL-4 when compared with Hib-inoculated rats. IL-12, IFN-γ, IL-10 and TGF-β exhibited similar patterns of induction in the brains of Hib- and S. pneumoniae-inoculated rats. At 18 h p.i., immunohistochemistry showed similar patterns of IL-1β, IL-4, IL-6 and IFN-γ as mRNA expression in the brains of Hib- and S. pneumoniae-inoculated rats. The differences of cytokine profiles induced by the two bacterial strains may imply that different immunomodulating approaches should be considered, depending on etiology.     

Counterimmunoelectrophoresis for early detection and rapid identification of Haemophilus influenzae type b and Streptococcus pneumoniae in blood cultures.

Counterimmunoelectrophortesis was shown to be a sensitive method for rapid detection of pneumococcal and Haemophilus influenzae type b antigens in blood cultures.     

Detection of Haemophilus influenzae and Streptococcus pneumoniae DNA in blood culture by a single PCR assay.

A multiplex PCR assay was developed to screen blood cultures from children in The Gambia with suspected pneumonia for the simultaneous detection of Haemophilus influenzae type b and Streptococcus pneumoniae isolates. Analysis of 295 blood cultures showed that PCR detected the organisms in all samples positive by culture in two samples infected with H. influenzae type b and four samples infected with S. pneumoniae that were culture negative, indicating that this method is sensitive for detecting these organisms in blood cultures.     

Relative importance of nasopharyngeal versus oropharyngeal sampling for isolation of Streptococcus pneumoniae and Haemophilus influenzae from healthy and sick individuals varies with age

Streptococcus pneumoniae and Haemophilus influenzae carriage is a useful index for measuring the emergence of resistance and outcome in vaccination trials. We performed a study to determine which sampling site, nasopharynx (NP) or oropharynx (OP), yields the highest rate of S. pneumoniae and H. influenzae isolation at different ages. Both NP and OP cultures were obtained from 216 children aged <60 months and their mothers. The total S. pneumoniae carriage rate was 68% among children and 15% among mothers (P < 0.001). Using NP alone for the isolation of S. pneumoniae would have missed 2, 2, and 42% and using OP alone would have missed 77, 66, and 45% of S. pneumoniae in children aged 0 to 23 months, 24 to 59 months, and mothers, respectively. Using NP cultures alone for H. influenzae would have missed 23, 24, and 81% of the isolates, respectively. The respective figures for H. influenzae isolation from OP alone are 38, 29, and 9%. In children, S. pneumoniae was carried mainly in the NP while H. influenzae was equally carried in the NP and OP. In mothers, S. pneumoniae was carried equally in the NP and OP while H. influenzae was carried significantly more often in the OP. In children, H. influenzae colonization increased during illness, mainly in the NP. Culturing only one site significantly reduced the recovery of H. influenzae at all ages. NP cultures for S. pneumoniae detected close to 100% of isolates in children but only 58% of isolates in mothers.     

Chemiluminescent response of polymorphonuclear leukocytes to Streptococcus pneumoniae and Haemophilus influenzae in suspension and adhered to glass.

We measured the luminol- and lucigenin-enhanced chemiluminescent response of human polymorphonuclear leukocytes (PMN) stimulated by various strains of Streptococcus pneumoniae and Haemophilus influenzae. In the absence of opsonin, phagocytosis of either bacterial species elicited good PMN response when the bacteria were adhered to a surface but minimal PMN response when they were in suspension. When 10% pooled human serum was used as a source of opsonin, a moderate to excellent chemiluminescent PMN response was elicited during phagocytosis of opsonized bacteria both in suspension and adhered to surface. We conclude that opsonin significantly enhances PMN chemiluminescence when a suspension-type assay is used and that opsonin-independent mechanisms play a significant role in the chemiluminescent response of PMN during phagocytosis of adherent bacteria.     

Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolated from the nasopharynges of asymptomatic children and molecular analysis of S. pneumoniae and H. influenzae strain replacement in the nasopharynx

Nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in 226 children in different settings (in a crèche [day care center], in an orphanage, and at home) during two seasons (winter and spring) was studied. The rates of carriage of S. pneumoniae and H. influenzae were markedly higher in the crèche and in the orphanage than in the home setting (e.g., 56.5, 63.3, and 25.9%, respectively, for S. pneumoniae in winter). Approximately 80% of the S. pneumoniae isolates identified in the crèche and in the orphanage belonged to the serotypes represented in the seven-valent pneumococcal vaccine, and 4.4% of the children were colonized by H. influenzae type b. Almost all H. influenzae isolates were fully susceptible to the antimicrobial agents tested, and only five (3.6%) produced beta-lactamase; in contrast, 100% of the M. catarrhalis isolates were beta-lactamase positive. Among S. pneumoniae isolates, 36.2% were nonsusceptible to penicillin (PNSP) and 11.8% were fully resistant to penicillin (PRP). All PNSP isolates were obtained from children at the crèche and at the orphanage but not among children brought up at home, and all PRP isolates showed a multiresistant phenotype. Colonization by PRP isolates correlated well with prior treatment with beta-lactams. For the majority of children colonized at both sampling times, strain replacement of S. pneumoniae and H. influenzae was observed; long-term colonization by a single strain was rare.