Dewan S. Billal

Rapid diagnosis of pneumococcal meningitis: implications for treatment and measuring disease burden.

Background: Streptococcus pneumoniae is the leading cause of childhood pneumonia and meningitis worldwide. Isolation of this organism, however, is uncommon in resource-poor countries, in part because of extensive use of prior antibiotics. A rapid, highly sensitive immunochromatographic test (ICT) for S. pneumoniae was evaluated for the diagnosis of meningitis. Methods: Cerebrospinal fluid (CSF) from 450 children with suspected meningitis was tested with ICT, and results were compared with CSF culture, latex agglutination test (LAT) and/or polymerase chain reaction (PCR). Serial CSF specimens from 11 patients were also evaluated for duration of positive results during effective antimicrobial therapy. Findings: All 122 cases of pyogenic pneumococcal meningitis positive either by culture (N = 87) or PCR (N = 35) were positive by ICT, yielding 100% (122 of 122) sensitivity. All purulent CSF specimens from patients with meningitis caused by other bacteria by culture (N = 149) or by LAT (N = 48) or those negative by culture, LAT and LytA and thus of unknown etiology (N = 20), and normal CSF specimens (N = 104) were negative by ICT. Thus the specificity of ICT also was 100% (321 of 321), although negativity of ICT was not confirmed by PCR, if it was positive for other organisms either by culture or LAT. Serotyping of S. pneumoniae strains revealed 28 different serotypes, indicating that outcome of ICT are independent of diverse capsular serotype of pneumococcus. Antigen was detected by ICT for at least 10 days after presentation, and 1 was still positive on day 20, which was longer than for either LAT or PCR. Interpretation: ICT for pneumococcal antigen in CSF is 100% sensitive and specific in diagnosing pyogenic pneumococcal meningitis and can detect ∼30% more pneumococcal meningitis cases than with culture alone. The simplicity of the test procedure and the longevity of CSF antigen detection suggest the potential utility of ICT to estimate the true burden of pneumococcal disease, as for Haemophilus influenzae type b using data from meningitis, and to guide selection of appropriate antibiotic treatment, especially in resource-poor countries with widespread prehospital antimicrobial use.

Clinical bacteriology and immunology in acute otitis media in children

Acute otitis media (AOM) is the most common disease seen in childhood. Streptococcus pneumoniae, non-typeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis are the most frequent pathogens of all AOM episodes. The high prevalence of drug-resistant pathogens such as penicillin-resistant S. pneumoniae (PRSP) and betalactamase producing or nonproducing ampicillin-resistant H. influenzae (BLPAR or BLNAR) is causing serious clinical problems worldwide. PRSP and BLNAR have become important risk factors for intractable clinical outcome of AOM. PRSP causes a three times higher incidence of intractable AOM than susceptible strains. BLNAR strains show penicillin-binding protein gene mutation and are not only resistant to ampicillin, but also have reduced susceptibility to cephalosporin. The resistant H. influenzae pathogen has shown clonal dissemination in Japan in ways different from those of penicillin-resistant S. pneumoniae. Protection against AOM due to these pathogens may depend on pathogen-specific antibodies. Pneumococcal capsular polysaccharides (PCPs) are type specific and poorly immunogenic in children younger than 2 years old. Approximately 50% of otitis-prone children showed subnormal levels of anti-PCP IgG2 antibody. In our immunological study in children with otitis media, however, otitis-prone children were not unusually vulnerable to infections except those resulting in otitis media. This fact seems to refute the presence of a broad immunological deficit in these children. Some pathogen-specific antibodies may be directed against protein immunogens such as pneumococcal surface protein A (PspA) of S. pneumoniae, P6 of NTHi, and UspA of M. catarrhalis. The levels of antibody to P6 of NTHi in healthy children were significantly higher than those in the otitis-prone children after the age of 18 months. In general, individual antibody levels in otitis-prone individuals did not have an age-dependent rise. The failure to develop a good antibody response to common antigens such as PspA and P6 may enable the pathogen to cause persistent or recurrent disease.

Genetic Characteristics and Clonal Dissemination of β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae Strains Isolated from the Upper Respiratory Tract of Patients in Japan

ABSTRACT We evaluated the recent prevalence of antimicrobial-resistant Haemophilus influenzae isolated from the upper respiratory tracts (URT) of patients in Japan. Mutations in the ftsI gene, which encodes penicillin binding protein 3 (PBP3), and the clonal dissemination of the resistant strains were also investigated. A total of 264 H. influenzae isolates were collected from patients with URT infections. According to the criteria of the Clinical and Laboratory Standards Institute for the susceptibility of H. influenzae to ampicillin (AMP), the isolates were distributed as follows: 161 (61.0%) susceptible strains (MIC ≤ 1 μg/ml), 37 (14.0%) intermediately resistant strains (MIC = 2 μg/ml), and 66 (25.0%) resistant strains (MIC ≥ 4 μg/ml). According to PCR-based genotyping, 172 (65.1%) of the isolates had mutations in the ftsI gene and were negative for the β-lactamase (bla) gene. These 172 isolates were thus defined as genetically β-lactamase-negative ampicillin-resistant (gBLNAR) strains. The ftsI mutant group included 98 (37.1%) strains with group I/II mutations in the variable mutated region (group I/II gBLNAR) and 74 (28.0%) strains with group III mutations in the highly mutated region (group III gBLNAR). Eighty-seven (33.0%) of the isolates were genetically β-lactamase-negative ampicillin-susceptible (gBLNAS) strains. The group III gBLNAR strains showed resistance to β-lactams. Only five strains (1.9%) were positive for a bla gene encoding TEM-type β-lactamase. The three clusters consisting of 16 strains found among the 61 BLNAR strains (MIC ≥ 4 μg/ml and without the bla gene) showed identical or closely related DNA restriction fragment patterns. Those isolates were frequently identified among strains with a MIC to AMP of 16 μg/ml. The current study demonstrates the apparent dissemination and spread of a resistant clone of H. influenzae among medical centers in Japan. The gBLNAR strains show a remarkable prevalence among H. influenzae isolates, with the prevalence increasing with time. This fact should be taken into account when treating URT infections.

Formation of biofilm by Haemophilus influenzae isolated from pediatric intractable otitis media

OBJECTIVES The aims of this study are to evaluate biofilm formation by nontypeable Haemophilus influenzae (NTHi) isolated from children with acute otitis media (AOM) and its relation with clinical outcome of the disease. METHODS Biofilm formations by NTHi clinical isolates from pediatric AOM patients were evaluated by a crystal violet microtiter plate and a 98 well pin-replicator assay with a confocal laser scanning microscopy (CLSM). Optical density values of clinical isolates were compared with a positive control and the ratio of clinical isolates to a positive control was defined as biofilm formation index (BFI). RESULTS 84.3% clinical isolates of NTHi were biofilm forming strains (BFI> or =0.4). The BFI represented the levels of biofilm formation and adherence on the surface. The identical strains isolated from both middle ear fluids (MEFs) and nasopharynx showed biofilm formation at the same level. The prevalence of biofilm forming isolates was significantly higher among the susceptible strains than resistant strains. The level of biofilm formation of NTHi isolated from AOM cases who was not improved by amoxicillin (AMPC) was significantly higher than that of NTHi isolated from AOM cases who was improved by AMPC. CONCLUSION We clearly showed the biofilm formation of clinical NTHi isolates from AOM children. In addition, the biofilm formed by NTHi would play an important role in persistent or intractable clinical course of AOM as a result of lowered treatment efficacy of antibiotics.

Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells.

OBJECTIVES The aim of this study is to examine the internalization of nontypeable Haemophilus influenzae (NTHi) into human epithelial cells. METHODS Bactericidal assay was applied to examine the effects of antibiotics against cell-adherent NTHi using HEp-2 cells. A trans-well chamber assay was applied to examine the internalization and penetration of NTHi using Detroit562 cells. RESULTS The adherence of NTHi to HEp-2 cells was noted after 2h of incubation. Azithromycin had a strong bactericidal effect against both cell-associated and non-adherent NTHi, while ceftriaxone did not show bactericidal effects on NTHi adhered to the HEp-2 cells. Three (60.0%) out of five NTHi isolates from the nasopharynx of children with intractable acute otitis media (AOM) internalized into and subsequently penetrated through the epithelial cells at various degrees. Azithromycin had a strong bactericidal effect against the cell-internalized NTHi, while ceftriaxone was bactericidal only against extracellular NTHi. CONCLUSION The potential of NTHi as the intracellular pathogen may contribute to the persistent existence of this pathogen that result in the prolonged and intractable clinical course of AOM. Azithromycin may be a therapeutically significant antibiotic for patients with prolonged respiratory tract infections due to NTHi.

Serotype Distribution and Penicillin Resistance of Streptococcus pneumoniae Isolates from Middle Ear Fluids of Pediatric Patients with Acute Otitis Media in Japan

ABSTRACT Out of 175 pneumococcal isolates from middle ear fluids, 26.3% were penicillin-resistant S. pneumoniae (PRSP). Serotypes 19F and 23F occurred most frequently among PRSP strains. The 7-valent pneumococcal conjugate vaccine (PCV) showed better coverage of PRSP strains (87.0%). The 7-valent PCV may reduce the prevalence of PRSP in Japan.

Antimicrobial resistance of Haemophilus influenzae isolated from the nasopharynx of Japanese children with acute otitis media

Conclusion. A high prevalence of penicillin-binding protein gene-mutated (PGM) strains of Haemophilus influenzae should be taken into account when treating otitis media in children. Objective. To evaluate the prevalence of β-lactamase-non-producing ampicillin-resistant strains of H. influenzae with mutations in the ftsI gene encoding penicillin-binding protein 3 (PBP3) among children with otitis media. Material and methods. A total of 644 nasopharyngeal isolates of H. influenzae were collected from pediatric acute otitis media patients with or without otitis media with effusion at the clinics of the Department of Otolaryngology—Head and Neck Surgery, Wakayama Medical University Hospital and 6 affiliated hospitals in Wakayama Prefecture between January 1999 and December 2003. MICs to ampicillin (AMP), cefdinir (CFD), cefaclor (CCL), cefpodoxime (CPD) and cefcapene (CFPN) were determined by a microbroth dilution method according to the recommendations of the National Committee for Clinical Laboratory Standards. Types of mutations in the PBP3 gene (ftsI) were evaluated by means of a polymerase chain reaction (PCR)-based genotyping method. The β-lactamase gene (bla) was also identified by means of PCR. Results. β-lactamase-producing (BLP) strains having the bla gene were identified in 16 isolates (2.5%). PGM strains were identified in 279 isolates (43.3%). There were 242 PGM1-non-BLP strains (37.6%) with mutations in the variable mutated locus of ftsI, 35 PGM2-non-BLP strains (5.4%) with mutations in the highly mutated locus of ftsI and 2 BLP-PGM strains (0.3%) with mutations in ftsI that produced β-lactamase. BLP-non-PGM strains producing β-lactamase without mutations in ftsI were identified in 14 isolates (2.2%). MICs of PGM1-non-BLP strains to AMP were 0.5–2.0 µg/ml. The MIC90 of CDN to the PGM1-non-BLP strains was the lowest (0.06 µg/ml). The proportion of PGM1-non-BLP strains increased rapidly during 1999–2002 and then decreased in 2003. In contrast, the proportion of PGM2-non-BLP strains increased in 2003.

Genotyping of Streptococcus pneumoniae and Haemophilus influenzae Isolated from Paired Middle Ear Fluid and Nasopharynx by Pulsed-Field Gel Electrophoresis

Twenty-eight isolates of Streptococcus pneumoniae and 30 isolates of Haemophilus influenzae from paired nasopharynx and middle ear fluids of 21 children with acute otitis media (AOM) were evaluated to determine genotypes by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE). Among the 28 isolates of S. pneumonaie, 21 isolates (75.0%) possessed mutations in the pbp1a,pbp2x, and pbp2b genes, and 7 isolates (25%) had mutations in the pbp2x gene. Nineteen isolates (67.9%) expressed the mefE gene, and 5 isolates (17.9%) possessed the ermB gene. Among the 30 isolates of H. influenzae, 5 isolates (16.7%) had mutations in pbp3 genes, 3 isolates (10.0%) produced β-lactamase, and 2 (6.7%) isolates possessed mutations both in the pbp3 gene and the β-lactamase gene. Ten out of the 14 pairs (71.4%) of the restriction fragment patterns of S. pneumoniae from paired nasopharynx and middle ear fluids were indistinguishable following PFGE analysis. The same patterns were identified among 5 children of unrelated families. The restriction fragment patterns of H. influenzae isolated by PFGE were also indistinguishable in 13 out of the 15 pairs (86.7%) of nasopharynx and middle ear fluids. The genetic similarity between nasopharyngeal and middle ear isolates suggests that the causative bacteria migrate from the nasopharynx into the middle ear cavity via the Eustachian tube. Some resistant strains might be prevalent. In children with AOM, the nasopharynx could have been colonized by a virulent strain of bacteria that replaced the benign, commensal bacteria and then progressed to the middle ear, where they caused AOM.

Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR.

The appropriate clinical applications of pneumococcal polysaccharide vaccines against recent increases in antimicrobial resistant Streptococcus pneumoniae (S. pneumoniae) urgently require accurate analytical methodologies for determining and characterizing the serotypes. The results of current immunological determinations of serotypes with anti-capsular polysaccharide-specific sera are difficult to interpret in terms of quellung changes of the pneumococci. In this study, we applied the multiplex PCR technique for the rapid identification of pneumococci and simultaneous rapid determinations of their serotypes and genotypes that directly correlated with antimicrobial susceptibilities from nasopharyngeal secretions (NPS). Serogroups 6, 19F and 23F were the predominant capsular types of S. pnuemoniae in the NPS samples. Strains of serotypes 19F and 23F frequently had mutations in pbp1a, pbp2x and pbp2b and expressed ermB and mefA; they also were mostly resistant to both penicillin G (PCG) and clarithromycin (CAM). Two NPS samples contained the strain of serotype 19F together with the strain of serotype 23F, although only the strain of serotype 19F was identified by a conventional bacterial culture. Pneumococci were identified in six NPS samples and their serotypes determined by the multiplex PCR, while a conventional bacterial culture failed to identify the pathogens. Our findings suggest that PCR-based serotyping and genotyping can provide an accurate and rapid distribution of pneumococcal serotypes and antimicrobial resistance. The relatively minor populations in the nasopharynx may be determined using molecular techniques.

Increase of Macrolide‐Resistant Streptococcus pneumoniae‐Expressing mefE or ermB Gene in the Nasopharynx among Children with Otitis Media

Objective: To evaluate prevalence of macrolide resistant strains and the genotypes of the resistance among Streptococcus pneumoniae isolated from the nasopharynx of children with otitis media.