The multiplex-PCR-based detection and genotyping of diarrhoeagenic Escherichia coli in diarrhoeal stools.

In several hospitals in Beirut, Lebanon, 77 isolates of Escherichia coli were successfully derived from the stools of patients with diarrhoeal diseases, by culture on MacConkey or MacConkey-sorbitol agar. When the isolates were screened, using a multiplex PCR, 14 (from 14 different patients) were each found positive for one of the various genes defining the enterotoxigenic (five), enteroinvasive (four), enteroaggregative (three) or enteropathogenic (two) groups. Genotyping of these 14 diarrhoeagenic isolates, by pulsed-field gel electrophoresis, indicated that all were genomically distinct with the exception of two of the enteroaggregative isolates (which were of the same genotype). The E. coli apparently involved in diarrhoeal disease in Beirut therefore belong to at least four different diarrhoeagenic groups and show strain variation within each group. Diarrhoea in the absence of diarrhoeagenic E. coli may be the result of infection with bacteria other than E. coli or viral or parasitic enteropathogens.

Comparative analysis between Pseudomonas aeruginosa genotypes and severity of symptoms in patients with unilateral or bilateral otitis externa.

Random amplified polymorphic DNA (RAPD) analysis was done on 32 isolates of Pseudomonas aeruginosa. These isolates were obtained from 22 patients who presented to the emergency room in a major medical center in Beirut, Lebanon, during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa. Patients had yellowish to greenish discharge, moderate to severe external auditory canal swelling, moderate to severe pain, and periauricular cellulitis. None of these patients had intrinsic predisposing factors. An ear swab was obtained from both ears of patients, cultured on trypticase soy agar. P. aeruginosa was identified on the basis of pyocyanine production and API identification kits. RAPD analysis was done by using two primers (10 mer and 21 mer primers) and appropriate PCR conditions on extracted DNA. Our data have shown 23 RAPD patterns (A-W) distributed among the 32 P. aeruginosa isolates. RAPD patterns were reproducible. Twenty of 32 isolates were recovered from 10 patients with bilateral otitis externa. The remaining 12 of 32 isolates were recovered from 12 different patients with unilateral otitis externa. Eleven RAPD patterns (A,B,C,D,E,F,H,I,R,U,V) were associated with severe clinical symptoms, including severe pain, severe external auditory canal swelling, periauricular cellulitis, and a yellowish discharge. The remaining RAPD patterns were not associated with severe infections. This denotes a possible association between certain genotypes and severity of symptoms.

Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group.

A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.

Heterogeneity of rRNA gene restriction patterns of multiresistant serotype 6B Streptococcus pneumoniae strains.

Three multiresistant serotype 6B Streptococcus pneumoniae strains were isolated from the middle ear fluids of children undergoing tympanostomy in Atlanta. Because multiresistant 6B pneumococci have been reported to spread from a single clone, the three isolates were compared with 13 other multiresistant 6B pneumococci by hybridization of endonuclease-restricted DNA fragments with a digoxigenin-labeled cDNA probe complementary to 16 and 23S rRNAs (ribotyping). The ear isolates were heterogeneous, whereas six of the other pneumococcal isolates were alike, indicating a need for additional studies to determine the possibility of clonal spread.

Safety and immunogenicity of meningococcal A and C polysaccharide conjugate vaccine in adults.

A meningococcal vaccine containing group A and C polysaccharides conjugated to CRM197 was evaluated in 50 adults. Vaccinees were entered into one of five groups: 30 adults received a single dose of either 22, 11, or 5.5 micrograms of the conjugated A-C vaccine; 10 received an approved meningococcal vaccine; and 10 received saline injections. Local and systemic reactions to vaccines were recorded, and immune responses were determined. The experimental meningococcal vaccine was well tolerated, with the most frequent reaction being pain at the injection site. Both A and C polysaccharide components of the experimental vaccine were highly immunogenic, and total antibody concentrations 1 month postvaccination were not significantly different from the mean antibody concentrations among adults given the approved meningococcal vaccine. In addition, significant rises in immunoglobulin G, A, and M antibodies to both A and C polysaccharides occurred. Antibody concentrations measured at 6 and 12 months postvaccination had declined but remained significantly higher than prevaccination concentrations. Postvaccination meningococcal group C functional antibody activity increased more than 600-fold for both the polysaccharide and the conjugate vaccines. Further studies of this conjugated meningococcal vaccine are indicated for young children and infants.

Moxalactam in the treatment of typhoid fever.

Moxalactam therapy was evaluated in 25 patients with typhoid fever. A satisfactory initial response was observed in all cases. Treatment for 3 days resulted in a much higher relapse rate (three of five patients) than did treatment for 5 days (one of nine patients). None of the 11 patients treated for 10 to 11 days relapsed. Moxalactam is effective in typhoid fever, but its use is better restricted to special indications.

Potential role of temocillin in the treatment of biliary sepsis.

Temocillin concentrations were determined in the gallbladder bile and/or common bile duct bile obtained intraoperatively from 20 patients, and in the T-tube bile of 5 postoperative patients. Blood samples were also obtained for determining the concomitant serum antibiotic concentrations. In 6 patients with cholelithiasis, but without common bile duct obstruction or acute infection, the mean temocillin concentrations were 890 mg/L in gallbladder bile and 1030 mg/L in common bile duct bile. In the group of 6 patients with common bile duct obstruction, the antibiotic concentrations ranged between 5.6 and 88 mg/L (mean 38.8 mg/L) in gallbladder bile and between 'undetectable' and 700 mg/L in common bile duct bile. In patients with biliary sepsis, a further reduction in temocillin bile concentrations was observed, and postoperatively, the T-tube bile temocillin concentrations were in the range of 21 to 460 mg/L (mean 130 mg/L). The clinical efficacy of temocillin in the 7 patients with acute cholecystitis was judged to be satisfactory. Our results suggest that temocillin may be considered as a potentially useful antibiotic in the treatment of patients with biliary tract sepsis caused by susceptible organisms.

Treatment of typhoid fever with cefamandole.

Cefamandole therapy was evaluated in nine patients with typhoid fever. Six patients, including all five who received the antibiotic by continuous intravenous drip (8.0 g daily), were cured. Dosage schedules resulting in maintenance of antibiotic concentrations in serum high above the MIC seemed to correlate well with treatment success.