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The dynamics of clarithromycin and azithromycin efficacy against pulmonary Haemophilus influenzae infection in rats were evaluated. Efficacy was measured by reduction in pulmonary H. influenzae burden on days 3 and 7 postinoculation. Clarithromycin therapy was effective on day 3 or 7 of therapy, while azithromycin was effective on day 7 but not on day 3 of therapy. Both macrolides produced marked efficacy against all six strains of H. influenzae tested, including four strains for which MICs were above the susceptible breakpoint (8 microgram/ml) concentration of clarithromycin. The two macrolides demonstrated markedly different pharmacokinetic characteristics, with clarithromycin present in both blood and tissue, while azithromycin was concentrated primarily in tissue. During pulmonary infection in rats, H. influenzae was found in both intracellular locations and an extracellular location in the lung. Blood concentrations of clarithromycin and azithromycin approximated human pharmacokinetics, and the blood concentrations for either macrolide rarely exceeded MICs for H. influenzae. At dosages producing blood concentrations similar to values achieved clinically, clarithromycin produced efficacy on day 3 of therapy, while both clarithromycin and azithromycin were equally effective on day 7. The different dynamics of clarithromycin and azithromycin suggest that length of therapy should be considered as a key parameter in evaluations of drug efficacy.

Fifty-four of 59 (91.5%) clarithromycin-resistant isolates of Helicobacter pylori from different patients possessed either the A2143G (formerly A2058G) or the A2144G (formerly A2059G) mutation in the gene encoding 23S rRNA. The A2143G mutation was significantly more likely to occur in isolates with MICs exceeding 64 mg/L (65% versus 30% with the A2144G mutation; P = 0.01). The majority (26 of 31; 83.9%) of isolates with the A2143G mutation had MICs exceeding 64 mg/L. Peptic ulcer disease recurred in a substantial proportion of patients infected with H. pylori strains containing either the A2143G or the A2144G mutation.

The National Committee for Clinical Laboratory Standards (NCCLS) methods for susceptibility testing of Haemophilus influenzae in Haemophilus test medium allow a pH range of 7.2 to 7.4. However, it is known that bacteria may appear to be less susceptible to macrolides at lower pHs. Forty-four strains of H. influenzae were tested for their susceptibilities to clarithromycin and azithromycin by the disk diffusion and broth microdilution methods. The isolates appeared to be less susceptible at pH 7.2 than at pH 7.4 by both methods. Clarithromycin was less active at pH 7.2 against 43% of the isolates by the disk diffusion method and against 52% of the isolates by the broth microdilution method. Similarly, azithromycin was less active at pH 7.2 against 41 and 45% of the isolates by the disk diffusion and broth microdilution methods, respectively. Forty-two isolates were classified as clarithromycin susceptible and all isolates were classified as azithromycin susceptible by the disk diffusion method, regardless of the medium pH. However, only 21 isolates were clarithromycin susceptible at pH 7.2 and 34 isolates were susceptible at pH 7.4 by the broth microdilution method, even though quality control results indicated valid testing at both pHs. This study indicated that the results of tests of the susceptibility of H. influenzae with clarithromycin and azithromycin are highly dependent on the pH of the medium. Test results and their interpretations varied even when the medium pH was within the NCCLS-approved range and, coupled with the current NCCLS breakpoint of 8 microg/ml in the case of clarithromycin, may explain some of the observed discordances between the disk diffusion and broth microdilution methods.

A clinical trial was conducted in Argentina to determine the efficacy of clarithromycin plus lansoprazole for the treatment of Helicobacter pylori in duodenal ulcers and non-ulcer dyspepsia. PCR-RFLP was conducted on an 820-bp amplified product of the ureC gene of H. pylori to determine the genetic heterogeneity of 83 pretreatment and 21 post-treatment isolates. Twelve different restriction patterns were observed when digested with Sau 3A or Hha I, resulting in 40 different RFLP types. Comparison of isolates before treatment to after treatment showed that 20 of 20 patients had the same RFLP type. In addition, the presence of the cytotoxin-associated gene (cagA) and the vacuolating gene (vacA) were determined. All pretreatment isolates were positive for vacA whereas 75% of the pretreatment isolates were positive for cagA. The results of this study indicate that a high degree of heterogeneity exists among H. pylori and that infection is not limited to a small number of RFLP types.

We have developed a rapid PCR-oligonucleotide ligation assay that can discriminate single base substitutions that are associated with clarithromycin resistance in Helicobacter pylori. Susceptible isolates were wild type at positions 2143 and 2144 (cognate to 2058 and 2059 in Escherichia coli), while 93% of the resistant isolates contained A-to-G mutations at either position and 7% of the isolates contained A-to-C mutations at position 2143. In addition, the MIC for 86% of the resistant isolates with an A2143 mutation was > or = 64 micrograms per ml, and that for 89% of the resistant isolates with an A2144 mutation was < or = 32 micrograms per ml.

Background: Transition mutations (A-G) at residue 2143, cognate to position 2058 in the Escherichia coli 23S rRNA gene, have been shown to confer resistance to macrolides in Helicobacter pylori. This study reports the finding that transversion mutations (A-C) can occur at 2143 as well., Materials and Methods: Three clarithromycin-resistant H. pylori isolated from three different patients after treatment with clarithromycin were analyzed for point mutations by cycle sequencing of a 163-bp amplified region surrounding residue 2143 within the conserved loop of the 23S rRNA gene., Results: Nucleotide sequence comparisons of a 163-bp amplified product revealed that A-C transversion mutations occurred at position 2143. H. pylori isolated from the patients prior to treatment were susceptible to clarithromycin and displayed no polymorphism at 2143., Conclusion: This is the first report to show that A-C transversion mutations at position 2143 can confer resistance to clarithromycin in H. pylori and further support the role that mutations at position 2143 play in conferring macrolide resistance in H. pylori.

Twelve clarithromycin-resistant Helicobacter pylori isolates (100% of resistant isolates examined) from seven different patients each contained an A-->G transition mutation within a conserved loop of 23S rRNA. A-->G transition mutations at positions cognate with Escherichia coli 23S rRNA positions 2058 and 2059 were identified. Clarithromycin-susceptible H. pylori isolates from 14 different patients displayed no polymorphisms in a conserved loop within domain V of 23S rRNA. The study is the first to report mutations in H. pylori associated with resistance to an antimicrobial agent used in established peptic ulcer treatment regimens.

The optimal method for the determination of the minimum inhibitory concentration (MIC) of antimicrobials against Helicobacter pylori has not been established. The epsilometer agar diffusion gradient test (E-Test; AB Biodisk, Solna, Sweden) was compared with broth microdilution, the reference method, and disk diffusion for the antimicrobial susceptibility testing of 122 clinical isolates of H. pylori to ampicillin, clarithromycin, and metronidazole. Isolates were considered to be resistant when the MIC values was > 8 micrograms/ml for either ampicillin or metronidazole and > 2 micrograms/ml for clarithromycin. For an individual isolate, the MICs for ampicillin and clarithromycin determined by broth microdilution and the E-test were highly reproducible, with replicate results being within +/- 1 log2 dilution. The correlation between the MICs determined by E-test and broth microdilution was excellent for both ampicillin and clarithromycin (90.1% and 88.5% were within +/- log2 dilution, and 98.3% and 96.7% of the values were within +/- 2 log2 dilution, respectively). In no instance did the interpretation of "sensitive" or "resistant" differ. Conversely, only 70.5% of the E-test results of metronidazole were within +/- 1 log2 dilution of the broth microdilution results. In addition, 15 (12.3%) of the H. pylori isolates interpreted as resistant by the E-test were sensitive by the broth microdilution method. All discrepancies occurred when the E-test MIC values fell between 8 and 32 micrograms/ml. The results of the ampicillin and clarithromycin disk diffusion assay correlated 100% with the results of the broth microdilution. However, these data suggest that when the E-test MIC results of metronidazole yield values between 8 and 32 micrograms/ml, the MIC should be reevaluated by another method.

A multi-country clinical trial was conducted in ten European countries to determine the efficacy of clarithromycin-omeprazole dual therapy for treating Helicobacter pylori infection in peptic ulcers. Gastric biopsies were cultured for H. pylori before and after treatment. PCR-RFLP was used to determine the genetic heterogeneity of 100 H. pylori isolates from pretreatment and posttreatment biopsies. An 820 bp amplified fragment of the ureC gene was digested with the restriction enzymes Sau3A and Hhal. Fourteen different Sau3A patterns and 15 different Hhal patterns were identified among the pretreatment isolates. In combination, 42 different RFLP types were identified. Comparison of isolates before treatment with those after treatment showed that five of ten patients on clarithromycin-omeprazole dual therapy had the same RFLP type and that all 12 patients on omeprazole therapy alone had the same RFLP type. All isolates were susceptible to clarithromycin prior to treatment, while seven of ten patients on clarithromycin-omeprazole therapy had H. pylori that was resistant to clarithromycin after therapy and 11 of 12 patients on omeprazole therapy had isolates susceptible to clarithromycin after treatment. In addition to PCR-RFLP typing, the presence of the cytotoxin-associated gene (cagA) and the vacuolating gene (vacA) was determined; 79% of the isolates were cagA-positive and all were vacA-positive. The results of this study indicate that infection of H. pylori in Europe is not restricted to a few RFLP types. [ABSTRACT FROM AUTHOR]