Rapid detection of clarithromycin resistant Helicobacter pylori from children's gastric biopsy specimens by polymerase chain reaction-restriction fragment length polymorphism / 中华儿科杂志

<p><b>OBJECTIVE</b>Infection with clarithromycin-resistant Helicobacter pylori (Hp) is often predictive of treatment failure. Susceptibility testing for Hp could guide therapy of Hp infections. However, agar dilution approved by the Clinical and Laboratory Standards Institute (CLSI) to test for antimicrobial susceptibility of Hp is time consuming (results are often not available in a week or more). So a more expeditious method is necessary. The purpose of this study was to evaluate polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) test performed directly on gastric biopsy specimen from children to detect 23S rRNA mutations (A2143G and A2144G) indicating clarithromycin resistance.</p><p><b>METHODS</b>All biopsy specimens were derived from patients presenting with upper gastrointestinal symptoms, submitted to endoscopy in the Affiliated Children's Hospital, Zhejiang University School of Medicine from September 2006 to February 2007. No patients had undergone eradication therapy. Thirty-nine samples randomly selected from positive specimens by rapid urease test, were homogenized in 500 microl brucella broth with 30% glycerol. The 200 microl homogenized fluid was used to purify genomic DNA with the kit according to the instructions provided by manufacturer, and the rest was used to isolate Hp strains by culturing. All the Hp isolates were tested for clarithromycin susceptibility with the agar dilution and classified as resistant if the minimum inhibitory concentrations (MIC) exceeded 1 microg/ml. Simultaneously, PCR-RFLP analysis was performed in order to identify 23S rRNA mutations (A2143G and A2144G). Finally, the two methods were compared by statistics. The agar dilution was used as a standard to determine the sensitivity and specificity of the PCR-RFLP assay.</p><p><b>RESULTS</b>Of the 39 samples, agar dilution and PCR-RFLP method respectively detected 13 (33.3%) and 14 (35.9%) clarithromycin-resistant gastric specimens. The sensitivity and specificity of PCR-RFLP for the detection of Hp in biopsy specimens were both 92%. The positive and negative predictive value was 85.7% and 96% respectively. No statistically significant difference was found between the two methods (chi2=0.06, P>0.05). The rate of Hp resistance to clarithromycin significantly increased compared with a previous report from the authors' hospital in 2004 (chi2=6.20, P<0.05).</p><p><b>CONCLUSIONS</b>Rising clarithromycin resistance rates were observed in children who visited the authors' hospital. PCR-RFLP test is reliable and rapid for detection of clarithromycin resistance directly on gastric biopsy specimen from children and may help choose appropriate antibiotic in Hp eradication therapy.</p>

Preliminary study on in-vitro induction of antibiotic resistance in Helicobacter pylori strains isolated from children / 中华儿科杂志

<p><b>OBJECTIVE</b>Many clinical studies indicated that Helicobacter pylori (Hp) strains rarely acquired resistance to amoxicillin but easily to clarithromycin and metronidazole. However, it was unclear whether the antibiotic resistance of Hp strains was induced or passively selected during long-term or frequent treatment with metronidazole, clarithromycin and amoxicillin. To compare the propensity of acquired resistance to antibiotics, Hp strains were exposed to amoxicillin, clarithromycin and metronidazole in vitro in this study.</p><p><b>METHODS</b>All Hp strains were clinical isolates, derived from biopsy specimens of patients taken during endoscopy in the Affiliated Children's Hospital, Zhejiang University School of Medicine from December 2004 to July 2005. To seek susceptible strains, the minimum inhibitory concentrations (MICs) of the three antibiotics were determined by using Epsilometer test (E-test) method. In vitro induction was carried out on serially doubling concentrations of antibiotics incorporated into agar. Isolates were also transferred at least three times on antimicrobial agent-free medium, followed by a redetermination of the final MICs to assess the stability of the selected resistance.</p><p><b>RESULTS</b>7 strains were exposed to antibiotics in vitro. After 6 - 17 passages on antibiotic plates, 7 and 3 strains respectively acquired resistance to metronidazole and clarithromycin, while none of the strains were resistant to amoxicillin. The inductive folds were different among three groups: 8 - 128 folds in metronidazole group; 1 - 256 folds in clarithromycin group; 2 - 16 folds in amoxicillin group. After three transfers on antimicrobial agent-free medium, the MICs decreased significantly in amoxicillin group (P < 0.05) but had no change in metronidazole group and clarithromycin group (P > 0.05).</p><p><b>CONCLUSIONS</b>The metronidazole resistance in Hp was easily selected. Strains resistant to clarithromycin could be selected, but the amoxicillin resistance could not be selected after in vitro induction for Hp isolated from children. The correlation between in vitro and in vivo outcomes suggests that acquired resistance was the main cause for the resistance in Hp strains. The laboratory results of in vitro antibiotic induction could help predict the actual rate of resistance and select appropriate antibiotics for treatment.</p>

Prevalence of resistance to metronidazole in Helicobacter pylori from children and induction of resistance in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the prevalence of resistance of Helicobacter pylori (H. pylori) to metronidazole (MTZ) and the distribution change of minimal inhibitory concentrations (MICs) in H. pylori from local children, evaluate the applicability of E-test for MIC determination, and display the propensity of acquired-resistance to MTZ after induction of resistance in vitro.</p><p><b>METHODS</b>One group of 44 H. pylori isolates obtained from Oct. 2002 to Nov. 2003 and another 83 H. pylori isolates obtained from Dec. 2004 to Jul. 2005 from the local children who underwent gastroscopy in the Children's Hospital affiliated to Zhejiang University Medical School and were diagnosed as H. pylori-associated gastritis or peptic ulcer were studied. Susceptibility was tested by agar dilution method or E-test method. In 11 randomly selected metronidazole-sensitive isolates (MTZ(S)), resistance was induced in vitro with MTZ.</p><p><b>RESULTS</b>The resistance rate was 31.8% (14/44) in the 44-islates obtained from Oct. 2002 to Nov. 2003 and 51.8% (43/83) (chi(2) = 4.64, P < 0.05) in 83-isolates obtained from Dec. 2004 to Jul. 2005, respectively. The distribution of MICs were < 0.125 - 128 mg/L and 0.25- > 256 mg/L, in which, the MIC(50) was 0.5 mg/L and 16 mg/L, the MIC(90) was 128 mg/L, respectively. Comparing to agar dilution method which is recommended by National Committee for Clinical Laboratory Standards (NCCLS) for MIC, E-test was significantly associated with agar dilution method (chi(2) = 32.38, P < 0.001). The sensitivity, specificity, agreement rate of E-test were 73.08%, 100%, 87.27%, respectively, while there were factors of 2(2) to 2(6) difference in MICs between the results obtained by E-test and agar dilution. For all the 11 MTZ(S) isolates inducted resistance in vitro with MTZ, 16 MICs were achieved through 7 - 9 (7.2 +/- 0.6) passages of induction in vitro, and 100% acquired-resistance to MTZ through 8 - 10 generations; as a result, 10 of 11 MTZ(S) isolates achieved stable high-level resistance (256 mg/L for 2 and > 256 mg/L for 8) and 1 stable 64 mg/L resistance to MTZ.</p><p><b>CONCLUSIONS</b>The prevalence of resistance to MTZ seems to be increasing in H. pylori from local children. To avoid missed diagnosis of H. pylori resistant to MTZ (MTZ(R)), agar dilution method was needed when detecting susceptibility of H. pylori to MTZ. Resistance to MTZ of H. pylori from children is readily induced in vitro.</p>