The aim of the work was to compare H. pylori clarithromycin-resistance according two methods. Etest was performed on H. pylori isolated from gastric biopsy samples. TaqMan Real-Time-PCR (RT-PCR) was performed on paraffin-embedded gastric biopsy samples of the same patients. Forty-seven out of 88 strains were resistant to clarithromycin by Etest, whereas RT-PCR detected this resistance on paraffin-embedded specimens of 50 patients. RT-PCR performed on paraffin-embedded biopsy specimens of 47 patients infected with H. pylori resistant to clarithromycin as detected by Etest, revealed the presence of a resistant strain only in 40 samples. RT-PCR performed on samples of 41 patients harbouring clarithromycin-susceptible H. pylori strains showed the presence of 31 susceptible and 10 resistant strains. RT-PCR detected 18 cases with heteroresistant status. The difference between the two tests in detecting clarithromycin-resistance was not statistically significant even if RT-PCR detected more resistant cases. The genotyping resistance on paraffin-embedded gastric biopsy specimens may be used to establish resistance to clarithromycin before the treatment when culture and susceptibility testing are not available. In case of failure of an empirical clarithromycin-based triple antimicrobial treatment, RT-PCR performed on paraffin-embedded biopsy sample will establish the primary resistance to clarithromycin. In addition, this test can be useful for epidemiological investigation as well as for monitoring the evolution of clarithromycin resistance along the time. The recommended first-line treatment for H. pylori infection is a proton pump inhibitor combined with amoxicillin and clarithromycin. Unfortunately, primary clarithromycin resistance is increasing worldwide, and it is regarded as the main factor reducing the efficacy of eradication therapy [1-3]. In vitro clarithromycin susceptibility testing are currently based on the agar dilution method or gradient diffusion susceptibility testing (Etest) performed on H. pylori isolated from biopsy specimens [4]. Even if agar dilution is the method recommended by Clinical Laboratory Standard Institute (CLSI), this method and Etest have yielded similar results [5]. Furthermore, the sensitivity of the culture of H. pylori from gastric biopsy samples may be reduced, even in expert hands, by strain fastidiousness, by low bacterial density due to past antibiotic treatments, by loss of viability or overgrowth of contaminating bacteria deriving from delayed transport of the specimens [4]. Recently, a polymerase chain reaction has been developed as an alternative tool for the detection of clarithromycin resistance not only on gastric biopsy specimens but also on paraffin-embedded gastric biopsy samples, thus offering the possibility of performing studies on archival materials [6-8]. This technique accurately assesses mutations in the peptidyltransferase region encoded in domain V of H. pylori 23S ribosomal RNA gene conferring clarithromycin resistance. Despite a dozen of point mutations having been identified, three of them – namely A2143G, A2142G, A2142C – have been shown to be responsible for 90% of cases of primary clarithromycin resistance in Western countries. Moreover, PCR is more sensitive in detecting mixed populations with clarithromycin susceptible and clarithromycin resistant strains (i.e. heteroresistant status) in the gastric biopsy samples [7-9]. The aim of this study was to compare clarithromycin resistance evaluated by Etest performed on H. pylori isolated from gastric biopsy samples with the results obtained by TaqMan Real-Time-PCR (RT-PCR) performed on paraffin-embedded gastric biopsy samples of the same patients.

Helicobacter pylori clarithromycin resistance detected by Etest and TaqMan real-time polymerase chain reaction: a comparative study.

MONNO, Rosa;SOLEO, Leonardo;
2012

Abstract

The aim of the work was to compare H. pylori clarithromycin-resistance according two methods. Etest was performed on H. pylori isolated from gastric biopsy samples. TaqMan Real-Time-PCR (RT-PCR) was performed on paraffin-embedded gastric biopsy samples of the same patients. Forty-seven out of 88 strains were resistant to clarithromycin by Etest, whereas RT-PCR detected this resistance on paraffin-embedded specimens of 50 patients. RT-PCR performed on paraffin-embedded biopsy specimens of 47 patients infected with H. pylori resistant to clarithromycin as detected by Etest, revealed the presence of a resistant strain only in 40 samples. RT-PCR performed on samples of 41 patients harbouring clarithromycin-susceptible H. pylori strains showed the presence of 31 susceptible and 10 resistant strains. RT-PCR detected 18 cases with heteroresistant status. The difference between the two tests in detecting clarithromycin-resistance was not statistically significant even if RT-PCR detected more resistant cases. The genotyping resistance on paraffin-embedded gastric biopsy specimens may be used to establish resistance to clarithromycin before the treatment when culture and susceptibility testing are not available. In case of failure of an empirical clarithromycin-based triple antimicrobial treatment, RT-PCR performed on paraffin-embedded biopsy sample will establish the primary resistance to clarithromycin. In addition, this test can be useful for epidemiological investigation as well as for monitoring the evolution of clarithromycin resistance along the time. The recommended first-line treatment for H. pylori infection is a proton pump inhibitor combined with amoxicillin and clarithromycin. Unfortunately, primary clarithromycin resistance is increasing worldwide, and it is regarded as the main factor reducing the efficacy of eradication therapy [1-3]. In vitro clarithromycin susceptibility testing are currently based on the agar dilution method or gradient diffusion susceptibility testing (Etest) performed on H. pylori isolated from biopsy specimens [4]. Even if agar dilution is the method recommended by Clinical Laboratory Standard Institute (CLSI), this method and Etest have yielded similar results [5]. Furthermore, the sensitivity of the culture of H. pylori from gastric biopsy samples may be reduced, even in expert hands, by strain fastidiousness, by low bacterial density due to past antibiotic treatments, by loss of viability or overgrowth of contaminating bacteria deriving from delayed transport of the specimens [4]. Recently, a polymerase chain reaction has been developed as an alternative tool for the detection of clarithromycin resistance not only on gastric biopsy specimens but also on paraffin-embedded gastric biopsy samples, thus offering the possibility of performing studies on archival materials [6-8]. This technique accurately assesses mutations in the peptidyltransferase region encoded in domain V of H. pylori 23S ribosomal RNA gene conferring clarithromycin resistance. Despite a dozen of point mutations having been identified, three of them – namely A2143G, A2142G, A2142C – have been shown to be responsible for 90% of cases of primary clarithromycin resistance in Western countries. Moreover, PCR is more sensitive in detecting mixed populations with clarithromycin susceptible and clarithromycin resistant strains (i.e. heteroresistant status) in the gastric biopsy samples [7-9]. The aim of this study was to compare clarithromycin resistance evaluated by Etest performed on H. pylori isolated from gastric biopsy samples with the results obtained by TaqMan Real-Time-PCR (RT-PCR) performed on paraffin-embedded gastric biopsy samples of the same patients.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11586/130163
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