Fast and Accurate Quantitative Detection of Helicobacter pylori and Identification of Clarithromycin Resistance Mutations in H . pylori Isolates from Gastric Biopsy Specimens by Real-Time PCR

Abstract

Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of H. pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density.Many diagnostic assays have been developed for Helicobacter pylori: culture, histology, rapid urease test, urea breath test, serology, stool antigen test, and molecular-based tests (24). Culture has the great advantage of permitting subsequent determination of the antimicrobial susceptibility of the strain isolated, in particular to macrolides. Indeed, the macrolide drug clarithromycin is the key component of many combination therapies used to eradicate H. pylori, and macrolide resistance is the most important cause of treatment failure (13, 22). However, disadvantages of culture include special conditions for specimen transportation, the use of complicated media with special conditions for maintenance, the need for special incubation conditions, and the length of time necessary to obtain a result (20).Clarithromycin resistance in H. pylori is due to the lack of binding of the macrolides to the 23S rRNA components of the bacterial ribosome due to modification of the target site by occurrence of a single spontaneous point mutation in the peptidyltransferase region of the 23S rRNA gene (17,25). Mutations A2142G and A2143G are the most often observed, with the A2142C mutation being much rarer (25). Other mutations (A2115G, G2141A, and T2717C) have been described but appear to be exceptional (10,1...

Quantitative real-time PCR for the clinical detection of Helicobacter pylori

“…Our results show that the sensitivity of the RT-PCR assay was higher than that of the standard reference methods RUT, bacteriological analysis and histology, supporting the findings of other workers (He et al, 2002;Lascols et al, 2003;Schabereiter-Gurtner et al, 2004). Indeed, 16 of the 81 "uninfected" patients actually gave large numbers of H. pylori DNA copies when assessed by RT-PCR.…”

“…Recently, five studies have shown that RT-PCR can be used to detect and quantify H. pylori DNA directly from gastric biopsy samples (He et al, 2002;Kobayashi et al, 2002;Lascols et al, 2003;Mikula et al, 2003;Schabereiter-Gurtner et al, 2004). Additionally, RT-PCR has been validated for the quantitative analysis of the low level of bacterial infection in immunized and experimentally infected mice, suggesting that this methodology may also be useful for the evaluation of new anti-H. pylori drugs and vaccines (Mikula et al, 2003).…”

“…Recently, real-time PCR (RT-PCR) quantitative assays have been developed to quantify H. pylori in gastric and stool samples (He et al, 2002;Kobayashi et al, 2002;Lascols et al, 2003;Mikula et al, 2003;SchabereiterGurtner et al, 2004). These methods allow rapid and accurate quantification of H. pylori and have increased in importance because they can facilitate the monitoring of Genetics and Molecular Biology, 30, 2, 431-434 (2007) Copyright by the Brazilian Society of Genetics.…”

PCR detection of clarithromycin-susceptible and -resistant Helicobacter pylori from formalin-fixed, paraffin-embedded gastric biopsies

“…Several previous reports have described the identification of clarithromycin-resistant H. pylori by PCR from clinical specimens, using cultures grown from gastric biopsies 12,18,19 and more recently from formalin-fixed, paraffin-embedded gastric biopsy tissue. 20 The use of a PCR assay to detect clarithromycinresistant H. pylori in formalin-fixed, paraffinembedded tissues offers several potential advantages over culture-based methods.…”

Antimicrobial susceptibility of Helicobacter pylori and mechanisms of clarithromycin resistance in strains isolated from patients in Uruguay

“…This study showed a resistance rate in the AD subset that fell into this category. Susceptibility testing could be implemented through culture and susceptibility tests; however, genotypic techniques permit detection of the organism as well as its virulence mechanisms and changes in virulence against this drug and others (32)(33)(34). For example, dual priming oligonucleotidebased multiplex PCR has been proposed to the detection of clarithromycin resistant strains of H. pylori in gastric samples (35).…”