Novel Real-Time PCR Assay for Detection of Helicobacter pylori Infection and Simultaneous Clarithromycin Susceptibility Testing of Stool and Biopsy Specimens

Abstract

A biprobe real-time PCR protocol, followed by hybridization melting point analysis, to detect point mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance was established and evaluated in a clinical study. Of 92 patients who underwent endoscopy, 45 were found to be H. pylori infected and invariably were also culture positive. Of the 45 isolates, 11 were shown to be resistant to clarithromycin by E-test. With respect to the detection of H. pylori infection, PCR showed sensitivities of 100% in biopsies and 98% in stool specimens and a specificity of 98% in both biopsy and stool samples. All clarithromycin-sensitive cases were identified as such by PCR in both biopsy and stool samples. Of the cases with a resistant strain, eight were identified as such in stool DNA and nine were identified in biopsy DNA. Failure of PCR to detect the resistant genotype in the biopsy DNA, stool DNA, or both (one case) was associated with mixed populations. In these cases, patients had not been treated for H. pylori infection before, and the sensitive population showed to be present in considerably higher numbers than the resistant population. In five of six cases in which infection with a resistant genotype only was identified by PCR, the patients had received clarithromycin-based eradication therapy in the past. Thus, the assay presented provides a highly accurate noninvasive method to detect H. pylori infection in stool and at the same time allows for culture-independent clarithromycin susceptibility testing.Helicobacter pylori is a gram-negative bacterium associated with different digestive diseases, such as gastritis, peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer (3). At present, several diagnostic assays for H. pylori detection are available (23). Invasive methods requiring gastric endoscopy include rapid urease testing, culture, histology, and molecular diagnostics. Noninvasive approaches include fecal antigen detection, serologic testing, and urea breath testing. During recent years, noninvasive methods gained in importance; however, no information on resistance against antibiotics can yet be obtained with these tests. Clarithromycin is an integral part of first-line therapies to treat H. pylori infection. As demonstrated by a meta-analysis of published data, susceptibility or resistance to clarithromycin result in eradication rates of 81 to 95% and 0 to 48%, respectively (8). Since clarithromycin is a widely used antimicrobial drug, the prevalence of clarithromycin-resistant H. pylori strains is increasing continuously.Resistance of H. pylori to clarithromycin is mainly due to an adenine-to-guanine transition at positions 2142 and 2143 and to an adenine-to-cytosine transversion at position 2142, which are included in the peptidyltransferase loop of the 23S rRNA (24). Recently, several PCR based methods, such as PCR-restriction fragment length polymorphism (17), PCR-DNA-enzyme immunoassay (13), reverse hybridization line probe assay (22), and real-time PCR...

New concepts of resistance in the treatment of Helicobacter pylori infections

“…Agar dilution is the recommended test of choice although the Etest has proven to provide clinically reliable except with metronidazole where it tends to overestimate the presence of resistance and must be confirmed by agar dilution methods. Molecular methods of detection of specific changes in the organisms' genome are alternative approaches to detection of resistance and in theory allow a more rapid detection of resistance as well as detection of resistance using stool or biopsy specimens 23,24 .…”

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

“…The lowest H. pylori DNA concentration detected was 10 fg, equivalent to 5 bacterial cells (Mikula et al, 2003;Schabereiter-Gurtner et al, 2004). In a range between 10 to 10 6 fg correlation coefficients, obtained by lin- ear regression analysis from all independent experiments, were higher than R 2 = 0.99, indicating highly efficient and reproducible reactions.…”

“…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).…”

Helicobacter pylori – Not Only a Gastric Pathogene?

“…Culture is currently accepted gold standard for the diagnosis of gastric H. pylori (Makristathis et al, 2004). This method achieve 80-90% sensitivity and 90-100% specificity rates (Hep 2003).…”