Methods For Detection In Helicobacter Pylori

Developments in this field are due to the following:

1. The clinical importance of this bacterium which is not only responsible for peptic ulcer disease but also gastric malignancies. H. pylori infection has been recognized by the International Agency on Research on Cancer as a type 1 carcinogen and is indeed the first infection to be involved in cancer in humans. Another cancer, gastric MALT lymphoma, can also be cured by eradicating the bacterium. 2. The key role of clarithromycin in eradication treatment: clarithromycin has low MICs, good diffusion, and limited influence of low pH. Nevertheless, clar-ithromycin resistance may occur and jeopardize the treatment success. The eradication rate of the main treatment combination (clarithromycin and amoxicillin plus a proton pump inhibitor bd for 7 days) decreases from 88% to 18% if the strain is resistant. The prevalence of the resistant strains in adults varies from less than 5% in Northern Europe to more than 15% in Southern Europe. It is even higher (20-25%) among children. The main risk factor for H. pylori resistance to clarithromycin is consumption of this drug, essentially for respiratory tract infections.[15]

As resistance is caused by a limited number of point mutations, it was possible to develop methods for their detection. The three major point mutations are A2143G (70%), A2142G (12%), and A2142C (3%) corresponding to A2059G, A2058G, and A2058C respectively in E. coli numbering. However, this last percentage is underestimated because in most of the studies, the A2142C mutation was not sought.[15]

PCR-Based Methods

These mutations can obviously be detected by sequencing, but many other PCR-based techniques have been developed (Table 2). The most widely used is PCR-RFLP with BsaI and BbsI.[8,16] A third enzyme (BceAl) allowing the detection of the A2142C mutation has recently been proposed.[17] However, these techniques have drawbacks, most are time consuming and are subject to the risk of PCR contaminations.

The main progress has been the application of real-time PCR and the fluorescence resonance energy transfer (FRET) chemistry. The first attempt concerning H. pylori was made on strains and was published in 1999.[18] Since then, it has been extended to both the detection of the bacterium and its resistance to clarithromycin, directly in gastric biopsies.[19-22] In our study,[21] we only tested sensitivity as biopsies were obtained from patients who had failed a first eradication therapy and were all H. pylori positive. Indeed, in only one case out of 200 (99.5% sensitivity) did we not obtain amplification products. The results of FRET-melting curve analysis (MCA) were compared to those of two phenotypical methods of susceptibility testing performed on the corresponding strains obtained by culture: Etest and agar dilution. The results of the two phenotypical methods agreed in 195 cases (98%), and among them the FRET-MCA was concordant in 188 (96.4%). Using agar dilution as the gold standard, sensitivity and specificity of FRET-MCA were 98.4% and 94.1%, respectively. The mutations identified by FRET-MCA were also compared to those obtained by PCR-RFLP performed on a subset of biopsy specimens

Table 2 Molecular methods for Helicobacter pylori testing of clarithromycin resistance

Based on amplification of 23S rRNA gene

- Sequencing

- RFLP (restriction fragment length polymorphism)

- OLA (oligonucleotide ligation assay)

- DEIA (DNA enzyme immunoassay)

- PHFA (preferential homoduplex formation assay)

- INNO-LiPA (line probe assay)

- Mismatch PCR

- DG-DGGE (double gradient-denaturing gradient gel electrophoresis)

- FRET-MCA (fluorescence resonance energy transfer-melting curve analysis)

Based on hybridization

- FISH assay (fluorescence in situ hybridization assay)

and a good agreement was found. In only three cases (1.5%) did we find a clarithromycin-resistant phenotype and a wild-type genotype only, suggesting either that mutations in the 23S rDNA were not detected by this method, or that other mechanisms not related to mutations may be involved.

Matsumura et al. using a similar biprobe assay reported a perfect sensitivity and specificity (100%) for H. pylori detection on 186 biopsies and also a perfect correlation with the PCR-RFLP results in 151 cases. However, few cultures were performed in this study and the A2142C mutation was not reported.[20]

The assay described by Lascols et al. is different for H. pylori detection as they used an LC-Red 640 primer as the sensor probe. Sensitivity and specificity of their assay tested on 196 biopsies, in comparison to histology and culture, were 97% and 94.6%, respectively. Regarding clarithromycin resistance determined for 59 of 66 H. pylori positive biopsies, the sensitivity and specificity were 91% and 100%, respectively.[22]

In contrast to the previous studies using a biprobe assay, Gibson et al.[18] and Chisholm et al.[19] used a monoprobe assay with SYBR green 1 as the quencher which transfers its energy to a probe labeled with Cy5. For H. pylori detection, Chisholm et al. reported a sensitivity of 85% and a specificity of 98% on 121 biopsies tested, 17 being H. pylori positive. Sensitivity and specificity for the detection of clarithromycin resistance seem inferior, and few resistant cases were tested.

The FRET-MCA also has the potential of detecting mixed infections, essentially wild type and a resistant mutant, which may not be seen by using phenotypical methods. Indeed, mixed infections represented 21% of the total and three genotypes were even detected in one case. A genotype can be detected when it represents at least 10% of the DNA mixture.

The other advantages of this assay are:

• Obtaining a very quick result (2 hr) with limited manpower which can be compared to the week's delay in obtaining phenotypical results

• Avoiding any handling post-PCR which is an important source of contamination in laboratories

• Avoiding the demanding transport conditions necessary to perform culture and susceptibility testing. Shipment by mail at ambient temperature is convenient.

The limitation of the assay described above[21] is the difficulty in separating the A2143G and A2142G mutations. The melting curves for the two genotypes differ only by 1°C (Fig. 1), which does not allow a reliable distinction in routine testing. Despite the fact that the A2142G mutation is usually associated with higher MICs

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