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See article on page 463
Testing for the presence of Helicobacter pylori and subsequent eradication of the infection is a key aspect in the management of dyspepsia, which is now the largest single area of cost for primary care in the United Kingdom.1Since the bacterium was discovered in 1982 and its role in the aetiology of peptic ulcer disease became established, significant advances have been achieved in antimicrobial treatment with current regimens enabling successful eradication in 85 to 90% of patients.2 3 The reasons for treatment failure in the remaining small but significant group of infected individuals is often not clearly established but resistance to metronidazole and to clarithromycin are generally considered to be the primary factors. Both of these antibiotics are widely used in current regimens for eradicating H pylori although rates of resistance vary significantly from 10 to 50% for metronidazole, and from 0 to 15% for clarithromycin, depending on the population group surveyed.4 For instance, in a recent study of dyspeptics attending an open access endoscopy clinic in mid-Essex, we found pretreatment rates of 6% for clarithromycin and 37% for metronidazole (L Teare, personal communication). The fact that resistance rates for clarithromycin may be gradually increasing is particularly worrying as it is a most useful antibiotic against H pylori because of its absorption and stability properties in the stomach. Consequently it is a key component in eradication regimens, particularly those for first line treatment in areas of high prevalence of metronidazole resistance.3
The mode of action of clarithromycin after penetrating the cell wall is to bind to ribosomes and disrupt protein synthesis. Decrease in the binding of clarithromycin to the ribosome is linked to the development of resistance which in turn is attributed to various point mutations in the two 23S rRNA genes of H pylori. These mutations were first reported in US isolates in 1996 by Versalovic and colleagues5 and since then have been confirmed in clinical isolates from France,4 Sweden,6Canada,7 and the Netherlands.8 The two most common mutations are those in which adenine residues are replaced by guanine at positions 2143 and 2144 (transition mutations A2143G and A2144G equivalent to Escherichia colicoordinates 2058 and 2059) although other rarer mutations may occur (e.g. A2116G and A2143C). Detection of the two main mutations were first based on the use of PCR assays combined with digestion with specific restriction endonucleases. Such molecular tests were advocated for speed as they could be completed on cultures within 24 hours whereas conventional microbiological culture based susceptibility testing (E-test or disc diffusion) requires up to four days. Subsequent technical developments have led to the availability of more rapid mutant specific probe based assays, namely a PCR-oligonucleotide ligation assay9 and a DNA enzyme linked immunoassay (DEIA).10 The optimal conditions were defined for each probe which discriminated single base variations and a complete correlation with culture methods was found.
Marais et al, in this issue (see page 463), have described a further novel development to the DEIA technique, in which mutant specific probes were used in a rapid (one day) laboratory assay that could be applied directly to gastric biopsy samples. This assay tested both for the presence of H pylori and for evidence of resistance to clarithromycin, but most importantly without the need for culture. The technique has the potential of providing a result within 24 hours whereas conventional culture from biopsy samples takes up to five days with a further three to four days for susceptibility testing. The method, which was based on the use of a colorimetric hybridisation assay with four probes—for the wild (sensitive) type and three resistant mutants—offered a significant improvement in reporting time as the result could be available within a day of endoscopy. When tested on 33 biopsy samples infected with resistant strains of H pylori, most mutations were either at A2144G or A2143G with a few at A2143C. For some samples, the results highlighted the presence of multiple genotypes with the A2143C mutation mostly present either with the wild type or with the A2143G mutation. These results were interpreted as possibly mixtures of strain types in the sample or as heterozygotic with respect to the two 23S rRNA gene copies.
The use of DNA amplification or probing technology, or both, in antibiotic resistance gene detection is a developing area of clinical bacteriology with already recognised applications for detection of resistance in Staphylococcus aureus (MRSA) and in Mycobacterium tuberculosis. The work of Marais and colleagues demonstrates how their time-saving and potentially cost effective approach can be applied toH pylori. It will be interesting to see to what extent this approach finds a place in the routine diagnostic laboratory, particularly as definitive therapy forH pylori depends on information about metronidazole as well as clarithromycin susceptibilities. At present, no DNA based assay is available for detecting metronidazole resistance, and as resistance to that antibiotic is widespread, susceptibility results are essential for prescribing.
In conclusion, this is an interesting new technical development, although in the short term it would seem unlikely to replace the need for culture based sensitivity testing in the clinical laboratory given the relatively sophisticated technical aspects involved combined with the need to establish rigorous quality indicators to satisfy accreditation requirements. At present there is no information on such mutations in strains isolated from UK patients, so the reliability of the DNA assay would also need to be assessed rigorously on local populations in view of the recognised genomic diversity ofH pylori.
See article on page 463
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