Developing the role of PCR for detection and characterisation of helicobacters in human infection

Chisholm, Stephanie Angela (2004). Developing the role of PCR for detection and characterisation of helicobacters in human infection. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f582

Abstract

This study aimed to explore and develop the role of PCR in the investigation of human Helicobacter infections. Overall performance of PCR assays (both conventional and real-time) was influenced by certain key factors, notably specimen transport conditions, DNA extraction methods, primers, and inherent target sequence variation. PCR-based detection and characterisation of non-viable H. pylori in gastric biopsies demonstrated the benefit of this approach as an alternative to culture, while the design and application of the first PCR test to detect both H. pylori and 'H. heilmannii'-like organisms (HHLOs) showed a higher rate of HHLO infection (2.3 %) than had been reported previously. The potential of PCR for further strain characterisation was demonstrated by determining clarithromycin susceptibility direct from gastric biopsy by a real-time PCR (LightCycler) assay for mutation detection. Development of an analogous test for metronidazole appears less feasible, as examination of a unique strain set suggested that, contrary to earlier reports, genetic mutations in rdxA and frxA may not be the principal resistance mechanism. Additionally, development of a novel multiplex PCR assay to determine vacA genotypes direct from gastric biopsies provided a useful tool to facilitate surveillance and confirmed that the s 1ml genotype is a common feature of peptic ulcer disease-associated isolates. Application of three novel LightCycler PCR assays to detect cagA tyrosine phosphorylation motifs (TPMs) demonstrated that TPM A was common in South East England. No significant associations were observed between disease status and cagA TPM. Extension of the PCR-based approach for non-invasive detection of H. pylori from stool specimens showed that it was less sensitive and less specific than stool antigen testing. Nevertheless, development of real-time nested PCRs that performed identically to conventional assays demonstrated the potential for next-day diagnosis from stools and offered the possibility of future screening of larger populations. Application of PCR to investigate extra-gastric chronic inflammatory conditions provided the first evidence of H. pylori DNA in the human bladder, although its significance as a causal factor in human disease remains unclear. The study provides new evidence to support wider use of conventional and real-time PCR-based assays as powerful tools for future investigation of gastric and extra-gastric Helicobacter infections.