Molecular Detection of Antibiotic Resistance in South African Isolates ofHelicobacter pylori

Rapid diagnosis and treatment ofHelicobacter pylori(H. pylori) presents a challenge. We aimed at investigating the presence ofH. pylori, susceptibility profile, and associated mutations in an effort to validate the effectiveness of GenoType HelicoDR assay inH. pylorityping in our environment. Two hundred and fifty-four biopsy specimens were cultured and DNA extracted from seventy-eight positive cultures using the Qiagen DNA extraction kit. The GenoType Helico DR which employs reverse hybridisation was used to confirm the presence ofH. pylori, determination of its susceptibility to antimicrobials, and detection of mutations conferring resistance to clarithromycin and fluoroquinolones. The organism was isolated from 168/254 (66.1 %) of the specimens by culture. Of the 78 strains used for further investigation, 12/78 (15.38%) were resistant to clarithromycin while 66/78 (84.61%) were susceptible. For fluoroquinolone, 70/78 (89.74%) strains were susceptible while 8 (10.26%) were resistant. Mutations were observed in 17 strains with A2147G being the most prevalent; A2146C and D91N were the least. The reverse hybridisation assay is an easy and fast technique in confirming the presence ofH. pylori, its antimicrobial profile, and associated mutations. Analysis regarding the suitability of this assay forH. pylorityping is warranted in other regions.

KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay

AimsTo evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips.MethodsThe strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input.ResultsThe strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent.ConclusionsThe strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.