9-100) and 100% specificity (95% CI, 71.6-100). The set of probes can discriminate the resistant and susceptible strains, even though they only have one mismatch. We next further tested the method using a selleck compound mixture of the four probes simultaneously in a multiplex detection (figure 1; A-C). In this case, the detection of point mutations was even more robust, which is possibly due to the fact that all probes target the same locus, and as such there is a competition effect between them. However,
with the mixture it is only possible to discriminate between clarithromycin resistant and clarithromycin sensitive strains, as opposed to the discrimination between point mutations that was conferred by using the probes separately. In practical terms and considering the application of PRMT inhibitor the PNA-FISH to the clinical setting, the mixture of probes introduces an important simplification to the method. Figure 1 PNA-FISH detection. A)-C) In smears: A) Susceptible strain in the red channel; B) Resistant strain in the same microscopic field in the green channel; C) Superimposition of both VX-809 channels. D)-K)
In gastric biopsy histological slides. D) Strain visualization using the Hp1 (A2143G) PNA probe; F) Hp2 (A2142G) PNA probe; H) Hp3 (A2142C) PNA probe; K) Hpwt (wild type strain) PNA probe; E),G),I) Visualization of the same microscopic field of D),F),H) with the red channel (negative controls for Hp1, Hp2 and Hp3); J) Visualization of the same microscopic learn more field of K) with the green
channel (negative control for Hpwt). Arrows indicate the presence of H. pylori infecting the gastric mucosa. (Original magnification × 600). Validation of the testing protocol in gastric biopsy slides for clinical application Considering the application of the PNA-FISH method in clinical settings, we used the developed PNA probes to identify and differentiate clarithromycin resistant and susceptible H. pylori strains in histological slides of gastric biopsy samples. Results clearly show that it is possible to discriminate susceptible from resistant H. pylori strains and, in the latter group, to detect the three different mutations, using fluorescence microscopy (figure 1; D-K). Taking into consideration the antibiogram as the gold standard, the PNA-FISH method showed specificity and sensitivity of 90.9% (95% CI, 57.1-99.5) and 84.2% (95% CI, 59.5-95.8), respectively (data not shown). These can probably be explained by the existence of another mechanism of resistance apart from the three point mutations assessed in this study. In fact, association between A2142C, A2142G and A2143G mutations and clarithromycin resistance was defined as approximately 84% in a world wide data compilation [3].