Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Effect of Weikangfu granule on the physiopathologic figure of precancerosis of gastric mucosa in patients of chronic gastritis with Pi-deficiency syndrome.

OBJECTIVE: To study the physiopathologic basis of Weikangfu Granule (WKFG) in treating precancerosis of gastric mucosa in patients of chronic gastritis with Pi-deficiency syndrome (CG-PDS). METHODS: One hundred and fifteen patients of CG-PDS who suffered from intestinal metaplasia (IM) and atypical hyperplasia (ATHP) of gastric mucosa, were divided into two groups. The treated group (n = 61) was treated by WKFG with its ingredients modified according to the syndrome type of patients. The control group (n = 54) was treated with Weishu granule. The histopathological and subcellular ultrastructural changes were detected by optical microscope, screening electronic microscope, transmission electronic microscope and histochemical staining; the nuclear and mitochondrial ultrastructure of gastric mucosa were analyzed with energy dispersion X-ray analyser and image analysis system. And the changes of cAMP, lipid peroxide (LPO), superoxide dismutase (SOD) before and after treatment in the treated group were measured and compared with those of the health control group consisting of 15 volunteers. RESULTS: The symptomatic and pathological therapeutic effect in the treated group were significantly superior to those in the control group (P < 0.05). The contents of Zn, Cu, cAMP, SOD and (3)H-TdR LCT in gastric mucosa of the treated group before treatment were all lower than those of the healthy control group, yet all these indexes markedly increased after treatment, while serum LPO level, which increased before treatment was lowered after treatment. All the changes showed statistical significance (P < 0.05 or P < 0.01). CONCLUSION: WKFG can reverse IM and ATHP in patients of CG-PDS, and the effect may be realized by way of increasing the level of Zn, Cu, cAMP and SOD in gastric mucosa, promoting cell differentiation, enhancing cellular immunity and reducing oxygen free radicals and lipid peroxidation.