Molecular assessment of clarithromycin resistant Helicobacter pylori strains using rapid and accurate PCR-RFLP method in gastric specimens in Iran

Currently, a seven-day, triple-drug regimen has been recommended as one of the first-line therapies for Helicobacter pylori management in which clarithromycin is a key component. Development of clarithromycin resistance leads to the long term assessment of the efficacy of clarithromycin in the triple-drug regimen. The aim of this study was to rapidly and directly assess clarithromycin resistance point mutations on gastric biopsy specimens by using PCR-RFLP method. Biopsy samples were obtained over a 6-months period of 2009, from 200 dyspeptic patients referred to Shahrekord University of Medical Sciences, Iran. Initially, rapid urease test was performed and then DNA was isolated from each tissue and used for molecular analysis such as PCR (for H. pylori diagnostic) and PCR-RFLP (for Cla resistance determination). RUT and PCR results showed that 164 (82%) of the patients were H. pylori-positive. Resistance was evaluated in 164 samples by using enzymes BsaI and MboII. Thirty nine (39) (23/78%) clarithromycin-resistant strains were detected which were identified as 15 (9.15%) A2143G, 15 (9.15%) A2142G and 9 (5.49%) mix strains. The results showed that PCR-RFLP method had a high accuracy to detect A2142G and A2143G mutations associated with resistance to clarithromycin in the minimum possible time.Key words: Helicobacter pylori, clarithromycin resistance, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

PP-005 Clarithromycin resistance assessment in Helicobacter pylori isolates by using 23S rRNA gene molecular markers

Background: H. pylori is a relatively fastidious and microaerophilic microorganism and therefore standard phenotypic susceptibility tests, even in the hands of experts, are slow and can take at least 10 14 days. Molecular based diagnostic assays by using molecular markers for resistance detection offer an attractive alternative approach to obtain susceptibilities to antibiotics with greater accuracy and speed, and the possibility of a same day result. The aim of this study is the assessment of clarithromycin resistance by using molecular markers. Methods: This cross-sectional descriptive study was performed on 200 gastric biopsy specimens which were obtained from patients undergoing upper gastrointestinal tract endoscopy in Hajar hospital of Shahrekord, by using TaqMan real-time PCR. Initially, H. pylori strains were identified by RUT and PCR. Then, by this regard that accumulation of mutations associated with resistance to clarithromycin were in the region between nucleotides 2142 2144 of 23S rRNA gene, the first probe was designed to be able the distinguish between sensitive and resistant strains. Finally four probes were designed that each be able to identify only one mutation associated with a particular level of clarithromycin resistance. Results: Out of 200 samples, 164 (82%) were H. pylori positive. Overall, clarithromycin susceptible strains were detected in 105 (64.02%) patients and clarithromycin resistance were detected in 59 (35.98%) which were identified as 4 (2.44%) A2144G, 26 (15.85%) A2143G, 15 (9.15%) A2143C and 20 (12.19%) A2142G point mutations. Purely resistant strains were detected in 38 (23.17%), while heteroresistant were found in the remaining 16 (9.76%) cases. Genotype of 5 (8.47%) strains was not detected. This data was confirmed by PCR-RFLP technique. Conclusion: Results showed that Real-time PCR assay in combination with molecular markers has high accuracy to simultaneously identify H. pylori and clarithromycin resistance types directly in gastric biopsy specimens in short time

Characterization of Isolated Hydatid Cyst from Slaughtered Livestock in Yasuj Industrial Slaughterhouse by PCR-RFLP

Background & aim: Given the existence of 10 different genotypes of the parasite Echinococcus granulosus from different hosts and intermediate and final impact of these genotypes in the life cycle of the parasite and its transmission to humans, the purpose of this study was to determine the molecular characterization of isolates of hydatid cysts in industrial slaughterhouses of Yasuj city. Methods: In this study, 93 animal isolates (56 goat, 31 sheep and 6 cattle) were collected from the industrial slaughterhouse of Yasuj city. The genomic DNA corresponding to protoscolices was extracted, using the standard Phenol–Chloroform method. The fragment of DNA-ITSI of each sample was assessed by PCR with designed primers of EgF, EgR and then amplified. Moreover, the PCR products were assessed by electrophoresis and digested by the Alu I and Rsa I enzymes. RFLP products were evaluated by electrophoresis. Results: Using a PCR test, rDNA-ITSI of all isolates of similar size bands and 1000 bp were obtained respectively.The patterns generated by RFLP using Alu I and Rsa I enzymes, showed Echinococcus granulosus G1 genotype in all isolates. Conclusion: This study showed that strain G1 is the predominant strain causing hydatid cysts in different organs of the animal in Yasuj

Enteroaggregative Escherichia Coli (EAEC) in South of Iran

Introduction:  The aim of the present study was to investigate the presence and the frequency of EAEC as etiologic agent of diarrhea in Shiraz. Enteroaggregative E. coli (EAEC) is increasingly recognized as a cause of often persistent diarrhoea in children and adults in both developing and developed countries, and have been identified as the cause of several outbreaks worldwide.   Materials and Method: A total of 715 stool samples were collected from patients with diarrhea in Shiraz. Diarrheagenic E. coli were isolated by biochemical tests and culture from 715 stool samples collected from different hospitals. Diarrheagenic E. coli strains isolated from diarrheal stool samples were examined for the detection of the aggR gene by Real time PCR and PCR method.   Results: In this study, a total of 101 (14.12%) diarrheagenic E. coli were isolated from 715 stool samples collected from different hospitals. Diarrheagenic E. coli were isolated much more frequently in the summer months than other season. Out of these 101 diarrheagenic E. coli identified, 5 were confirmed as EAEC in patient. The high prevalence of EAEC isolates was also found in watery diarrhea.   Conclusion: We therefore, recommend the routine isolation and identification of EAEC strains from patient with diarrhea in all the clinical laboratories and other pathotype diarrhoeagenic E. coli in Iran.   Keywords: Diarrhea, Enteroaggregative Escherichia coli (EAEC), Real-Time PCR