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ABSTRACT Background and Aim: Isoniazid is one of the essential first line drugs in TB treatment. Resistance rate to this drug is increasing in many parts of the world. Mutations in KatG and inhA genes are frequently the cause of resistance of mycobacterium TB to INH. The aim of this study was to find a precise method for early detection of mutations associated with resistance of mycobacterium TB to INH. Materials and Methods: After performing sensitivity tests, presence of mutations in special loci of katG and inhA genes in 90 specimens obtained from positive cultures of TB patients was investigated. PCR-RLFP method was used to detect mutations in katG 315 codon. The PCR product resulting from the reproduction of this genetic segment (620 bp) was digested by restriction enzyme MspI. To identify mutations in inhA gene, MAS-PCR technique was used. Results: 34.5% of the INH resistant strains had Thr315 phenotype and 65.5% had Ser 315 phenotype. Thr 315 is 100% specific for determination of INH resistant isolates. Among 52 resistant isolates, 34.6% had Arg and 65.4% had Leu in codon 463. The frequencies of mutations in the (-15C → T) locus of inhA gene were 20% and 16.7% in MDR and Non MDR isolates respectively. Conclusions: By using PCR-RFLP with restrictive enzyme and MAS PCR mutations in codon 315 of KatG gene and promoter location of inhA gene can be identified. These methods are simpler and cheaper than other methods and provide early accurate and reliable results. Key words: Mycobacterium tuberculosis, Isoniazid resistance, PCR-RFLP, KatG, inhA. Conflict of Interest: Nill Received: Dec 30, 2009 Accepted: Feb 7, 2010

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ABSTRACT Background and Aim: Current therapeutic regimens used for eradication of H.pylori consist of two antibiotics with proton pump inhibitors and bismuth compounds. Clarithromycin is the most important antibiotic used for treatment of H.pylori infection in Iran. The aim of this study was to identify clarithromycin resistance in H.pylori strains and evaluate role of A2143G, A2142G and A2142C of 23SrRNA point mutations in producing resistance to this drug. Material and Methods: This was a cross-sectional study which included 263 patients who had referred to endoscopy department of Hajar hospital, in Shahrekord city, in 2007. Biopsy samples were cultured on brucella agar medium and incubated. For identification of H.pylori gram stain, urease, catalase, oxidase tests were used and PCR method was used to identify Urec gene. Standard method of NCLSI (National Clinical and Laboratory Standards Institute) was used for assessment of clarithromycin resistance. Specific primers and restriction enzymes BsaI and MboII were used to detect A2143G and A2142G mutations by PCR-RFLP method. Also for determination of A2142C mutation, specific primers and PCR method were used. Results: Isolation of 84 strains of H.pylori (31.94%) was confirmed by means of PCR method. Among 19 (22.62%) of clarithromycin resistant strains, 13 (68.40%), 3 (15.78%) and 2 (10.52%) had A2143G, A2142G, A2142C mutations respectively and one unknown mutation was detected in 23SrRNA gene. Conclusion: Because of considerable resistance to clarithromycin, precise diagnosis of this mutation by molecular approach in other parts of the country seems necessary. Key words: H.pylori, clarithromycin, 23SrRNA, PCR-RFLP Conflict of Interest: Nill Received: Nov 2, 2009 Accepted: March 28, 2010

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Background and Aim: There are few studies on the isolation of Brucella abortus and Brucella melitensis from patients´ sera. The aim of this study was simultaneous isolation of pathogenic brucella species from human serum samples by multiplex PCR method. Material andMethods: 50 blood and serum samples isolated from patients with clinically suspected brucellosis were inoculated into brucella agar medium and we used multiplex-PCR, with three primers to detect brucella species. We incubated 0.5 ml of serum in Brucella broth for 72 hours at 37 °C in 5% carbon dioxide. To confirm the results of PCR, the PCR products were restricted by restriction enzymes, TaqI and RsaI. Result: From 50 blood samples 4 (8%) cultures were positive. Using biochemical tests and after determination of the characteristics of the positive cases, they were identified as B. melitensis. After multiplex PCR, 9 cases (18%) were positive and 41 cases (82%) were negative. Among the positive sera, B. melitensis was identified in 7 cases (78%) and B. abortus in 2 cases (22%). Of 50 blood samples, 5 (10%) were positive and 45 (90%) were negative for B. melitensis. All the results were confirmed by PCR-RFLP. Conclusion: Our study showed that multiplex PCR method was simple, rapid and is more sensitive for isolation of brucella from serum (especially B. abortus and B. melitensis) in comparison to complete blood. Keywords: Brucella spp., Multiplex-PCR, Serum blood, PCR-RFLP Received: Jul 7, 2014 Accepted: Dec 10, 2014

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Background and Aim: Nowadays, the importance of pathogenicity of non-tuberculosis mycobacteria is well known. Generally, this group, in addition to the respiratory system can cause lymph nodes, skin, soft tissue and bone disorders. Identification of Mycobacterium by culture and biochemical tests may take several weeks and may not be useful for definitive diagnosis. PCR-RFLP (PRA) technique of the hsp65 gene using HaeIII and BstEII enzymes is a precise method for species differentiation, in comparison to phenotypic methods. It is a quick and inexpensive method for detection of mycobacterial species. This study aimed to assess the prevalence of non-tuberculous mycobacteria isolated from the patients referring to tuberculosis center (TB) of Kashan University of Medical Sciences.

Material and Method: The study included 106 patients who had been referring to TB Center of Kashan University of Medical Sciences, from 1391 to 1394. The samples were tested by biochemical diagnostic tests. At the same time identification of the strains was made by use of PRA. Amplification of 441-bp fragment was performed by PRA for detection of hsp65 gene. The PCR products were digested with HaeIII and BsteII enzymes and analysis was performed on the basis of electrophoresis.

Results: Molecular analysis showed non-tuberculosis mycobacteria in 4(8.3%) sputum samples,i.e. one positive sample (o.9 %)  for every one of the following strains: M. abscessus, M. senegalense, M. fortuitum and M.kansasii.  

Conclusion: The results of this study showed that some cases of tuberculosis in Kashan are due to non-tuberculosis mycobacteria. Also use of PRA analysis of hsp65 gene for clinical specimens is a rapid and useful tool for identification of species of mycobacterium which is helpful for early diagnosis, treatment and control of tuberculosis.  

Keywords: Mycobacterium tuberculosis complex, Non-tuberculosis mycobacteria, PCR-RFLP ، hsp65.

Received: Jul 31, 2016      Accepted: Sep 5, 2016

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Background and Aim: Malassezia yeast can become pathogenic under certain conditions in humans. Malassezia has various species which can cause a number of skin diseases, such as pityriasis versicolor (tinea versicolor), seborrheic dermatitis and folliculitis and even systemic infections. In the present study, Malassezia species isolated from the patients with pityriasis versicolor were identified by PCR-PFLP molecular method.
Material and Methods: In this study, the scraping specimens of the trunk and scalp of the patients with pityriasis versicolor were cultured on Dixon agar medium. Finally, one-hundred Malassezia colonies were obtained. The genomic DNA was extracted by phenol–chloroform method and then was studied by use of PCR-PFLP molecular method. D1-D2 segment in the area of 26srDNA of ITS gene was proliferated by specific primers and then the PCR products were exposed to CfoI restrictive enzyme.
Results: Among 100 Malassezia colonies, the most common Malassezia species were; M. globosa (44%), M. globosa/M. restricta (27%), M. restricta (11%), M. sympodialis (7%), M. sympodialis/M. restricta (4%), M. globosa/M. restricta/M. furfur (1%), M. sympodialis/M. restricta/ M. globosa (1%) and unknown species (5%).
Conclusion: The dominant species isolated from patients with pityriasis versicolor were M. globosa, M. restricta and M. sympodialis, respectively. Thirty-three percent of the specimens had more than one Malassezia species. Therefore, rapid PCR-RFLP method is recommended for identification of Malassezia species in epidemiological studies and production of more effective medicines.
Key words: Malassezia; Pityriasis versicolor; PCR-RFLP.
 
Received: Oct 1, 2016      Accepted: Feb 11, 2017

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Background and Aim: Infections, particularly lung infections, pose significant challenges for patients in the intensive care unit (ICU) and play a crucial role in patient morbidity and mortality. Candida, Aspergillus, and Cryptococcus are key contributors to fungal infections in these patients. This study aimed to determine the prevalence of fungal colonization, specifically Candida species, in hospitalized ICU patients.

Materials and Methods: Fifty bronchoalveolar lavage (BAL) samples from both right and left lungs were collected from twenty-five patients admitted to hospitals in Fasa. Candida strains were isolated and identified in 23 samples (46%) using PCR-RFLP and sequencing. The in vitro susceptibility of these 23 Candida species to four triazole antifungal drugs was tested using the broth microdilution method.

Results: Candida albicans was the most frequently isolated species (n = 9, 39.1%), followed by C. krusei (n = 4, 17.4%), C. tropicalis (n = 3, 13.1%), C. glabrata and C. dubliniensis (n = 2 each, 8.7%), and C. orthopsilosis, C. guilliermondii, and C. famata (n = 1 each, 4.3%). All Candida species were susceptible to the four triazoles, except for four C. krusei strains, which were resistant to fluconazole and showed susceptibility dose-dependent (SDD) response to itraconazole, and two C. glabrata strains, which were SDD to both fluconazole and itraconazole.

Conclusion: Given the rising number of patients at risk for fungal infections and the potential for resistant fungal colonization in ICU settings, it is crucial to prioritize the investigation of these infections and the implementation of advanced diagnostic methods.