Evaluation of mRNA expression levels of cyp51a and mdr1, candidate genes for voriconazole resistance in Aspergillus flavus

Background: Voriconazole Resistance (VRC-R) in Aspergillus flavus isolates impacts the management of aspergillosis, since azoles are the first choice for prophylaxis and therapy. However, to the best of our knowledge, the mechanisms underlying voriconazole resistance are poorly understood. Objectives: The present study was designed to evaluate mRNA expression levels of cyp51A and mdr1 genes in voriconazole resistant A. flavus by a Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique. Materials and Methods: Five A. flavus isolates with resistance to VRC were examined by a RT-PCR approach. Results: Four out of five isolates revealed cyp51A and mdr1 mRNA overexpression. Interestingly, the isolate, which was negative for cyp51A and mdr1 mRNA expression showed a high voriconazole Minimum Inhibitory Concentration (MIC). Furthermore, a computational-based analysis predicted that voriconazole resistance could be mediated through cooperation with a network protein interaction. Conclusions: Our experimental and in silico findings may provide new insight in the complex molecular pathways of drug resistance and also could assist design an efficient therapeutic strategy for aspergillosis treatment. © 2015 Ahvaz Jundishapur University of Medical Sciences

Expression of efflux pumps and fatty acid activator one genes in azole resistant Candida glabrata isolated from immunocompromised patients

Acquired azole resistance in opportunistic fungi causes severe clinical problems in immunosuppressed individuals. This study investigated the molecular mechanisms of azole resistance in clinical isolates of Candida glabrata. Six unmatched strains were obtained from an epidemiological survey of candidiasis in immunocompromised hosts that included azole and amphotericin B susceptible and azole resistant clinical isolates. Candida glabrata CBS 138 was used as reference strain. Antifungal susceptibility testing of clinical isolates was evaluated using Clinical and Laboratory Standards Institute (CLSI) methods. Complementary DNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technology, semiquantitative RT-PCR, and sequencing were employed for identification of potential genes involved in azole resistance. Candida glabrata Candida drug resistance 1 (CgCDR1) and Candida glabrata Candida drug resistance 2 (CgCDR2) genes, which encode for multidrug transporters, were found to be upregulated in azole-resistant isolates (�2-fold). Fatty acid activator 1 (FAA1) gene, belonging to Acyl-CoA synthetases, showed expression in resistant isolates �2-fold that of the susceptible isolates and the reference strain. This study revealed overexpression of the CgCDR1, CgCDR2, and FAA1 genes affecting biological pathways, small hydrophobic compounds transport, and lipid metabolism in the resistant clinical C.glabrata isolates. © 2016 Tehran University of Medical Sciences. All rights reserved

Identification of Azole Resistance Markers in Clinical Isolates of Candida tropicalis Using cDNA-AFLP Method

Background: Global reports have highlighted the increasing prevalence of Candida tropicalis infections as well as organism's drug resistance. This study aimed at identifying azole resistance markers in clinical isolates of C. tropicalis, which will be a great resource for developing new drugs. Methods: Two susceptible and resistant isolates of C. tropicalis were recovered from an epidemiological investigation of candidiasis in immunocompromised patients. C. tropicalis ATCC 750 was used as reference strain. Antifungal susceptibility to fluconazole and itraconazole was determined using Clinical and Laboratory Standards Institute (CLSI) method. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology and real-time reverse-transcriptase (RT) PCR were used for identification of potential genes involved in azole resistance of C. tropicalis clinical isolates. Results: Five genes encoding the following enzymes were identified as superoxide dismutase (SOD) implicated in antioxidant defense, ornithine aminotransferase (OAT), acetyl ornithine aminotransferase (ACOAT), adenosylmethionine-8-amino-7-oxononanoate aminotransferase (DAPA AT), and 4-aminobutyrate aminotransferase (ABAT)-belonging to pyridoxal phosphate (PLP) dependent enzymes and acting in an important physiological role in many fungal-cell cycles. Real-time RT-PCR confirmed mRNA level of the aforementioned genes. Conclusion: Our findings showed that factors such as PLP-dependent enzymes and SOD might be implicated in drug resistance in C. tropicalis clinical isolate. Therefore, further studies are required to explore the accurate biological functions of the mentioned genes that would be helpful for effective drug development. © 2016 Wiley Periodicals, Inc

Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tehran, Iran

BACKGROUND: Pityriasis versicolor is a superficial infection of the stratum corneum which caused by a group of yeasts formerly named pityrosporium. The taxonomy of these lipophilic yeasts has recently been modified and includes seven species referred as Malassezia. The aim of this study is to compare the distribution of Malassezia species isolated from pityriasis versicolor lesions and those isolated from healthy skins. METHODS: Differentiation of all malassezia species performed using morphological features and physiological test including catalase reaction, Tween assimilation test and splitting of esculin. RESULTS: In pityriasis versicolor lesions, the most frequently isolated species was M. globosa (53.3%), followed by M. furfur (25.3%), M. sympodialis(9.3%), M. obtusa (8.1%) and M. slooffiae (4.0%). The most frequently isolated species in the skin of healthy individuals were M. globosa, M. sympodialis, M. furfur, M. sloofiae and M. restricta which respectively made up 41.7%, 25.0%, 23.3%, 6.7% and 3.3% of the isolated species. CONCLUSIONS: According to our data, M. globosa was the most prevalent species in the skin of healthy individuals which recovered only in the yeast form. However, the Mycelial form of M. globosa was isolated as the dominant species from pityriasis versicolor lesions. Therefore, the role of predisposing factors in the conversion of this yeast to mycelium and its subsequent involvement in pityriasis versicolor pathogenicity should be considered

A survey on Candida colonization prevalence in patients with gastritis, duodenitis and peptic ulcer

Background: Prolonged antiacid and antibiotic usage in gasterointestinal diseases may predispose candidial colonization in GI tract. In order to isolate and diagnose of candida infections in patients with gastritis, duodenitis, gastric ulcer and duodenal ulcer, this study have been planned. Methods: We studied 300 biopsy specimens of patients referred to hospital, 51.7% of the patients were male and the others were female. The isolated fungi were identified by direct examination and culture of specimens. Results: Forthy four cases of yeasts were isolated in this investigation. Isolated yeasts have been identified as follows: 26 cases of C.albicance , I case C.tropicalis, 2 cases of C.krusei, and finally 1 case of unknown yeast. Conclusion: All the patients had a positive history of long lasting antacid taking for gastric ulser or gastritis. Candidiasis must be investigated in patients with gastritis, duodenitis and gastric ulcer, who are refractory to classic therapies and also in patients who have the chronic disease

Efficiency of PCR Method for Screening Pulmonary Tuberculosis Patients under DOTs Protocol Therapy

Tuberculose has increased in the recent years. Use of a sensitive screening method, can be one of the influential parameters in reduction of pulmonary tuberculosis. Current screening method in the laboratories is based on observation of bacilli in stained smear by Ziel-Neelsen procedure. The aim of this research was to study the PCR efficiency in all confirmed patients of Hamadan province. Twenty eight patients were registered as having pulmonary tuberculosis by the tuberculosis comity of the Hamadan province. All these patients had positive results for staining and culture. Amonst these registered patients, we could access only to the 12 patients during the study. Sputums were collected from early stage of diagnosis and continued during treatment at the end of each month. All these patients were under the treatment by the DOTs protocols. All samples were tested by PCR and the results were compared with Ziehl-Neelsen staining method that is recommended procedure by WHO. Compared results showed none of the samples were positive by staining method at the end of second month up to the last month of treatment, while PCR changed to negative gradually. It was negative in 5 and 4 patients at the end of the third and forth month, respectively. Specimens of the three remanied patients were continuously positive up to the end of treatment period. Gradually changing PCR results to negative in three forth of studied patients means it can be applicable screening tools, but one forth remained positive cases needs more study for the evaluation of target gene role and efficiency of the test