Voriconazole resistance genes in Aspergillus flavus clinical isolates.

OBJECTIVE: The present study was designed to discover novel biomarkers involved in voriconazole resistance in clinical isolates of Aspergillus flavus. MATERIALS AND METHODS: Two voriconazole non-wild-type and two voriconazole-wild-type A. flavus clinical isolates were selected to evaluate possible molecular mechanism involved in A. flavus resistance to voriconazole using the mutation assessment, Quantitative real- time PCR of cyp51A and cyp51C genes and complementary DNA- amplified fragment length polymorphism technique. RESULTS: No mutations were seen in the cyp51A and cyp51C genes in voriconazole non-wild-type isolates compared to wild- type and reference strains. Regarding to mRNA expression results, no changes were observed in expression fold of cyp51A and cyp51C mRNA expression level in first non- wild- type isolate compared to wild-type isolate. For second isolate cyp51C mRNA expression level was down regulated (5.6 fold). The set of genes including ABC fatty acid transporter XM- 002375835 and aldehydereductase XM- 002376518 and three unknown functional genes were identified. Based on results, the over-expression of AKR1 and ABC fatty acid transporter in the voriconazole non- wild- type isolates suggests these genes could represent a novel molecular marker linked to the voriconazole resistance in A. flavus. CONCLUSION: The results obtained in this study showed a novel finding as the authors identified AKR1 and ABC fatty acid transporter genes as possible voriconazole target genes in Iranian clinical isolates of A. flavus.

Synthesis, characterization and antifungal activity of a novel formulated nanocomposite containing Indolicidin and Graphene oxide against disseminated candidiasis.

OBJECTIVE: Candidiasis is one of the most opportunistic fungal infections in immunocompromised patients. The emergence of multidrug-resistant Candida species necessitates the development of novel antifungal agents. Seeking to the discovery of natural antifungal agents, this study aimed to synthesize a novel formulated nanocomposite containing Indolicidin (IN), antimicrobial peptide, and Graphene oxide (GO), kind of nanomaterial, against Candida growth using in vitro and in vivo experiments for the first time. METHODS: The formulated nanocomposite (GO-IN) synthetized and was characterized using scanning electron microscopy, X-ray power diffraction, and fourier transform infrared method analysis. The in vitro antifungal activity of fluconazole (FLU), GO, IN, and GO-IN was determined against Candida albicans (C. albicans) compared to control groups, cell cytotoxicity assay on human intestinal epithelial cells (IEP) and hemolytic activities were performed. Moreover, in vivo experiments of nanocomposite were assessed in BALB/c mice. RESULTS: Our results showed that nanocomposite had the highest inhibitory effect against C. albicans (MIC 3.12µg/mL) compared with flu (MIC 4µg/mL), IN (MIC 12.5µg/mL), and GO (MIC 6.25µg/mL). Viability of human intestinal cell line at the MIC concentration (3.12µg/mL) of nanocomposite (GO-IN) was detected as 60% (P<0.05). The results of hemolytic activity showed that nanocomposite cause 2.73% of red blood cell membrane damage. For in vivo experiments, infected mice were successfully treated with GO-IN once a day within 7 days. GO-IN treated group eliminated the Candida infection in the spleen and liver of BALB/c mice (P=0.001) similar to fluconazole. There was no significant difference in histological manifestations between flu and GO-IN groups. CONCLUSION: This study suggests that synergistic combination of GO and IN provide a new option, representing a potential therapeutic efficiency against disseminated candidiasis in an animal model as well as might be used as adjunct therapy in the management of candidiasis. However, further investigation is needed to evaluate the efficacy of the nanocomposite.

Iranian HIV/AIDS patients with oropharyngeal candidiasis: identification, prevalence and antifungal susceptibility of Candida species.

Oropharyngeal candidiasis is the commonest mucocutaneous infection in HIV-positive individuals. Herein, samples were taken from oral cavities of 150 HIV-infected patients and cultured on Sabouraud-dextrose agar; 89 (59·3%) of 150 patients had positive culture for Candida and presented clinical sign of classical oral candidiasis. Totally, 102 morphologically distinct colonies were isolated from Candida positive cultures and subsequently identified by polymerase chain reaction and sequencing assay, presenting the following frequency: 54 C. albicans (52·9%), 16 C. dubliniensis (15·7%), 12 C. tropicalis (11·8%), 9 C. glabrata (8·8%), 7 C. kefyr (6·9%) and 4 C. africana (3·9%). Additionally, multiple Candida species were co-isolated from 13·5% (12/89) patients. Regarding the antifungal susceptibility test, which was performed by CLSI protocol (M27-A3/M27-S3), all Candida isolates were susceptible to amphotericin B and caspofungin, while some of them were resistant to fluconazole (17·6%; 16 C. albicans, 1 C. dubliniensis and 1 C. glabrata), itraconazole (16·7%; 15 C. albicans, 1 C. dubliniensis and 1 C. tropicalis) and voriconazole (5·9%; 5 C. albicans and 1 C. tropicalis). Collectively, our findings reinforce the urgent necessity to find new therapeutic agents to treat oral candidiasis in HIV-positive patients, especially due to the high incidence of azole-resistant Candida strains and the increased frequency of non-C. albicans species. SIGNIFICANCE AND IMPACT OF THE STUDY: The Candida species recovered from oral cavity of 150 Iranian HIV/AIDS patients and their antifungal susceptibility profiles were reported. Candida albicans was the commonest Candida species, followed by C. dubliniensis, C. tropicalis, C. glabrata, C. kefyr and C. africana. All Candida isolates were susceptible to amphotericin B and caspofungin, while resistance to azoles was detected. The growing drug-resistance profile reported in clinical isolates of C. albicans and non-C. albicans strains is a serious problem in hospitals worldwide. Consequently, the suitable antifungal choice to treat the HIV/AIDS population with oral candidiasis needs to be rethought and new therapeutic options must urgently arise.

Overexpression of MDR-1 and CDR-2 genes in fluconazole resistance of Candida albicans isolated from patients with vulvovaginal candidiasis.

BACKGROUND AND PURPOSE: Candida albicans (C. albicans) is an opportunistic fungus that can colonize women's mucosal epithelial cell surfaces, causing vulvovaginitis in specific circumstances. The major genes contributing to drug resistance in C. albicans are the candida drug resistance (CDR) and multi drug resistance (MDR) genes. The purpose of this study was to evaluate the CDR-2 and MDR-1 gene expression patterns in C. albicans strains isolated from patients with recurrent vulvovaginal candidiasis. MATERIALS AND METHODS: In this study, 40 isolates of fluconazole-resistant C. albicans were cultured on Sabouraud dextrose agar. These isolates were collected from women with vulvovaginitis who were referred to a clinic in Tehran, Iran, and transferred to a mycology laboratory. Then, RNA was extracted from the isolates using phenol-chloroform and glass beads, and the complementary DNA (cDNA) was synthetized. To detect the semi-quantitative expression of CDR-2 and MDR-1 genes, the reverse transcriptase-PCR (RT-PCR) technique was performed using specific primers. RESULTS: Our findings indicated that of the 40 C. albicans isolates, 35 (87.5%) strains were positive for mRNA of the CDR-2 gene, 32 (80%) strains expressed mRNA of the MDR-1 gene, and 30 (75%) strains were confirmed to express mRNA of both the CDR-2 and MDR-1 genes simultaneously using the RT-PCR assay. CONCLUSION: According to the obtained results, the expression rates of CDR-2 and MDR-1 genes were high in fluconazole-resistant C. albicans isolates, which can cause treatments to fail and result in chronic infections. Inhibiting these important genes using novel or natural agents can help with the treatment of chronic and recurrent vaginitis.

Fractionation and identification of the allergic proteins in Aspergillus species.

BACKGROUND AND PURPOSE: Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of Aspergillus strains including A. fumigatus, A. flavus, and A. niger could be mentioned. In this study, we aimed to separate allergic proteins from Aspergillus strains and determine their identity. MATERIALS AND METHODS: Standard species of Aspergillus strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freeze-thawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods. RESULTS: Among the studied species, A. fumigatus showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE-reactive proteins was confirmed to be 45 kDa mycelia catalase. CONCLUSION: Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to Aspergillus species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase.

Characterization and identification of candiduria due to Candida species in diabetic patients.

BACKGROUND AND PURPOSE: The presence of Candida yeasts in urine, known as candiduria, is an indicator of infection or colonization of the urinary tract by Candida species. This condition in diabetic patients can be hazardous due to diminished immune system response. The objective of this study was to investigate the incidence of candiduria in diabetic patients and to identify its causative agents. Furthermore, the demographic and laboratory (HbA1c, urine glucose and pH, urine culture colony count, and fasting blood sugar) data and their possible associations with candiduria were investigated. MATERIALS AND METHODS: This cross-sectional, descriptive study was performed on 305 diabetic patients referred to the diabetes research center, Hamedan, Iran, during April 2015 to September 2015. Urine and blood specimens were collected and urine analysis, urine culture, FBS, and HbA1c tests were performed. Positive cases were subjected to colony count and the causative agents were subsequently identified through the routine identification tests, as well as colony color in CHROMagar Candida medium, and the assimilation patterns in API 20 C auxanographic method. RESULTS: Among the 305 cases, 38 (%12.5) were positive for candiduria. Causative agents were identified as Candidaglabrata (n=19, 50%), C. albicans (n=12, 31.6%), C. krusei (n=4, 10.5%), C. tropicalis (n=2, 5.3%), andC. kefyr (n=1, 2.6%). According to the results of the statistical analyses, there were significant association between candiduria and female gender, high FBS and urine glucose, uncontrolled diabetes (HbA1c ≥8), and acidic urine pH (P<0.05). CONCLUSION: Considering the high incidence rate of candiduria in diabetic patients, control of diabetes, predisposing factors, and causal relationships between diabetes and candiduria should be highlighted.

Implantation phaeohyphomycosis caused by a non-sporulating Chaetomium species.

We report the case of a 66-year-old Iranian woman with a phaeohyphomycotic cyst (approximately 3×2.5cm in size) on the right lateral side of the neck. She had dysphagia and hoarseness, without any pain. She complained about discharge of black liquid on the skin and irritation. Histological examination of biopsy fragments from the lesions showed septate, branched brown hyphae. The fungus was cultured, but sporulation remained absent from 4- week-old cultures on Sabouraud dextrose agar (SDA), malt extract agar (MEA), potato dextrose agar (PDA), and water agar with sterile filter paper. Identification with the genus Chaetomium was achieved by sequencing the internal transcribed spacer (ITS) and the small subunit (SSU) domains of the rDNA gene and comparison with sequences held at GenBank and at the Centraalbureau voor Schimmelcultures (CBS). Sequencing of the SSU rRNA gene reveals this strain as belonging to the genus Chaetomium. The sequence of ITS did not fully match with any sequence of available ex-type strains of Chaetomium, Thielavia, Madurella and Papulaspora and hence might belong to an undescribed species. However, without diagnostic morphological features the taxon cannot be introduced as a novel member of the genus Chaetomium. After local excision of the cyst and antifungal therapy with ketoconazole (200mg twice a day), the lesion regressed and healed completely.