Antifungal activity of green-synthesized curcumin-coated silver nanoparticles alone and in combination with fluconazole and itraconazole against Candida and Aspergillus species.

Background and Purpose: Regarding the wide-spectrum antimicrobial effects of curcumin and silver, this study aimed to evaluate the antifungal activity of green-synthesized curcumin-coated silver nanoparticles (Cur-Ag NPs) against a set of Candida and Aspergillus species. Materials and Methods: Cur-Ag NPs were synthesized by mixing 200 µL of curcumin solution (40 mM) and 15 mL of deionized water. The mixture was stirred for 3-5 min, followed by the addition of 2.5 mL of silver nitrate solution (2.5 mM). The resulting solution was incubated for 3 days. Antifungal susceptibility of 30 fungal isolates of Aspergillus and Candida to fluconazole and itraconazole, as well as the activity of Cur-Ag NPs against the isolates, were determined, both alone and in combination, using broth microdilution according to the Clinical and Laboratory Standards Institute guidelines. Results: Cur-Ag NPs demonstrated promising antifungal activity, particularly against Candida species. The geometric mean value of the minimum inhibitory concentration of Cur-Ag NPs was significantly lower than that of fluconazole for all the studied fungi. Similarly, it was lower than those of itraconazole in C. albicans and A. fumigatus. The minimum fungicidal concentrations of Cur-Ag NPs were markedly better than those of fluconazole but still inferior to those of itraconazole. Conclusion: Cur-Ag NPs demonstrated indisputable antifungal activity and great potential that can be harnessed to combat fungal infections, particularly those caused by azole-resistant strains of Aspergillus and Candida.

Anti-biofilm properties of eucalyptol in combination with antifungals against Candida albicans isolates in patients with hematological malignancy.

Oral candidiasis is a fungal infection caused mainly by Candida albicans and it is a major problem among hematologic malignancy patients. Biofilm formation is an attributable factor to both virulence and drug resistance of Candida species. The aim of the study was to evaluate the biofilm-producing ability of oral C. albicans isolates and to evaluate the inhibitory activity of eucalyptol on Candida biofilm, alone and in combination with antifungal agents. Samples were collected from the oral cavity of 106 patients with hematologic malignancy. The isolated yeasts were identified by PCR-sequencing. Then C. albicans isolates were analyzed for their biofilm-producing ability by crystal violet staining and MTT assay. The minimum biofilm inhibition concentrations (MBIC) of eucalyptol, amphotericin B, itraconazole, and nystatin and the in vitro interaction of eucalyptol with these drugs were tested according to CLSI-M-27-A3 protocol and checkerboard methods, respectively. From 106 patients, 50 (47.2%) were confirmed for oral candidiasis [mean ± SD age 39 ± 14 years; female 31 (62%) and male 19 (38%)]. C. albicans was isolated from 40 of 50 (80%) patients. From 40 C. albicans isolates, 24 (60%) and 16 (40%) were moderate and weak biofilm producer, respectively. The geometric mean MBIC of amphotericin B, itraconazole, nystatin and eucalyptol were 3.93 µg/mL, 12.55 µg/mL, 0.75 µg/mL and 798 µg/mL, respectively. Eucalyptol interacted synergistically with amphotericin B, itraconazole and nystatin against 12.5, 10, and 22.5% of isolates, respectively. Eucalyptol demonstrated promising activity against biofilm of C. albicans when tested alone or combined with antifungal drugs.

HLA-DRB1 alleles as predisposing and resisting factor in women suffering from vulvovaginal candidiasis.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a vaginal mucosal infection that usually affects women in their reproductive age. When the signs of VVC persist on a daily basis or last for a long time and repeat at least three times per year, the disease is considered chronic and recurrent. OBJECTIVE: The purpose of this study was to evaluate the frequency of HLA-DRB1 alleles in patients with recurrent vulvovaginal candidiasis (RVVC). STUDY DESIGN: 120 patients with RVVC and 136 age-matched healthy controls underwent low-resolution HLA-DRB typing performed using the polymerase chain reaction-sequence-specific primers (PCR-SSP) technique. RESULTS: In the present work, we studied different genes that encode HLA-DRB (HLA-DRB1 / HLA-DRB3 / HLA-DRB4 / HLA-DRB5) and showed that HLA-DRB1×14, found in 25% of the patients. In the present study, the significant frequency of HLA-DRB1×10 in the control group suggests a resistant role of this allele to RVVC infections CONCLUSIONS: In the HLA-DRB region, the DRB1×14 allele showed a higher frequency in the patients with RVVC than in the controls. Moreover, the higher frequency of DRB1×10 observed in the controls than in the patients with RVVC. These results demonstrate the HLA-DRB1 alleles are in relation with both susceptibility and immunity factors in RVVC infection and possible susceptible role of HLA-DRB1×14.

Acute invasive fungal rhinosinusitis: Molecular identification and update in management of frozen section biopsy.

The clinical diagnosis of Acute Invasive Fungal Rhinosinusitis (AIFRS) is technically difficult because it presents with non-exclusive and nonspecific clinical symptoms. Laboratory confirmation (usually via histopathologic techniques such as formalin-fixed paraffin-embedded (FFPE)) is necessary but it is time-consuming, despite the urgent need for timely diagnosis of AIFRS for effective management. This study aimed to investigate the sensitivity and specificity of the GMS frozen-section biopsy in the diagnosis of AIFRS and compare the same with that of different tissue staining methods to provide valid decision-grounds that may guide clinicians in prompt diagnosis of acute fungal invasive rhinosinusitis. A cross-sectional study was conducted in the Medical Mycology Laboratory, Faculty of Medicine, Iran University of Medical Sciences between 2018 and 2020 on 200 patients with suspected AIFRS referred to Baqiyatallah and Imam Khomeini Hospital, Tehran. All patients were subjected to diagnostic nasal endoscopy and computed tomography (CT) scan of paranasal sinuses. Magnetic resonance imaging (MRI) was done in cases of suspected intracranial extension. After screening by routine mycological examination, the diagnosis was confirmed using complementary molecular methods. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the frozen-section biopsy were also compared with FFPE. Of the 200 suspect patients, 47 cases (23.5%) met the criteria for AIFRS. Species of the genus Aspergillus were the predominant 27 (57.4%) followed by Mucorales species 10 (21.3%), and Fusarium spp 3 (6.4%). Also, 3 cases (6.4%) of co-infection due to Aspergillus/Rhizopus were reported. The accuracy, sensitivity, specificity, PPV, and NPV of frozen section assessments were 99.5%, 97.9%, 100%, 100% and 99.3%, respectively. For GMS frozen-section alone, sensitivity, specificity, NPV, and PPV was 100%. Overall, the calculated accuracy of FFPE was 98.5%, sensitivity was 94%, specificity was 100%, PPV was 100%, and NPV was 98.1%. Examination of the frozen-section biopsy is a highly predictive tool for a rapid and effective diagnosis of patients with suspected AIFRS. We observed that GMS frozen-section is a fast and reliable exam to confirm the diagnosis of fungal invasion, with good accuracy, sensitivity, and specificity compared to the gold-standard FFPE biopsy.

Antifungal Activity of Capric Acid, Nystatin, and Fluconazole and Their In Vitro Interactions Against Candida Isolates from Neonatal Oral Thrush.

Due to the increasing resistance of various Candida species to azole drugs, particularly fluconazole, it would be of significant importance to look for alternative therapies. The aim of this study was to investigate the antifungal activity of capric acid and its in vitro interactions with nystatin and fluconazole against Candida isolates. A total of 40 Candida isolates (C. albicans, 36; C. kefyr, 2; C. tropicalis, 1; C. glabrata, 1) collected from the oral cavity of neonates with oropharyngeal candidiasis and a reference strain of C. albicans (ATCC 10231) were used in this study. Antifungal activity of capric acid and two comparator antifungal drugs, namely fluconazole and nystatin, was tested according to CLSI M27-A3/M60 method. The in vitro interaction between capric acid with fluconazole and nystatin was determined following a checkerboard method and results were interpreted using fractional inhibitory concentration index. Nystatin had the lowest minimum inhibitory concentrations (range, 0.125-8 µg/mL; geometric mean [GM], 0.6229 µg/mL) followed by fluconazole (range, 0.5-16 µg/mL; GM, 1.9011 µg/mL) and capric acid (range, 128-2,048 µg/mL; GM, 835.9756 µg/mL). When tested in combination, capric acid with fluconazole demonstrated synergistic, indifferent, and antagonistic interactions in 3 (7.317%), 24 (58.536%), and 14 (34.146%) cases, respectively. For combination of capric acid with nystatin, synergistic, indifferent, and antagonistic interactions were observed in 1 (2.439%), 19 (46.341%), and 21 (51.219%) cases, respectively. All cases of synergistic interactions were against resistant or susceptible dose-dependent isolates. Fluconazole, nystatin, and capric acid seem to be more effective when they are used alone compared with their combination. However, their combination might be effective on resistant isolates.

The effect of Candida cell wall beta-glucan on treatment-resistant LL/2 cancer cell line: in vitro evaluation.

Candida albicans (C. albicans) cell wall beta-glucan has been considered as a potential agent in the treatment of cancers due to its anti-tumor properties. Therefore, in the present study, we investigated the anti-cancer effects of Candida cell wall beta-glucan on Lewis lung carcinoma cell line (LL/2) cells. Beta-glucan of C. albicans cell wall was extracted. LL/2 cell line was cultured, then sphere cells and parental cells were exposed to the different concentrations of beta-glucan extracted from C. albicans (10-6000 µg/ml), for 24, 48 and 72 h. Cytotoxicity of beta-glucan was assayed by MTT test, then RNA extracted from cells population (treated and untreated cells), cDNA synthetized and expression level of Sox2, Oct4, C-myc, Nanog genes were also investigated using Real-time methods. At optimal concentrations of 800 and 1000 µg/ml, the extracted beta-glucan showed a significant cytotoxic effect on both parental and sphere cell populations (p < 0.05). Real-time PCR analysis revealed a decreased expression of Oct4 and Sox2 genes in treatment of cells with beta-glucan compared with control group. Since the extracted beta-glucan showed an inhibitory effect on the expression of Oct4 and Sox2 genes involved in LL/2 metastasis, therefore, beta-glucan can be considered as an anti-tumor agent because of its anti-metastatic properties, however, more in vitro and in vivo studies are needed to provide further evidence on this topic in the future.

Low prevalence of antifungal resistant Candida africana, in the C. albicans complex causing vulvovaginal candidiasis.

The Candida (C.) albicans complex includes C. albicans, C. dubliniensis, C. stellatoidea, and C. africana, with the last mentioned as an important emerging agent of vulvovaginal candidiasis (VVC). The aim of the study was to identify C. africana and C. dubliniensis and assess their drug susceptibility in vaginitis. One-hundred Candida isolates of the C. albicans complex from women diagnosed with vaginitis and from vaginal samples in the culture collection of a medical mycology laboratory were examined. Species of the C. albicans complex were identified with conventional and molecular methods using polymerase chain reaction (PCR) for amplification and sequencing of the internal transcribed spacer (ITS) region, PCR for partial amplification of hyphal wall protein 1 (HWP1) gene and duplex PCR. The effects of antifungal drugs were evaluated according to standard broth microdilution protocols. Ninety-seven C. albicans (97%) and three C. africana (3%) isolates were identified. Results of susceptibility testing revealed one isolate of C. africana to be resistant to both clotrimazole and fluconazole, and one showed reduced susceptibility to itraconazole. Identification of Candida species especially C. africana in vaginitis is crucial, there are varying levels of resistance to antifungal drugs.

Investigating the expression of ALS2 and ALS9 genes along with allele frequency of ALS9 in patients with vulvovaginal candidiasis.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a vaginal mucosal infection that usually infects women in their reproductive age. When the signs of VVC persist on a daily basis or last for a long time and repeat at least three times per year, the disease is considered chronic and recurrent. OBJECTIVES: The purpose of this study was to determine the expression rate of 2 genes responsible for adhesion and virulence of candida in RVVC patients using Real-time PCR, and comparing them together and assess the presence or absence of ALS9-2 allele in these patients. PATIENTS/METHODS: The vaginal discharge was collected from 120 women aged (22-55) attending lolagar hospital which were all diagnosed with RVVC and 120 age-matched healthy controls. The expression rate of ALS2 and ALS 9 genes was quantified using real-time PCR. PCR method was used for Identification of ALS9 gene alleles. RESULTS: Results showed an increase in ALS2 gene expression and a decrease in ALS9 gene expression, comparing to basic level and standard sample. 42.5% (51 of total 120 samples) contained the small allele. CONCLUSIONS: The significant difference in expression rates of ALS2 and ALS9 genes indicates their different roles in making morphogenesis changes during the virulence of Candida albicans. Emergence of heterogeneous form and detection of ALS's short allele in invasive form of fungi proves the significant pathogenic role of this allele, specially when attached to mucosal tissue. Invasive and recurrent form of the disease can be accompanied by genetic-morphologic changes in fungi. Considering the form of this disease and the reduction in ALS9 gene expression, it can be concluded that this gene plays a significant role in attachment and initiation of the pathogenic phase.

Incidence and spectrum of yeast species isolated from the oral cavity of Iranian patients suffering from hematological malignancies.

Background: Oral candidiasis (OC) has a profound effect on the life quality of immunocompromised patients, such as those undergoing chemotherapy. Objective: Systematic investigation of clinical outcome and microbiological features of yeast isolates recovered from the oral cavity of 150 Iranian patients with hematological malignancies. Design: MALDI-TOF MS, 21-plex PCR, and rDNA sequencing were used for identification. Antifungal susceptibility testing (broth microdilution, CLSI M27-A3/S4) and genotypic diversity of yeast isolates (amplified fragment length polymorphism) were assessed. Results: Nystatin treatment resulted in 70% therapeutic failure and administration of 150 mg fluconazole (FLZ) + nystatin for patients with OC relapse showed 70% clinical failure. Previous history of OC was significantly correlated with FLZ treatment requirement and nystatin failure (P = 0.005, α < 0.05). Candida albicans (80.3%) and Kluyveromyces marxianus (C. kefyr) (12.7%) were the two most prevalent yeast species isolated. FLZ and AMB exhibited the highest geometric mean values. 21-PCR showed 98.9% agreement with MALDI-TOF MS. K. marxianus isolates had the same genotype, while C. albicans isolates grouped in 15 genotypes. Conclusions: Marked rate of therapeutic failure of nystatin necessitated OC treatment with systemic antifungals. K. marxianus was the second most prevalent yeast and 21-plex PCR could be considered as an inexpensive identification tool.

Differential role of gpaB and sidA gene expressions in relation to virulence in Aspergillus species from patients with invasive aspergillosis

Abstract The virulence genes in invasive aspergillosis (IA) have not been analyzed adequately. The present study was designed to evaluate the expression of gpaB and sidA genes, which are important virulence genes in Aspergillus spp. from bronchoalveolar lavage (BAL) samples. Direct examination and culture on Czapek Agar and Sabouraud Dextrose Agar media were performed for 600 BAL specimens isolated from patients with possible aspergillosis. A Galactomannan ELISA assay was also carried out. The expression levels of the gpaB and sidA genes in isolates were analyzed using quantitative real-time PCR (qRT-PCR). We identified 2 species, including Aspergillus flavus (A. flavus) and Aspergillus fumigatus (A. fumigatus) in 25 positive samples for invasive aspergillosis as validated using GM-ELISA. A. flavus is the main pathogen threatening transplant recipients and cancer patients worldwide. In this study, A. flavus had low levels of the gpaB gene expression compared to A. fumigatus (p = 0.006). The highest sidA expression was detected in transplant recipients (p = 0.05). There was no significant correlation between sidA expression and underlying disease (p = 0.15). The sidA and gpaB gene expression patterns may provide evidence that these virulence genes play important roles in the pathogenicity of Aspergillus isolates; however, there are several regulatory genes responsible for the unexpressed sidA and gpaB genes in the isolates.

Effect of 2-Phenylethanol as Antifungal Agent and Common Antifungals (Amphotericin B, Fluconazole, and Itraconazole) on Candida Species Isolated from Chronic and Recurrent Cases of Candidal Vulvovaginitis.

The antifungal effects of 2-phenylethanol are clearly visible through its intervention in Candida morphogenesis. Chronic and recurrent vulvovaginitis, however, does not respond to this standard experimental therapy; therefore, the study presented in this article investigated the effect of common antifungal drugs (amphotericin B [AMB], fluconazole [FLU], and itraconazole [ITC]), in combination with 2-phenylethanol, on the Candida species isolated from cases of chronic and recurrent vulvovaginitis, thereby allowing the recommendation of a more appropriate treatment option. Forty isolates from patients with chronic and recurrent vaginal candidiasis were investigated in this experimental study. The specimens were examined by direct microscopy, culturing, and PCR to identify the species. The antifungal effects of 2-phenylethanol and conventional drugs, both alone and in combination, were determined in duplicate. Finally, the findings were analyzed. In this study, 40 strains of Candida species were identified, whose agents were Candida albicans (95%) and Candida africana (5%). After 48 h, the minimum inhibitory concentration (MIC) range of the 2-phenylethanol was 800-3,200 µg/mL. Also, in the final study on the MIC levels of common antifungal drugs, AMB (0.42 µg/mL) had the lowest MIC, FLU (40.51 µg/mL) had the highest MIC, and the combination of ITC and 2-phenylethanol had the lowest fractional inhibitory concentration index (FICI) of any of the combinations (FICI range, 0.26-1.03). Combining FLU and ITC with 2-phenylethanol can effectively increase their antifungal effect.

Differential role of gpaB and sidA gene expressions in relation to virulence in Aspergillus species from patients with invasive aspergillosis.

The virulence genes in invasive aspergillosis (IA) have not been analyzed adequately. The present study was designed to evaluate the expression of gpaB and sidA genes, which are important virulence genes in Aspergillus spp. from bronchoalveolar lavage (BAL) samples. Direct examination and culture on Czapek Agar and Sabouraud Dextrose Agar media were performed for 600 BAL specimens isolated from patients with possible aspergillosis. A Galactomannan ELISA assay was also carried out. The expression levels of the gpaB and sidA genes in isolates were analyzed using quantitative real-time PCR (qRT-PCR). We identified 2 species, including Aspergillus flavus (A. flavus) and Aspergillus fumigatus (A. fumigatus) in 25 positive samples for invasive aspergillosis as validated using GM-ELISA. A. flavus is the main pathogen threatening transplant recipients and cancer patients worldwide. In this study, A. flavus had low levels of the gpaB gene expression compared to A. fumigatus (p=0.006). The highest sidA expression was detected in transplant recipients (p=0.05). There was no significant correlation between sidA expression and underlying disease (p=0.15). The sidA and gpaB gene expression patterns may provide evidence that these virulence genes play important roles in the pathogenicity of Aspergillus isolates; however, there are several regulatory genes responsible for the unexpressed sidA and gpaB genes in the isolates.

Molecular Identification and Antifungal Susceptibility Pattern of Non-albicans Candida Species Isolated from Vulvovaginal Candidiasis

Background: Vulvovaginal candidiasis (VVC) is an important health problem caused by Candida spp. The aim of this study was molecular identification, phylogenetic analysis, and evaluation of antifungal susceptibility of non-albicans Candida isolates from VVC. Methods: Vaginal secretion samples were collected from 550 vaginitis patients at Sayyad Shirazi Medical and Educational Center of Gorgan (Golestan Province, Iran) from May to October 2015. Samples were analyzed using conventional mycological and molecular approaches. Clinical isolates were analyzed with specific PCR using CGL primers, and the internal transcribed spacer region and the D1-D2 domain of the large-subunit rRNA gene were amplified and sequenced. Susceptibility to amphotericin B, fluconazole, itraconazole, and clotrimazole was determined by the guidelines of the Clinical and Laboratory Standard Institute. Results: In total, 35 non-albicans Candida isolates were identified from VVC patients. The isolates included 27 strains of Candida glabrata (77.1%), 5 Candida krusei (Pichia kudriavzevii; 14.3%), 2 Candida kefyr (Kluyveromyces marxianus; 5.7%), and 1 Candida lusitaniae (Clavispora lusitaniae; 2.9%). The fungicides itraconazole and amphotericin B were effective against all species. One isolate of C. glabrata showed resistance to fluconazole and clotrimazole, and 26 isolates of C. glabrata indicated dose-dependent susceptibility to fluconazole. C. lusitaniae was susceptible in a dose-dependent manner to fluconazole and resistant to clotrimazole. Conclusion: Non-albicans Candida spp. are common agents of vulvovaginitis, and C. glabrata is the most common species in the tested patients.

The first report of onychomycosis caused by Cryptococcus friedmannii (Naganishia friedmannii) a basidiomycetous yeast.

Yeasts are common etiologic agents of onychomycosis. This study reported a case of onychomycosis due to Cryptococcus friedmannii (Naganishia friedmannii). This yeast was isolated of the right great toenail of 57-year-old man. Microscopic examination of nail scrapings showed budding cells with thin capsule. Sequence analyzes of the internal transcribed spacer regions was closely related to Cryptococcus friedmannii. The results of susceptibility testing showed the Cryptococcus friedmannii to be sensitive to fluconazole, itraconazole and amphotericin B.

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, semi-quantitative 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.

The First Case of Total Dystrophic Onychomycosis Caused by Aspergillus clavatus Resistant to Antifungal Drugs.

Onychomycosis is a common fungal infection of nails which is mainly caused by dermatophyte species and less often by yeasts and non-dermatophyte molds. We present a case of onychomycosis due to Aspergillus clavatus for the first time worldwide. The patient was an immunocompetent 32-year-old woman who identified with Psoriasis of the nail. The presence of A. clavatus in a nail sample was confirmed using microscopic and culture analysis followed by PCR of the ß-tubulin gene. After antifungal susceptibility test, it is revealed that the isolate was resistant to the majority of common antifungal drugs, but finally the patient was treated with itraconazole 200 mg daily. A. clavatus and drug-resistant A. clavatus have not previously been reported from onychomycosis.

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.

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.

Identification and Sequencing of Candida krusei Aconitate Hydratase Gene Using Rapid Amplification of cDNA Ends Method and Phylogenetic Analysis.

BACKGROUND: The production and development of an effective fungicidal drug requires the identification of an essential fungal protein as a drug target. Aconitase (ACO) is a mitochondrial protein that plays a vital role in tricarboxylic acid (TCA) cycle and thus production of energy within the cell. OBJECTIVES: The current study aimed to sequence Candida krusei ACO gene and determine any amino acid residue differences between human and fungal aconitases to obtain selective inhibition. MATERIALS AND METHODS: Candida krusei (ATCC: 6258) aconitase gene was determined by 5'Rapid Amplification of cDNA Ends (RACE) method and degenerate Polymerase Chain Reaction (PCR) and analyzed using bioinformatics softwares. RESULTS: One thousand-four hundred-nineteen nucleotide of C. krusei aconitase gene were clarified and submitted in Genbank as a partial sequence and then taxonomic location of C. krusei was determined by nucleotide and amino acid sequences of this gene. The comparison of nucleotide and amino acid sequences of Candida species ACO genes showed that C. krusei possessed characteristic sequences. No significant differences were observed between C. krusei and human aconitases within the active site amino acid residues. CONCLUSIONS: Results of the current study indicated that aconitase was not a suitable target to design new anti-fungal drugs that selectively block this enzyme.