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

Specific identification and antifungal susceptibility pattern of clinically important dermatophyte species isolated from patients with dermatophytosis in Tehran, Iran

Background: With regard to the increasing number of antifungal-resistant dermatophytes, the requirement for precise identification of causative agents of infections and antifungal susceptibility test is vital. Antifungal susceptibility testing of dermatophytes plays a pivotal role in managing dermatophytosis. The current study aimed at determining antifungal susceptibility profile of 161 important dermatophyte species isolated from Iranian patients. Methods: The current descriptive, cross sectional study was conducted on 508 clinically suspected samples of dermatophytosis collected and identified by conventional methods. All dermatophyte isolates were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The susceptibility of dermatophyte strains to two routine antider-matophyte agents (terbinafine and griseofulvin) was evaluated using micro-dilution method according to CLSI (the clinical and laboratory standards institute) M38-A2 guidelines. Trichophyton rubrum PTCC 5143 and Candida krusei ATCC 6258 were used as quality controls. Results: Among 161 dermatophyte isolates, T. interdigitale was reported as the most frequent species isolated from patients using PCR-RFLP and Microsporum ferruginum was the least isolated species. The minimum inhibitory concentration (MIC) values of griseofulvin and terbinafine were ranged 0.0312-8 and 0.008-4 µg/mL, respectively. The most susceptible and resistant species to griseofulvin were T. interdigitale (MIC = 0.0312 µg/mL) and T. interdigitale/T. rubrum (MIC = 8 µg/mL), respectively. The results indicated that T. verrucosum (MIC = 0.008 µg/mL) was the most susceptible species to terbinafine, whereas T. interdigitale and T. rubrum were the most resistant species to it (MIC = 4 µg/mL). Conclusions: The obtained results assist clinicians to monitor the trend and be able to choose effective medications to treat patients with dermatophytosis, especially in countries such as Iran, where dermatophytosis is still a public health problem. © 2018, Archives of Clinical Infectious Diseases

Molecular investigation of etiologic agents causing vulvovaginal candidiasis

Background: Vulvovaginal candidiasis (VVC) is an ordinary infection caused by Candida species. Meanwhile, a shift towards non-albicans Candida (NAC) species has been detected in VVC patients. Objectives: This study aimed at molecular identification of Candida isolates, causing VVC. Methods: Vaginal secretion samples of 320 non-pregnant vaginitis patients at Shahid Akbar-Abadi Obstetrics and Gynecology Hospital in Tehran (Iran) were collected. Samples were evaluated using mycological and molecular approaches. Vaginitis isolates were analyzed with the PCR using NL1 and NL4 primers, and the D1/D2 region of the large-subunit rRNA gene was amplified and sequenced. Results: In total, 100 Candida isolates were identified from VVC and recurrent vulvovaginal candidiasis (RVVC). Candida albicans was the most frequent (51), followed by C. glabrata (36), C. krusei (Pichia kudriavzevii) (8), and C. kefyr (Kluyveromyces marxianus) (5). 51 and 49 of isolates had C. albicans and NAC, respectively. Conclusions: Candida albicans and C. glabrata were the most common agents of vulvovaginal candidiasis. NAC spp. (49) was found as an important agent associated with VVC. © 2020, Author(s)

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. © 2018 Sociedade Brasileira de Microbiologi

Study on synergistic effect of 2-phenylethanol and clotrimazole on candida species isolated from patients with chronic vulvovaginal candidiasis

Background: 2-phenylethanol is a colorless and aromatic compound with antimicrobial effects which is used extensively in perfumes and cosmetics, as well as in the food industry. Chronic vulvovaginal candidiasis is a vulvovaginal inflammation which is caused by Candida spp. Resistance to clotrimazole which is one of the most common drugs in the treatment of this disease was reported in many patients. In order to improve the treatment, the effect of 2-phenyl ethanol was investigated in combination with clotrimazole on Candida species isolated from chronic vulvovaginal candidiasis. Methods: This interventional study was performed in Iran University of Medical Sciences from February, 2016 until December, 2016 on Candida species isolated from women with chronic candidial vulvovaginitis who had been referred to Lolagar Hospital of Tehran. All specimens were examined by direct microscopy, culturing on Candida CHROMagar medium (to primary identification), sabouraud dextrose agar medium (to preservation the isolates) and determining the internal transcribed spacer (ITS) sequence (in order to final determination of Candida species). Then clotrimazole and 2-phenyl ethanol alone and in combination, was examined on isolated species, according to Clinical and Laboratory Standards Institute (CLSI) M27-A3 protocol (micro-broth dilution method). Finally, findings were analyzed. Results: From 40 detected strains of Candida species in this study, 95 were Candida albicans and 5 were Candida africana. The mean minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of clotrimazole were 24.73±28.87 μg/ml and 30.18±33.004 μg/ml, respectively and the mean MIC and MFC of 2-phenylethanol were 2580±932.38 μg/ml and 3200±1403.29 μg/ml, respectively. The MIC50 and MIC90 of clotrimazole were 16 and 64 μg/ml, respectively. The MIC50 and MIC90 of 2-phenylethanol were both 3200 μg/ml. Most of the isolates were resistant to clotrimazole (82.5). In combination test, the mean MIC of 2-phenylethanol and clotrimazole alone were 3200±0 μg/ml and 56±40.16 μg/ml, respectively. The fractional inhibitory concentration index (FICI) range was 0.14-0.37. Also, there was a significant difference between clotrimazole MIC values alone and in combination (P= 0.021). Conclusion: The synergistic effect was observed in combination of clotrimazole and 2-phenylethanol. © 2018, Tehran University of Medical Sciences. All rights reserved

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. © 2020 Microbiology; Mycology; Microbial genomics; DNA sequencing; Antimicrobial; Women's health; Candida albicans complex; Candida dubliniensis; Candida africana; Drug susceptibility; Vulvovaginal candidiasis; HWP1. © 202

Study on synergistic effect of 2-phenylethanol and clotrimazole on candida species isolated from patients with chronic vulvovaginal candidiasis

Background: 2-phenylethanol is a colorless and aromatic compound with antimicrobial effects which is used extensively in perfumes and cosmetics, as well as in the food industry. Chronic vulvovaginal candidiasis is a vulvovaginal inflammation which is caused by Candida spp. Resistance to clotrimazole which is one of the most common drugs in the treatment of this disease was reported in many patients. In order to improve the treatment, the effect of 2-phenyl ethanol was investigated in combination with clotrimazole on Candida species isolated from chronic vulvovaginal candidiasis. Methods: This interventional study was performed in Iran University of Medical Sciences from February, 2016 until December, 2016 on Candida species isolated from women with chronic candidial vulvovaginitis who had been referred to Lolagar Hospital of Tehran. All specimens were examined by direct microscopy, culturing on Candida CHROMagar medium (to primary identification), sabouraud dextrose agar medium (to preservation the isolates) and determining the internal transcribed spacer (ITS) sequence (in order to final determination of Candida species). Then clotrimazole and 2-phenyl ethanol alone and in combination, was examined on isolated species, according to Clinical and Laboratory Standards Institute (CLSI) M27-A3 protocol (micro-broth dilution method). Finally, findings were analyzed. Results: From 40 detected strains of Candida species in this study, 95 were Candida albicans and 5 were Candida africana. The mean minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of clotrimazole were 24.73±28.87 μg/ml and 30.18±33.004 μg/ml, respectively and the mean MIC and MFC of 2-phenylethanol were 2580±932.38 μg/ml and 3200±1403.29 μg/ml, respectively. The MIC50 and MIC90 of clotrimazole were 16 and 64 μg/ml, respectively. The MIC50 and MIC90 of 2-phenylethanol were both 3200 μg/ml. Most of the isolates were resistant to clotrimazole (82.5). In combination test, the mean MIC of 2-phenylethanol and clotrimazole alone were 3200±0 μg/ml and 56±40.16 μg/ml, respectively. The fractional inhibitory concentration index (FICI) range was 0.14-0.37. Also, there was a significant difference between clotrimazole MIC values alone and in combination (P= 0.021). Conclusion: The synergistic effect was observed in combination of clotrimazole and 2-phenylethanol. © 2018, Tehran University of Medical Sciences. All rights reserved

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 nonalbicans 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. Conclusions: Non-albicans Candida spp. are common agents of vulvovaginitis, and C. glabrata is the most common species in the tested patients. © 2018, Pasteur Institute of Iran. All rights reserved