In Vitro Activity of Amphotericin B in Combination with Statins against Clinical and Environmental Rhizopus oryzae Strains.

BACKGROUND: Mucormycosis is an acute and invasive fungal infection with a high mortality rate. Mucorales are less sensitive than other types of fungi to most antifungal agents. Amphotericin B (AMB) is one treatment option for this infection, but in recent studies, the antifungal activity of statins against Mucorales was shown. Therefore, therapy that combines AMB with these agents may have better effects in management of patients with mucormycosis. We evaluated the in vitro activity of AMB alone and in combination with statins, against Mucorales. METHODS: Susceptibility profiles of AMB alone and in combination with two statins, atorvastatin (ATO) and lovastatin (LOV) determined against clinical (n: 15) and environmental (n: 5) Rhizopus oryzae isolates, obtained between Jan 2009 and Oct 2016 from patients with uncontrolled diabetes mellitus and cancer referred to the Department of Parasitology and Medical Mycology of Tehran University of Medical Sciences, Tehran, Iran. It was performed by microdilution method, based on the Clinical and Laboratory Standard Institute (CLSI) M38-A2 guideline. RESULTS: All clinical and environmental isolates were susceptible to AMB (MIC≤1 µg/mL). The results of the interactions between AMB and the two statins were positive. The AMB-ATO (GM: 0.13 µg/Ml) combination produced greater activity than the AMB-LOV (GM: 0.26 µg/mL) combination. AMB, in combination with ATO and LOV, reacts positively against clinical and environmental R. oryzae isolates. CONCLUSION: This combination strategy may lead to more effective treatment of mucormycosis and fewer side effects using low dose of AMB.

Candiduria in Hospitalized Patients and Identification of Isolated Candida Species by Morphological and Molecular Methods in Ilam, Iran.

BACKGROUND: Candiduria in hospitalized patients may represent contamination, colonization, or urinary tract infections. On the other hand, candidemia and upper urinary tract infection could be the complications of candiduria. The aim of this study was to determine candiduria in hospitalized patients and identify isolated Candida species by conventional and molecular methods. METHODS: This cross-sectional study was conducted on hospitalized patients in Imam Khomeini and Mostafa Khomeini hospitals in Ilam, western Iran from Jan to Dec 2016. Urine samples of hospitalized patients were collected during a period of 4 months for diagnosis of candiduria. Primary identification was done by conventional methods. PCR profile was carried out using phenol-chloroform method and confirmed using restriction fragment length polymorphism (PCR-RFLP) technique by MspI restriction enzyme. RESULTS: Candiduria was diagnosed in 18 (9.2%) cases from a total of 195 patients. Isolated yeasts were identified as C. albicans (n: 13), C. glabrata (n: 5), and C. parapsilosis (n: 1) in the one case both C. albicans and C. glabrata were isolated from a urine sample. CONCLUSION: Candida urinary tract infection is becoming increasingly common in hospitalized patients but, differentiation fungal colonization from infection and identification of etiologic agents for optimal treatment is necessary.

MGL_3741 gene contributes to pathogenicity of Malassezia globosa in pityriasis versicolor.

Dihydroxyacid dehydratase (DHAD) is a key enzyme in biosynthetic pathway of isoleucine and valine. This pathway is absent in human but exists in various organisms such as fungi. Using RNA-seq analysis in this study, we identified MGL_3741gene which encodes DHAD protein in Malassezia globosa (M. globosa). Furthermore, we found that mentioned gene is homologous to the Ustilago maydis, Saccharomyces cerevisiae, Aspergillus flavus, and Aspergillus fumigatus ILV3P. For understanding the probable role of this gene in pathogenicity of M. globosa, we applied Real-time PCR to investigate the differentially expressed of the MGL_3741 gene in healthy and pathogenic states. Our results indicate a significant difference between two mentioned stats. These results revealed that ILV3-like gene in M. globosa can be related to the pathogenicity of this yeast.

Increased Urine Interleukin-17 and Interleukin-22 Levels in Patients With Candidal Urinary Tract Infection.

INTRODUCTION: Candiduria is common in the hospitalized patients. This study aimed to quantify interleukin (IL)-17 and IL-22 levels in urine of candiduric patients. MATERIALS AND METHODS: A case-control study was conducted on inpatients at Hashemi Nejad Kidney Center. Thirty-four patients were identified with Candida species in their urine samples (> 103 colony-forming units per milliliter and presence of Candida species only). Urine samples with concomitant infections were excluded. Thirty-four patients with negative direct examination and culture were included as the control patients. Interleulin-17 and IL-22 levels were measured in the lyophilized and nonlyophilized urine. The relevant cytokine titers of the two groups were compared, and the association of cytokine elevation and candiduria was investigated. RESULTS: The majority of the candiduric patients were from the intensive care and urology units of women. Only 4 patients (11.7%) manifested fever and dysuria. Massive leukocyturia was observed in 4 patients. Candida glabrata was the most commonly isolated species (44%). Levels of the urine IL-17 and IL-22 were significantly elevated in the candiduric patients, when compared to the noncandiduric controls. While an increased IL-17 level was significantly associated with candiduria (odds ratio, 1.09; 95% confidence interval, 1.003 to 1.17; P = .04), an increased IL-22 level was not. The results showed that lyophilized urine samples maximized the detection power of urinary cytokines. CONCLUSIONS: Our results indicated that direct examination, fungal urine culture, and investigation of urine IL-17 and IL-22 levels are useful tools for diagnosis of Candida urinary tract infection.

Inhibitory effect of vitamin C on Aspergillus parasiticus growth and aflatoxin gene expression.

BACKGROUND AND PURPOSE: Aflatoxin is known as one of the most important mycotoxins threatening human life. This toxin is produced by Aspergillus species, which is the common cause of agricultural product contamination. The use of organic compounds has been always considered for the inhibition of fungal growth and toxin production. Regarding this, the aim of the present study was to investigate the effect of vitamin C on the rate of fungal growth, aflR gene expression, and toxin production. MATERIALS AND METHODS: For the purpose of the study, first, Aspergillus parasiticus ATCC15517 was cultured in Sabouraud dextrose agar medium containing vitamin C at concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.1 mg/ml and temperature of 28°C for 72 h. Then, the amount of aflatoxin produced in the presence of vitamin C was measured through high performance liquid chromatography. Finally, by extracting the DNA of the cultured samples, the aflR gene expression level was evaluated by means of real-time polymerase chain reaction at different concentrations of vitamin C. RESULTS: The results showed that mycelium deformation was started at the vitamin C concentration of 50 mg/ml, and that only fungal spores were observed at higher concentrations. The levels of total aflatoxin and its subsets, namely B1, B2, G1, and G2, in the presence of vitamin C were estimated as 5.9, 1.9, 0.2, 3.5, and 0.3 ppm, respectively. On the other hand, these values were respectively obtained as 207.5, 73.6, 4.5, 123.4, and 6 ppm in the absence of vitamin C. Measurement of the expression level of aflR genes showed that the level of gene expression decreased to 68% and up to 81% at the vitamin C concentrations of 25 and 50 mg/ml, respectively. CONCLUSION: This study showed that vitamin C, as a human-compatible compound, could be considered a good agent to protect agricultural products against fungal aflatoxin.

The Utilization of RNA Silencing Technology to Mitigate the Voriconazole Resistance of Aspergillus Flavus; Lipofectamine-Based Delivery.

Purpose: Introducing the effect of RNAi in fungi to downregulate essential genes has made it a powerful tool to investigate gene function, with potential strategies for novel disease treatments. Thus, this study is an endeavor to delve into the silencing potentials of siRNA on cyp51A and MDR1 in voriconazole-resistant Aspergillus flavus as the target genes. Methods: In this study, we designed three cyp51A-specific siRNAs and three MDR1-specific siRNAs and after the co-transfection of siRNA into Aspergillus flavus, using lipofectamine, we investigated the effect of different siRNA concentrations (5, 15, 25, 50nM) on cyp51A and MDR1 expressions by qRT-PCR. Finally, the Minimum Inhibitory Concentrations (MICs) of voriconazole for isolates were determined by broth dilution method. Results: Cyp51A siRNA induced 9, 22, 33, 40-fold reductions in cyp51A mRNA expres-sion in a voriconazole-resistant strain following the treatment of the cells with concentrations of 5, 15, 25, 50nM siRNA, respectively. Identically, the same procedure was applied to MDR1, even though it induced 2, 3, 4, 10-fold reductions. The results demonstrated a MIC for voriconazole in the untreated group (4µg per ml), when compared to the group treated with cyp51A-specific siRNA and MDR1-specific siRNA, both at concentrations of 25 and 50nM, yielding 2µg per ml and 1µg per ml when 25 nM was applied and 2µg per ml and 0.5µg per ml when the concentration doubled to 50 nM. Conclusion: In this study, we suggested that siRNA-mediated specific inhibition of cyp51A and MDR1 genes play roles in voriconazole-resistant A.flavus strain and these could be apt target genes for inactivation. The current study promises a bright prospect for the treatment of invasive aspergillosis through the effective deployment of RNAi and gene therapy.

Regulation of ERG3, ERG6, and ERG11 Genes in Antifungal-Resistant isolates of Candida parapsilosis

Background: Candida parapsilosis is one of the five common strains of yeasts involved in invasive candidiasis. The expression analysis of sterol biosynthesis pathway genes, which are associated with resistance, can assist the better understanding of antifungal resistance mechanisms. Method: The antifungal susceptibility of 120 clinical C. parapsilosis isolates was examined. The changes in the gene expression related to resistance were analyzed. Results: Eight strains were resistant to fluconazole (FLC), itraconazole (ITC), and amphotericin B (AMB). The regulation variations included increased mRNA levels of ERG3, ERG6, and ERG11 and decreased mRNA levels of ERG3 and ERG6 in response to FLC. ERG11 mRNA level increases in response to ITC and AMB. Conclusion: The mechanism of resistance to azoles in C. parapsilosis is very similar to C. Albicans. This feature may help to design new treatment strategy for candidiasis.

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.

Antifungal Susceptibility Analysis of Clinical Isolates of Candida parapsilosis in Iran.

BACKGROUND: Candida parapsilosis is an emergent agent of invasive fungal infections. This yeast is one of the five most widespread yeasts concerned in invasive candidiasis. C. parapsilosis stands out as the second most common yeast species isolated from patients with bloodstream infections especially in neonates with catheter. Recently several reports suggested that its reduced susceptibility to azoles and polyene might become a cause for clinical concern, although C. parapsilosis is not believed to be intensely prone to the development of antifungal resistance. METHODS: In the present report, One hundred and twenty clinical isolates of C. parapsilosis complex were identified and differentiated by using PCR-RFLP analysis. The isolates were then analyzed to determine their susceptibility profile to fluconazole (FLU), itraconazole (ITC) and amphotericin B. The minimum inhibitory concentration (MIC) results were analyzed according to the standard CLSI guide. RESULTS: All of isolates were identified as C. parapsilosis. No C. metapsilosis and C. orthopsilosis strains were found. Evaluation of the antifungal susceptibility profile showed that only three (2.5%) C. parapsilosis were resistant to fluconazole, three (2.5%) C. parapsilosis were resistant to itraconazole and two (1.7%) C. parapsilosis were amphotericin B resistant. CONCLUSION: Profiles in clinical isolates of C. parapsilosis can provide important information for the control of antifungal resistance as well as distribution and susceptibility profiles in populations.

Sporothrix schenckii complex in Iran: Molecular identification and antifungal susceptibility.

Sporotrichosis is a global subcutaneous fungal infection caused by the Sporothrix schenckii complex. Sporotrichosis is an uncommon infection in Iran, and there have been no phenotypic, molecular typing or antifungal susceptibility studies of Sporothrix species. This study aimed to identify nine Iranian isolates of the S. schenckii complex to the species level using colony morphology, carbohydrate assimilation tests, and PCR-sequencing of the calmodulin gene. The antifungal susceptibilities of these Sporothrix isolates to five antifungal agents (amphotericin B (AMB), voriconazole (VRC), itraconazole (ITC), fluconazole (FLC), and terbinafine (TRB)) were also evaluated according to the M27-A3 and M38-A2 protocols of the Clinical and Laboratory Standards Institute for yeast and mycelial phases, respectively. Five of seven clinical isolates were identified as S. schenckii, and two clinical and two environmental isolates were identified as S. globosa. This is the first report of S. globosa in Iran. There was significant agreement (73%) between the results of the phenotypic and genotypic identification methods. TRB and ITC were the most effective antifungals against the Sporothrix isolates. The minimum inhibitory concentration (MIC) values of TRB for the yeast and mycelial phases of S. schenckii differed significantly. There was also a significant difference in the minimum fungicidal concentration (MFC) values of AMB and TRB for the two phases. Considering the low efficacy of VRC and FLC and the wide MIC ranges of AMB (1-16 µg/ml and 1-8 µg/ml for yeast and mycelial forms, respectively) observed in the present study, in vitro antifungal susceptibility testing should be performed to determine appropriate therapeutic regimens.

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.

Detection of Fungal Elements in Atherosclerotic Plaques Using Mycological, Pathological and Molecular Methods.

BACKGROUND: The aim of this study was to detect fungi in atherosclerotic plaques and investigate their possible role in atherosclerosis. METHODS: Coronary atherosclerotic plaques specimen were obtained from patients with atherosclerosis. Direct examination, culture, histopathology study, PCR and sequencing were performed to detect/identify the mycotic elements in the plaques. Age, sex, smoking, obesity, hypertension, hyperlipidemia, family history of heart diseases and diabetes were considered and data were analyzed using Chi Square test by SPSS version 15. RESULTS: A total of 41 specimens were analyzed. Direct examination for fungal elements was negative in all cases but in culture only one specimen grew as a mold colony. The presence of fungal elements were confirmed in 6 and 2 tissue sections stained by Gomori methenamine silver and Hematoxylin and Eosin methods, respectively. Using PCR, 11 cases were positive for fungi. The DNA sequence analysis of six positive specimens which were randomly selected revealed fungi as Candida albicans (n=3), Candida guilliermondii (n=2) and Monilia sp. (n=1). CONCLUSION: A significant association between the presence of fungi in atherosclerotic plaques and severity of atherogenesis and atherosclerotic disease was not found. This could be due to limited numbers of patients included in our study. However, the presence of fungal elements in 26.8% of our specimens is considerable and the results does not exclude the correlation between the presence of fungi with atherosclerosis and coronary artery disease.

In Vitro Activity of Caspofungin Against Fluconazole-Resistant Candida Species Isolated From Clinical Samples in Iran.

BACKGROUND: Candida spp. is the most common organisms involved in fungal infections in the high risk patients. It causes the greatest number of invasive candidiasis. Fluconazole is effective in treating mucosal candidiasis. However, resistance to fluconazole and other azoles antifungal drugs is an important clinical problem to treat candidiasis. Caspofungin is more effective against Candida species such as some azoles-resistant isolates. OBJECTIVES: The current study aimed to investigate the susceptibilities of clinical fluconazole-resistant and fluconazole - susceptible dose- dependent Candida species to caspofungin. MATERIALS AND METHODS: In the Minimum Inhibitory Concentration (MIC) test, 207 Candida species and other yeasts isolated from Iranian patients (each isolated from a high-risk patient) were evaluated. The yeasts were differentiated by standard mycological methods, CHROM agar Candida, and verified by API20C.AUX. In vitro susceptibilities were determined using Broth Micro Dilution (BMD) method described in the Clinical Laboratory Standards Institute M27-A3. MICs were noted after 24 and 48 hours of incubation. RESULTS: The most frequently isolated species were Candida albicans (52.2%), C. glabrata (24.6%), followed by C. tropicalis (7.7%) and C. krusei (3.4%). MICs of caspofungin against 87% of C. albicans and 90% of C. glabrata and C. tropicalis isolates were 2 µg/mL and for C. krusei were 4 µg/mL, respectively. The results revealed that only 20 out of 207 isolates (9.7%) were non-sensitive to caspofungin. Caspofungin non-susceptible isolates were isolated from the patients with cancer, diabetes and AIDS; and not in the species isolated from patients with other underlying diseases. CONCLUSIONS: Caspofungin appears more effective in vitro against Iranian fluconazole-resistant Candida isolates and some other yeasts.

Detection of Aspergillus flavus and A. fumigatus in Bronchoalveolar Lavage Specimens of Hematopoietic Stem Cell Transplants and Hematological Malignancies Patients by Real-Time Polymerase Chain Reaction, Nested PCR and Mycological Assays.

BACKGROUND: Pulmonary aspergillosis (PA) is one of the most serious complications in immunocompromised patients, in particular among hematopoietic stem cell transplants (HSCT) and patients with hematological malignancies. OBJECTIVES: The current study aimed to evaluate the incidence of PA and utility of molecular methods in HSCT and patients with hematological malignancies, four methods including direct examination, culture, nested polymerase chain reaction (PCR) and real-time PCR were performed on bronchoalveolar lavage (BAL) specimens in Tehran, Iran. PATIENTS AND METHODS: During 16 months, 46 BAL specimens were obtained from individuals with allogeneic HSCT (n = 18) and patients with hematological malignancies (n = 28). Direct wet mounts with 20% potassium hydroxide (KOH) and culture on mycological media were performed. The molecular detection of Aspergillus fumigatus and A. flavus was done by amplifying the conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA by nested-PCR and the ß-tubulin gene by TaqMan real-time PCR. RESULTS: Seven (15.2%) out of 46 specimens were positive in direct examination and showed branched septate hyphae; 11 (23.9%) had positive culture including eight (72.7%) A. flavus and three (27.3%) A. fumigatus; 22 (47.8%) had positive nested-PCR and eight (17.4%) had positive real-time PCR. The incidence of invasive pulmonary aspergillosis (IPA) in these patients included proven IPA in 1 (2.2%), probable IPA in 10 (21.7%), possible IPA in 19 (41.3%) and not IPA in 16 cases (34.8%). CONCLUSIONS: The incidence of IPA in allogeneic HSCT and patients with hematological malignancies was relatively high and A. flavus was the most common cause of PA. As molecular methods had higher sensitivity, it may be useful as screening methods in HSCT and patients with hematological malignancies, or to determine when empirical antifungal therapy can be withheld.

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.

Restriction analysis of ß-tubulin gene for differentiation of the common pathogenic dermatophytes.

BACKGROUND: Identification of dermatophytes at the species level, relying on macro- and microscopic properties of the colonies is time-consuming, questioned in many circumstances, and requires considerable expertise. In this study, we examined the potency of a new genetic marker, ß-tubulin (BT2) gene, for differentiation of dermatophytes in an in silico and experimental restriction fragment length polymorphism (RFLP) profile. METHODS: The BT2 sequences of dermatophyte species were retrieved from GenBank and analyzed using bioinformatics softwares to choose suitable restriction enzyme(s). Forty reference culture collections and 100 clinical isolates were PCR-amplified using the primers T1 and Bt2b and consequently subjected to virtual RFLP analysis. The dermatophytes were identified according to specific lengths of bands in agarose gel electrophoresis. RESULTS: After digestion of partially amplified ß-tubulin gene with the restriction enzyme FatI, three dermatophyte species, that is, Microsporum gypseum, M. canis, and Trichophyton verrucosum yielded unique restriction maps while the remaining species including T. interdigitale, T. rubrum, T. tonsurans, T. schoenleinii, and T. violaceum, were identified by further restriction digestion by Alw21I, MwoI, and HpyCH4V endonucleases. The length of RFLP products was same as of those expected by computer analysis. CONCLUSION: The two-step BT2 restriction mapping used in this study is an effective tool for reliable differentiation of the clinically relevant species of dermatophytes.

In-vitro Activity of 10 Antifungal Agents against 320 Dermatophyte Strains Using Microdilution Method in Tehran.

Dermatophyte fungi are the etiologic agents of skin infections commonly referred to as ringworm. These infections are not dangerous but as a chronic cutaneous infections they may be difficult to treat and can also cause physical discomfort for patients. They are considered important as a public health problem as well. No information is available regarding the efficacy of antifungal agents against dermatophytes in Tehran. Therefore, in this study we evaluated the efficacy of 10 systemic and topical antifungal medications using CLSI broth microdilution method (M38-A). The antifungal agents used included griseofulvin, terbinafine, itraconazole, ketoconazole, fluconazole, voriconazole, clotrimazole, ciclopirox olamine, amorolfine and naftifine.Fifteen different species of dermatophytes which were mostly clinical isolates were used as follows; T. mentagrophytes, T. rubrum, E. floccosum, M. canis, T. verrucosum, T. tonsurans, M. gypseum, T. violaceum, M. ferruginum, M. fulvum, T. schoenleinii, M. racemosum, T. erinacei, T. eriotrephon and Arthroderma benhamiae. The mean number of fungi particles (conidia) inoculated was 1.25 ×104 CFU/mL. Results were read after 7 days of incubation at 28 °C. According to the obtained results,itraconazole and terbinafine showed the lowest and fluconazole had the greatest MIC values for the most fungi tested. Based on the results, it is necessary to do more research and design a reliable standard method for determination of antifungal susceptibility to choose proper antibiotics with fewer side effects and decrease antifungal resistance and risk of treatment failure.

Pneumocystis jirovecii Colonization in Non-HIV-Infected Patients Based on Nested-PCR Detection in Bronchoalveolar Lavage Samples.

BACKGROUND: Pneumocystis jirovecii causes Pneumocystis pneumonia (PCP) in immunocompromised patients with a high rate of morbidity and mortality. Colonization with this fungus may stimulate pulmonary inflammation or lead to PCP in susceptible patients. The epidemiology of this infection and routs of its transmission has poorly studied in Iran. We examined Pneumosystis colonization in patients with various lung underlying diseases. METHODS: Bronchoalveolar lavage (BAL) fluids of 458 patients with different underlying diseases or pulmonary signs were collected between August 2010 and January 2012. Patients were divided into four groups: transplant recipients, malignant patients, immunosuppressive drug recipients and patients with other different lung diseases. A sensitive nested-PCR method targeted 18S ribosomal RNA gene was used for investigating P. jirovecii in the specimens. RESULTS: P. jirovecii DNA was detected in 57 out of 458 (12.5%) BAL samples by nested-PCR. Colonization rate in malignant patients, transplant recipients, immunosuppressive therapy recipients and patients with other various lung diseases was 21.7%, 20.3%, 12.7% and 7.3%, respectively. The enzyme BanI cuts all PCR products producing fragments with the size of 228 and 104 base pair. This finding as well as sequencing of four random positive samples validated and reconfirmed the PCR results. P. jirovecii cysts were found in 5 out of 57 PCR positive samples. CONCLUSION: A significant number of patients with pulmonary diseases were colonized by P. jirovecii that can develop to PCP in these patients or they may transmit the fungus to other susceptible patients.

Overexpression of aldo-keto-reductase in azole-resistant clinical isolates of Candida glabrata determined by cDNA-AFLP.

BACKGROUND: Candida glabrata causes significant medical problems in immunocompromised patients. Many strains of this yeast are intrinsically resistant to azole antifungal agents, and treatment is problematic, leading to high morbidity and mortality rates in immunosuppressed individuals. The primary goal of this study was to investigate the genes involved in the drug resistance of clinical isolates of C. glabrata. METHODS: The clinical isolates of C. glabrata were collected in an epidemiological survey of candidal infection in immunocompromised patients and consisted of four fluconazole and itraconazole resistant isolates, two fluconazole and itraconazole sensitive isolates, and C. glabrata CBS 138 as reference strain. Antifungal susceptibility patterns of the organisms were determined beforehand by the Clinical and Laboratory Standards Institute (CLSI). The potential gene(s) implicated in antifungal resistance were investigated using complementary DNA- Amplified Fragment Length Polymorphism (cDNA-AFLP). Semi-quantitative RT-PCR was carried out to evaluate the expression of gene(s) in resistant isolates as compared to sensitive and reference strains. RESULTS AND CONCLUSIONS: The aldo-keto-reductase superfamily (AKR gene) was upregulated in the resistant clinical isolates as assessed by cDNA-AFLP. Semi-quantitative RT-PCR revealed AKR mRNA expression approximately twice that seen in the sensitive isolates. Overexpression of the AKR gene was associated with increased fluconazole and itraconazole resistance in C. glabrata. The data suggest that upregulation of the AKR gene might give a new insight into the mechanism of azole resistance.