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Background and purpose: Among the different categories of antifungal drugs, azole antifungals have received more attention due to their broad spectrum, high potency, and acting on specific target enzyme. Fluconazole is one of these drugs that as a bis-triazole, was the first triazole drug to enter the market. New-generation antifungal drugs such as albaconazole and voriconazole have been designed by replacing one of the triazole rings of fluconazole with other heterocycles. Based on this strategy, in this research, a new derivative of fluconazole in which a triazole residue of fluconazole is replaced by 4-benzylpiperazine dithiocarbamate was synthesized and its effects were evaluated in vitro. Materials and methods: The desired final compound (6) was obtained from the reaction of two key intermediates, oxirane, and N-benzylpiperazine, in the presence of carbon disulfide and triethylamine in ethanol. To prepare the oxirane derivative, the reaction of a ketonic compound namely 1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl) ethan-1-one with trimethylsulfoxonium iodide was used. On the other hand, for the synthesis of N-benzyl piperazine, benzyl chloride was refluxed with the excess of piperazine. It should be noted that the purification of the compounds was done with common methods of extraction and recrystallization and there is no need for chromatography. The structure of the final synthesized compound was confirmed by infrared (IR) nuclear magnetic resonance (NMR) and mass spectrometry (MS). The antifungal activity of the synthesized compound was investigated in comparison with fluconazole against several species of pathogenic fungi sensitive to fluconazole, including Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, and Candida tropicalis, as well as fluconazole-resistant isolates, and the minimum inhibitory concentration (MIC) was determined in vitro by broth micro-dilution method. In addition, the hemolytic effect on red blood cells and cytotoxicity on HepG2 cells were investigated for this compound. The docking mode of the target compound with lanosterol 14α-demethylase (CYP51) enzyme was also studied. Results: Preliminary investigation of antifungal effects on ten fungi strains sensitive to fluconazole showed that the target compound with MIC values between 0.063 and 1 μg/ml has strong antifungal effects on C. albicans, C. glabrata, C. parapsilosis, C. krusei, and C. tropicalis. In general, its antifungal activity was 4-64 times more than that of fluconazole. Also, the compound showed a good anti-Candida profile against fluconazole-resistant species. While the MIC values of fluconazole against C. albicans and C. krusei isolates were equal to or greater than 64 μg/ml, compound 6 effectively inhibited the growth of C. albicans and C. krusei resistant to fluconazole at concentrations of 8 or 16 μg/ml. The MIC value of this compound against C. parapsilosis was equal to 0.5 μg/ml. The results of in vitro hemolysis and cytotoxicity tests also showed that this compound is as safe for human cells as fluconazole. The docking study showed that the designed compound can interact more tightly with the lanosterol 14α-demethylase enzyme due to the presence of a benzylpiperazine dithiocarbamate scaffold in its structure. Conclusion: According to the obtained results, the compound synthesized in this study is a suitable candidate for conducting in vivo tests and other preclinical and pharmacokinetic tests to discover a new antifungal drug.

Background and Purpose: Candida auris is a multidrug-resistant yeast that rapidly spreads, making it the leading Candidate for the next pandemic. One main leading cause of emerging resistant C. auris isolates is nonsynonymous mutations. This study aimed to detect the Y132F mutation, one of the most important azole resistance-associated mutations in the ERG-11 gene of C. auris, by developing a reliable high-resolution melt (HRM)-based method.Materials and Methods: Five C. auris isolates from Iran, plus three control isolates from other Clades were used in the study. The antifungal susceptibility testing through micro broth dilution was performed to recheck their susceptibility to three azole antifungals,including fluconazole, itraconazole, and voriconazole. Moreover, the polymerase chain reaction (PCR) sequencing of the ERG-11 gene was performed. Following the bioinformatic analysis and HRM-specific primer design, an HRM-based assay was developed and evaluated to detect ERG-11 mutations.Results: The minimum inhibitory concentrations of fluconazole among Iranian C. auris isolates ranged from 8 to 64 μg/mL. The PCR-sequencing of the ERG-11 gene and bioinformatic analyses revealed the mutation of Y132F, a substitution consequence of A to T on codon 395 in one fluconazole-resistant isolate (IFRC4050). The developed HRM assay successfully differentiated the targeted single nucleotide polymorphism between mutant and wild types (Melting temperature [Tm]: 81.79 ℃ - cycle threshold [CT]: 20.06 for suspected isolate). For both mutant and non-mutant isolates, the mean Tm range was 81.79-82.39 °C and the mean CT value was 20.06-22.93. These results were completely in accordance with the findings of DNA sequencing.Conclusion: The fast-track HRM-based method successfully detected one of the most common mechanisms of resistance in the ERG-11 gene of C. auris within 3 h. Finally, the development of more panels of HRM assays for the detection of all azole resistance mutations in C. auris ERG-11 is recommended to expand the scope of the field and facilitate the elaboration of rapid and accurate methods of antifungal resistance assessment.

Candida auris, an emerging non-albicans multidrug-resistant yeast, has become a significant cause of invasive candidiasis in healthcare settings. So far, data on the metabolites of C. auris in different clades are minimal, and no studies have focused on clade V metabolites. Therefore, Gas chromatography–mass spectrometry (GC-MS) was used for the metabolomic profiling of clade I C. auris compared with fluconazole-resistant and susceptible C. auris in clade V strains. GC-MS chromatography revealed 28, 22, and 30 compounds in methanolic extracts of the fluconazole-susceptible and fluconazole-resistant C. auris clade V and C. auris clade I strain, respectively. Some compounds, such as acetamide and metaraminol, were found in fluconazole-susceptible and resistant C. auris clade V and clade I. N-methyl-ethanamine and bis(2-ethylhexyl) phthalate metabolites were found in both fluconazole -susceptible and resistant C. auris clade V, as well as 3-methyl-4-isopropylphenol, 3,5-bis(1,1-dimethyl)-1,2-benzenediol, and diisostyl phthalate metabolites in both fluconazole resistant C. auris clade V and I. Identifying these metabolites contributes to understanding the morphogenesis and pathogenesis of C. auris, highlighting their potential role in antifungal drug resistance and the control of fungal growth. However, further experiments are warranted to fully comprehend the identified metabolites’ regulatory responses, and there may be potential challenges in translating these findings into clinical applications.

Abstract Regarding the important role of the urease enzyme as a virulence factor in urease-positive microorganisms in this study, new series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were designed and synthesized. All compounds evaluated against urease enzyme exhibiting IC50 values of 0.87 ± 0.09 to 8.32 ± 1.21 µM as compared with thiourea as the positive control (IC50 = 22.54 ± 2.34 µM). The kinetic evaluations of 6a as the most potent derivative recorded a competitive type of inhibition. Molecular dynamic simulations of the 6a derivative were also conducted, showing that 6a occupied the active site with closed state. Antimicrobial activities of all derivatives were performed, and 6f (R = 3-Cl), 6g (R = 4-Cl), and 6h (R = 3,4-diCl) analogs demonstrated significant antifungal activities with MIC values of 1, 2, and 0.5 µg/mL compared with fluconazole with MIC = 2 µg/mL. Synthesized analogs also exhibited potent urease inhibitory activities against C. neoformans (IC50 = 83.7–118.7 µg/mL) and P. mirabilis (IC50 = 74.5–113.7 µg/mL), confirming their urease inhibitory potential. The results demonstrated that the designed scaffold could be considered a suitable pharmacophore to develop potent urease inhibitors.

Virtually all Candida species linked to clinical candidiasis are capable of forming highly resistant biofilms on different types of surfaces, which poses an additional significant threat and further complicates therapy of these infections. There is a scarcity of antifungal agents, and their effectiveness, particularly against biofilms, is limited. Here we provide a historical perspective on antifungal agents and therapy of Candida biofilms. As we reflect upon the past, consider the present, and look towards the future of antifungal therapy of Candida biofilms, we believe that there are reasons to remain optimistic, and that the major challenges of Candida biofilm therapy can be conquered within a reasonable timeframe.

Background and Purpose: Wickerhamomyces myanmarensis is a new opportunistic yeast previously named Pichai myanmarensis, which belongs to the order Saccharomycetales. Since its discovery, one environmental isolate of W. myanmarensis has been reported from Myanmar, and one clinical sample from Iran.Case Report:We report a case of bloodstream infection related to an implantable venous access port. W. myanmarensis was isolated from patient's blood after chemotherapy, which was meant to control and heal T-cell lymphoblastic lymphoma. Broth dilution minimum inhibitory concentrations were performed according to the CLSI M27-A3 document. The patient recovered with intravenous voriconazole and was discharged with the recommended prescription of oral voriconazole as a maintenance drug.Conclusion: So far, only one case of W. myanmarensis fungemia has been reported in the world in 2019. This is the second case of bloodstream infection with this yeast from a patient undergoing chemotherapy in Iran.

Background and Purpose: Scedosporium species are ubiquitous environmental fungi,which are considered emerging agents that trigger disease in humans and animals. Thepresent study aimed to determine Scedosporium dehoogii strain isolated from paddy field soil samples using semi-selective media and evaluate its antifungal susceptibility profile.Materials and Methods: Three paddy field soil samples were collected during aninvestigation for the isolation of Scedosporium species in Mazandaran province, Iran.Morphological and molecular analyses based on ITS-rDNA sequencing were performed. Furthermore, in vitro antifungal susceptibility testing for conventional drugs and novel imidazole (luliconazole) was performed based on Clinical and Laboratory Standards Institute M38-A3 guidelines.Results: In this study, S. dehoogii was isolated from the soil in paddy fields. Based onthe results, itraconazole and luliconazole showed the least and most antifungal activityagainst this isolate, respectively.Conclusion: Based on the findings, molecular identification was essential fordistinguishing the species of S. dehoogii. Remarkably, luliconazole showed potent activity against this strain.

Candida auris has emerged globally as a multidrug-resistant pathogen causing outbreaks in health care facilities. Whole genome sequencing (WGS) analysis has identified four major clades, while earlier WGS data from a single Iranian isolate suggested the existence of a potential fifth clade. Here, we confirm the existence of this fifth clade by providing WGS data of another four Iranian isolates. These clade V isolates differed less than 100 single-nucleotide polymorphisms (SNPs) between each other, while they were separated from the other clades by more than 200,000 SNPs. Two of these isolates were resistant to fluconazole and were found to harbour mutations in the TAC1b and ERG11 genes.

Candida auris is a newly emerging multidrug-resistant fungal pathogen considered to be a serious global health threat. Due to diagnostic challenges, there is no precise estimate for the prevalence rate of this pathogen in Iran. Since 2019, only six culture-proven C. auris cases have been reported from Iran, of which, five belonged to clade V and one to clade I. Herein, we report a case of otomycosis due to C. auris from 2017 in a 78-year-old man with diabetes mellitus type II without an epidemiological link to other cases or travel history. Short tandem repeat genotyping and whole genome sequencing (WGS) analysis revealed that this isolate belonged to clade I of C. auris (South Asian Clade). The WGS single nucleotide polymorphism calling demonstrated that the C. auris isolate from 2017 is not related to a previously reported clade I isolate from Iran. The presence of this retrospectively recognized clade I isolate also suggests an early introduction from other regions or an autochthonous presence. Although the majority of reported C. auris isolates worldwide are resistant to fluconazole and, to a lesser extent, to echinocandins and amphotericin B, the reported clade I isolate from Iran was susceptible to all antifungal drugs.

Background and Purpose: The frequency and genetic diversity of black fungi in environmental and clinical settings have not been fully studied in Iran. This study aimed to identify and evaluate intra- and inter-species DNA sequence variation and also understand the phylogenetic relationships of melanized fungi and relatives isolated from different geographical regions of Iran.Materials and Methods: In total, 111 clinical and environmental strains of dematiaceous fungi were isolated, and their internal transcribed spacer ribosomal DNA(rDNA) regions were sequenced and analyzed.Results: An inter-species nucleotide sequence diversity rate of 1 to 464 nucleotides was observed between the species. Intra-species differences were found in the strains of Alternaria alternata, Cladosporium cladosporioides, Alternaria tenuissima, Curvularia spicifera, Aureobasidium pullulans, Curvularia hawaiiensis, Neoscytalidium dimidiatum,Alternaria terricola, Alternaria chlamydospora, Didymella glomerata, and Drechslera dematioidea by 0–59, 0–22, 0–4, 0–4, 0–3, 0–2, 0–2, 0–2, 0–2, 0–1, and 0–1 nt, respectively.Conclusion: The internal transcribed spacer rDNA is useful for the discrimination of several taxa of dematiaceous fungi. However, a better understanding of the taxonomy of species of Alternaria requires a larger rDNA region or a library of other gene sequences.

Background and purpose: Candidiasis is an opportunistic infection caused by Candida species which occurs following the host´s immunological weakness. Restrictions in treatment of fungal diseases such as drug resistance and side-effects increased interest towards exploring new drugs, especially herbal medicines. The objective of this study was to compare the antifungal effects of methanolic extract of Rhus Coriaria and nystatin on Candida albicans strains. Materials and methods: In this experimental-in vitro study, the antifungal effect of Sumac methanolic extract on Candida alicens strains was assessed by minimum inhibitory concentration (MIC) test and broth microdilution method. In addition, MIC was performed on these strains using nystatin. Results: The methanolic extract of Rhus Coriaria did not show the ability to inhibit Candida albicans strains at5 low concentrations. It exerted its inhibitory effect at least at 512 mg/L, while the effective range of nystatin was 0.016-0.125 mg/L. In addition, results of MIC, MIC50, MIC90, and G mean revealed lower values which indicated the higher inhibitory potency of this drug. Conclusion: Methanolic extract of sumac exhibited inhibitory effect on Candida albicans at high concentrations and could be used in traditional medicine more broadly against Candida albicans yeast. However, the effect is weaker than that of nystatin.

Background and Purpose: Because of the growing incidence of Aspergillus infection,typing methods of Aspergillus species are increasingly being used. Accordingly,studying the spread and population dynamics of strains isolating from clinical and environment, from a single host to large scale ecosystems is definitely needed. In the current study, we carried out a genetic analysis of nine microsatellite loci in isolates from different regions of Iran to compare and explore the genetic diversity between environmental and clinical A. fumigatus strains. Materials and Methods: Sixty-six clinical (n=43) and environmental (n= 23) isolates of A. fumigatus, have collected from six cities of Iran. All A. fumigatus isolates identified based on macroscopic and microscopic characters, the ability to grow at above 45°C, and confirmed using DNA sequencing of the partial b-tubulin gene.Sixty-six A. fumigatus isolates were subjected by microsatellite typing using three separate multiplex PCRs with a panel of nine short tandem repeats (STR) to evaluate the genetic relatedness. Results: The STR typing of 66 A. fumigatus isolates revealed 38 distinct genotypes distributed among environmental and clinical isolates. We identified 12 clones including 40 different isolates representing 60% of all isolates tested, which each clone included 2–7 isolates. Conclusion: The STR typing is considered as a valuable tool with excellent discriminatory power to study the molecular epidemiology and genotypic diversity of A.fumigatus isolates. These findings show that the high genetic diversity observed of Iranian A. fumigatus isolates with those outside Iran and formed a separate cluster.

Background and Purpose: Candida auris, as a new characterized pathogenic yeast,has attracted remarkable attention in the recent decade due to its rapid global emergence and multidrug resistance traits. This unique species is able to cause nosocomial outbreaks and tolerate adverse conditions; however, it has been mostly misidentified by conventional methods. Case report: This report aimed to describe the first fluconazole-resistant case of C.auris otitis in an immunocompetent patient in Iran. The isolate showed minimum inhibitory concentration of ≥ 32 μg/ml for fluconazole; however, the patient was treated with topical clotrimazole and miconazole with no recurrence. Conclusion: This was the second strain of C. auris isolated from otitis in Iran which was fluconazole-resistant, unlike the first Iranian isolate.

Several prolonged and significant outbreaks of dermatophytosis caused by Trichophyton indotineae, a new emerging terbinafine-resistant species, have been ongoing in India in recent years, and have since spread to various countries outside Asia. Miltefosine, an alkylphosphocholine, is the most recently approved drug for the treatment of both visceral and cutaneous leishmaniasis. Miltefosine in vitro activity against terbinafine-resistant and susceptible T. mentagrophytes/T. interdigitale species complex, including T. indotineae, is limited. The current study aimed to assess miltefosine’s in vitro activity against dermatophyte isolates, which are the most common causes of dermatophytosis. Miltefosine, terbinafine, butenafine, tolnaftate, and itraconazole susceptibility testing was performed using Clinical and Laboratory Standards Institute broth microdilution methods (CLSI M38-A3) against 40 terbinafine-resistant T. indotineae isolates and 40 terbinafine-susceptible T. mentagrophytes/T. interdigitale species complex isolates. Miltefosine had MIC ranges of 0.063–0.5 µg/mL and 0.125–0.25 µg/mL against both terbinafine-resistant and susceptible isolates. In terbinafine-resistant isolates, the MIC50 and MIC90 were 0.125 µg/mL and 0.25 µg/mL, respectively, and 0.25 µg/mL in susceptible isolates. Miltefosine had statistically significant differences in MIC results when compared to other antifungal agents (p-value 0.05) in terbinafine-resistant strains. Accordingly, the findings suggest that miltefosine has a potential activity for treating infections caused by terbinafine-resistant T. indotineae. However, further studies are needed to determine how well this in vitro activity translates into in vivo efficacy.

Background and Purpose: The present study aimed to evaluate the effect of cyproconazole, the most used fungicide in Iranian wheat farms, on the induction of voriconazole resistance in Aspergillus fumigatus isolates. Materials and Methods: A collection of 20 clinical and environmental isolates were selected for investigation of the in vitro activity of fungicides. The minimum inhibitory concentrations (MICs) were determined by the documented broth microdilution method M38-A2 (CLSI, 2008). Induction experiments were performed and the possibly induced isolate(s) were subjected to antifungal susceptibility testing, sequencing of the CYP51A promoter, and full coding gene. Furthermore, CYP51-protein homology modeling and docking modes were evaluated using SWISS-MODEL (https://swissmodel.expasy.org/) and SEESAR software (version 9.1). Results: Among 10 susceptible isolates, only one strain showed a high MIC value against voriconazole (MIC=4μg/ml) after 25 passages. Nevertheless, sequencing of the CYP51A promoter and full coding gene did not reveal any mutations. Cyproconazole, which has three nitrogen atoms in the aromatic ring, coordinated to the iron atom of heme through a hydrogen bond contact to residue Lys147 present in the active site of the A. fumigates Cyp51 homology model. Conclusion: Cyproconazole is being applied extensively in wheat farms in Iran. According to the results, cyproconazole may not play a key role in the induction of azole resistance in the isolates through the environmental route. However, the potential ability of the fungicide to induce medically triazole-resistant strains over a long period of application should not be neglected.

Introduction: Candida parapsilosis (C. parapsilosis) is a common non-albicans Candida species ranked as the second common cause of bloodstream infections. Azole resistance and elevated echinocandin MICs have been reported for these fungi. This study was conducted to determine the interactions between azoles and echinocandins against C. parapsilosis species complex. Materials and methods: Fifteen fluconazole-resistant clinical isolates of C. parapsilosis complex were included: C. parapsilosis sensu stricto (n = 7), C. orthopsilosis (n = 5) and C. metapsilosis (n = 3). The activity of azoles (fluconazole, itraconazole) and echinocandins (anidulafungin, micafungin) alone and in combination was determined using checkerboard broth microdilution. The results were determined based on the fractional inhibitory concentration index (FICI). Results: In vitro combination of fluconazole with anidulafungin was found to be synergistic (FICI 0.07–0.37) and decreased the MIC range from 4–64 μg/mL to 0.5–16 μg/mL for fluconazole and from 2–8 μg/mL to 0.125–1 μg/mL for anidulafungin. Similarly, interactions of fluconazole with micafungin (FICI 0.25–0.5), itraconazole with anidulafungin (FICI 0.15–0.37) and itraconazole with micafungin (FICI 0.09–0.37) were synergistic. Conclusion: The combination of fluconazole and itraconazole with either anidulafungin or micafungin demonstrated synergistic interactions against C. parapsilosis species complex, especially against isolates with elevated MIC values. However, the use of these combinations in clinical practice and the clinical relevance of in vitro combination results remain unclear.

Abstract The COVID-19 pandemic has become the leading societal concern. The pandemic has shown that the public health concern is not only a medical problem, but also affects society as a whole; so, it has also become the leading scientific concern. We discuss in this treatise the importance of bringing the world’s scientists together to find effective solutions for controlling the pandemic. By applying novel research frameworks, interdisciplinary collaboration promises to manage the pandemic’s consequences and prevent recurrences of similar pandemics.

Background and Purpose: Emergence and development of antifungal drug resistance in Candida species constitute a serious concern. Candida auris as an emerging multidrug-resistant fungus is the most important public health threat with high levels of mortality and morbidity. Almost all C. auris isolates are resistant to fluconazole, and there have been reports of elevated minimum inhibitory concentrations (MICs) to amphotericin B and echinocandins. To overcome the growing challenge of antifungal resistance, a valuable alternative option would be the use of drug combination. Materials and Methods: The present study evaluated the in vitro combination of nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, diclofenac and aspirin with fluconazole against fluconazole-resistant C. auris in comparison to other fluconazole-resistant Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei originating from patients with candidiasis. Results: The MIC ranges of fluconazole-ibuprofen and fluconazole-diclofenac decreased from 32-256 to 32-128 and 16-256 μg/ml, respectively and remained the same for fluconazole-aspirin against C. auris. However, the combination of fluconazole with ibuprofen resulted in a synergistic effect for 5 strains, including C. albicans (n=2), C. tropicalis (n=1), C. glabrata (n=1), and C. krusei (n=1), by decreasing the MIC of fluconazole by 2-3 log2 dilutions. Conclusion: Although the interaction of NSAIDs with fluconazole was not synergistic against fluconazole-resistant C. auris isolates, no antagonism was observed for any combinations. Therefore, combination with newer azole agents needs to be conducted.

Background and Purpose: Although the mechanism of action for echinocandins is known, the physiological mechanisms by which these antifungal agents cause cell death via the classical apoptotic pathways are not well-defined yet. Regarding this, the present study aimed to evaluate the mechanisms of caspofungin-induced Candida glabrata cell death. Materials and Methods: For the purpose of the study, the minimum inhibitory concentration (MIC) of caspofungin against C. glabrata (ATCC 90030) was determined using the broth microdilution reference method (CLSI M27-A2 and M27-S4). The annexin V and propidium iodide staining was performed to determine the way through which caspofungin acts against C. glabrata (i.e., through the induction of apoptosis and/or necrosis). Additionally, the possible effect of caspofungin on inducing the expression of two apoptotic genes, namely MCA1 and NUC, was studied using the real-time polymerase chain reaction assay. Results: According to the obtained MIC value (0.5 μg/mL), C. glabrata, exposed to 0.25, 0.5, and 1 μg/mL of caspofungin, exhibited the features of late apoptosis/necrosis after 18 h of incubation. Furthermore, the use of 0.25, 0.5, and 1 μg/ml caspofungin induced apoptosis (early/late) in 14.67%, 17.04%, and 15.89% of the cells, respectively. The results showed a significant difference between the percentages of early-apoptotic cells at the three concentrations (p Conclusion: As the findings of the present study indicated, caspofungin promoted both necrosis and apoptosis of C. glabrata cells at concentrations higher than or equal to the MIC value.

Background and Purpose: Candidemia is a life-threatening fungal infection with significant mortality and morbidity in neutropenic individuals, immunosuppressive chemotherapy recipients, and broad-spectrum antibiotics consumers. The epidemiology and antifungal susceptibility testing of non-albicans Candida species have been poorly studied. These species are characterized by low susceptibility to azoles and echinocandins. Herein, we report the first pediatric case of candidemia due to C. guilliermondii in Iran and review the literature on fungemia caused by C. guilliermondii. Case report: We presented the first candidemia case due to Candida guilliermondii in a 4-month-old male infant with neuroblastoma in Iran. This study also involves a comprehensive literature review on fungemia caused by C. guilliermondii during a period of 18 years (i.e., 2000-2018) to discuss the epidemiology, clinical features, and treatment of this disease. The literature review resulted in the identification of 501 cases of candidemia caused by C. guilliermondii. Most of the patients were adults and had multiple risk factors. However, the main risk factors were significantly related to cancer chemotherapy, followed by central venous catheter use and Intensive Care Unit admission. Mortality rate due to this disease had a range of 3.4-66.6%; in this regard, the patients with cancer had the highest mortality rate. Conclusion: Given the high mortality of candidemia, the early diagnosis of this infection and timely initiation of antifungal therapy significantly improve the patients’ survival rate and result in better outcomes. Consequently, it is highly recommended to monitor the local epidemiology of this life-threatening infection and raise awareness in this regard.

Fusarium species are filamentous fungi that cause a variety of infections in humans. Because they are commonly resistant to many antifungal drugs currently available in clinical settings, research into alternative targets in fungal cells and therapeutic approaches is required. The antifungal activity of miltefosine and four comparators, amphotericin B, voriconazole, itraconazole, and caspofungin, were tested in vitro against a collection of susceptible and resistant clinical (n = 68) and environmental (n = 42) Fusarium isolates. Amphotericin B (0.8 μg/mL) had the lowest geometric mean (GM) MICs/MECs values followed by miltefosine (1.44 μg/mL), voriconazole (2.15 μg/mL), caspofungin (7.23 μg/mL), and itraconazole (14.19 μg/mL). Miltefosine was the most effective agent against Fusarium isolates after amphotericin B indicating that miltefosine has the potential to be studied as a novel treatment for Fusarium infections.

Prompt and targeted antifungal treatment has a positive impact on the clinical outcome of mucormycosis; however, current diagnostic tools used in histopathology laboratories often fail to provide rapid results. Rapid culture-based strategies for early diagnosis of Mucorales infections, which may influence treatment decisions, are urgently needed. Herein, we evaluated a microculture assay for the early diagnosis of mucormycosis in an immunocompetent murine model of disseminated infection, by comparing it with traditional diagnostic methods. The assay specificity was assessed using blood (n = 90) and tissue (n = 90) specimens obtained from mice infected with Rhizopus arrhizus using different inoculum sizes [1 × 104, 1 × 105, and 1 × 106 colony forming units (CFUs)/mouse] and blood (n = 15) and tissue specimens (n = 15) from uninfected mice. Surprisingly, 26 of 90 (28.9%) blood samples revealed positive results by microculture, whereas all blood samples were negative when assayed by conventional culture. The overall positive conventional culture rate for the mouse tissue (kidney) samples was 31.1% (28/90). The calculated sensitivity for kidney microculture was 98.8% [95% confidence interval (CI) 96.6–100], with an assay specificity of 100%. Hence, the microculture assay may be useful for rapid culturing and diagnosis of mucormycosis caused by R. arrhizus directly in blood and tissue samples. Hence, this method may allow for the timely administration of an appropriate treatment.

Abstract Background Opportunistic infections due to Candida species occur frequently in cancer patients because of their inherent immunosuppression. The aim of the present study was to investigate the epidemiology of yeast species from the oral cavity of patients during treatment for oncological and haematological malignancies. Methods MALDI-TOF was performed to identify yeasts isolated from the oral cavity of 350 cancer patients. Moreover, antifungal susceptibility testing was performed in according to CLSI guidelines (M27-A3). Results Among 162 yeasts and yeast-like fungi isolated from the oral cavity of cancer patients, Candida albicans was the most common species (50.6%), followed by Candida glabrata (24.7%), Pichia kudriavzevii (Candida krusei (9.9%)), Candida tropicalis (4.3%), Candida dubliniensis (3.7%), Kluyveromyces marxianus (Candida kefyr (3.7%)) and Candida parapsilosis (1%). In addition, uncommon yeast species i.e., Saprochaete capitata, Saccharomyces cerevisiae, Clavispora lusitaniae (C. lusitaniae) and Pichia kluyveri (C. eremophila) were recovered from oral lesions. Oral colonization by C. albicans, non-albicans Candida species and uncommon yeasts were as follow; 55%, 44% and 1%, whereas oral infection due to C. albicans was 33.3%, non-albicans Candida species 60.6%, and uncommon yeasts 6.1%. Poor oral hygiene and xerostomia were identified as independent risk factors associated with oral yeast colonization. The overall resistance to fluconazole was 11.7% (19/162). Low MIC values were observed for anidulafungin for all Candida and uncommon yeast species. Conclusions This current study provides insight into the prevalence and susceptibility profiles of Candida species, including emerging Candida species and uncommon yeasts, isolated from the oral cavity of Iranian cancer patients. The incidence of oral candidiasis was higher amongst patients with hematological malignancies. The majority of oral infections were caused by non-albicans Candida species which were often more resistant to anti-fungal agents. Our findings suggest that anidulafungin should be used as antifungal of choice for prophylaxis in clinically high-risk patients with documented oral colonization or infection.

Background and Purpose: Candida parapsilosis is a predominant species found in nosocomial infection, particularly in hospitalized patients. The molecular epidemiology of the clinical strains of this species has not been well studied. The present study was performed with the aim of investigating the microsatellite genotyping of Candida parapsilosis among the Iranian clinical isolates. Materials and Methods: This study was conducted on 81 independent clinical C. parapsilosis isolates that were genotyped by using a panel of six microsatellite markers. Results: The short tandem repeat (STR) typing of clinical C. parapsilosis isolates demonstrated 68 separate genotypes, among which 57 genotypes were observed once and the remaining 11 cases were identified for multiple times. The Simpson’s diversity index for the panel of combined six markers yielded a diversity index of 0.9951. The heterogeneity was observed among the Iranian and the Netherlands clinical C. parapsilosis isolates. Conclusion: As the findings indicated, the clinical C. parapsilosis isolates from Iran showed a high genetic diversity. It can be concluded that molecular epidemiology could be useful for screening during outbreak investigation where C. parapsilosis is involved.

Abstract Background Antifungal susceptibility testing is a subject of interest in the field of medical mycology. The aim of the present study were the distributions and antifungal susceptibility patterns of various Candida species isolated from colonized and infected immunocompromised patients admitted to ten university hospitals in Iran. Methods In totally, 846 Candida species were isolated from more than 4000 clinical samples and identified by the API 20 C AUX system. Antifungal susceptibility testing was performed by broth microdilution method according to CLSI. Results The most frequent Candida species isolated from all patients was Candida albicans (510/846). The epidemiological cutoff value and percentage of wild-type species for amphotericin B and fluconazole in Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei were 0.5 μg/ml (95%) and 4 μg/ml (96%); 1 μg/ml (95%) and 8 μg/ml (95%); 0.5 μg/ml (99%) and 19 μg/ml (98%); and 4 μg/ml (95%) and 64 μg/ml (95%), respectively. The MIC90 and epidemiological cutoff values to posaconazole in Candida krusei were 0.5 μg/ml. There were significant differences between infecting and colonizing isolates of Candida tropicalis in MIC 90 values of amphotericin B, and isolates of Candida glabrata in values of amphotericin B, caspofungin, and voriconazole (P

Background and purpose: The choice of antifungal agent in treatment of Candida urinary tract infections (CUTI) is dependent on the site of infection, the underlying disease of the patient, and the pharmacokinetics/pharmacodynamics (PK/PD) of the agent. This study aimed to perform a review of antifungal therapy for CUTI. Materials and methods: Data was obtained by a search for full-text articles in Medline, PubMed, Embase, Scopus, Web of Science, Science Direct, Google Scholar, Magiran, Irandoc, and Iran Medex published from 1994 until 2016. The search keywords included urinary tract infections, Candida species, diagnosis, and treatment. Results: Fluconazole is the drug of choice for prophylaxis and treatment of CUTI due to low toxicity, high solubility, and wide tissue distribution. Although flucytosine is concentrated in urine and has potent activity against Candida species, treatment is restricted because of its toxicity and expansion of resistance when it is used alone. In addition, amphotericin B is an active drug against most Candida species (except resistant C. krusei strains). Other azoles and echinocandins are not effective for treating CUTI due to the minimum excretion of the active compound into the urine. However, a localized renal infection followed by blood spreading might be treated by echinocandins because of its effective tissue concentrations. Conclusion: We presented diagnostic tests and treatment protocols of CUTI, but new surveillance protocols and diagnostic strategies for control and prevention of CUTI in critically ill patients are essential.

Occurrence of non-Candida albicans Candida (NCAC) species that are associated with elevated MIC values and therapeutic failures are increasing. As a result, timely and accurate means of identification to the species level is becoming an essential part of diagnostic practices in clinical settings. In this study, 301 clinically isolated yeast strains recovered from various anatomical sites [Blood (n = 145), other sites (n = 156)] were used to assess the accuracy and practicality of API 20C AUX and 21-plex PCR compared to MALDI-TOF MS and large subunit rDNA (LSU rDNA). MALDI-TOF MS correctly identified 98.33% of yeast isolates, 100% of top five Candida species, 95.7% of rare yeast species, while 1.3% of isolates were misidentified. API 20C AUX correctly identified 83.7% of yeast isolates, 97.2% of top five Candida species, 61.8% of rare yeast species, while 16.2% of yeast isolates were misidentified. The 21-plex PCR, accurately identified 87.3% of yeast isolates, 100% of top five Candida species, 72% of rare yeast species, but it misidentified 1.3% of rare yeast species while 9.9% of whole yeast isolates were not identified. The combination of rapidity of 21-plex PCR and comprehensiveness of API 20C AUX, led to correct identification of 92% of included yeast isolates. Due to expensiveness of MALDI-TOF MS and sequencing, this combination strategy could be the most accurate and inexpensive alternative identification strategy for developing countries. Moreover, by the advent and development of cost-effective, reliable, and rapid PCR machines that cost 130 US dollars, 21-plex could be integrated in routine laboratories of developing and resource-limited countries to specifically identify 95% causative agents of yeast-related infections in human. Databases of MALDI-TOF MS, API 20C AUX, and the number of target species identified by 21-plex require further improvement to keep up with the diverse spectrum of yeast species.

Invasive candidiasis remains one of the most prevalent systemic mycoses, and several studies have documented the presence of mixed yeast (MY) infections. Here, we describe the epidemiology, clinical, and microbiological characteristics of MY infections causing invasive candidiasis in a multicenter prospective study. Thirty-four centers from 14 countries participated. Samples were collected in each center between April to September 2018, and they were sent to a reference center to confirm identification by sequencing methods and to perform antifungal susceptibility testing, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 6895 yeast cultures were identified and MY occurred in 150 cases (2.2%). Europe accounted for the highest number of centers, with an overall MY rate of 4.2% (118 out of 2840 yeast cultures). Of 122 MY cases, the most frequent combinations were Candida albicans/C. glabrata (42, 34.4%), C. albicans/C. parapsilosis (17, 14%), and C. glabrata/C. tropicalis (8, 6.5%). All Candida isolates were susceptible to amphotericin B, 6.4% were fluconazole-resistant, and two isolates (1.6%) were echinocandin-resistant. Accurate identification of the species involved in MY infections is essential to guide treatment decisions.

Background and Purpose: Vulvovaginal candidiasis is a frequent disease affecting approximately more than %75 of all childbearing women at least once in their lifetime by overgrowth of opportunistic Candida species. Recurrent vulvovaginal candidiasis (RVVC) is common in otherwise healthy individuals. Several risk factors were reported to contribute to RVVC susceptibility. A polymorphism in Dectin-1 (Y238X, rs16910526 ) was identified in patients with RVVC and hypothesized that genetic factors play an important role in susceptibility to RVVC. Herein, we aimed to survey the polymorphisms in the Dectin-1 gene, linked to susceptibility to RVVC. Materials and Methods: In the current study, blood samples were obtained from 25 patients who had frequent vulvovaginal candidiasis relapses and were diagnosed as RVVC. In addition, blood cultures were obtained from control group comprising of healthy individuals (n=25) with no history of RVVC, vaginal discharge, or itching on the day of examination. Dectin-1 Y238X gene polymorphism was investigated using Bi-PASA and DNA sequencing. Results: The analysis revealed that all of the patients were wild-type homozygous for Dectin-1 Y238X polymorphisms. None of the individuals showed heterozygous or mutant homozygous Dectin-1 polymorphism. Conclusion: No significant correlations were observed between the susceptibility to RVVC and Dectin-1 Y238X polymorphism in the Iranian population, which was not previously studied.

Background and Purpose: Candida albicans is the most common causative agent of candidiasis. Candidiasis management is dependent on the immune status of the host, severity of disease, and the choice of antifungal drug. Antifungals, specifically triazoles, are widely administered for the treatment of invasive fungal infections. Herein, we aimed to evaluate the in vitro susceptibility of C. albicans isolates to fluconazole (FLZ), itraconazole (ITZ), voriconazole (VRZ), amphotericin B (AMB), and Caspofungin (CAS). Materials and Methods: A total of 44 clinical strains of C. albicans were collected from 36 patients admitted to four hospitals in Mazandaran Province, Iran. The in vitro antifungal susceptibility testing was performed based on the Clinical and Laboratory Standards Institute methods. Results: Generally, 34 isolates were susceptible to all the five antifungal drugs, while four isolates were susceptible or susceptible dose-dependent (SDD) and six isolates were SDD or resistant to these antifungals. The lowest minimum inhibitory concentration (MIC; 0.016 μg/ml) belonged to AMB and the highest MIC was for FLZ (16 μg/ml). The lowest MIC50 (0.063 μg/ml) was related to ITZ and the lowest MIC90 (0.25 μg/ml) pertained to CAS; in addition, the highest MIC50 (1 μg/ml) and MIC90 (4 μg/ml) were for FLZ. Four of the isolates showed resistance to both FLZ and VRZ, separately, and five isolates were resistant to ITZ. Caspofungin showed potent activity against more than 95% of the C. albicans isolates. Conclusion: Overall, we reported 9.1% resistance to FLZ and VRZ, 11.3% resistance to ITZ and AMB, and 4.6% resistance to caspofungin. Our finding is in agreement with previous observations proposing that C. albicans isolates develop resistance to some antifungal drugs such as FLZ since they are widely used as prophylaxis.

Background and Purpose: Candidiasis is a major fungal infection, and Candida albicans is the major cause of infections in humans. The Clinical and Laboratory Standards Institute (CLSI) developed new breakpoints for antifungal agents against C. albicans. In this multi-center study, we aimed to determine the drug susceptibility profile of C. albicans, isolated from Iranian population according to new species-specific CLSI. Materials and Methods: Clinical samples were cultured on Sabouraud dextrose agar and were incubated at room temperature for seven days. The isolates were transferred to Professor Alborzi Clinical Microbiology Research Center, Shiraz, Iran. C. albicans were identified by using API 20C AUX system. Broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of amphotericin B, caspofungin, voriconazole, fluconazole, posaconazole, itraconazole, and ketoconazole, based on CLSI document M27-S4 and new breakpoints for some azoles and caspofungin. Results: Overall, 397 C. albicans were isolated from patients admitted to ten university hospitals in Iran. The MIC90 of the isolates to amphotericin B, caspofungin, voriconazole, fluconazole, posaconazole, itraconazole, and ketoconazole were 0.125, 0.125, 0.125, 1, 0.064, 0.5, and 0.125 μg/ml, and rates of resistance were 0.5%, 0.3%, 3.8%, 2.8%, and 2.5% for amphotericin B, caspofungin, voriconazole, fluconazole, and itraconazole, respectively. Conclusion: According to our data, fluconazole is the drug of choice for management of patients at risk for systemic candidiasis throughout the region, since it is cost-effective with low side effects.

Azole-resistance in Aspergillus fumigatus is a worldwide medical concern complicating the management of aspergillosis (IA). Herein, we report the clonal spread of environmental triazole resistant A. fumigatus isolates in Iran. In this study, 63 A. fumigatus isolates were collected from 300 compost samples plated on Sabouraud dextrose agar supplemented with itraconazole (ITR) and voriconazole (VOR). Forty-four isolates had the TR34/L98H mutation and three isolates a TR46/Y121F/T289A resistance mechanism, while two isolates harbored a M172V substitution in cyp51A. Fourteen azole resistant isolates had no mutations in cyp51A. We found that 41 out of 44 A. fumigatus strains with the TR34/L98H mutation, isolated from compost in 13 different Iranian cities, shared the same allele across all nine examined microsatellite loci. Clonal expansion of triazole resistant A. fumigatus in this study emphasizes the importance of establishing antifungal resistance surveillance studies to monitor clinical Aspergillus isolates in Iran, as well as screening for azole resistance in environmental A. fumigatus isolates.

Autosomal recessive deficiency in the caspase recruitment domain containing protein 9 (CARD9) results in susceptibility to fungal infections. In the last decade, infections associated with CARD9 deficiency are more reported due to the advent of genome sequencing. The aim of this study was to evaluate the frequency, geographic distribution and nature of mutations in patients with CARD9 deficiency. We identified 60 patients with 24 mutations and different fungal infections. The presence of the homozygous (HMZ) p.Q295X (c.883C > T) and HMZ p.Q289X (c.865C > T) mutations were associated with an elevated risk of candidiasis (OR: 1.6; 95% CI: 1.18–2.15; p = 0.004) and dermatophytosis (OR: 1.85; 95% CI: 1.47–2.37; p < 0.001), respectively. The geographical distribution differed, showing that the main mutations in African patients were different Asian patients; HMZ p.Q289X (c.865C > T) and HMZ p.Q295X (c.865C > T) accounted for 75% and 37.9% of the African and Asian cases, respectively. The spectrum of CARD9 mutations in Asian patients was higher than in African. Asia is the most populous continent in the world and may have a greater genetic burden resulting in more patients with severe fungal infections. The presence of a high diversity of mutations revealing 24 distinct variations among 60 patients emphasize that the unique genetic alteration in CARD9 gene may be associated with certain geographical areas.

Candida auris, C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii are closely related and highly multidrug resistant yeast pathogens. The high cost and low accuracy of current diagnostics may underestimate their prevalence, especially in medical resource-limited regions. In this study, we used 172 C. auris stains and its relatives and 192 other fungal strains to establish and validate a novel multiplex end-point PCR. A prospective and a retrospective clinical screenings using this assay were further performed in China and Iran respectively. We identified the first isolate of C. pseudohaemulonii in China and the first isolate of C. haemulonii in Iran from 821 clinical isolates in total, without any false positive. Animal models of C. auris and C. haemulonii were established for validation. The overall positive rates of the assay for mice blood and tissue were 28.6 and 92.9%, respectively. Compared with previously developed assays, our assay is more available and affordable to the developing countries, and may contribute to a better understanding of the epidemiology of C. auris and its relatives in these regions.

Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied.Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene.Results: The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4% of all tested isolates, 6.2% of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V.Conclusions:Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4% of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10%, resistance against voriconazole was rare and absent for itraconazole.

Four major clades of Candida auris have been described, and all infections have clustered in these 4 clades. We identified an isolate representative of a potential fifth clade, separated from the other clades by >200,000 single-nucleotide polymorphisms, in a patient in Iran who had never traveled outside the country.

Background and Purpose: Chronic obstructive pulmonary disease (COPD) has been recognized as a risk factor for invasive aspergillosis. Airway colonization by Aspergillus species is a common feature of chronic pulmonary diseases. Nowadays, the incidence of COPD has increased in critically ill patients. The aim of the present study was to isolate and identify Aspergillus colonies in the respiratory tract of COPD patients. Materials and Methods: This study was performed on 50 COPD patients, who were aged above 18 years, and were in intensive care units of three hospitals in Sari, Iran, for at least six days. All the samples obtained from sputum, bronchoalveolar lavage, and tracheal aspirates were cultured for fungi each week. According to the conventional techniques, Aspergillus isolates were initially based on growth and standard morphological characteristics. To confirm the identification of grown Aspergillus, the partial beta-tubulin gene was sequenced using specific primers. Results: A total of 50 patients, who met our inclusion criteria, were enrolled in the study during 2012-14. The results showed that 27 (54%) and 23 (46%) of the participants were male and female, respectively. The majority of the patients developed dyspnea followed by hemoptysis, chest pain, and high fever. Corticosteroids and broad-spectrum antibacterial agents were administered to 75% and 80% of the patients, respectively. Based on the conventional and molecular approaches, A. fumigatus (seven cases 43.7%), A. flavus (five cases 31.2%), A. niger (one case 6.2%), A. terreus (one case 6.2%), A. orezea (one case 6.2%), and A. tubingensis (one case 6.2%) were recovered. Conclusion: Recovery of Aspergillus species from the respiratory tract of COPD patients with pneumonia indicates two possibilities: either colonization or invasive aspergillosis

Background and Purpose: Candida species are the most common organisms involved in superficial fungal infections, worldwide. Although econazole is among the most frequently used topical formulations for the treatment of candidiasis, no information is available regarding the susceptibility profiles of Candida species in Iran. Materials and Methods: In vitro susceptibility of 100 clinical Candida isolates belonging to 6 species from superficial candidiasis of Iran towards to econazole was compared with three other common antifungal agents including itraconazole, fluconazole, and miconazole. Minimum inhibitory concentrations (MICs) values were analyzed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A3 document. All isolates were previously identified to the species level, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on ITS region. Results: The MIC of econazole, itraconazole, miconazole, and fluconazole were within the range of 0.016-16, 0.032-16, 0.016-16, and 0.25-64 g/ml, respectively. In general, econazole and miconazole were more active against Candida isolates, compared to the other two agents. Conclusion: The present study demonstrated that for Candida albicans isolates, miconazole and econazole had the best effect, but in non-albicans Candida species, itraconazole and miconazole displayed more activity than other antifungal agents.

ABSTRACT BACKGROUND AND OBJECTIVE: The number of fungal species on earth is estimated at about 1.5 million species, among which about 400 species belong to animal and human pathogens. Human pathogenic fungal species are mainly isolated from plant hosts. Studies on multi-host fungi have shown that with changes in physiological pathogenicity, these fungi are able to change their host according to the circumstances they are in. Horizontal gene transfer may play an important role in the evolution of fungal virulence in human hosts. METHODS: In this retrospective study, Pubmed, Medline, Scopus, Google Scholar, Elsevier, Irandoc, Iranmedex, Magiran, SID, and MEDLIB databases were searched thoroughly. MeSH keywords in our search included the evolution of virulence, pathogenic fungi, human pathogenic fungi, pathogenic plant fungi, horizontal gene transfer, and limited host plants. The related articles, published during 1992-2010, were extracted and retrospectively studied. FINDINGS: Molecular studies on multi-host fungi confirm the hypothesis of pathogenic fungal evolution from plant hosts to human hosts. The present study evaluated the recent findings on the origin of human pathogenic fungi and host changes from plant to human hosts. CONCLUSION: By comparing the content and structure of genomes and genes in pathogenic fungi, with different host ranges, penetration methods, and pathogenicity, we will have a better understanding of pathogenic genes and the processes involved in the evolution of the disease.

Background and Purpose: Invasive aspergillosis (IA) is one of the most common life-threatening fungal infections among the critically ill patients including intensive care unit (ICU) patients. Delayed diagnosis and therapy may lead to poor outcomes. Diagnosis may be facilitated by a test for molecular biomarkers, i.e. detection of galactomannan (GM) antigen based on enzyme immunoassay, which is of increasing interest in the clinical settings for the diagnosis of IA. In the present study, we assessed GM testing of bronchoalveolar lavage (BAL) fluid as a tool for early diagnosis of IA among ICU patients who were at risk for developing IA. Material and Methods: A prospective study was performed in ICU patients with underlying predisposing conditions for IA between August 2010 and September 2011. BAL samples for direct microscopic examination, culture, and GM detection were obtained once or twice weekly. GM in BAL levels was measured using the Platellia Aspergillus EIA test kit. According to modified European Organization for the Research and Treatment of Cancer/ Mycoses Study Group (EORTC/MSG) criteria, patients were classified as having probable or possible IA. Results: Out of 43 suspected patients to IA, 13 (30.2%) cases showed IA. According to the criteria presented by EORTC/MSG, they were categorized as: 4 cases (30.8%) of possible IA and 9 (69.2%) of probable IA. Out of 21 BAL samples from patients with IA, 11 (52.4%) had at least one positive BAL GM index. Using a cutoff index of 0.5, the sensitivity and specificity, positive and negative predictive values of GM detection in BAL fluid were 100%, 85.7%, 65.7% and 96%, respectively. The sensitivity and specificity was 73% and 92.7% at cutoff ≥1.0, respectively. In 6 of 13 IA cases, BAL culture or direct microscopic examination remained negative, whereas GM in BAL was positive. Conclusion: Our data have revealed that the sensitivity of GM detection in BAL was better than that of conventional tests. It seems that GM detection in BAL is beneficial to establish or exclude the early diagnosis of IA in ICU patients.

Chronic obstructive pulmonary disease (COPD) is associated with a chronic inflammatory response in airways and lung parenchyma that results in significant morbidity and mortality worldwide. Cigarette smoking considered as an important risk factor plays a role in pathogenesis of disease. Pneumocystis jirovecii is an atypical opportunistic fungus that causes pneumonia in immunosuppressed host, although the low levels of its DNA in patients without signs and symptoms of pneumonia, which likely represents colonization. The increased prevalence of P. jirovecii colonization in COPD patients has led to an interest in understanding its role in the disease. P. jirovecii colonization in these patients could represent a problem for public health since colonized patients could act as a major reservoir and source of infection for susceptible subjects. Using sensitive molecular techniques, low levels of P. jirovecii DNA have been detected in the respiratory tract of certain individuals. It is necessary to elucidate the role of P. jirovecii colonization in the natural history of COPD patients in order to improve the clinical management of this disease. In the current review paper, we discuss P. jirovecii colonization in COPD patients.

ABSTRACT Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16 ), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature “C. neoformans species complex” and “C. gattii species complex.” Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

Background: Although Candida albicans remains the most common fungal isolate from clinical specimens, many studies have detected a shift towards non-albicans Candida species. Despite worrying clinical pictures associated with latter species, there is little information regarding its susceptibility patterns against currently available antifungal agents, with only a small number of strains having been studied. Methods: We evaluated the in vitro antifungal susceptibilities of clinical isolates of C. orthopsilosis already identified by two-steps PCR-RFLP and reconfirmed by sequence analysis of entire ITS rDNA region, to six antifungal drugs. Results: The resulting MIC50 and MIC90 for all strains (n=18) were in increasing order, as follows: posaconazole (0.016 & 0.063 μg/ml); itraconazole (0.031 & 0.125 μg/ml); amphotericin B (0.5 & 1 μg/ml); fluconazole (0.25 & 0.5 μg/ml) and caspofungin (4 & 8 μg/ml). A uniform pattern of the MIC ranges was seen for amphotericin B, fluconazole, itraconazole, and posaconazole, while a widest range and the highest MICs were observed for caspofungin. Conclusion: Although we emphasis on the careful species designation of the clinical isolates of Candida, the antifungal susceptibility patterns of these clinically important organisms may have an application in clinical and epidemiological setting and deserve the implementation of local surveillance programs to monitor.

Despite the introduction of new antifungal agents, resistances to antifungal therapy continue to increase and outcome of invasive fungal infections treatment is frequently suboptimal. A large amount of the recent effort in antifungal drug discovery has focused on a limited set of targets with functions known or expected to be important for fungal viability and virulence. A variety of techniques can be used to identify fungal genes of interest. Gene expression profiling, RNA mediated gene silencing and insertional mutagenesis are three main molecular genetics technologies used to identify and validate antifungal drug targets. The term RNA interference (RNAi) refers to a cellular process by which a sequence-specific double-stranded RNA (dsRNA) inhibits the expression of a gene. This mechanism is strongly conserved in eukaryotes and has been documented to be existed in different fungal species such as Candida albicans, Aspergillus nidulans and Penicillium marneffei. Many vital and virulence genes have been successfully knocked down using RNAi technology. RNAi can be regarded as a promising approach for discovery of new gene targets for the design of fungus-specific antifungal agents. Here we discuss about a novel approach and its application in designing new molecular antifungal targets.

Background: Due to the predisposing conditions in patients with cystic fibrosis (CF) caused by defective mucociliary clearance facilitating colonization and invasion with Candida species has dramatically increased. Traditional methods for identifying problems are imminent and time-consuming. Therefore, molecular techniques utilizing amplification of target DNA provide quick and precise methods for the diagnosis and identification of Candida species. Therefore, the aim of the current study was to identify the most medically common isolated Candida species from the air way of CF patients by PCR-RFLP and amplification of HWP1 gene. Materials and Methods: A total of 42 CF patients presenting symptoms who referred to pediatric respiratory diseases research center were screened for the presence of Candida spp. The isolates initially were phenotypically identified and confirmed by molecular approaches based on restriction fragment length polymorphism ( PCR -RFLP) for the discrimination of C. albicans of non-albicans and the amplification of HWP1 gene for the discrimination of C. albicans from C. dubliniensis and C. africana was conducted. Results: The results show that C. albicans was the most frequently isolated species (83.8%) followed by non-albicans included C. parapsilosis (7.1%), C. glabrata (3.2%), and C. tropicalis (3.2%). The restriction patterns of each Candida species were perfectly specific. Since MspI could not discriminate between the three morphological related species, C. albicans, C. dubliniensis and C. africana, we used PCR amplification of HWP1 gene, which (7.1%) species from C. albicans identified as C. dubliniensis, however C. africana strains were not found. Conclusion: The present study found that C. albicans as predominant species wereisolated from the CF patients. It could be concluded that molecular diagnostic methods are reliable and would be useful for the identification of medically important Candida species in clinical samples . Therefore, considerable attention has been paid to the prevention and treatment of microbial growth, which has resulted in the improvement of patient management.

Invasive aspergillosis (IA) are a major complication in immunocompromized patients where can be serious and rapidly fatal. Early diagnosis and early appropriate antifungal treatment is important in reducing mortality and morbidity. But despite many efforts to develop detection methods, the diagnosis of IA still remains challenging and current conventional methods are limited for adequate diagnosis. New rapid methods which can detect IA early in the course of disease with high sensitivity and specificity are needed since treating these infections at an early stage. Using molecular methods for Aspergillus species identification can be a cost-effective, rapid, discriminatory, and objective approach for delineating Aspergillus species in a clinical microbiology laboratory. PCR techniques for the diagnosis of IA have been studied for more than a decade and are still considered investigational, however until now PCR is not included in current EORTC/MSG diagnostic criteria.

Candidiasis, the main opportunistic fungal infection has been increased over the past decades. This study aimed to characterize C.albicans species complex (C.albicans, C.dubliniensis, and C.africana) isolated from patients with respiratory infections by molecular tools and in vitro antifungal susceptibilities by using broth microdilution method according to CLSI M27-A3 guidelines. Totally, 121 respiratory samples were collected from patients with respiratory infections. Of these, 83 strains were germ tube positive and green colonies on chromogenic media, so initially identified as C.albicans species complex and subsequently were classified as C.albicans (89.15%), C.dubliniensis (9.63%), and C.africana (1.2%) based on PCR-RFLP and amplification of hwp1 gene. Minimum inhibitory concentration (MICs) results showed that all tested isolates of C.albicans complex were highly susceptible to triazole drugs. However, caspofungin had highest activity against C.albicans, C.dubliniensis, and C.africana. Our findings indicated the variety of antifungal resistance of Candida strains in different areas. These results may increase the knowledge about the local distribution of the mentioned strains as well as their antifungal susceptibility pattern which play an important role in appropriate therapy.

Introduction: Recently, the incidence of fungal infections dramatically increased with an appearance of many novel species due to different criteria. Therefore, several molecular methods have been established for identification of these agents which cause human and animal disorder based on genomic DNA. Among these methods, RAPD-PCR technique is a powerful discriminative method based on amplifies target genomic DNA sequence by short random primers (arbitrary short primers) with low annealing temperature (36˚C) for discrimination and identification in the species level. Materials and Methods: All clinical strains were originated oropharyngeal lesions of cancer patients from four Mazandaran University Hospitals in Iran. These strains were previously identified by phenotypic methods such as colony on CHROM-agar Candida medium, germ-tube formation in horse serum and chlamydospore formation. In this study RAPD-PCR technique was used to amplify hyper variable inter-repeat DNA sequences using oligonucleotide primers specific microsatellite (GACA) 4 for identifying, clustering and take into account the genetic correlation of 30 clinical isolates. Results: The results in this study showed that the RAPD-PCR by using of arbitrary short primer was able to amplify hyper variable inter repeated DNA sequences with classifying the isolates. RAPD patterns showed genetically inter speciation relationship in the best possible way and PCR-fingerprinting with primer (GACA) 4, was able to discriminate both C. albicans with other Candida species based on size and number of bands. Conclusion: We concluded that, regarding to the previous studies which have been reported misidentification by conventional mycological method for identifying Candida species, RAPD-DNA method is able to discriminate Candida species by using of a single primer. However, determination of differences and accurate assessment of genetic distances in the RAPD technique was generally limited.