Aspergillus Fumigatus Isolates Research Articles

Azole resistance among clinical isolates of Aspergillus fumigatus in Lima-Peru

Azole resistance among Aspergillus fumigatus isolates, which is mainly related to mutations in the cyp51A gene, is a concern because it is rising, worldwide disseminated, and associated with treatment failure and death. Data on azole resistance of aspergillus from Latin American countries is very scarce and do not exist for Peru. Two hundred and seven Aspergillus clinical isolates collected prospectively underwent mycology and molecular testing for specie identification, and 143 isolates were confirmed as A. fumigatus sensu stricto (AFSS). All AFSS were tested for in vitro azole susceptibility, and resistant isolates underwent PCR amplification and sequencing of the whole cyp51A gene and its promoter. The in vitro susceptibility showed a minimal inhibitory concentration (MIC) range, MIC50 and MIC90 of 0.125 to >16, 0.25, and 0.5μg/ml for itraconazole; 0.25 to 2, 0.5, and 0.5μg/ml for voriconazole; and 0.003 to 1, 0.06, and 0.125μg/ml for posaconazole. Three isolates (2%) showed resistance to itraconazole and exhibited different mutations of the cyp51A gene. One isolate harbored the mutation M220K, while a second one exhibited the G54 mutation plus a modification in the cyp51A gene promoter. The third isolate, from an azole naive patient, presented an integration of a 34-bp tandem repeat (TR34) in the promoter region of the gene and a substitution of leucine 98 by histidine (L98H). The three source patients had a diagnosis or suspicion of chronic pulmonary aspergillosis.

Posaconazole therapy in children with cystic fibrosis and Aspergillus-related lung disease.

There is emerging evidence for the role of posaconazole in the management of Aspergillus-related cystic fibrosis (CF) lung disease. The tolerability and efficacy of posaconazole in paediatric CF is not well established. We report a prospective study over a fifty-three month period evaluating the safety, tolerability, and efficacy of posaconazole in pediatric CF. Fourteen children (seven males, median age 13 years, range 3-17 years) received a total of twenty-three courses of posaconazole (13 oral suspension and 10 tablet formulation). Of these patient episodes, nine received posaconazole for emerging or active allergic bronchopulmonary aspergillosis (ABPA) and two required a combination of posaconazole and systemic corticosteroids for difficult-to-treat ABPA. A subgroup of patients (n = 12) with persistent isolates of Aspergillus fumigatus, in the absence of serological markers of ABPA, received posaconazole monotherapy for pulmonary exacerbations not responding to conventional broad-spectrum antibiotic treatment. Posaconazole levels, full blood count, electrolytes, and liver function were monitored on day 7 of treatment and then monthly. Posaconazole was well tolerated in all but three patients. Therapeutic plasma levels >1mg/l were achieved in all receiving the tablet formulation in comparison to 60% on the liquid preparation. There was a modest but significant improvement in FEV1 (% predicted) demonstrated for the cohort as a whole (p = 0.015) following posaconazole therapy. Posaconazole is well tolerated in children as young as six years old, improvements in lung function are observed, and therapeutic plasma levels are readily achieved in patients taking the tablet formulation and in adherent patients taking the liquid formulation.

The validation of VIPcheck™ plates to screen Aspergillus fumigatus isolates for phenotypic resistance to triazole antifungal agents in St. James’s Hospital, Dublin

Triazole resistance is an emerging problem in Aspergillus fumigatus (AF) resulting in failure of azole therapy. Triazole resistant AF is acquired through one of two routes – previous exposure to triazole therapy or an environmental source. In vitro antifungal susceptibility testing (AFST) on all AF strains isolated in a microbiology laboratory would be both labour intensive and impractical. A method to screen for triazole resistance would be more favourable. VIPcheck™ plates provide a simple agar based screening method. Each 4-well plate contains a growth control (GC) well and 3 wells containing itraconazole (4 mg l−1), voriconazole (2 mg l−1) and posaconazole (0.5 mg l−1). Briefly, 25 µl of a 0.5-2 McF suspension AF is inoculated into each well and plates are read after 48 h incubation at 37 °C. Any growth in a triazole containing well is suggestive of resistance. Currently in SJH, AFST is carried out using gradient strips (Liofilchem™) and results are interpreted using EUCAST breakpoints. We validated the VIPcheck™ plates with the intention to include this screening method as part of our AFST for AF isolated from clinical samples. A total of 18 isolates (clinical and environmental) of AF were tested using the VIPcheck™ plates (n=2 wild type, n=18 resistant to ≥1 triazole drug as previously determined by AFST and/or molecular methods). The wild type isolates showed growth only in the GC well while the resistant strains all showed growth in one or more of the triazole containing wells. Our results suggest that the VIPcheck™ plate is a reliable screening method for triazole resistance.

Activity of isavuconazole and other triazole derivatives against clinical isolates of Aspergillus fumigatus

Aspergillus fumigatus is the most frequent pathogen of the genus Asperillus, which is highly susceptible to triazole derivatives, especially to isavuconazole and voriconazole. Many countries face a growing problem of infections due to A. fumigatus showing acquired resistance to one or several triazoles. In medical centres, monitoring the susceptibility of isolated Aspergillus spp. is recommended. Aim: The aim of this study was to collect and test triazole susceptibility of Aspergillus fumigatus obtained from clinical samples, which were investigated in diagnostic laboratories located in Wrocław, Warszawa and Ruda Śląska (Poland). In addition, 5 resistant A. fumigatus strains with TR34/L98H mutation were included. Material/Methods: The microdilution method, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was applied to test susceptibility to isavuconazole (ISV), voriconazole (VOR), posaconazole (POS) and itraconazole (ITR). Results: During a period of 24 months, a total number of 75 A. fumigatus isolates were collected. Most of the strains were obtained from lower respiratory tract specimens (58/75; 77%), from patients hospitalized on pulmonology (41%) or intensive care and surgery units (29%). No isolate resistant to ISV or other triazoles was found. The minimal inhibitory concentration (MIC) value of ISV ranged from 0.125 to 1 mg/L (mean 0.4 ±0.15 mg/L) in triazole susceptible isolates, whereas among triazole-resistant strains, three showed a MIC of 8 mg/L and two had a MIC of 4 mg/L. Conclusions: A. fumigatus isolates carrying the mutation TR34/L98H are cross-resistant to ISV. The acquired resistance is very rare in our region (0-4%), which supports of use of triazole derivatives (VOR, ISV) in the therapy of aspergillosis.

Preexposure to Isavuconazole Increases the Virulence of Mucorales but Not Aspergillus fumigatus in a Drosophila melanogaster Infection Model.

Breakthrough mucormycosis in patients receiving isavuconazole prophylaxis or therapy has been reported. We compared the impact of isavuconazole and voriconazole exposure on the virulence of clinical isolates of Aspergillus fumigatus and different Mucorales species in a Drosophila melanogaster infection model. In contrast to A. fumigatus, a hypervirulent phenotype was found in all tested Mucorales upon preexposure to either voriconazole or isavuconazole. These findings may contribute to the explanation of breakthrough mucormycosis in isavuconazole-treated patients.

Prospective multicenter surveillance of clinically isolated Aspergillus species revealed azole-resistant Aspergillus fumigatus isolates with TR34/L98H mutation in the Kyoto and Shiga regions of Japan.

The prevalence of azole-resistant Aspergillus fumigatus (ARAF) in Japan is unclear. We aimed to investigate the epidemiology of clinically isolated Aspergillus species and the frequency of azole resistance in Aspergillus species, particularly Aspergillus fumigatus, in the Kyoto and Shiga regions of Japan. Strains of clinically isolated Aspergillus species were prospectively collected from nine acute care hospitals. Species identification was performed by DNA sequence analysis, and all strains were subjected to antifungal susceptibility testing. Sequencing of the Aspergillus cyp51A gene and promoter region and genotyping by short tandem repeats were performed for ARAF isolates. A total of 149 strains were collected, and 130 strains were included for the subsequent analysis after the exclusion of duplicate isolates. The most commonly isolated species was Aspergillus fumigatus, accounting for 43.1% (56 isolates) overall, and seven (12.7%) of 55 strains of A. fumigatus were azole-resistant. Azole-resistance of other Aspergillus species were also found that two (22.2%) of nine strains of A. tubingensis and two (28.6%) of seven strains of A. flavus were azole-resistant. DNA sequence analysis of the ARAF strains revealed that two carried the cyp51A TR34/L98H mutation, one carried G448S, one carried M220I, and three had no relevant mutations (wild type). Genotyping and phylogenetic analyses showed that the TR34/L98H strains were clustered with the strains from the Netherlands and France. These data suggest the emergence of ARAF with TR34/L98H in Japan, and continuous surveillance will be important to identify trends in resistance.

Phenotypic plasticity and the evolution of azole resistance in Aspergillus fumigatus; an expression profile of clinical isolates upon exposure to itraconazole

BackgroundThe prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole.ResultsAfter analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus.ConclusionsOur data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any “hard-wired” genetic mutations arise.

Low frequency of itraconazole resistance found among Aspergillus fumigatus originating from poultry farms in Southwest Poland

AimThe aim of this study was to investigate the collection of avian Aspergillus fumigatus isolates for the presence of triazole resistance. Material and methodThe study was performed on 60 A. fumigatus isolates cultured from lung tissue samples from chicken (25), geese (17), turkeys (13) and ducks (5). The samples were obtained from 40 different farms located in the Southwest Poland and were collected in the period of September 2015 to November 2016. The EUCAST microdilution method, with the use of three concentrations of itraconazole (ITR) (1, 0.5, and 0.25mg/L), was used to screen the susceptibility of all isolates. Additionally, the selected 20 isolates were tested with eleven concentrations ranging 0.015–16mg/L of ITR, voriconazole, posaconazole and isavuconazole. ResultsMost tested isolates (59/60) were susceptible to ITR (MIC≤0.5mg/L). One isolate showed elevated MIC for ITR (16mg/L), as well as voriconazole (4mg/L), izavuconazole (4mg/L), and posaconazole (0.5mg/L). This isolate was identified on the basis of DNA analysis as A. fumigatus carrying TR34/L98H mutation. All of the ITR-susceptible isolates under study were also susceptible to other triazoles. ConclusionObtained results indicated a low frequency (1.6%) of A. fumigatus resistant to triazoles among avian isolates from the Southwest regions of Poland.

First Clinical Isolation of an Azole-Resistant Aspergillus fumigatus Isolate Harboring a TR46 Y121F T289A Mutation in South America.

One of the most recently described Aspergillus fumigatusCYP51A-mediated azole resistance mechanisms is TR46 Y121F T289A. Clinical A. fumigatus strains harboring these substitutions have been reported worldwide, with the exception of South America. We describe the first clinical A. fumigatus strain with this resistance mechanism isolated from an Argentinian patient. The strain was isolated in 2009 (1 year after the first-described mutant in United States), demonstrating that these alleles were scattered worldwide earlier than previously thought.

Comparative genotyping and phenotyping of Aspergillus fumigatus isolates from humans, dogs and the environment

BackgroundAspergillus fumigatus is a ubiquitous saprotrophic fungus and an opportunistic pathogen of humans and animals. Humans and animals can inhale hundreds of A. fumigatus spores daily. Normally this is harmless for humans, but in case of immunodeficiency, invasive pulmonary aspergillosis (IPA) can develop with a high mortality rate. A. fumigatus also causes non-invasive mycoses like sino-nasal aspergillosis (SNA) in dogs.ResultsIn this study we compared A. fumigatus isolates from humans with suspected IPA, dogs with SNA, and a set of environmental isolates. Phylogenetic inference based on calmodulin (CaM) and beta-tubulin (benA) sequences did not reveal A. fumigatus sub-groups linked to the origin of the isolates. Genotyping and microsatellite analysis showed that each dog was infected by one A. fumigatus genotype, whereas human patients had mixed infections. Azole resistance was determined by antifungal susceptibility testing and sequencing of the cyp51A gene. A total of 12 out of 29 human isolates and 1 out of 27 environmental isolates were azole resistant. Of the azole resistant strains, 11 human isolates showed TR34/L98H (n = 6) or TR46/Y121F/T289A (n = 5). Phenotypically, isolates from dogs were more variable in growth speed and morphology when compared to those isolated from human and the environment.ConclusionsA. fumigatus from dogs with SNA are phenotypically very diverse in contrast to their environmental and human counterparts.Phenotypic variability can be induced during the chronic infection process in the sinus of the dogs. The basis of this heterogeneity might be due to genomic differences and/or epigenetic variations.Differences in dogs is a could be a result of within-host adaption and might be triggered by environmental factors in the sinus, however this hypothesis still needs to be tested.

High prevalence of triazole resistance in clinical Aspergillus fumigatus isolates in a specialist cardiothoracic centre

To evaluate the prevalence of triazole-resistant Aspergillus fumigatus and common molecular cyp51A polymorphisms amongst clinical isolates in a specialised cardiothoracic centre in London, UK. All A. fumigatus isolates were prospectively analysed from April 2014 to March 2016. Isolates were screened with a four-well VIPcheck™ plate to assess triazole susceptibility. Resistance was confirmed with a standard microbroth dilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Triazole-resistant A. fumigatus isolates were subjected to a mixed-format real time polymerase chain reaction (RT-PCR) assay (AsperGenius®) to detect common cyp51A alterations. We identified 167 clinical A. fumigatus isolates from 135 patients. Resistance to at least one azole antifungal drug was confirmed in 22/167 (13.2%) of isolates from 18/135 (13.3%) patients, including 12/74 (16.2%) patients with cystic fibrosis (CF). The highest detection rate of azole-resistant A. fumigatus was among the 11- to 20-y age group. All triazole-resistant isolates (n = 22) were resistant to itraconazole, 18 showed cross-resistance to posaconazole and 10 displayed reduced susceptibility to voriconazole. No pan-azole-resistant A. fumigatus was identified. TR34/L98H was identified in 6/22 (27.3%) of azole-resistant isolates and detectable in 5/12 (42%) patients with CF. In our specialist cardiothoracic centre, the prevalence of triazole-resistant A. fumigatus is alarmingly high (13.2%). The majority of azole-resistant isolates were from patients with CF. We found a higher prevalence of the environmentally driven mutation TR34/L98H in our A. fumigatus isolates than in published UK data from other specialist respiratory centres, which may reflect differing patient populations managed at these institutions.

Airborne Aspergillus fumigatus spore concentration during demolition of a building on a hospital site, and patient risk determination for invasive aspergillosis including azole resistance

Invasive aspergillosis (IA) in immunocompromised patients has been associated with demolition in or adjacent to hospitals. In recent years, azole-resistant clinical isolates of Aspergillus fumigatus, the most common agent of IA, have emerged in Western Europe and are spreading globally. To determine the potential risk of IA, including azole resistance, in patients caused by demolition of a hospital building. Air sampling before, during and after demolition, screening for azole resistance, genotyping of non-susceptible isolates, and comparing those with strains from patients with azole-resistant IA during demolition. Mean concentrations of A.fumigatus spores did not differ significantly between the three periods before [17.5colony-forming units (cfu)/m³], during (20.8cfu/m³) (P=0.26) and after (17.7cfu/m³) demolition (P=0.33). No significant difference in IA cases documented by clinicians was found when comparing the timeframe of demolition with the previous year (44 vs 42 cases). Thirty of 200 A.fumigatus isolates (15%) showed azole resistance. Genotyping by microsatellite polymerase chain reaction of the azole-resistant environmental and clinical isolates showed a polyclonal distribution. The results suggest that with implemented preventive measures, there is no increased risk for IA, including azole resistance, in immunocompromised patients during outdoor demolition work. Further prospective studies are needed to confirm these findings.

Rezafungin (CD101) demonstrates potent in vitro activity against Aspergillus, including azole-resistant Aspergillus fumigatus isolates and cryptic species.

Rezafungin is an investigational echinocandin under development for the treatment and prevention of invasive fungal infections, with a long half-life in humans (∼130 h) and potent in vitro activity against Aspergillus spp. Our objective was to further evaluate its activity against Aspergillus fumigatus isolates, including azole-resistant isolates and cryptic Aspergillus spp. Clinical isolates of Aspergillus were used, including 15 WT and 31 azole-resistant A. fumigatus, 11 Aspergillus lentulus, 5 each of Aspergillus thermomutatus and Aspergillus udagawae and 11 Aspergillus calidoustus. Minimum effective concentrations (MECs) and MICs of rezafungin, caspofungin, micafungin, posaconazole and voriconazole were determined by CLSI M38-A2 broth microdilution. Differences in geometric mean (GM) MEC/MIC values were assessed for significance by ANOVA. Rezafungin GM MECs for A. fumigatus were 0.024 and 0.043 mg/L for WT and azole-resistant isolates, respectively. Rezafungin was also active against cryptic species, including A. lentulus (0.016 mg/L), A. calidoustus (0.044 mg/L), A. thermomutatus (MEC range ≤0.015-0.25 mg/L) and A. udagawae (≤0.015-0.03 mg/L). This activity was similar to that of caspofungin and micafungin with the exception of A. calidoustus, against which rezafungin was more potent than caspofungin (GM MEC 0.044 versus 0.468 mg/L; P < 0.0001). Rezafungin demonstrated potent in vitro activity against Aspergillus spp., including azole-resistant A. fumigatus isolates and cryptic species with elevated posaconazole and voriconazole MICs. Additional studies are warranted to determine whether the in vitro activity translates into in vivo efficacy against infections caused by resistant Aspergillus isolates.

Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates.

The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

Prospective survey of azole drug resistance among environmental and clinical isolates of Aspergillus fumigatus in a French University hospital during major demolition works

ObjectivesRecent studies have reported the emerging worldwide problem of azole drug resistance of A. fumigatus isolates. The aim of this study was to evaluate the antifungal susceptibilities of A. fumigatus isolates recovered from air and clinical samples collected in a French University hospital (Lyon), which underwent major deconstruction works over a one year-period. MethodsA daily surveillance of fungal contamination was implemented during 11-months. Environmental survey was realized by air samplings, outdoor and indoor, with an automatic agar sampler. In parallel, surveillance of IA infection cases was conducted by epidemiological investigation. Environmental and clinical isolates of A. fumigatus were identified by conventional methods and β-tubulin sequencing. Susceptibility testing of A. fumigatus isolates against Itraconazole (ITZ), Voriconazole (VCZ) was performed using Etest method. ResultsA total of 3885 air samples (1744 outdoor samples and 2141 indoor samples) were collected. From the 3073 identified colonies of A. fumigatus, 400 A. fumigatus isolates were tested for their susceptibility to ITZ and VCZ, including 388 isolates coming from the environment (indoor n:157, outdoor n:231) and 12 isolates coming from clinical samples. All the 400 isolates were susceptible to azoles (≤1μg/mL). ConclusionsNo environmental reservoir of A. fumigatus azole resistant strains was found in our hospital which was undergoing major demolition works. Further studies with larger number of A. fumigatus clinical isolates and environmental isolates from agricultural areas and healthcare establishments are needed to better appreciate the occurrence and prevalence of azole resistance.

Surveillance for azoles resistance in Aspergillus spp. highlights a high number of amphotericin B-resistant isolates.

Aspergillus spp. are the most common invasive mould infection and are responsible for high mortality. Aspergillus fumigatus is currently of interest because resistance to azole antifungals has emerged. The Campinas University Hospital (HC-UNICAMP) receives high-risk patients susceptible to opportunistic infections but there have been no reports of resistant A.fumigatus. This study aimed to assess the susceptibility profile of Aspergillus isolates, specifically looking for azole resistance. ITS and β-tubulin DNA sequencing was performed on 228 sequential clinical isolates. Broth microdilution susceptibility testing was performed for all isolates. A.fumigatus represented 74% of the isolates followed by Aspergillus flavus (12%). Nine A.fumigatus isolates from 9 different patients showed high MIC values to at least 1 azole, but cyp51A polymorphisms were detected in only 6 isolates and none correlated with known resistance mutations. The most troubling observation was that the minimum inhibitory concentration for amphotericin B was elevated (≥2mgL-1 ) in 87% of patients with A.flavus isolates and 43% with Aspergillusfumigatus isolates. Given that amphotericin B is used to treat azole-resistant infections, these data highlight the need for continuous surveillance in Aspergillus for all antifungal resistance to implement correct treatment strategies for the management of these pathogens.

In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus from different geographical regions

In vitro interaction of voriconazole with micafungin was evaluated against 33 clinical Aspergillus fumigatus isolates, including azole-resistant (n=31) and -susceptible (n=2) isolates. Interaction was synergistic for only 1 resistant isolate carrying the TR34/L98H mutation. No antagonistic effects were observed for 96.8% of azole-resistant isolates.

Pesticide behavior in paddy fields and development of azole-resistant Aspergillus fumigatus: Should we be concerned?

Tricyclazole as a common fungicide wildly used to control rice blast disease in the Asian country may induce azole resistance in Aspergillus fumigatus isolates. The main reason of the acquired azole resistance is probably environmental exposure through wide fungicide use in agriculture. The present study was conducted with the aim of investigating the current status of the azole-resistant A. fumigatus obtained from the paddy fields with exposure to tricyclazole. A total of 108 soil samples were collected from four different locations of paddy fields in Mazandaran Province, Iran. Pure fungal colonies were initially identified based on the conventional tools, and then reconfirmed by using DNA sequencing of the partial ß-tubulin gene. In addition, the in vitro antifungal susceptibility was determined using the Clinical and Laboratory Standards Institute document (CLSI) M38-A2. The identification of the mutations in the CYP51A gene was accomplished by the implementation of the polymerase chain reaction amplification assay on the selected isolates. Overall, 31 of 108 (28.7%) isolates were identified as A.fumigatus, four (3.7%) of which were recognized as azole-resistant with MICs of itraconazole ≥8μg/ml and voriconazole ≥4μg/ml. Only two out of the four azole-resistant A. fumigatus isolates harboured TR34/L98H variant and the other two isolates were identified as azole-resistant without any CYP51A gene mutations. However, other point mutations (TR46/Y121F/T289A) were not detected in the CYP51A gene. The high molecular structure similarity between environmental and medical triazoles may result in the selection of resistance mechanisms. Nonetheless, one might conclude that tricyclazole with different molecular structures against medical azoles induces azole-resistance in A.fumigatus isolates. The behavior of such pesticides as tricyclazole in the rice paddy fields would have an effective role in the development of azole-resistance that requires detailed information.

Sequencing of Gliotoxin Genes in Clinical and Environmental Aspergillus fumigatus isolates in Iraq

Gliotoxin is an important virulence factors in Aspergillus fumigatus. The biosynthesis of this mycotoxin is regulated and expressed by the presence of gliP genes. This study aimed to identify Aspergillus fumigatus isolates in clinical and environmental sources with glip genes using conventional PCR and sequence. To achieve this, DNA was isolated from twenty A. fumigatus isolates using commercial kit. The range of the DNA extracted was 65-210 ng/μl with a purity of 1.5-1.9. Species identification of the A. fumigatus isolates was achieved to a high specificity by using tailored primer. The results showed that all isolates had positive results to the primer and all isolates were able to produce gliotoxin. PCR detected the gliotoxin genes, glip in five isolates. The five PCR product samples were sent for sequence analysis and 25 µl (10 pmol) from the forward primer. The results of all the samples indicated have a single band of the desired product of gliP gene of A.fumigatus and the samples sent for sequencing related to molecular weight 190 bp.

Bioethanol Production from Fungal Treated Rice Husks Fermented with Bakers Saccharomyces cerevisiae and Yeast Isolates from Palm Wine

This research was necessitated by the quest to create a useful product from wastes (bio-ethanol from rice husk). This if successful will serve two purposes; first, help reduce wastes in the environment and to create wealth from waste. Separated isolates of Trichophyton soudanense, Trichophyton rubrum, Trichophyton mentagrophyte, Aspergillus oryzae, Aspergillus niger and Aspergillus fumigatus were obtained from husks of processed rice undergoing decomposition for more than 8 months. Husks of freshly processed rice were pretreated by autoclave boiling for 20 minutes at a temperature of 121°C after mixing in Mandle’s media. The experimental test samples consisted of co-culture combinations and monocultures individually inoculated into various measured heat treated husks; additional control groups were also made. Non-reducing sugar, reducing sugar and total sugar were assayed at the seventh day following hydrolysis. The resulting filtrates of the various husks (treated and control experimental units) were subjected to 7 days fermentation with yeasts from palm wine as well as bakers’ yeasts. Values of the result indicated highest trends in the following treatments: T. mentagrophyte treated husks with soluble reducing sugar value of 2.66 ± 0.14 g/L, A. fumigatus treated husk with soluble non reducing sugar value of 18.08 ± 2.61%, co-culture of T. soudanense and T. rubrum treated husks gave total sugar value of 20.53 ± 2.73%. Fermented A. oryzae treated husk filtrate inoculated with palmwine yeasts had optimal bio-ethanol yield (120.82 ± 0.39 g/L) followed by A. oryzae and T. soudanense treated husks fermented with bakers yeasts with 60.60 ± 0.10 g/L bioethanol. Recognizable yields of bioethanol from palm wine yeast fermented husk as well as sugar from other treated husks were obtained.