Marcin G. Fraczek

High-frequency Triazole Resistance Found In Nonculturable Aspergillus fumigatus from Lungs of Patients with Chronic Fungal Disease

BACKGROUND Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. METHODS Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., we assessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotide polymorphisms (SNPs) associated with triazole resistance. RESULTS Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with culture or microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of 42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] and M220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%) with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequate plasma drug concentrations around the time of sampling. CONCLUSIONS The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy.

The cdr1B efflux transporter is associated with non-cyp51a-mediated itraconazole resistance in Aspergillus fumigatus

OBJECTIVES Recent increases in triazole resistance in Aspergillus fumigatus have been attributed primarily to target site (cyp51A) mutations. A recent survey of resistant isolates in Manchester showed that >50% of resistant isolates had no mutation in cyp51A or its promoter. We investigated the mechanisms of resistance in clinical azole-resistant isolates without cyp51A mutations. METHODS Twelve azole-resistant isolates, 10 of which were itraconazole resistant, were studied. Bioinformatic comparisons between Candida albicans efflux genes and A. fumigatus genome data identified 20 putative azole transporter genes. Basal and azole-induced expression of these genes and cyp51A was quantified using RT-PCR with comparison with clinical azole-susceptible isolates. Function of high basal or itraconazole-induced expression transporters was tested by gene knockout in azole-susceptible and azole-resistant isolates. RESULTS All susceptible strains showed minimal basal expression of cdr1B compared with 8 of 10 azole-resistant strains with high basal expression of this gene (>5-fold), 3 of which showed >30-fold increased expression. Knockout of this gene resulted in a 4-fold reduction in itraconazole, posaconazole and voriconazole MICs for a susceptible clinical isolate and a 4-fold reduction in itraconazole susceptibility in a clinical resistant isolate. One strain showed a >500-fold induction of cyp51A. No increase in basal expression or expression after induction was seen for the 18 remaining putative transporters. CONCLUSIONS The reasons behind the shift away from target site mutation in azole-resistant isolates from Manchester are unknown. The modest change in expression of cdr1B in azole-susceptible strains implies that only study of resistant isolates will lead to further understanding of resistance mechanisms in A. fumigatus.

Comparative genomics of fungal allergens and epitopes shows widespread distribution of closely related allergen and epitope orthologues

BackgroundAllergy is a common debilitating and occasionally life threatening condition. The fungal kingdom contains a number of species that produce a wide range of well defined protein allergens although the vast majority of fungal species have unknown allergenic potential. The recent genome sequencing of a variety of fungi provides the opportunity to assess the occurrence of allergen orthologues across the fungal kingdom. Here we use comparative genomics to survey the occurrence of allergen orthologues in fungi.ResultsA database of 82 allergen sequences was compiled and used to search 22 fungal genomes. Additionally we were able to model allergen structure for representative members of several highly homologous allergen orthologue classes. We found that some allergen orthologue classes that had predicted structural congruence to allergens and allergen epitopes were ubiquitous in all fungi. Other allergen orthologues classes were less well conserved and may not possess conserved allergen epitope orthologues in all fungi. A final group of allergen orthologues, including the major allergens Asp f 1 and Alt a 1, appear to be present in only a limited number of species.ConclusionThese results imply that most fungi may possess proteins that have potential to be allergens or to cross react with allergens. This, together with the observation that important allergens such as Asp f 1 are limited to genera or species, has significant implications for understating fungal sensitization, and interpreting diagnosis and management of fungal allergy.

Volume dependency for culture of fungi from respiratory secretions and increased sensitivity of Aspergillus quantitative PCR

Diagnosis of aspergillosis is often difficult. We compared fungal yields from respiratory specimens using the Health Protection Agency standard culture method (BSOP57), a higher volume undiluted culture method Mycology Reference Centre Manchester (MRCM) and Aspergillus quantitative real time polymerase chain reaction (qPCR). Sputum, bronchial aspirate and bronchoalveolar lavage (BAL) samples (total 23) were collected from aspergillosis patients. One fraction of all samples was cultured using the MRCM method, one BSOP57 and one was used for qPCR. The recovery rate for fungi was significantly higher by MRCM (87%) than by BSOP57 (8.7%) from all 23 specimens. Sputum samples were 44% positive by MRCM compared to no fungi isolated (0%) by BSOP57. Bronchial aspirates were 75% positive by MRCM and 0% by BSOP57. BAL samples were positive in 20% by MRCM and 10% by BSOP57. qPCR was always more sensitive than culture (95.6%) from all samples. In general, over 100 mould colonies (81 Aspergillus fumigatus) were grown using the MRCM method compared with only one colony from BSOP57. This study provides a reference point for standardisation of respiratory sample processing in diagnostic laboratories. Culture from higher volume undiluted respiratory specimens has a much higher yield for Aspergillus than BSOP57. qPCR is much more sensitive than culture and the current UK method requires revision.

Mannose-binding lectin genotype and serum levels in patients with chronic and allergic pulmonary aspergillosis

Several studies suggest mannose‐binding lectin (MBL) deficiency is associated with various manifestations of aspergillosis. MBL serum levels and function are genetically determined, but levels rise during inflammation. We address the relative frequency of deficient genotypes, the relationship between serum level and genotype and both age and disease manifestations in patients with chronic pulmonary (CPA) and allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitization (SAFS). DNA was extracted from blood samples, and MBL2 genotyping was performed using the INNO‐LiPA MBL2 kit. Serum MBL concentrations were determined using ELISA. One hundred and eight patients were evaluated, 70 (65%) with CPA, 38 (35%) with allergic disease (ABPA, SAFS or undefined) and 13 (12%) had both CPA and ABPA. The mean MBL serum level was 1849 μg L−1 and did not differ between groups. Forty subjects (37%) had exon 1 genotypes producing nonfunctional MBL (A/B, A/C, A/D and O/O), a frequency not different from published normal controls. A/A subjects with CPA had higher levels (2981 μg L−1) compared with allergic A/A subjects (2202 μg L−1) (pc0.012). No single haplotype, genotype or allele was significantly related to any aspergillosis phenotype. Worse breathlessness was associated with higher MBL levels among A/A subjects (P = 0.009) and conversely nonfunctional genotypes. Mean MBL values were higher in those with an Medical Research Council (MRC) breathlessness score of 5 compared with those with and MRC score of 1 (P = 0.023). A/A allergic subjects (n = 27) in this study were ∼11 years younger than allergic A/O subjects (n = 11, P = 0.02). Subjects with worse respiratory status or more severe CPA had higher MBL serum levels (P = 0.023; P = 0.034). Bronchiectasis was not associated with MBL levels in CPA or allergic aspergillosis. MBL genotype and serum level modulate progression of aspergillosis.

An Improved Model of the Aspergillus fumigatus CYP51A Protein

ABSTRACT Azole resistance is an increasing clinical problem for Aspergillus fumigatus, with the majority of published resistance arising from mutations in the azole target gene CYP51A. Previous structural studies of this protein have suffered from a nonorthologous, low-homology template for homology modeling. Here we present a new model based on the human CYP51A orthologue that provides a higher-quality model for A. fumigatus CYP51A.

Aspergillus fumigatus proteases, Asp f 5 and Asp f 13, are essential for airway inflammation and remodelling in a murine inhalation model.

In susceptible individuals, exposure to Aspergillus fumigatus can lead to the development of atopic lung diseases such as allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitization (SAFS). Protease allergens including Asp f 5 and Asp f 13 from Aspergillus fumigatus are thought to be important for initiation and progression of allergic asthma.

Non-coding RNAs and disease: the classical ncRNAs make a comeback.

Many human diseases have been attributed to mutation in the protein coding regions of the human genome. The protein coding portion of the human genome, however, is very small compared with the non-coding portion of the genome. As such, there are a disproportionate number of diseases attributed to the coding compared with the non-coding portion of the genome. It is now clear that the non-coding portion of the genome produces many functional non-coding RNAs and these RNAs are slowly being linked to human diseases. Here we discuss examples where mutation in classical non-coding RNAs have been attributed to human disease and identify the future potential for the non-coding portion of the genome in disease biology.

Occurrence of azole-resistant species of Aspergillus in the UK environment

The aim of this study was to survey environmental isolates of Aspergillus resistant to azoles in azole-treated and naïve areas to determine whether resistance could be related to azole treatment history. Aspergillus fumigatus was sampled from the centre of a large city and from fields with known azole history. Azole resistance was determined and sequencing was performed to identify strains and mutations in the cyp51A gene. Azole resistance was detected in azole-treated field isolates but not in urban isolates (P=0.038). In addition, an azole-resistant isolate of Neosartorya fischeri was isolated. These results support the hypothesis that agricultural azole use may lead to resistance in environmental fungi of clinical importance. We report the first environmental UK TR34/L98H isolate of A. fumigatus.

Aspergillus fumigatus allergen expression is coordinately regulated in response to hydrogen peroxide and cyclic AMP

BackgroundA. fumigatus has been associated with a wide spectrum of allergic disorders such as ABPA or SAFS. It is poorly understood what allergens in particular are being expressed during fungal invasion and which are responsible for stimulation of immune responses. Study of the dynamics of allergen production by fungi may lead to insights into how allergens are presented to the immune system.MethodsExpression of 17 A. fumigatus allergen genes was examined in response to various culture conditions and stimuli as well as in the presence of macrophages in order to mimic conditions encountered in the lung.ResultsExpression of 14/17 allergen genes was strongly induced by oxidative stress caused by hydrogen peroxide (Asp f 1, -2, -4, -5, -6, -7, -8, -10, -13, -17 and -18, all >10-fold and Asp f 11, -12, and -22, 5-10-fold) and 16/17 allergen genes were repressed in the presence of cAMP. The 4 protease allergen genes (Asp f -5, -10, -13 and -18) were expressed at very low levels compared to the comparator (β-tubulin) under all other conditions examined. Mild heat shock, anoxia, lipid and presence of macrophages did not result in coordinated changes in allergen gene expression. Growth on lipid as sole carbon source contributed to the moderate induction of most of the allergen genes. Heat shock (37°C > 42°C) caused moderate repression in 11/17 genes (Asp f 1, -2, -4, -5, -6, -9, -10, -13, -17, -18 and -23) (2- to 9-fold), which was mostly evident for Asp f 1 and -9 (~9-fold). Anaerobic stress led to moderate induction of 13/17 genes (1.1 to 4-fold) with one, Asp f 8 induced over 10-fold when grown under mineral oil. Complex changes were seen in gene expression during co-culture of A. fumigatus with macrophages.ConclusionsRemarkable coordination of allergen gene expression in response to a specific condition (oxidative stress or the presence of cAMP) has been observed, implying that a single biological stimulus may play a role in allergen gene regulation. Interdiction of a putative allergen expression induction signalling pathway might provide a novel therapy for treatment of fungal allergy.