Pan-azole Resistance Research Articles

Pan-azole resistance in clinical Aspergillus fumigatus isolates carrying TR34/L98H from birds and mammals in Belgium

Aspergillosis causes significant health risks to both birds and mammals. The outcome of these infections is often poor due to delayed diagnosis and treatment failure. We investigated 152 cases of aspergillosis from birds and mammals in Belgium. Most samples originated from the taxonomic orders Artiodactyla (40.1 %) and Columbiformes (19.7 %). Five isolates (3.3 %) showed phenotypical resistance against at least one medical azole. Three of these isolates were pan-azole resistant bearing the TR34/L98H mutation. The predominance of this resistance mutation supports an environmental route for exposure and resistance selection, highlighting the importance of the One Health concept.

Candida auris – Comparison of sensititre YeastOne and Vitek 2 AST systems for antifungal susceptibility testing – A single centre experience

IntroductionCandida auris is emerging as an important cause of candidemia and deep seated candidal infection. We compared the susceptibility results of bloodstream Candida auris isolates by Vitek 2 with Sensititre YeastOne (SYO) method. MethodsForty-seven C. auris blood stream isolates were simultaneously tested for AFST by Vitek 2 and SYO. ResultsAll strains were resistant to Fluconazole. 25.5% isolates showed pan-azole resistance. In comparison with SYO, lower MICs for voriconazole were noted with Vitek 2 (VME rate 76.1%). All strains were sensitive to anidulafungin and micafungin by SYO. For micafungin, Vitek 2 demonstrated higher MICs and an ME rate of 23.5%. Susceptibility interpretation of caspofungin by SYO was challenged by development of ‘Eagle effect’ resulting in sensitivity of 28.2%. We studied the evolution of caspofungin ‘Eagle effect’ with SYO by serial hourly MIC readings and noted that paradoxical growth commenced at 21 hrs of incubation. Compared to SYO, Vitek 2 showed higher resistance rate to Amphotericin B with ME rate of 25.6%. ConclusionLaboratories using commercial AFST systems for Candida auris need to be aware of the possibility of ME and VME for amphotericin B and voriconazole respectively with Vitek 2 and ‘Eagle effect’ for caspofungin with SYO.

Azole resistance mechanisms and population structure of the human pathogen Aspergillus fumigatus on retail plant products.

Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic fungus that is life-threatening to the immunocompromised. Triazole-resistant A. fumigatus was found in patients without prior treatment with azoles, leading researchers to conclude that resistance had developed in agricultural environments where azoles are used against plant pathogens. Previous studies have documented azole-resistant A. fumigatus across agricultural environments, but few have looked at retail plant products. Our objectives were to determine if azole-resistant A. fumigatus is prevalent in retail plant products produced in the United States (U.S.), as well as to identify the resistance mechanism(s) and population genetic structure of these isolates. Five hundred twenty-five isolates were collected from retail plant products and screened for azole resistance. Twenty-four isolates collected from compost, soil, flower bulbs, and raw peanuts were pan-azole resistant. These isolates had the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all known to underly pan-azole resistance, as well as WT alleles, suggesting that non-cyp51A mechanisms contribute to pan-azole resistance in these isolates. Minimum spanning networks showed two lineages containing isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant analysis of principle components identified three primary clusters. This is consistent with previous studies detecting three clades of A. fumigatus and identifying pan-azole-resistant isolates with TR alleles in a single clade. We found pan-azole resistance in U.S. retail plant products, particularly compost and flower bulbs, which indicates a risk of exposure to these products for susceptible populations and that highly resistant isolates are likely distributed worldwide on these products.IMPORTANCEAspergillus fumigatus has recently been designated as a critical fungal pathogen by the World Health Organization. It is most deadly to people with compromised immune systems, and with the emergence of antifungal resistance to multiple azole drugs, this disease carries a nearly 100% fatality rate without treatment or if isolates are resistant to the drugs used to treat the disease. It is important to determine the relatedness and origins of resistant A. fumigatus isolates in the environment, including plant-based retail products, so that factors promoting the development and propagation of resistant isolates can be identified.

Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States.

Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in eight states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in seven states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades, and TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow indicating recombination between clades highlighting the potential for azole-resistance to continue spreading in the U.S.IMPORTANCEAspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris, and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for 20 years and has recently been reported in patients in the United States (U.S.). Until this study, we did not know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study, we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus which is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.

Pan-azole-resistant Meyerozyma guilliermondii clonal isolates harbouring a double F126L and L505F mutation in Erg11.

Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied. The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, invitro experiments on fitness cost and virulence were carried out. Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission. In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species.

Detection of azole-resistant Aspergillus fumigatus in the environment from air, plant debris, compost, and soil.

Aspergillus fumigatus is a ubiquitous fungus, a saprophyte of plants, and an opportunistic pathogen of humans. Azole fungicides are used in agriculture to control plant pathogens, and azoles are also used as a first line of treatment for aspergillosis. The continued exposure of A. fumigatus to azoles in the environment has likely led to azole resistance in the clinic where infections result in high levels of mortality. Pan-azole resistance in environmental isolates is most often associated with tandem-repeat (TR) mutations containing 34 or 46 nucleotides in the cyp51A gene. Because the rapid detection of resistance is important for public health, PCR-based techniques have been developed to detect TR mutations in clinical samples. We are interested in identifying agricultural environments conducive to resistance development, but environmental surveillance of resistance has focused on labor-intensive isolation of the fungus followed by screening for resistance. Our goal was to develop assays for the rapid detection of pan-azole-resistant A. fumigatus directly from air, plants, compost, and soil samples. To accomplish this, we optimized DNA extractions for air filters, soil, compost, and plant debris and standardized two nested-PCR assays targeting the TR mutations. Sensitivity and specificity of the assays were tested using A. fumigatus DNA from wild type and TR-based resistant isolates and with soil and air filters spiked with conidia of the same isolates. The nested-PCR assays were sensitive to 5 fg and specific to A. fumigatus without cross-reaction with DNA from other soil microorganisms. Environmental samples from agricultural settings in Georgia, USA were sampled and tested. The TR46 allele was recovered from 30% of samples, including air, soil and plant debris samples from compost, hibiscus and hemp. These assays allow rapid surveillance of resistant isolates directly from environmental samples improving our identification of hotspots of azole-resistant A. fumigatus.

P001 Characteristics and dynamics of azole-resistant Aspergillus fumigatus variants emerging over a 28-year period in the Netherlands

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMBackground Aspergillus fumigatus, a globally distributed opportunistic pathogen, is the main cause of invasive aspergillosis, especially in immunocompromised patients with high mortality. The emergence of azole-resistant A. fumigatus isolates has been a significant concern worldwide and an important clinical problem.ObjectivesWe aim to determine the presence of variants in a large collection of clinical A. fumigatus isolates from the Netherlands, if the number of variants increased over time and if the presence of additional short nucleotide polymorphisms (SNPs) or tandem repeats (TR) variations impacted on the triazole phenotype.MethodsThe Radboud University Medical Center has collected 11 813 clinical A. fumigatus isolates since 1994. The collection includes isolates cultured from patients admitted to our own center, isolates sent from other hospitals for identification and in vitro susceptibility testing, and isolates sent from five university medical centers and five teaching hospitals that contribute to the national Aspergillus resistance surveillance. The genotypes were detected by Cyp51A Sanger sequencing. All isolates were subjected to in vitro susceptibility testing using the EUCAST microdilution reference method. Minimal inhibitory concentrations (MICs) were determined for itraconazole, voriconazole, posaconazole, in all isolates and for isavuconazole in isolates cultured in 2015 and thereafter.ResultsIn total, 1826 A. fumigatus isolates harbored azole-resistant mutations in the Cyp51A-gene with 92 genotypes. Tandem Repeat-associated resistance genotypes accounted for 55.43% of the variants and were involved in 1728 isolates (94.63%). TR34/L98H and TR46/Y121F/T289A resistance mutations remained dominant, and increasingly additional SNPs in the Cyp51A-gene or changes to the gene promoter were observed. The G448S mutation was relatively common and present in various genetic backgrounds. This SNP was most often found in isolates harboring the TR46 resistance mechanism (8 variants) and was also observed in two variants in the TR34 genetic background. TR34 and TR46 resistance mutations are associated with 1170 (64.07%) isolates that exhibited a pan-azole resistance phenotype, 547 (29.96%) a multi-azole resistance phenotype, and 75 (4.11%) resistance to a single azole. TR34/L98H confers high itraconazole resistance, while T289A confers high voriconazole resistance in the TR46 background. Isolates with a G448S point mutation show high MICs for both voriconazole and itraconazole. The TR34/L98H/T289A/G448S isolate showed low itraconazole MICs but high voriconazole resistance, and mutations in the promoter region, TR34/C-86 G/L98H, and (T-66 G)/TR34/L98H variants, showed increased voriconazole and isavuconazole MIC compared with the parent phenotype. TR46/Y121F/M172I/T289A/G448S variant was observed with an increased itraconazole (GM MIC 16 mg/L, 1→16 mg/l) and decreased voriconazole (GM MIC 18.664 mg/l, 4→16 mg/l) compared with the parent MIC of TR46/Y121F/T289A, while TR92/Y121F/M172I/T289A/G448S and TR46/Y121F/ T289A/G448S variants showed the consistent MIC distribution with parent genotype. The variants with more combination mutations showed pan-azole resistance with increased MIC distribution.ConclusionOur survey showed a significant increase in resistance genotypes in clinical A. fumigatus over a period of 28 years. Azoles resistance phenotypes vary from resistant variants in clinical isolates; it is an implication for clinical A. fumigatus infection treatment options and antifungal stewardship.

Deletion of cox7c Results in Pan-Azole Resistance in Aspergillus fumigatus.

ABSTRACTIn Aspergillus fumigatus, the most prevalent resistance to azoles results from mutational modifications of the azole target protein Cyp51A, but there are non-cyp51A mutants resistant to azoles, and the mechanisms underlying the resistance of these strains remain to be explored. Here, we identified a novel cytochrome c oxidase, cox7c (W56*), nonsense mutation in the laboratory and found that it caused reduced colony growth and resistance to multiantifungal agents. Meanwhile, we revealed that cold storage is responsible for increased tolerance of conidia to itraconazole (ITC) stress, which further advances azole-resistant mutations (cryopreservation→ITC tolerance→azole resistance). The deletion or mutation of cox7c results explicitly in resistance to antifungal-targeting enzymes, including triazoles, polyenes, and allylamines, required for ergosterol synthesis, or resistance to fungal ergosterol. A high-performance liquid chromatography (HPLC) assay showed that the cox7c knockout strain decreased intracellular itraconazole concentration. In addition, the lack of Cox7c resulted in the accumulation of intracellular heme B. We validated that an endogenous increase in, or the exogenous addition of, heme B was capable of eliciting azole resistance, which was in good accordance with the phenotypic resistance analysis of cox7c mutants. Furthermore, RNA sequencing verified the elevated transcriptional expression levels of multidrug transport genes. Additionally, lower itraconazole-induced reactive oxygen species generation in mycelia of a cox7c-deletion strain suggested that this reduction may, in part, contribute to drug resistance. These findings increase our understanding of how A. fumigatus’s direct responses to azoles promote fungal survival in the environment and address genetic mutations that arise from patients or environments.

Evidence for the agricultural origin of resistance to multiple antimicrobials in Aspergillus fumigatus, a fungal pathogen of humans.

Pathogen resistance to clinical antimicrobial agents is an urgent problem. The fungus Aspergillus fumigatus causes 300,000 life-threatening infections in susceptible humans annually. Azoles, which are widely used in both clinical and agricultural settings, are currently the most effective treatment, but resistance to clinical azoles is emerging worldwide. Here, we report the isolation and analysis of azole-sensitive and azole-resistant A. fumigatus from agricultural environments in the southeastern United States (USA) and show that the USA pan-azole-resistant isolates form a clade with pan-azole-resistant isolates from the United Kingdom, the Netherlands, and India. We show that several pan-azole-resistant isolates from agricultural settings in the USA and India also carry alleles with mutations conferring resistance to agricultural fungicides from the benzimidazole (MBC) and quinone outside inhibitor (QoI) classes. We further show that pan-azole-resistant A. fumigatus isolates from patients in clinical settings in the USA, India, and the Netherlands also carry alleles conferring resistance to MBC and QoI agricultural fungicides. The presence of markers for resistance to agricultural-use fungicides in clinical A. fumigatus isolates is strong evidence for an agricultural origin of pan-azole resistance in patients. The presence of multiple fungicide-resistance alleles in agricultural and clinical isolates further suggests that the unique genetics of the pan-azole-resistant clade enables the evolution and/or persistence of antimicrobial resistance mutations leading to the establishment of multifungicide-resistant isolates.

938. Azole Resistant Aspergillus Species in Lung Transplant Recipients: A 10 Year Experience

BackgroundInvasive aspergillosis (IA) causes significant morbidity and mortality in lung transplant (LTx) recipients. Antifungal resistance in Aspergillus species is on the rise globally with specific concern in Europe related to the TR34/L98H mutation in the cyp51A enzyme that induces pan-azole resistance. Azole exposure is a known risk factor for development of resistant Aspergillus, but this is less well described in LTx population.MethodsWe reviewed the electronic medical records of LTx patients with respiratory cultures positive for Aspergillus species known to be inherently resistant or any Aspergillus species tested to be resistant to one or more triazole from 2010 to 2019. For available isolates, Sanger sequencing was performed on cyp51A with primers targeting the promoter region and 3 known hotspot areas.ResultsTwenty eight patients met inclusion criteria and 2.7% (28/1026) Aspergillus isolates were azole-resistant during study period (Figure 1). Median time from LTx to resistant Aspergillus growth was 196 days (range 14 - 3146). There was a cluster of positive cultures within 1-year post-Tx period (13/28). Azole exposure varied, from 7 to 2443 days (median 128). There was no change in incidence over the study period. The most common species was Aspergillus calidoustus (Figure 2). Twenty cases were deemed colonization, vs 5 probable IFI and 3 proven IFI. Mortality of IFI with resistant Aspergillus was 38% (3/8), higher than azole-susceptible IA (p=0.05). Twelve isolates were available for sequencing; none carried TR34/L98H mutation. There was wide variation in mutations, ranging from 1 to 12 point mutations in the cyp51 enzyme, many of them SNPs previously described as engendering an azole resistant phenotype (Figure 3). Aspergillosis-free Survival by Resistant and Susceptible Isolates Development of azole-resistant aspergillus colonization/infection was rare relative to common occurrence of colonization/infection with susceptible isolates in the lung transplant recipient populationAzole Resistant Aspergillus Species Isolated from Lung Transplant Recipients The most commonly isolated species were A. calidoustus and ustus. Only 4/28 isolates were A. fumigatus.Azole Resistant Aspergillus Genotypic Analysis Sequenced isolates showed wide variability in point mutations. M220V, G54, and G448 were the only mutations observed more than once. 3 isolates were wild type.ConclusionAzole resistant Aspergillus infections remain an uncommon problem in LTx. The majority of isolates were deemed colonization, but mortality was high when IFI was present. Most isolates had mutations within the hot spot regions of cyp51A known to induce azole resistance. There were no TR34/L98H mutants found in our patient population.Disclosures Minh-Hong Nguyen, MD, Merck (Grant/Research Support)

Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance.

Aspergillus fumigatus is an opportunistic human pathogen that causes aspergillosis, a spectrum of environmentally acquired respiratory illnesses. It has a cosmopolitan distribution and exists in the environment as a saprotroph on decaying plant matter. Azoles, which target Cyp51A in the ergosterol synthesis pathway, are the primary class of drugs used to treat aspergillosis. Azoles are also used to combat plant pathogenic fungi. Recently, an increasing number of azole-naive patients have presented with pan-azole–resistant strains of A. fumigatus. The TR34/L98H and TR46/Y121F/T289A alleles in the cyp51A gene are the most common ones conferring pan-azole resistance. There is evidence that these mutations arose in agricultural settings; therefore, numerous studies have been conducted to identify azole resistance in environmental A. fumigatus and to determine where resistance is developing in the environment. Here, we summarize the global occurrence of azole-resistant A. fumigatus in the environment based on available literature. Additionally, we have created an interactive world map showing where resistant isolates have been detected and include information on the specific alleles identified, environmental settings, and azole fungicide use. Azole-resistant A. fumigatus has been found on every continent, except for Antarctica, with the highest number of reports from Europe. Developed environments, specifically hospitals and gardens, were the most common settings where azole-resistant A. fumigatus was detected, followed by soils sampled from agricultural settings. The TR34/L98H resistance allele was the most common in all regions except South America where the TR46/Y121F/T289A allele was the most common. A major consideration in interpreting this survey of the literature is sampling bias; regions and environments that have been extensively sampled are more likely to show greater azole resistance even though resistance could be more prevalent in areas that are under-sampled or not sampled at all. Increased surveillance to pinpoint reservoirs, as well as antifungal stewardship, is needed to preserve this class of antifungals for crop protection and human health.

Genome-Wide Association Analysis for Triazole Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is a ubiquitous fungus and the main agent of aspergillosis, a common fungal infection in the immunocompromised population. Triazoles such as itraconazole and voriconazole are the common first-line drugs for treating aspergillosis. However, triazole resistance in A. fumigatus has been reported in an increasing number of countries. While most studies of triazole resistance have focused on mutations in the triazole target gene cyp51A, >70% of triazole-resistant strains in certain populations showed no mutations in cyp51A. To identify potential non-cyp51A mutations associated with triazole resistance in A. fumigatus, we analyzed the whole genome sequences and triazole susceptibilities of 195 strains from 12 countries. These strains belonged to three distinct clades. Our genome-wide association study (GWAS) identified a total of six missense mutations significantly associated with itraconazole resistance and 18 missense mutations with voriconazole resistance. In addition, to investigate itraconazole and pan-azole resistance, Fisher’s exact tests revealed 26 additional missense variants tightly linked to the top 20 SNPs obtained by GWAS, of which two were consistently associated with triazole resistance. The large number of novel mutations related to triazole resistance should help further investigations into their molecular mechanisms, their clinical importance, and the development of a comprehensive molecular diagnosis toolbox for triazole resistance in A. fumigatus.

VATS Trisegmentectomy Resection of Aspergilloma with Pan-Azole Resistance?

Abstract Pulmonary aspergillosis is a relatively rare disease and its management is sometimes difficult. Surgical resection is indicated in selected patients. We present a nationally unreported case (to the best of our knowledge) of VATS anatomical sublobar lung resection for isolated left upper lobe aspergilloma in a 55-year-old male with history of chronic pulmonary aspergillosis (CPNA). As adhesions prove to be a significant intra-operative obstacle, VATS segmentectomy can be ambitious, however this can be considered for simple aspergilloma in absence of severe pleural adhesions.

Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain.

A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

The Multi-Fungicide Resistance Status of Aspergillus fumigatus Populations in Arable Soils and the Wider European Environment.

The evolution and spread of pan-azole resistance alleles in clinical and environmental isolates of Aspergillus fumigatus is a global human health concern. The identification of hotspots for azole resistance development in the wider environment can inform optimal measures to counteract further spread by minimizing exposure to azole fungicides and reducing inoculum build-up and pathogen dispersal. We investigated the fungicide sensitivity status of soil populations sampled from arable crops and the wider environment and compared these with urban airborne populations. Low levels of azole resistance were observed for isolates carrying the CYP51A variant F46Y/M172V/E427K, all belonging to a cluster of related cell surface protein (CSP) types which included t07, t08, t13, t15, t19, and t02B, a new allele. High levels of resistance were found in soil isolates carrying CYP51A variants TR34/L98H and TR46/Y121F/T289A, all belonging to CSP types t01, t02, t04B, or t11. TR46/Y121F/M172V/T289A/G448S (CSP t01) and TR46/Y121F/T289A/S363P/I364V/G448S (CSP t01), a new haplotype associated with high levels of resistance, were isolated from Dutch urban air samples, indicating azole resistance evolution is ongoing. Based on low numbers of pan-azole resistant isolates and lack of new genotypes in soils of fungicide-treated commercial and experimental wheat crops, we consider arable crop production as a coldspot for azole resistance development, in contrast to previously reported flower bulb waste heaps. This study also shows that, in addition to azole resistance, several lineages of A. fumigatus carrying TR-based CYP51A variants have also developed acquired resistance to methyl benzimidazole carbamate, quinone outside inhibitor and succinate dehydrogenase (Sdh) inhibitor fungicides through target-site alterations in the corresponding fungicide target proteins; beta-tubulin (F200Y), cytochrome b (G143A), and Sdh subunit B (H270Y and H270R), respectively. Molecular typing showed that several multi-fungicide resistant strains found in agricultural soils in this study were clonal as identical isolates have been found earlier in the environment and/or in patients. Further research on the spread of different fungicide-resistant alleles from the wider environment to patients and vice versa can inform optimal practices to tackle the further spread of antifungal resistance in A. fumigatus populations and to safeguard the efficacy of azoles for future treatment of invasive aspergillosis.

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens.

BackgroundInfections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited.ObjectivesTo develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates.MethodsMethod validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017–March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing.ResultsThe cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens.ConclusionsThe pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

Nationwide surveillance of azole-resistant Aspergillus fumigatus environmental isolates in Greece: detection of pan-azole resistance associated with the TR46/Y121F/T289A cyp51A mutation.

Acquired azole resistance (AR) in Aspergillus fumigatus emphasizes the importance of the One Health multisectorial approach. The prevalence of azole-resistant A. fumigatus in the environment of Greece is unknown. Between October 2016 and September 2017, a total of 716 soil samples were collected from 23 provinces and screened for AR using azole-containing agar plates. Recovered isolates were macro-/microscopically identified and colonies were counted. Azole susceptibility testing of A. fumigatus species complex (SC) isolates was performed (EUCAST E.DEF9.3.1). Azole-resistant A. fumigatus isolates were subjected to confirmatory molecular identification and sequencing of the cyp51A gene. No yeasts were recovered, while multiple moulds grew on 695 (97%) samples. Overall, zygomycetes (most non-Mucor genera) grew on 432 (60%) samples, while Aspergillus spp. grew on 500 (70%) [410 (57%) Aspergillus niger SC; 120 (17%) Aspergillus terreus SC; 101 (14%) A. fumigatus SC; 34 (5%) Aspergillus flavus SC]. The mean ± SD soil load of Aspergillus spp. was 2.23 ± 0.41 log10 cfu/g (no differences among species). No azole-resistant non-A. fumigatus spp. isolate was detected. Itraconazole, voriconazole, isavuconazole and posaconazole MIC50/MIC90 (MIC range) of A. fumigatus SC strains were 0.25/0.5 (0.25 to >8), 0.5/1 (0.25 to >8), 1/1 (0.125 to >8) and 0.06/0.125 (0.06-1) mg/L, respectively. Overall, 1/500 (0.2%) of Aspergillus isolates, and 1/101 (1%) of A. fumigatus SC isolates, was pan-azole-resistant (itraconazole, voriconazole, isavuconazole and posaconazole MIC >8, >8, >8 and 1 mg/L, respectively). The resistant isolate was recovered from organically grown raisin grapes treated with homemade compost and it was an A. fumigatus sensu stricto isolate harbouring the TR46/Y121F/T289A mutation. The soil's load was higher compared with azole-susceptible strains (3.74 versus 2.09 log10 cfu/g). This is the first known report of environmental pan-azole-resistant A. fumigatus in Greece. Since data on Greek clinical isolates are lacking, this finding must alarm the systematic local surveillance of AR in medical settings.

A Novel Combination of CYP51A Mutations Confers Pan-Azole Resistance in Aspergillus fumigatus.

The treatment of invasive and chronic aspergillosis involves triazole drugs. Its intensive use has resulted in the selection of resistant isolates, and at present, azole resistance in Aspergillus fumigatus is considered an emerging threat to public health worldwide. The aim of this work is to uncover the molecular mechanism implicated in the azole resistance phenotype of three Aspergillus fumigatus clinical strains isolated from an Argentinian cystic fibrosis patient under long-term triazole treatment. Strain susceptibilities were assessed, and CYP51A gene sequences were analyzed. Two of the studied Aspergillus fumigatus strains harbored the TR34-L98H allele. These strains showed high MIC values for all tested triazoles (>16.00 μg/ml, 1.00 μg/ml, 1.00 μg/ml, and 2.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The third strain had a novel amino acid change (R65K) combined with the TR34-L98H mutations. This new mutation combination induces a pan-azole MIC augment compared with TR34-L98H mutants (>16 μg/ml, 4.00 μg/ml, 4.00 μg/ml, and 8.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The strain harboring the TR34-R65K-L98H allele showed no inhibition halo when voriconazole susceptibility was evaluated by disk diffusion. The effect of these mutations in the azole-resistant phenotype was confirmed by gene replacement experiments. Transformants harboring the TR34-L98H and TR34-R65K-L98H alleles mimicked the azole-resistant phenotype of the clinical isolates, while the incorporation of the TR34-R65K and R65K alleles did not significantly increase azole MIC values. This is the first report of the TR34-L98H allele in Argentina. Moreover, a novel CYP51A allele (TR34-R65K-L98H) that induces a pan-azole MIC augment is described.

Aspergillus fumigatus and pan-azole resistance: who should be concerned?

Although clinical outcomes in the treatment of aspergillosis have markedly improved with the availability of newer triazoles, the development of resistance to these antifungals, especially in Aspergillus fumigatus, is a growing concern. The purpose of this review is to provide an update on azole resistance mechanisms and their epidemiology in A. fumigatus, the clinical implications of azole resistance, and to discuss future treatment options against azole-resistant aspergillosis. Resistance may develop through either patient or environmental azole exposure. Environmental exposure is the most prevalent means of resistance development, and these isolates can cause disease in various at-risk groups, which now include those with influenza, and potentially COVID-19. Although current treatment options are limited, newer therapies are in clinical development. These include agents with novel mechanisms of action which have in vitro and in vivo activity against azole-resistant A. fumigatus. Azole-resistant A. fumigatus is an emerging threat that hampers our ability to successfully treat patients with aspergillosis. Certain geographic regions and patient populations appear to be at increased risk for this pathogen. As new patient groups are increasingly recognized to be at increased risk for invasive aspergillosis, studies to define the epidemiology and management of azole-resistant A. fumigatus are critically needed. While treatment options are currently limited, new agents under clinical development may offer hope.

Chronic Pulmonary Aspergillosis: Notes for a Clinician in a Resource-Limited Setting Where There Is No Mycologist.

Chronic pulmonary aspergillosis (CPA) is a spectrum of several progressive disease manifestations caused by Aspergillus species in patients with underlying structural lung diseases. Duration of symptoms longer than three months distinguishes CPA from acute and subacute invasive pulmonary aspergillosis. CPA affects over 3 million individuals worldwide. Its diagnostic approach requires a thorough Clinical, Radiological, Immunological and Mycological (CRIM) assessment. The diagnosis of CPA requires (1) demonstration of one or more cavities with or without a fungal ball present or nodules on chest imaging, (2) direct evidence of Aspergillus infection or an immunological response to Aspergillus species and (3) exclusion of alternative diagnoses, although CPA and mycobacterial disease can be synchronous. Aspergillus antibody is elevated in over 90% of patients and is the cornerstone for CPA diagnosis. Long-term oral antifungal therapy improves quality of life, arrests haemoptysis and prevents disease progression. Itraconazole and voriconazole are alternative first-line agents; voriconazole is preferred for patients with contra-indications to itraconazole and in those with severe disease (including large aspergilloma). In patients co-infected with tuberculosis (TB), it is not possible to treat TB with rifampicin and concurrently administer azoles, because of profound drug interactions. In those with pan-azole resistance or intolerance or progressive disease while on oral triazoles, short-term courses of intravenous liposomal amphotericin B or micafungin is used. Surgery benefits patients with well-circumscribed simple aspergillomas and should be offered earlier in low-resource settings.