Azole-naive Patients Research Articles

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.

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.

Azole resistance and cyp51A mutation screening in Aspergillus fumigatus in Mexico.

Fungicide exposure in the environment has driven the emergence of azole-resistant Aspergillus fumigatus worldwide. A screening test allows identification of resistant isolates. We screened clinical samples for azole-resistant Aspergillus through azole-containing agar plates and identified mutations in the cyp51A gene of A. fumigatus. Aspergillus isolates from clinical samples collected in a tertiary care centre from 2014 to 2017 were screened for azole resistance. Samples were subcultured in azole-containing agar plates. Isolates with a positive screening test were subject to DNA extraction, DNA amplification and sequencing of the cyp51A gene (coding and promoter regions). Clinical data were obtained from medical records. We screened 43 Aspergillus isolates from 39 patients for azole resistance. Three isolates from three patients grew on azole-containing agar plates: two A. fumigatus and one Aspergillus flavus. PCR analysis and cyp51A sequencing identified the TR34/L98H mutation in both A. fumigatus isolates. The prevalence of cyp51A mutations among A. fumigatus was 8.3% (2/24). Both patients with TR34/L98H mutants were azole naive and presented with invasive aspergillosis; one had multiple myeloma and the other was a liver retransplant recipient. They suffered progressive disease and failed voriconazole therapy. To the best of our knowledge, this is the first report of azole-resistant A. fumigatus with the TR34/L98H mutation in two azole-naive patients with refractory invasive aspergillosis in Mexico.

Azole-resistant Aspergillus fumigatus harboring TR34/L98H, TR46/Y121F/T289A and TR53 mutations related to flower fields in Colombia

Resistance to triazoles in Aspergillus fumigatus has been reported in azole-naive patients in Europe, Asia, Australia and North America. This resistance has been linked to fungicide-driven mutations in the cyp51A gene and its promoter region. We investigated the presence of environmental azole-resistant A. fumigatus strains related to the use of azole fungicides in Colombia. Soil samples were collected from flower beds, flower fields and public gardens from the outskirts, suburbs and city centre of Bogotá. Out of the 86 soil samples taken, 17 (19.8%) grew A. fumigatus of whom eight (9.3%) contained 40 strains able to grow on azole-containing itraconazole and/or voriconazole supplemented media. All but one triazole-resistant strains were isolated from soil samples collected from flower fields and flower beds (39/40). Importantly, the majority had the TR46/Y121F/T289A, TR34/L98H, and TR53 molecular resistance mechanisms and one azole resistant strain had a wild-type cyp51A gene. Soil samples from flower fields and beds contained 4 azole fungicides (penconazole, difenoconazole, tetraconazole and tebuconazole) above the limit of detection. Our findings underline the need for extensive investigations to determine azole-resistant A. fumigatus prevalence in both clinical and environmental samples in other regions of Latin America.

Azole resistance surveillance in Aspergillus fumigatus: beneficial or biased?

Azole resistance is a growing concern with Aspergillus fumigatus, and may cause increased mortality in patients with azole-resistant invasive aspergillosis (IA). Microbial surveillance has been recognized as a fundamental component of resistance management. Surveillance information may be used to inform decisions regarding health services and research funding allocation, to guide local infection control in hospitals and communities, and to direct local and national drug policies and guidelines. Azole resistance frequencies have been based on screening of unselected A. fumigatus isolates, on the number of azole-resistant cases within a cohort of patients with a specific Aspergillus disease, or on analysis of patients within a specific risk group. The various surveillance approaches differ in their aims, as well as in their associated advantages and drawbacks. Nevertheless, a wide range of azole resistance frequencies has been reported, partly due to the denominator used. As most azole resistance is believed to develop in the environment and, as a consequence, azole-naive patients may present with azole-resistant aspergillosis, experts recommended a 10% resistance frequency threshold above which the standard treatment choice, i.e. voriconazole, should be reconsidered. We believe that local resistance rates based on Aspergillus disease and/or risk group should be leading for decisions regarding empirical antifungal therapy in specific units. In addition, patient factors should be considered, such as admission to the ICU. Collecting valid surveillance data may be challenging in azole resistance due to numerous factors that present potential biases. Surveillance research may benefit from further standardization, which may be facilitated through the recently instituted International Society for Human and Animal Mycology (ISHAM) Aspergillus Resistance Surveillance Working Group.

Corrigendum to “First detection of Aspergillus fumigatus azole resistant strain due to Cyp51A TR46/Y121F/T289A in an azole-naive patient in Spain” [New Microbes New Infect 6 (2015) 33–34

[This corrects the article DOI: 10.1016/j.nmni.2015.04.005.].

First detection of Aspergillus fumigatus azole-resistant strain due to Cyp51A TR46/Y121F/T289A in an azole-naive patient in Spain

We report the first isolation of a voriconazole-resistant Aspergillus fumigatus strain harbouring the azole resistance mechanism TR46/Y121F/T289A, recovered from an azole-naive patient in Spain with chronic obstructive pulmonary disease. This new finding in Spain suggests the spread of this resistance mechanism and reinforces the need for antifungal susceptibility surveillance.

Aspergillus fumigatus harbouring the sole Y121F mutation shows decreased susceptibility to voriconazole but maintained susceptibility to itraconazole and posaconazole.

Voriconazole, itraconazole and posaconazole are members of the azole family and widely used for the treatment of aspergillosis. They act by inhibiting the activity of the fungal Cyp51A enzyme. The emergence of environmental azole-resistant Aspergillus fumigatus strains raises major concerns for human health. Recently, a new cyp51A-mediated resistance mechanism (namely TR46/Y121F/T289A) was described in clinical samples and patient-frequented environmental sites. In an azole-naive patient, we isolated an A. fumigatus strain that was not susceptible to voriconazole but was susceptible to itraconazole and posaconazole. A molecular analysis indicated a single Y121F substitution without the TR46 or T289A alterations, which to our knowledge has never been reported. Structure modelling and molecular dynamics offered an explanation for the resistance profile consistent with the structural differences between the three azoles. Taken together, these observations suggest an original mechanism conferring resistance to azoles mediated by cyp51A of environmental origin. This uncommon susceptibility pattern might represent a 'missing link' between the wild-type A. fumigatus and the fully azole-resistant strain harbouring the TR46/Y121F/T289A mutations.

First detection of TR46/Y121F/T289A and TR34/L98H alterations in Aspergillus fumigatus isolates from azole-naive patients in Denmark despite negative findings in the environment.

Azole-resistant Aspergillus fumigatus harboring the TR34/L98H or TR46/Y121F/T289A alterations is increasingly found in Europe and Asia. Here, we present the first clinical cases of TR46/Y121/T289A and three cases of TR34/L98H outside the cystic fibrosis (CF) population in Denmark and the results of environmental surveys. Four patients (2012 to 2014) with 11 A. fumigatus and 4 Rhizomucor pusillus isolates and 239 soil samples (spring 2010 and autumn 2013, respectively) with a total of 113 A. fumigatus isolates were examined. Aspergillus isolates were screened for azole resistance using azole-containing agar. Confirmatory susceptibility testing was done using the EUCAST microbroth dilution EDEF 9.1 reference method. For relevant A. fumigatus isolates, CYP51A sequencing and microsatellite genotyping were performed. Three patients harbored TR34/L98H isolates. Two were azole naive at the time of acquisition and two were coinfected with wild-type A. fumigatus or R. pusillus isolates, complicating and delaying diagnosis. The TR46/Y121F/T289A strain was isolated in 2014 from a lung transplant patient. Genotyping indicated that susceptible and resistant Aspergillus isolates were unrelated and that no transmission between patients occurred. Azole resistance was not detected in any of the 113 soil isolates. TR34/L98H and TR46/Y121F/T289A alterations appear to be emerging in the clinical setting in Denmark and now involve azole-naive patients. Two recent soil-sampling surveys in Denmark were unable to indicate any increased prevalence of azole-resistant A. fumigatus in the environment. These findings further support the demand for real-time susceptibility testing of all clinically relevant isolates and for studies investigating the seasonal variation and ecological niches for azole-resistant environmental A. fumigatus.

Azole resistance in Aspergillus fumigatus

Reports from the end of the 2000s forced the medical community to take azole resistance in Aspergillus fumigatus into account. Not only patients with chronic aspergillus disease, who develop resistance during long-term azole treatment, but also azole-naive patients are at risk, owing to the presence of resistant strains in the environment. The purpose of this review is to overview the latest findings concerning the origin, evolution, and implications of azole resistance in A. fumigatus. TR34/L98H is the predominant resistance mechanism of environmental origin in A. fumigatus. Recent epidemiological data show that this mechanism is an expanding problem, with reports from China, Iran, and India. However, the TR34/L98H strains from the Middle East are genotypically different from the European isolates; their emergence is, therefore, not due to simple geographical spread of the 'European' isolates. A new environmental resistance mechanism, TR46/Y121F/T289A, was detected in the Netherlands, conferring voriconazole resistance. In patients chronically treated with triazoles, the spectrum of resistance has become more diverse, with the emergence of non-CYP51A-mediated mechanisms. Central registration of treatment and outcome data of patients with resistant aspergillus disease are needed. Azole resistance in A. fumigatus is evolving to a global health problem.

Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus

Azole resistance is an emerging problem in the opportunistic mould Aspergillus fumigatus. The triazoles are the most important agents for the management of Aspergillus diseases in humans. Selection for acquired resistance may occur in the hospital setting through exposure to high doses of azoles during azole therapy, but evidence is accumulating that A. fumigatus may become resistant to medical triazoles through environmental exposure to fungicides. The recovery of A. fumigatus isolates resistant to the medical triazoles from azole-naive patients as well as from the environment strongly indicates an environmental route of resistance selection. Molecule alignment studies have identified five fungicides that share a very similar molecule structure with the medical triazoles, and thus may have selected for mechanisms that confer resistance to both groups of compounds. It is important to explore further the presumed fungicide-driven route of resistance selection in order to implement effective preventive measures as the prevalence of azole resistance in A. fumigatus continues to increase and causes major challenges in the management of azole-resistant Aspergillus diseases.

Azole-Resistant Invasive Aspergillosis: Relationship to Agriculture

Azole resistance in Aspergillus fumigatus has been increasingly reported particularly over the last decade. Two routes of acquisition are described: selection of resistance during long term azole therapy in the clinical setting, and primary acquisition of resistant isolates from the environment due to the considerable use of azole fungicides in agriculture and for material preservation. Three specific resistance genotypes have been found in azole naive patients. Two of these have also been found in the environment and are characterized by a tandem repeat in the promoter region of the target gene coupled with point mutation(s) in CYP51A (TR34/L98H and TR46/Y121F/T289A). In the third a single target enzyme alteration (G432S) is found. These resistant “environmental” strains have been detected in many West-European countries as well as in the Asia-Pacifics. Noticeably, these two continents account for the highest fungicide use in the global perspective (37 % and 24 %, respectively). Among the 25 azole fungicides, five have been associated with the potential to select for the TR34/L98H genotype; three of these are among those most frequently used. Although the number of antifungal fungicide compounds and classes available is impressive compared to the armamentarium in human medicine, azoles will remain the most important group in agriculture due to superior field performance and significant resistance in fungal pathogens to other compounds. Hence, further spread of environmental resistant Aspergillus genotypes may occur and will depend on the fitness of each resistant phenotype and the pattern of azole fungicide use.

Comment on: Low prevalence of resistance to azoles in Aspergillus fumigatus in a French cohort of patients treated for haematological malignancies

Azole resistance is an emerging clinical problem in patients with Aspergillus infections, primarily those caused by Aspergillus fumigatus. Alanio et al. 1 describe the results of a study that investigated the prevalence of azole resistance in 118 clinical Aspergillus isolates obtained from a cohort of 89 patients with haematological malignancy. They found only one azole-resistant A. fumigatus isolate, which corresponded to a prevalence of 0.85%. 1 Although the low prevalence of resistance may be reassuring, the circumstances surrounding the resistant isolate are a cause of great concern. The azole-resistant isolate was cultured from an azole-naive patient, which precludes resistance development through azole therapy. The only other possibility is that the patient acquired the resistant isolate from the environment, as A. fumigatus is …

Non-albicans Candida is the most common cause of candidemia in pediatric cancer patients.

Candidemia is a serious infection that can severely complicate the care of children with cancer. We sought to determine the spectrum of Candida species in children with cancer, since effective therapy may depend on the species involved. A retrospective review of candidemia episodes in our pediatric oncology patients over a 9-year period was conducted. During this period azole prophylaxis was not routine in this group. 38 episodes of candidemia were identified: C. albicans 29%, C. tropicalis 26%, C. parapsilosis 24%, C. krusei 8%, C. glabrata 8%, and C. lusitaniae 5%. Non-albicans Candida was common in patients not receiving azole prophylaxis. Species typically susceptible to azoles were common among patients not using azoles. Death attributed to the fungal infection occurred in 21% of episodes, with nearly all the deaths occurring in patients with C. albicans and C. tropicalis. C. albicans is not the predominant species in pediatric oncology patients experiencing candidemia, even in azole-naive patients.