Emergence Of Azole Resistance Research Articles

In Vitro Interaction of Voriconazole and Anidulafungin against Triazole-Resistant Aspergillus fumigatus

Voriconazole is the recommended drug of first choice to treat infections caused by Aspergillus fumigatus. The efficacy of voriconazole might be hampered by the emergence of azole resistance. However, the combination of voriconazole with anidulafungin could improve therapeutic outcomes in azole-resistant invasive aspergillosis (IA). The in vitro interaction between voriconazole and anidulafungin was determined against voriconazole-susceptible and voriconazole-resistant (substitutions in the cyp51A gene, including single point [M220I and G54W] and tandem repeat [34-bp tandem repeat in the promoter region of the cyp51A gene in combination with substitutions at codon L98 and 46-bp tandem repeat in the promoter region of the cyp51A gene in combination with mutation at codons Y121 and T289] mutations) clinical A. fumigatus isolates using a checkerboard microdilution method with spectrophotometric analysis and a viability-based XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} assay within 2 h of exposure after 24 and 48 h of incubation at 35 °C to 37 °C. Fractional inhibitory concentration (FIC) indexes (FICis) were determined using different MIC endpoints and Bliss independence analysis performed based on the response surface calculation of the no-drug interaction. Significant synergistic interactions obtained based on measuring the FIC index were dependent on the MIC endpoint, in which FICs were inversely related to voriconazole and anidulafungin MICs and were influenced by the CYP51A genotype. A statistically significant difference was observed between FIC indexes of isolates harboring tandem repeat mutations and wild-type controls (P = 0.006 by one-way analysis of variance [ANOVA]), indicating that synergy is decreased in azole-resistant strains. Our results indicated that a combination of voriconazole and anidulafungin might be effective against infections caused by both azole-susceptible and azole-resistant A. fumigatus isolates, but the combination could possibly be less effective in voriconazole-resistant strains with high MICs. Studies in vivo and in vitro-in vivo correlation investigations are required to validate the potential synergy of voriconazole and anidulafungin.

Pulmonary fungal infections

This review details some of the advances that have been made in the recent decade in the diagnosis, treatment and epidemiology of pulmonary fungal infections. These advances have occurred because of increasing knowledge regarding the fungal genome, better understanding of the structures of the fungal cell wall and cell membrane and the use of molecular epidemiological techniques. The clinical implications of these advances are more rapid diagnosis and more effective and less toxic antifungal agents. For example, the diagnosis of invasive pulmonary aspergillosis, as well as histoplasmosis and blastomycosis, has improved with the use of easily performed antigen detection systems in serum and bronchoalveolar lavage fluid. Treatment of angioinvasive moulds has improved with the introduction of the new azoles, voriconazole and posaconazole that have broad antifungal activity. Amphotericin B is less frequently used, and when used is often given as lipid formulation to decrease toxicity. The newest agents, the echinocandins, are especially safe as they interfere with the metabolism of the fungal cell wall, a structure not shared with humans cells. Epidemiological advances include the description of the emergence of Cryptococcus gattii in North America and the increase in pulmonary mucormycosis and pneumonia due to Fusarium and Scedosporium species in transplant recipients and patients with haematological malignancies. The emergence of azole resistance among Aspergillus species is especially worrisome and is likely related to increased azole use for treatment of patients, but also to agricultural use of azoles as fungicides in certain countries.

Isolation of multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR/L98H mutations in the cyp51A gene in India

Azole resistance in Aspergillus fumigatus isolates impacts on the management of aspergillosis since azoles are primary agents used for prophylaxis and therapy. We report the emergence of resistance to triazoles in two A. fumigatus isolates from patients in Delhi, India. One hundred and three A. fumigatus isolates, collected from 85 patients suspected of bronchopulmonary aspergillosis during 2005-10, were investigated for susceptibility to itraconazole, voriconazole, posaconazole and isavuconazole. We undertook a mixed-format real-time PCR assay for the detection of mutations leading to triazole resistance in A. fumigatus. The resistant isolates were compared with 25 Dutch TR/L98H-positive isolates by microsatellite analysis. Of the 103 A. fumigatus isolates tested, only 2 had high MIC values of itraconazole (>16 mg/L), voriconazole (2 mg/L), posaconazole (2 mg/L) and isavuconazole (8 mg/L). The resistant A. fumigatus isolates exhibited the TR/L98H genotype and showed identical patterns by microsatellite typing, but were different from 25 Dutch TR/L98H isolates. We report for the first time from India the occurrence of TR/L98H mutations in the cyp51A gene (responsible for reduced azole susceptibility) in two A. fumigatus isolates from patients with chronic respiratory disease who had not previously been exposed to azoles. The presence of TR/L98H is consistent with a route of resistance development through exposure to azole compounds in the environment. Given the emergence of azole resistance in environmental strains, continued surveillance of resistance in clinical A. fumigatus strains is desirable for successful therapy of aspergillosis.

Chronic aspergillus infections of the respiratory tract: diagnosis, management and antifungal resistance

Chronic pulmonary aspergillosis (CPA) is a relatively rare, slowly progressive pulmonary syndrome due to Aspergillus spp. that requires specific knowledge in terms of disease entity, diagnosis, management and azole resistance. This review focuses on the recent understanding of CPA entity and the emergence of azole resistance in CPA. Due to complexities related to patients' background and limited pathological evidence, the disease entity of CPA was incomprehensive and numerous names were previously used. The disease entities and nomenclature of subtypes of CPA have recently been proposed, though previous literature had grouped several different forms of CPA together. Recent advances in the methodology of susceptibility testing have indicated increasing azole resistance in Aspergillus spp. CPA is potentially involved in producing azole resistance and associated with poor response to azoles. As there are few publications regarding CPA, there are still many unanswered questions. However, updating of disease entity will promote the clinical and basic research in this field. Moreover, the emergence of antifungal drug resistance of Aspergillus is becoming a major concern. Thus, more evidence and research regarding drug resistance are required to improve the outcome of CPA.

Recent experience with fungaemia: change in species distribution and azole resistance

Owing to a rise in frequency and change in pattern of cases with fungaemia at our tertiary care centre, we conducted a prospective study for 4 months to understand the epidemiology and outcome of this infection. Detailed case histories including management protocol and outcomes were noted. Records of 140 cases with fungaemia (27.1% adult and 72.9% paediatric patients) were analysed. Although C. tropicalis was the most common yeast isolated, significantly higher isolation of C. guilliermondii (30.4%) and C. pelliculosa (17.6%) was noted in paediatric patients; and C. albicans (26.3%) and C. glabrata (10.5%) in adult patients. Rare species isolated included C. ustus (0.7%) and Trichosporon asahii (2.1%). Mortality was high (56.9% and 47.4%, respectively), in both groups of patients. Resistance to azoles (fluconazole, itraconazole, voriconazole) emerged in C. albicans (12.5-18.8%) and C. tropicalis (10.2-13.6%). Antifungal susceptibility testing report modified the therapy from fluconazole to amphotericin B in 8 patients; 5 survived. In conclusion, the study highlighted the rise of non-albicans Candida species in our hospital with differential distribution in paediatric and adult wards and emergence of azole resistance.

Emergence of Azole Resistance in Aspergillus fumigatus and Spread of a Single Resistance Mechanism

BackgroundResistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007.Methods and FindingsWe investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which 69% harboured the TR/L98H substitution, and in six isolates originating from four other countries.ConclusionsAzole resistance has emerged in A. fumigatus and might be more prevalent than currently acknowledged. The presence of a dominant resistance mechanism in clinical isolates suggests that isolates with this mechanism are spreading in our environment.