invasive aspergillosis ( ia ) is a life - threatening infection in immunocompromised patients associated with severe mortality . aspergillus fumigatus is the most common species recovered from cases of ia ( 90% of ia cases involving the lungs ) . triazole antifungals , such as itraconazole , posaconazole , and voriconazole , are first - line drugs in prophylaxis and treatment of ia . however , during the past decades , a number of clinical failures of ia management due to triazole - resistant a. fumigatus have been reported , which brings new challenges to the clinical treatment of ia . a global survey in the year 2005 showed that there was no itraconazole - resistant a. fumigatus in the 331 isolates that were examined . however , in the year 2009 , 43 strains of a. fumigatus from a total of 637 ( 6.75% ) isolates were determined to have an itraconazole minimum inhibitory concentrations ( mics ) of 2 mg / l . the major mechanism of triazole resistance in a. fumigatus involves mutations in the cyp51a gene encoding 14-demethylase . some specific point mutations , such as g54 , m220 , i266 , s297 , and l98h amino acid substitution in combination with a 34-base pair tandem repeat ( tr ) in the promoter region , have been identified as causes of triazole resistance . tr / l98h , considered as an intrinsic resistance mechanism , has been identified mainly in netherlands and china . at present , a few studies have focused on the prevalence of triazole resistance among a. fumigatus clinical isolates in china . thus , we investigated the in vitro triazole susceptibilities of a large collection of a. fumigatus clinical isolates and analyzed cyp51a mutations in the triazole - resistant a. fumigatus . a. fumigatus clinical isolates were obtained from four large tertiary hospitals ( jinling hospital , medical school of nanjing university ; drum tower hospital , medical school of nanjing university ; the first affiliate hospital of nanjing medical university ; and zhongda hospital , southeast university ) in nanjing , china , between august 2012 and july 2015 . all isolates were identified as a. fumigatus by macroscopic and micromorphological characteristics , thermotolerance at 48c , and molecular identification . the strains of a. fumigatus american type culture collection 204305 were included as quality controls . itraconazole ( janssen pharmaceutica nv , turnhoutseweg , belgium ) , voriconazole ( pfizer inc . , dun laoghaire , ireland ) , and posaconazole ( merck sharp & dohme pty . ltd . , new south wales , australia ) were provided in powder form . itraconazole , voriconazole , and posaconazole were dissolved in 100% dimethyl sulfoxide and stored at 80c . the in vitro antifungal susceptibility testing was performed by broth microdilution according to the european committee on antimicrobial susceptibility testing ( eucast ) reference method . itraconazole , voriconazole , and posaconazole were diluted to the required concentrations in rpmi with 2% of glucose stock solutions . spore suspensions were prepared to a final working inoculum of 25  10 cfu / ml . microdilution plates were incubated at 35c for 48 h. the endpoints for mics were defined as the lowest drug concentration that caused complete growth inhibition . mics > 2 mg / l for itraconazole and voriconazole and > 0.25 mg / l for posaconazole were considered resistant ; mics 1 mg / l for itraconazole and voriconazole and 0.125 mg / l for posaconazole were considered susceptible . for resistant isolates , the forward primer 5-atggtgccgatgctatgg-3 and reverse primer 5-agtttcagggactcctttc-3 were used in polymerase chain reaction amplification . sequence analysis was determined on an abi 3730xl dna sequencer ( applied biosystems , inc . sequences were compared to a wild type a. fumigatus strain ( genbank accession no . af . a. fumigatus clinical isolates were obtained from four large tertiary hospitals ( jinling hospital , medical school of nanjing university ; drum tower hospital , medical school of nanjing university ; the first affiliate hospital of nanjing medical university ; and zhongda hospital , southeast university ) in nanjing , china , between august 2012 and july 2015 . all isolates were identified as a. fumigatus by macroscopic and micromorphological characteristics , thermotolerance at 48c , and molecular identification . the strains of a. fumigatus american type culture collection 204305 were included as quality controls . itraconazole ( janssen pharmaceutica nv , turnhoutseweg , belgium ) , voriconazole ( pfizer inc . , dun laoghaire , ireland ) , and posaconazole ( merck sharp & dohme pty . ltd . , new south wales , australia ) were provided in powder form . itraconazole , voriconazole , and posaconazole were dissolved in 100% dimethyl sulfoxide and stored at 80c . the in vitro antifungal susceptibility testing was performed by broth microdilution according to the european committee on antimicrobial susceptibility testing ( eucast ) reference method . itraconazole , voriconazole , and posaconazole were diluted to the required concentrations in rpmi with 2% of glucose stock solutions . spore suspensions were prepared to a final working inoculum of 25  10 cfu / ml . microdilution plates were incubated at 35c for 48 h. the endpoints for mics were defined as the lowest drug concentration that caused complete growth inhibition . mics > 2 mg / l for itraconazole and voriconazole and > 0.25 mg / l for posaconazole were considered resistant ; mics 1 mg / l for itraconazole and voriconazole and 0.125 mg / l for posaconazole were considered susceptible . for resistant isolates , the entire cyp51a gene and its promoter were amplified as previously described . the forward primer 5-atggtgccgatgctatgg-3 and sequence analysis was determined on an abi 3730xl dna sequencer ( applied biosystems , inc . , sequences were compared to a wild type a. fumigatus strain ( genbank accession no . af . over a period of 4 years from august 2012 to july 2015 , a total of 126 a. fumigatus clinical isolates from 126 patients with proven or probable ia were collected and analyzed . figure 1 shows the distribution of mic values for itraconazole , voriconazole , and posaconazole according to the eucast broth microdilution procedure . a total of 4 a. fumigatus isolates ( 3.17% ) were confirmed to be itraconazole resistant with mics of 8 mg / l , and one isolate ( 0.8% ) was confirmed to be voriconazole resistant and posaconazole resistant , with mics of 4 minimal inhibitory concentrations distributions of itraconazole , voriconazole , and posaconazole in clinical isolates of aspergillus fumigatus . the mic values , cyp51a substitutions , and characteristics of triazole - resistant isolates of a. fumigatus are shown in table 1 . one isolate from a chronic obstructive pulmonary disease ( copd ) patient underwent itraconazole therapy had the m220i mutation . moreover , another itraconazole - resistant isolate did not have a substitution in the cyp51a gene . characteristics of triazole - resistant isolates of aspergillus fumigatus copd : chronic obstructive pulmonary disease ; sle : systemic lupus erythematosus ; nhl : non - hodgkin lymphoma ; bmd : bone marrow depression ; dm : diabetes mellitus ; ipa : invasive pulmonary aspergillosis ; cas : caspofungin ; mic : minimal inhibitory concentration ; itz : itraconazole ; vrz : voriconazole ; psz : posaconazole ; tr : tandem repeat ; wt : wild type . in this study , we investigated the prevalence of triazole resistance and examined cyp51a mutations among a. fumigatus clinical isolates from patients with proven or probable ia in nanjing , china . this rate is similar to that reported in germany ( 3.2% ) but lower than data from some previous studies : from a total of 497 a. fumigatus isolates , in a worldwide survey from 62 medical centers , 5.8% showed triazole resistance , and similarly , a rate of 5.3% was shown in a nationwide multicenter study from netherlands and 5.5% in copenhagen , denmark . astonishingly , a rate of 14% and 20% of a. fumigatus clinical isolates were noted as having triazole antifungal resistance in the year 2008 and 2009 , respectively , in manchester . nevertheless , the prevalence rate in the present study is higher compared to spain ( 2.5% ) and india ( 1.7% ) . mutations in the cyp51a gene encoding 14-demethylase were the major mechanisms of triazole resistance in a. fumigatus . in the present study , some studies suggested that the tr / l98h mutation acquired in the environment due to azole fungicides usage in agriculture . we found that one itraconazole - resistant isolate harbored the tr34/l98h mutation was cross - resistance to both voriconazole and posaconazole . similarly , multiazole - resistant isolates with tr34/l98h mutations were detected in asia as well as in europe . in this study , the itraconazole - resistant isolate with the m220i mutation was considered as acquired resistance as a result of prolonged treatment with itraconazole . other specific point mutations , such as g54 , i266 , and s297 , have been identified as primary causes of triazole - acquired resistance . however , in this study , one itraconazole - resistant isolate was without substitution in the cyp51a gene . tashiro et al . showed that 43% of triazole - resistant isolates did not contain a substitution in the cyp51a gene . therefore , cyp51a gene mutation is not the only mechanism of triazole resistance in a. fumigatus . other possible mechanisms ( e.g. , hape mutation , efflux pumps , cholesterol import by a. fumigatus ) might contribute to triazole resistance . we only investigated a. fumigatus clinical isolates from patients with proven or probable ia in four hospitals in nanjing ; therefore , the results might not be representative . the epidemiological data on the resistance of triazole to a. fumigatus were still insufficient , and the mechanisms of triazole resistance have not been fully investigated . in conclusion , this study confirmed the occurrence of triazole - resistant a. fumigatus clinical isolates , with a 3.17% resistance rate in nanjing , china . triazole - resistant a. fumigatus might develop through different mechanisms , which brings new challenges to the clinical treatment of ia . larger studies are needed to better investigate triazole - resistant a. fumigatus in different regions of china and elucidate the underlying mechanisms of triazole resistance . this work was supported by a grant from the national natural science foundation of china ( no . this work was supported by a grant from the national natural science foundation of china ( no .
background : during the past decades , the incidence of invasive aspergillosis ( ia ) caused by aspergillus fumigatus has increased dramatically . 
 the aims of this study were to investigate the susceptibility of clinical isolates of a. fumigatus to triazole and the underlying cyp51a mutations in triazole - resistant a. fumigatus.methods:a total of 126 a. fumigatus clinical isolates from 126 patients with proven or probable ia were obtained from four large tertiary hospitals in nanjing , china , between august 2012 and july 2015 . 
 the determination of minimal inhibitory concentrations ( mics ) for itraconazole , voriconazole , and posaconazole was performed by broth microdilution according to the european committee on antimicrobial susceptibility testing reference method.results:a total of 4 a. fumigatus isolates ( 3.17% ) were confirmed to be itraconazole resistant , with mics of 8 mg / l , and one isolate ( 0.8% ) was confirmed to be voriconazole resistant and posaconazole resistant , with mics of 4 mg / l and 0.5 mg / l , respectively . 
 we found that two of the 4 isolates of triazole - resistant a. fumigatus had the l98h amino acid substitution in combination with a 34-base pair tandem repeat in the promoter region , one isolate had an m220i mutation , and another itraconazole - resistant isolate did not have a substitution in the cyp51a gene.conclusions:this study shows that triazole - resistant a. fumigatus clinical isolates are present in nanjing , china , which is a new challenge to the clinical management of ia .