Genetic Diversity And In Vitro Antifungal Susceptibility Of Clinical Aspergillus Isolates From Two Teaching Hospitals In Shanghai,China

BackgroundApproximately 300 species have been defined within the filamentous fungus genus Aspergillus.Among Aspergillus species,a majority have been described only during the asexual stage.A few species,such as A.fumigatus,have been defined in both sexual and asexual stages.Aspergillus isolates are ubiquitously distributed in humid areas and agricultural environments,such as grains,moldy bread,decayed wood.With an extensive use of clinical immunosuppressive and broad-spectrum antimicrobial agents,the incidences of opportunistic fungal infections including invasive aspergillosis have been increasingfordecades.Generally,invasiveaspergillosismainlyaffects immunocompromised hosts.The infections are difficult to diagnose quickly and have an extremely high mortality rate.A.fumigatus is the most common Aspergillusspecies.However,the number of infections caused by non-A.fumigatus and zole-resistant A.fumigatus has increased.Different Aspergillus species have various levels of sensitivity to antifungal drugs.The traditional culture methods for Aspergillus cannot accurately identify species,which may delay diagnosis and treatment.At present studies that analyze the genetic diversity of clinical Aspergillus isolates and monitor susceptibility the antifungal are scarce for most regions in China,including Shanghai.For this reason,this study was performed on Aspergillus isolates from deep clinical samples from two tertiary hospitals in Shanghai.We analyzed the genetic diversity of clinical Aspergillus isolates and monitored the antifungal drug susceptibility in vitro using the latest molecular marker techniques and standardized drug susceptibility monitoring methods.This work provides valuable reference and research basis for improving clinical diagnosis and treatment of aspergillosis in China.ObjectiveFirst,to analyze the genetic diversity of Aspergillus by identifying the Aspergillus species and explore the composition of Aspergillus species in Shanghai.Second,to test the antifungal susceptibility of Aspergillus species in vitro as a means of guiding clinical medication regimens.Third,to select the strains of drug-resistant Aspergillus and detect the mutation sites of zole-resistant A.fumigatus.MethodsPart one:Aspergillus isolates from two large tertiary hospitals in Shanghai were collected,separated,and cultured.We identified Aspergillus species by amplifying the genes encoding ITS,β-tubulin,and calmodulin.Weconstructed phylogenetic trees of three single-gene sequences（ITS,β-tubulin,calmodulin）and three combined gene sequences,to compare the advantages and disadvantages of these three single-gene sequences in identifying Aspergillus isolates.Part two:Using a CLSI M38-A2 reference method,we tested the antifungal susceptibility of Aspergillusisolates and selected the drug-resistant isolates.Part three:We amplified and sequenced the cyp51A gene of zole-resistant A.fumigatus.We then aligned the cyp51A gene from zole-resistant A.fumigatus with one from a triazole-susceptible isolate（GenBank accession AF338659）using BioEdit to determine the mutation sites of zole-resistant A.fumigatus.ResultsPart one:The causative pathogens of invasive aspergillosis in Shanghai include A.fumigatus,A.flavus,A.niger,A.tubingensis,A.sydowii,A.terreus,A.nidulans,and A.tamarii.Among these species,A.fumigatus（54.6%;59/108）was the most common.The main site of infection of invasive aspergillosis was found to be the respiratory system（90.7%;98/108）,in which A.fumigatus（58.2%;57/98）wasthe most common causative pathogen.However,in infections at other sites,other strains（75%;6/8）outnumberA.fumigatus（25%;2/8）.Part two:Among antifungal drugs,the echinocandin drugs(MEC₅₀ 0.03μg/ml;MEC₉₀ 0.25μg/ml)have the greatest antifungal activity against Aspergillus spp.,followed by azole(MIC₅₀ 0.5μg/ml;MIC₉₀ 2μg/ml)and polyenes drugs(MIC₅₀ 1μg/ml;MIC₉₀2μg/ml).Among the echinocandin drugs,anidulafungin(MEC₅₀ 0.03μg/ml;MEC₉₀0.03μg/ml)has the most pronounced antifungal activity;among the azole drugs,posaconazole(MIC₅₀ 0.25μg/ml;MIC₉₀ 0.5μg/ml)has the most pronounced antifungal activity.In addition,the number of Aspergillus strains,which showed the decreased antifungal activity to Amphotericin B,are not a minority（34.3%MIC≥2μg/ml）.Part three:The rate of triazole resistance in A.fumigatus isolates is 6.8%,and we here report the first detected TR46/Y121F/T289A mutation to be found in Shanghai.We also found two zole-resistant A.fumigatus containing F46Y,G89G,M172V,N248T,and D255E mutation sites.ConclusionIn Shanghai,the causative pathogens of invasive aspergillosis were found to include the following eight species:A.fumigatus,A.flavus,A.niger,A.tubingensis,A.sydowii,A.terreus,A.nidulans,and A.tamarii.Among these species,A.fumigatus is still the most common causative pathogen.Among these antifungal drugs,echinocandin drugs showed the most pronounced antifungal activity against Aspergillus spp.,followed by azole and polyenes drugs.The rate of triazole resistance in A.fumigatus isolates is mildly increasing,and there are rich diversity of related zole-resistant mutation sites,and our study is the first to report detection of the TR46/Y121F/T289A mutation in Shanghai,which suggested that there are multiple origins and evolutionary pathways for zole-resistant isolates.These findings may be very important to the diagnosis and treatment of invasive aspergillosis and to systematic,long-term monitoring of the clinical and environmental isolates of A.fumigatus.