In Vitro Susceptibilities Of Clinical Isolates Of Aspergillus Species Obtained In Nanjing, China And The Resistance Mechanisms Of Aspergillus Fumigatus To Itraconazole

Invasive aspergillosis(IA)is an opportunistic infection caused by fungi of the genus Aspergillus.Aspergillus fumigatusis is the most common species recovered from cases of IA.Other commonly recovered species are Aspergillus flavus,Aspergillus niger,Aspergillus terreus and so on.The morbidity of IA has been on a sharp rise for the past few decades,and there have been Aspergillus resistance to antifungal.Nevertheless,the susceptibility testing of Aspergillus in vitro is not routinely carried in hospitals.The susceptibility testing method of Aspergillus in vitro,including dilution,disk diffusion method,E-test method and so on,is not uniform.Dilution method has high accuracy and good repeatability.Currently,there are two dilution methods for Aspergillus,European Committee on Antimicrobial Susceptibility Testing(EUCAST)and Clinical and Laboratory Standards Institute(CSLI)reference methods.The azoles inhibit Aspergillus cell membranes by inhibiting 14-a-demethylase.It leads to the biosynthesis block of lanosterol to ergosterol in the Aspergillus membrane,thereby inhibiting the growth of Aspergillus.The study confirmed that the mechanism of Aspergillus fumigatus resistance to azoles mostly due to cyp51A mutations,including amino acid substitutions at codon 54,220 caused intrinsic resistance and TR/L98H caused acquired resistance.At present,there was no epidemiology report of antifungal agents resistance in clinical Aspergillus in NanJing,China.Thus,we investigated the sensitivity distributions for amphotericin B,itraconazole,voriconazole,caspofungin and micafungin against clinical Aspergillus in Nanjing,China.And the resistance mechanism of the azole-resistant Aspergillus was analyzedChapter 1,The susceptibility survey of clinical isolates of Aspergillus in NanJing,ChinaObjective:To investigate the sensitivity distributions for antifungal agents of clinical isolates of Aspergillus in Nanjing and compare the differences microdilution assay of EUCAST and CLSI.Materials and Methods:The clinical Aspergillus were obtained from 5 hospitals in Nanjing during 2012?2013.All strains were confirmed by macroscopic colony morphology,microscopic morphology and molecular identification and stored in 10%glycerol broth at-80 ?.The susceptibilities were determined for amphotericin B,voriconazole,itraconazole,caspofungin and micafungin according to the EUCAST(E.DEF 9.1)and CLSI(M38-A2)methods.The essential agreement(EA),categorical agreement(CA),major errors(ME)and very major errors(VME)of two methods were compared.Results:A total of 116 clinical Aspergillus spp.isolates(81 of Aspergillu fumigatus,28 of Aspergillu flavus and 7 of Aspergillu niger)from 116 patients obtained from 5 hospitals in Nanjing during 2012?2013 were tested.Three isolates of Aspergillus fumigatus were founded itraconazole-resistant.The EA between two methods was 96.3%?100%.The CA was 98.8%,the ME was 0-1.2%and the VME was 0 for Aspergillus fumigatus to voriconazole.The CA was 100%,the ME and the VME was 0 for Aspergillus fumigatus and Aspergillus niger to amphotericin B,and Aspergillus fumigatus and Aspergillus flavus to itraconazole.Conclusion:The percentage of itraconzole-resistant Aspergillus fumigatus strains was 3.7%.There was no amphotericin B-resistant Aspergillus strains.Caspofungin and micafungin showed excellent efficacy for antifungal agents.There was consistent between the EUCAST and CLSI methods in vitro susceptibility against Aspergillus isolates.Chapter 2,The resistance mechanisms of Aspergillus fumigatus to itraconazoleObjective:To analyze cyp51A mutations in the itraconazole-resistant Aspergillus strains.Materials and Methods:The characteristics of the three patients with invasive aspergillosis infected by itraconazole-resistant Aspergillus fumigatus were retrospected.The cyp51A gene sequence was analysed in the itraconazole-resistant strains.Sequence analysis was determined on an ABI 3730XL DNA sequencer.Sequence alignments were performed with a reference cyp51A sequence(GenBank accession no.AF338659)to identify mutations.Results:Three patients infected by itraconazole-resistant Aspergillus fumigatus were all died.Two of the three isolates had a tandem repeat of 34 bp sequence in the promoter region and mutation L98H(TR/L98H),The mutation M220I was found in another isolate.Conclusion:The intrinsic resistance and acquired resistance were found in the itraconazole-resistant strains of Aspergillus fumigatus in Nanjing.The outcome infected by itraconazole-resistant Aspergillus fumigatus was poor.