| Aspergillus is widely found in the natural environment and can be rapidly spread by spore forms,resulting in a variety of chronic and acute aspergillosis.In recent years,the incidence of invasive aspergillosis(IA)has shown an upward trend,with invasive pulmonary aspergillosis(IPA)being the most common with a mortality rate of more than 50%.Among the Aspergillus species causing invasive pulmonary aspergillosis,A.fumigatus infection is the most common,accounting for more than 90%of the incidence rate,followed by Aspergillus flavus,Aspergillus niger,etc.Although antifungal drugs such as triazoles play an important role in the treatment of invasive pulmonary aspergillosis,the mortality rate remains high,and there are reports of treatment failure due to triazole antifungal drug resistance to A.fumigatus infection.A multi-country study by ARTEMIS showed that the resistance rate of A.fumigatus to triazole antifungal drugs was 5.8%,and similar data were 5.3%in the Netherlands and 5.5%in Denmark.The detection rate of triazole-resistant A.fumigatus in the UK is relatively high and the resistance rates in 2008 and 2009 were 14%and 20%,respectively.In 2015,a multi-center survey in the Netherlands showed that the detection rate of triazole-resistant A.fumigatus was 5%to 10%,but the triazole-resistant rate of high-risk patients was as high as 30%.It can be seen that the detection rate of triazole-resistant A.fumigatus strains is on the rise.The action of the triazole antifungal agent on A.fumigatus is in the cell membrane,and the biosynthesis of lanosterol to ergosterol in the A.fumigatus cell membrane is blocked by inhibiting 14α-demethylase,thereby inhibiting the growth of Aspergillus.A mutation in the coding gene Cyp51A of A.fumigatus lanosterol 14α-demethylase,such as 54 and 220 mutations,can cause acquired resistance to triazole antifungal drugs;A series of tandem repeats(TR)of the upstream promoter region of A.fumigatus such as the TR34/L98H mutation,is considered to be a natural resistance mechanism responsible for the resistance of triazole antifungal drugs.However,a multi-country study by ARTEMIS showed that 3 of the 11 strains of itraconazole-resistant A.fumigatus strains did not detect the Cyp51A gene mutation.It can be seen that the mutation of A.fumigatus Cyp51A gene is not the only mechanism for its resistance to triazole antifungal drugs.Studies have found that Heat shock protein 90(Hsp90)is essential for the survival of eukaryotes and plays a role in fungal resistance.Hsp90 is accompanied by calcineurin(CaN)to form a signal transduction network that is required for the formation and maintenance of drug resistance.Hsp90 inhibitors or calcineurin inhibitors block the formation of these signaling networks,prevent the developmentof drug resistance,reduce the virulence of A.fumigatus strains,and eliminate them even if they are resistant.The Hsp90 inhibitor geldanamycin is most commonly used for antitumor therapy,but its widespread use has been limited due to its toxic side effects.Its derivative,17-allylamino-17-desmethoxygeldanamycin(17-AAG),has lower hepatotoxie side effects and stronger antitumor activity.However,the antifungal activity of 17-AAG against triazole-resistant A.fumigatus strains is still unclear.In vitro drug sensitivity testing of Aspergillus has not been routinely carried out in various hospitals,and there is still a lack of in vitro drug sensitivity data for A.fumigatus clinical isolates in this region.Therefore,the main purpose of this study is to:1.Collect clinical isolates of A.fumigatus in this region,test their in vitro drug susceptibility to triazole antifungal drugs,screen resistant strains of A.fumigatus,and explore the mechanism of drug resistance.2.The antifungal activity of 17-AAG against triazole-resistant A.fumigatus clinical strains was tested by in vitro and in vivo experiments,and the value of 17-AAG in the application of triazole-resistant A.fumigatus strains was determined.Part Ⅰ Screening of triazole drug resistance in clinical isolates of Aspergillus fumigatus and preliminary study on its drug resistance mechanismObjective:To detect the in vitro sensitivity of clinical isolates of A.fumigatus to triazole antifungal drugs,and explore the mutation of Cyp51A gene in resistant strains of A.fumigatus.Materials and Methods:The clinical isolates of A.fumigatus were collected from five third-grade hospitals in Nanjing and Changzhou from August 2012 to July 2015.All A.fumigatus strains were confirmed by colony morphology,microscopic characteristics or molecular biological identification,and stored at 10%glycerol-80℃.The in vitro susceptibility of A.fumigatus strains to itraconazole,voriconazole and posaconazole was tested according to the European Antimicrobial Drugs Committee(EUCAST)microdilution assay.The clinical data of patients with triazole-resistant A.fumigatus strains were retrospectively analyzed.The resistant A.fumigatus strain Cyp51A was extracted and amplified.The Cyp51A gene was sequenced by the ABI 3730XL DNA sequencer and compared with the standard sequence of GenBank AF.338659 A.fumigatus Cyp51A gene to confirm its mutation.Results:A total of 126 strains of A.fumigatus clinical isolates were collected and confirmed by identification.Four strains of A.fumigatus strains(3.17%)were detected to be resistant to itraconazole and the minimum inhibitory concentration(MIC)was 16mg/L;one of the itraconazole resistant A.fumigatus strains(0.8%)was resistant to both voriconazole and posaconazole,and the minimum inhibitory concentrations were 4mg/L and 0.5mg/L,respectively.Among the four strains of triazole-resistant A.fumigatus,two strains of Cyp51A gene had TR34/L98H mutation,one strain had M220I mutation,and one strain did not detect Cyp51A gene mutation.Of the 4 patients infected with the triazole-resistant A.fumigatus strain,3 patients died and 1 patient survived with a mortality rate as high as 75%.Conclusion:There are clinical isolates of triazole-resistant A.fumigatus in this region,and the resistance rate of itraconazole is higher than that of voriconazole and posaconazole.The detection rate of drug-resistant A.fumigatus clinical isolates is still lower than other countries.The A.fumigatus Cyp51A gene mutation is not the only mechanism for its resistance to triazole antifungal drugs.The emergence of triazole-resistant A.fumigatus clinical isolates poses new challenges for the treatment of invasive pulmonary aspergillosis.Part Ⅱ Effect of 17-AAG on Antifungal Activity of Triazole-resistant Aspergillus fumigatus Clinical StrainObjective:To detect the antifungal activity of 17-AAG against triazole-resistant A.fumigatus clinical strains in vitro and in vivo,and to clarify the value of 17-AAG in the application of triazole-resistant A.fumigatus strains.Materials and Methods:The minimum inhibitory concentration(MIC)or minimum effective concentration(MEC)of four triazole-resistant A.fumigatus isolates on itraconazole,voriconazole,posaconazole,17-AAG,CsA and FK506 in the first part was detected by EUCAST method.Fractional inhibitory concentration(FIC)of itraconazole,voriconazole or posaconazole and 17-AAG,CsA or FK506 was detected by the checkerboard method.A rat model of invasive pulmonary aspergillosis infected with triazole-resistant A.fumigatus was constructed and treated with voriconazole,17-AAG and FK506 alone or in combination.The survival of the first day to the 15th day after infection was observed.The GM test on the 3rd and 6th day after infection was detected.The lung fungal burden of rats was tested and the histopathology was analyzed.Results:The minimum inhibitory concentrations of four strains of triazole-resistant A.fumigatus to itraconazole,voriconazole and posaconazole were 16 mg/L,0.5～2 mg/L and 0,25～2mg/L,respectively;for 17-AAG The minimum effective concentration ranges of CsA and FK506de were 8 to 32 mg/L,8 to 32mg/L,and 0.125 to 0.5mg/L,respectively.Except for one strains of A.fumigatus which showed indifferent in itraconazole combined with CsA,the fractional inhibitory concentrations of itraconazole,voriconazole or posaconazole with 17-AAG,CsA or FK506 were all synergistic or cumulative.The survival time of rats in group D(voriconazole+17-AAG 20mg/kg)was longer than that in group A(voriconazole)(P=0.044)and group E(voriconazole+17-AAG 60mg/kg)(P=0.011).The survival time of rats in group H(voriconazole+FK506 30ng/kg)and group I(voriconazole+FK506 90ng/kg)was not statistically different compared to group A(voriconazole)(P=0.402 and P=0.267).The GM test value of group D(voriconazole+17-AAG 20mg/kg)on day 6 was significantly lower than that of group B(17-AAG 20mg/kg)and group C(17-AAG 60mg/kg)(P<0.05).However,there was no significant difference compared with group A(voriconazole)(P=0.431).The lung fungal burden of rats in group D(voriconazole+17-AAG 20mg/kg)was significantly lower than that in group A(voriconazole)(P=0.032),group B(17-AAG 20 mg/kg)(P=0.019)and group C(17-AAG 60mg/kg)(P=0.011),but there was no significant difference compared with group E(voriconazole+17-AAG 60 mg/kg)(P=0.109).Pathological sections of group D(voriconazole +17-AAG 20mg/kg)and group A(voriconazole)showed alveolar integrity and neutrophil infiltration,with few to moderate amounts of A.fumigatus spores.Pathological sections of the lungs of group H(voriconazole+FK506 30ng/kg)and group J(PBS)showed disappearance of alveolar structure,exudation of bronchioles and perivascular edema,and a large number of A.fumigatus spores.Conclusion:17-AAG,CsA and FK506 have good antifungal activity against triazole-resistant A.strains in vitro,and the combination of antifungal drugs can enhance the antifungal activity.Low-dose 17-AAG(20mg/kg)combined with voriconazole could significantly prolong the survival time,reduce the blood GM test and the lung fungal burden of rats with azole-resistant invasive pulmonary aspergillosis.Lung histopathology was positively correlated with the severity of lung lesions in IPA rats.Low-dose 17-AAG(20mg/kg)alone,high-dose 17-AAG(60mg/kg)and FK506 alone or in combination with voriconazole could not effectively improve the survival time of IPA rats.Although 17-AAG provides a new idea for the treatment of triazole-resistant A.fumigatus,its safety and effectiveness still need to be further explored. |
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