Aspergillus Surveillance And Molecular Epidemiology Of Environment And Patients In Transplantation Department And Intensive Care Unit

Background and ObjectivesAspergillus is ubiquitous in the environment and capable of causing allergic, aspergilloma and invasive aspergillosis(IA) in immunocompromised patients.The incidence of life-threatening invasive aspergillus infection has been increasing with bone or organ transplant,the using of high-dose corticosteroids or immunosuppressive therapies etc. Although the immune status of host is thought to be the major contributor to the establishment of infection,air Aspergillus load and its fluctuations in the hospital environment are expected indirectly to influence the incidence of hospital-acquired Aspergillus infection,as well as the predominant specie.Much of our current understanding about the transmission of IA is based on information gathered from outbreak investigations. Nosocomial outbreaks of IA have become a well-recognized complication of construction or renovation work in or near hospital wards in which high-risk patients are housed.These reports have highlighted the fact that hospital air is often contaminated with Aspergillus spores,and they have contributed to the current perception that most cases of IA in immunocompromised persons are hospital acquired.Invasive Aspergillus infections are hard to diagnose at an early state because their clinical presentation is usually non-specific. The traditional diagnostic evidence derived from mycological cultivation or histological demonstration of fungi within tissue samples is difficult to obtain.Despite appropriate antifungal therapy,the prognosis of IA remains very poor and the overall mortality is higher than 50%.Amphotericin B is the first agent for the initial treatment of Aspergillus infection.Some data indicate that itraconazole and terbinafine are two agents licensed for treatment of Aspergillus infection.But with the resistance of Aspergillus increasing,the treatment of choice for infected patients has become outstanding problem.In order to prevent and cure the invasive Aspergillus infection,we surveillance the Aspergillus load and species distribution in environment and patients of transplantation department and intensive care unit in Southwest hospital,and the relationship with season, temperature,humidity,ventilation and personnel activities are also analyzed.Aspergillus isolated from environment and patients were genotyped to determine the origin of infection by random amplification of polymorphic DNA(RAPD).We also investigate the activities of amphotericin B(AraB),itraconazole(ICZ),terbinafine(TBF),fluconazole(FCZ) and 5-flucytosine(5-FC) alone and interaction in combination against Aspergillus and analyze the relationship of genotype and antifungal susceptibility.Methods and Results1.Air,surfaces and tap water were sampled twice a month from liver transplantation department(LTD),cerebral surgery intensive care unit(CSICU) and central intensive care unit(CICU) using LWC-1 air-samplers.The results showed that air fungal load was 123.63cfu/m~3,139.90cfu/m~3,7cfu/m~3 and 214cfu/m~3 in LTD,CSICU,CICU and outdoor respectively.Air fungal load was higher in CSICU and LTD than CICU(P＜0.01).The five most prevalent fungi collected from air and surface was Penicillium spp.,Cladospcrium spp.,Alternaria spp.,Aspergillus spp.and Saccharomyces spp.in turn.The fungal load in CSICU and LTD was correlated with the average temperature and the average humidity,but the correlation between air fungal load and personnel activities weren't observed.The fungal load of surfaces was 0.181cfu/cm~2,0.110cfu/cm~2 and 0.211cfu/cm~2 in LTD, CSICU and CICU respectively.The difference of fungal contamination between different departments was not statistically significant(P＞0.05).The fungal load was 0.035cfu/cm~2, 0.145cfu/cm~2,0.156cfu/cm~2 and 0.706cfu/cm~2 at treatment unit,tap,air conditioning vent and inlet respectively.The fungal load of air conditioning vent was higher than other surfaces and tap was lower than other surfaces.The fungal load of water was 2.gcfu/500ml, 2.86cfu/500ml and 2.94cfu/500ml in LTD,CSICU and CICU respectively.The five most prevalent fungi collected from water were Saccharomyces spp.,Candida spp.,Aspergillus spp.,Penicillium spp.and Rhodotorula spp.in turn.2.Air,surfaces and tap water were sampled twice a month from LTD;CSICU and CICU.Clinic sample of nose,pharynx and sputum collected from patients.The results showed that mean total Aspergillus was 12cfu/m~3,10.75cfu/m~3,0cfu/m~3 and 20cfu/m~3 at LTD,CSICU,CICU and outdoor respectively.Air fungal load was lower in CICU than others(P＜0.05).The five most prevalent Aspergillus species collected inside the hospital were Aspergillus flavus,Aspergillus fumigatus,Aspergillus niger,Aspergillus versicolor and Aspergillus clavatus.The fungal loads in CSICU and LTD were correlated with personnel activities,but the the correlation between air fungal load and humidity or temperature weren't observed.The Aspergillus loads of surfaces were 0.02cfu/cm~2,0.010cfu/cm~2 and 0.037cfu/cm~2 in LTD,CSICU and CICU respectively.The Aspergillus load of surfaces was higher in CICU than in others(P＜0.05).The Aspergillus loads were 0.006cfu/cm~2,0.013cfu/cm~2, 0.013cfu/cm~2 and 0.122cfu/cm~2 at treatment unit,trap,air conditioning vent and inlet respectively.The Aspergillus loads of air conditioning vent was higher than other surfaces and tap were lower than other surfaces.The Aspergillus loads of water were 0.68cfu/500ml, 0.5cfu/500ml and 0.34cfu/500ml in LTD,CSICU and CICU respectively.The most prevalent fungi collected from water were Aspergillus flavus,Aspergillus niger,Aspergillus fumigatus and Aspergillus versicolor in turn.Thirty-three Aspergillus flavus and three Aspergillus fumigatus species were isolated from nose,pharynx and sputum of five patients.Aspergillus flavus isolated from environment and patients were genotyped to determine the origin of infection by random amplification of polymorphic DNA(RAPD).RAPD analysis demonstrated that strains isolated from patients in CSICU were identical to environmental strain.Strains isolated from patients in ICU were different to environment strain,but strains isolated from two patients were identical.3.NCCLS M38-A protocol was employed to investigate the activities of amphotericin B(AraB),itraconazole(ICZ),terbinafine(TBF),fluconazole(FCZ) and 5-flucytosine (5-FC) alone and interaction in combination against Aspergillus.In test alone,the MIC endpoints were determine of AraB,TBF,ICZ as 100%growth reduction compared with turbidity produced by the control wall,FCZ and 5-FC were as≥50%growth reduction,but 100%growth reduction when they were given in combination.Drug interactions were classified on the basis of the fractional inhibitory concentration(FIC) index.The interaction was defined as synergic if the FIC index was 0.50,additive if the FIC index was＞0.50～1.0, indifferent if the FIC index was＞1.0～2.0 and antagonistic if the FIC index was＞2.0.The results showed that MIC of TBF was highest for Aspergillus fumigatus(1.578μg/ml).MIC of ICZ was lowest for Aspergillus flavus(0.104μg/ml).MIC of AraB was lowest for Aspergillus niger(0.094μg/ml) and highest for Aspergillusflavus(1.809μg/ml).MIC of FCZ and 5-FC were much higher than other drug for Aspergillus(25.77μg/ml,3.1μg/ml respectively).Much of clinic isolates were resistant to FCZ,ICZ,AmB and 5-FC.The combination of ICZ-TBF and ICZ-AmB were synergic or additive against most Aspergillus (FIC=0.13～2.5,FIC=0.25～3 respectively).The combination of TBF and AmB were additive against most Aspergillus(FIC=0.28～4.99).The combination of FCZ and 5-FC with other drugs were antagonistic or indifferent(FIC=0.28～16.7,FIC=0.25～16 respectively).The relationship between genotype of Aspergillus and its sensitivity to antifungal drugs was analysed by dendrogram according to RAPD patterns.The results showed that there were differences among sensitivity to TBF of the genotypes of Aspergillus fumigatus and to AmB of the genotypes of Aspergillus niger.But no statistically difference was found among antifungal angents of the genotypes of Aspergillusflavus.Conclusions1.It demonstrated the fungus was found in the environment of the hospital including air,surface and water.The air fungal load varies throughout the year.The crest-time was May to June and September to October.The correlation between air fungal load and temperature,humidity was observed.Air fungal load was lower in CICU and higher in CSICU.2.Aspergillus contamination was found in LTD,CSICU and CICU.Clinic and environmental strain from CSICU have identical genotype;the infection may be from hospital environment.3.The sensitivity of antifungal agents were different when test alone to different species of Aspergillus.The combination of ICZ-TBF and ICZ-AmB in vitro displayed a potent synergic or additive against most Aspergillus compared with the combination of other drugs.The correlation between sensitivity to antifungal drugs and genotypes of Aspergillus was observed.