invasive fungal infections ( ifd ) are increasingly recognized and represent a primary cause of morbidity and mortality in critically ill patients [ 1 - 4 ] . a variety of factors , including immunosuppressive agents , broad - spectrum antibiotics , and antineoplastic agents influence the incidence and severity of ifds .
transplant and haematopoietic stem cell transplant recipients , intensive care unit and surgical patients display the population at risk [ 1,4 - 7 ] .
the introduction of voriconazole , posaconazole and echinocandins ( caspofungin , micafungin and anidulafungin ) improved the therapeutic option for treatment of invasive aspergillosis ( ia ) .
although the outcome of ia is largely influenced by the state of immunosuppression , factors related to the fungus also play a role . until recently ,
species identification was sufficient to guide antifungal therapy , but the emergence of acquired resistance limits the use of species identification for predicting activity of antifungal agents .
aspergilli , less susceptible to antifungals emerged and acquired resistance to azoles have been found mainly in aspergillus fumigatus ; this has launched a new era in handling aspergillosis .
this article reviews the epidemiology and antifungal resistance against azoles and candins with particular emphasis on aspergillus species .
in a 4-year - study pagano et al .   showed that 64% of ifds in patients with haematological malignancies were caused by moulds and among them 90% were due to aspergillus species .
overall , the incidence of ia varies according to underlying diseases , pathogen [ lf ] and geographic location ; rates of up to 7% are reported in europe .
mortality rates for ia are high and vary according to patient population , ranging from 38% in patients with acute myelogenous leukaemia , from 50 - 60% in patients with organ transplantation and from 70 - 85% in other immunosuppressed patients [ 11 - 16 ] .
although a. fumigatus still represents the leading cause of ia , species like aspergillus terreus and aspergillus flavus become more frequent . these non - a .
fumigatus may be intrinsically resistant to antifungal agents ( eg a. ustus )   and the clinical presentation and evolution of ia may differ from commonly observed a. fumigatus infections [ 18 - 23 ] . maximizing the efforts
the mic represents the lowest drug concentration that results in a notably reduction or complete lack of fungal growth . in vitro resistance
secondary resistance is generated following exposure to an antifungal and may be associated with an altered gene expression .
clinical resistance is defined as the failure to eradicate an infection despite the administration of an adequate antifungal .
such failures can be attributed to a combination of the host , the pathogen and the drug . using the european committee for antibiotic susceptibility testingg ( eucast ) methodology breakpoints
were recently proposed for a .fumigatus and itraconazole , voriconazole and posaconazole ; for itraconazole and voriconazole , < 2 mg / l ( susceptible ) , 2 mg / l ( intermediate ) and > 2 mg / l ( resistant ) ; for posaconazole , < 0.25 , 0.5 and > 0.5 mg / l respectively .
it is suggested to differentiate between single - azole , pan - azole and multi - azole resistance ( see table 1 ) , the majority of infections is associated with clinical failure when treated with the ascertained agent . azole resistance in a. fumigatus   mics = minimal inhibitory concentrations
yet reports from the netherlands and manchester display an alarmingg increase of azole resistance in a. fumzgatus since 1998 .
the first published case of itraconazole - resistance in a. fumigatus appeared in 1997 ; in 2000 , a survey testing over 900 isolates showed a 2% prevalence of itc resistance in manchester . in 2007 the percentage of patients with an azole - resistant a. fumigatus increased up to 15% . in the netherlands azole resistance
increased dramatically from 2.5% in 2000 , to 4.9% in 2002 , to 6.6% in 2004 and to 10% in 2009 .
this represents an increasing frequency of 6% per year and is an issue due to the limited number of antifungals .
overall , azole - resistance differs from country to country and occurred sporadically in belgium , denmark , france , sweden , spain and norway [ 33 - 39 ] . in spain ,
the prevalence is about 2% among clinical a. fumigatus and in austria about 0% . in the usa ,
species with mics of voriconazole and posaconazole > 2 mg / l remain rare , less than 1% .
the clinical presentation and disease evolution may be related to the underlying genotypes in a. fumigatus ( table 2 ) .
clinical overview of azole resistance in a. fumigatus   abpa = allergic bronchopulmonary aspergillosis azole drug resistance in a. fumigatus has been reported both , before and after drug exposure ; acquired resistance appears to develop through treatment of patients or through exposure of isolates to azole fungicides in the environment .
these findings have major implications for clinical practice especially as fungal drug resistance is an acute issue due to the limited number of antifungal compounds .
experts expect that triazole resistance in this haploid , sparingly sexual worldwide airborne fungus will increase .
key elements in the management of patients will be an accurate speciation of aspergillus species and the performance of in vitro susceptibility testing for an approbiate antifungal treatment .
presently we do not have exact data on the prevalence of azole - resistance aspergilli in germany , but seems to be rather low than high .
where invasive candidiasis was once the predominant type of invasive fungal infections , invasive mould infections have become increasingly important , including those caused by unusual pathogens .
aspergillus species are the most frequent mould pathogens , but the number of infections caused by previously rare pathogens , such as the zygomycetes and fusarium species , is increasing .
the reasons for the shift in the epidemiology are multifactorial , but are a result , at least in part , of the increased use of extensive voriconazole and echinocandins as prophylaxis / treatment . as a result of growing numbers of immune - suppressed patients with risk factors , the patient populations for ia will expand and will include patients with haematological malignancy , icu intervention , pulmonary disease [ e.g.chronic obstructive pulmonary disease ( copd ) and asthma ] , sot recipients and patients with solid tumours .
much less is currently known about echinocandin resistance in aspergillus , in part because susceptibility testing is not routinely performed and because the methods suffer from technical difficulties and suboptimal reproducibility ; breakthrough infections with a. fumigatus showing high minimum effective concentrations have been reported sporadically .
so far , the selection pressure of candins has risen and the development of resistance is presumed to be inevitable .
these antifungals bind to lanosterol 14--demethylase ( 14--dm , or cyp51p ) which is encoded by the erg11 genes .
such step leads to depletion of ergosterol and an accumulation of lanosterol and other toxic 14--methylated sterols .
several pathomechanisms account for azole resistance in a. fumigatus ; these include a modification of target enzymes , an increased expression of drug efflux mechanisms , an overexpression of target enzymes , an incorporation of exogenous cholesterol , an overexpression of hsp90 and of a sterole - regulatory element binding protein .
the resistance phenotype depends on the amino - acid substitution and more than one azole can be affected .
azole - resistant isolates have been reported as multidrug resistant , multiazole resistant , azole cross - resistant   and multiple - triazole resistant   isolates . in most cases
azole resistance has been associated with point mutations in cyp51a , which represents the target enzyme of the azoles ; hot spots at codons 54 , 98 and 220 are most frequently characterized [ 26,33,35,48 - 50 ] .
interestingly , other mutations have been found in azole susceptible strains and so are unlikely to be associated with resistance .
the resistance mechanisms differ between the dutch and british azole - resistant isolates ; in the netherlands , the presence of a single resistance mechanism ( denoted by tr / l98h , a point mutation at codon 98 accompanied by a tandem repeat in the promoter region ) , was found in over 90% of clinical a. fumigatus isolates .
by contrast , several cyp51a mutations are present in the uk strains   and no prevalence of any one alteration
. the reasons for this might be due to differences in the patient population from which the isolates originate .
azole - resistance may develop due to exposure of a. fumigatus to azole fungicides for plant protection .
howard et al . suggest that the reasons of the widespread increase of azole - resistance in the uk may be part of long - term azole drug exposures in patients .
echinocandins are a unique class which block the -(1,3)-d - glucan synthesis by inhibiting - ( 1,3)-d - glucan synthase .
much less is currently known about echinocandin resistance in aspergillus and only few clinical isolates associated with treatment failures have been investigated . in such isolate mutation in the fks1 target gene
was not detected , but expression of the fks1 gene was found to be upregulated . manipulated or laboratory - selected strains with various degrees of caspofungin resistance have been described .
some of these strains have been found to have mutations in the ecm33 gene ( afuecm33 ) , encoding cell wall proteins .
strains with mutations in the fks1 gene encoding a subunit of the -1,3-d - glucan synthase enzyme have been generated .
in other resistant aspergillus mutants the glucan synthase exhibited a wild - type affks1 gene sequence , where the function , level , and the enzyme itself were susceptible to caspofungin .
for example , aspergillus niger is much more susceptible to echinocandins than other species probably in charge of its different cell - wall composition .
aspergillus lentulus isolates are less susceptible to caspofungin , although they maintain susceptibility to anidulafungin and micafungin .
the analysis of the a. lentulus fks sequence did not reveal a polymorphism at any of the known hot - spot regions of the gene .
cross - resistance patterns are closely linked with the position of the mutation in the cyp51a gene .
isolates with mutations at codon 54 remain voriconazole susceptible although cross - resistant to posaconazole .
cross - resistance patterns in isolates with m220 alterations appear to be unpredictable , particularly with respect to voriconazole .
the risk of cross - resistance between the azole compounds is high , in one report 74% and 65% of itraconazole resistant isolates were cross - resistant to posaconazole and voriconazole respectively . between itraconazole , voriconazole and ravuconazole cross - resistance
was demonstrated in 10 clinical isolates of a. fumigatus obtained from patients with long - term exposure to itraconazole or voriconazole .
also , broad - spectrum cross - resistance among all the azoles has been shown in a. fumigatus in a patient receiving prolonged itraconazole prophylaxis .
overall , there is a limited number of reported cases that help us to understand the clinical impact of azole resistance on clinical outcome .
for example , in a small case series of patients with ia and no respond to voriconazole , treatment with posaconazole was successful in 50% of infections . on the other hand in animal model of ia caused by an itraconazole - resistant a. fumigatus strain ,
the potential frequency of cross - resistance amongst echinocandins in aspergillus species is still unclear and has not been investigated in detail . at present , there is no evidence that the activity and efficacy of other antifungal compounds , such as the polyenes and echinocandins , is attenuated in azole - resistant isolates .
 aspergilli , less susceptible to antifungals emerge and resistance to azoles have been found mainly in aspergillus fumigatus ; this has launched a new phase in handling aspergillosis . 
 resistant strains have currently been reported from belgium , canada , china , denmark , france , norway , spain , sweden , the netherlands , uk and the usa . 
 centres in the uk ( manchester ) and the netherlands ( nijmegen ) have described particularly high frequencies ( 15 and 10% respectively ) , and a significant increase in azole resistance in recent years . 
 the reason of this high incidence may be due to long term azole therapy in patients with chronic aspergillosis in manchester , and due to high use of agricultural azoles in nijmegen . 
 the primary underlying mechanism of resistance is as a result of alterations in the cyp51a target gene , with a variety of mutations found in clinical isolates and one genotype identified in the environmental ( lh98 ) . 
 reports on well documented in vitro and in vivo resistance to echinocandins are rare for aspergillus species and resistance may be under - diagnosed as susceptibility testing is less frequently performed due to technical reasons . 
Introduction
Epidemiology of invasive aspergillosis
Epidemiology of azole and candine resistance in clinical 
Mechanism of azole-resistance in 
Mechanism of echinocandin-resistance in 
Cross resistance among azoles and echinocandins