REVIEW
10.1111/1469-0691.12513
Update on antifungal resistance in Aspergillus and Candida M. C. Arendrup Unit of Mycology and Parasitology, Department Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark
Abstract Antifungal resistance in Candida and Aspergillus may be either intrinsic or acquired and may be encountered in the antifungal drug exposed but also the antifungal drug-na€ıve patient. Prior antifungal treatment confers a selection pressure and notoriously raises the awareness of possible resistance in patients failing therapy, thus calling for susceptibility testing. On the contrary, antifungal resistance in the drug-na€ıve patient is less expected and therefore more challenging. This is particularly true when it concerns pathogens with acquired resistance which cannot be predicted from the species identification itself. This scenario is particularly relevant for A. fumigatus infections due to the increasing prevalence of azole-resistant isolates in the environment. For Candida, infections resistance is most common in the context of increasing prevalence of species with intrinsic resistance. Candida glabrata which has intrinsically reduced susceptibility to fluconazole is increasingly common particularly among the adult and elderly population on the Northern Hemisphere where it may be responsible for as many as 30% of the blood stream infections in population-based surveillance programmes. Candida parapsilosis is prevalent in the paediatric setting, at centres with increasing echinocandin use and at the southern or pacific parts of the world. In the following, the prevalence and drivers of intrinsic and acquired resistance in Aspergillus and Candida will be reviewed. Keywords: Acquired resistance, Aspergillus, azoles, Candida, echinocandins, intrinsic resistance, susceptibility testing Article published online: 21 December 2013 Clin Microbiol Infect 2014; 20 (Suppl. 6): 42–48
Corresponding author: M. C. Arendrup, Unit of Mycology, Dept. Microbiology and Infection Control, Statens Serum Institut, Building 43/317, Copenhagen, Denmark E-mail: [email protected] Invited review for Clin Microbiol Infection: based on a lecture given at the ESCMID conference Invasive Fungal Infections, Rome Jan 1-18, 2013.
Introduction Resistance in Aspergillus and Candida has been increasingly investigated and reported because standards for susceptibility testing and associated breakpoints became available and as a consequence of the increased use of antifungal compounds [1–4]. Resistant infection can be encountered in the antifungal drug-exposed patient due to selection of intrinsically resistant species or isolates with acquired resistance belonging to species that are normally susceptible. In both cases, resistance may be expected as any antimicrobial therapy is associated with a selection pressure and therefore risk of resistance.
Resistance can, however, also be encountered in the antifungal drug-na€ıve patient and, again, can be due either to infection with intrinsically resistant species or to isolates with acquired resistance. Whereas resistance due to intrinsically resistant species can be diagnosed through correct species identification, detection of isolates with acquired resistance is more demanding and requires appropriate and carefully performed susceptibility testing and endpoint interpretation [5]. New tools for rapid species identification of Candida species including the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), fluorescence in situ hybridization (FISH), etc. have improved correct species identification at clinical microbiological routine
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases
CMI
Arendrup
laboratories. However, various challenges are still associated with performance and interpretation of susceptibility testing even though commercial tests are now available for most antifungal compounds and clinical breakpoints have been established [1,2,6–11]. Firstly, performance requires expertise to obtain reproducible results, second the standardization of commercial methods against the reference test is not perfect for all drug–bug combinations leading to misclassification of susceptibility test results when reference breakpoints are adopted, and finally for Aspergillus, such tests are not routinely performed. Hence, the greatest diagnostic challenges related to resistant infections lay with the correct and timely detection of infections due to isolates with acquired resistance and particularly so in the drug-na€ıve patients where, historically, resistance has not been expected.
Resistance in Aspergillus From an epidemiological point of view, less is known about the true prevalence of resistant Aspergillus infections than about resistant infections for most other organisms. This is due to the fact that most routine laboratories do not susceptibility test their Aspergillus isolates and many laboratories find species (or even genus) identification of aspergilli difficult. Additionally, national surveillance programmes are lacking. Intrinsic resistance
Aspergillus fumigatus is by far the most common species causing human infection. The wild-type isolates are susceptible to all the licensed mould active azoles (Table 1) and echinocandins [3,4,12]. However, the A. fumigatus species complex includes more than 30 sibling species which cannot be differentiated morphologically from one another or from A. fumigatus. Several of these have been isolates from humans and been shown to be intrinsically resistant to one or more antifungals (Table 2) [12–15]. Notably, however, a simple temperature
Antifungal resistance in Aspergillus and Candida
43
TABLE 2. Intrinsic resistance (R) and variable (V) susceptibility in Aspergillus
Aspergillus section fumigati A. fumigatiaffinis A. lentulus N. pseudofischeri A. viridinutans N. udagawae A. terreus (and A. alabamensis) A. flavus A. versicolor (and A. sydowi) A. calidoustus A. allilaceus
AMB
Azoles
R R V R R R R R
R R R R R (vor) R V R
V
Echinocandins
V
V V
Data compiled from [12–15]. Vor, Voriconazole.
tolerance test (growth at temperatures higher than 48°C) can discriminate between A. fumigatus and the sibling species. Intrinsic amphotericin B resistance has been recognized in A. terreus for many decades, but also A. flavus and other less common species have reduced susceptibility to amphotericin B [3]. Importantly, a number of these rare Aspergillus species are also resistant to azoles and in some cases also to echinocandins posing obvious challenges for patient management (Tables 1 and 2). Hence, careful species identification is mandatory for clinically important isolates. Acquired resistance in AF-na€ıve patients
Azole-resistant A. fumigatus infections have been reported in a number of countries. Most reports derive from Europe and involve a single molecular resistance mechanism consisting of a 34-bp tandem repeat TR34 in the promotor region of the azole target CYP51A gene and a point mutation in the target gene itself leading to an L98H amino acid substitution (TR34/L98H). This resistance mechanisms has been detected in isolates deriving from Austria, Belgium, Denmark, Germany, France, the Netherlands, Norway, Spain, Sweden and the UK [16–23] and outside Europe in China, India and Iran [24,25] (Verweij, personal communication). Notably, 61% of the global market share of agricultural fungicides is used in these Western
TABLE 1. Intrinsic susceptibility pattern and epidemiological cut-off values (ECOFFs) for Aspergillus species and mould active azoles. Data compiled from [3,4,12] Aspergillus (S/I/R (ECOFF))a calidoustus Amphotericin B Itraconazole Posaconazole Voriconazole
IE R R R
(NDb) (NDb) (NDb) (NDb)
flavus
fumigatus
nidulans
niger
terreus
versicolor
IE S IE IE
S S S S
NDc S (1 mg/L) IE (0.5 mg/L) S (1 mg/L)
S IE IE IE
R S S IE
NDb NDb NDb NDc
(4 mg/L) (1 mg/L) (0.5 mg/L) (2 mg/L)
(1 mg/L) (1 mg/L) (0.25 mg/L) (1 mg/L)
(1 mg/L) (4 mg/L) (0.5 mg/L) (2 mg/L)
(4 mg/L) (0.5 mg/L) (0.25 mg/L) (2 mg/L)
ECOFF: epidemiological cut-off values; IE: insufficient evidence to suggest whether this species is a good target for the compound in question. MICs are higher than for Aspergillus fumigatus, but there is insufficient clinical data to suggest whether this translates into poorer efficacy; ND: not done due to insufficient amount of data for ECOFF setting (low number of MIC values or data set). a S/I/R: intrinsically susceptible/intermediate/resistant. b MIC range higher than that for A. fumigatus. c MIC range similar to that for A. fumigatus.
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
44
Clinical Microbiology and Infection, Volume 20 Supplement 6, June 2014
European and Asia Pacific regions, and thus, the prevalence of TR34/L98H correlates with the geographical use of these agents [23]. Two additional resistant genotypes have been demonstrated among isolates from the European environment or from azole-na€ıve patients, namely isolates harbouring the TR46/Y121F/T289A or the G432S alterations [26,27]. Alterations involving tandem repeats in the promotor region of CYP51A and amino acid changes at codons Y121, T289 and G432 have similarly been found in azole pesticide-resistant isolates of the common plant pathogen Mycosphaerella graminicola, suggesting these codons are hot spots for possible selection via the agricultural azoles [23]. The true prevalence of the environmental type resistant isolates is largely unknown. In the Netherlands, they accounted for approx. 10% in 2007, in Danish soil samples for 8% and in Danish patients approx. 4% in 2009–11 [19,28,29].
CMI
G138 and M220) found an astonishingly high rate of resistance markers (55%) in samples from patient mainly suffering from chronic forms of aspergillosis in the UK [17,45]. However, using the same technique for Danish patients at risk for acute or chronic forms of aspergillosis and undergoing bronchoscopy with galactomannan antigen determination, 3.3 and 4.3% resistance was detected by molecular and conventional susceptibility tests, respectively [46]. It is not fully understood why these two studies reported such different rates of resistance, but a reasonable and conservative estimate of the global prevalence of azole resistance in Aspergillus appears to be 3–6%. This still raises concern as resistance was a very uncommon event before the turn of the millennium and as resistance is found also in drug-na€ıve patients.
Antifungal Resistance in Candida
Acquired resistance in AF-exposed patients
A number of reports have documented the selection of azole resistance during treatment. Most reports concern long-term treatment in patients with allergic or chronic forms of aspergillosis [29–37]. The majority of isolates harbour mutations in the target gene CYP51A, but other mechanisms are important as well, including efflux pumps [38] and target gene upregulation encoded by a mutation in the hapE gene [31,39]. Acquired azole resistance has also been reported in A. terreus and A. flavus, which poses obvious challenges regarding clinical management of related infections as these two species have intrinsically reduced susceptibility to amphotericin B [40,41]. Acquired resistance to amphotericin B in Aspergillus has not been described so far, and only a single case of mechanistically characterized acquired echinocandin resistance has been reported [42]. Susceptibility testing of Aspergillus and echinocandins is, however, even more challenging than susceptibility testing of azoles and amphotericin B due to the lack of total inhibition in vitro and therefore no clear endpoint. Hence, acquired resistance to echinocandins may be underdiagnosed and indeed breakthrough cases have been described in patients receiving echinocandin therapy [43,44]. Size of the problem
A global study involving 19 countries reported overall prevalence of azole resistance of 3.2% and ranging from 0 to 25.5% among the participating centres [16]. Notably, no centres testing at least 100 isolates failed to detect resistant isolates. It is well known that culture is an insensitive diagnostic test, and hence, the molecular approach with direct detection in patient samples is attractive. A recent study using direct molecular detection of A. fumigatus and of alterations on the codons most commonly involved in azole resistance (G54, L98,
The epidemiology of candidaemia is particularly well documented in the Nordic countries due to ongoing nationwide surveillance programmes [47–50]. Such programmes provide precise figures for the burden of disease in entire populations, without centre bias, but are less sensitive with respect to the significance of acquired resistance. This is a consequence of the study designs, as epidemiological studies include only the initial isolate typically at the time of the lowest antifungal exposure. This is a sound strategy from an epidemiological point of view as otherwise the data set would be skewed due to repeated isolates. With this in mind, the following section summarizes the contemporary and available data on resistance in Candida. Intrinsic resistance
The intrinsic susceptibility pattern of the various Candida species is summarized in Table 3. Candida glabrata and C. parapsilosis represent the two most prevalent species with reduced azole and echinocandin susceptibility, respectively, and their prevalence is shown in a global perspective in Fig. 1. Candida glabrata is particularly prevalent on the Northern Hemisphere with incidence rates as high as 30% in some centres. It is significantly more common in the elderly population, whereas the opposite is true for C. parapsilosis [47]. Other drivers of selection of resistant species include prior antifungal drug exposure where choice of compound as well as dosing (suboptimal) and duration (more than a week) are important [51–53]. Finally, a recent report has suggested that various antibacterial compounds may also drive the selection of resistance. The underlying mechanism may be altered gut flora or the low-grade antifungal effect exerted by some antibacterial compounds, such as metronidazole [54]. Increasing use of echinocandins has been linked
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
CMI
Arendrup
TABLE 3. Intrinsic susceptibility pattern for Candida species. EUCAST breakpoints have been established for the common Candida species allowing classification if wild-type isolates into S, I and R categories. For the uncommon Candida species, breakpoints have not been established. For these species, an ‘X’ denotes that the MICs for the antifungal compound are elevated compared with those for Candida albicans AMB
ECS
FCO
S S
S S
S S
C. glabrata
S
S
I
C. krusei C. parapsilosis C. tropicalis
S S S
S I S
R S S
X X X
X
Common Candida species C. albicans C. dubliniensis
Uncommon Candida species C. lusitaniae C. fermentati C. guilliermondii C. metapsilosis
Comments
Closely related to C. albicans; fluconazole resistance acquired more easily Efflux pumps often induced during azole therapy
X
C. orthopsilosis C. cifferrii C. inconspicua C. humicula C. lambica C. lipolytica C. norvegensis C. palmioleophila C. rugosa C. valida S. cerevisiaea
Closely related to C. parapsilosis Closely related to C. parapsilosis
X X X X X X X X X X X
Closely related to C. glabrata
Antifungal resistance in Aspergillus and Candida
45
Various reports suggest intrinsic resistance may be emerging. Pfaller et al. reported that high-level fluconazole resistance (MIC ≥16 mg/L), and thus clearly above the susceptibility breakpoint of 2 mg/L, was rare in C. albicans and C. dubliniensis isolates (2.0% and 3.9%, respectively) but found in as many as 6.8% and 9.0% of the C. parapsilosis and C. tropicalis isolates, respectively, among a quarter of a million isolates from 41 countries [58]. Oxman et al. reported that 19% of their Candida infections involved fluconazole-resistant isolates (again with MICs ≥16 mg/L) at two tertiary cancer centres in the US and notably that in a third of these cases, resistance was acquired rather than intrinsic [59]. Such a finding questions the use of species identification alone as a driver of antifungal treatment. Finally, 13% of fluconazole-resistant C. glabrata (MIC ≥64 mg/ L) were cross-resistant to echinocandins among isolates collected in 2006–10, whereas no such isolates were found in the period 2001–4 at the same institution [60]. As stated earlier, acquired resistance is rarely reported in epidemiological surveillance programmes of candidaemia. Interestingly, notable discrepancies were found between the resistance rates among deep-seated isolates and mucosal isolates in a recent study of voriconazole susceptibility [61]. Similarly, we noted fluconazole MICs above the EUCAST susceptibility in 15% and 20% of mucosal C. albicans and C. dubliniensis isolates in Denmark (M.C. Arendrup, Personal observation). Taken together, mounting evidence suggests acquired resistance may be an emerging and underdiagnosed entity. Possible link to agricultural azole use?
ECS: echinocandins; FCO: fluconazole. Anamorphic state is C. robusta.
a
to an increasing proportion of blood stream infections due to C. parapsilosis [55,56]. It is important to be aware of the fact, however, that a considerable usage of oral antifungals takes place outside the hospital [57]. For example, more than two-thirds of the systemic azole use is prescribed in the primary healthcare sector in Denmark and presumably for rather benign conditions like toenail infection (itraconazole), vaginitis (fluconazole and itraconazole) and various skin conditions (e.g. pityriasis) [57]. As invasive infection derive from the colonizing Candida flora, this may deserve more attention with respect to antibiotic stewardship policies [57]. Acquired resistance
Acquired resistance is less common than intrinsic resistance. One of the reasons is that fungi do not exchange resistance genes via plasmids, and hence, selection of resistance takes place in the individual patient during individual antifungal use.
Whereas the link between agricultural use of azole pesticides and the emergence of azole-resistant Aspergillus has been suggested beyond reasonable doubt, it is less clear whether agricultural pesticides may also be drivers of azole resistance in Candida. Three observations, however, suggest this may be a relevant concern. The first one concerns susceptibility in Candida isolated from the surface of fruit harvested from non-organic vs. organic farms. Not surprisingly, fluconazole MICs were higher for Candida isolated from non-organic fruit (16–64 mg/L vs. 1–8 mg/L) [62]. Furthermore, in the same study, the fungal pesticide myclobutanil was shown to induce efflux pump expression in C. glabrata in vitro [62]. The second observation concerns C. tropicalis isolates with reduced susceptibility to fluconazole found in unrelated patients at various Japanese hospitals [63]. These isolates were found to share indistinguishable pulse field gel electrophoresis patterns and to belong to the same diploid sequence genotypes as C. tropicalis with reduced fluconazole susceptibility found in soil samples and to possess reduced susceptibility to agricultural pesticides [63]. Finally, a wild Brazilian
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
46
CMI
Clinical Microbiology and Infection, Volume 20 Supplement 6, June 2014
(a)
(b)
FIG. 1. Global percentages of Candida glabrata (a) and C. parapsilosis (b) among blood stream isolates. Data compiled from [57,65–70].
porcupine with documented invasive candidiasis due to a C. albicans isolate with efflux pump-mediated high-level azole resistance was recently found [64]. Although the evidence is limited and circumstantial, it cannot be excluded that the use of agricultural azoles may drive antifungal resistance in Aspergillus, but also in Candida.
Conclusion and Future Perspectives In conclusion, resistant Candida and Aspergillus infection are increasingly encountered in the antifungal drug-na€ıve patient due to increasing incidence rates of C. glabrata (particularly at the northern hemisphere), C. parapsilosis (particularly at centres with a high echinocandin use) and due to increasing proportion of environmental A. fumigatus isolates harbouring azole resistance mechanisms. In vivo selection of acquired resistance during medical treatment is increasingly reported for Candida in patients typically receiving more than 3 weeks of echinocandin therapy and in patients with chronic forms of aspergillosis on long-term azole treatment. In numbers, 20–30% of candidaemia cases today involve species with intrinsic resistance to either fluconazole or echinocandins. This is a significant change, as C. albicans (which is intrinsically susceptible to all drug classes)
typically accounted for at least 85% of the cases in the prefluconazole, pre-echinocandin era. Azole resistance in Aspergillus overall is probably around 4% but increasing. The drivers of this changing epidemiology are antifungal use in the hospitals but importantly also in the primary healthcare sector, and particularly for Aspergillus the high use for plant protection. In any case, we only see the tip of the iceberg due to challenges associated with antifungal susceptibility testing, the design of epidemiological surveillance programmes and the infrequent performance of susceptibility testing of aspergilli.
Acknowledgements This paper is based upon an oral presentation given at the Invasive Fungal Infection ESCMID conference held in Rome January 2013.
Transparency Declaration M.C. Arendrup has received research grants and acted as speaker for Astellas, Gilead, MSD and Pfizer, and has been a consultant for Gilead, MSD and Pcovery.
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
CMI
Arendrup
References 1. Arendrup MC, Cuenca-Estrella M, Lass-Fl€ orl C, Hope W. EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST). Clin Microbiol Infect 2012; 18: E246–E247. 2. Rodriquez Tudela JL, Donnelly JP et al. EUCAST technical note on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia-forming moulds. Clin Microbiol Infect 2008; 14: 982–984. 3. Arendrup MC, Cuenca-Estrella M, Lass-Flo¨rl C, Hope WW. EUCAST technical note on Aspergillus and amphotericin B, itraconazole, and posaconazole. Clin Microbiol Infect 2012; 18: E248–E250. 4. Hope WW, Cuenca-Estrella M, Lass-Flo¨rl C, Arendrup MC. EUCAST technical note on Voriconazole and Aspergillus spp. Clin Microbiol Infect 2013; 19: E278–E280. 5. Arendrup MC, Chryssanthou E, Gaustad P, Koskela M, Sandven P, Fernandez V. Diagnostics of fungal infections in the Nordic countries: we still need to improve!. Scand J Infect Dis 2007; 39: 337–343. 6. Arendrup MC, Rodriguez-Tudela JL, Lass-Flo¨rl C et al. EUCAST technical note on anidulafungin. Clin Microbiol Infect 2011; 17: E18–E20. 7. Arendrup MC, Pfaller MA. Caspofungin Etest susceptibility testing of Candida species: risk of misclassification of susceptible isolates of C. glabrata and C. krusei when adopting the revised CLSI caspofungin breakpoints. Antimicrob Agents Chemother 2012; 56: 3965–3968. 8. Astvad KM, Perlin DS, Johansen HK, Jensen RH, Arendrup MC. Evaluation of caspofungin susceptibility testing by the new Vitek 2 AST-YS06 yeast card using a unique collection of FKS wild-type and hot spot mutant isolates, Including the five most common Candida species. Antimicrob Agents Chemother 2013; 57: 177–182. 9. Arendrup MC, Cuenca-Estrella M, Lass-Flo¨rl C, Hope WW. The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST). EUCAST technical note on Aspergillus and amphotericin-B, itraconazole, and posaconazole. Clin Microbiol Infect 2012; 17: E248–E250. 10. Arendrup MC, Cuenca-Estrella M, Lass-Flo¨rl C, Hope WW. The European Committee on Antimicrobial Susceptibility Testing – Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST). EUCAST technical note on Candida and Micafungin, Anidulafungin and Fluconazole. Mycoses 2014; doi: 10.1111/myc.12170. 11. Lass-Flo¨rl C, Arendrup MC, Rodriguez-Tudela JL et al. EUCAST technical note on Amphotericin B. Clin Microbiol Infect 2011; 17: E27– E29. 12. Varga J, Houbraken J, van der Lee HAL, Verweij PE, Samson RA. Aspergillus calidoustus sp. nov., causative agent of human infections previously assigned to Aspergillus ustus. Eukaryot Cell 2008; 7: 630–638. 13. van der Linden JW, Warris A, Verweij PE. Aspergillus species intrinsically resistant to antifungal agents. Med Mycol 2011; 49 (Suppl 1): S82–S89. 14. Alcazar-Fuoli L, Mellado E, astruey-Izquierdo A, Cuenca-Estrella M, Rodriguez-Tudela JL. Aspergillus section fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob Agents Chemother 2008; 52: 1244–1251. 15. Perlin DS, Mellado E. Antifungal mechanisms of action and resistance. In: Latge JP, Steinbach WJ, eds. Aspergillus fumigatus and aspergillosis. Washington: ASM Press, 2009; 457–466. 16. van der Linden J, Arendrup MC, Verweij PE. SCARE. Interscience Conference on Antimicrobial Agents and Chemotherapy 2011; Abstr. M-490. 17. Denning DW, Park S, Lass-Flo¨rl C et al. High-frequency triazole resistance found in nonculturable Aspergillus fumigatus from lungs of patients with chronic fungal disease. Clin Infect Dis 2011; 52: 1123– 1129.
Antifungal resistance in Aspergillus and Candida
47
18. Snelders E, van der Lee HA, Kuijpers J et al. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med 2008; 5: e219. 19. Mortensen KL, Mellado E, Lass-Flo¨rl C, Rodriguez-Tudela JL, Johansen HK, Arendrup MC. Environmental study of Azole-Resistant Aspergillus fumigatus and other aspergilli in Austria, Denmark, and Spain. Antimicrob Agents Chemother 2010; 54: 4545–4549. 20. Verweij PE, Snelders E, Kema GH, Mellado E, Melchers WJ. Azole resistance in Aspergillus fumigatus: a side-effect of environmental fungicide use? Lancet Infect Dis 2009; 9: 789–795. 21. Mellado E, Garcia-Effron G, Alcazar-Fuoli L et al. A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob Agents Chemother 2007; 51: 1897–1904. 22. Rath PM, Buchheidt D, Spiess B, Arfanis E, Buer J, Steinmann J. First reported case of azole-resistant Aspergillus fumigatus due to the TR/ L98H mutation in Germany. Antimicrob Agents Chemother 2012; 56: 6060–6061. 23. Stensvold CR, Jørgensen LN, Arendrup MC. Azole-resistant invasive aspergillosis: relationship to agriculture. Curr Fungal Infect Rep 2012; 6: 178–191. 24. Lockhart SR, Frade JP, Etienne KA, Pfaller MA, Diekema DJ, Balajee SA. Azole resistance in Aspergillus fumigatus isolates from the ARTEMIS global surveillance study is primarily due to the TR/L98H mutation in the cyp51A gene. Antimicrob Agents Chemother 2011; 55: 4465–4468. 25. Chowdhary A, Kathuria S, Randhawa HS, Gaur SN, Klaassen CH, Meis JF. Isolation of multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR/L98H mutations in the cyp51A gene in India. J Antimicrob Chemother 2012; 67: 362–366. 26. Kuipers S, Bruggemann RJ, de Sevaux RG et al. Failure of posaconazole therapy in a renal transplant patient with invasive aspergillosis due to Aspergillus fumigatus with attenuated susceptibility to posaconazole. Antimicrob Agents Chemother 2011; 55: 3564–3566. 27. Alanio A, Sitterle E, Liance M et al. Low prevalence of resistance to azoles in Aspergillus fumigatus in a French cohort of patients treated for haematological malignancies. J Antimicrob Chemother 2011; 66: 371–374. 28. Verweij PE, Howard SJ, Melchers WJ, Denning DW. Azole-resistance in Aspergillus: proposed nomenclature and breakpoints. Drug Resist Updat 2009; 12: 141–147. 29. Mortensen KL, Jensen RH, Johansen HK et al. Aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: a laboratory-based study with focus on Aspergillus fumigatus azole resistance. J Clin Microbiol 2011; 49: 2243–2251. 30. Morio F, Aubin GG, Danner-Boucher I et al. High prevalence of triazole resistance in Aspergillus fumigatus, especially mediated by TR/ L98H, in a French cohort of patients with cystic fibrosis. J Antimicrob Chemother 2012; 67: 1870–1873. 31. Arendrup MC, Mavridou E, Mortensen KL et al. Development of azole resistance in Aspergillus fumigatus during azole therapy associated with change in virulence. PLoS ONE 2010; 5: e10080. 32. Howard SJ, Cerar D, Anderson MJ et al. Azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis 2009; 15: 1068–1076. 33. Howard SJ, Webster I, Moore CB et al. Multi-azole resistance in Aspergillus fumigatus. Int J Antimicrob Agents 2006; 28: 450–453. 34. Howard SJ, Pasqualotto AC, Denning DW. Azole resistance in allergic bronchopulmonary aspergillosis and Aspergillus bronchitis. Clin Microbiol Infect 2010; 16: 683–688. 35. Bueid A, Howard SJ, Moore CB et al. Azole antifungal resistance in Aspergillus fumigatus: 2008 and 2009. J Antimicrob Chemother 2010; 65: 2116–2118. 36. Camps SMT, van der Linden JWM, Li Y et al. Rapid induction of multiple resistance mechanisms in Aspergillus fumigatus during azole
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
48
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47. 48.
49.
50.
51.
52.
53.
54.
Clinical Microbiology and Infection, Volume 20 Supplement 6, June 2014
therapy: a case study and review of the literature. Antimicrob Agents Chemother 2012; 56: 10–16. Chen J, Li H, Li R, Bu D, Wan Z. Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially isolated from a patient with lung aspergilloma. J Antimicrob Chemother 2005; 55: 31–37. Slaven JW, Anderson MJ, Sanglard D et al. Increased expression of a novel Aspergillus fumigatus ABC transporter gene, atrF, in the presence of itraconazole in an itraconazole resistant clinical isolate. Fungal Genet Biol 2002; 36: 199–206. Camps SMT, Dutilh BE, Arendrup MC et al. Discovery of a hapE mutation that causes azole resistance in Aspergillus fumigatus through whole genome sequencing and sexual crossing. PLoS ONE 2012; 7: e50034. Arendrup MC, Jensen RH, Grif K et al. In vivo emergence of Aspergillus terreus with reduced azole susceptibility and a Cyp51a M217I alteration. J Infect Dis 2012; 206: 981–985. Liu W, Sun Y, Chen W et al. The T788G mutation in the cyp51C gene confers voriconazole resistance in Aspergillus flavus causing aspergillosis. Antimicrob Agents Chemother 2012; 56: 2598–2603. Arendrup MC, Perkhofer S, Howard SJ et al. Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echinocandins. Antimicrob Agents Chemother 2008; 52: 3504–3511. Arendrup MC, Garcia-Effron G, Buzina W et al. Breakthrough Aspergillus fumigatus and Candida albicans double infection during caspofungin treatment: laboratory characteristics and implication for susceptibility testing. Antimicrob Agents Chemother 2009; 53: 1185–1193. Madureira A, Bergeron A, Lacroix C et al. Breakthrough invasive aspergillosis in allogeneic haematopoietic stem cell transplant recipients treated with caspofungin. Int J Antimicrob Agents 2007; 30: 551–554. Garcia-Effron G, Dilger A, Alcazar-Fuoli L, Park S, Mellado E, Perlin DS. Rapid detection of triazole antifungal resistance in Aspergillus fumigatus. J Clin Microbiol 2008; 46: 1200–1206. Zhao Y, Stensvold CR, Perlin DS, Arendrup MC. Azole resistance in Aspergillus fumigatus from bronchoalveolar lavage fluids of patients with chronic diseases. J Antimicrob Chemother 2013; 68: 1497–1504. Arendrup MC, Bruun B, Christensen JJ et al. National surveillance of fungemia in Denmark 2004-2009. J Clin Microbiol 2011; 49: 325–334. Asmundsdottir LR, Erlendsdottir H, Gottfredsson M. Nationwide study of candidemia, antifungal use, and antifungal drug resistance in iceland, 2000 to 2011. J Clin Microbiol 2013; 51: 841–848. Poikonen E, Lyytikainen O, Anttila VJ et al. Secular trend in candidemia and the use of fluconazole in Finland, 2004-2007. BMC Infect Dis 2010; 10: 312. Sandven P, Bevanger L, Digranes A, Haukland HH, Mannsaker T, Gaustad P. Candidemia in Norway (1991 to 2003): results from a nationwide study. J Clin Microbiol 2006; 44: 1977–1981. Lortholary O, Desnos-Ollivier M, Sitbon K, Fontanet A, Bretagne S, Dromer F. Recent exposure to caspofungin or fluconazole influences the epidemiology of candidemia: a prospective multicenter study involving 2,441 patients. Antimicrob Agents Chemother 2011; 55: 532–538. Arendrup MC, Sulim S, Holm A et al. Diagnostic issues, clinical characteristics, and outcomes for patients with fungemia. J Clin Microbiol 2011; 49: 3300–3308. Shah DN, Yau R, Lasco TM et al. Impact of prior inappropriate fluconazole dosing on isolation of fluconazole-nonsusceptible Candida species in hospitalized patients with candidemia. Antimicrob Agents Chemother 2012; 56: 3239–3243. Ben-Ami R, Olshtain-Pops K, Krieger M et al. Antibiotic exposure as a risk factor for fluconazole-resistant Candida bloodstream infection. Antimicrob Agents Chemother 2012; 56: 2518–2523.
CMI
55. Forrest GN, Weekes E, Johnson JK. Increasing incidence of Candida parapsilosis candidemia with caspofungin usage. J Infect 2008; 56: 126– 129. 56. Sipsas NV, Lewis RE, Tarrand J et al. Candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001-2007): stable incidence but changing epidemiology of a still frequently lethal infection. Cancer 2009; 115: 4745–4752. 57. Arendrup MC, Dzajic E, Jensen RH et al. Epidemiological changes with potential implication for antifungal prescription recommendations for fungaemia: data from a nationwide fungaemia surveillance programme. Clin Microbiol Infect 2013; 19: e343–e353. 58. Pfaller MA, Diekema DJ, Gibbs DL et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida Species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion. J Clin Microbiol 2010; 48: 1366–1377. 59. Oxman DA, Chow JK, Frendl G et al. Candidaemia associated with decreased in vitro fluconazole susceptibility: is Candida speciation predictive of the susceptibility pattern? J Antimicrob Chemother 2010; 65: 1460–1465. 60. Pfaller MA, Castanheira M, Lockhart SR, Ahlquist AM, Messer SA, Jones RN. Frequency of decreased susceptibility and resistance to echinocandins among fluconazole-resistant bloodstream isolates of Candida glabrata. J Clin Microbiol 2012; 50: 1199–1203. 61. Cuenca-Estrella M, Gomez-Lopez A, Cuesta I, Zaragoza O, Mellado E, Rodriguez-Tudela JL. Frequency of voriconazole resistance in vitro among Spanish clinical isolates of Candida spp. According to breakpoints established by the Antifungal Subcommittee of the European Committee on Antimicrobial Susceptibility Testing. Antimicrob Agents Chemother 2011; 55: 1794–1797. 62. Crump KR, Edlinds TD. Agricultural Fungicides May Select for Azole Antifungal Resistance in Pathogenic Candida. 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC. Oct 30-Nov 2 2004; M-1684. 63. Yang YL, Lin CC, Chang TP et al. Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole. PLoS ONE 2012; 7: e34609. 64. Castelo-Branco DS, Brilhante RS, Paiva MA et al. Azole-resistant Candida albicans from a wild Brazilian porcupine (Coendou prehensilis): a sign of an environmental imbalance? Med Mycol 2013; 51: 555–560. 65. Arendrup MC. Epidemiology of invasive candidiasis. Curr Opin Crit Care 2010; 16: 445–452. 66. Cleveland AA, Farley MM, Harrison LH et al. Changes in incidence and antifungal drug resistance in candidemia: results from population-based laboratory surveillance in Atlanta and Baltimore, 2008–2011. Clin Infect Dis 2012; 55: 1352–1361. 67. Pfaller M, Neofytos D, Diekema D et al. Epidemiology and outcomes of candidemia in 3648 patients: data from the Prospective Antifungal Therapy (PATH Alliance(R)) registry, 2004-2008. Diagn Microbiol Infect Dis 2012; 74: 323–331. 68. Ben-Ami R, Rahav G, Elinav H et al. Distribution of fluconazole-resistant Candida bloodstream isolates among hospitals and inpatient services in Israel. Clin Microbiol Infect 2013; 19: 752–756. 69. Das I, Nightingale P, Patel M, Jumaa P. Epidemiology, clinical characteristics, and outcome of candidemia: experience in a tertiary referral center in the UK. Int J Infect Dis 2011; 15: e759–e763. 70. Zhang XB, Yu SJ, Yu JX, Gong YL, Feng W, Sun FJ. Retrospective analysis of epidemiology and prognostic factors for candidemia at a hospital in China, 2000-2009. Jpn J Infect Dis 2012; 65: 510–515.
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
10.1111/1469-0691.12513
Update on antifungal resistance in Aspergillus and Candida M. C. Arendrup Unit of Mycology and Parasitology, Department Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark
Abstract Antifungal resistance in Candida and Aspergillus may be either intrinsic or acquired and may be encountered in the antifungal drug exposed but also the antifungal drug-na€ıve patient. Prior antifungal treatment confers a selection pressure and notoriously raises the awareness of possible resistance in patients failing therapy, thus calling for susceptibility testing. On the contrary, antifungal resistance in the drug-na€ıve patient is less expected and therefore more challenging. This is particularly true when it concerns pathogens with acquired resistance which cannot be predicted from the species identification itself. This scenario is particularly relevant for A. fumigatus infections due to the increasing prevalence of azole-resistant isolates in the environment. For Candida, infections resistance is most common in the context of increasing prevalence of species with intrinsic resistance. Candida glabrata which has intrinsically reduced susceptibility to fluconazole is increasingly common particularly among the adult and elderly population on the Northern Hemisphere where it may be responsible for as many as 30% of the blood stream infections in population-based surveillance programmes. Candida parapsilosis is prevalent in the paediatric setting, at centres with increasing echinocandin use and at the southern or pacific parts of the world. In the following, the prevalence and drivers of intrinsic and acquired resistance in Aspergillus and Candida will be reviewed. Keywords: Acquired resistance, Aspergillus, azoles, Candida, echinocandins, intrinsic resistance, susceptibility testing Article published online: 21 December 2013 Clin Microbiol Infect 2014; 20 (Suppl. 6): 42–48
Corresponding author: M. C. Arendrup, Unit of Mycology, Dept. Microbiology and Infection Control, Statens Serum Institut, Building 43/317, Copenhagen, Denmark E-mail: [email protected] Invited review for Clin Microbiol Infection: based on a lecture given at the ESCMID conference Invasive Fungal Infections, Rome Jan 1-18, 2013.
Introduction Resistance in Aspergillus and Candida has been increasingly investigated and reported because standards for susceptibility testing and associated breakpoints became available and as a consequence of the increased use of antifungal compounds [1–4]. Resistant infection can be encountered in the antifungal drug-exposed patient due to selection of intrinsically resistant species or isolates with acquired resistance belonging to species that are normally susceptible. In both cases, resistance may be expected as any antimicrobial therapy is associated with a selection pressure and therefore risk of resistance.
Resistance can, however, also be encountered in the antifungal drug-na€ıve patient and, again, can be due either to infection with intrinsically resistant species or to isolates with acquired resistance. Whereas resistance due to intrinsically resistant species can be diagnosed through correct species identification, detection of isolates with acquired resistance is more demanding and requires appropriate and carefully performed susceptibility testing and endpoint interpretation [5]. New tools for rapid species identification of Candida species including the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), fluorescence in situ hybridization (FISH), etc. have improved correct species identification at clinical microbiological routine
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases
CMI
Arendrup
laboratories. However, various challenges are still associated with performance and interpretation of susceptibility testing even though commercial tests are now available for most antifungal compounds and clinical breakpoints have been established [1,2,6–11]. Firstly, performance requires expertise to obtain reproducible results, second the standardization of commercial methods against the reference test is not perfect for all drug–bug combinations leading to misclassification of susceptibility test results when reference breakpoints are adopted, and finally for Aspergillus, such tests are not routinely performed. Hence, the greatest diagnostic challenges related to resistant infections lay with the correct and timely detection of infections due to isolates with acquired resistance and particularly so in the drug-na€ıve patients where, historically, resistance has not been expected.
Resistance in Aspergillus From an epidemiological point of view, less is known about the true prevalence of resistant Aspergillus infections than about resistant infections for most other organisms. This is due to the fact that most routine laboratories do not susceptibility test their Aspergillus isolates and many laboratories find species (or even genus) identification of aspergilli difficult. Additionally, national surveillance programmes are lacking. Intrinsic resistance
Aspergillus fumigatus is by far the most common species causing human infection. The wild-type isolates are susceptible to all the licensed mould active azoles (Table 1) and echinocandins [3,4,12]. However, the A. fumigatus species complex includes more than 30 sibling species which cannot be differentiated morphologically from one another or from A. fumigatus. Several of these have been isolates from humans and been shown to be intrinsically resistant to one or more antifungals (Table 2) [12–15]. Notably, however, a simple temperature
Antifungal resistance in Aspergillus and Candida
43
TABLE 2. Intrinsic resistance (R) and variable (V) susceptibility in Aspergillus
Aspergillus section fumigati A. fumigatiaffinis A. lentulus N. pseudofischeri A. viridinutans N. udagawae A. terreus (and A. alabamensis) A. flavus A. versicolor (and A. sydowi) A. calidoustus A. allilaceus
AMB
Azoles
R R V R R R R R
R R R R R (vor) R V R
V
Echinocandins
V
V V
Data compiled from [12–15]. Vor, Voriconazole.
tolerance test (growth at temperatures higher than 48°C) can discriminate between A. fumigatus and the sibling species. Intrinsic amphotericin B resistance has been recognized in A. terreus for many decades, but also A. flavus and other less common species have reduced susceptibility to amphotericin B [3]. Importantly, a number of these rare Aspergillus species are also resistant to azoles and in some cases also to echinocandins posing obvious challenges for patient management (Tables 1 and 2). Hence, careful species identification is mandatory for clinically important isolates. Acquired resistance in AF-na€ıve patients
Azole-resistant A. fumigatus infections have been reported in a number of countries. Most reports derive from Europe and involve a single molecular resistance mechanism consisting of a 34-bp tandem repeat TR34 in the promotor region of the azole target CYP51A gene and a point mutation in the target gene itself leading to an L98H amino acid substitution (TR34/L98H). This resistance mechanisms has been detected in isolates deriving from Austria, Belgium, Denmark, Germany, France, the Netherlands, Norway, Spain, Sweden and the UK [16–23] and outside Europe in China, India and Iran [24,25] (Verweij, personal communication). Notably, 61% of the global market share of agricultural fungicides is used in these Western
TABLE 1. Intrinsic susceptibility pattern and epidemiological cut-off values (ECOFFs) for Aspergillus species and mould active azoles. Data compiled from [3,4,12] Aspergillus (S/I/R (ECOFF))a calidoustus Amphotericin B Itraconazole Posaconazole Voriconazole
IE R R R
(NDb) (NDb) (NDb) (NDb)
flavus
fumigatus
nidulans
niger
terreus
versicolor
IE S IE IE
S S S S
NDc S (1 mg/L) IE (0.5 mg/L) S (1 mg/L)
S IE IE IE
R S S IE
NDb NDb NDb NDc
(4 mg/L) (1 mg/L) (0.5 mg/L) (2 mg/L)
(1 mg/L) (1 mg/L) (0.25 mg/L) (1 mg/L)
(1 mg/L) (4 mg/L) (0.5 mg/L) (2 mg/L)
(4 mg/L) (0.5 mg/L) (0.25 mg/L) (2 mg/L)
ECOFF: epidemiological cut-off values; IE: insufficient evidence to suggest whether this species is a good target for the compound in question. MICs are higher than for Aspergillus fumigatus, but there is insufficient clinical data to suggest whether this translates into poorer efficacy; ND: not done due to insufficient amount of data for ECOFF setting (low number of MIC values or data set). a S/I/R: intrinsically susceptible/intermediate/resistant. b MIC range higher than that for A. fumigatus. c MIC range similar to that for A. fumigatus.
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
44
Clinical Microbiology and Infection, Volume 20 Supplement 6, June 2014
European and Asia Pacific regions, and thus, the prevalence of TR34/L98H correlates with the geographical use of these agents [23]. Two additional resistant genotypes have been demonstrated among isolates from the European environment or from azole-na€ıve patients, namely isolates harbouring the TR46/Y121F/T289A or the G432S alterations [26,27]. Alterations involving tandem repeats in the promotor region of CYP51A and amino acid changes at codons Y121, T289 and G432 have similarly been found in azole pesticide-resistant isolates of the common plant pathogen Mycosphaerella graminicola, suggesting these codons are hot spots for possible selection via the agricultural azoles [23]. The true prevalence of the environmental type resistant isolates is largely unknown. In the Netherlands, they accounted for approx. 10% in 2007, in Danish soil samples for 8% and in Danish patients approx. 4% in 2009–11 [19,28,29].
CMI
G138 and M220) found an astonishingly high rate of resistance markers (55%) in samples from patient mainly suffering from chronic forms of aspergillosis in the UK [17,45]. However, using the same technique for Danish patients at risk for acute or chronic forms of aspergillosis and undergoing bronchoscopy with galactomannan antigen determination, 3.3 and 4.3% resistance was detected by molecular and conventional susceptibility tests, respectively [46]. It is not fully understood why these two studies reported such different rates of resistance, but a reasonable and conservative estimate of the global prevalence of azole resistance in Aspergillus appears to be 3–6%. This still raises concern as resistance was a very uncommon event before the turn of the millennium and as resistance is found also in drug-na€ıve patients.
Antifungal Resistance in Candida
Acquired resistance in AF-exposed patients
A number of reports have documented the selection of azole resistance during treatment. Most reports concern long-term treatment in patients with allergic or chronic forms of aspergillosis [29–37]. The majority of isolates harbour mutations in the target gene CYP51A, but other mechanisms are important as well, including efflux pumps [38] and target gene upregulation encoded by a mutation in the hapE gene [31,39]. Acquired azole resistance has also been reported in A. terreus and A. flavus, which poses obvious challenges regarding clinical management of related infections as these two species have intrinsically reduced susceptibility to amphotericin B [40,41]. Acquired resistance to amphotericin B in Aspergillus has not been described so far, and only a single case of mechanistically characterized acquired echinocandin resistance has been reported [42]. Susceptibility testing of Aspergillus and echinocandins is, however, even more challenging than susceptibility testing of azoles and amphotericin B due to the lack of total inhibition in vitro and therefore no clear endpoint. Hence, acquired resistance to echinocandins may be underdiagnosed and indeed breakthrough cases have been described in patients receiving echinocandin therapy [43,44]. Size of the problem
A global study involving 19 countries reported overall prevalence of azole resistance of 3.2% and ranging from 0 to 25.5% among the participating centres [16]. Notably, no centres testing at least 100 isolates failed to detect resistant isolates. It is well known that culture is an insensitive diagnostic test, and hence, the molecular approach with direct detection in patient samples is attractive. A recent study using direct molecular detection of A. fumigatus and of alterations on the codons most commonly involved in azole resistance (G54, L98,
The epidemiology of candidaemia is particularly well documented in the Nordic countries due to ongoing nationwide surveillance programmes [47–50]. Such programmes provide precise figures for the burden of disease in entire populations, without centre bias, but are less sensitive with respect to the significance of acquired resistance. This is a consequence of the study designs, as epidemiological studies include only the initial isolate typically at the time of the lowest antifungal exposure. This is a sound strategy from an epidemiological point of view as otherwise the data set would be skewed due to repeated isolates. With this in mind, the following section summarizes the contemporary and available data on resistance in Candida. Intrinsic resistance
The intrinsic susceptibility pattern of the various Candida species is summarized in Table 3. Candida glabrata and C. parapsilosis represent the two most prevalent species with reduced azole and echinocandin susceptibility, respectively, and their prevalence is shown in a global perspective in Fig. 1. Candida glabrata is particularly prevalent on the Northern Hemisphere with incidence rates as high as 30% in some centres. It is significantly more common in the elderly population, whereas the opposite is true for C. parapsilosis [47]. Other drivers of selection of resistant species include prior antifungal drug exposure where choice of compound as well as dosing (suboptimal) and duration (more than a week) are important [51–53]. Finally, a recent report has suggested that various antibacterial compounds may also drive the selection of resistance. The underlying mechanism may be altered gut flora or the low-grade antifungal effect exerted by some antibacterial compounds, such as metronidazole [54]. Increasing use of echinocandins has been linked
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
CMI
Arendrup
TABLE 3. Intrinsic susceptibility pattern for Candida species. EUCAST breakpoints have been established for the common Candida species allowing classification if wild-type isolates into S, I and R categories. For the uncommon Candida species, breakpoints have not been established. For these species, an ‘X’ denotes that the MICs for the antifungal compound are elevated compared with those for Candida albicans AMB
ECS
FCO
S S
S S
S S
C. glabrata
S
S
I
C. krusei C. parapsilosis C. tropicalis
S S S
S I S
R S S
X X X
X
Common Candida species C. albicans C. dubliniensis
Uncommon Candida species C. lusitaniae C. fermentati C. guilliermondii C. metapsilosis
Comments
Closely related to C. albicans; fluconazole resistance acquired more easily Efflux pumps often induced during azole therapy
X
C. orthopsilosis C. cifferrii C. inconspicua C. humicula C. lambica C. lipolytica C. norvegensis C. palmioleophila C. rugosa C. valida S. cerevisiaea
Closely related to C. parapsilosis Closely related to C. parapsilosis
X X X X X X X X X X X
Closely related to C. glabrata
Antifungal resistance in Aspergillus and Candida
45
Various reports suggest intrinsic resistance may be emerging. Pfaller et al. reported that high-level fluconazole resistance (MIC ≥16 mg/L), and thus clearly above the susceptibility breakpoint of 2 mg/L, was rare in C. albicans and C. dubliniensis isolates (2.0% and 3.9%, respectively) but found in as many as 6.8% and 9.0% of the C. parapsilosis and C. tropicalis isolates, respectively, among a quarter of a million isolates from 41 countries [58]. Oxman et al. reported that 19% of their Candida infections involved fluconazole-resistant isolates (again with MICs ≥16 mg/L) at two tertiary cancer centres in the US and notably that in a third of these cases, resistance was acquired rather than intrinsic [59]. Such a finding questions the use of species identification alone as a driver of antifungal treatment. Finally, 13% of fluconazole-resistant C. glabrata (MIC ≥64 mg/ L) were cross-resistant to echinocandins among isolates collected in 2006–10, whereas no such isolates were found in the period 2001–4 at the same institution [60]. As stated earlier, acquired resistance is rarely reported in epidemiological surveillance programmes of candidaemia. Interestingly, notable discrepancies were found between the resistance rates among deep-seated isolates and mucosal isolates in a recent study of voriconazole susceptibility [61]. Similarly, we noted fluconazole MICs above the EUCAST susceptibility in 15% and 20% of mucosal C. albicans and C. dubliniensis isolates in Denmark (M.C. Arendrup, Personal observation). Taken together, mounting evidence suggests acquired resistance may be an emerging and underdiagnosed entity. Possible link to agricultural azole use?
ECS: echinocandins; FCO: fluconazole. Anamorphic state is C. robusta.
a
to an increasing proportion of blood stream infections due to C. parapsilosis [55,56]. It is important to be aware of the fact, however, that a considerable usage of oral antifungals takes place outside the hospital [57]. For example, more than two-thirds of the systemic azole use is prescribed in the primary healthcare sector in Denmark and presumably for rather benign conditions like toenail infection (itraconazole), vaginitis (fluconazole and itraconazole) and various skin conditions (e.g. pityriasis) [57]. As invasive infection derive from the colonizing Candida flora, this may deserve more attention with respect to antibiotic stewardship policies [57]. Acquired resistance
Acquired resistance is less common than intrinsic resistance. One of the reasons is that fungi do not exchange resistance genes via plasmids, and hence, selection of resistance takes place in the individual patient during individual antifungal use.
Whereas the link between agricultural use of azole pesticides and the emergence of azole-resistant Aspergillus has been suggested beyond reasonable doubt, it is less clear whether agricultural pesticides may also be drivers of azole resistance in Candida. Three observations, however, suggest this may be a relevant concern. The first one concerns susceptibility in Candida isolated from the surface of fruit harvested from non-organic vs. organic farms. Not surprisingly, fluconazole MICs were higher for Candida isolated from non-organic fruit (16–64 mg/L vs. 1–8 mg/L) [62]. Furthermore, in the same study, the fungal pesticide myclobutanil was shown to induce efflux pump expression in C. glabrata in vitro [62]. The second observation concerns C. tropicalis isolates with reduced susceptibility to fluconazole found in unrelated patients at various Japanese hospitals [63]. These isolates were found to share indistinguishable pulse field gel electrophoresis patterns and to belong to the same diploid sequence genotypes as C. tropicalis with reduced fluconazole susceptibility found in soil samples and to possess reduced susceptibility to agricultural pesticides [63]. Finally, a wild Brazilian
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
46
CMI
Clinical Microbiology and Infection, Volume 20 Supplement 6, June 2014
(a)
(b)
FIG. 1. Global percentages of Candida glabrata (a) and C. parapsilosis (b) among blood stream isolates. Data compiled from [57,65–70].
porcupine with documented invasive candidiasis due to a C. albicans isolate with efflux pump-mediated high-level azole resistance was recently found [64]. Although the evidence is limited and circumstantial, it cannot be excluded that the use of agricultural azoles may drive antifungal resistance in Aspergillus, but also in Candida.
Conclusion and Future Perspectives In conclusion, resistant Candida and Aspergillus infection are increasingly encountered in the antifungal drug-na€ıve patient due to increasing incidence rates of C. glabrata (particularly at the northern hemisphere), C. parapsilosis (particularly at centres with a high echinocandin use) and due to increasing proportion of environmental A. fumigatus isolates harbouring azole resistance mechanisms. In vivo selection of acquired resistance during medical treatment is increasingly reported for Candida in patients typically receiving more than 3 weeks of echinocandin therapy and in patients with chronic forms of aspergillosis on long-term azole treatment. In numbers, 20–30% of candidaemia cases today involve species with intrinsic resistance to either fluconazole or echinocandins. This is a significant change, as C. albicans (which is intrinsically susceptible to all drug classes)
typically accounted for at least 85% of the cases in the prefluconazole, pre-echinocandin era. Azole resistance in Aspergillus overall is probably around 4% but increasing. The drivers of this changing epidemiology are antifungal use in the hospitals but importantly also in the primary healthcare sector, and particularly for Aspergillus the high use for plant protection. In any case, we only see the tip of the iceberg due to challenges associated with antifungal susceptibility testing, the design of epidemiological surveillance programmes and the infrequent performance of susceptibility testing of aspergilli.
Acknowledgements This paper is based upon an oral presentation given at the Invasive Fungal Infection ESCMID conference held in Rome January 2013.
Transparency Declaration M.C. Arendrup has received research grants and acted as speaker for Astellas, Gilead, MSD and Pfizer, and has been a consultant for Gilead, MSD and Pcovery.
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
CMI
Arendrup
References 1. Arendrup MC, Cuenca-Estrella M, Lass-Fl€ orl C, Hope W. EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST). Clin Microbiol Infect 2012; 18: E246–E247. 2. Rodriquez Tudela JL, Donnelly JP et al. EUCAST technical note on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia-forming moulds. Clin Microbiol Infect 2008; 14: 982–984. 3. Arendrup MC, Cuenca-Estrella M, Lass-Flo¨rl C, Hope WW. EUCAST technical note on Aspergillus and amphotericin B, itraconazole, and posaconazole. Clin Microbiol Infect 2012; 18: E248–E250. 4. Hope WW, Cuenca-Estrella M, Lass-Flo¨rl C, Arendrup MC. EUCAST technical note on Voriconazole and Aspergillus spp. Clin Microbiol Infect 2013; 19: E278–E280. 5. Arendrup MC, Chryssanthou E, Gaustad P, Koskela M, Sandven P, Fernandez V. Diagnostics of fungal infections in the Nordic countries: we still need to improve!. Scand J Infect Dis 2007; 39: 337–343. 6. Arendrup MC, Rodriguez-Tudela JL, Lass-Flo¨rl C et al. EUCAST technical note on anidulafungin. Clin Microbiol Infect 2011; 17: E18–E20. 7. Arendrup MC, Pfaller MA. Caspofungin Etest susceptibility testing of Candida species: risk of misclassification of susceptible isolates of C. glabrata and C. krusei when adopting the revised CLSI caspofungin breakpoints. Antimicrob Agents Chemother 2012; 56: 3965–3968. 8. Astvad KM, Perlin DS, Johansen HK, Jensen RH, Arendrup MC. Evaluation of caspofungin susceptibility testing by the new Vitek 2 AST-YS06 yeast card using a unique collection of FKS wild-type and hot spot mutant isolates, Including the five most common Candida species. Antimicrob Agents Chemother 2013; 57: 177–182. 9. Arendrup MC, Cuenca-Estrella M, Lass-Flo¨rl C, Hope WW. The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST). EUCAST technical note on Aspergillus and amphotericin-B, itraconazole, and posaconazole. Clin Microbiol Infect 2012; 17: E248–E250. 10. Arendrup MC, Cuenca-Estrella M, Lass-Flo¨rl C, Hope WW. The European Committee on Antimicrobial Susceptibility Testing – Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST). EUCAST technical note on Candida and Micafungin, Anidulafungin and Fluconazole. Mycoses 2014; doi: 10.1111/myc.12170. 11. Lass-Flo¨rl C, Arendrup MC, Rodriguez-Tudela JL et al. EUCAST technical note on Amphotericin B. Clin Microbiol Infect 2011; 17: E27– E29. 12. Varga J, Houbraken J, van der Lee HAL, Verweij PE, Samson RA. Aspergillus calidoustus sp. nov., causative agent of human infections previously assigned to Aspergillus ustus. Eukaryot Cell 2008; 7: 630–638. 13. van der Linden JW, Warris A, Verweij PE. Aspergillus species intrinsically resistant to antifungal agents. Med Mycol 2011; 49 (Suppl 1): S82–S89. 14. Alcazar-Fuoli L, Mellado E, astruey-Izquierdo A, Cuenca-Estrella M, Rodriguez-Tudela JL. Aspergillus section fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob Agents Chemother 2008; 52: 1244–1251. 15. Perlin DS, Mellado E. Antifungal mechanisms of action and resistance. In: Latge JP, Steinbach WJ, eds. Aspergillus fumigatus and aspergillosis. Washington: ASM Press, 2009; 457–466. 16. van der Linden J, Arendrup MC, Verweij PE. SCARE. Interscience Conference on Antimicrobial Agents and Chemotherapy 2011; Abstr. M-490. 17. Denning DW, Park S, Lass-Flo¨rl C et al. High-frequency triazole resistance found in nonculturable Aspergillus fumigatus from lungs of patients with chronic fungal disease. Clin Infect Dis 2011; 52: 1123– 1129.
Antifungal resistance in Aspergillus and Candida
47
18. Snelders E, van der Lee HA, Kuijpers J et al. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med 2008; 5: e219. 19. Mortensen KL, Mellado E, Lass-Flo¨rl C, Rodriguez-Tudela JL, Johansen HK, Arendrup MC. Environmental study of Azole-Resistant Aspergillus fumigatus and other aspergilli in Austria, Denmark, and Spain. Antimicrob Agents Chemother 2010; 54: 4545–4549. 20. Verweij PE, Snelders E, Kema GH, Mellado E, Melchers WJ. Azole resistance in Aspergillus fumigatus: a side-effect of environmental fungicide use? Lancet Infect Dis 2009; 9: 789–795. 21. Mellado E, Garcia-Effron G, Alcazar-Fuoli L et al. A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob Agents Chemother 2007; 51: 1897–1904. 22. Rath PM, Buchheidt D, Spiess B, Arfanis E, Buer J, Steinmann J. First reported case of azole-resistant Aspergillus fumigatus due to the TR/ L98H mutation in Germany. Antimicrob Agents Chemother 2012; 56: 6060–6061. 23. Stensvold CR, Jørgensen LN, Arendrup MC. Azole-resistant invasive aspergillosis: relationship to agriculture. Curr Fungal Infect Rep 2012; 6: 178–191. 24. Lockhart SR, Frade JP, Etienne KA, Pfaller MA, Diekema DJ, Balajee SA. Azole resistance in Aspergillus fumigatus isolates from the ARTEMIS global surveillance study is primarily due to the TR/L98H mutation in the cyp51A gene. Antimicrob Agents Chemother 2011; 55: 4465–4468. 25. Chowdhary A, Kathuria S, Randhawa HS, Gaur SN, Klaassen CH, Meis JF. Isolation of multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR/L98H mutations in the cyp51A gene in India. J Antimicrob Chemother 2012; 67: 362–366. 26. Kuipers S, Bruggemann RJ, de Sevaux RG et al. Failure of posaconazole therapy in a renal transplant patient with invasive aspergillosis due to Aspergillus fumigatus with attenuated susceptibility to posaconazole. Antimicrob Agents Chemother 2011; 55: 3564–3566. 27. Alanio A, Sitterle E, Liance M et al. Low prevalence of resistance to azoles in Aspergillus fumigatus in a French cohort of patients treated for haematological malignancies. J Antimicrob Chemother 2011; 66: 371–374. 28. Verweij PE, Howard SJ, Melchers WJ, Denning DW. Azole-resistance in Aspergillus: proposed nomenclature and breakpoints. Drug Resist Updat 2009; 12: 141–147. 29. Mortensen KL, Jensen RH, Johansen HK et al. Aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: a laboratory-based study with focus on Aspergillus fumigatus azole resistance. J Clin Microbiol 2011; 49: 2243–2251. 30. Morio F, Aubin GG, Danner-Boucher I et al. High prevalence of triazole resistance in Aspergillus fumigatus, especially mediated by TR/ L98H, in a French cohort of patients with cystic fibrosis. J Antimicrob Chemother 2012; 67: 1870–1873. 31. Arendrup MC, Mavridou E, Mortensen KL et al. Development of azole resistance in Aspergillus fumigatus during azole therapy associated with change in virulence. PLoS ONE 2010; 5: e10080. 32. Howard SJ, Cerar D, Anderson MJ et al. Azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis 2009; 15: 1068–1076. 33. Howard SJ, Webster I, Moore CB et al. Multi-azole resistance in Aspergillus fumigatus. Int J Antimicrob Agents 2006; 28: 450–453. 34. Howard SJ, Pasqualotto AC, Denning DW. Azole resistance in allergic bronchopulmonary aspergillosis and Aspergillus bronchitis. Clin Microbiol Infect 2010; 16: 683–688. 35. Bueid A, Howard SJ, Moore CB et al. Azole antifungal resistance in Aspergillus fumigatus: 2008 and 2009. J Antimicrob Chemother 2010; 65: 2116–2118. 36. Camps SMT, van der Linden JWM, Li Y et al. Rapid induction of multiple resistance mechanisms in Aspergillus fumigatus during azole
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
48
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47. 48.
49.
50.
51.
52.
53.
54.
Clinical Microbiology and Infection, Volume 20 Supplement 6, June 2014
therapy: a case study and review of the literature. Antimicrob Agents Chemother 2012; 56: 10–16. Chen J, Li H, Li R, Bu D, Wan Z. Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially isolated from a patient with lung aspergilloma. J Antimicrob Chemother 2005; 55: 31–37. Slaven JW, Anderson MJ, Sanglard D et al. Increased expression of a novel Aspergillus fumigatus ABC transporter gene, atrF, in the presence of itraconazole in an itraconazole resistant clinical isolate. Fungal Genet Biol 2002; 36: 199–206. Camps SMT, Dutilh BE, Arendrup MC et al. Discovery of a hapE mutation that causes azole resistance in Aspergillus fumigatus through whole genome sequencing and sexual crossing. PLoS ONE 2012; 7: e50034. Arendrup MC, Jensen RH, Grif K et al. In vivo emergence of Aspergillus terreus with reduced azole susceptibility and a Cyp51a M217I alteration. J Infect Dis 2012; 206: 981–985. Liu W, Sun Y, Chen W et al. The T788G mutation in the cyp51C gene confers voriconazole resistance in Aspergillus flavus causing aspergillosis. Antimicrob Agents Chemother 2012; 56: 2598–2603. Arendrup MC, Perkhofer S, Howard SJ et al. Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echinocandins. Antimicrob Agents Chemother 2008; 52: 3504–3511. Arendrup MC, Garcia-Effron G, Buzina W et al. Breakthrough Aspergillus fumigatus and Candida albicans double infection during caspofungin treatment: laboratory characteristics and implication for susceptibility testing. Antimicrob Agents Chemother 2009; 53: 1185–1193. Madureira A, Bergeron A, Lacroix C et al. Breakthrough invasive aspergillosis in allogeneic haematopoietic stem cell transplant recipients treated with caspofungin. Int J Antimicrob Agents 2007; 30: 551–554. Garcia-Effron G, Dilger A, Alcazar-Fuoli L, Park S, Mellado E, Perlin DS. Rapid detection of triazole antifungal resistance in Aspergillus fumigatus. J Clin Microbiol 2008; 46: 1200–1206. Zhao Y, Stensvold CR, Perlin DS, Arendrup MC. Azole resistance in Aspergillus fumigatus from bronchoalveolar lavage fluids of patients with chronic diseases. J Antimicrob Chemother 2013; 68: 1497–1504. Arendrup MC, Bruun B, Christensen JJ et al. National surveillance of fungemia in Denmark 2004-2009. J Clin Microbiol 2011; 49: 325–334. Asmundsdottir LR, Erlendsdottir H, Gottfredsson M. Nationwide study of candidemia, antifungal use, and antifungal drug resistance in iceland, 2000 to 2011. J Clin Microbiol 2013; 51: 841–848. Poikonen E, Lyytikainen O, Anttila VJ et al. Secular trend in candidemia and the use of fluconazole in Finland, 2004-2007. BMC Infect Dis 2010; 10: 312. Sandven P, Bevanger L, Digranes A, Haukland HH, Mannsaker T, Gaustad P. Candidemia in Norway (1991 to 2003): results from a nationwide study. J Clin Microbiol 2006; 44: 1977–1981. Lortholary O, Desnos-Ollivier M, Sitbon K, Fontanet A, Bretagne S, Dromer F. Recent exposure to caspofungin or fluconazole influences the epidemiology of candidemia: a prospective multicenter study involving 2,441 patients. Antimicrob Agents Chemother 2011; 55: 532–538. Arendrup MC, Sulim S, Holm A et al. Diagnostic issues, clinical characteristics, and outcomes for patients with fungemia. J Clin Microbiol 2011; 49: 3300–3308. Shah DN, Yau R, Lasco TM et al. Impact of prior inappropriate fluconazole dosing on isolation of fluconazole-nonsusceptible Candida species in hospitalized patients with candidemia. Antimicrob Agents Chemother 2012; 56: 3239–3243. Ben-Ami R, Olshtain-Pops K, Krieger M et al. Antibiotic exposure as a risk factor for fluconazole-resistant Candida bloodstream infection. Antimicrob Agents Chemother 2012; 56: 2518–2523.
CMI
55. Forrest GN, Weekes E, Johnson JK. Increasing incidence of Candida parapsilosis candidemia with caspofungin usage. J Infect 2008; 56: 126– 129. 56. Sipsas NV, Lewis RE, Tarrand J et al. Candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001-2007): stable incidence but changing epidemiology of a still frequently lethal infection. Cancer 2009; 115: 4745–4752. 57. Arendrup MC, Dzajic E, Jensen RH et al. Epidemiological changes with potential implication for antifungal prescription recommendations for fungaemia: data from a nationwide fungaemia surveillance programme. Clin Microbiol Infect 2013; 19: e343–e353. 58. Pfaller MA, Diekema DJ, Gibbs DL et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida Species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion. J Clin Microbiol 2010; 48: 1366–1377. 59. Oxman DA, Chow JK, Frendl G et al. Candidaemia associated with decreased in vitro fluconazole susceptibility: is Candida speciation predictive of the susceptibility pattern? J Antimicrob Chemother 2010; 65: 1460–1465. 60. Pfaller MA, Castanheira M, Lockhart SR, Ahlquist AM, Messer SA, Jones RN. Frequency of decreased susceptibility and resistance to echinocandins among fluconazole-resistant bloodstream isolates of Candida glabrata. J Clin Microbiol 2012; 50: 1199–1203. 61. Cuenca-Estrella M, Gomez-Lopez A, Cuesta I, Zaragoza O, Mellado E, Rodriguez-Tudela JL. Frequency of voriconazole resistance in vitro among Spanish clinical isolates of Candida spp. According to breakpoints established by the Antifungal Subcommittee of the European Committee on Antimicrobial Susceptibility Testing. Antimicrob Agents Chemother 2011; 55: 1794–1797. 62. Crump KR, Edlinds TD. Agricultural Fungicides May Select for Azole Antifungal Resistance in Pathogenic Candida. 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC. Oct 30-Nov 2 2004; M-1684. 63. Yang YL, Lin CC, Chang TP et al. Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole. PLoS ONE 2012; 7: e34609. 64. Castelo-Branco DS, Brilhante RS, Paiva MA et al. Azole-resistant Candida albicans from a wild Brazilian porcupine (Coendou prehensilis): a sign of an environmental imbalance? Med Mycol 2013; 51: 555–560. 65. Arendrup MC. Epidemiology of invasive candidiasis. Curr Opin Crit Care 2010; 16: 445–452. 66. Cleveland AA, Farley MM, Harrison LH et al. Changes in incidence and antifungal drug resistance in candidemia: results from population-based laboratory surveillance in Atlanta and Baltimore, 2008–2011. Clin Infect Dis 2012; 55: 1352–1361. 67. Pfaller M, Neofytos D, Diekema D et al. Epidemiology and outcomes of candidemia in 3648 patients: data from the Prospective Antifungal Therapy (PATH Alliance(R)) registry, 2004-2008. Diagn Microbiol Infect Dis 2012; 74: 323–331. 68. Ben-Ami R, Rahav G, Elinav H et al. Distribution of fluconazole-resistant Candida bloodstream isolates among hospitals and inpatient services in Israel. Clin Microbiol Infect 2013; 19: 752–756. 69. Das I, Nightingale P, Patel M, Jumaa P. Epidemiology, clinical characteristics, and outcome of candidemia: experience in a tertiary referral center in the UK. Int J Infect Dis 2011; 15: e759–e763. 70. Zhang XB, Yu SJ, Yu JX, Gong YL, Feng W, Sun FJ. Retrospective analysis of epidemiology and prognostic factors for candidemia at a hospital in China, 2000-2009. Jpn J Infect Dis 2012; 65: 510–515.
ª2013 The Author Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20 (Suppl. 6), 42–48
