Epidemiology and Resistance in Aspergillus and other Moulds

Epidemiology and Resistance in Aspergillus and other Moulds Nathan Wiederhold, PharmD Associate Professor Director, Fungus Testing Laboratory Invasive Mould Infections Often opportunistic Associated with significant morbidity and mortality in immunocompromised hosts Hematologic malignancies HSCT recipients (allogeneic & autologous) SOT recipients (lung, heart, liver, kidney, etc) Limited treatment options for some Order Mucorales Fusarium species / Lomentospora species Knowledge of fungal taxonomy increasing Diagnostic strategies some what lacking IFIs Organ Transplant Recipients TRANSNET organ transplant recipient database (00 006) Proven or probable IFIs from 5 U.S. centers Invasive Fungal Infections Surveillance Cohort (0 IFI in 063 pts) Incidence Cohort (79 IFI in 633 pts) Invasive candidiasis 5.9% 56% Invasive aspergillosis.%.% Cryptococcosis.0% 6.7% Other moulds 6.5%.% Unspecified moulds.0%.% Endemic fungi 5.3%.% Mucormycosis.3%.% -month cumulative incidence for any IFI 3.% (0.7% for IA; ~0.% other moulds) Pappas et al. Clin Infect Dis 00;50:0-. IMI Lung Transplant Recipients 73 lung transplant recipients at of 5 TRANSNET SOT centers 3 (.%) developed invasive mould infections (00 006) IMIs occurred median months post-transplant Majority late onset (>90 days) 6 months for mucormycosis Doligalski et al. Am J Transpl 0;:3-333. Species Percent Aspergillus 7.7% Other moulds 6.% No culture.9% 3.5% Mucorales.% Other moulds = Acremonium, Alternaria, Chrysosporium, Cladosporium, Exophiala, Microascus, Ochroconis, Paecilomyces, Paraphaeosphaeria, Penicillium, Phaeoacremonium, Phialemonium, Rhinocladiella, Scopulariopsis, Trichoderma (several phaeoid species)

IFIs HSCT Recipients TRANSNET organ transplant recipient database (00 006) Proven or probable IFIs from U.S. centers Invasive Fungal Infections Surveillance Cohort (93 IFI in 76 pts) Incidence Cohort (7 IFI in 639 pts) Invasive aspergillosis 3% % Invasive candidiasis % 30% Mucormycosis.0% 9.0% Other moulds 7.0% 5.0% Unspecified moulds 6.0% 6.0% Fusariosis 3.0% 3.0% Endemic fungi 0.6% 0.% -month cumulative incidence for any IFI 3.% (0.9% - 3.% range by site) Kontoyiannis et al. Clin Infect Dis 00;50:09-00. IFI HSCT PATH Alliance 3 medical centers U.S. & Canada, July 00 to September 007 50 proven/probable IFI in 3 HSCT patients Invasive Fungal Infection Proven/Probable Week Mortality Invasive aspergillosis 59.% 35.9% Invasive candidiasis.%.9% Mucormycosis 7.% 6.3% Other moulds 6.% 0% Other Moulds Mucormycosis Invasive Aspergillosis Invasive Candidiasis Neofytos et al. Clin Infect Dis 009; : 65-73. 0 0 0 60 0 00 Proportion Clinical Outcome Complete/Partial Stable Worse Unknown TRANSNET Phaeohyphomycosis Included in the other moulds category 56 of 9 (.6%) IFI in TRANSNET cohort Similar divided between HSCT & SOT recipients Pulmonary infections in HSCT recipients (57.7% vs. 6.7%) Overall 90 day mortality higher HSCT (% vs. 0%) Cutaneous infections in SOT recipients (53.3% vs. 3.%) Species (n = 56 isolates total) No. Isolates (%) No. Deaths (%) Alternaria (3.%) 6 (33.3%) Exophiala 6 (0.7%) 0 Cladophialophora 5 (.9%) (0%) Scopulariopsis 5 (.9%) (0%) Curvularia (7.%) (5%) Phialemonium (7.%) (5%) Exserohilum 3 (5.%) (33.3%) McCarty et al. Med Mycol 05;53:0-6. Aspergillosis Acute invasive aspergillosis Estimated ~00,000 cases/year Leading cause of invasive mould infections SOT recipients (TRANSNET) Lung transplant recipients (TRANSNET) HSCT recipients (TRANSNET & PATH Alliance) Leading cause IFI in HSCT recipients Chronic pulmonary aspergillosis Estimated >3 million people infected worldwide Common with underlying lung disease Tuberculosis, sarcoidosis Denning et al. Bull World Health Organ 0;9:6-7. Denning et al. Eur Respir J 03;:6-66. Denning et al. Med Mycol 03;5:36-370.

Percent Resistant Percent Resistant 5/5/07 Ubiquitous mould Microbiology >00 spp. Aspergillus Common causes of infections in humans A. flavus - A. terreus - A. niger - A. fumigatus Conidia inhaled and may evade upper respiratory defenses - Germinate into angioinvasive hyphae local tissue damage hemorrhage, infarction coagulative necrosis Antifungal Activity vs. A. fumigatus Parameter Itraconazole Voriconazole Posaconazole MIC Range <0.03 >6 μg/ml 0.06 - >6 μg/ml <0.03 >6 μg/ml MIC50 0.5 μg/ml 0.5 μg/ml 0.5 μg/ml MIC90 μg/ml μg/ml 0.5 μg/ml GM MIC 0.537 μg/ml 0.56 μg/ml 0.9 μg/ml 0 5 0 5 0.5% Itraconazole (n = 6) A. fumigatus Overall Resistance 3.6%.06% Voriconazole (n = 76) Posaconazole (n = 3) 0 5 0 5 0 A. fumigatus Resistance Matched Isolates (N = 396) 5.% 3.76%.00% Itraconazole Voriconazole Posaconazole Wiederhold et al. Advances Against Aspergillosis 0. Azole Resistance in Aspergillus Modification of Target Enzyme Point mutations in CYP5A (gene encoding for demethylase azole target) observed in A. fumigatus clinical and laboratory isolates with azole resistance Position of point mutation determines azole resistance Some pan-azole, other affect voriconazole & isavuconazole, other affect posaconazole Historically observed with chronic azole exposure Patients with chronic pulmonary aspergillosis (Manchester, UK) Mann et al. AntimicrobAgents Chemother 003;7:557-5. Mellado et al. AntimicrobAgents Chemother 00;:77. Howard et al. Emerg Infect Dis 009;5:06-076. Seyedmousavi et al. Drug Resist Updat 0;7:37-50. Environmental Exposure to Azoles Azole-resistant IA identified in patients without prior azole exposure in parts of Europe Indoor environment in hospitals & direct proximity to medical centers Fields where azole fungicides used Used in agriculture to combat crop failure & other products to prevent rotting Mechanisms of azole resistance in environmental isolates & azole-naïve patients TR 3 /L9H TR 6 /YF/T9A TR 53 Snelders et al. Appl Environ Microbiol 009;75:053-3057. Chowdhary et al. PLoS One 0;7:e57. van der Linder et al. Clin Infect Dis 03;57:53-50. Chowdhary et al. J Antimicrob Chemother 0;69:555-557. Chowdhary et al. J Antimicrob Chemother 0;69:979-93. 3

Worldwide Issue Continent/Country Percent Resistance Isolate Source Europe (G5, M, GS, G3, TR 3/L9H, TR 6/YF/T9A, TR 53) Belgium 5.7% Clinical France 0.9 0.6% Clinical Germany. % Clinical & Environmental Netherlands. 0% Clinical & Environmental Poland.3% Clinical Spain.% Clinical Turkey 0.% Clinical United Kingdom 6.6 % Clinical North & South America (G5, M, GS, G3, TR 3/L9H, TR 6/YF/T9A, TR 53) United States 0.6.% Clinical Colombia (TR 53) 3.3% Environmental Africa (TR 3/L9H, TR 6/YF/T9A) Tanzania 3.9% Environmental Asia & Australia (G5, M, GS, TR 3/L9H, TR 6/YF/T9A) Australia.6% Clinical China, India, Iran, Japan, Kuwait, Pakistan.9.% Clinical & Environmental Rivero-Menendez J Fungi 06;; doi:0.3390/jof0300. Azole Resistance - FTL Results 0 of 6 isolates confirmed to be A. fumigatus & with elevated azole MICs had CYP5A mutations TR 3 /L9H or TR 6 /YF/T9A mutations each found in two isolates First TR 6 /YF/T9A mutation (00) predates first one found in Europe (the Netherlands, 009) Patients with proven disease & without travel history 6 azole resistant isolates without CYP5A mutation Wiederhold et al. J Clin Microbiol 06;:6-7. Other Mechanisms of Resistance Mechanism Overexpression CYP5B Efflux pumps Cholesterol import GOF mutation in HapE Comments Cyp5p encoded by cyp5a & cyp5b CYP5A important in regulating -α-demethylase activity CYP5B associated with growth rate & cell maintenance Role CYP5B in azole resistance unclear ~50 ABC family transporters & ~300 MFS genes predicted in A. fumigatus Overexpression of CDRB, AfuMDR, AfuMDR, AfuMDR3, AfuMDR (biofilm resistance) & ATRF associated with azole resistance in vitro Little known about efflux pumps and in vivo resistance Import of exogenous cholesterol as substitute for ergosterol after azole treatment SrbA (sterol-regulatory element binding protein) plays role in resistance HapE is a CCAAT-binding transcription factor complex subunit PL mutation in HapE-complex & binding to CCAAT-box in promoter region can induce CYP5A expression Aspergillus Antifungal Resistance Two phenomena associated with resistance to antifungal agents in IFIs caused by Aspergillus. Secondary azole-resistance in A. fumigatus isolates. Increase in number of infections due to non-a. fumigatus isolates Emerging species with primary resistance to azoles Mellado et al. J Clin Microbiol 00;39:3-3. Bueid et al. J Antimicrob Chemother 03;6:5-5. Chamilos & Kontoyiannis Drug Resist Updat 005;:3-35. Rajendran et al. Antimicrob Agents Chemother 0;55:09-097. Ferreira et al Med Mycol 005;3(suppl ):S33-39. Slaven et al. Fungal Genet Biol 00;36:99-06. Fraczek et al. J Antimicrob Chemther 03;6:6-96. Willger et al. PLoS Pathog 00;:e00000. Camps et al. PLoS One 0;7:E5003. Goncalves et al. Mycoses 06;59:9-9.

Cryptic Aspergillus Species Leading causes of IA: A. fumigatus (5% 60%) A. flavus (6% 0%) A. terreus (% 0%) A. niger (% - 9%) Molecular tools led to description of new species Cryptic or sibling species difficult to differentiate by classic means (phenotype/morphology alone) TRANSNET (U.S.: 00 006) - % cryptic species among Aspergillus isolates A. lentulus (.%), A. udagawae (.%), A. tubingensis (.%), A. calidoustus (.%) FILPOP (Spain: Oct. 00 & May 0).5% cryptic species among 33 Aspergillus isolates A. lentulus (.%), A. alliaceus (.%), A. tubingensis (7.9%), A. calidoustus (.%) Rivero-Menendez J Fungi 06;; doi:0.3390/jof0300. Balajee et al. J Clin Microbiol 009;7:33-3. Kontoyiannis et al. Clin Infect Dis 00;50:09-00. Pappas et al. Clin Infect Dis 00;50:0-. Alastruey-Izquierdo et al. Antimicrob Agents Chemother 03;57:330-337. Aspergillus Section Fumigati (aka A. fumigatus Species Complex) 5 phylogenetically distinct species Difficult to distinguish phenotypically Unstable morphology Identification requires molecular/proteinomic means DNA sequences analysis (β-tubulin, calmodulin) MALDI-TOF Balajee et al. Stud Mycol 007;59:39-6. Sugui et al. J Clin Microbiol 0;5:3707-37. Samson et al. Stud Mycol 0;7:-73. Aspergillus Section Fumigati (aka A. fumigatus Species Complex) At least 5 reported to have caused disease in humans Aspergillus species A. felis, A. fumigatiaffinis, A. fumigatus, A. fumisynnematus, A. lentulus, A. novofumigatus, A. parafelis, A. pseudofelis, A. pseudoviridinutans, A. viridinutans Previously Neosartorya species A. fischeri (N. fischeri), A. hiratsukae (N. hiratsukae), A. thermomutatus (N. pseudofischeri), and A. udagawae (N. udagawae) Several with reduced azole susceptibility Species AMB ITR VOR A. fumigatus A. lentulus A. viridinutans 0.5 A. felis A. parafelis >6 A. pseudofelis >6 A. pseudoviridinutans >6 Infections include: IPA, osteomyelitis, peritonitis, cerebral aspergillosis, etc. Sugui et al. J Clin Microbiol 0;5:3707-37. Balajee et al. J Clin Microbiol 005;3:5996-5999. Matsumoto et al. Pediatr Nephrol 00;7:3-5. Jarv et al. 00; J Clin Microbiol 00;:95-9. Zbinden et al. Transpl Infect Dis 0;:E60-63. Ghebremedhin et al. J Med Microbiol 009:5:67-6. Other Aspergillus Sections/Species Flavi Section Species Antifungal Susceptibility Nidulantes Nigri Terrei A. flavus A. alliaceus A. nidulans A. delacroxii A. quadrilineatus A. niger A. tubingensis A. terrei A. alabamensis A. citrinoterreus Reduced susceptibility to AMB and echinocandins Variable AMB susceptibility Azole susceptible Variable azole susceptibility Reduced AMB susceptibility Usti A. calidoustus Reduced AMB and azole susceptibility Versicolores A. sydowii A. versicolor Variable AMB susceptibility & reduced azole susceptibility NOT A COMPREHENSIVE LIST! 5

Mucormycosis Study Prevalence IMI Rank TRANSNET - SOT.3% TRANSNET - Lung SOT.% 3 TRANSNET - HSCT.0% Johns Hopkins (HSCT & SOT).3 -.9% & 3 UT MD Anderson (Hematol. malignancies - autopsy).5% France. cases/million population/year Spain 0. cases/million population/year California.7 cases/million population/year Pappas et al. Clin Infect Dis 00;50:0-. Doligalski et al. Am J Transpl 0;:3-333. Kontoyiannis et al. Clin Infect Dis 00;50:09-00. Neofytos et al. Transpl Infect Dis 03;5:33-. Lewis et al. Mycoses 03;56:63-65. Bitar et al. Emerg Infect Dis 009:5:395-0. Rees et al. Clin Infect Dis 99;7:3-7. Pagano et al. Haematologica 00;6:6.70. Causative Agents Mucormycosis Genera Apophysomyces Cunninghamella Lichtheimia (formerly Absidia) Mucor Rhizomucor Rhizopus Saksenaea Representative Species (not comprehensive) A. elegans, A. trapeziformis, A. variabilis C. bertholletiae, C. echinulata L. corymbifera, L. ornata, L. ramosa M. circinelloides (f. circinelloides & f. janssenii), M. irregularis R. pusillus R. arrhizus, R. microsporus, R. schipperae S. erythrospora, S. oblongispora, S. vasiformis Rhizopus arrhizus (oryzae) Major cause of mucormycosis in United States 55% mucormycosisinfections in PATH Alliance HSCT patients Very aggressive; high morbidity & mortality (immunocompromised) Difficult to treat with limited options (increasing) R. arrhizus var. arrhizus & R. arrhizus var. delemar Is Rhizopus delemar a Separate Species? (R. arrhizus var. delemar OR R. delemar) Phylogenetically distinct ITS & multilocus analysis In agreement with other studies R. arrhizus able to produce lactic acid R. delemar lack ldha gene; cannot produce lactic acid fumaric and malic acid Abe et al. Mycologia 007;99:7-7. Dolatabadi et al. Mycoses 0;57(Suppl. 3):0-7. Species Clinical Significance? Isolate Number VT-6 MIC (mg/l) R. arrhizus var. arrhizus RA 0.5 R. arrhizus var. arrhizus RA R. arrhizus var. arrhizus RA5 0.5 R. arrhizus var. arrhizus RA 0.5 R. arrhizus var. arrhizus RA9 0.5 R. arrhizus var. arrhizus RA0 R. arrhizus var. arrhizus RA* R. arrhizus var. delemar RA3 >3 R. arrhizus var. delemar RA >3 R. arrhizus var. delemar RA6 R. arrhizus var. delemar RA7 >3 R. arrhizus var. delemar RA 6 Antifungal R. arrhizus R. delemar Amphotericin B 0. 0.39 Itraconazole 0.7.6 Posaconazole 0. 0.9 Fungus Testing Laboratory Internal Results (0 months data - 3 R. arrhizus var. arrhizus & 9 var. delemar) Gebremariam et al. Antimicrob Agents Chemother 05;59:75-77. 6

MIC (mg/ml) MIC (mg/ml) MIC (mg/ml) MIC (mg/ml) 5/5/07 Scedosporiosis Invasive infections primarily in immunocompromised hosts Breakthrough infections in persistently neutropenic and/or lymphopenic hosts Enters through sinopulmonary route & causes pulmonary infection; difficult to distinguish from invasive aspergilosis Common colonizers of cystic fibrosis patients Infections can occur in survivors of neardrowning events Pulmonary infections with dissemination to the CNS Species apiospermum aurantiacum boydii dehoogii minutispora desertorum Lomentospora prolificans Species Comment Previously considered to be anamorph of Pseudallescheria boydii Maybe less susceptible to posaconazole Reduced AMB & azole susceptibility Previously Pseudallescheria boydii Rare Reduced voriconazole susceptibility Previously Pseudallescheria minutispora Previously Pseudallescheria desertorum Previously prolificans (now separate genus) Highly drug resistant species cannot be reliable differentiated by morphology Requires DNA sequence analysis or MALDI-TOF MALDI-TOF Dendrogram (FTL Internal Data) Gilgado et al. J Clin Microbiol 00:6:766-77. Lackner et al. Fungal Diversity 0;67:-0. Antifungal Activity S. apiospermum 3 6 0.5 0.5 0. 0.06 0.03 0.05 0.00 0.00 F903 Amphotericin Caspofungin Posaconazole Voriconazole S. boydii 3 6 0.5 0.5 0. 0.06 0.03 0.05 0.00 0.00 F903 Amphotericin Caspofungin Posaconazole Voriconazole S. aurantiacum & S. dehoogii 3 6 0.5 0.5 0. 0.06 0.03 0.05 0.00 0.00 F903 Amphotericin Caspofungin Posaconazole Voriconazole L. prolificans 3 6 0.5 0.5 0. 0.06 0.03 0.05 0.00 0.00 F903 Amphotericin Caspofungin Posaconazole Voriconazole Fusariosis Significant cause of morbidity and mortality in immunocompromised hosts Usually invasive and disseminated (bloodstream) Neutropenic patients with acute leukemia Severe T-cell deficiency (corticosteroids for GVHD) Poor prognosis (although improving) Species F903 Amphotericin Caspofungin Posaconazole Voriconazole S. apiospermum (3) 0.079 3.0 5.70.9 S. boydii (5) 0.06 5.59 7.6.3 0.630 S. aurantiacum & S. dehoogii (6 & ) 0.93.76 >.00.00 L. prolificans (7) 0.6 >6 > >6 >6 Fothergill et al. ECCMID 06. Nucci et al. Clin Infect Dis 00;3:37-. Nucci et al. Clin MicrobiolInfect 0;0:50-55. Period : 95-000 Period : 00-0 7

Fusarium Species Human infections can be caused by species complexes Etiologic agents Fusarium solani spp. Complex (FSSC) for majority of Fusarium oxysporum spp. Complex (FOSC) infections Fusarium fujikuroi spp. Complex (FFSC) Fusarium chlamydosporum spp. Complex (FCSC) Fusarium dimerum spp. Complex (FDSC) Fusarium incarnatum-equiseti spp. Complex (FIESC) Fusarium sambucinum spp. Complex (FSAMSC) Fusarium tricinctum spp. Complex (FTSC) O Donnell et al. J Clin Microbiol 007;5:35-. Fusarium solani Species Complex Composed of numerous haplotypes, many of which are now considered separate species Those that cause infections in humans F. petroliphilum (haplotype ) F. keratoplasticum (haplotype ) F. falciforme (haplotype 3+) F. solani (haplotype 5) Other species/haplotypes cause disease in animals & plants Major plant pathogen Identification to species level requires MLST EFα, RPB www.fusariumdb.org/ Clinical relevance of different species in FSSC unknown (most clinical laboratories do not identify to this level) O Donnell et al. Fungal Genet Biol 03;5:0-3. Summary Invasive mould infections significant causes of morbidity & mortality in immunocompromised patients Aspergillus, Mucorales,, & Fusarium species Other moulds can also cause disease in these populations Although new antifungals are available & under development, continued work needed due to resistance (intrinsic & acquired) Epidemiology & clinical understanding of causes of IMI infections changing due to use of molecular/ proteinomic assays for species identification