Invasive aspergillosis in immunosuppressed patients is difficult to diagnose, is problematic to treat, and results in a high mortality rate ( 1 ). Chronic and allergic pulmonary and sinus aspergillosis are increasingly recognized in numerous clinical settings. Treatment with itraconazole, voriconazole, and, recently, posaconazole is the backbone of therapy for these conditions because azoles are the only licensed class of oral drugs for treatment of aspergillosis ( 2 , 3 ). Amphotericin B and caspofungin are licensed intravenous agents for invasive aspergillosis but have limited utility for chronic and allergic aspergillosis. Itraconazole resistance in Aspergillus spp. was first reported in 1997 in 3 clinical isolates obtained from California in the late 1980s ( 4 ); since then, only a few clinical cases have been published ( 5 – 9 ). The emergence of itraconazole resistance alone is of concern, but widespread azole cross-resistance would be devastating because oral treatment would not be effective. The primary mechanism of resistance described for A. fumigatus clinical isolates is mutation in the target protein. The cyp51A gene encodes the target of azoles, lanosterol 14α-demethylase, and this enzyme catalyzes a step in the biosynthetic pathway of ergosterol (an essential cell membrane component of filamentous fungi). Mutations in the open reading frame of the cyp51A gene can result in structural alterations to the enzyme, which in turn may inhibit binding of drugs. Mutational hotspots confirmed to cause resistance have been characterized in the gene at codons 54 ( 6 , 10 – 13 ), 220 ( 6 , 14 , 15 ), and 98 ( 16 – 18 ). Other mutations in the cyp51A gene have been reported, and additional resistance mechanisms have been postulated ( 11 , 19 , 20 ). The environmental or antifungal pressures driving azole resistance are unclear because few clinical azole-resistant Aspergillus strains have been studied in any detail; many reports simply describe individual patient cases. In this study, we investigated the frequency of A. fumigatus itraconazole resistance in a referral laboratory collection, defined the resulting azole cross-resistance pattern, identified mutations in the cyp51A gene, and investigated any epidemiologic links between resistant isolates. Materials and Methods Isolates Isolates deposited in the Regional Mycology Laboratory Manchester (RMLM) culture collection (between 1992 and 2007) were identified as A. fumigatus by macro- and micromorphologic characteristics. All isolates were screened for growth at 50oC, thus confirming A. fumigatus and excluding A. lentulus. Aspergilli were subcultured onto Sabouraud glucose agar (Oxoid, Basingstoke, UK) for 48 h at 37°C. Thirty-four azole-resistant and 5 susceptible isolates from 17 patients were studied from the RMLM collection (prefixed F); 36 isolates were respiratory specimens, 1 was cerebral, and 2 were from unknown sites. In addition, 18 azole-resistant isolates from a single aspergilloma case-patient (prefixed A, patient 3) collected at autopsy were also investigated. Patients Pertinent details from patients were extracted from the clinical records. All but 6 were under the care of 1 investigator (D.W.D.). Information was collected on underlying disease(s), type of aspergillosis, antifungal treatment, azole plasma levels, and characteristics of therapeutic failure. Susceptibility Testing Susceptibilities were determined by a modified European Committee on Antimicrobial Susceptibility Testing (EUCAST) method ( 21 ). The modification was a lower final inoculum concentration (0.5 × 105 as opposed to 1–2.5 × 105 CFU/mL). Isolates were tested at a final drug concentration range of 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015 mg/L against itraconazole (Research Diagnostics Inc, Concord, MA, USA), voriconazole (Pfizer Ltd, Sandwich, UK), posaconazole (Schering-Plough, Kenilworth, NJ, USA), and amphotericin B (Sigma, Poole, UK). RPMI-1640 (Sigma) was supplemented to 2% glucose (Sigma). Inocula were prepared in phosphate-buffered saline with 0.05% Tween 80 (Sigma); Aspergillus spores were counted on a hemacytometer and adjusted to a final concentration of 5 × 104 CFU/mL. Inocula were loaded into flat-bottomed microtiter plates (Costar Corning, Lowell, MA, USA) and incubated at 37oC for 48 h. A no-growth end point was determined by eye. MIC testing was performed on RMLM isolates in triplicate, and a consensus mean was derived (median or mode). Susceptibilities of the aspergilloma isolates were determined once, except for 6 that were tested 3 times. Values of >8 mg/L were classed as 16. Clinical or epidemiologic breakpoints/cutoffs have not been declared by the Clinical and Laboratory Standards Institute (CLSI) or EUCAST for azoles and Aspergillus spp. However, proposed epidemiologic cutoff values have been mooted for the latter ( 22 ), and we have recently proposed clinical breakpoints ( 23 ). Cutoffs used in this study were itraconazole and voriconazole >2 mg/L and posaconazole >0.5 mg/L (we have not defined an intermediate zone of susceptibility). Sequencing DNA was extracted by using commercially available kits (FastDNA Kit, Q-biogene, Cambridge, UK; Ultraclean Soil DNA Isolation Kit, MO BIO Laboratories Inc., Cambridge; and DNeasy plant tissue kit, QIAGEN, Crawley, UK). The entire coding region of the cyp51A gene was amplified as previously described ( 7 ), except 3 mmol/L MgCl2 was used and both strands were sequenced using 8 primers ( 7 ). Twelve of the aspergilloma (A) isolates were sequenced with only 1 primer, covering the region of interest in this case. Sequences were aligned against the sequence from an azole-susceptible strain (GenBank accession no. AF338659), and mismatches were identified by using AlignX (VectorNTI; Invitrogen, Paisley, UK). Mutations were confirmed by repeating the PCR and sequencing both strands by using the closest 2 primers. Isolates with an alteration in the cyp51A gene at codon 98 were also investigated for promoter modifications by sequencing this region ( 17 ). GenBank accession numbers for the cyp51A sequences determined in this study are EU807919−EU807922 and FJ548859−FJ548890. Microsatellite Typing Six microsatellite loci (3A, 3B, 3C, 4A, 4B, 4C) were amplified as previously described ( 24 ). Initially some amplicons were sequenced, whereas later ones were sized by using capillary electrophoresis on an ABI PRISM 3130×l Genetic Analyzer (Applied Biosystems, Warrington, UK). Electrophoresis data were analyzed by using Peak Scanner Software version 1.0 (Applied Biosystems); amplicon sizes were adjusted by using a correction factor derived from sequenced alleles to determine the actual sizes of alleles ( 25 ). Concatenated multilocus microsatellite genotypes were created for each isolate and used to generate allele-sharing genetic distance matrices, D AS. Here, D AS = 1 – (the total number of shared alleles at all loci / n), where n is the total number of loci compared ( 26 ). Subsequently, phylogenetic comparisons using 5 of the loci (not 3B) were performed with the software PAUP* 4.0 (www.paup.csit.fsu.edu) by using the neighbor-joining algorithm with the minimum-evolution option active. The strength of support for relationships was assessed by using 1,000 bootstrap resamples of the dataset. Results Susceptibility The susceptibility of 519 A. fumigatus RMLM culture collection isolates was determined. All isolates were tested for susceptibility against itraconazole and amphotericin B; 456 and 118 isolates were also tested against voriconazole and posaconazole, respectively. Subsequently, all itraconazole-resistant isolates were tested against voriconazole and posaconazole. Geometric means, ranges, MIC50 (median MIC), and MIC90 (90% of the isolates tested had a MIC at or below this level) values are shown in Table 1. Amphotericin B susceptibility was retained in the 34 itraconazole-resistant isolates tested. Of these, 65% (22) were cross-resistant to voriconazole and 74% (25) were cross-resistant to posaconazole. We did not identify any isolates that were resistant to voriconazole or posaconazole while remaining susceptible to itraconazole. Table 1 MICs for 519 Aspergillus fumigatus isolates from RMLM culture collection, 1992–2007* Isolate group (no. isolates) Susceptibility results, mg/L Itraconazole Voriconazole Posaconazole Amphotericin B GM
(range) MIC50/ MIC90 GM
(range) MIC50/ MIC90 GM
(range) MIC50/ MIC90 GM
(range) MIC50/ MIC90 RMLM collection (519), 1992–2007 0.46
( 8) 0.25/2 0.92
(0.125–>8) 1/2 0.22
(0.03–>8) 0.125/2 0.34
(0.06–2) 0.25/1 Azole resistant (34) 16.0
(>8) >8/>8 3.69
(0.125–>8) 4/>8 1.70
(0.125–>8) 1/>8 0.22†
(0.06–0.5) 0.25/0.5 Percentage resistant 100% 65% 74% 0% Aspergilloma (18) 16.0
(>8) >8/>8 2.16
(0.5–4.0) 4/4 1.92
(0.125–>8) 1/>8 0.10‡
(0.06–0.125) 0.125/ 0.125 *RMLM, Regional Mycology Laboratory Manchester; GM, geometric mean. Values >8 mg/L were classed as 16 mg/L for GM analysis. See also the Appendix Table.
†n = 28.
‡n = 6. Five percent of 400 isolates were resistant to itraconazole (when duplicate isolates from the same patient with similar susceptibility profiles were removed from the analysis). The overall frequency of itraconazole resistance in this collection (with repeat specimens included) was 7% (n = 519). The first case of azole resistance in this collection was seen in 1999. The frequency of resistance since 2004 (8%) has increased significantly (Fisher exact test, p 5.0 mg/L) documented at steady state during their treatment course (online expanded version of Table 2, available from www.cdc.gov/EID/content/15/7/1068-T2.htm). The infections in patient 1 (treated with voriconazole only for 18 months) failed therapy, and the 1 isolate identified had MICs of >8 mg/L for both itraconazole and voriconazole. Table 2 Clinical information for 17 patients with azole resistant Aspergillus fumigatus infections* Patient no. Age, y/sex City No. isolates Aspergillus disease Other diseases, y Daily dose, duration Serum azole levels, mg/L† Outcome Survival 1 50/F Cambridge, UK 1 CCPA with aspergilloma Breast cancer, 1990; M. malmoense pulmonary tuberculosis, 1999 and 2005 Vori 200–400 mg, 18 mo ND Clinical and radiological failure Alive 2 21/F Copenhagen, Denmark 1 ABPA CF, concomitant bacterial colonization with Staphylococcus aureus and Achromobacter Itra 200 mg, 14 mo (plus previous courses) ND Unknown Alive 3 40/F Manchester, UK 2‡ CCPA with aspergilloma, then CFPA Pulmonary TB with residual bilateral UL scarring and LUL cavity, 1986; smoke inhalation, 1989 Itra 400 mg, 90 mo 15.0–26.0§ Clinical failure Died 4 72/M Manchester, UK 3 CCPA with aspergilloma COPD, squamous cell carcinoma with LUL segmentectomy, 1992 Itra 400 mg, >2 mo 2.9–11.3 No improvement Died 5 43/M Montreal, Quebec, Canada 2 Cerebral aspergillosis, Nov 1998 AML-M2, 1997; RUL lobectomy, 1997; AlloHSCT, 1998; GVHD Itra 400 mg, 4 mo ND Regression of cerebral abscess, then IPA with respiratory failure Died 6 60/M Manchester, UK 2 CCPA with aspergilloma COPD, M. szulgai pulmonary infection, 2003; celiac disease Itra 200–400 mg, 1 mo 25.6 No improvement Alive 12 29/F Manchester, UK (Malawi origin) 1 CCPA with 2 aspergillomas Pulmonary TB, 1995; HIV positive, HAART Itra 400 mg, 18 mo 2.5–8.4 Improvement then progression Alive 13 64/M Preston, UK 4 CCPA with aspergilloma COPD, bronchiectasis, M. avium pulmonary infection, 2002 and 2006 Itra 600 mg, 10 mo 2.6–4.5 Progression Alive 14 42/M Birkenhead, UK 1 CCPA with LUL aspergilloma Sarcoidosis, COPD, celiac disease; aspergilloma removed as part of left lung transplant, 2007¶ Itra 400 mg, 11 mo 13.8–17.8 Unchanged, switched to vori Unknown 15 68/F Wirral, UK 1 Sputum isolate Cardiac transplant for congestive cardiomyopathy, 1999; chronic cough; 2007; polymyalgia rheumatica, hiatal hernia, oesophagitis Not documented NA Not assessable Alive 16 12/F Liverpool, UK 1 Sputum isolate Unknown Unknown Unknown Unknown Unknown 17 43/M Manchester, UK 1 Sputum isolate Unknown Unknown Unknown Unknown Unknown *CCPA , chronic cavitary pulmonary aspergillosis; M., Mycobacterium; vori, voriconazole; ND, not determined; ABPA, allergic bronchopulmonary aspergillosis; CF, cystic fibrosis; itra, itraconazole; CFPA, chronic fibrosing pulmonary aspergillosis; TB, tuberculosis; UL, upper lobe; LUL, left upper lobe; COPD, chronic obstructive pulmonary disease; AML, acute myeloid leukemia; RUL, right upper lobe; AlloHSCT, allogeneic haematopoietic stem cell transplant; GVHD, graft versus host disease; IPA, invasive pulmonary aspergillosis; AVR, aortic valve replacement; posa, posaconazole; HAART, highly active antiretroviral therapy.
†Determined by bioassay (target range 5–15 mg/L).
‡Plus aspergilloma isolates studied, taken at autopsy.
§Received a generic formulation of itra, resulting in lower concentrations (i.e., 4.6 mg/L) and then probably was noncompliant at end of treatment period.
¶Successfully completed with vori treatment. Of the 14 patients with available data, 2 had invasive disease; 9 had chronic disease with >1 aspergillomas; 2 had allergic bronchopulmonary aspergillosis; and 1 had Aspergillus bronchitis. At least 5 of the patients died of progressive infection, despite alternative therapies for some. Mutations in the cyp51A Gene A summary of Cyp51A amino acid substitutions and azole cross-resistance patterns identified in 34 resistant isolates from our clinical culture collection is shown in Table 3 and listed by line in the Appendix Table. The sequences of all 5 azole-susceptible isolates examined were identical to that of a previously published cyp51A gene sequence from an azole-susceptible isolate (AF338659). No cyp51A mutations were found in 3 itraconazole-resistant isolates (from 2 patients). In addition to the L98H substitution, 2 isolates from 2 patients had a 34-bp sequence that was duplicated in the promoter region ( 16 , 17 ) of the cyp51A gene. One isolate had 2 amino acid substitutions, H147Y and G448S. Three isolates from 2 patients had the same 6 mutations, 3 nonsynonymous ones (F46Y, M172V, E427K), along with 3 synonymous (silent) alterations at codons 89, 358, and 454 (data not shown), and an isolate from a third patient had additional mutations (N248T, D255E) as well as these 6. Four novel mutations were found (H147Y, P216L, Y431C, and G434C). The isolate bearing the P216L mutation was resistant to itraconazole and posaconazole, whereas the isolates with Y431C and G434C showed pan-azole resistance phenotypes. Table 3 Cyp51A amino acid substitutions and associated cross-resistance patterns in azole-resistant RMLM Aspergillus fumigatus isolates* Cyp51A codon No. patients No. isolates Amino acid substitutions MIC, mg/L† Itraconazole Voriconazole Posaconazole F46‡ 3 4‡ Y >8 2–4 0.125–0.5 G54 4 5 E, R, V >8 0.125–1 1–>8 L98+TR 2 2 H >8 8 1–2 G138 1 10 C >8 8–>8 2–>8 H147§ 1 1§ Y >8 >8 0.5 M172‡ 3 4‡ V >8 2–4 0.125–0.5 P216 1 1 L >8 1 1 M220 3 4 K, T >8 1–4 0.5–>8 N248‡ 1 1 T >8 2 0.25 D255‡ 1 1 E >8 2 0.25 E427‡ 4 5‡ G, K >8 2–4 0.125–0.5 Y431 1 1 C >8 4 1 G434 1 1 C >8 4 1 G448 2 2 S >8 >8 0.5–1 No substitutions 2 3 NA >8 2–8 0.25–1 *RMLM, Regional Mycology Laboratory Manchester; TR, tandem repeat in cyp51A promoter; NA, not applicable. Synonymous mutations not shown. Some mutations are associated with resistance but may not be causal (see text).
†Putative cut-off values for resistance are itraconazole and voriconazole >2 mg/L and posaconazole >0.5 mg/L.
‡F46Y found with M172V and E427K in 4 isolates along with 3 silent mutations. E427G seen alone in 1 isolate. N248 and D255 found in combination with 46/172/427 in 1 isolate.
§Found with G448S in 1 of 2 isolates. Patient 3 had 2 respiratory samples taken while she was alive, in addition to 18 aspergilloma isolates sampled at autopsy. All isolates were resistant to itraconazole (>8 mg/L), and 1 of 2 different mutations at codon 220 was detected in the cyp51A gene. Isolates with a methionine-to-lysine substitution were highly cross-resistant to voriconazole (4 mg/L) and posaconazole (>8 mg/L), whereas those with an alteration to threonine had variable voriconazole (0.5–4 mg/L) and posaconazole (0.125–1 mg/L) MICs. Microsatellite Typing The relatedness of isolates obtained from patients 3, 4, 5, 6, 8, 9, and 13 were compared by microsatellite typing (Figure 2). The isolates from 5 patients consisted of a susceptible/resistant pair, whereas an overlapping group of 4 patients had more than 1 cyp51A mutation. All isolates were from the lower respiratory tract, except the resistant isolate from patient 5, which was from a cerebral lesion. Figure 2 Unrooted phylogenetic tree showing the genetic relationship of isolates from 7 patients.The genetic relationship of these isolates is shown in relation to each other and to 18 other isolates. AF numbers belong to a collection of >200 isolates, held in Manchester, UK. ATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures; FGSC, Fungal Genetics Stock Center. Bootstrap values >90 only are shown. Scale bar indicates nucleotide substitutions per site. Multiple isolates from 5 of 7 patients had identical or nearly identical genotypes. The isolates from 2 of these 5 patients (3 and 6) differed by 1 and 2 trinucleotide repeat units, respectively, at the most polymorphic locus (3A). Three matched sets (isolates pre- and postdevelopment of resistance) were identified, where resistance almost certainly evolved from an originally susceptible strain. Figure 2 shows an unrooted tree of the phylogenetic relationships, derived from 5 of the 6 microsatellite markers, for the isolates from these 7 patients plus 18 A. fumigatus isolate controls. Only bootstrap values >90 are shown. Strains from these 7 patients are distributed among other clinical isolates; statistically supported clustering is not evident. Therefore, none of the azole-resistant isolates have been transmitted from patient to patient, indicating that they have all evolved independently from different original strains. The only statistically supported clades contain isolates that only differ from each other by 1 of the 5 markers. Discussion Itraconazole resistance and azole cross-resistance in Aspergillus spp. have been reported infrequently, which suggests that they are infrequent events to date. A contributing factor to this low prevalence has been variability in testing between laboratories. Since the initial report of resistance in isolates collected before the licensure of itraconazole, substantial improvements in susceptibility testing methods that allow confidence in reported azole MICs have been implemented. Recommended methods are now promulgated by the CLSI method M38-A2 ( 27 ) and EUCAST ( 21 ), and work is ongoing to establish internationally agreed interpretative cutoffs ( 22 ) and clinical breakpoints ( 23 ). By using such methods, some researchers have documented and published the frequency of itraconazole resistance in clinical A. fumigatus isolates ( 8 , 28 – 32 ); frequency ranged between 2% and 6%. However, most of these studies included fewer isolates ( 1 aspergillomas, which may explain the high frequency of resistance in our center. Because surgery is not an option for most patients with chronic cavitary pulmonary aspergillosis, these patients usually require long-term (if not lifelong) antifungal therapy, under which conditions as we have shown, strains of A. fumigatus may evolve resistance. Another contributory explanation could be our systematic application of susceptibility testing of Aspergillus spp. isolates in all cases in which treatment is to be given. In 6 of 10 patients, steady state itraconazole plasma level data were at or above minimum therapeutic levels (i.e., <5 mg/L), as determined by bioassay ( 38 , 39 ). Low plasma levels of itraconazole were attributable to limited bioavailability in some patients, low doses (i.e., 200 mg daily, the standard UK registered dose), drug interactions in patients with concomitant atypical mycobacterial infection, and use of generic itraconazole ( 40 ). Low plasma levels of itraconazole, in combination with the high proportion of patients in this study with prior azole exposure (13 out of 14), indicates that resistance primarily emerged during or after azole therapy. Our observations are of concern on several fronts. We found a sudden rise in the frequency of azole resistance in A. fumigatus since 2004, and many isolates showed cross-resistance between all the currently licensed azole options. Clinical data indicate that resistance has occurred during and after azole therapy in all but 1 of these cases. The infections caused by azole-resistant isolates fail therapy or at best do not respond. The molecular epidemiology shows that resistance evolved in infecting strains within the lung, rather than by superinfection with a resistant stain from the environment. Because azoles are the only useful class of oral drugs for aspergillosis (and many other serious filamentous fungal infections), clinical management of these chronically infected cases is therefore problematic. Vigilance is called for to identify azole-resistant aspergilli, and novel classes of oral antifungal would be welcome for those infected with azole-resistant strains. Supplementary Material Appendix Table Isolates, MICs, cyp51A mutations, and molecular similarity, by patient*