Summary
We found an increase in triazole minimum inhibitory concentrations (MICs) of 290 clinical Aspergillus isolates following the introduction of Aspergillus-potent triazoles. This was associated with prior azole exposure and seen only in Aspergillus fumigatus. There was no correlation of MIC with clinical outcome.
Keywords: aspergillosis, azole, resistance, hematology, in vitro.
Abstract
Background.
Azole-resistant aspergillosis in high-risk patients with hematological malignancy or hematopoietic stem cell transplantation (HSCT) is a cause of concern.
Methods.
We examined changes over time in triazole minimum inhibitory concentrations (MICs) of 290 sequential Aspergillus isolates recovered from respiratory sources during 1999–2002 (before introduction of the Aspergillus-potent triazoles voriconazole and posaconazole) and 2003–2015 at MD Anderson Cancer Center. We also tested for polymorphisms in ergosterol biosynthetic genes (cyp51A, erg3C, erg1) in the 37 Aspergillus fumigatus isolates isolated from both periods that had non-wild-type (WT) MICs. For the 107 patients with hematologic cancer and/or HSCT with invasive pulmonary aspergillosis, we correlated in vitro susceptibility with 42-day mortality.
Results.
Non-WT MICs were found in 37 (13%) isolates and was only low level (MIC <8 mg/L) in all isolates. Higher-triazole MICs were more frequent in the second period and were Aspergillus-species specific, and only encountered in A. fumigatus. No polymorphisms in cyp51A, erg3C, erg1 genes were identified. There was no correlation between in vitro MICs with 42-day mortality in patients with invasive pulmonary aspergillosis, irrespective of antifungal treatment. Asian race (odds ratio [OR], 20.9; 95% confidence interval [CI], 2.5–173.5; P = .005) and azole exposure in the prior 3 months (OR, 9.6; 95% CI, 1.9–48.5; P = .006) were associated with azole resistance.
Conclusions.
Non-WT azole MICs in Aspergillus are increasing and this is associated with prior azole exposure in patients with hematologic cancer or HSCT. However, no correlation of MIC with outcome of aspergillosis was found in our patient cohort.
Invasive aspergillosis (IA) is a life-threatening mycosis predominantly caused by Aspergillus fumigatus. Triazole antifungal drugs represent the first line of therapy. Azoles block the ergosterol biosynthesis pathway via the inhibition of the cyp51A (ERG11) gene, which encodes the enzyme responsible for converting lanosterol to ergosterol [1]. Although environmental factors have been suggested to be the key factors driving the global emergence of Aspergillus resistance to azoles [2], triazole resistance is a growing concern in tertiary care centers, which care for a high volume of immunocompromised patients [3]. Alterations in the cyp51A gene leading to amino acid substitutions in the target enzyme 14-α lanosterol demethylase are considered the primary mechanisms leading to azole resistance [4–6], although non–cyp51A-mediated resistance has been reported [7]. To that end, we have analyzed Aspergillus respiratory isolates recovered from MD Anderson Cancer Center to determine changes in in vitro susceptibility to triazoles from 2 nonoverlapping periods: period A: 1999–2002, before the introduction of the potent Aspergillus-active triazoles (voriconazole [VRC] and posaconazole [PCZ]); and period B: 2003–2015. In addition, we sought to determine if any changes in susceptibility were species-, triazole-, and/or host-dependent, and their clinical implications.
METHODS
Study Design and Purposes
We prospectively collected all Aspergillus species clinical isolates recovered from respiratory sources (sputum, tracheal aspirates, bronchoalveolar lavage [BAL] fluid, and lung biopsy) in patients treated at MD Anderson Cancer Center between January 1999 and December 2015. Isolates were then screened for the presence of triazole resistance with 4-well plates that contained multiple azoles, as previously described (primary screen [3]), followed by confirmation of resistant isolates by the microdilution method (Clinical and Laboratory Standards Institute [CLSI] M38-A2 microdilution method) [8]. We then performed a retrospective case-control study in adult patients (≥18 years of age) with underlying hematologic malignancy and/or hematopoietic stem cell transplantation (HSCT) who met the definition of proven or probable invasive pulmonary aspergillosis (IPA) as per the revised European Organization for Research and Treatment of Cancer/Infectious Disease Group and the National Institute of Allergy and Infectious Diseases Mycosis Study Group (EORTC/MSG) criteria to assess potential risk factors and implications of an azole non–wild-type (WT) phenotype. We defined as cases patients with IPA caused by an azole non-WT Aspergillus isolate and as controls those patients with IPA caused by WT Aspergillus isolates. If different Aspergillus species were isolated on multiple dates from the same patient, they were analyzed as different cases. We excluded patients who had different Aspergillus species isolated on the same date and patients with solid tumor and nonmalignant diseases. For each patient, we collected detailed clinical information (see Supplementary Data). The results of minimum inhibitory concentrations (MICs) were not available to the treating physician. Therefore, no decisions regarding type/intensity of antifungal treatment, future courses of chemotherapy, or interruption of chemotherapy were made based on the results of in vitro susceptibility testing.
Definitions
In this study, we focused on 4 Aspergillus species (A. fumigatus, A. flavus, A. terreus, and A. niger) that account for >95% of cases with invasive aspergillosis in our center (D. P. Kontoyiannis, unpublished data). Although CLSI susceptibility breakpoints are yet to be established for Aspergillus species [4, 9], we defined azole susceptibility as WT and non-WT based on the MIC values at or above proposed epidemiological cutoff values (ECVs) defining the upper limit of susceptibility for the WT isolate population, as proposed by the CLSI (Supplementary Table 1) [8] (see below). We defined mono-, multi-, and pan-azole resistance as resistance to a single agent, to >1 but not all agents, and to all available active azoles such as itraconazole (ITC), VRC, PCZ, and isavuconazole (ISA), respectively [4, 10]. We defined high level of resistance as MIC ≥8 µg/mL against any of the azoles tested [5]. Invasive pulmonary aspergillosis was defined according to the revised EORTC/MSG consensus criteria [11].
The sampling date of fungal culture was defined as day 0 (D0). History of neutropenia was defined as an absolute neutrophil count of <500 cells/μL for at least 10 days, within 4 weeks of D0. Lymphopenia was defined as an absolute lymphocyte count <500 cells/μL, and malnutrition as a serum albumin level <3 g/dL at the time of D0. Patients who received at least 1 dose of a triazole (fluconazole, ITC, VRC, PCZ alone, or in any combination) given for prophylaxis or empiric therapy within 12 weeks of D0 were considered azole-exposed patients [12]. History of immunosuppressive therapy was defined by patients receiving calcineurin inhibitors, monoclonal antibodies, nucleoside analogues, and corticosteroids (at least 0.3 mg/kg/day of prednisone equivalent for at least 3 weeks) within the 3 months of D0. IPA-attributable mortality was defined as death in a patient with documented radiographic, mycological, or histological findings suggestive of active IPA at the time of death who did not have response to treatment.
Outcomes
We evaluated all-cause mortality at 42 days after D0. Comparison of mortality was made following triazole-based (triazole alone [VRC or PCZ] or triazole plus echinocandin) treatment to liposomal amphotericin B (L-AmB)–based treatment (L-AmB alone, L-AmB plus echinocandin, or L-AmB plus triazole), each used for at least 3 days within 14 days before and after identification of IPA caused by a non-WT Aspergillus isolate. Risk factors associated with mortality in IPA, both in case and control groups, were assessed.
Aspergillus Resistance Primary Screen
A screen of the collected molds was performed using the 4-well multidish method to determine growth on ITC, VRC, and PCZ agar (VIPcheck, Nijmegen, the Netherlands) [13] (Supplementary Methods). The primary screening for resistance was done in duplicate. Each plate was read separately. Molds that did not exhibit resistance (only growth in control well) “failed” the screen and were recorded as pan-susceptible. Molds that exhibited resistance “passed” the primary screen and were more stringently tested per the CLSI M38-A2 broth microdilution antifungal susceptibility testing method. Resistance was defined as per the CLSI M59 Guidance Document ECVs (Supplementary Table 1); however, as PCZ ECVs are currently not defined by CLSI for A. fumigatus, 0.25 μg/mL was used as previously established [8].
Microdilution MIC Testing
For non-WT isolates based on the primary screen, we performed broth microdilution susceptibility testing according to CLSI document M38-A2 [8].
Molecular Characterization of Genes Involved in the Ergosterol Biosynthetic Pathway
Conidia from the 37 A. fumigatus non-WT isolates both periods were collected from potato dextrose agar slants after 2 days of growth at 37°C, and DNA was extracted following the protocol published by Calera et al [14]. The extracted DNA was subjected to amplification of the complete protein coding regions together with the promoter regions of the erg1 (AFUA_5G07780), cyp51A (AFUA_4G06890), and erg3C (AFUA_8G01070) genes. The primers used for polymerase chain reaction (PCR) amplification and sequencing of the aforementioned genes are listed in Supplementary Table 2 and were specific for A. fumigatus (details regarding PCR amplification are shown in the Supplementary Methods). Sequences were assembled and edited using the SeqMan Pro and EditSeq software packages (Lasergene 13.0, DNAStar, Inc, Madison, Wisconsin). The sequences of the reference genes were retrieved from AspGD (http://www.aspergillusgenome.org/).
Statistical Methods
Categorical variables were compared using χ2 or Fisher exact test, as appropriate. Continuous variables were compared using Wilcoxon rank-sum test. If a significant difference was detected, the odds ratio and 95% confidence interval were calculated. Two logistic regression analyses were performed to (1) identify the risk factors for IPA caused by non-WT isolates, and (2) to identify the factors that were independently associated with IPA patients’ mortality within 42 days after the date of Aspergillus culture (Supplementary Methods). Last, survival curves were estimated using the Kaplan-Meier method and compared by the log-rank test for patients with azole-susceptible and azole non-WT IPA, respectively. Survival curves adjusted for the factors we identified that were associated with mortality were estimated and compared using a Cox proportional hazards model. All the tests were 2-sided with a significance level of .05. The data analyses were performed using JMP version 11.0 and SAS version 9.3 software (SAS Institute, Cary, North Carolina).
RESULTS
We identified 290 respiratory cultures positive for Aspergillus species. Of these, 51 (18%) isolates were found to grow on VIPcheck plates and when retested by the CLSI microdilution method, 37 (13%) were found to have non-WT MICs. Non-WT MICs were only low level (MIC <8 µg/mL) for any azole and for all isolates tested. None of the isolates were found to be ISA resistant; therefore, isolates were defined as either pan- susceptible, ITR-resistant, PCZ-resistant, VRC-resistant, or multiresistant. A complete list of isolates by Aspergillus species can be found in (Figure 1). Overall, the number of Aspergillus pan-susceptible isolates decreased (167/183 [91.3%] vs 86/107 [80.4%]; P = .0102) and multiresistant isolates increased (14/183 [7.7%] vs 21/107 [19.6%]; P = .004). When comparing isolates before and after 2002, a statistically significant decrease was observed in the incidence of pan-triazole–susceptible isolates of A. fumigatus (89/97 [91.8%] vs 41/53 [77.4%]; P = .021). Likewise, the prevalence of multiresistant isolates statistically significantly increased for A. fumigatus (7/97 [7.2%] vs 12/53 [22.6%]; P = .009). No statistically significant changes in isolate resistance were observed for A. flavus, A. niger, or A. terreus (Figure 1).
Figure 1.
Patterns of susceptibility among Aspergillus species between 1999–2002 and 2003–2015 (*P < .05). Monoresistant are resistant isolates against itraconaozle or posaconazole and multiresistant are those resistant to itraconazole, posaconazole, and voriconazole.
To investigate potential genetic mutations associated with resistance in A. fumigatus, we sequenced 3 key genes involved in the ergosterol biosynthesis (cyp51A, erg3C, and erg1) for all 37 non-WT isolates obtained from both study periods [15]. We found 5–6 amino acid changes in the Cyp51A protein in all but 1 (isolate 7, which presented a WT sequence) of the 37 clinical isolates analyzed, but these changes were not reported to be associated with azole resistance [16, 17]. Regarding erg3C and erg1, the majority of the isolates showed a WT sequence for both genes. There were 2 exceptions: Isolate 11 showed an amino acid change in Erg3C (L306I) and isolates 19 and 25 showed an amino acid change in Erg1 (P121H) (Table 1).
Table 1.
Gene Sequencing of the Ergosterol Biosynthetic Pathway for the Aspergillus fumigatus Isolates
| Isolate Number | Collection Year | cyp51A a | erg1 a | erg3C a |
|---|---|---|---|---|
| 1 | 2003 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 2 | 2000 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 3 | 2012 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 4 | 2002 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 5 | 2002 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 6 | 2002 | Y46F, V172M, I242V, T248N, E255D, K427E | WT | WT |
| 7 | 2000 | WT | WT | WT |
| 8 | 2002 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 9 | 2013 | T248N, E255D | WT | WT |
| 10 | 2012 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 11 | 2003 | Y46F, V172M, T248N, E255D, K427E | WT | L306I |
| 12 | 2011 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 13 | 2002 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 14 | 2003 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 15 | 2014 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 16 | 2010 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 17 | 2001 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 18 | 2012 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 19 | 2013 | Y46F, V172M, T248N, E255D, K427E | P121H | WT |
| 20 | 2015 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 21 | 2001 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 22 | 2003 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 23 | 2014 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 24 | 2011 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 25 | 2014 | Y46F, V172M, T248N, E255D, K427E | P121H | WT |
| 26 | 2014 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 27 | 2011 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 28 | 2002 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 29 | 2012 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 30 | 2010 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 31 | 2003 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 32 | 2002 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 33 | 2012 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 34 | 2002 | Y46F, V172M, I242V, T248N, E255D, K427E | WT | WT |
| 35 | 2003 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 36 | 2002 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
| 37 | 2010 | Y46F, V172M, T248N, E255D, K427E | WT | WT |
Abbreviation: WT, wild type.
aReference strain used = Af293.
Of the 290 patients with respiratory secretions positive for Aspergillus, 135 had underlying hematologic cancer or HSCT. Of these, 112 (83%) met the criteria for IPA, and after excluding 5 patients with mixed infections, 107 patients with IPA were eligible for analysis (Supplementary Figure 1). The clinical characteristics of these patients and the comparison between cases (azole non-WT group) and control are shown in (Table 2). Of all of the clinical factors analyzed in a logistic regression, 3 demonstrated independent statistical significance for predicting azole-resistant IPA: Asian race (P = .004), previous azole exposure history (P = .006), and culture source being BAL fluid (P = .046) (Table 3). As BAL was more frequently performed in period B (80% of IPA patients vs 54% in patients diagnosed in period A; P = .005) and because of the fact that the number of Aspergillus pan-susceptible isolates decreased during period B, we performed another multivariate logistic regression analysis, which showed that after adjusting for the timing of IPA diagnosis (year 1999–2002 vs 2003–2015), fungal cultures from BAL were not more significantly associated with azole-resistant IPA (P = .09). No significant differences in 42-day mortality were seen in 19 cases compared to 88 controls (Figure 2). MIC of the isolates to either ITC, VRC, PCZ, or ISA had no effect on 42-day mortality in patients with IPA (Table 4). Supplementary Table 3 compares patients with different outcome at 42 days. Multivariate logistic regression analysis showed that history of neutropenia (P = .03), lymphopenia (P = .017), and intensive care unit (ICU) stay at diagnosis (P < .001), along with the period of IPA diagnosis (P = .008), were independent prognostic factors for death at 42 days (Table 5). Importantly, the type of treatment (triazole based vs L-AmB based) was not associated with 42-day mortality following culture diagnosis of IPA between the azole non-WT group and azole-susceptible group (Table 5 and Supplementary Table 3).
Table 2.
Comparison of Clinical Characteristics Between Patients With Invasive Pulmonary Aspergillosis Caused With an Azole Non–Wild-typea Isolate With Those Infected With a Susceptible Isolate
| Characteristics | All Patients (n = 107) | Azole-Susceptible Group (n = 88) | Azole-Resistant Group (n = 19) | P Valueb | OR (95% CI) |
|---|---|---|---|---|---|
| Age, y, median (range) | 57 (18–87) | 57 (20–87) | 63 (18–78) | .93 | … |
| Male sex | 67 (63) | 55 (63) | 12 (63) | .96 | … |
| Weight, kg, median (range) | 73 (34.8–147) | 72 (40.5–147) | 75.1 (34.8–115) | .79 | … |
| Race | |||||
| White | 83 (78) | 70 (80) | 13 (68) | .01 | … |
| Black | 8 (7) | 6 (7) | 2 (11) | … | … |
| Hispanic | 10 (9) | 10 (11) | 0 (0) | … | … |
| Asianc | 6 (6) | 2 (2) | 4 (21) | .009 | 11.5 (1.9–68.3) |
| Culture specimend | |||||
| Sputum | 17 (16) | 17 (19) | 0 (0) | .04 | …m |
| BAL fluid | 71 (66) | 55 (63) | 16 (84) | .07 | … |
| Othere | 19 (18) | 16 (18) | 3 (16) | >.99 | … |
| Aspergillus species | |||||
| A. fumigatus | 52 (49) | 41 (47) | 11 (58) | .37 | … |
| A. terreus | 27 (25) | 24 (27) | 3 (16) | .39 | … |
| A. flavus | 18 (17) | 13 (15) | 5 (26) | .31 | … |
| A. niger | 10 (9) | 10 (11) | 0 (0) | .20 | … |
| Hematologic malignancy | |||||
| AML | 37 (35) | 27 (31) | 10 (53) | .07 | … |
| ALL | 6 (6) | 4 (5) | 2 (11) | .29 | … |
| CML | 10 (9) | 9 (10) | 1 (5) | .69 | … |
| CLL | 14 (13) | 13 (15) | 1 (5) | .46 | … |
| MM | 9 (8) | 9 (10) | 0 (0) | .36 | … |
| MDS | 8 (7) | 8 (9) | 0 (0) | .35 | … |
| Lymphoma | 23 (22) | 18 (20) | 5 (26) | .55 | … |
| HSCT | 46 (43) | 39 (44) | 7 (37) | .55 | … |
| Autologous | 12/46 (26) | 10/39 (26) | 2/7 (29) | … | … |
| Allogeneic | 34/46 (74) | 29/39 (74) | 5/7 (71) | … | … |
| Malignancy status | |||||
| Active | 73/100 (73) | 61/82 (74) | 12/18 (67) | .56 | … |
| Remission | 27/100 (27) | 21/82 (26) | 6/18 (33) | … | |
| Underlying medical condition | |||||
| Diabetes mellitus | 12 (11) | 10 (11) | 2 (11) | >.99 | … |
| CKD | 2 (2) | 1 (1) | 1 (5) | .33 | … |
| CHF | 3 (3) | 3 (3) | 0 (0) | >.99 | … |
| Chronic lung disease (COPD) | 12 (11) | 11 (13) | 1 (5) | .69 | … |
| Liver disease | 3 (3) | 3 (3) | 0 (0) | >.99 | … |
| Immunosuppressantsf | |||||
| Calcineurin inhibitorsg | 23 (21) | 18 (20) | 5 (26) | .55 | … |
| Monoclonal antibodiesh | 11 (10) | 7 (8) | 4 (21) | .10 | … |
| Nucleoside analoguesi | 35/105 (33) | 28/86 (33) | 7 (37) | .72 | … |
| Corticosteroids | 28 (26) | 24 (27) | 4 (21) | .78 | … |
| History of neutropenia | 31 (29) | 23 (26) | 8 (42) | .16 | … |
| ICU at diagnosis | 16 (15) | 11 (13) | 5 (26) | .16 | … |
| Cavitation in radiology | 18 (17) | 14 (16) | 4 (21) | .74 | … |
| Previous azole exposurej | 62 (58) | 46 (52) | 16 (84) | .011 | 4.9 (1.3–17.9) |
| Median total duration, d (IQR) | 99 (35–330) | 104 (54–335) | 82 (25–269) | .60 | … |
| >30 d | 47/62 (76) | 35/46 (76) | 12/16 (75) | >.99 | … |
| >90 d | 32/62 (52) | 25/46 (54) | 7/16 (44) | .47 | … |
| FCZ alone | 34 (32) | 24 (27) | 10 (53) | .03 | 3.0 (1.1–8.2) |
| Mold-active triazolek | 16 (15) | 11 (13) | 5 (26) | .16 | … |
| Previous exposure of FCZ | 48 (45) | 35 (40) | 13 (68) | .02 | 3.3 (1.1–9.4) |
| Exposure type | |||||
| Continuous | 47/48 (98) | 35/35 (100) | 12/13 (92) | .27 | … |
| Discontinuous | 1/48 (2) | 0/35 (0) | 1/13 (8) | … | |
| Median duration of FCZ, d (IQR) | 80 (27–263) | 75 (26–260) | 86 (35–293) | .64 | … |
| Median dose of FCZ, mg (IQR) | 16800 (8000–53000) | 16800 (8000–52400) | 16300 (9800–67600) | .83 | … |
| Previous exposure of ITC | 14 (13) | 13 (15) | 1 (5) | .27 | … |
| Exposure type | |||||
| Continuous | 13/14 (93) | 12/13 (92) | 1/1 (100) | >.99 | … |
| Discontinuous | 1/14 (7) | 1/13 (8) | 0/1 (0) | … | |
| Median duration of ITC, d (IQR) | 228 (62–426) | 335 (94–426) | 7 (7–7) | .14 | … |
| Median dose of ITC, mg (IQR) | 103000 (24000–170400) | 103000 (24000–170400) | 1400 (1400–1400) | .14 | … |
| Previous exposure of VRC | 12 (11) | 9 (10) | 3 (16) | .44 | … |
| Exposure type | |||||
| Continuous | 11/12 (92) | 8/9 (89) | 3/3 (100) | >.99 | … |
| Discontinuous | 1/12 (8) | 1/9 (11) | 0/3 (0) | … | |
| Median duration of VRC, d (IQR) | 30 (11–136) | 48 (11–175) | 21 (14–39) | .85 | … |
| Median dose of VRC, mg (IQR) | 12000 (4400–54400) | 24700 (4400–70000) | 8400 (7000–15600) | .85 | … |
| Previous exposure of PCZ | 7 (7) | 4 (5) | 3 (16) | .10 | … |
| Exposure type | |||||
| Continuous | 6/7 (86) | 4/4 (100) | 2/3 (67) | .43 | … |
| Discontinuous | 1/7 (14) | 0/4 (0) | 1/3 (33) | … | |
| Median duration of PCZ, d (IQR) | 36 (17–126) | 59 (25–142) | 36 (2–126) | .86 | … |
| Median dose of PCZ, mg (IQR) | 25500 (10800–75600) | 25950 (19550–53000) | 10800 (900–75600) | .38 | … |
| Time period | |||||
| 1999–2002 | 57 (53) | 51 (58) | 6 (32) | .037 | … |
| 2003–2015 | 50 (47) | 37 (42) | 13 (68) | 3.0 (1.04–8.6) | |
| Outcome at 6 wkl | |||||
| Total mortality | 37/102 (36) | 30/83 (36) | 7 (37) | .95 | … |
| Death attributable to IA | 33/102 (32) | 26/83 (31) | 7 (37) | .64 | … |
Data are presented as No. (%) unless otherwise indicated.
Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; BAL, bronchoalveolar lavage; CHF, congestive heart failure; CI, confidence interval; CKD, chronic kidney disease; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; COPD, chronic obstructive pulmonary disease; FCZ, fluconazole; HSCT, hematopoietic stem cell transplantation; IA, invasive aspergillosis; ICU, intensive care unit; IQR, interquartile range; ITC, itraconazole; MDS, myelodysplastic syndrome; MM, multiple myeloma; OR, odds ratio; PCZ, posaconazole; VRC, voriconazole.
aAny level of in vitro azole nonsusceptibility (Clinical and Laboratory Standards Institute criteria).
b P values of tests comparing azole-susceptible vs azole-resistant groups.
cAsian included 2 susceptible (1 Filipino, 1 Vietnamese) and 4 resistant (1 Vietnamese, 1 Japanese, 1 Chinese, and 1 unknown) cases. All but 1 patients were born in the United States.
dThese patients had pneumonic feature in clinical and laboratory evidence.
eOthers included tracheal aspiration (2), lung tissue (4), skin and soft tissue (4), sinus (7), pleural effusion (1), and peripheral blood (1).
fDrugs used within 12 weeks of culture date.
gCalcineurin inhibitors included cyclosporine and tacrolimus.
hMonoclonal antibodies included tumor necrosis factor α blockers, alemtuzumab, and other cytotoxic monoclonals.
iNucleoside analogues included cytarabine, fluorouracil, gemcitabine, and methotrexate.
jAzole exposure within 12 weeks prior to culture date of the Aspergillus species.
kMold-active triazole includes azole-susceptible group (ITC alone, 5 [6%]; VCZ alone, 4 [5%]; PCZ alone 1 [1%]; FCZ and VCZ, 1 [1%]) and azole-resistant group (ITC alone, 1 [5%]; PCZ alone, 1 [5%]; FCZ and VCZ, 2 [11%]; FCZ and PCZ, 1 [5%]).
lWe excluded 5 patients who were lost to follow-up before outcome was known within 6 weeks after date of culture.
mOdds ratio estimate was not computed due to zero cells.
Table 3.
Independent Risk Factors for Invasive Pulmonary Aspergillosis Caused by Aspergilli With a Non–Wild-Type Azole Minimum Inhibitory Concentration
| Predictor | OR (95% CI) | P Value |
|---|---|---|
| Asian race | 20.9 (2.5–173.5) | .0048 |
| Culture specimen–BAL fluid | 4.4 (1.03–18.6) | .046 |
| Previous azole exposure historya | 9.6 (1.9–48.5) | .0063 |
Abbreviations: BAL, bronchoalveolar lavage; CI, confidence interval; OR, odds ratio.
aAzole exposure within 12 weeks prior to culture date of the Aspergillus species.
Figure 2.
Survival curves for patients who had either azole wild-type or azole non–wild-type invasive pulmonary aspergillosis (IPA). A, Kaplan-Meier survival curve. B, Adjusted Kaplan-Meier survival curve (adjusted for history of neutropenia, lymphopenia, intensive care unit admission, and time period of IPA diagnosis).
Table 4.
Crude Mortality Within 42 Days in Patients With Invasive Pulmonary Aspergillosis According to Various Minimum Inhibitory Concentration Cutoffs of the Aspergillus Isolatesa
| MIC Cutoff, µg/mL | Rate of Mortality Within 42 Days, no./No. (%) | ||
|---|---|---|---|
| MIC ≤ Cutoff | MIC > Cutoff | P Valuec | |
| Itraconazole | |||
| 1 | 30/83 (36) | 7/19 (37) | .95 |
| 2 | 33/91 (36) | 4/11 (36) | >.99 |
| 3 | 35/95 (37) | 2/7 (29) | >.99 |
| 4 | 37/102b (36) | NA | … |
| Voriconazole | |||
| 1 | 30/86 (35) | 7/16 (44) | .50 |
| 2 | 36/98 (37) | 1/4 (25) | >.99 |
| 3 | 36/100 (36) | 1/2 (50) | >.99 |
| Posaconazole | |||
| 0.25 | 31/85 (36) | 6/17 (35) | .93 |
| 0.5 | 34/93 (37) | 3/9 (33) | >.99 |
| 1 | 36/97 (37) | 1/5 (20) | .65 |
| 2 | 37/102 (36) | NA | … |
| Isavuconazole | |||
| 1 | 37/102 (36) | NA | … |
Abbreviations: MIC, minimum inhibitory concentration; NA, not applicable.
aClinical and Laboratory Standards Institute criteria.
bWe excluded 5 patients who were lost to follow-up before outcome was known within 6 weeks after date of culture.
cUnivariate analysis.
Table 5.
Comparing Patients According to Death Status Within 42 Days After the Respiratory Culture for Aspergillus
| Characteristics | Alive (n = 65) | Death (n = 37) | P Value | OR (95% CI)a |
|---|---|---|---|---|
| Age, y, median (range) | 58 (18–87) | 56 (22–82) | .72 | … |
| Male sex | 41 (63) | 24 (65) | .86 | … |
| Race | ||||
| White | 51 (78) | 28 (76) | .86 | … |
| Black | 4 (6) | 3 (8) | … | |
| Hispanic | 7 (11) | 3 (8) | … | |
| Asian | 3 (5) | 3 (8) | … | |
| Culture specimen | ||||
| Sputum | 10 (15) | 7 (19) | .65 | … |
| BAL fluid | 46 (71) | 22 (59) | .24 | … |
| Otherb | 9 (14) | 8 (22) | .31 | … |
| Aspergillus species | ||||
| A. fumigatus | 34 (52) | 16 (43) | .38 | … |
| A. terreus | 15 (23) | 9 (24) | .89 | … |
| A. flavus | 8 (12) | 10 (27) | .10 | … |
| A. niger | 8 (12) | 2 (5) | .32 | … |
| EORTC criteria of IPA | ||||
| Proven | 7 (11) | 9 (24) | .07 | … |
| Probable | 58 (89) | 28 (76) | … | |
| Hematologic malignancy | ||||
| AML | 21 (32) | 14 (38) | .57 | … |
| ALL | 3 (5) | 3 (8) | .67 | … |
| CML | 6 (9) | 4 (11) | >.99 | … |
| CLL | 9 (14) | 5 (14) | .96 | … |
| MM | 6 (9) | 3 (8) | >.99 | … |
| MDS | 5 (8) | 3 (8) | >.99 | … |
| Lymphoma | 15 (23) | 5 (14) | .24 | … |
| HSCT | 30 (46) | 13 (35) | .28 | … |
| Autologous | 8/30 (27) | 4/13 (31) | … | |
| Allogeneic | 22/30 (73) | 9/13 (69) | … | |
| Malignancy status | ||||
| Active | 40/59 (68) | 30/36 (83) | .10 | … |
| Remission | 19/59 (32) | 6/36 (17) | … | |
| Underlying medical conditions | ||||
| Diabetes | 8 (12) | 4 (11) | >.99 | … |
| CKD | 2 (3) | 0 (0) | .53 | … |
| CHF | 3 (5) | 0 (0) | .55 | … |
| Lung disease (COPD) | 9 (14) | 3 (8) | .53 | … |
| Liver disease | 2 (3) | 1 (3) | >.99 | … |
| History of neutropenia | 13 (20) | 17 (46) | .006 | 3.4 (1.4–8.3) |
| Prolonged use of corticosteroidsc | 19 (29) | 9 (24) | .59 | … |
| ICU at diagnosis | 1 (2) | 15 (41) | <.001 | 43.6 (5.4–349.8) |
| Laboratory findingsd | ||||
| Neutropenia (ANC <500 cells/µL) | 21/60 (35) | 23 (62) | .009 | 3.1 (1.3–7.1) |
| Lymphopenia (ALC <500 cells/µL) | 28/60 (47) | 30 (81) | .001 | 4.9 (1.9–12.9) |
| Albumin, <3.0 g/dL | 33/54 (61) | 31/33 (94) | <.001 | 9.9 (2.1–45.6) |
| Time period | ||||
| 1999–2002 | 29 (45) | 24 (65) | .049 | … |
| 2003–2015 | 36 (55) | 13 (35) | 0.4 (.2–1.0) | |
| In vitro azole susceptibility | ||||
| ITC, non-WT | 12 (18) | 7 (19) | .95 | … |
| VRC, non-WT | 6 (9) | 6 (16) | .34 | … |
| PCZ, non-WT | 10 (15) | 6 (16) | .91 | … |
| Any azole, non-WT | 12 (18) | 7 (19) | .95 | … |
| Treatment within 14 d before culture result | ||||
| Azole basede | 20/63 (32) | 11/36 (31) | .90 | … |
| L-AmB basedf | 32/63 (51) | 25/36 (69) | .07 | … |
| Treatment within 14 d after culture result | ||||
| Azole based | 36 (55) | 9 (24) | .002 | 0.3 (.1–.6) |
| L-AmB based | 30 (46) | 25 (68) | .037 | 2.4 (1.05–5.7) |
Data are presented as No. (%) unless otherwise indicated.
Abbreviations: ALC, absolute leukocyte count; ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; ANC, absolute neutrophil count; BAL, bronchoalveolar lavage; CHF, congestive heart failure; CI, confidence interval; CKD, chronic kidney disease; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; COPD, chronic obstructive pulmonary disease; EORTC, European Organization for Research and Treatment of Cancer; HSCT, hematopoietic stem cell transplantation; ICU, intensive care unit; IPA, invasive pulmonary aspergillosis; IQR, interquartile range; ITC, itraconazole; L-AmB, liposomal amphotericin B; MDS, myelodysplastic syndrome; MM, multiple myeloma; OR, odds ratio; PCZ, posaconazole; VRC, voriconazole; WT, wild type.
aOdds ratio for mortality; univariate analysis.
bOthers included tracheal aspiration (2), lung tissue (4), skin and soft tissue (3), sinus (6), pleural effusion (1), and peripheral blood (1).
cCorticosteroid used at least 0.3 mg/kg/day of prednisone equivalent dose at least 3 weeks within 12 weeks before culture date.
dThese results were detected at culture date of Aspergillus species.
eAzole-based treatment indicates azole alone or azole plus echinocandin treatment.
fL-AmB–based treatment indicates L-AmB alone, L-AmB plus echinocandin, or L-AmB plus azole treatment.
DISCUSSION
Here we report the largest experience to date with the trends of aspergilli susceptibility to triazoles in a tertiary care cancer center. There are several advantages in our study. It compared the prevalence of azole resistance for all Aspergillus-active triazoles (ITC, VRC, PCZ, and the recently introduced ISA) in the era before and after the introduction of anti-Aspergillus potent new triazoles (VRC, PCZ) in our center. We also evaluated if there were polymorphisms in key ergosterol biosynthetic genes (erg1, erg3C, cyp51A) associated with non-WT azole phenotype in A fumigatus isolates. In addition, we identified risk factors associated with azole non-WT MICs and attempted to correlate in vitro susceptibility with crude 42-day mortality after the diagnosis of IPA. We found that since the widespread use of anti-Aspergillus potent new triazoles, the rates of multiresistant Aspergillus isolates have increased significantly in our institution, and this was Aspergillus species specific, as rates of multidrug resistance have increased only in A. fumigatus.
Importantly, of 37 non-WT A. fumigatus isolates, there were no cyp51A-dependent mutations identified to explain this resistance, including the prominent TR46/Y121F/T289A. This is in contrast to previous reports, most derived with patients without cancer who had chronic aspergillosis syndromes and chronic azole exposure that implicated mutations in ergosterol pathway encoding genes, more importantly cyp51A, the target of azole activity [18]. Other mechanisms, such as drug efflux or even reduced azole import to maintain low intracellular azole levels, could be operating in clinical isolates, at least in hematology patients [19], but were not explored in this study. Interestingly, we found that preexposure to Aspergillus-inactive fluconazole was associated with nonsusceptibility to Aspergillus-active triazoles (Table 2), in accordance with experimental data [20]. Deciphering the mechanisms of such resistance should be the focus of further investigation and it might be that the mechanisms may differ in patients with chronic lung disease and chronic azole exposure and in highly immunosuppressed patients with more episodic and at times intermittent azole exposure. Whether antineoplastic drugs, with or without azole exposure, result in Aspergillus resistance due to unconventional mutations (eg, changes in DNA repair mechanisms induced by alkylating agents) or upregulation of unknown drug pumps is an intriguing yet unproven possibility. Finally, whether whole- genome sequencing of A. fumigatus, an organism with extreme genetic variability and versatility [21], rather than pathway- specific integration for mutations would be more helpful, requires further study. Such studies could help decipher why resistance was not observed in A. terreus, A. niger, or A. flavus.
In an unbiased assessment of outcome (42-day mortality post-D0) and in contrast to “classic” host risk factors for poor outcome (eg. neutropenia, ICU stay; Supplementary Table 3), we found no correlation between elevated MICs and mortality. Of note, no decisions regarding type/intensity of antifungal treatment were made by the treating physician based on knowledge of the MICs, and this adds to the impartial analysis of these complex data. Assessment of mortality following the diagnosis of aspergillosis in this time frame is considered more appropriate as several competing causes of death exist in this patient population [22]. CLSI has not defined clinical breakpoints for Aspergillus species, and the European Committee on Antimicrobial Susceptibility Testing has defined limited species and drug values [23]. What determines WT and non-WT is currently based only on epidemiological and probabilistic concepts based on antifungal pharmacokinetic/pharmacodynamic relationships [8]. Whether the lack of differences in survival in IPA caused by susceptible vs nonsusceptible aspergilli can be due to practice patterns such as the frequent use of combination therapy in our cohort (data not shown) or to biological factors such as compensatory evolutionary genetic changes and subsequent “fitness cost” of the latter, or a combination of both, requires further study [24]. However, complex issues, involving both adaption in the host and Aspergillus persistence, may play a role in terms of response of patients with IPA caused by aspergilli having low level of resistance [24]. In particular, sufficient serum levels of VRC or PCZ in our patients could have overcome low levels of resistance, as indicated by preclinical (pharmacokinetic modeling, animal model studies) data [25]. In fact, VRC or PCZ levels were above proposal target troughs among the few patients in our study who had IPA caused by a non-WT isolate during therapeutic drug monitoring (data not shown). Finally, the correlation of Asian race with azole resistance was unexpected and requires further study. Only one of these patients came from Japan; the rest were born in the United States. Whether immunogenetic or pharmacogenetic (azole metabolism) factors underlie such correlation would require more study in large registries.
Our study had several weaknesses. It was a retrospective, single-institution, uncontrolled study that spanned 2 decades. Importantly, no environmental isolates were collected. Environmental surveys are required to decipher whether azole-resistant aspergillosis is a result of drug selection pressure or an environmental acquisition, at least for resistant isolates having the TR46/Y121F/T289A mutation [26]. Our data were too limited in terms of number and heterogeneity of treatment scenarios to dissect the relative contribution of each azole (type, dose, sequence of use, monotherapy vs combination therapy, effect of neutrophil recovery in neutropenic patients) to the development of non-WT MICs. The fact we did not collect serial isolates is another limitation; however, availability of serial isolates is rather uncommon in patients with hematologic cancer and microbiologically proven IPA. Importantly, only 1 colony was tested and this might be suboptimal, as there might be a variability in resistance profile of a population in a clinical sample and because azole-nonsusceptible and -susceptible aspergilli could coexist [27]. Finally, our results cannot be extrapolated to other patient populations at risk (eg, solid organ recipients) or patients with earlier IPA where molecular methods can diagnose both the disease and determine the mechanism of resistance [28].
To conclude, culture recovery of aspergilli with non-WT MICs to triazole appears to be increasing in our tertiary care cancer center. However, the clinical implications of this finding could not be shown in our study; perhaps such nonsusceptibility is still low level, and the number of episodes tested was relatively small. The hematology population might be a “niche” for increased rates of resistance, although culture-proven IPA might be the tip of the iceberg, in view of the relative low frequency of positive respiratory cultures. As in vitro growth of Aspergillus is poorly reflective of the in vivo growth conditions, the optimal practice of susceptibility testing needs to be revisited.
Supplementary Data
Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Supplementary Material
Notes
Acknowledgments. D. P. K. acknowledges the Frances King Black Endowed Professorship for Cancer Research. D. P. K. dedicates this work to the loving memory of his twin brother Kostas, who recently succumbed to cancer.
Financial support. This work was supported by Pfizer, Inc (educational grant to D. P. K.) and the MD Anderson Cancer Center (Institutional Core Grant CA16672). D. S. P was funded by grants AI109025 from the National Institutes of Health and Astellas Pharma; A. G. M. was funded by grant R01 CA180279 from the National Institutes of Health; A. G. M. and A. M. T. were supported by the John S. Dunn Foundation.
Potential conflicts of interest. D. P. K. has received research support from Pfizer and Astellas, and has received honoraria from Merck, Astellas, Pfizer, Cidara, Inc, Amplyx, Inc, and F2G, Inc. J. F. M. has received research grants from Astellas and Basilea, and speaker’s fees from Gilead Sciences, Merck, Pfizer, and United Medical. P. E. V. has received research grants from Gilead Sciences, Astellas, Pfizer, Merck, F2G, and Basilea, and speaker’s fees from Astellas, Merck, Gilead Sciences, Pfizer, and BioRad. R. E. L. has research support from Gilead Inc, and honoraria from Merck, Gilead, and Basilea. D. S. P. has received research support, honoraria, and/or consulting fees and has served on advisory boards for Pfizer, Astellas, Merck, Cidara, Synexis, F2G, Myconostica, Amplyx, and Matinas; is an adviser to Global Action Fund for Fungal Infections; and was a consultant to the Bill & Melinda Gates Foundation. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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