Abstract
We compared EUCAST and CLSI antifungal susceptibility testing (AFST) methods for triazoles and amphotericin B against 124 clinical Mucorales isolates. The EUCAST method yielded MIC values 1- to 3-fold dilutions higher than those of the CLSI method for amphotericin B. The essential agreements between the two methods for triazoles were high, i.e., 99.1% (voriconazole), 98.3% (isavuconazole), and 87% (posaconazole), whereas it was significantly lower for amphotericin B (66.1%). Strategies for harmonization of the two methods for Mucorales AFST are warranted.
TEXT
Mucormycosis is a life-threatening fungal infection caused by fungi belonging to the subphylum Mucoromycotina and order Mucorales (1). The etiologic agents of mucormycosis include the genera Rhizopus, Mucor, Lichtheimia, Cunninghamella, Rhizomucor, and Apophysomyces among others (2, 3). The disease is the second most common mold infection in hematologic malignancies and organ transplantation and is increasingly reported in patients with uncontrolled diabetes or ketoacidosis (1, 2). Notably, diagnosis is very difficult (4), and outbreaks of mucormycosis in hospitals (5), among victims of natural disasters such as the Joplin tornado (6), and among soldiers following combat-related injuries (7), as well as food-borne outbreaks in healthy individuals due to Mucor circinelloides (8), highlight the potential of Mucorales to cause severe infections. Despite aggressive antifungal therapy and, in selected cases, extensive surgical debridement, the overall mortality due to mucormycosis remains unacceptably high (9). Primary antifungal therapies for mucormycosis are amphotericin B (AMB) lipid formulations, whereas open-label salvage studies suggest posaconazole (POS) as an option for patients who are refractory to or intolerant of polyenes (10, 11). Furthermore, recent data also demonstrated the activity of isavuconazole (ISAV), which was approved by the U.S. Food and Drug Administration on 6 March 2015 for the primary treatment of invasive aspergillosis and mucormycosis, against Mucorales in vitro and in vivo, adding a second triazole antifungal for the therapy of patients with mucormycosis (12–14). Different species of Mucorales show differences in their in vitro susceptibilities to AMB, POS, and voriconazole (VRC) (15–17). So far, reported in vitro antifungal susceptibility testing (AFST) of Mucorales is mainly based on the Clinical and Laboratory Standards Institute broth microdilution method (CLSI BMD) (18), and recently, a multicentric study based on CLSI susceptibility data proposed epidemiologic cutoff values (ECV) for the detection of AMB, POS, and itraconazole resistance among the 4 most commonly encountered and clinically relevant species of Mucorales (19). In addition to the CLSI BMD method, the other international standard method for AFST and surveillance of antifungal resistance is the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method (20). Limited EUCAST AFST data and a lack of data comparing the two reference BMD methods for Mucorales prompted us to study the essential agreement (EA) between the 2 standardized methods for testing AMB and triazoles against Mucorales. Furthermore, we determined the potency of ISAV for a large collection of 124 molecularly characterized, clinically relevant Mucorales.
(This work was presented at the 55th Interscience Conference on Antimicrobial Agents and Chemotherapy and International Congress of Chemotherapy, San Diego, CA, 17 to 21 September 2015 [21].)
Of the 124 isolates, 79 were from hospitals in Delhi, India, and 45 were from the Netherlands. They originated from various clinical specimens, viz., tissue biopsy specimens, lung aspirates, sputa, sinus aspirates, bronchoalveolar lavage specimens, and endotracheal aspirates. The isolates were identified by sequencing the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (22). The sequences were aligned, and species identification was performed using GenBank basic local alignment search tool (BLAST) searches. Furthermore, all of the isolates were subjected to AFST using CLSI BMD as described in the CLSI standard M38-A2 (18) and using the EUCAST method as outlined in the EUCAST document EDef 9.2 (20). The antifungals tested include AMB (Sigma, St. Louis, MO, USA), VRC (Pfizer, Groton, CT, USA), POS (Merck, Whitehouse Station, NJ, USA), and ISAV (Basilea Pharmaceutica, Basel, Switzerland). The final concentrations of drugs used for the two methods ranged from 0.03 to 16 μg/ml for AMB and VRC and 0.015 to 8 μg/ml for ISAV and POS. The inoculum was prepared from mold colonies subcultured on potato dextrose agar plates at 35°C for 5 days. In CLSI BMD, RPMI 1640 containing 0.2% glucose buffered to a pH of 7 with 0.165 mol/liter 3-N-morpholinepropanesulfonic acid (Sigma-Aldrich, Germany) was used with a final inoculum of 0.5 to 2.5 × 104 CFU/ml. However, for the EUCAST method, RPMI 1640 medium with 2% glucose and a final inoculum of 1 to 2.5 × 105 CFU/ml were used. Drug-free and mold-free controls were included, and microtiter plates were incubated at 35°C; MIC values were determined visually after 24 h of incubation. However, isolates showing poor growth at 24 h were read after 48 h of incubation. Control strains Paecilomyces variotii (ATCC MYA-3630), Aspergillus flavus (ATCC 204304), and Aspergillus fumigatus (ATCC 204305) were used in each test. The MICs were the lowest drug concentrations that showed 100% inhibition of growth. The MIC results of the two methods were statistically analyzed using SPSS version 20.0 (SPSS, Chicago, IL, USA). Discrepancies of more than ±2 log2 dilutions were used to calculate the EA between the two methods.
ITS sequencing identified Mucorales to the species level. These results are presented in Table 1. The most common Mucorales were species of Rhizopus (n = 74), including Rhizopus arrhizus, Rhizopus microsporus, and Rhizopus stolonifer. Other Mucorales genera included Lichtheimia (n = 13), Rhizomucor (n = 8), Mucor (n = 7), Apophysomyces (n = 3), Cunninghamella (n = 4), and Syncephalastrum (n = 15). Table 1 summarizes the in vitro susceptibilities of 124 isolates of Mucorales to AMB, ISAV, POS, and VRC as determined by the two methods at 24 h. However, a few isolates of R. stolonifer (n = 3), Apophysomyces elegans (n = 2), Lichtheimia ramosa (n = 1), and Syncephalastrum racemosum (n = 1) yielded poor growth at 24 h, and MICs were recorded at 48 h. Overall, AMB showed the most potent activity among all drugs tested with the two methods. The geometric mean (GM) MICs of AMB tested by the EUCAST method (0.33 μg/ml) were >3-fold dilutions higher than those of the CLSI method (0.09 μg/ml), which was statistically significant (P < 0.0001). Barring a solitary isolate of Cunninghamella elegans, all other Mucorales had AMB MICs of <1 μg/ml by each of the two methods. Notably, the AMB MIC90 values were 1- to 2-fold dilutions higher using the EUCAST method, except for the MIC90 of R. arrhizus (CLSI method, 0.25 μg/ml; EUCAST method, 2 μg/ml), which was >2-fold dilutions higher. Also, the GM MICs of POS (CLSI method, 0.9 μg/ml; EUCAST method, 2.1 μg/ml) and ISAV (CLSI method, 2.8 μg/ml; EUCAST method, 4.8 μg/ml) were within 2-fold dilutions higher using the EUCAST method than with the CLSI method. VRC exhibited no activity against Mucorales (GM MICs of 10.46 μg/ml by the CLSI method and 15.21 μg/ml by the EUCAST method). Overall, the EAs (MICs of more than ±2 log2 dilutions) between the two methods for testing AMB and POS, which are commonly used antifungals for therapy of mucormycosis, were variable, i.e., 66.1% for AMB and 87% for POS. However, high EAs were noted for ISAV (98.3%) and VRC (99.1%). This report represents the most extensive comparison of the two reference BMD methods for the testing of Mucorales to date. Previously, a solitary report on the in vitro activity of ISAV against 345 Mucorales isolates collected from 8 countries demonstrated that EUCAST guidelines appeared to generate modal MICs 2-fold higher than those generated using CLSI guidelines (23). However, these observations were based on data from a single center, which tested only 36 Mucorales isolates (23). Of the discrepancies (>2 log2 dilutions) noted between the EUCAST and CLSI results in the present study, the MIC values generated by EUCAST methods were higher than those obtained by CLSI methods in 61 (49%) instances (n = 42 AMB, n = 16 POS, n = 2 ISAV, n = 1 VRC). The largest number of discrepancies observed while comparing the EUCAST and CLSI methods occurred with R. arrhizus (n = 24, 39.3%) and R. microsporus (n = 11, 18%) isolates tested against AMB. Also, 5 and 6 discrepant results for POS were observed with R. arrhizus and R. microsporus, respectively. It is pertinent to mention here that although all of the isolates of R. arrhizus and R. microsporus showed growth at 24 h by the two methods, an overall more luxuriant growth was seen with the EUCAST method than with the CLSI method, which may be an explanation for the discrepant results in these two species. Furthermore, 6% of Mucorales isolates other than R. arrhizus and R. microsporus did not grow at 24 h using the CLSI method in the present study. Overall, using the CLSI method, 34% (n = 16/47) of R. arrhizus isolates and 26% (6/23) of R. microsporus isolates and about 65% of R. arrhizus isolates (62%, 29/47) and R. microsporus isolates (65%, 15/23) showed POS MICs above the proposed ECV (1 μg/ml) (Table 2). Notably, 27.6% (n = 13/47) of the R. arrhizus isolates and 39.1% (n = 9/23) of the R. microsporus isolates tested with POS were categorized as non-wild-type (non-WT) strains by EUCAST methods and as WT strains by CLSI methods (19). Similarly, 2 isolates each of Lichtheimia corymbifera and Mucor circinelloides tested with POS were categorized as non-WT strains by the EUCAST method and as WT strains by the CLSI method. Overall, 30.6% (38/124) and 58% (72/124) of the isolates had POS MICs of >1 μg/ml according to CLSI and EUCAST methods, respectively. ISAV showed variable activity across all of the Mucorales tested (CLSI method, 0.125 to 8 μg/ml; EUCAST method, 0.5 to 8 μg/ml), with low GM MICs observed for R. arrhizus (CLSI method, 1.76 μg/ml; EUCAST method, 3.2 μg/ml), R. microsporus (CLSI method, 2.32 μg/ml; EUCAST method, 4.65 μg/ml), and S. racemosum (CLSI method, 2.4 μg/ml; EUCAST method, 4 μg/ml). There are no breakpoints of POS and ISAV defined for any of the Mucorales, but the median plasma trough level in the phase 3 SECURE trial of ISAV (NCT00412893) was 3.9 μg/ml, which is much higher than that on record for POS. In addition, a higher area under the concentration-time curve (AUC) exposure is observed for ISAV (100 mg · h/ml) than with POS (15 to 35 mg · h/ml). In conclusion, as demonstrated previously with Aspergillus spp. (24), the susceptibility results obtained by the two reference methods were comparable when testing the triazoles for Mucorales. However, future studies focusing on the harmonization between the two methods are warranted.
TABLE 1.
In vitro susceptibilities of Mucorales (n = 124) to amphotericin B, posaconazole, isavuconazole, and voriconazole as determined by the CLSI and EUCAST broth microdilution methods
| Genus (no. of isolates tested) | Species (no. of isolates tested) | Parameter | Test result (μg/ml) for: |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| AMB |
POS |
ISAV |
VRC |
|||||||
| CLSI | EUCAST | CLSI | EUCAST | CLSI | EUCAST | CLSI | EUCAST | |||
| Rhizopus (74) | arrhizus var. delemar (25) | GM | 0.05 | 0.24 | 1.43 | 3.78 | 5.42 | 6.96 | 10.55 | 15.56 |
| MIC50 | 0.03 | 0.25 | 1 | 4 | 8 | 8 | 16 | 16 | ||
| MIC90 | 0.25 | 0.5 | 8 | 8 | 8 | 8 | 16 | 16 | ||
| Range | 0.03–0.5 | 0.03–0.5 | 0.125–8 | 0.5–8 | 1–8 | 2–8 | 4–16 | 8–16 | ||
| arrhizus var. arrhizus (22) | GM | 0.07 | 0.40 | 0.66 | 1.45 | 1.76 | 3.20 | 8.25 | 15.02 | |
| MIC50 | 0.06 | 0.5 | 0.5 | 1 | 1 | 4 | 8 | 16 | ||
| MIC90 | 0.25 | 2 | 8 | 8 | 8 | 8 | 16 | 16 | ||
| Range | 0.03–0.5 | 0.06–2 | 0.125–8 | 0.5–8 | 0.5–8 | 1–8 | 4–16 | 8–16 | ||
| microsporus (23) | GM | 0.11 | 0.53 | 0.78 | 2.70 | 2.32 | 4.65 | 9.02 | 15.06 | |
| MIC50 | 0.125 | 0.5 | 1 | 4 | 2 | 8 | 16 | 16 | ||
| MIC90 | 1 | 1 | 2 | 8 | 4 | 8 | 16 | 16 | ||
| Range | 0.03–1 | 0.125–1 | 0.125–8 | 0.5–8 | 0.5–8 | 1–8 | 1–16 | 8–16 | ||
| stolonifer (4) | Range | 0.03–0.06 | 0.125–0.5 | 0.125–0.5 | 0.125–1 | 0.25–1 | 0.5–2 | 4–8 | 8–16 | |
| Mucor (7) | circinelloides (5) | Range | 0.03–0.125 | 0.125–0.5 | 0.125–1 | 0.5–8 | 1–8 | 2–8 | 8–16 | 16 |
| ramosus (1) | MIC | 0.25 | 0.25 | 1 | 1 | 8 | 8 | 16 | 16 | |
| velutinosus (1) | MIC | 0.06 | 0.125 | 2 | 8 | 2 | 8 | 16 | 16 | |
| Lichtheimia (13) | ramosa (7) | Range | 0.06–1 | 0.125–1 | 0.5–8 | 0.5–8 | 0.5–8 | 1–8 | 8–16 | 8–16 |
| corymbifera (6) | Range | 0.06–0.5 | 0.125–1 | 0.125–8 | 0.5–8 | 2–8 | 2–8 | 8–16 | 16 | |
| Rhizomucor (8) | pusillus (7) | Range | 0.06–1 | 0.06–1 | 0.25–8 | 1–8 | 1–8 | 4–8 | 8–16 | 16 |
| miehei (1) | MIC | 1 | 2 | 0.125 | 2 | 8 | 8 | 16 | 16 | |
| Cunninghamella (4) | bertholletiae (3) | Range | 0.25–1 | 0.5–1 | 8 | 8 | 8 | 8 | 16 | 16 |
| elegans (1) | MIC | 2 | 8 | 8 | 8 | 8 | 8 | 16 | 16 | |
| Apophysomyces (3) | elegans (2) | Range | 0.06–0.125 | 0.125–0.5 | 0.25–0.5 | 1–2 | 0.25–4 | 8 | 4–16 | 16 |
| variabilis (1) | MIC | 0.06 | 0.5 | 0.5 | 1 | 4 | 8 | 8 | 16 | |
| Syncephalastrum (15) | racemosum (15) | GM | 0.06 | 0.15 | 0.62 | 1.14 | 2.40 | 4 | 11.05 | 16 |
| MIC50 | 0.06 | 0.125 | 0.5 | 1 | 4 | 4 | 16 | 16 | ||
| MIC90 | 0.125 | 0.5 | 4 | 8 | 8 | 8 | 16 | 16 | ||
| Range | 0.03–0.5 | 0.03–1 | 0.125–8 | 0.25–8 | 0.125–8 | 1–8 | 4–16 | 16 | ||
TABLE 2.
Distribution of MICs of amphotericin B, posaconazole, voriconazole, and isavuconazole against genera of Mucorales (n = 124) as determined by the CLSI and EUCAST broth microdilution methods
| Genus (no. of isolates tested) | Species (no. of isolates tested) | Drug | Test method | No. of isolates with an MIC (μg/ml) of: |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | ||||
| Rhizopus (74) | arrhizus var. arrhizus (22) | AMB | CLSI | 10 | 3 | 4 | 4 | 1 | |||||
| EUCAST | 1 | 4 | 4 | 8 | 2 | 3 | |||||||
| POS | CLSI | 4 | 5 | 4 | 3 | 2 | 1 | 3 | |||||
| EUCAST | 8 | 6 | 2 | 6 | |||||||||
| ISAV | CLSI | 6 | 6 | 2 | 2 | 6 | |||||||
| EUCAST | 5 | 5 | 4 | 8 | |||||||||
| VRC | CLSI | 5 | 11 | 6 | |||||||||
| EUCAST | 2 | 20 | |||||||||||
| arrhizus var. delemar (25) | AMB | CLSI | 14 | 4 | 3 | 3 | 1 | ||||||
| EUCAST | 1 | 3 | 7 | 14 | |||||||||
| POS | CLSI | 1 | 1 | 3 | 10 | 3 | 2 | 5 | |||||
| EUCAST | 1 | 3 | 5 | 4 | 12 | ||||||||
| ISAV | CLSI | 2 | 3 | 3 | 17 | ||||||||
| EUCAST | 2 | 1 | 22 | ||||||||||
| VRC | CLSI | 4 | 7 | 14 | |||||||||
| EUCAST | 1 | 24 | |||||||||||
| microsporus (23) | AMB | CLSI | 4 | 6 | 6 | 4 | 1 | 2 | |||||
| EUCAST | 1 | 3 | 11 | 8 | |||||||||
| POS | CLSI | 3 | 3 | 3 | 8 | 4 | 2 | ||||||
| EUCAST | 5 | 3 | 3 | 1 | 11 | ||||||||
| ISAV | CLSI | 1 | 5 | 7 | 8 | 2 | |||||||
| EUCAST | 1 | 5 | 5 | 12 | |||||||||
| VRC | CLSI | 1 | 5 | 5 | 12 | ||||||||
| EUCAST | 2 | 21 | |||||||||||
| stolonifer (4) | AMB | CLSI | 2 | 2 | |||||||||
| EUCAST | 1 | 1 | 2 | ||||||||||
| POS | CLSI | 1 | 1 | 2 | |||||||||
| EUCAST | 1 | 1 | 2 | ||||||||||
| ISAV | CLSI | 2 | 1 | 1 | |||||||||
| EUCAST | 1 | 2 | 1 | ||||||||||
| VRC | CLSI | 1 | 3 | ||||||||||
| EUCAST | 3 | 1 | |||||||||||
| Mucor (7) | circinelloides (5) | AMB | CLSI | 1 | 2 | 2 | |||||||
| EUCAST | 3 | 1 | 1 | ||||||||||
| POS | CLSI | 2 | 3 | ||||||||||
| EUCAST | 1 | 1 | 1 | 2 | |||||||||
| ISAV | CLSI | 2 | 1 | 2 | |||||||||
| EUCAST | 2 | 3 | |||||||||||
| VRC | CLSI | 2 | 3 | ||||||||||
| EUCAST | 5 | ||||||||||||
| ramosus (1) | AMB | CLSI | 1 | ||||||||||
| EUCAST | 1 | ||||||||||||
| POS | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| ISAV | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| VRC | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| velutinosus (1) | AMB | CLSI | 1 | ||||||||||
| EUCAST | 1 | ||||||||||||
| POS | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| ISAV | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| VRC | CLSI | 1 | |||||||||||
| EUCAST | |||||||||||||
| Lichtheimia (13) | corymbifera (6) | AMB | CLSI | 2 | 1 | 2 | 1 | ||||||
| EUCAST | 1 | 1 | 3 | 1 | |||||||||
| POS | CLSI | 2 | 1 | 1 | 2 | ||||||||
| EUCAST | 1 | 1 | 2 | 2 | |||||||||
| ISAV | CLSI | 1 | 2 | 3 | |||||||||
| EUCAST | 1 | 5 | |||||||||||
| VRC | CLSI | 1 | 5 | ||||||||||
| EUCAST | 1 | 5 | |||||||||||
| ramosa (7) | AMB | CLSI | 1 | 4 | 2 | ||||||||
| EUCAST | 1 | 2 | 2 | 2 | |||||||||
| POS | CLSI | 2 | 3 | 1 | 1 | ||||||||
| EUCAST | 3 | 1 | 1 | 2 | |||||||||
| ISAV | CLSI | 4 | 2 | 1 | |||||||||
| EUCAST | 1 | 2 | 4 | ||||||||||
| VRC | CLSI | 1 | 6 | ||||||||||
| EUCAST | 7 | ||||||||||||
| Rhizomucor (8) | pusillus (7) | AMB | CLSI | 2 | 2 | 2 | 1 | ||||||
| EUCAST | 1 | 1 | 1 | 3 | 1 | ||||||||
| POS | CLSI | 1 | 1 | 1 | 1 | 3 | |||||||
| EUCAST | 2 | 2 | 3 | ||||||||||
| ISAV | CLSI | 1 | 1 | 1 | 4 | ||||||||
| EUCAST | 1 | 6 | |||||||||||
| VRC | CLSI | 1 | 6 | ||||||||||
| EUCAST | 7 | ||||||||||||
| miehei (1) | AMB | CLSI | 1 | ||||||||||
| EUCAST | 1 | ||||||||||||
| POS | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| ISAV | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| VRC | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| Cunninghamella (4) | bertholletiae (3) | AMB | CLSI | 1 | 1 | 1 | |||||||
| EUCAST | 1 | 2 | |||||||||||
| POS | CLSI | 3 | |||||||||||
| EUCAST | 3 | ||||||||||||
| ISAV | CLSI | 3 | |||||||||||
| EUCAST | 3 | ||||||||||||
| VRC | CLSI | 3 | |||||||||||
| EUCAST | 3 | ||||||||||||
| elegans (1) | AMB | CLSI | 1 | ||||||||||
| EUCAST | 1 | ||||||||||||
| POS | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| ISAV | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| VRC | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| Apophysomyces (3) | elegans (2) | AMB | CLSI | 1 | 1 | ||||||||
| EUCAST | 1 | 1 | |||||||||||
| POS | CLSI | 1 | 1 | ||||||||||
| EUCAST | 1 | 1 | |||||||||||
| ISAV | CLSI | 1 | 1 | ||||||||||
| EUCAST | 2 | ||||||||||||
| VRC | CLSI | 1 | 1 | ||||||||||
| EUCAST | 2 | ||||||||||||
| variabilis (1) | AMB | CLSI | 1 | ||||||||||
| EUCAST | 1 | ||||||||||||
| POS | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| ISAV | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| VRC | CLSI | 1 | |||||||||||
| EUCAST | 1 | ||||||||||||
| Syncephalastrum (15) | racemosum (15) | AMB | CLSI | 5 | 5 | 4 | 1 | ||||||
| EUCAST | 1 | 2 | 7 | 2 | 2 | 1 | |||||||
| POS | CLSI | 1 | 6 | 1 | 5 | 2 | |||||||
| EUCAST | 3 | 1 | 7 | 2 | 2 | ||||||||
| ISAV | CLSI | 1 | 6 | 2 | 6 | ||||||||
| EUCAST | 3 | 2 | 3 | 7 | |||||||||
| VRC | CLSI | 2 | 4 | 9 | |||||||||
| EUCAST | 15 | ||||||||||||
ACKNOWLEDGMENTS
This study was made possible by a grant from Basilea Pharmaceutica, Basel, Switzerland.
We thank Ilse Curf-breuker for expert technical assistance.
J.F.M. received grants from Astellas, Basilea, and Merck. He has been a consultant to Astellas, Basilea, and Merck and has received speaker's fees from Merck and Gilead. All other authors declare no conflicts of interest.
The authors alone are responsible for the content and writing of the paper.
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