Diagnostic Microbiology and Infectious Disease Evaluation of fluconazole resistance mechanisms in Candida albicans clinical isolates from HIV-infected patients in Brazil Gustavo H. Goldmana,*, Ma´rcia Eliana da Silva Ferreiraa, Everaldo dos Reis Marquesa, Marcela Savoldia, David Perlinb, Steven Parkb, Patricio Christian Godoy Martinezc, Maria Helena S. Goldmand, Arnaldo L. Colomboc aFaculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil bPublic Health Research Institute, USA cDivision of Infectious Diseases, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil dFaculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil Received 15 January 2004; accepted 8 April 2004 Abstract
In this study, we describe resistance mechanisms in fluconazole-resistant isolates of C. albicans isolated from AIDS patients from nine Brazilian hospitals. These mechanisms include the presence of point mutations in the ERG11 gene and overexpression of ERG11, andseveral genes encoding efflux pumps, as measured by quantitative real-time reverse transcriptase polymerase chain reaction. Severalfluconazole-resistant strains had multiple mechanisms of resistance. Four mutations previously described, Y132F, K143R, E266D, andV437I, were identified among the strains, whereas some isolates contained more than one mutation. Fourteen novel mutations wereidentified. Interestingly, all Brazilian fluconazole-resistant isolates showed homozygosity at mating-type loci (MTL) associated withfluconazole resistance. This is the first comprehensive assessment at molecular level of mechanisms of fluconazole resistance in C. albicansisolates from South America. 2004 Elsevier Inc. All rights reserved.
1. Introduction
that causes oral, vaginal, and systemic infections Triazole drugs such as fluconazole and The frequency of life-threatening fungal infections is itraconazole are commonly used to treat Candida infections.
rising worldwide. Considering that most patients infected However, resistant strains often emerge during long-term or with opportunistic fungal agents have AIDS or neoplastic and/or degenerative diseases, it is clear that effective anti- mechanisms of fluconazole resistance have been identified so far in these strains: (I) alterations in the drug target (14-␣-sterol demethylase, the product of the ERG11 gene), with improvements in performance and standardization of which results in an increased level of production of the antifungal susceptibility testing, have drawn attention to the enzyme or in its reduced binding affinity for fluconazole, problem of antifungal drug resistance. It is now well estab- and (II) a reduced level of intracellular fluconazole, which lished that antifungal agents foster clinical and epidemio- correlates with the overexpression of the CDR1 and CDR2 logical situations that are analogous to those found with genes encoding transporters of the ABC family and of the antibiotic-resistant bacteria (For a review, see MDR1 and FLU1 genes coding for major facilitators The predominant cause of fungal infections in hospital- ized patients remains Candida albicans, a pathogenic yeast It has already beenobserved that multiple mechanisms of fluconazole resis-tance can arise in a single C. albicans isolate * Corresponding author. Tel.:ϩ0055-016-6024280/81; fax:ϩ0055- E-mail address: [email protected] (G.H. Goldman).
In this study, we evaluate resistance mechanisms of flu- 0732-8893/04/$ – see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.diagmicrobio.2004.04.009 G.H. Goldman et al. / Diagnostic Microbiology and Infectious Disease 50 (2004) 25–32 Table 1Patient demographics, MIC values of clinical isolates, and ERG11 sequence analysis for isolate collection a Isolates were obtained from nine hospitals throughout Southeast Brazil.
NA, not available; M, male; F, female; ND, not detected; NE, not evaluated; FLC, fluconazole; ITRA, itraconazole; KTC, ketoconazole.
conazole-resistant strains of C. albicans isolated from AIDS different patient and was epidemiologically distinct. After patients from different medical institutions in Brazil. Our being identified by conventional methods data show that some of the fluconazole-resistant strains have all the strains were frozen and maintained in the diverse mechanisms of resistance, including the presence of yeast stock collection of the Laboratorio Especial de Mico- point mutations in the ERG11 gene and overexpression of logia-UNIFESP for different periods of time. Isolates were ERG11, and several genes encoding efflux pumps, as mea- maintained in solid yeast-peptone-glucose (YEPD: 1% sured by quantitative real-time RT-PCR. To our knowledge, yeast extract, 2% Bacto peptone, and 2% D-glucose, 2% this is the first assessment at molecular level of fluconazole resistance mechanisms in C. albicans isolates from SouthAmerica.
2. Materials and methods
Antifungal susceptibility testing was performed by using the National Committee for Laboratory Standards (NCCLS) 2.1. C. albicans strains and cell culture reference broth microdilution Referencepowders of fluconazole, and itraconazole and ketoconazole, The C. albicans isolates used in this study represent a were kindly provided by the manufacturers: Pfizer Pharma- collection of 20 strains from 9 different hospitals that were ceutical Group (New York, NY, USA) and Janssen (Titus- obtained from AIDS patients with oral or esophageal can- ville, NJ, USA), respectively. Amphotericin B was obtained didiasis who received fluconazole during a 6- to 12-month from Sigma. Breakpoints and interpretative criteria were period (See Each isolate was obtained from a based on the as described by White et al.
G.H. Goldman et al. / Diagnostic Microbiology and Infectious Disease 50 (2004) 25–32 Table 2List of primers and fluorescent probes used in this work C. albicans genes(NCBI accession number) 5Ј-FAM-CGA CCC GAG GTG CTG CCA TGT TCT TTG GGG TCG-Dabcyl-3Ј 5Ј-FAM-CCGTGGTGGGTGGATGCACTGGACAATTCCACGG-Dabcyl-3Ј 5Ј-FAM-CGACCCGTCTGCCATTGAATCTTTGGGGGTCG-Dabcyl-3Ј 5Ј-FAM-CCGTGGTGGGAAAGTTTCTAAAGGGGTTCCACGG-Dabcyl-3Ј 5Ј-CCGTGGAGTCCTTGTTTGGCCACTGGTGCCACGG-Dabcyl-3Ј 5Ј-FAM-CCGCTGAAAATTTTATATTGTGCATCTGCAGCGG-Dabcyl-3Ј MTLa F
MTLa (AF167163)
5Ј-ACGCGTCGACAATATGGCTATTGTTGAAACTGTC-3Ј 6-FAM, 6-carboxyfluorescein; Dabcyl, 4-(4Ј-dimethylaminophenylazo) benzoic acid succinimidyl ester; TAMRA, 6-carboxy-N,N,NЈ,NЈ-tetramethylrho- a Taq-Man probe (an oligonucleotide 5Ј-terminally labeled with a reporter fluorophor like fluorescein and labeled internally or 3Ј-terminally with a quencher); all the other probes are molecular beacons (are oligonucleotides labeled on both ends; one end is attached to a reporter fluorophor, and the otherend is attached to a quencher). For a review, see Controls for these experiments included previously minute, and 68°C for 2 minutes, followed by an extension characterized drug-susceptible isolates 5737 and 5568.
step at 68°C for 10 minutes. After the reaction, the approx-imately 1.7-kb PCR product was purified with a QiagenPCR cleanup kit and inserted into TOPO TA cloning kit 2.3. Sequencing of the ERG11 gene (Invitrogen) following manufacturer’s instructions. Se-quencing reactions were prepared using BigDyeTM Termi- The entire open reading frame of the ERG11 gene en- nator Cycle Sequencing (Applied Biosystems) and primers coding lanosterol 14␣- demethylase was sequenced from all described in The nucleotide sequences were deter- C. albicans isolates. The open reading frame was polymer- mined in both strands by primer elongation with an ase chain reaction (PCR) amplified using Taq DNA plati- ABI3100 automated DNA sequencer (Applied Biosystems).
num polymerase high fidelity (Invitrogen) and primers de- Sequence data were compared with a published ERG11 scribed in Table 2. PCR conditions were as follows: 94°C for 2 minutes and 35 times 94°C for 1 minute, 55°C for 1 G.H. Goldman et al. / Diagnostic Microbiology and Infectious Disease 50 (2004) 25–32 2.4. PCR analysis of the (mating type locus) MTL locus azoles (fluconazole, itraconazole, and ketoconazole) are sum-marized in The 20 C. albicans isolates included 9 that The PCR protocol used to analyze the MTL locus was the were resistant to fluconazole (MICs Ն64 ␮g/mL), 6 that were following: 94°C for 5 minutes, 35 times 94°C for 45 seconds, susceptible-dose dependent (S-DD for MICs of 16 and 32 55°C for 30 seconds, and 72°C for 1 minute, and an extension ␮g/mL), and 5 that were susceptible to fluconazole (MICs Ͻ8 step at 72°C for 10 minutes. The presence of MTLa was
␮g/mL). Regarding itraconazole, 1 isolate was considered re- ascertained by amplifying MTLa1 and the associated gene
sistant to itraconazole (MIC Ն1 ␮g/mL), 10 were considered PAPa The presence of MTL␣ was
as S-DD (MICs of 0.25 and 0.5 ␮g/mL), and 9 were suscep- ascertained by amplifying MAT␣1 and MAT␣2 and the asso- tible to itraconazole (MIC Ͼ 0.25 Ͻ0.5) All of them ciated gene PAP␣. These three different genes, MTL␣1, were susceptible to amphotericin B (MICs Ͻ1 ␮g/mL).
MTL␣2, and MTLa1, are the C. albicans homologues of Sac-
Clinical breakpoints for ketoconazole have not been pro- charomyces cerevisiae mating-type genes MAT␣1, MAT␣2, posed, but they are likely to be close to the breakpoints for and MATa1, respectively
itraconazole, because the drug concentrations achievable inblood are similar for both drugs when similar doses are ad- 2.5. DNA and RNA isolation, and real-time RT-PCR ministered We have considered an MIC ofՆ1 ␮g/mL to define ketoconazole resistance in this study, Yeast cells were grown to logarithmic phase in 125-mL susceptible-dose dependent (S-DD) for MICs of 0.25 and 0.5 Erlenmeyer flasks containing 25 mL of YEPD at 32°C with ␮g/mL, and finally MICs Յ0.125 for susceptible isolates.
constant shaking. DNA was prepared using glass beads Considering the mentioned breakpoints for ketoconazole, 11 out of 20 isolates were S-DD/resistant to ketoconazole.
scriptase polymerase chain reaction (RT-PCR) experiments, shows that 12 out 15 isolates S-DD or resistant the different isolates were propagated in YEPD medium and to fluconazole were also considered as S-DD (9 for itra and harvested while growing in antifungal drug-free medium in keto) or resistant (1 for itra and 2 for keto) to the other logarithmic phase. The cells were washed thoroughly with azoles. Otherwise, only 1 out of 5 isolates susceptible to sterile water, disrupted by vortexing with glass beads; then fluconazole exhibited an MIC value compatible with S-DD.
total RNA was extracted with Trizol (Life Technologies, Naturally occurring ERG11 mutations identified in C. USA). To verify RNA integrity, 20 micrograms of RNA albicans azole-resistant isolates are clustered in three dif- from each treatment were fractionated in a 2.2-M formal- fuse hot-spot regions in the primary sequence, including dehyde, 1.2% agarose gel, stained with ethidium bromide, amino acid regions 105 to 165, 266 to 287, and 405 to 488 and visualized with ultraviolet light. The presence of intact 28S and 18S ribosomal RNA bands (semiquantitatively in a of the entire ERG11 from our isolates was performed. As 2:1 ratio) was used as a criterion to determine whether the expected from unrelated clinical isolates, frequent silent RNA was degraded. RNAse-free DNAse treatment was mutations that do not change the protein sequence were identified (data not shown). The sequence alterations that The absence of DNA contamination after the RNAse-free resulted in changes in the protein sequences are listed in DNAse treatment was verified by PCR amplification of the Mutations that change the protein sequence were ACT1 gene. cDNA was synthesized by using the Super- not identified in the susceptible isolates 5737 and 5568.
Script reverse transcriptase (Gibco, BRL).
Several mutations, located in the three hot spots, were All the RT-PCR reactions were performed using an ABI observed in all the isolates, except for isolates 15, 16, 17, Prism 7700 Sequence Detection System (Perkin-Elmer Ap- 69, and 86, which did not show any mutation. Isolate 61 plied Biosystem, USA). The Taq- ManR PCR Reagent kit contained five mutations (K99T, G303D, L305P, G307S, was used for PCR reactions. The thermal cycling conditions and G450E). Isolate 8 contained three mutations (V130I, comprised an initial step at 50°C for 2 min, followed by 10 E266D, and V488I), whereas isolates 23, 33, 36, 51, and 68 minutes at 95°C, and 40 cycles at 95°C for 15 seconds and contained two mutations (Y132F and F145L; K145E and 60°C for 1 min. The reactions and calculations were per- P503L; Y79C and T199I; R265G and K342R; I253V and V437I, respectively). Isolates 14, 19, and 21 contained a single mutation (F380S, K143R, and H283D, respectively).
We did not find in these isolates mutations in only one allele(i.e., heterozygous for the mutation), but only point muta- 3. Results
tions in both alleles (i.e., homozygous for the mutation).
3.1. Antifungal susceptibility testing of clinical isolates 3.2. ERG11, CDR1, CDR2, MDR1, and FLU1 genes and sequencing of ERG11 genes The minimal inhibitory concentration (MIC) values ob- Overexpression of the genes ERG11, CDR1, CDR2, tained for 20 C. albicans isolates with the three different MDR1, and FLU1 has been linked to fluconazole resistance G.H. Goldman et al. / Diagnostic Microbiology and Infectious Disease 50 (2004) 25–32 Table 3ERG11 and efflux transporter gene mRNA expression levels in the clinical isolates as assessed by real-time RT-PCR a cDNA levels were calculated relative to those of the average cDNA levels of the isolates 5737 and 5568. The numbers represent the averages of 3 replicates, with the ranges shown in parentheses.
indicates that multiple mechanisms are operating to confer resistance in our clinical isolates by using real-time RT- fluconazole resistance in these isolates. However, in isolate PCR The cDNA levels of the different genes were 23, which is resistant to fluconazole and SDD to ketocon- normalized using the ACT1 gene (encoding actin); compa- azole, we were not able to see any of these genes overex- rable results were observed when the PMA1 gene (encoding pressed. Taken together, our results suggest that there is no the Hϩ-ATPase) was used as a normalizer gene (data not clear correlation among different levels of expression of the shown). Because a matched set of isolates was not available for this collection of fluconazole- resistant isolates, themRNA expression levels of isolates 5737 and 5568 were 3.3. PCR screen of the MTL loci of clinical isolates of used as controls. illustrates the number of times each respective gene was expressed greater than the averageexpression of the same gene in the control isolates (where the mRNA expression levels were given a value of 1.0).
When this criterion is used, the isolates that are resistant to fluconazole albicans. An analysis of 46 fluconazole expressed ERG11 at much higher levels (11.2 to 14.4 times) than the average in isolates 14, 16, 17, 68, and 72. The CDR1 gene was more highly expressed in isolates gous for the either a/a or ␣/␣), 22% of the
16, 68, and 72, whereas the CDR2 gene was more highly latter were investigated the MTL loci of expressed in the isolates 14, 16, 17, 33, 68, and 72. MDR1 our clinical amplifying three MTL genes: was more highly expressed in the isolate 33, whereas FLU1 MTLa1, 2. PCR fragments from MTLa1 was more highly expressed in isolates 8, 36, and 72. Con- and MTL1 for 10 of the 20 clinical isolates sidering the isolates SSD to fluconazole, the ERG11 is more expressed in the isolate 85, whereas the CDR1 gene is more expressed in the isolates 21 and 85. The CDR2 is more amplify MTL1 (Fig. 1A). None of these 10 isolates ho- expressed in the isolates 21, 69, 85, and 86. MDR1 is more mozygous at the MTL locus (MTLhom) were fluconazole expressed in the isolates 19 and 69, whereas the FLU1 is more expressed in the isolate 19. Interestingly, some iso- of the loss of heterogeneity, the presence of PAPa and lates that show susceptibility to fluconazole, such as 51, 55, PAP␣ was determined by PCR in the ten MTLhom strains and 61, show higher expression of MDR1 and CDR2, (Fig. 1B). The four strains missing MLT1 (MTLhom) were CDR2, and ERG11, respectively. Taken together, our results found to also lack MTL2. In addition, the PAPa gene was show that some isolates concomitantly overexpressed dif- present in only one of these MTLahom strains. The PAP␣ ferent genes involved in drug resistance in C. albicans. This gene was not present in all four MTL␣hom. The fact that the G.H. Goldman et al. / Diagnostic Microbiology and Infectious Disease 50 (2004) 25–32 Fig. 1. PCR screen of MTL genotype in clinical isolates. DNA fragments from within the MTLa1, MTL1, and MTL2 genes (A) and PAPa and PAP␣ (B)were amplified using genomic DNA from 20 clinical isolates and visualized on EtBr-stained gels. The order of the isolates is based on their MIC: I ϭ Ͻ0.25 ␮g/ml fluconazole (isolates 5568 and 5737); II ϭ 4 ␮g/ml (isolates 51 and 55); III ϭ 8 ␮g/ml (isolate 61); IV ϭ 16 ␮g/ml (isolates 19 and 86); V ϭ32 ␮g/ml (isolates 15, 21, 69, and 85); VI ϭ 64 ␮g/ml (isolates 8, 16, 17, 33, and 68); and VII ϭ Ͼ 64 ␮g/ml (isolates 14, 23, 36, and 72). Interestingly, allBrazilian fluconazole resistant isolates showed homozygosity at mating type (MTL) loci associated with fluconazole resistance.
loci-specific PAP and MTL genes do not segregate in these The fourth hot spot (comprising D116E, F126L, K128T, 10 MTLhom strains indicates that the loss of heterozygosity G129A, Y132H, K143R, F145L, K147R, A149V, and does not extend through the entire 9-Kb MTL locus in these D153E) is located in the region between the BЈ and C strains. Our results suggest that homozygosity at either helices that have been postulated to be involved in inhibitor- locus is associated with fluconazole resistance.
or substrate-induced structural changes. The mutationsidentified in C. albicans fluconazole-resistant isolates indi-cate that azole resistance in fungi develops in protein re- 4. Discussion
gions involved in orchestrating the passage of CYP51pthrough different conformational stages rather than in resi- The clinical isolates used in this study were screened for dues directly contacting the triazole. We were able to iden- the currently characterized molecular mechanisms of azole tify four mutations previously described as being involved resistance, and to our knowledge, this represents the first in azole resistance in C. albicans, Y132F (Y132H), K143R, time such an analysis has been performed on South Amer- ican C. albicans isolates. Naturally occurring ERG11 mu- Some of our isolates contained more than one mu- tations in C. albicans azole-resistant clinical isolates can be tation, and 14 novel mutations were identified among them.
divided into four hot-spot regions on the basis of their These novel mutations are being further characterized to association with different structural regions observed in the validate that they actually mediate fluconazole resistance.
recently described MTCYP51 structure However, their presence suggests that the clinical environ- The first hot spot (comprising G464S, G465S, and R476K) ment in Brazil can select novel mutations in C. albicans associates with the N-terminal part of the cysteine pocket. A second hot spot is mapped to the C-terminus of the G helix Real-time RT-PCR assays were used to obtain more and H helix, and a third hot spot (comprising F72L, F105L, accurate data on gene expression in C. albicans. This ap- S495F, and T229A) associates with the domain interface.
proach is preferable over conventional Northern blot anal- G.H. Goldman et al. / Diagnostic Microbiology and Infectious Disease 50 (2004) 25–32 ysis, because of the narrower linear range associated with PDR1 and PDR3, which positively influence expression of radioactively labeled probes, and has been successfully used drug transporter genes such as PDR5 in the characterization of gene expression of sigma factor genes in Mycobacterium tuberculosis has previously shown that gene conversion and, more recently, in the quantitative expression of or mitotic recombination associated with the ERG11 gene, ABC transporter encoding genes in Aspergillus spp. located on chromosome 5, is associated with azole resis- quantitative expression of two ABC transporters, CDR1 and fluconazole-resistant strains of C. albicans, there was a CDR2, and two MFS transporters, MDR1 and FLU1, and higher proportion of homozygotes for the mating-type locus also ERG11. Some clinical isolates showed increased (MTL) than in a collection of fluconazole- sensitive isolates, mRNA expression of the ERG11 gene, which encodes the suggesting the possibility that when cells become MTL homozygous, they acquire intrinsic drug resistance. ously shown, in a collection of fluconazole-resistant and used an opposite strategy to investigate this pos- -susceptible isolates, considerable variation in the levels of sibility. Instead of looking for fluconazole- resistant strains ERG11 mRNA expression, but this expression did not seem of C. albicans in a heterogeneous population of MTL ho- to be related to the azole resistance of the isolates. However, mozygotes and heterozygotes, drug susceptibility was mea- ERG11 overexpression has been found in many other flu-conazole-resistant C. albicans isolates compared with sured in a collection of isolates selected for MTL homozy- gosity. The majority of these isolates had not been exposed expression of ERG11 from C. albicans has been shown to to antifungal drugs. The level of drug susceptibility was confer a fivefold enhanced resistance to fluconazole in S. compared between spontaneously generated MTL-homozy- cerevisiae Therefore, constitutive gous progeny and their MTL- homozygous parent strains, ERG11 overexpression may contribute to fluconazole resis- which had not been exposed to antifungal drugs. The results tance in our clinical C. albicans isolates.
demonstrate that naturally occurring MTL-homozygous An important mechanism of fluconazole resistance is strains are not intrinsically more drug resistant, supporting reduced intracellular accumulation of the drug. the hypothesis that either the higher incidence of MTL demonstrated that many fluconazole-resistant, homozygosity involved a drug resistance gene linked to the clinical C. albicans isolates displayed strongly increased MTL locus, or that MTL-homozygous strains may be better mRNA levels of CDR1 or MDR1 compared to the parental at developing drug resistance upon exposure to drug than strains and accumulated less intracellular fluconazole.
MTL-heterozygous strains. We have found that the Brazil- CDR2 was also observed in fluconazole-resistant clinical C. ian fluconazole-resistant isolates are homozygous at either albicans isolates Recently, a gene locus associated with fluconazole resistance.
that is homologous to MDR1, FLU1, has been isolated by its In summary, we demonstrated that (i) the same mecha- ability to confer fluconazole resistance on hypersusceptible nisms of fluconazole resistance already described in Euro- pean and North American C. albicans isolates are present expression of CDR1, CDR2, and MDR1 genes has been also in Brazilian isolates, and (ii) in spite of higher mRNA shown to be the most frequent mechanism of fluconazole expression levels for ERG11 and transporter genes, and different mutations in the fluconazole-resistant isolates, clinical isolates showed increased mRNA expression of there was no linear relationship between these phenomena these transporters. In addition, three clinical isolates also and MIC values. Putative resistance mechanisms can be showed increased mRNA expression of FLU1. None of the found even in susceptible isolates. This suggests that these transporter genes was induced in the clinical isolate 23.
mechanisms alone are not sufficient, and most probably However, this isolate has a F145L mutation in the ERG11gene that may be responsible for the observed fluconazole there are other putative mechanisms of resistance not yet resistance. Only two fluconazole-resistant isolates were described that could provide a better correlation with drug truly resistant to itraconazole or ketoconazole, but six of them were considered as S-DD to itraconazole and/or keto-conazole. This reduced drug susceptibility could be due tothe high levels of mRNA expression of the transporter genes Acknowledgments
exhibited by most of these isolates.
Some clinical isolates, such as 68 and 72, showed in- We thank the Fundac¸a˜o de Amparo a Pesquisa do Estado creased mRNA expression of more than one transporter de Sa˜o Paulo and the Conselho Nacional de Desenvolvi- gene. These clinical isolates could harbor dominant muta- mento Cientifico e Tecnolo´gico, Brazil, for financial sup- tions that resulted in concomitant constitutive transcrip- port, and Dr. P. Magee for the oligonucleotide sequences of tional activation of the transporter genes. Such behavior is the mating-type loci genes. We also thank the two anony- well known for the S. cerevisiae transcriptional activators mous reviewers for critical reading of the manuscript.
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1 School of Biological Sciences, Victoria University, P.O. Box 600, Wellington. Present address: Science Directorate, Department of Conservation, P.O. Box 10420, Wellington. 2 Forest Research Institute, P.O. Box 31-011, Christchurch. Present address: Advocacy and Extension Directorate, Department of Conservation, P.O. Box 10420, Wellington. GEOGRAPHIC PATTERNS OF GENETIC VARIATION INBRUSHTAIL

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