A 5-year-old neutered female toy Poodle chronically treated with systemic and topical azoles to control recurrent Malassezia dermatitis/otitis was presented because of the loss of treatment efficacy. Minimum Inhibitory Concentrations (MICs) obtained in vitro for various azoles (especially itraconazole and ketoconazole) against Malassezia strains isolated from the dog were increased by several-fold compared with MICs obtained for control isolates. These results reinforced the assumption based on clinical observation, i.e. the development of azole resistance Keywords: Malassezia pachydermatis, Dog, Resistance, Azole

Andrea Peano

Mario Pasquetti

Martina Angileri

Michela De Lucia

Medical Mycology Case Reports

Pasquetti, Mario

Peano, Andrea

Institutional Research Information System University of Turin

Contents lists available at ScienceDirectMedical Mycology Case Reportsjournal homepage: www.elsevier.com/locate/mmcrAzole resistance of Malassezia pachydermatis causing treatment failure in adogMartina Angileria, Mario Pasquettib, Michela De Luciaa, Andrea Peanob,⁎a San Marco Veterinary Clinic, Via dell’Industria 3, 35030 Veggiano, ItalybDipartimento di Scienze Veterinarie, Università di Torino, Largo Paolo Braccini 2, 10095 Grugliasco, ItalyA R T I C L E I N F OKeywords:Malassezia pachydermatisDogResistanceAzolesA B S T R A C TA 5-year-old neutered female toy Poodle chronically treated with systemic and topical azoles to control recurrentMalassezia dermatitis/otitis was presented because of the loss of treatment efficacy. Minimum InhibitoryConcentrations (MICs) obtained in vitro for various azoles (especially itraconazole and ketoconazole) againstMalassezia strains isolated from the dog were increased by several-fold compared with MICs obtained for controlisolates. These results reinforced the assumption based on clinical observation, i.e. the development of azoleresistance1. IntroductionMalassezia pachydermatis is a lipophilic yeast that colonizes thestratum corneum and mucosal sites of dogs. Favorable growth condi-tions in the local environment allow excessive multiplication of thisorganism, which may then function as an opportunistic secondary pa-thogen [1]. Dogs may present with Malassezia otitis, dermatitis (eitherlocalized or generalized) or both. Clinical signs are variable and includeerythema, alopecia, mild–to-severe pruritus, greasy exudation andscaling. Secondary lesions include excoriations, lichenification, hy-perpigmentation and exudation. In generalized cases, a rancid odor iscommonly reported [1].The yeast overgrowth is generally secondary to an ongoing disorder,predominantly hypersensitivity diseases (especially atopy), keratiniza-tion defects, recurrent bacterial pyoderma and endocrine diseases(especially hypothyroidism) [1]. Both in the case of dermatitis andotitis, a hypersensitivity response to the yeast itself is likely to occur inmany allergic dogs [1]. Breed predisposition appears to be an importantrisk factor and cases without apparent underlying causes have also beenreported [2].Therapeutic options for treating Malassezia otitis/dermatitis includesystemic and/or topical antifungal agents belonging to various che-mical classes. Azole compounds, which inhibit the fungal biosynthesisof ergosterol by interacting with sterol-14α-demethylase [3], are widelyemployed [1]. Various antiseptics, such as chlorhexidine, are also ef-fective treatments [1].The possibility of developing azole resistance by M. pachydermatishas been frequently claimed in the literature on the basis of in vitrotests (e.g.in the studies by Nijima et al. [4] and Jesus et al.[5]), but onlyone case of in vivo resistance (i.e. treatment failure) has been reported,to date, in the dog [6]. Here we describe another case of M. pachy-dermatis that displayed in vivo resistance to azole compounds.2. CaseA 2.5-year-old neutered female toy Poodle was evaluated (day 0) forsevere, recurrent, generalized Malassezia dermatitis and otitis. Possiblepredisposing causes (allergic dermatitis, hypothyroidism, leishmaniasis,ecto-parasite infestation etc.) were investigated and ruled out. Due tothe “idiopathic” nature of the yeast overgrowth, the following protocolwas adopted: itraconazole oral solution (Itrafungol® Eli Lilly Italia Spa,Sesto Fiorentino, Italy) 5mg/kg twice a week on 2 consecutive days;2% chlorhexidine and 2% miconazole shampoo (Malaseb®, DechraVeterinary Products A/S, Denmark) once a week; ear drops containingmiconazole, prednisone and polymyxin B (Surolan®, Elanco AnimalHealth; Basingstoke, UK), applied twice a day for 2 weeks wheneverneeded. This protocol allowed an excellent control of clinical signs, aswell as a reduction in the number of yeast cells detected on skin and earcytological samples. The treatment protocol had to be maintained on along-term basis, since dermatological problems recurred when with-drawal was attempted.About two years and a half later (day+ 900), although the therapyhad not been changed, severe scaling dermatitis, pruritus and offensiveodor developed, associated with an increase in the number of yeast cellshttps://doi.org/10.1016/j.mmcr.2018.12.004Received 28 November 2018; Received in revised form 17 December 2018; Accepted 20 December 2018⁎ Corresponding author.E-mail address: andrea.peano@unito.it (A. Peano).Medical Mycology Case Reports 23 (2019) 58–61Available online 21 December 20182211-7539/ © 2018 Published by Elsevier B.V. on behalf of International Society for Human and Animal Mycology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).Tin the skin and ear cytological samples. Treatment frequency was in-creased, as follows: itraconazole oral solution 5mg/kg once daily; 2%chlorhexidine and 2% miconazole shampoo every 2 days. However,there was neither improvement in dermatological signs, nor a reductionin the number of yeast cells in skin and ear cytological samples.The dog was presented for a new evaluation (day+930). Thedermatological picture consisted of numerous small white and yellowscales distributed over the entire surface of the body, including thelimbs and the head, which were loosely attached to the hairs or stuck tothe skin surface, partial alopecia together with pruritus and markedoffensive odor (Fig. 1). Bilateral ceruminous otitis was also present(Fig. 2). Numerous Malassezia yeasts (> 30× high-power field) weredetected on multiple skin and ear cytological samples (Fig. 3).Parasitic diseases and concurrent bacterial or dermatophyte infec-tions were ruled out based on the administration of selamectin spot on(Stronghold® Zoetis 1348 Louvain-la-Neuve, Belgium) skin scrapings,direct hair examination, cytology and fungal cultures.The worsening of an unidentified predisposing condition, a con-comitant immunosuppressive disease, or the presence of azole re-sistance were considered as possible causes for the loss of treatmentefficacy. Concomitant systemic or dermatological problems were notdetected despite extensive clinical and laboratory investigations, in-cluding complete blood cell count, serum biochemistry panel, ur-inalysis, haemostatic profile, thyroid hormones and TSH measurement.In order to investigate possible azole resistance, acetate tape and swabsamples were collected from different body areas and both ear canalsand sent to a mycological research laboratory (day+ 940).All samples, cultured on Sabouraud dextrose agar (SDA) withchloramphenicol and gentamycin at 30 °C, yielded a profuse growth ofyeast colonies with microscopic appearance typical ofM. pachydermatis.The isolates employed for the in vitro tests (see below) were depositedin the collection of the Westerdijk Institute (formerly the FungalBiodiversity Center – CBS) of Utrecht, where identification was con-firmed at the molecular level.2.1. Antifungal susceptibility testing (day+ 950)Three isolates were tested for the susceptibility to the followingantifungal agents:- miconazole (MCZ), clotrimazole (CTZ) and terbinafine (TER) using amicroplate broth-dilution (MiB) method modified from the re-ference method for in vitro testing of yeasts by the Clinical andLaboratory Standard Institute (CLSI) [7] (method described indocument S1).- fluconazole (FCZ) and posaconazole (PSZ) using a commercial kit(E-test®, bioMérieux) (method described in document S1).- itraconazole (ITZ) and ketoconazole (KTZ), using both methods.Results were expressed as MICs (minimum inhibitory concentrationsof a drug able to inhibit fungal growth) [7]. For comparison, a referencestrain (strain CBS 1879, considered the M. pachydermatis “type strain”)was included in the tests. For ITZ, KTZ, MCZ and CTZ, MICs were alsocompared with MICs – available in the database of the laboratory -previously obtained for 10 isolates of the yeast coming from dogs neversubjected to antifungal therapies.MICs of different azoles – in particular of ITZ, KTZ and MCZ – wereincreased by several-fold compared with MICs obtained for the controlisolates (Table 1; Fig. 4).These results reinforced the assumption based on clinical observa-tion, i.e. the development of azole resistance.Topical and systemic azoles were replaced with TER (Lamisil®,Novartis Pharma AG; Basel, Switzerland; 30mg/Kg per os once a day)[8,9]. However, during the following month (day + 960–990), clinicalsigns worsened. Oral TER was thus suspended and an exclusively to-pical approach was attempted with 4% chlorhexidine digluconateshampoo (Chlorexyderm® 4%, ICF, Cremona, Italy) performed every 2days and an otic gel containing TER, dexamethasone and florphenicolFig. 1. Severe generalized scaling dermatitis due to M. pachydermatis in a 5-year-old neutered female toy Poodle. A large amount of scales has been shed onthe surface of the consulting table.Fig. 2. Scaling dermatitis with very mild erythema of the inner aspect of thepinna in the same dog as in picture 1. Ceruminous otitis was also present.Fig. 3. Numerous Malassezia yeasts visible on skin cytological sample (adhesivetape preparation, 40×) from a toy Poodle dog with severe generalized scalingdermatitis (Bar 10 µm).M. Angileri et al. Medical Mycology Case Reports 23 (2019) 58–6159(Osurnia®, Elanco Animal Health; Basingstoke, UK, twice a day). After 1month (day + 1020), skin and ear lesions had significantly improved,but moderate pruritus and offensive odor were still present. Un-fortunately, at that time cytology was not performed. Since then, dif-ferent shampoos containing chlorhexidine, miconazole, zinc gluconateand colloidal sulphur are being employed on a rotating basis, with a faircontrol of the clinical signs. Moreover, during rechecks, a large numberof Malassezia yeasts are always detected at cytology.Eleven months later (day+1350), other isolates were obtained inculture and tested in vitro. MICs for these isolates were consistentlyhigher than those for the control isolates (Table 1).3. DiscussionThis case confirms what has been reported recently [6], namely thatazole resistance can be the cause of treatment failure in a dog withMalassezia dermatitis/otitis. The previous report of resistance [6]concerned a 15-year old Miniature Dachshund dog in Japan, whereresistance was suspected because of the lack of response despite 23 daysof once-daily oral therapy with ITZ (8mg/kg) and weekly shampooingwith 2% miconazole/2% chlorhexidine shampoo. The clinical historyregarding the dog was not provided, in particular nothing was saidabout previous antifungal treatments [6]. On the contrary, our dog wasfollowed over an extended period, which allowed us to document that aprolonged exposure (years of treatment) to azole agents was required todevelop resistance.Antifungal susceptibility testing for M. pachydermatis must be in-terpreted with caution, as reference methods are not available [10]. Toovercome this limit we used two methods (i.e. MiB and E-test) [11–13]and we compared the MICs of the isolates from the index case with theMICs obtained for some field isolates and a reference strain of the yeast.Not unexpectedly, the absolute values were somewhat different be-tween the two methods (Table 1), but the same trend was observed, i.e.MICs of different azoles were increased for the six isolates comparedwith the control ones. One isolate in particular (strain CBS 15216) washighly resistant to MCZ and ITZ (where, in some cases, no MIC wasdetected, Fig. 4). These findings suggest that the skin of a given dogmay be colonized by strains of M. pachydermatis with different anti-fungal susceptibility profiles. This possibility has already been shownfor different bacterial species involved in canine otitis externa [14].Interestingly, azole agents that had not been used in this dog (PSZ,FCZ, KTZ) showed a reduced activity in vitro, suggesting a possiblecross-resistance of M. pachydermatis to different azoles. This phenom-enon has been hypothesized in previous studies based on in vitro results[5].Oral TER was ineffective in controlling clinical signs, despite therebeing a less marked difference between the MICs of the isolates and thereference strain (Table 1). This could be due to the lower concentrationsof TER in canine stratum corneum or sebum compared to serum [15].However, TER was active when employed topically, which can be ex-plained by the concentration of active principle in the commercialformulation (Osurnia®), which greatly exceeds the MIC, being in theorder of mg/ml.The fact that chlorhexidine only allowed partial control of clinicalsigns is surprising as an antiseptic should also maintain its activityagainst an azole-resistant strain of M. pachydermatis. This possible in-congruence could be explained by considering that the dog sufferedfrom an “idiopathic” form of Malassezia dermatitis and otitis, and insuch cases topical therapy alone is often insufficient [2].Another important consideration is that the MICs for the isolatessampled at a later date were consistently higher than those for thecontrol isolates (Table 1). This may indicate that resistance in M. pa-chydermatis, once established, is stable.Very little is known regarding possible mechanisms of resistance toazoles in M. pachydermatis. Iatta et al. [16] reported that resistance maydepend on increased efflux by pumps, particularly those belonging tothe “major facilitator superfamily”. The underlying cause may also beTable 1MICs (µg/ml) for six isolates ofM. pachydermatis obtained from the toy Poddle dog, a reference strain and 10 isolates of the yeast coming from dogs never subjected toantifungal therapies.ITZ KTZ MCZ TER CTZ PSZ FCZNumber Provenance MiB E-test MiB E-test MiB MiB MiB E-test E-testCBS 15214a skin (abdomen) 0.5 0.75 2 0.75 16 1 32 1.5 > 256CBS 15215a skin (dorsum) 1 0.75 2 1 16 1 16 1.5 128CBS 15216a right ear 8 > 32 16 1.5 > 32 4 16 4 > 256CBS 15208b right ear 1 0.75 4 1 16 1 32 2 128CBS 15212b skin (neck) 1 0.5 4 0.75 16 2 32 1.5 > 256CBS 15211b skin (left thigh) 1 0.75 4 0.5 16 2 32 1.5 > 256CBS 1879 reference strain 0.03 0.125 0.25 0.125 2 0.5 4 0.25 12/ dogs never subjected to antifungal therapiesc 0.016–0.06 0.023–0.094 0.03–0.125 0.19–0.125 1–4 / 1–16 / /a,b = obtained from the toy Poddle dog, a) on day +940; b) at the occasion of a follow up visit (day+1350); c = for these isolates (n=10) the range of MICsobtained is provided.Number= the accession number assigned by the fungal collection of the Westerdijk Institute (formerly the Fungal Biodiversity Center – CBS) of Utrecht.MiB=microplate broth-dilution method. ITZ= Itraconazole; MCZ=Miconazole; TER=Terbinafine; KTZ=Ketoconazole; CTZ=Clotrimazole;PSZ=Posaconazole; FCZ=Fluconazole.Fig. 4. Results of the E-test assay for two isolates of M. pachydermatis againstitraconazole. Left: reference strain CBS 1079 (MIC 0.125 µg/ml); right: isolateCBS 15216 - sampled from the toy Poddle dog - (MIC> 32 µg/ml).M. Angileri et al. Medical Mycology Case Reports 23 (2019) 58–6160due to mutations in the gene encoding the enzyme sterol-14α-de-methylase. These mutations were found in the resistant strain isolatedin Japan [6]. This theory could benefit from the availability of the re-sistant strains we have deposited in a culture collection, which can beused for further experiments.In conclusion, this case shows that the development of azole re-sistance of clinical relevance is possible for strains of M. pachydermatisharbored by dogs. However, it is important to emphasize that the un-ique conditions that lead to resistance (an unusually long azole treat-ment necessary to treat an “idiopathic” and chronic overgrowth ofMalassezia) are uncommon in clinical practice.AcknowledgementsNone.Conflict of interestThere are none.Appendix A. Supplementary materialSupplementary data associated with this article can be found in theonline version at doi:10.1016/j.mmcr.2018.12.004.References[1] T.-A. Chen, P.B. Hill, The biology of Malassezia organisms and their ability to in-duce immune responses and skin disease, Vet. Dermatol. 16 (2005) 4–26.[2] R. Bond, E.A. Ferguson, C.F. Curtis, J.M. Craig, D.H. Lloyd, Factors associated withelevated cutaneous Malassezia pachydermatis populations in dogs with pruritic skindisease, J. Small Anim. Pract. 37 (1996) 103–107.[3] H. Vanden Bossche, M. Engelen, F. Rochette, Antifungal agents of use in animalhealth–chemical, biochemical and pharmacological aspects, J. Vet. Pharmacol.Ther. 26 (2003) 5–29.[4] M. Nijima, R. Kano, M. Nagata, A. Hasegawa, H. Kamata, An azole-resistant isolateof Malassezia pachydermatis, Vet. Microbiol. 149 (2011) 288–290.[5] F.P.K. Jesus, C. Lautert, R.A. Zanette, D.L. Mahl, M.I. Azevedo, M.L. Machado, et al.,In vitro susceptibility of fluconazole-susceptible and -resistant isolates of Malasseziapachydermatis against azoles, Vet. Microbiol. 152 (2011) 161–164.[6] R. Kano, S. Yokoi, N. Kariya, K. Oshimo, H. Kamata, Multi-azole-resistant strain ofMalassezia pachydermatis isolated from a canine Malassezia dermatitis, Med. Mycol.(2018).[7] S. Arikan, Current status of antifungal susceptibility testing methods, Med. Mycol.45 (2007) 569–587.[8] M.S. Rosales, R. Marsella, G. Kunkle, B.L. Harris, C.F. Nicklin, J. Lopez, Comparisonof the clinical efficacy of oral terbinafine and ketoconazole combined with cepha-lexin in the treatment of Malassezia dermatitis in dogs - a pilot study, Vet. Dermatol.16 (2005) 171–176.[9] J. Guillot, E. Bensignor, F. Jankowski, W. Seewald, R. Chermette, J. Steffan,Comparative efficacies of oral ketoconazole and terbinafine for reducing Malasseziapopulation sizes on the skin of Basset Hounds, Vet. Dermatol. 14 (2003) 153–157.[10] A. Peano, M. Pasquetti, P. Tizzani, E. Chiavassa, J. Guillot, E. Johnson,Methodological Issues in Antifungal Susceptibility Testing of Malassezia pachy-dermatis, J. Fungi 3 (2017) 37.[11] C. Cafarchia, L.A. Figueredo, R. Iatta, V. Colao, M.T. Montagna, D. Otranto, In vitroevaluation of Malassezia pachydermatis susceptibility to azole compounds using E-test and CLSI microdilution methods, Med. Mycol. 50 (2012) 795–801.[12] A. Peano, M. Beccati, E. Chiavassa, M. Pasquetti, Evaluation of the antifungal sus-ceptibility of Malassezia pachydermatis to clotrimazole, miconazole and thiabenda-zole using a modified CLSI M27-A3 microdilution method, Vet. Dermatol. 23 (2012)131–135 (, e29).[13] M. Pasquetti, E. Chiavassa, P. Tizzani, P. Danesi, A. Peano, Agar diffusion proce-dures for susceptibility testing of Malassezia pachydermatis: evaluation of Mueller-Hinton Agar Plus 2% glucose and 0.5 µg/ml methylene blue as the test medium,Mycopathologia 180 (2015) 153–158.[14] C.A. Graham-Mize, E.J. Rosser, Comparison of microbial isolates and susceptibilitypatterns from the external ear canal of dogs with otitis externa, J. Am. Anim. Hosp.Assoc. 40 (2004) 102–108.[15] J.R. Gimmler, A.G. White, R.A. Kennis, C. Cruz-Espindola, D.M. Boothe,Determining canine skin concentrations of terbinafine to guide the treatment ofMalassezia dermatitis, Vet. Dermatol. 26 (2015).[16] R. Iatta, M.R. Puttilli, D. Immediato, D. Otranto, C. Cafarchia, The role of drugefflux pumps in Malassezia pachydermatis and Malassezia furfur defence againstazoles, Mycoses 60 (3) (2016) 178–182.M. Angileri et al. Medical Mycology Case Reports 23 (2019) 58–6161

English

Azole resistance of Malassezia pachydermatis causing treatment failure in a dog

Directory of Open Access Journals

https://iris.unito.it/bitstream/2318/1690124/1/1-s2.0-S2211753918301441-main.pdf

Azole resistance of Malassezia pachydermatis causing treatment failure in a dog

Abstract

Similar works

Full text

Available Versions

Institutional Research Information System University of Turin

Directory of Open Access Journals