840686 research-article2019 VDIXXX10.1177/1040638719840686Quantification of M. pachydermatis in samples by qPCRPuig et al. Full Scientific Report Journal of Veterinary Diagnostic Investigation Quantification of Malassezia pachydermatis 2019, Vol. 31(3) 440­–447 © 2019 The Author(s) Article reuse guidelines: by real-time PCR in swabs from the sagepub.com/journals-permissions DOI: 10.1177/1040638719840686 https://doi.org/10.1177/1040638719840686 jvdi.sagepub.com external ear canal of dogs Laura Puig, Gemma Castellá,1 F. Javier Cabañes Abstract. Malassezia pachydermatis is part of the normal microbiota of canine skin and external ear canal, and is also associated with otitis externa in dogs. Laboratory detection of Malassezia otitis relies on the presence of elevated numbers of the yeast on cytologic examination of otic exudate. Although cytology has high specificity, it has low sensitivity, resulting in false-negatives and posing a challenge for clinicians to accurately diagnose Malassezia otitis. We developed a quantitative PCR (qPCR) to detect and quantify M. pachydermatis yeasts and validate the method with swabs from external ear canals of dogs. Our qPCR uses the β-tubulin gene, a single-copy gene, as a target. The limit of quantification was established as 0.18 ng/ reaction, equivalent to 2.0 × 104 genome equivalents (gEq). Swabs from healthy dogs yielded quantification values of ≤2.7 × 104 gEq in the qPCR, whereas swabs from dogs with otitis yielded quantification values of ≥2.5 × 105 gEq. Our qPCR assay provides accurate quantification of M. pachydermatis yeasts from swab samples from dogs, is more sensitive than cytology, and could be used to monitor response to treatment. Our assay could also be valuable in a research setting to better understand the pathogenesis of M. pachydermatis. Key words: β-tubulin; dogs; Malassezia pachydermatis; otitis; quantitative PCR Introduction susceptibility testing allows monitoring for the emergence of azole-resistant strains of M. pachydermatis.20 A comparison The lipophilic yeast Malassezia pachydermatis is a normal of cytologic examination and fungal culture concluded that inhabitant of healthy canine skin and mucosae.17 The skin cytologic examination has good specificity but low sensitiv- population of M. pachydermatis on dogs can overgrow and ity.11 Consequently, there is a need for a specific, sensitive, act as an opportunistic pathogen, causing dermatitis and oti- precise, and rapid method to detect and quantify M. pachy- tis externa in animals affected with atopic dermatitis, inter- dermatis yeasts from dogs with otitis externa. trigo, endocrinopathies, and primary keratinization defects. Several qPCR assays have been developed to detect and These concurrent diseases can occur without necessarily quantify the most frequently isolated Malassezia species favoring the yeast infection.6 Factors that favor proliferation from human skin, mainly M. globosa and M. restricta.1,15,32,33 of M. pachydermatis and the transition from a commensal to Those assays usually amplified rRNA genes, which have a pathogen on canine skin have not been fully clarified, been shown to be present in multiple copies in the genomes although they seem related to skin disturbances by physical, of ascomycete and basidiomycete fungi,3,28,31 and were chemical, and immunologic mechanisms.36 designed to study the distribution of Malassezia species on Otitis externa is a common and multifactorial cutaneous human skin rather than the absolute quantification of these disorder affecting up to 20% of dogs seen by veterinarians.2 yeasts. Otitis externa associated with M. pachydermatis is often We developed a fast, sensitive, and accurate technique to characterized by the presence of a waxy, moist, and dark exu- reliably detect and quantify M. pachydermatis yeasts from date, with erythema and pruritus, and lesions that are often the external ear canal of dogs. We selected the β-tubulin gene colonized by an increased number of M. pachydermatis.17 as an alternative qPCR target to the rRNA genes because it Diagnosis of otitis externa caused by M. pachydermatis is based on the observation of compatible lesions, the response to antifungal therapy, and the presence of elevated numbers Veterinary Mycology Group, Department of Animal Health and of the yeast by direct microscopic observation.6 Microbio- Anatomy, Universitat Autònoma de Barcelona, Bellaterra, Catalonia, Spain. logic culture is not routinely performed, and it is required 1 Corresponding author: Gemma Castellá, Grup de Micologia only when direct microscopy is negative in animals with sus- Veterinària, Departament de Sanitat i d’Anatomia Animals, Facultat de pected infections.11 However, quantitative culture has been Veterinària, Universitat Autònoma de Barcelona, E-08193 Bellaterra, suggested to have diagnostic value.4,5 Moreover, culture and Barcelona, Spain. Gemma.Castella@uab.cat Quantification of M. pachydermatis in samples by qPCR 441 Table 1. Malassezia spp. strains used, including the host species and location from which they were retrieved, β-tubulin sequence, and melting temperature (Tm) of the PCR product. Species Strain Host Location β-tubulin GenBank accession (genotype) Tm (°C) M. pachydermatis CBS1879 Dog Ear KC573803 (I) 79.4 CBS1884 Dog Ear KU313727 (II) 79.6 CBS6535 Dog Ear KC573803 (I) 79.5 MA 13 Dog Ear KC573803 (I) 79.6 MA 52 Dog Ear KU313727 (II) 80.3 MA 56 Dog Ear KU313727 (II) 80.3 MA 107 Goat Ear KU313728 (III) 79.8 MA 140 Cat Ear KU313729 (IV) 79.3 MA 195 Dog Ear KC573803 (I) 79.3 MA 280 Dog Ear KU313728 (III) 80.4 MA 312 Cat Ear KU313730 (V) 79.8 MA 356 Dog Ear KU313731 (VI) 80.1 MA 475 Pig Ear KU313732 (VII) 80.1 MA 579 Cat Skin KU313729 (IV) 79.4 MA 1382 Dog Ear KU313733 (VIII) 80.2 M. arunalokei CBS13387 Human Scalp MF185816 — M. brasiliensis CBS 14135 Parrot Beak KR872311 — M. caprae CBS10434 Goat Ear KC573795 — M. cuniculi CBS11721 Rabbit Skin KC573808 — M. dermatis CBS9169 Human Skin KC573796 — M. equina CBS9969 Horse Skin KC573798 — M. furfur CBS1878 Human Skin KC573799 — M. globosa CBS7966 Human Skin KC573806 78.6 M. japonica CBS9431 Human Skin KC573801 — M. nana CBS9557 Cat Ear HM594270 — M. obtusa CBS7876 Human Skin KC573802 70 and 79.3 M. psittaci CBS 14136 Parrot Beak KR872310 — M. restricta CBS7877 Human Skin KC573807 — M. slooffiae CBS7956 Pig Skin KC573805 80.1 M. sympodialis CBS7222 Human Skin KC573797 — M. yamatoensis CBS9725 Human Skin KC573804 — Dash (—) = no melting peak detected. CBS = Centraalbureau voor Schimmelcultures, Westerdijk Fungal Biodiversity Institute, Utrech, The Netherlands; MA = culture collection of the Veterinary Mycology group, Universitat Autònoma de Barcelona, Bellaterra, Catalonia, Spain. has been shown to be present only in a single copy in closely Eleven swabs were collected from the external ear canals related species including M. globosa,38 and it has also been of 7 healthy dogs with no visible signs of otitis externa. proposed as a phylogenetic marker for the differentiation of Eleven swabs were obtained from the external ear canal of Malassezia species.12 9 dogs in which otitis caused by M. pachydermatis was diagnosed by veterinary clinicians based on otoscopic examination (presence of inflammation, a waxy brown Materials and methods exudate, pruritus) and elevated numbers of yeast by direct microscopic observation. Also, 2 swabs from 1 dog with Strains and samples otitis externa caused by M. pachydermatis that had been We tested 15 M. pachydermatis strains representative of the treated topically for 15 d with miconazole, polymyxin B, 8 β-tubulin genotypes27 of this species with the developed and prednisolone (Surolan; Elanco España, Madrid, Spain) assay (Table 1), in order to ensure amplification of all geno- were processed. Samples from healthy dogs without otitis types of the target gene. Also, 16 strains, each representing a were obtained from the dog pound of the Autonomous different Malassezia species, were tested to assess the speci- University of Barcelona (UAB; Spain) following proce- ficity of the qPCR. dures approved by Ethics Committee on Animal and Human To validate the assay, 24 swabs from the external ear Experimentation from UAB and Generalitat de Catalunya canal of 17 dogs were analyzed. When possible, both ears (approval CEEAH 1240; date of approval 28 February were sampled regardless of the health status of the ear. 2012). Samples from cases of Malassezia otitis were 442 Puig et al. obtained during routine veterinary procedures and with ver- qPCR development bal owner consent with the collaboration of veterinary clini- We developed our qPCR following the defined criteria of cians from the Veterinary Teaching Hospital of the UAB. the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.8 The Microbiology assays were performed (Applied Biosystems 7500 real- In all cases, 2 swabs were obtained from the affected ear of time system; Thermo Fisher Scientific, Waltham, MA) the same dog. One swab was maintained at −20°C and using SYBR Green chemistry with the absolute quantifica- used for qPCR analysis. The second swab was used for tion method. classical microbiologic processing and cytologic examina- Optimal concentrations of the forward and reverse prim- tion. Briefly, one side of the swab was streaked on a Sab- ers were determined by testing concentrations of 100– ouraud glucose agar (SGA) plate (Oxoid, Madrid, Spain), 800 nM. Concentrations giving the lowest quantification with 0.05 g of chloramphenicol, and on a 5% sheep blood cycle (Cq) values for the standards were selected: 300 nM agar plate (BD Difco, Madrid, Spain). Plates were incu- for the forward primer and 500 nM for the reverse primer. bated at 37°C with 5% carbon dioxide for 5 d, and colony- The optimized reaction mix (20 µL final volume) contained forming units (CFU) were counted. M. pachydermatis was 10 µL of 2× SYBR Green PowerUp PCR master mix identified by microscopic morphology and by the ability to (Applied Biosystems, Foster City, CA), 0.45 µL of 13.3 μM grow on SGA.16 The other side of each swab was rolled on forward primer, 0.75 µL of 13.3 μM reverse primer, 3.8 µL a clean glass slide and stained (Diff-Quick stain; Microp- of deionized DNase- and RNase-free water, and 5 µL of tic, Barcelona, Spain). The stained slides were examined template DNA. Runs were performed using the following microscopically (1,000× magnification). Yeast cells with thermal cycling conditions: 1 cycle of 50°C for 2 min, 1 morphology compatible with M. pachydermatis were cycle of 95ºC for 2 min, 30 amplification cycles of 95°C for counted in 10 random fields, and the presence of other 15 s (denaturing step), and 60°C for 1 min (annealing-exten- fungi and bacteria were also noted. sion step and data collection). Next, a melting curve analy- sis was performed, with a gradual increase of temperature from 60°C to 95°C. DNA extraction To achieve absolute quantification of the samples, the M. pachydermatis strains were grown on SGA; strains of the standard curve method was used. A standard curve of other Malassezia species were grown on modified Dixon genomic DNA (gDNA) extracted from the neotype strain of agar.16 After 5 d of incubation at 32°C, DNA was extracted M. pachydermatis CBS1879 was constructed, including 8 from yeast colonies (FastDNA spin kit protocol, FastPrep ten-fold dilutions, from 180 ng to 0.018 pg of DNA. The FP-24 instrument; MP Biomedicals, Biolink, Barcelona, quantity and purity of the gDNA were determined by spec- Spain). DNA was extracted from swabs (UCP Pathogen kit; trometry (NanoDrop 2000; Thermo Scientific, Barcelona, Qiagen, Madrid, Spain) according to the protocol recom- Spain). The amount of gDNA was extrapolated to genome mended for swab samples and using pre-treatment with equivalents (gEq), considering that β-tubulin is a single- mechanical disruption. A sterile swab was processed under copy gene in the M. pachydermatis genome, the size of the same conditions as the external ear canal samples in order which is ~8.15 Mbp.35,37 to control cross-contamination. DNA was stored at −20°C Efficiency was assessed in every run, based on the corre- until used as template in the qPCR. lation coefficient (r2) and slope values from the standard curve, and sensitivity was evaluated with the limit of quanti- fication (LOQ; the lowest concentration of DNA amplified Primer design from the standard curve). The linear dynamic range was Primers were designed to amplify a fragment of the β-tubulin determined (highest to lowest gDNA dilution amplified from gene of M. pachydermatis. Sequences used for primer design the standard curve).8 were obtained in a previous study in which 8 genotypes of To test for inhibition of the qPCR, gDNA standards this gene were described among M. pachydermatis strains.27 were spiked with randomly selected samples from dogs. Primers MPFRT-2 (5’-CGGACGAGACGTTCTGCATT-3’) The Cq values of spiked standards were compared to the and MPRRT-2 (5’-TTGAGTGTGCGGAAGCAGAT-3’) Cq values obtained from the same non-spiked standards to were designed (Primer Express v.3.0; Applied Biosystems, ensure that amplification was equivalent. Non-template Foster City, CA). Primer dimers and secondary structure for- controls (NTCs), in which DNA was substituted by water mation were evaluated (Primer Express v.3.0; IDT UNAFold in the reaction mix, allowed verification that no contami- online tool, https://eu.idtdna.com/calc/analyzer). Both prim- nation occurred and no primer dimers were formed. In ers were checked against the NCBI database using Primer- each run, all samples were run in triplicate, including the BLAST to confirm specificity. standards and NTCs. Quantification of M. pachydermatis in samples by qPCR 443 Statistical analysis Microbiology and validation of the qPCR using Statistical analyses were performed (Minitab 17 statistical clinical samples software; Minitab, State College, PA). A multiple regres- The presence and amount of M. pachydermatis yeasts of sion analysis was used to study the relationship between the samples from the external ear canal of dogs were also qPCR quantification values and values obtained by cyto- assessed by plate counting and cytologic examination (Table logic examination and plate counts. The Ryan–Joiner nor- 2). Samples from dogs without otitis yielded no growth of M. mality test was applied to determine whether data followed pachydermatis or plate counts of 0–8 CFU/plate (median: 3 a normal distribution. Differences in cytologic examina- CFU/plate); yeast cytology was negative in most of the sam- tion, CFU, and qPCR quantification values between healthy ples (8 of 11; range: 0–2; median: 0 cells/field). In samples dogs and dogs with Malassezia otitis externa were tested by from dogs with Malassezia otitis, cytology revealed 1–5 M. Kruskal–Wallis test. Samples with a qPCR value less than pachydermatis cells/field (median: 2 cells/field); plate counts the detection limit were assigned to 2 × 104 gEq value for were 10–250 CFU/plate (median: 210 CFU/plate). In most of statistical analysis. Samples from dog 8 and dog 17 were the samples (7 of 11), bacterial colonies also grew on blood excluded from the statistical analysis. The significance agar plates, mostly gram-positive cocci. level for all statistical analyses was set at p ≤ 0.05. In the developed qPCR, quantification values of 2.0 to 2.7 × 104 gEq were obtained from samples from healthy dogs Results (median: 2.0 × 104 gEq); values from 2.5 × 105 to 1.9 × 106 gEq (median: 3.8 × 105 gEq) were obtained from samples Primer design and specificity from dogs with otitis caused by M. pachydermatis (Table 2). Multiple regression analysis showed that the relationship (R2 The primers amplified a 61-bp amplicon with no identity = 67.0%) between qPCR quantification and cytologic exam- to the dog genome. A BLAST search against the NCBI ination and plate counts in the model was statistically sig- database showed no identity to other commensal and nificant (p < 0.001). Statistically significant differences in pathogen bacteria or fungi that can be found in the ear cytologic examination, plate counts, and qPCR quantifica- canals of dogs. In addition to the β-tubulin gene from M. tion values were found between samples from healthy dogs pachydermatis, the next sequence matches belonged to and dogs with otitis externa (Table 3). organisms that are not related with domestic carnivores, Melting curve analyses provided a single peak in all cases, such as a mushroom (Coprinopsis argentea), animals with Tms of 79.4–80.4°C (Fig. 1). Amplification of NTCs (Drosophila navojoa, Cyprinus carpio, Esox lucius), and a was not detected, and inhibition was not detected in gDNA plant (Nelumbo nucifera). standards spiked with samples from dogs. The primers consistently amplified the DNA from all tested M. pachydermatis strains. Melting curve analyses yielded a single peak in all M. pachydermatis strains at Discussion 79.3–80.4°C depending on the genotype (Table 1). No detectable amplification of other Malassezia species was The external ear canal of dogs can be colonized by a variety observed, except for M. globosa, M. slooffiae, and M. of bacterial and fungal species. In previous massively paral- obtusa. The M. globosa strain showed a single peak with a lel sequencing studies, it was found that the predominant clearly different melting temperature (Tm) of 78.6°C; the bacteria of the external ear canal of dogs are members of M. obtusa strain gave a melting curve with 2 peaks. For the phyla Proteobacteria, Actinobacteria, and Firmicutes,30 and M. slooffiae strain, a single dissociation peak with a Tm of some of the predominant fungi correspond to members of 80.1°C was obtained. genera Alternaria, Cladosporium, Epicoccum, and Crypto- coccus, as well as Malassezia spp.21 A BLAST search showed that our primers did not match any of the deposited sequences qPCR performance of any of the organisms found in dog ear canals in 2 prior A fragment of the β-tubulin gene from M. pachydermatis studies.21,31 Our designed primers showed amplification of was consistently amplified in a linear dynamic range of 4 all tested M. pachydermatis strains with consistent Tms ten-fold dilutions of gDNA, from 2.0 × 107 to 2.0 × 104 gEq. within 1°C of variation. Although a slight range of variation The standard curve yielded r2 values > 0.990 in all runs, can occur in melting curve analyses depending on factors and slope values of approximately −3.40 (Supplementary such as SYBR Green or DNA concentrations, an interval Fig. 1). All replicates were amplified with high precision, variation of ±1°C among Tms is considered to be acceptable with <1 Cq of variation among them and standard devia- for specificity determination.7,22,29 Amplification of the 16 tion values <0.4. The LOQ was established as 0.18 ng/ other Malassezia species was not detected, except for M. glo- reaction, equivalent to 2.0 × 104 gEq, which corresponds to bosa, M. obtusa, and M. slooffiae. Although these 3 species the lowest standard dilution amplified and accurately provided Tms similar to M. pachydermatis strains, M. glo- quantified (Cq ≥ 28). bosa and M. obtusa could be differentiated by melting curve 444 Puig et al. Table 2. Results obtained in samples from dogs with otitis externa by Malassezia pachydermatis and healthy dogs, including cytologic examination, plate counts, and qPCR quantification values, expressed as genome equivalents (gEq). Swab Dog/ear Otitis† Cells/field‡ CFU/plate gEq 1 1, left No 0 3 <2.0 × 104¦ 2 1, right No 0 8 <2.0 × 104¦ 3 2, right No 0 0 <2.0 × 104¦ 4 3, left No 0 6 <2.0 × 104¦ 5 3, right No 0 3 <2.0 × 104¦ 6 4, left No 0 0 <2.0 × 104¦ 7 4, right No 0 0 <2.0 × 104¦ 8 5, left No 1 1 <2.0 × 104¦ 9 5, right No 2 4 2.7 × 104 10 6, right No 1 4 <2.0 × 104¦ 11 7, right No 0 0 <2.0 × 104¦ 12 8, left Yes NA 179 1.9 × 106 13 8, right Yes NA 91 7.3 × 105 14# 9, left Yes 1 10 3.4 × 105 15# 10, left Yes 1 57 3.8 × 105 16# 11, left Yes 3 >250 5.4 × 105 17 12, left Yes 2 134 7.9 × 105 18 12, right Yes 1 40 1.2 × 106 19# 13, left Yes 2 223 1.1 × 106 20# 14, NA Yes 5 210 2.7 × 105 21# 15, NA Yes 2 >250 2.5 × 105 22# 16, NA Yes 4 >250 3.0 × 105 23§ 17, left Yes 1 0 6.8 × 104 24§ 17, right Yes 1 0 <2.0 × 104¦ CFU = colony-forming units; NA = no available data. † Diagnosed as otitis externa caused by M. pachydermatis when the sample was taken from the external ear canal of the dog. ‡ Average number of cells/field of morphology compatible with M. pachydermatis, counted in 10 fields by direct observation, at 1,000× magnification. § Samples from different ears of the same dog, which had been treated for 2 wk with antifungal medication because of otitis caused by M. pachydermatis. ¦ Quantification value below the limit of detection of the developed assay. # Bacterial growth on blood agar plates. Table 3. Mean values and standard deviation for cytologic Some qPCR methods have been developed for quantification examination, plate counts, and qPCR quantification values, of different Malassezia species from human skin. In these expressed as genome equivalents (gEq), between samples from methods, LOQ ranged from 102 to 105 amplicon copies per healthy dogs and dogs with otitis externa caused by Malassezia reaction depending on the Malassezia species detected.26,33 pachydermatis. All of these qPCR assays are based on multi-copy genes and, Healthy dogs Dogs with otitis p value therefore, are more sensitive than single-copy genes, although quantification is less accurate.18 Yeast/field 0.364 ± 0.674 2.333 ± 1.414 0.001 Statistically significant quantitative differences were CFU/plate 2.636 ± 2.730 158.2 ± 99.33 0.000 gEq 2.1 × 104 ± 2.1 × 103 5.7 × 105 ± 3.7 × 105 0.000 found by all of the methods used (cytologic examination, cul- ture, and qPCR) in samples from dogs according to their health status (healthy or otitis caused by M. pachydermatis). analysis. Nonetheless, amplification of these species should Samples from healthy dogs yielded plate counts ≤8 CFU/ not be considered as a disadvantage of our technique. M. glo- plate, a maximum of 2 M. pachydermatis cells/field on cyto- bosa, M. obtusa, and M. slooffiae have never been isolated logic examination, and qPCR quantification values of ≤2.7 × from dogs using culture-based methods9,13,34 or detected by 104 gEq. Samples from dogs with otitis yielded plate counts molecular-based methods,19 to our knowledge. There is only ≥10 CFU/plate, 1–5 M. pachydermatis cells/field on cytologic one report of M. obtusa associated with canine otitis examination, and qPCR quantification values of ≥2.5 × externa.14 However, this isolate was identified only by phe- 105 gEq. No colonies were grown on plates from the swabs notypic characteristics,14 and a subsequent molecular analy- obtained from a dog that had been treated for M. pachyderma- sis showed the isolate to be M. nana.10 tis otitis externa, and qPCR quantification values similar to Our qPCR assay has a LOQ of 0.18 ng of M. pachyderma- the LOQ were obtained from these samples. M. pachyderma- tis DNA, which corresponded to 2.0 × 104 gEq/reaction. tis is considered a secondary cause that contributes to otitis Quantification of M. pachydermatis in samples by qPCR 445 Figure 1. A. Amplification plot (delta Rn = normalized reporter value [Rn] – Rn of baseline signal; green horizontal line = baseline) and B. melting curve analysis of standard 3 (1.8 ng gDNA/reaction, equivalent to 2.0 × 105 genome equivalents) and 4 swab samples from dogs (8, 11, 12, 16), after 30 cycles. Swabs 12 and 16 were obtained from dogs in which Malassezia pachydermatis otitis externa was diagnosed; swabs 8 and 11 were obtained from healthy dogs. only in the abnormal ear or in combination with predisposing breeds.6 Although it is not known whether there is a thresh- or perpetuating factors.25 old population density needed for infection, the population For some authors, the observation of >5–10 cells of mor- of M. pachydermatis in dogs with skin disease can be phology compatible with M. pachydermatis in several micro- increased 100–10,000 fold.6 An accurate quantification of M. scopic fields or >70 CFU per sample from ear specimens of pachydermatis yeasts in swab samples from dogs can be dogs should be considered as an abnormal increase of Malas- achieved with our qPCR method. sezia.2,11,17,23 However, these proposed guidelines are not In some cases in our study, results from cytology, cul- generally accepted because a small population of the yeast ture, and qPCR did not correlate. This disagreement could might cause disease in sensitized animals, and variations in be explained in part by the fact that cytology has low sen- population sizes have been observed between different sitivity.11 Also, diverse factors can affect the number of M. 446 Puig et al. pachydermatis colonies grown on a culture plate. As an References example, M. pachydermatis has a tendency to form clumps, 1. Akaza N, et al. Cutaneous Malassezia microbiota of healthy meaning colonies could have originated from a clump of subjects differs by sex, body part and season. J Dermatol cells instead of a single yeast cell.24 Moreover, CFU reflect 2010;37:786–792. the number of live yeast, whereas the qPCR will give the 2. Angus JC. Otic cytology in health and disease. Vet Clin North total amount of the target gene detected, including both Am Small Anim Pract 2004;34:411–424. live and dead yeast. 3. Black J, et al. Determining fungi rRNA copy number by PCR. 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