Molecular identification of Trichophyton benhamiae in Strasbourg, France: a 9-year retrospective study

Molecular identification of Trichophyton benhamiae in Strasbourg, France: a 9-year retrospective... Abstract Trichophyton benhamiae is a zoophilic dermatophyte transmitted to humans mostly from guinea pigs and occasionally other animals. It presents two distinct phenotypes: yellow and white. T. benhamiae was formerly known as Trichophyton species of Arthroderma benhamiae; it was considered part of the T. mentagrophytes species complex, and some authors have incorrectly described the yellow phenotype of T. benhamiae as T. mentagrophytes var. porcellae. Identification of T. benhamiae has been difficult, as it was described under more than three names, two phenotypes, and in several different possible host species. During the past 15 years, human infections due to this dermatophyte have been increasingly reported all over the world. In order to better understand the local epidemiology of T. benhamiae and to compare it to other European countries, we performed a 9-year retrospective study in the Strasbourg University Hospital. We studied 41 dermatophytes (38 isolated from humans and 3 from guinea pigs) identified as T. mentagrophytes var. porcellae or A. benhamiae from January 2008 to December 2016 and verified their identification by ITS (Internal Transcribed Spacer) sequencing. ITS sequencing was performed in 35 of the 41 strains, and they were identified as T. benhamiae (33), T. bullosum (1), and T. eriotrephon (1). The other six remaining strains were identified according to morphology as T. mentagrophytes var. porcellae, name incorrectly used since 2010 for the yellow phenotype of T. benhamiae. ITS sequencing is recommended for accurate identification of this dermatophyte and the culture phenotype (yellow or white) should be specified. Trichophyton benhamiae, Arthroderma benhamiae, guinea pig, zoophilic dermatophyte, T. mentagrophytes var. porcellae, T. bullosum Introduction Dermatophytes are a group of keratinophilic filamentous fungi that can infect animals and humans. Dermatophytosis, commonly called ringworm, is usually limited to hair, nails, and the keratinized layers of the skin.1 These mycoses are transmissible diseases and can clinically range from mild to severe, depending on the host's immune status, the strain virulence, and other environmental factors.1 According to their habitat, dermatophytes can be divided into anthropophilic (human), zoophilic (animal) and geophilic (soil)1. The latter two types tend to cause more inflammatory human lesions that the former. Dermatophytes belong to four genera: Epidermophyton, Trichophyton, Microsporum, and Nannizzia, with only the first two being truly anthropophilic.2 The genus Trichophyton is the one most frequently isolated from humans, especially in regions with a temperate climate. Trichophyton benhamiae (comb. nov.)2 is a zoophilic species transmitted to humans mostly from guinea pigs and occasionally rabbits, cats and dogs. During the past 15 years, it has been described in animals in Japan, Europe, and the United States.3,4,5,6,7 Two phenotypes have been described for T. benhamiae: yellow and white.8,9,10,11,12,13,14 The yellow phenotype strains are downy, with a pleated mycelium, a yellow-orange reverse, and a slow growth rate. Sporulation is poor on Sabouraud agar, with rare microconidia and no macroconidia or spiral hyphae. Subcultures on other media (potato dextrose agar, diluted Sabouraud agar or M40Y) can enhance sporulation.8,10 The main differential diagnosis is Microsporum canis, also often macroscopically yellow, but presenting 6–12 celled macroconidia, with thick cell walls and thinner septa.15 The white phenotype strains are powdery to floccose, with a yellow, orange, or brown reverse and a rapid growth rate. Microconidia are numerous, spherical to clavate; macroconidia are sparse, 3–8 celled, smooth- and thin-walled, clavate to cigar-shaped; spiral hyphae are occasionally present.2,10 The main differential diagnosis for the T. benhamiae white phenotype is T. mentagrophytes, which presents numerous spherical microconidia and frequent spiral hyphae, aside from the clavate to cigar-shaped macroconidia. In the past years, the nomenclature of dermatophytes has undergone some changes. Initially, species were defined according to clinical data, and morphological and physiological characteristics. Thus, T. benhamiae was initially known as Trichophyton sp. of Arthroderma benhamiae20,21,22 and it was considered to be part of the T. mentagrophytes9,21,22,23,24,25,26,27,28,29 species complex. But dermatophytes presenting different phenotypes can have the same genotype, or vice versa. This is perfectly illustrated by the fact that in 2010 Contet-Audonneau and Leyer invalidly introduced the name T. mentagrophytes var. porcellae for the already described yellow phenotype Trichophyton sp. of A. benhamiae.8 The rapid development of molecular methods in the past 20 years has revolutionized the dermatophyte taxonomy. Based on sequencing the ITS ribosomal DNA region, seven clades have been described. The upper clade A comprises the Trichophyton species, with clade A-1 corresponding to T. mentagrophytes. Trichophyton sp. of Arthroderma benhamiae is no longer considered part of the T. mentagrophytes species complex; it became T. benhamiae (comb. nov.), which formed the A-2 clade together with T. schoenleinii and T. verrucosum. Clade A-3 is represented by the zoophilic species T. bullosum.2 Having more than three different names, two different phenotypes, and several different possible hosts rendered difficult the identification of T. benhamiae. This dermatophyte has been reported since 2001 from humans in Japan,3,16 Switzerland,4,10,17 Germany,5,14,18 France,8 Belgium,9 and the United Kingdom19. In recent years in Japan, T. benhamiae has become the second most frequent dermatophyte after M. canis and a study performed in Germany between March 2010 and March 2013 showed that T. benhamiae had already become the most frequent zoophilic dermatophyte responsible of human infections, with a prevalence of 2.9%.13,14 In the Medical Mycology laboratory of the Strasbourg University Hospital we look for dermatophytes in 1400 to 1750 samples per year, and rare or interesting strains are stored in a mycology bank since 2008. We performed a 9-year retrospective study, in order to better understand the local epidemiology of T. benhamiae and to compare it to that of the other European countries or regions. Methods Strains Strains were isolated in our laboratory from clinical samples sent to our diagnostic laboratory in sterile Petri dishes, or from strains sent to us for identification by external sources (private laboratories or smaller hospitals). For each sample of sufficient quantity, a direct examination with KOH 30% and culture on in-house slant media (Sabouraud Chloramphenicol Dextrose Agar and Sabouraud Chloramphenicol Dextrose Cycloheximide Agar) were performed. The cultures were incubated at 27°C for 4 to 6 weeks and examined twice a week. No direct examination was performed for strains sent to us for identification. In-house media plates (potato-dextrose agar, water agar, Borelli's lactrimel agar, and diluted Sabouraud dextrose agar) were used for subcultures of all the strains. They were incubated at 27°C for 3 to 10 days and examined twice a week. The urea hydrolysis activity was tested using Christensen's urea broth test.30 Subcultures of the strains were inoculated in 1 ml of ready-to-use Christensen's urea broth (Sigma-Aldrich) incubated at 27°C and examined after 3 and 7 days. All 41 strains were identified morphologically (T. mentagrophytes var. porcellae), and 35 were confirmed by DNA sequencing (33 A. benhamiae/T. benhamiae, 1 T. bullosum and 1 T. eriotrephon). The remaining six strains were not available for sequencing at the time of this study. DNA extraction, amplification, and sequencing Confirmation of the initial identification was performed retrospectively by sequencing the Internal Transcribed Spacer (ITS) region of the ribosomal DNA using primers ITS1 (5΄-TCCGTAGGTGAACCTGCGG-3΄) and ITS4 (5΄-TCCTCCGCTTATTGATATGC-3΄) as previously described.31 Briefly, DNA was extracted and purified directly from fungal colonies with a Qiagen QIAamp® DNA mini kit according to manufacturer's instructions. The polymerase chain reaction (PCR) mixture (40 μl) included 3 mM MgCl2 (Qiagen, Germany), 200 μM of each deoxynucleoside triphosphate (dNTP) (Euromedex, France), 0.2 μM of each primer, and 0.2 μM of Hotstar Taq DNA polymerase (Qiagen, Germany). The thermal cycler (Applied BioSystems, Foster City, CA, USA) was set for initial denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 95°C for 1 min, annealing for 1 min at 54°C, and extension for 2 min at 72°C. A final extension step at 72°C for 7 min was included at the end of the amplification. The PCR products were electrophoresed in 2% agarose (Eurogentec, Belgium) for 30 min at 150 V and viewed in gel documentation Gel Doc EZ System (BioRad, France) and stored at −20°C until they were sent (to GATC Biotech, Germany) for ITS sequencing. Resulting sequences were compared to GenBank, CBS, ISHAM and EMBL databases. Phylogenetic analysis ITS sequences from our strains were aligned with reference strains belonging to Arthroderma benhamiae complex using MUSCLE (Mega® 6.0 software), and the best model for phylogenetic analyses was identified using default setting. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura 3-parameter model. The percentage of trees in which the associated taxa clustered together is shown above the branches (10000 replicates). Initial trees for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The rate variation model allowed for some sites to be evolutionarily invariable. Evolutionary analyses were conducted in MEGA6. Trichophyton rubrum strains IHEM13800 (JQ407179) and ATCC28188 (AF170472) were used as outgroup. Results Between 2008 and 2016 we received 1390 (2009) to 1732 (2015) samples per year for dermatophytes analysis. About 50% had positive cultures for dermatophytes, moulds, and yeasts. About 10% of the samples were positive for zoophilic dermatophytes, mostly T. mentagrophytes, M. canis and T. benhamiae (Table 1). Table 1. Main zoophilic dermatophytes identified in our laboratory from 2008 to 2016. Year  Total samples  Positive samples (%)  T. mentagrophytes (%)  T. benhamiae (%)  M. canis (%)  T. verrucosum (%)  2008  1698  996 (58.65)  98 (9.84)  3 (0.3)  4 (0.4)  1 (0.1)  2009  1390  787 (56.61)  97 (12.32)  1 (0.13)  5 (0.63)  1 (0.12)  2010  1432  794 (55.44)  71 (8.94)  3 (0.37)  6 (0.75)  1 (0.1)  2011  1577  846 (53.44)  64 (7.56)  9 (1.06)  7 (0.82)  1 (0.12)  2012  1382  701 (50.72)  68 (9.7)  6 (0.85)  4 (0.57)  0  2013  1452  717 (49.38)  75 (10.46)  3 (0.41)  14 (1.95)  2 (0.28)  2014  1602  763 (47.62)  60 (7.86)  9 (1.18)  3 (0.4)  0  2015  1732  797 (46)  63 (7.9)  2 (0.25)  7 (0.87)  0  2016  1720  786 (45.69)  63 (8.01)  3 (0.38)  15 (1.65)  1 (0.12)  2008–2016  13985  7187 (51.4)  658 (9.15)  40 (0.55)  65 (0.9)  7 (0.09)  Year  Total samples  Positive samples (%)  T. mentagrophytes (%)  T. benhamiae (%)  M. canis (%)  T. verrucosum (%)  2008  1698  996 (58.65)  98 (9.84)  3 (0.3)  4 (0.4)  1 (0.1)  2009  1390  787 (56.61)  97 (12.32)  1 (0.13)  5 (0.63)  1 (0.12)  2010  1432  794 (55.44)  71 (8.94)  3 (0.37)  6 (0.75)  1 (0.1)  2011  1577  846 (53.44)  64 (7.56)  9 (1.06)  7 (0.82)  1 (0.12)  2012  1382  701 (50.72)  68 (9.7)  6 (0.85)  4 (0.57)  0  2013  1452  717 (49.38)  75 (10.46)  3 (0.41)  14 (1.95)  2 (0.28)  2014  1602  763 (47.62)  60 (7.86)  9 (1.18)  3 (0.4)  0  2015  1732  797 (46)  63 (7.9)  2 (0.25)  7 (0.87)  0  2016  1720  786 (45.69)  63 (8.01)  3 (0.38)  15 (1.65)  1 (0.12)  2008–2016  13985  7187 (51.4)  658 (9.15)  40 (0.55)  65 (0.9)  7 (0.09)  View Large Table 1. Main zoophilic dermatophytes identified in our laboratory from 2008 to 2016. Year  Total samples  Positive samples (%)  T. mentagrophytes (%)  T. benhamiae (%)  M. canis (%)  T. verrucosum (%)  2008  1698  996 (58.65)  98 (9.84)  3 (0.3)  4 (0.4)  1 (0.1)  2009  1390  787 (56.61)  97 (12.32)  1 (0.13)  5 (0.63)  1 (0.12)  2010  1432  794 (55.44)  71 (8.94)  3 (0.37)  6 (0.75)  1 (0.1)  2011  1577  846 (53.44)  64 (7.56)  9 (1.06)  7 (0.82)  1 (0.12)  2012  1382  701 (50.72)  68 (9.7)  6 (0.85)  4 (0.57)  0  2013  1452  717 (49.38)  75 (10.46)  3 (0.41)  14 (1.95)  2 (0.28)  2014  1602  763 (47.62)  60 (7.86)  9 (1.18)  3 (0.4)  0  2015  1732  797 (46)  63 (7.9)  2 (0.25)  7 (0.87)  0  2016  1720  786 (45.69)  63 (8.01)  3 (0.38)  15 (1.65)  1 (0.12)  2008–2016  13985  7187 (51.4)  658 (9.15)  40 (0.55)  65 (0.9)  7 (0.09)  Year  Total samples  Positive samples (%)  T. mentagrophytes (%)  T. benhamiae (%)  M. canis (%)  T. verrucosum (%)  2008  1698  996 (58.65)  98 (9.84)  3 (0.3)  4 (0.4)  1 (0.1)  2009  1390  787 (56.61)  97 (12.32)  1 (0.13)  5 (0.63)  1 (0.12)  2010  1432  794 (55.44)  71 (8.94)  3 (0.37)  6 (0.75)  1 (0.1)  2011  1577  846 (53.44)  64 (7.56)  9 (1.06)  7 (0.82)  1 (0.12)  2012  1382  701 (50.72)  68 (9.7)  6 (0.85)  4 (0.57)  0  2013  1452  717 (49.38)  75 (10.46)  3 (0.41)  14 (1.95)  2 (0.28)  2014  1602  763 (47.62)  60 (7.86)  9 (1.18)  3 (0.4)  0  2015  1732  797 (46)  63 (7.9)  2 (0.25)  7 (0.87)  0  2016  1720  786 (45.69)  63 (8.01)  3 (0.38)  15 (1.65)  1 (0.12)  2008–2016  13985  7187 (51.4)  658 (9.15)  40 (0.55)  65 (0.9)  7 (0.09)  View Large In our 9-year survey, 41 strains isolated from separate samples (38 human and 3 animal) and identified at that time as T. mentagrophytes var. porcellae or A. benhamiae were diagnosed in our laboratory by morphology or molecular identification. As this study is retrospective, some information is missing in some cases (Table 2). Table 2. List of strains used in this study. No.  Sex  Age  Year  Lesion localization  Contact with animals  Animal lesion  phenotype  Microscopy  Urea broth  Initial identification  ITS sequencing  GenBank Accession no.  Culture collection ID  Source  1  F  9  2008  Thorax (back)  NI  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885203  0801m210208  Our department  2  F  8  2008  NI  Rabbit  NI  White  abundant μ  +  A. benhamiae  T. benhamiae  KY885204  0802m180906  Our department  3  F  15  2008  Thigh  NI  NI  White  sterile  NP  A. benhamiae  T. bullosum  KY885205  0805m150877  External source  4  M  9  2008  Cheek  Cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885206  0810m040057  Our department  5  F  12  2009  Arm  NI  NI  Yellow  few μ  NP  A benhamiae  T. benhamiae  KY885207  0906m180356  External source  6  M  24  2009  Beard sycosis  NI  NI  White  μ and macro  +  A. benhamiae  T. eriotrephon  KY885208  0912m230081  Our department  7  M  8  2010  NI  NI  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885209  1001m210429  External source  8  F  4  2010  Face  Guinea pig, pony, cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885210  1005m220254  Our department  9  F  8  2010  Scalp kerion  Guinea pig  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885211  1012m200864  Our department  10  F  15  2011  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885212  1102m170903  External source  11  F  12  2011  Shoulder  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885213  1102m250627  External source  12  F  5  2011  Thorax (sternum)  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885214  1102m280391  External source  13  F  5  2011  Pubis, groin  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885215  1104m210816  External source  14  F  20  2011  Arm  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885216  1105m260453  External source  15  F  10  2011  Face, thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885217  1105m270378  External source  16  F  11  2011  Thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1108m030060  Our department  17  F  12  2011  Thorax  Guinea pig, hamster, gerbil, kitten  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885218  1110m250836  External source  18  M  12  2011  Abdomen  Rabbit  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885219  1111m220427  External source  19  F  7  2012  Scalp kerion  Guinea pig, rabbit, cow, dog, horse  NI  White  μ and macro  +  A. benhamiae  T. benhamiae  KY885220  1204m110484  Our department  20  F  36  2012  Thorax (nipple)  NI  NI  Yellow  some μ  ++  T. m. var porcellae  T. benhamiae  KY885221  1205m290936  External source  21  F  33  2012  Calf, knee  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885222  1206m110884  Our department  22  F  22  2012  Arm  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  NP  NA  1208m080872  Our department  23  F  26  2012  Arm  Guinea pig  Yes  Yellow  few μ  −  T. m. var porcellae  T. benhamiae  KY885223  1208m170694  Our department  24  M  60  2012  Forearm  NI  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885224  1211m220440  External source  25  M  4  2013  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885225  1308m120592  Our department  26  F  43  2013  Leg  NI  NI  Yellow  few μ  +  T. m. var porcellae  NP  NA  1310m090513  External source  27  A  NA  2013  NI  NA  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885226  1310m160506  External source  28  A  NA  2014  NI  NA  Yes  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885227  1401m280504  External source  29  F  12  2014  Thorax (back)  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885228  1403m070728  Our department  30  F  49  2014  Face, neck  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885229  1405m100253  Our department  31  F  8  2014  Scalp, face  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885230  1405m170464  Our department  32  F  6  2014  Thorax  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885231  1405m191015  Our department  33  F  15  2014  NI  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885232  1410m030306  External source  34  F  2  2014  Scalp  NI  NI  Yellow  few μ  −  T. m. var porcellae  NP  NA  1410m160928  Our department  35  F  15  2014  Thigh  NI  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885233  1411m170810  Our department  36  A  NA  2014  NI  NA  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  GRA Sév Spik  Our department  37  M  9  2015  NI  Guinea pig  NI  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1506m020295  External source  38  F  54  2015  Arm  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885234  1507m100694  Our department  39  F  11  2016  Eyelid  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885235  1607m060843  Our department  40  F  52  2016  Lip  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885236  1608m220245  Our department  41  M  43  2016  Elbow, knee  NI  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885237  1611m220253  Our department  No.  Sex  Age  Year  Lesion localization  Contact with animals  Animal lesion  phenotype  Microscopy  Urea broth  Initial identification  ITS sequencing  GenBank Accession no.  Culture collection ID  Source  1  F  9  2008  Thorax (back)  NI  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885203  0801m210208  Our department  2  F  8  2008  NI  Rabbit  NI  White  abundant μ  +  A. benhamiae  T. benhamiae  KY885204  0802m180906  Our department  3  F  15  2008  Thigh  NI  NI  White  sterile  NP  A. benhamiae  T. bullosum  KY885205  0805m150877  External source  4  M  9  2008  Cheek  Cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885206  0810m040057  Our department  5  F  12  2009  Arm  NI  NI  Yellow  few μ  NP  A benhamiae  T. benhamiae  KY885207  0906m180356  External source  6  M  24  2009  Beard sycosis  NI  NI  White  μ and macro  +  A. benhamiae  T. eriotrephon  KY885208  0912m230081  Our department  7  M  8  2010  NI  NI  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885209  1001m210429  External source  8  F  4  2010  Face  Guinea pig, pony, cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885210  1005m220254  Our department  9  F  8  2010  Scalp kerion  Guinea pig  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885211  1012m200864  Our department  10  F  15  2011  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885212  1102m170903  External source  11  F  12  2011  Shoulder  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885213  1102m250627  External source  12  F  5  2011  Thorax (sternum)  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885214  1102m280391  External source  13  F  5  2011  Pubis, groin  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885215  1104m210816  External source  14  F  20  2011  Arm  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885216  1105m260453  External source  15  F  10  2011  Face, thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885217  1105m270378  External source  16  F  11  2011  Thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1108m030060  Our department  17  F  12  2011  Thorax  Guinea pig, hamster, gerbil, kitten  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885218  1110m250836  External source  18  M  12  2011  Abdomen  Rabbit  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885219  1111m220427  External source  19  F  7  2012  Scalp kerion  Guinea pig, rabbit, cow, dog, horse  NI  White  μ and macro  +  A. benhamiae  T. benhamiae  KY885220  1204m110484  Our department  20  F  36  2012  Thorax (nipple)  NI  NI  Yellow  some μ  ++  T. m. var porcellae  T. benhamiae  KY885221  1205m290936  External source  21  F  33  2012  Calf, knee  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885222  1206m110884  Our department  22  F  22  2012  Arm  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  NP  NA  1208m080872  Our department  23  F  26  2012  Arm  Guinea pig  Yes  Yellow  few μ  −  T. m. var porcellae  T. benhamiae  KY885223  1208m170694  Our department  24  M  60  2012  Forearm  NI  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885224  1211m220440  External source  25  M  4  2013  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885225  1308m120592  Our department  26  F  43  2013  Leg  NI  NI  Yellow  few μ  +  T. m. var porcellae  NP  NA  1310m090513  External source  27  A  NA  2013  NI  NA  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885226  1310m160506  External source  28  A  NA  2014  NI  NA  Yes  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885227  1401m280504  External source  29  F  12  2014  Thorax (back)  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885228  1403m070728  Our department  30  F  49  2014  Face, neck  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885229  1405m100253  Our department  31  F  8  2014  Scalp, face  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885230  1405m170464  Our department  32  F  6  2014  Thorax  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885231  1405m191015  Our department  33  F  15  2014  NI  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885232  1410m030306  External source  34  F  2  2014  Scalp  NI  NI  Yellow  few μ  −  T. m. var porcellae  NP  NA  1410m160928  Our department  35  F  15  2014  Thigh  NI  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885233  1411m170810  Our department  36  A  NA  2014  NI  NA  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  GRA Sév Spik  Our department  37  M  9  2015  NI  Guinea pig  NI  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1506m020295  External source  38  F  54  2015  Arm  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885234  1507m100694  Our department  39  F  11  2016  Eyelid  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885235  1607m060843  Our department  40  F  52  2016  Lip  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885236  1608m220245  Our department  41  M  43  2016  Elbow, knee  NI  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885237  1611m220253  Our department  A, animal; F, female; M, male; NA, not applicable; NI, no information; NP, not performed; μ, microconidia; macro, macroconidia; −, negative; ±, equivocal; +, weakly positive; ++, positive. T. m., T. mentagrophytes. View Large Table 2. List of strains used in this study. No.  Sex  Age  Year  Lesion localization  Contact with animals  Animal lesion  phenotype  Microscopy  Urea broth  Initial identification  ITS sequencing  GenBank Accession no.  Culture collection ID  Source  1  F  9  2008  Thorax (back)  NI  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885203  0801m210208  Our department  2  F  8  2008  NI  Rabbit  NI  White  abundant μ  +  A. benhamiae  T. benhamiae  KY885204  0802m180906  Our department  3  F  15  2008  Thigh  NI  NI  White  sterile  NP  A. benhamiae  T. bullosum  KY885205  0805m150877  External source  4  M  9  2008  Cheek  Cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885206  0810m040057  Our department  5  F  12  2009  Arm  NI  NI  Yellow  few μ  NP  A benhamiae  T. benhamiae  KY885207  0906m180356  External source  6  M  24  2009  Beard sycosis  NI  NI  White  μ and macro  +  A. benhamiae  T. eriotrephon  KY885208  0912m230081  Our department  7  M  8  2010  NI  NI  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885209  1001m210429  External source  8  F  4  2010  Face  Guinea pig, pony, cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885210  1005m220254  Our department  9  F  8  2010  Scalp kerion  Guinea pig  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885211  1012m200864  Our department  10  F  15  2011  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885212  1102m170903  External source  11  F  12  2011  Shoulder  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885213  1102m250627  External source  12  F  5  2011  Thorax (sternum)  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885214  1102m280391  External source  13  F  5  2011  Pubis, groin  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885215  1104m210816  External source  14  F  20  2011  Arm  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885216  1105m260453  External source  15  F  10  2011  Face, thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885217  1105m270378  External source  16  F  11  2011  Thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1108m030060  Our department  17  F  12  2011  Thorax  Guinea pig, hamster, gerbil, kitten  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885218  1110m250836  External source  18  M  12  2011  Abdomen  Rabbit  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885219  1111m220427  External source  19  F  7  2012  Scalp kerion  Guinea pig, rabbit, cow, dog, horse  NI  White  μ and macro  +  A. benhamiae  T. benhamiae  KY885220  1204m110484  Our department  20  F  36  2012  Thorax (nipple)  NI  NI  Yellow  some μ  ++  T. m. var porcellae  T. benhamiae  KY885221  1205m290936  External source  21  F  33  2012  Calf, knee  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885222  1206m110884  Our department  22  F  22  2012  Arm  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  NP  NA  1208m080872  Our department  23  F  26  2012  Arm  Guinea pig  Yes  Yellow  few μ  −  T. m. var porcellae  T. benhamiae  KY885223  1208m170694  Our department  24  M  60  2012  Forearm  NI  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885224  1211m220440  External source  25  M  4  2013  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885225  1308m120592  Our department  26  F  43  2013  Leg  NI  NI  Yellow  few μ  +  T. m. var porcellae  NP  NA  1310m090513  External source  27  A  NA  2013  NI  NA  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885226  1310m160506  External source  28  A  NA  2014  NI  NA  Yes  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885227  1401m280504  External source  29  F  12  2014  Thorax (back)  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885228  1403m070728  Our department  30  F  49  2014  Face, neck  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885229  1405m100253  Our department  31  F  8  2014  Scalp, face  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885230  1405m170464  Our department  32  F  6  2014  Thorax  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885231  1405m191015  Our department  33  F  15  2014  NI  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885232  1410m030306  External source  34  F  2  2014  Scalp  NI  NI  Yellow  few μ  −  T. m. var porcellae  NP  NA  1410m160928  Our department  35  F  15  2014  Thigh  NI  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885233  1411m170810  Our department  36  A  NA  2014  NI  NA  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  GRA Sév Spik  Our department  37  M  9  2015  NI  Guinea pig  NI  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1506m020295  External source  38  F  54  2015  Arm  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885234  1507m100694  Our department  39  F  11  2016  Eyelid  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885235  1607m060843  Our department  40  F  52  2016  Lip  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885236  1608m220245  Our department  41  M  43  2016  Elbow, knee  NI  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885237  1611m220253  Our department  No.  Sex  Age  Year  Lesion localization  Contact with animals  Animal lesion  phenotype  Microscopy  Urea broth  Initial identification  ITS sequencing  GenBank Accession no.  Culture collection ID  Source  1  F  9  2008  Thorax (back)  NI  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885203  0801m210208  Our department  2  F  8  2008  NI  Rabbit  NI  White  abundant μ  +  A. benhamiae  T. benhamiae  KY885204  0802m180906  Our department  3  F  15  2008  Thigh  NI  NI  White  sterile  NP  A. benhamiae  T. bullosum  KY885205  0805m150877  External source  4  M  9  2008  Cheek  Cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885206  0810m040057  Our department  5  F  12  2009  Arm  NI  NI  Yellow  few μ  NP  A benhamiae  T. benhamiae  KY885207  0906m180356  External source  6  M  24  2009  Beard sycosis  NI  NI  White  μ and macro  +  A. benhamiae  T. eriotrephon  KY885208  0912m230081  Our department  7  M  8  2010  NI  NI  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885209  1001m210429  External source  8  F  4  2010  Face  Guinea pig, pony, cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885210  1005m220254  Our department  9  F  8  2010  Scalp kerion  Guinea pig  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885211  1012m200864  Our department  10  F  15  2011  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885212  1102m170903  External source  11  F  12  2011  Shoulder  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885213  1102m250627  External source  12  F  5  2011  Thorax (sternum)  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885214  1102m280391  External source  13  F  5  2011  Pubis, groin  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885215  1104m210816  External source  14  F  20  2011  Arm  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885216  1105m260453  External source  15  F  10  2011  Face, thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885217  1105m270378  External source  16  F  11  2011  Thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1108m030060  Our department  17  F  12  2011  Thorax  Guinea pig, hamster, gerbil, kitten  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885218  1110m250836  External source  18  M  12  2011  Abdomen  Rabbit  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885219  1111m220427  External source  19  F  7  2012  Scalp kerion  Guinea pig, rabbit, cow, dog, horse  NI  White  μ and macro  +  A. benhamiae  T. benhamiae  KY885220  1204m110484  Our department  20  F  36  2012  Thorax (nipple)  NI  NI  Yellow  some μ  ++  T. m. var porcellae  T. benhamiae  KY885221  1205m290936  External source  21  F  33  2012  Calf, knee  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885222  1206m110884  Our department  22  F  22  2012  Arm  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  NP  NA  1208m080872  Our department  23  F  26  2012  Arm  Guinea pig  Yes  Yellow  few μ  −  T. m. var porcellae  T. benhamiae  KY885223  1208m170694  Our department  24  M  60  2012  Forearm  NI  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885224  1211m220440  External source  25  M  4  2013  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885225  1308m120592  Our department  26  F  43  2013  Leg  NI  NI  Yellow  few μ  +  T. m. var porcellae  NP  NA  1310m090513  External source  27  A  NA  2013  NI  NA  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885226  1310m160506  External source  28  A  NA  2014  NI  NA  Yes  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885227  1401m280504  External source  29  F  12  2014  Thorax (back)  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885228  1403m070728  Our department  30  F  49  2014  Face, neck  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885229  1405m100253  Our department  31  F  8  2014  Scalp, face  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885230  1405m170464  Our department  32  F  6  2014  Thorax  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885231  1405m191015  Our department  33  F  15  2014  NI  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885232  1410m030306  External source  34  F  2  2014  Scalp  NI  NI  Yellow  few μ  −  T. m. var porcellae  NP  NA  1410m160928  Our department  35  F  15  2014  Thigh  NI  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885233  1411m170810  Our department  36  A  NA  2014  NI  NA  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  GRA Sév Spik  Our department  37  M  9  2015  NI  Guinea pig  NI  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1506m020295  External source  38  F  54  2015  Arm  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885234  1507m100694  Our department  39  F  11  2016  Eyelid  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885235  1607m060843  Our department  40  F  52  2016  Lip  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885236  1608m220245  Our department  41  M  43  2016  Elbow, knee  NI  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885237  1611m220253  Our department  A, animal; F, female; M, male; NA, not applicable; NI, no information; NP, not performed; μ, microconidia; macro, macroconidia; −, negative; ±, equivocal; +, weakly positive; ++, positive. T. m., T. mentagrophytes. View Large No M. canis were included in this study because all of the strains had typical macroconidia that made them easy to differentiate from the yellow phenotype of T. benhamiae. We had no T. concentricum or T. eriotrephon in our collections, and all of our T. verrucosum isolates were already confirmed by ITS sequencing. Only 35 of 41 strains were available for retrospective study in our mycology bank; for these, the morphological identification was verified by sequencing the ITS region of the ribosomal DNA. The six remaining strains had been identified according to morphology as T. mentagrophytes var. porcellae, name used incorrectly for the yellow phenotype Trichophyton sp. of A. benhamiae (Figs 1 and 2). Figure 1. View largeDownload slide Flow chart of the 41 strains included in this study. Figure 1. View largeDownload slide Flow chart of the 41 strains included in this study. Figure 2. View largeDownload slide Macroscopy and microscopy of T. benhamiae. Up: Macroscopy of white (left) and yellow (right) phenotypes of T. benhamiae. Potato-dextrose-agar slant media, diluted Sabouraud dextrose agar (two upper Petri dishes) and Borelli's lactrimel agar (two lower Petri dishes). Low-left: Microscopy of the white phenotype of T. benhamiae (magnification 400 ×): numerous micro- and macroconidia. Low-right: Microscopy of the yellow phenotype of T. benhamiae (magnification 400 ×): absent macroconidia, few microconidia. Figure 2. View largeDownload slide Macroscopy and microscopy of T. benhamiae. Up: Macroscopy of white (left) and yellow (right) phenotypes of T. benhamiae. Potato-dextrose-agar slant media, diluted Sabouraud dextrose agar (two upper Petri dishes) and Borelli's lactrimel agar (two lower Petri dishes). Low-left: Microscopy of the white phenotype of T. benhamiae (magnification 400 ×): numerous micro- and macroconidia. Low-right: Microscopy of the yellow phenotype of T. benhamiae (magnification 400 ×): absent macroconidia, few microconidia. Strain 3 had a white phenotype and was identified by ITS sequencing as T. bullosum (Table 2, Fig. 1, Table 3). This result was confirmed by the phylogenetic analysis presented in Figure 3. Strain 3 had been isolated in 2008 from a thigh lesion of a 15-year-old girl, presented a white phenotype and a sterile microscopy; no information concerning animal contact was known. Table 3. Results of GenBank and CBS comparison for our 35 ITS sequenced strains. Strains from this study  BLASTN first hits  Score  Identity  Query cover  Organism  GenBank definition  Strain 6  GenBank EU181452.1  1230 bits (666)  100%  99%  T. erinacei  A. benhamiae NCPF 431  672 bp  GenBank KJ606083.1  1229 bits (665)  99%  99%  Trichophyton cf. erinacei ATCC 24552  Trichophyton cf. erinacei ATCC 24552  ITS sequencing: A. benhamiae / T. erinacei  GenBank JN134091.1  1203 bits (651)  99%  100%  T. erinacei  T. erinacei isolate 379  Phylogeny: T. eriotrephon  GenBank EU622882.1  1203 bits (651)  99%  100%  T. benhamiae  A. benhamiae AMC 07101    GenBank JX413539  1044.49  98.958%  100%  T. erinacei  A. benhamiae CS783  Strain 3  CBS 131645  1041.32  100%  100%  T. bullosum BCCM/IHEM 24321    656 bp  CBS 363.35 GenBank FM992675.1  1038.32  100%  99.695%  T. bullosum LP 770    ITS sequencing and phylogeny: T. bullosum  GenBank LN589975.1  1197 bits (648)  99%  99%  T. bullosum CCF 4831      CBS 557.50 GenBank FM992676.1  958.02  100%  92%  T. bullosum No. 493      GenBank KU257463.1    99%  100%  T. benhamiae SU901378    White phenotype  GenBank KJ606075.1    99%  100%  A. benhamiae ATCC MYA-4681    Strains: 1, 2, 9, 19  GenBank JN134088.1    99%  100%  T. benhamiae 269    T. benhamiae  CBS 112370    99.201%  100%  A. benhamiae CHUV 2352/02    Yellow phenotype T. benhamiae  GenBank KU257464.1    99%  100%  T. benhamiae SU209346      GenBank AB458216.1    99%  100%  A. benhamiae IFM 54424    Strains: 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 28, 29, 30, 31, 32, 33, 35, 38, 39, 40, 41  GenBank AF170457.1    99%  100%  A. benhamiae RV 26680      CBS 624.66 GenBank JX122282.1    99%  100%  A. benhamiae ATCC 16782      CBS 623.66 GenBank JX122281.1    99%  100%  A. benhamiae ATCC 16781    Yellow phenotype T. benhamiae  GenBank KU257464.1    97%  99%  T. benhamiae SU209346      GenBank KX092365.1    97%  99%  T. benhamiae ATCC 42873    Strain 27  CBS 624.66    97%  99%  A. benhamiae ATCC 16782    Strains from this study  BLASTN first hits  Score  Identity  Query cover  Organism  GenBank definition  Strain 6  GenBank EU181452.1  1230 bits (666)  100%  99%  T. erinacei  A. benhamiae NCPF 431  672 bp  GenBank KJ606083.1  1229 bits (665)  99%  99%  Trichophyton cf. erinacei ATCC 24552  Trichophyton cf. erinacei ATCC 24552  ITS sequencing: A. benhamiae / T. erinacei  GenBank JN134091.1  1203 bits (651)  99%  100%  T. erinacei  T. erinacei isolate 379  Phylogeny: T. eriotrephon  GenBank EU622882.1  1203 bits (651)  99%  100%  T. benhamiae  A. benhamiae AMC 07101    GenBank JX413539  1044.49  98.958%  100%  T. erinacei  A. benhamiae CS783  Strain 3  CBS 131645  1041.32  100%  100%  T. bullosum BCCM/IHEM 24321    656 bp  CBS 363.35 GenBank FM992675.1  1038.32  100%  99.695%  T. bullosum LP 770    ITS sequencing and phylogeny: T. bullosum  GenBank LN589975.1  1197 bits (648)  99%  99%  T. bullosum CCF 4831      CBS 557.50 GenBank FM992676.1  958.02  100%  92%  T. bullosum No. 493      GenBank KU257463.1    99%  100%  T. benhamiae SU901378    White phenotype  GenBank KJ606075.1    99%  100%  A. benhamiae ATCC MYA-4681    Strains: 1, 2, 9, 19  GenBank JN134088.1    99%  100%  T. benhamiae 269    T. benhamiae  CBS 112370    99.201%  100%  A. benhamiae CHUV 2352/02    Yellow phenotype T. benhamiae  GenBank KU257464.1    99%  100%  T. benhamiae SU209346      GenBank AB458216.1    99%  100%  A. benhamiae IFM 54424    Strains: 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 28, 29, 30, 31, 32, 33, 35, 38, 39, 40, 41  GenBank AF170457.1    99%  100%  A. benhamiae RV 26680      CBS 624.66 GenBank JX122282.1    99%  100%  A. benhamiae ATCC 16782      CBS 623.66 GenBank JX122281.1    99%  100%  A. benhamiae ATCC 16781    Yellow phenotype T. benhamiae  GenBank KU257464.1    97%  99%  T. benhamiae SU209346      GenBank KX092365.1    97%  99%  T. benhamiae ATCC 42873    Strain 27  CBS 624.66    97%  99%  A. benhamiae ATCC 16782    View Large Table 3. Results of GenBank and CBS comparison for our 35 ITS sequenced strains. Strains from this study  BLASTN first hits  Score  Identity  Query cover  Organism  GenBank definition  Strain 6  GenBank EU181452.1  1230 bits (666)  100%  99%  T. erinacei  A. benhamiae NCPF 431  672 bp  GenBank KJ606083.1  1229 bits (665)  99%  99%  Trichophyton cf. erinacei ATCC 24552  Trichophyton cf. erinacei ATCC 24552  ITS sequencing: A. benhamiae / T. erinacei  GenBank JN134091.1  1203 bits (651)  99%  100%  T. erinacei  T. erinacei isolate 379  Phylogeny: T. eriotrephon  GenBank EU622882.1  1203 bits (651)  99%  100%  T. benhamiae  A. benhamiae AMC 07101    GenBank JX413539  1044.49  98.958%  100%  T. erinacei  A. benhamiae CS783  Strain 3  CBS 131645  1041.32  100%  100%  T. bullosum BCCM/IHEM 24321    656 bp  CBS 363.35 GenBank FM992675.1  1038.32  100%  99.695%  T. bullosum LP 770    ITS sequencing and phylogeny: T. bullosum  GenBank LN589975.1  1197 bits (648)  99%  99%  T. bullosum CCF 4831      CBS 557.50 GenBank FM992676.1  958.02  100%  92%  T. bullosum No. 493      GenBank KU257463.1    99%  100%  T. benhamiae SU901378    White phenotype  GenBank KJ606075.1    99%  100%  A. benhamiae ATCC MYA-4681    Strains: 1, 2, 9, 19  GenBank JN134088.1    99%  100%  T. benhamiae 269    T. benhamiae  CBS 112370    99.201%  100%  A. benhamiae CHUV 2352/02    Yellow phenotype T. benhamiae  GenBank KU257464.1    99%  100%  T. benhamiae SU209346      GenBank AB458216.1    99%  100%  A. benhamiae IFM 54424    Strains: 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 28, 29, 30, 31, 32, 33, 35, 38, 39, 40, 41  GenBank AF170457.1    99%  100%  A. benhamiae RV 26680      CBS 624.66 GenBank JX122282.1    99%  100%  A. benhamiae ATCC 16782      CBS 623.66 GenBank JX122281.1    99%  100%  A. benhamiae ATCC 16781    Yellow phenotype T. benhamiae  GenBank KU257464.1    97%  99%  T. benhamiae SU209346      GenBank KX092365.1    97%  99%  T. benhamiae ATCC 42873    Strain 27  CBS 624.66    97%  99%  A. benhamiae ATCC 16782    Strains from this study  BLASTN first hits  Score  Identity  Query cover  Organism  GenBank definition  Strain 6  GenBank EU181452.1  1230 bits (666)  100%  99%  T. erinacei  A. benhamiae NCPF 431  672 bp  GenBank KJ606083.1  1229 bits (665)  99%  99%  Trichophyton cf. erinacei ATCC 24552  Trichophyton cf. erinacei ATCC 24552  ITS sequencing: A. benhamiae / T. erinacei  GenBank JN134091.1  1203 bits (651)  99%  100%  T. erinacei  T. erinacei isolate 379  Phylogeny: T. eriotrephon  GenBank EU622882.1  1203 bits (651)  99%  100%  T. benhamiae  A. benhamiae AMC 07101    GenBank JX413539  1044.49  98.958%  100%  T. erinacei  A. benhamiae CS783  Strain 3  CBS 131645  1041.32  100%  100%  T. bullosum BCCM/IHEM 24321    656 bp  CBS 363.35 GenBank FM992675.1  1038.32  100%  99.695%  T. bullosum LP 770    ITS sequencing and phylogeny: T. bullosum  GenBank LN589975.1  1197 bits (648)  99%  99%  T. bullosum CCF 4831      CBS 557.50 GenBank FM992676.1  958.02  100%  92%  T. bullosum No. 493      GenBank KU257463.1    99%  100%  T. benhamiae SU901378    White phenotype  GenBank KJ606075.1    99%  100%  A. benhamiae ATCC MYA-4681    Strains: 1, 2, 9, 19  GenBank JN134088.1    99%  100%  T. benhamiae 269    T. benhamiae  CBS 112370    99.201%  100%  A. benhamiae CHUV 2352/02    Yellow phenotype T. benhamiae  GenBank KU257464.1    99%  100%  T. benhamiae SU209346      GenBank AB458216.1    99%  100%  A. benhamiae IFM 54424    Strains: 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 28, 29, 30, 31, 32, 33, 35, 38, 39, 40, 41  GenBank AF170457.1    99%  100%  A. benhamiae RV 26680      CBS 624.66 GenBank JX122282.1    99%  100%  A. benhamiae ATCC 16782      CBS 623.66 GenBank JX122281.1    99%  100%  A. benhamiae ATCC 16781    Yellow phenotype T. benhamiae  GenBank KU257464.1    97%  99%  T. benhamiae SU209346      GenBank KX092365.1    97%  99%  T. benhamiae ATCC 42873    Strain 27  CBS 624.66    97%  99%  A. benhamiae ATCC 16782    View Large Figure 3. View largeDownload slide Maximum likelihood phylogenetic tree (MEGA® 6.0 software) based on ITS sequences of Arthroderma benhamiae complex using T92+G+I model, with 10,000 bootstrap replications. Bootstrap values above 70% are shown, Trichophyton rubrum was used as outgroup. A: animal strain (2); *: type strains. Figure 3. View largeDownload slide Maximum likelihood phylogenetic tree (MEGA® 6.0 software) based on ITS sequences of Arthroderma benhamiae complex using T92+G+I model, with 10,000 bootstrap replications. Bootstrap values above 70% are shown, Trichophyton rubrum was used as outgroup. A: animal strain (2); *: type strains. Strain 6 had a white phenotype, and ITS sequencing showed 99–100% identity with A. benhamiae / T. erinacei strains (Table 3). However, the phylogenetic analysis with high bootstrap values presented in Figure 3 showed that it belongs to the T. eriotrephon clade. This strain had been isolated from a beard sycosis of a 24-year-old man in 2009 and no information concerning animal contact was given. Microscopically, it presented numerous microconidia and some macroconidia; urease activity was weekly positive (Table 2). The remaining 33 sequenced strains were confirmed as A. benhamiae or T. benhamiae. One animal strain (strain 27) only had 97% identity with three strains of T. benhamiae (Table 3). The other 32 strains all have 99–100% identity with strains of T. benhamiae, including the type strain. In sum, 36 of the total 39 T. benhamiae/T. mentagrophytes var. porcellae strains were isolated from humans and three from guinea pig samples (Fig. 1). And 35 of the 39 T. benhamiae/T. mentagrophytes var. porcellae strains (89.7%) belong to the yellow phenotype and four (10.3%) to the white phenotype. All white phenotype strains were isolated from human samples (strains 1, 2, 9, and 19); they all clustered together and separately from the yellow ones in the phylogenetic analysis presented in Figure 3. With regards to the 36 human T. benhamiae strains, 25 (69.4%) were from children with ages ranging from 2 to 15 years; the lesions were mostly inflammatory and located on the thorax or abdomen (seven), scalp (six), face (five), arms (two), leg (one), and groin (one). At least two children had more than one lesion (face and thorax, face and scalp). For four children, the clinical data were not precise (only “skin lesion” was specified). The 11 adults (30.6% of T. benhamiae strains) had ages ranging from 20 to 89 and presented lesions on arms (six), legs (four), thorax (one), lip (one), scalp (one), face and neck (one). Only two human strains come from two members (children) of the same family (strains 31 and 32). Both strains were of the T. benhamiae yellow phenotype, presented weakly positive urease activity but did not cluster next to one another in the dendrogramm presented in Figure 3. In nine of the 36 human T. benhamiae infections (25%), contact with an animal could not be established at the time of diagnosis, mostly because the interrogatory had been performed out of our department and no detailed information was transmitted to our laboratory. In sum, 27 out of 36 T. benhamiae patients (75%) had been in contact with an animal. And 21 of these 27 patients had been in contact only with guinea pigs; three patients had been in contact with other animals besides guinea pigs (guinea pig, pony, and cat for patient 8; guinea pig, hamster, gerbil, and kitten for patient 17; and guinea pig, rabbit, cow, dog, and horse for patient 19). Seven of the 24 guinea pigs were symptomatic, but we did not receive samples from these animals. Three patients with no guinea pig contact had been in contact with rabbits (patient 2 and patient 18) and patient 4 with a cat (Table 2). Three T. benhamiae strains isolated from guinea pigs were sent to us for identification. These animals were symptomatic but were not pets of any of the patients included in this article. They were all yellow type T. benhamiae. Only 32 T. benhamiae strains of the total of 39 were tested for urease activity: eight were positive, 19 were weakly positive, five were equivocal, and two were negative (Table 2 and Fig. 4). Figure 4. View largeDownload slide Christensen's urea broth test. From left to right: negative control, equivocal reaction (strain 11), weakly positive reaction (strain 10), positive reaction (strain 40). Figure 4. View largeDownload slide Christensen's urea broth test. From left to right: negative control, equivocal reaction (strain 11), weakly positive reaction (strain 10), positive reaction (strain 40). Regarding the incidence of T. benhamiae and M. canis, the former was more frequent than the latter in only 3 of the 9-year survey (Table 1): 2011 (1.06% vs 0.82%), 2012 (0.85% vs 0.57%), and 2014 (1.18% vs 0.4%). Discussion Most of the dermatophytes are cosmopolite, but some are confined to specific regions or areas of the globe or are associated with certain animals. Population migrations, changes in lifestyle, improvement of hygiene, and practice of more physical activity are changing the geographical distribution of dermatophytosis. Another important factor influencing the epidemiology of these infections is the increasing number and variety of pets. Lately, under the influence of fashion and mediatisation, these are no longer limited to cats and dogs. Reptiles, mice, rabbits, guinea pigs or ferrets have become equally common9,25,32. Children and staff working in pet shops are populations especially at risk of contracting an infection transmitted by these no longer exotic animals.32,33,34,35,36 More than adults, children are at risk of developing an infection due to zoophilic dermatophytes because of their increased outdoors activity and preferred close contact with pets and other animals.33 This was also evidenced by our cases, with infections concerning mostly children (25 out of 38 patients = 65.78%). The adults’ lesions were mostly situated on the exposed parts of the skin (arms and legs), whereas lesions on thorax, abdomen, scalp, face, groin, thigh, or shoulder, were predominant in children, probably due to petting and playing with their animals. T. benhamiae is transmitted mainly by guinea pigs but also occasionally by other animals.4,18,21,25,37 In our survey, three patients had been in contact with other animals besides guinea pigs (pony, cat, rabbit, cow, dog, and horse) and three patients had been in contact only with animals other than guinea pigs (rabbit and cat). These data correspond to those of the literature, since other hosts have recently been identified for T. benhamiae, such as cats, dogs, rabbits, mice, rats, foxes, and more rarely degus or porcupines.6,10,13,38,39 Lesions caused by T. benhamiae tend to be highly inflammatory. The animals are usually asymptomatic; when apparent, typical lesions are circumscribed areas of alopecia with erythema, scaling and crusting.4 In 27 of our 36 T. benhamiae human cases (75%), contact with guinea pigs has been established. Out of these 27 cases, the animals were confirmed symptomatic in only seven cases (25.92%). Khettar et al. in 20129 and Bloch et al. in 201640 have investigated pet shops in the city of Nancy (Eastern France) and found that 1/2 of the guinea pigs in 2012 and 2/3 in 2016 were carriers of T. benhamiae (morphological identification), most of them asymptomatic. The percentages of carriers for hosts other than guinea pigs have not been investigated. Transmission can take place directly by contact with the animals (even if asymptomatic) but also via soil, animal hairs, and scales. The infectivity could be as long as 2 years.8 These are elements that add to the spreading of the infection, since the animal is neither identified as a potential source of infection for its entourage, nor isolated or treated. In terms of incidence, in most regions with a temperate climate, M. canis remains the second most frequently isolated zoophilic dermatophyte37 after T. mentagrophytes. It is mainly transmitted by cats and dogs33,41 and determines lesions that are not very inflammatory, unlike most of the other zoophilic dermatophytes. The observed frequency of T. benhamiae infection has been increasing recently in Japan,11,16 Switzerland,4,10,17 Germany,5,14,18,42,43,44 France,8 Belgium,9 the Netherlands,37 and Chile36. In recent years in Japan11,16T. benhamiae has become the second most frequent dermatophyte after M. canis, and a study performed in Germany between March 2010 and March 2013 showed that T. benhamiae had already become the most frequent zoophilic dermatophyte responsible of human infections with a prevalence of 2.9%.13,14 In the past 9 years in our laboratory we diagnosed a total of 658 strains of T. mentagrophytes, 65 strains of M. canis and 39 strains of T. benhamiae/T. mentagrophytes var. porcellae (Tables 1 and 2). T. mentagrophytes remains the most frequent zoophilic dermatophyte (incidence around 9%). M. canis comes in second (incidence around 1%) and T. benhamiae third (incidence around 0.5%, varying between 0.25% and 1.18%). From our 9-year survey we can conclude (Table 1) that T. benhamiae was the third most frequent zoophilic dermatophyte after T. mentagrophytes and M. canis in our laboratory, except for 2011 (1.06% vs 0.82%), 2012 (0.85% vs 0.57%), and 2014 (1.18% vs 0.4%), when it came second, surpassing M. canis. This seems to be mostly due to the increased number of guinea pigs as popular pets and to the fact that up to 2/3 of these animals can be carriers of T. benhamiae, even if asymptomatic.9,40 Microscopical differential diagnosis between M. canis and the yellow phenotype of T. benhamiae can be straightforward when rough-walled spindle-like macroconidia are present for the former and only few microconidia for the latter. Recently, Brasch and Wodarg14 described loop or circuit-like mycelial junctions for T. benhamiae, which could make morphological identification easier. Easy and fast methods, like the one proposed by Mayser et al. in 201345 can be very useful. The authors used chromogenic media CandiSelect™ 4 (Bio Rad, France), which allowed differentiation of the two dermatophytes after a few hours of incubation: the medium turned pink or purple for M. canis and turquoise-green for T. benhamiae.45 The chromogenic medium used routinely in our laboratory is ChromID Candida™ (bioMérieux, France), but it does not allow the differentiation of M. canis and T. benhamiae described by Mayser et al. for CandiSelect™ 4. When both M. canis and the yellow phenotype of T. benhamiae present as sterile mycelia, they are difficult to differentiate and ITS sequencing is recommended. Differential diagnosis between T. mentagrophytes and the white phenotype of T. benhamiae can be problematic, considering their similar macroscopy and microscopy.15 Since these two species also show overlapping host specificity, ITS sequencing is the only method that can provide reliable identification. Christensen urea broth test for T. benhamiae has been described as negative,8 weakly positive,45 positive,13 or variable.14 Our results confirm the variability observed by Brasch and Wodarg14; 32 strains out of the 39 T. benhamiae strains were tested for urease activity: eight were positive, 19 were weakly positive, five were equivocal, and two were negative. Considering the subjective interpretation of this test, the different types of tests available and especially the strain variability of the urea hydrolysis activity, this test is less and less used and/or reported in the literature. Recently, mass spectrometry has been used for the identification of T. benhamiae, with great correlations when compared to PCR.28,46 This technique is now widely available in routine and hospital laboratories but some problems persist: sporulation of the mould is needed, extraction protocols are not standardized, databases need to be regularly updated, and most of them are expensive and/or contain a limited number of dermatophytes in general and of T. benhamiae isolates in particular. Dermatophyte species identification can be performed or confirmed by DNA sequencing, most frequently targeting the ITS region of the ribosomal DNA.2,21,25,27 Multilocus DNA sequencing has recently been used to revise the classification and taxonomy of dermatophytes.2 De Hoog et al. showed that dermatophyte taxonomy has reached an acceptable level of stability. ITS sequencing can also reveal unexpected results: in our study, one of the strains we suspected of being a white phenotype of T. benhamiae proved to be T. bullosum, a zoophilic dermatophyte rarely isolated from the coat of horses and possibly donkeys. Only two cases have been described in the literature: one from a forearm lesion of a 21-year-old male in rural France47 and one from a saddle-area lesion of a 6-year-old male horse in the Czech Republic.48 As shown by the dendrogram presented in Figure 3, the ITS sequence of our T. bullosum strain clustered with the other two available strains. This newly described species is closely related to T. verrucosum and T. eriotrephon and systematic molecular identification of the dermatophytes comprised in these genera could give us more insight into the real prevalence of this species. Another strain we suspected of being a white phenotype of T. benhamiae was strain 6, isolated from a beard sycosis of a 24-year-old man in 2009 (no information concerning animal contact was available). ITS sequencing showed a 99–100% identity with A. benhamiae and T. erinacei strains (Table 3), but the phylogenetic analysis with high bootstrap value presented in Figure 3 places this strain in the T. eriotrephon clade, next to the type strain. There are at least two limiting factors in our case: the sequence length (672 bp) and having used only one marker for the phylogenetic analysis presented in Figure 3. All four white phenotype T. benhamiae strains were isolated from human samples (strains 1, 2, 9, and 19); they all cluster together and separately from the yellow ones in the phylogenetic analysis presented in Figure 3. The phylogenetic analysis shows clusters of A. benhamiae as well as T. benhamiae. We think this is due to ongoing updates taking place in the different databases following the new taxonomy of dermatophytes.2 These efforts of the scientific community will be useful in deepening the knowledge of local epidemiologies and will improve communication between scientists. T. benhamiae is a zoophilic dermatophyte diagnosed as an agent of human infection with increased frequency in the past years on three continents (Asia, Europe, South America). Our study shows that ITS sequencing is necessary for accurate identification of both phenotypes (white or yellow) of the species. The new taxonomy2 should simplify identifying T. benhamiae and monitoring the epidemiology of this zoophilic dermatophyte. Acknowledgments We thank private laboratories and private dermatologists in the Strasbourg area for contributing patient samples and fungal strains, and the technicians from the Medical Mycology Laboratory of the Strasbourg University Hospital for their technical assistance. 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Med Mycol . 2011; 52: 291– 295. Google Scholar CrossRef Search ADS   23. Takashio M. Observations on African and European strains of Arthroderma benhamiae. Int J Dermatol . 1974; 13: 94– 101. Google Scholar CrossRef Search ADS PubMed  24. Makimura K, Mochizuki T, Hasegawa A, Uchida K, Saito H, Yamaguchi H. Phylogenetic classification of Trichophyton mentagrophytes complex strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol . 1998; 36: 2629– 2633. Google Scholar PubMed  25. Fréalle E, Rodrigue M, Gantois N et al.   Phylogenetic analysis of Trichophyton mentagrophytes human and animal isolates based on MnSOD and ITS sequence comparison. Microbiol Read Engl . 2007; 153: 3466– 3477. Google Scholar CrossRef Search ADS   26. Li HC, Bouchara J-P, Hsu MM-L, Barton R, Su S, Chang TC. Identification of dermatophytes by sequence analysis of the rRNA gene internal transcribed spacer regions. J Med Microbiol . 2008; 57: 592– 600. Google Scholar CrossRef Search ADS PubMed  27. Symoens F, Jousson O, Planard C et al.   Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species complex. Int J Med Microbiol . 2011; 301: 260– 266. Google Scholar CrossRef Search ADS PubMed  28. Packeu A, Hendrickx M, Beguin H, Martiny D, Vandenberg O, Detandt M. Identification of the Trichophyton mentagrophytes complex species using MALDI-TOF mass spectrometry. Med Mycol . 2013; 51: 580– 585. Google Scholar CrossRef Search ADS PubMed  29. Chollet A, Cattin V, Fratti M, Mignon B, Monod M. Which fungus originally was Trichophyton mentagrophytes? Historical review and illustration by a clinical case. Mycopathologia . 2015; 180: 1– 5. Google Scholar CrossRef Search ADS PubMed  30. Kane J, Fischer JB. The differentiation of Trichophyton rubrum and T. mentagrophytes by use of Christensen's urea broth. Can J Microbiol . 1971; 17: 911– 913. Google Scholar CrossRef Search ADS PubMed  31. White TJ, Bruns T, Lee S et al.   Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ et al.  , editors. PCR Protocols: A Guide to Methods and Applications . New York: Academic Press, 1990: 315– 322. Google Scholar CrossRef Search ADS   32. Quinet B. [Zoonoses in children from new pets]. Med Mal Infect . 2005; 35: S117– 120. Google Scholar CrossRef Search ADS PubMed  33. Reichert-Pénétrat S, Contet-Audonneau N, Barbaud A, Schurra JP, Fortier B, Schmutz JL. Epidemiology of dermatophytoses in children living in northeast France: a 5-year study. Pediatr Dermatol . 2002; 19: 103– 105. Google Scholar CrossRef Search ADS PubMed  34. Shiraki Y, Hiruma M, Matsuba Y et al.   A case of tinea corporis caused by Arthroderma benhamiae (teleomorph of Tinea mentagrophytes) in a pet shop employee. J Am Acad Dermatol . 2006; 55: 153– 154. Google Scholar CrossRef Search ADS PubMed  35. 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Molecular identification and classification of Trichophyton mentagrophytes complex strains isolated from humans and selected animal species. Mycoses . 2015; 58: 119– 126. Google Scholar CrossRef Search ADS PubMed  40. Bloch M, Cavignaux R, Debourgogne A, Dorin J, Machouart M, Contet-Audonneau N. [From guinea pig to man: Tinea outbreak due to Trichophyton mentagrophytes var. porcellae in pet shops in Nancy (France)]. J Mycol Medicale . 2016; 26: 227– 232. Google Scholar CrossRef Search ADS   41. Czaika VA, Lam P-A. Trichophyton mentagrophytes cause underestimated contagious zoophilic fungal infection. Mycoses . 2013; 56: 33– 37. Google Scholar CrossRef Search ADS PubMed  42. Budihardja D, Freund V, Mayser P. Widespread erosive tinea corporis by Arthroderma benhamiae in a renal transplant recipient: case report. Mycoses . 2010; 53: 530– 532. Google Scholar CrossRef Search ADS PubMed  43. Hiernickel C, Wiegand C, Schliemann S et al.   [Trichophyton species of Arthroderma benhamiae: Clinical therapeutic aspects of a new pathogen in dermatology]. Hautarzt Z Dermatol Venerol Verwandte Geb . 2016; 67: 706– 711. Google Scholar CrossRef Search ADS   44. Brasch J, Beck-Jendroschek V, Voss K, Uhrlaß S, Nenoff P. [Arthroderma benhamiae strains in Germany: Morphological and physiological characteristics of the anamorphs]. Hautarzt Z Dermatol Venerol Verwandte Geb . 2016; 67: 700– 705. Google Scholar CrossRef Search ADS   45. Mayser P, Budihardja D. A simple and rapid method to differentiate Arthroderma benhamiae from Microsporum canis. J Dtsch Dermatol Ges . 2013; 11: 322– 327. Google Scholar PubMed  46. de Respinis S, Tonolla M, Pranghofer S, Petrini L, Petrini O, Bosshard PP. Identification of dermatophytes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Med Mycol . 2013; 51: 514– 521. Google Scholar CrossRef Search ADS PubMed  47. Sitterle E, Frealle E, Foulet F et al.   Trichophyton bullosum: a new zoonotic dermatophyte species. Med Mycol . 2012; 50: 305– 309. Google Scholar CrossRef Search ADS PubMed  48. Lyskova P, Hubka V, Petricakova A, Dobias R, Cmokova A, Kolarik M. Equine Dermatophytosis due to Trichophyton bullosum, a poorly known zoophilic dermatophyte masquerading as T. verrucosum. Mycopathologia . 2015; 180: 407– 419. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Molecular identification of Trichophyton benhamiae in Strasbourg, France: a 9-year retrospective study

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10.1093/mmy/myx100
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Abstract

Abstract Trichophyton benhamiae is a zoophilic dermatophyte transmitted to humans mostly from guinea pigs and occasionally other animals. It presents two distinct phenotypes: yellow and white. T. benhamiae was formerly known as Trichophyton species of Arthroderma benhamiae; it was considered part of the T. mentagrophytes species complex, and some authors have incorrectly described the yellow phenotype of T. benhamiae as T. mentagrophytes var. porcellae. Identification of T. benhamiae has been difficult, as it was described under more than three names, two phenotypes, and in several different possible host species. During the past 15 years, human infections due to this dermatophyte have been increasingly reported all over the world. In order to better understand the local epidemiology of T. benhamiae and to compare it to other European countries, we performed a 9-year retrospective study in the Strasbourg University Hospital. We studied 41 dermatophytes (38 isolated from humans and 3 from guinea pigs) identified as T. mentagrophytes var. porcellae or A. benhamiae from January 2008 to December 2016 and verified their identification by ITS (Internal Transcribed Spacer) sequencing. ITS sequencing was performed in 35 of the 41 strains, and they were identified as T. benhamiae (33), T. bullosum (1), and T. eriotrephon (1). The other six remaining strains were identified according to morphology as T. mentagrophytes var. porcellae, name incorrectly used since 2010 for the yellow phenotype of T. benhamiae. ITS sequencing is recommended for accurate identification of this dermatophyte and the culture phenotype (yellow or white) should be specified. Trichophyton benhamiae, Arthroderma benhamiae, guinea pig, zoophilic dermatophyte, T. mentagrophytes var. porcellae, T. bullosum Introduction Dermatophytes are a group of keratinophilic filamentous fungi that can infect animals and humans. Dermatophytosis, commonly called ringworm, is usually limited to hair, nails, and the keratinized layers of the skin.1 These mycoses are transmissible diseases and can clinically range from mild to severe, depending on the host's immune status, the strain virulence, and other environmental factors.1 According to their habitat, dermatophytes can be divided into anthropophilic (human), zoophilic (animal) and geophilic (soil)1. The latter two types tend to cause more inflammatory human lesions that the former. Dermatophytes belong to four genera: Epidermophyton, Trichophyton, Microsporum, and Nannizzia, with only the first two being truly anthropophilic.2 The genus Trichophyton is the one most frequently isolated from humans, especially in regions with a temperate climate. Trichophyton benhamiae (comb. nov.)2 is a zoophilic species transmitted to humans mostly from guinea pigs and occasionally rabbits, cats and dogs. During the past 15 years, it has been described in animals in Japan, Europe, and the United States.3,4,5,6,7 Two phenotypes have been described for T. benhamiae: yellow and white.8,9,10,11,12,13,14 The yellow phenotype strains are downy, with a pleated mycelium, a yellow-orange reverse, and a slow growth rate. Sporulation is poor on Sabouraud agar, with rare microconidia and no macroconidia or spiral hyphae. Subcultures on other media (potato dextrose agar, diluted Sabouraud agar or M40Y) can enhance sporulation.8,10 The main differential diagnosis is Microsporum canis, also often macroscopically yellow, but presenting 6–12 celled macroconidia, with thick cell walls and thinner septa.15 The white phenotype strains are powdery to floccose, with a yellow, orange, or brown reverse and a rapid growth rate. Microconidia are numerous, spherical to clavate; macroconidia are sparse, 3–8 celled, smooth- and thin-walled, clavate to cigar-shaped; spiral hyphae are occasionally present.2,10 The main differential diagnosis for the T. benhamiae white phenotype is T. mentagrophytes, which presents numerous spherical microconidia and frequent spiral hyphae, aside from the clavate to cigar-shaped macroconidia. In the past years, the nomenclature of dermatophytes has undergone some changes. Initially, species were defined according to clinical data, and morphological and physiological characteristics. Thus, T. benhamiae was initially known as Trichophyton sp. of Arthroderma benhamiae20,21,22 and it was considered to be part of the T. mentagrophytes9,21,22,23,24,25,26,27,28,29 species complex. But dermatophytes presenting different phenotypes can have the same genotype, or vice versa. This is perfectly illustrated by the fact that in 2010 Contet-Audonneau and Leyer invalidly introduced the name T. mentagrophytes var. porcellae for the already described yellow phenotype Trichophyton sp. of A. benhamiae.8 The rapid development of molecular methods in the past 20 years has revolutionized the dermatophyte taxonomy. Based on sequencing the ITS ribosomal DNA region, seven clades have been described. The upper clade A comprises the Trichophyton species, with clade A-1 corresponding to T. mentagrophytes. Trichophyton sp. of Arthroderma benhamiae is no longer considered part of the T. mentagrophytes species complex; it became T. benhamiae (comb. nov.), which formed the A-2 clade together with T. schoenleinii and T. verrucosum. Clade A-3 is represented by the zoophilic species T. bullosum.2 Having more than three different names, two different phenotypes, and several different possible hosts rendered difficult the identification of T. benhamiae. This dermatophyte has been reported since 2001 from humans in Japan,3,16 Switzerland,4,10,17 Germany,5,14,18 France,8 Belgium,9 and the United Kingdom19. In recent years in Japan, T. benhamiae has become the second most frequent dermatophyte after M. canis and a study performed in Germany between March 2010 and March 2013 showed that T. benhamiae had already become the most frequent zoophilic dermatophyte responsible of human infections, with a prevalence of 2.9%.13,14 In the Medical Mycology laboratory of the Strasbourg University Hospital we look for dermatophytes in 1400 to 1750 samples per year, and rare or interesting strains are stored in a mycology bank since 2008. We performed a 9-year retrospective study, in order to better understand the local epidemiology of T. benhamiae and to compare it to that of the other European countries or regions. Methods Strains Strains were isolated in our laboratory from clinical samples sent to our diagnostic laboratory in sterile Petri dishes, or from strains sent to us for identification by external sources (private laboratories or smaller hospitals). For each sample of sufficient quantity, a direct examination with KOH 30% and culture on in-house slant media (Sabouraud Chloramphenicol Dextrose Agar and Sabouraud Chloramphenicol Dextrose Cycloheximide Agar) were performed. The cultures were incubated at 27°C for 4 to 6 weeks and examined twice a week. No direct examination was performed for strains sent to us for identification. In-house media plates (potato-dextrose agar, water agar, Borelli's lactrimel agar, and diluted Sabouraud dextrose agar) were used for subcultures of all the strains. They were incubated at 27°C for 3 to 10 days and examined twice a week. The urea hydrolysis activity was tested using Christensen's urea broth test.30 Subcultures of the strains were inoculated in 1 ml of ready-to-use Christensen's urea broth (Sigma-Aldrich) incubated at 27°C and examined after 3 and 7 days. All 41 strains were identified morphologically (T. mentagrophytes var. porcellae), and 35 were confirmed by DNA sequencing (33 A. benhamiae/T. benhamiae, 1 T. bullosum and 1 T. eriotrephon). The remaining six strains were not available for sequencing at the time of this study. DNA extraction, amplification, and sequencing Confirmation of the initial identification was performed retrospectively by sequencing the Internal Transcribed Spacer (ITS) region of the ribosomal DNA using primers ITS1 (5΄-TCCGTAGGTGAACCTGCGG-3΄) and ITS4 (5΄-TCCTCCGCTTATTGATATGC-3΄) as previously described.31 Briefly, DNA was extracted and purified directly from fungal colonies with a Qiagen QIAamp® DNA mini kit according to manufacturer's instructions. The polymerase chain reaction (PCR) mixture (40 μl) included 3 mM MgCl2 (Qiagen, Germany), 200 μM of each deoxynucleoside triphosphate (dNTP) (Euromedex, France), 0.2 μM of each primer, and 0.2 μM of Hotstar Taq DNA polymerase (Qiagen, Germany). The thermal cycler (Applied BioSystems, Foster City, CA, USA) was set for initial denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 95°C for 1 min, annealing for 1 min at 54°C, and extension for 2 min at 72°C. A final extension step at 72°C for 7 min was included at the end of the amplification. The PCR products were electrophoresed in 2% agarose (Eurogentec, Belgium) for 30 min at 150 V and viewed in gel documentation Gel Doc EZ System (BioRad, France) and stored at −20°C until they were sent (to GATC Biotech, Germany) for ITS sequencing. Resulting sequences were compared to GenBank, CBS, ISHAM and EMBL databases. Phylogenetic analysis ITS sequences from our strains were aligned with reference strains belonging to Arthroderma benhamiae complex using MUSCLE (Mega® 6.0 software), and the best model for phylogenetic analyses was identified using default setting. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura 3-parameter model. The percentage of trees in which the associated taxa clustered together is shown above the branches (10000 replicates). Initial trees for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The rate variation model allowed for some sites to be evolutionarily invariable. Evolutionary analyses were conducted in MEGA6. Trichophyton rubrum strains IHEM13800 (JQ407179) and ATCC28188 (AF170472) were used as outgroup. Results Between 2008 and 2016 we received 1390 (2009) to 1732 (2015) samples per year for dermatophytes analysis. About 50% had positive cultures for dermatophytes, moulds, and yeasts. About 10% of the samples were positive for zoophilic dermatophytes, mostly T. mentagrophytes, M. canis and T. benhamiae (Table 1). Table 1. Main zoophilic dermatophytes identified in our laboratory from 2008 to 2016. Year  Total samples  Positive samples (%)  T. mentagrophytes (%)  T. benhamiae (%)  M. canis (%)  T. verrucosum (%)  2008  1698  996 (58.65)  98 (9.84)  3 (0.3)  4 (0.4)  1 (0.1)  2009  1390  787 (56.61)  97 (12.32)  1 (0.13)  5 (0.63)  1 (0.12)  2010  1432  794 (55.44)  71 (8.94)  3 (0.37)  6 (0.75)  1 (0.1)  2011  1577  846 (53.44)  64 (7.56)  9 (1.06)  7 (0.82)  1 (0.12)  2012  1382  701 (50.72)  68 (9.7)  6 (0.85)  4 (0.57)  0  2013  1452  717 (49.38)  75 (10.46)  3 (0.41)  14 (1.95)  2 (0.28)  2014  1602  763 (47.62)  60 (7.86)  9 (1.18)  3 (0.4)  0  2015  1732  797 (46)  63 (7.9)  2 (0.25)  7 (0.87)  0  2016  1720  786 (45.69)  63 (8.01)  3 (0.38)  15 (1.65)  1 (0.12)  2008–2016  13985  7187 (51.4)  658 (9.15)  40 (0.55)  65 (0.9)  7 (0.09)  Year  Total samples  Positive samples (%)  T. mentagrophytes (%)  T. benhamiae (%)  M. canis (%)  T. verrucosum (%)  2008  1698  996 (58.65)  98 (9.84)  3 (0.3)  4 (0.4)  1 (0.1)  2009  1390  787 (56.61)  97 (12.32)  1 (0.13)  5 (0.63)  1 (0.12)  2010  1432  794 (55.44)  71 (8.94)  3 (0.37)  6 (0.75)  1 (0.1)  2011  1577  846 (53.44)  64 (7.56)  9 (1.06)  7 (0.82)  1 (0.12)  2012  1382  701 (50.72)  68 (9.7)  6 (0.85)  4 (0.57)  0  2013  1452  717 (49.38)  75 (10.46)  3 (0.41)  14 (1.95)  2 (0.28)  2014  1602  763 (47.62)  60 (7.86)  9 (1.18)  3 (0.4)  0  2015  1732  797 (46)  63 (7.9)  2 (0.25)  7 (0.87)  0  2016  1720  786 (45.69)  63 (8.01)  3 (0.38)  15 (1.65)  1 (0.12)  2008–2016  13985  7187 (51.4)  658 (9.15)  40 (0.55)  65 (0.9)  7 (0.09)  View Large Table 1. Main zoophilic dermatophytes identified in our laboratory from 2008 to 2016. Year  Total samples  Positive samples (%)  T. mentagrophytes (%)  T. benhamiae (%)  M. canis (%)  T. verrucosum (%)  2008  1698  996 (58.65)  98 (9.84)  3 (0.3)  4 (0.4)  1 (0.1)  2009  1390  787 (56.61)  97 (12.32)  1 (0.13)  5 (0.63)  1 (0.12)  2010  1432  794 (55.44)  71 (8.94)  3 (0.37)  6 (0.75)  1 (0.1)  2011  1577  846 (53.44)  64 (7.56)  9 (1.06)  7 (0.82)  1 (0.12)  2012  1382  701 (50.72)  68 (9.7)  6 (0.85)  4 (0.57)  0  2013  1452  717 (49.38)  75 (10.46)  3 (0.41)  14 (1.95)  2 (0.28)  2014  1602  763 (47.62)  60 (7.86)  9 (1.18)  3 (0.4)  0  2015  1732  797 (46)  63 (7.9)  2 (0.25)  7 (0.87)  0  2016  1720  786 (45.69)  63 (8.01)  3 (0.38)  15 (1.65)  1 (0.12)  2008–2016  13985  7187 (51.4)  658 (9.15)  40 (0.55)  65 (0.9)  7 (0.09)  Year  Total samples  Positive samples (%)  T. mentagrophytes (%)  T. benhamiae (%)  M. canis (%)  T. verrucosum (%)  2008  1698  996 (58.65)  98 (9.84)  3 (0.3)  4 (0.4)  1 (0.1)  2009  1390  787 (56.61)  97 (12.32)  1 (0.13)  5 (0.63)  1 (0.12)  2010  1432  794 (55.44)  71 (8.94)  3 (0.37)  6 (0.75)  1 (0.1)  2011  1577  846 (53.44)  64 (7.56)  9 (1.06)  7 (0.82)  1 (0.12)  2012  1382  701 (50.72)  68 (9.7)  6 (0.85)  4 (0.57)  0  2013  1452  717 (49.38)  75 (10.46)  3 (0.41)  14 (1.95)  2 (0.28)  2014  1602  763 (47.62)  60 (7.86)  9 (1.18)  3 (0.4)  0  2015  1732  797 (46)  63 (7.9)  2 (0.25)  7 (0.87)  0  2016  1720  786 (45.69)  63 (8.01)  3 (0.38)  15 (1.65)  1 (0.12)  2008–2016  13985  7187 (51.4)  658 (9.15)  40 (0.55)  65 (0.9)  7 (0.09)  View Large In our 9-year survey, 41 strains isolated from separate samples (38 human and 3 animal) and identified at that time as T. mentagrophytes var. porcellae or A. benhamiae were diagnosed in our laboratory by morphology or molecular identification. As this study is retrospective, some information is missing in some cases (Table 2). Table 2. List of strains used in this study. No.  Sex  Age  Year  Lesion localization  Contact with animals  Animal lesion  phenotype  Microscopy  Urea broth  Initial identification  ITS sequencing  GenBank Accession no.  Culture collection ID  Source  1  F  9  2008  Thorax (back)  NI  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885203  0801m210208  Our department  2  F  8  2008  NI  Rabbit  NI  White  abundant μ  +  A. benhamiae  T. benhamiae  KY885204  0802m180906  Our department  3  F  15  2008  Thigh  NI  NI  White  sterile  NP  A. benhamiae  T. bullosum  KY885205  0805m150877  External source  4  M  9  2008  Cheek  Cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885206  0810m040057  Our department  5  F  12  2009  Arm  NI  NI  Yellow  few μ  NP  A benhamiae  T. benhamiae  KY885207  0906m180356  External source  6  M  24  2009  Beard sycosis  NI  NI  White  μ and macro  +  A. benhamiae  T. eriotrephon  KY885208  0912m230081  Our department  7  M  8  2010  NI  NI  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885209  1001m210429  External source  8  F  4  2010  Face  Guinea pig, pony, cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885210  1005m220254  Our department  9  F  8  2010  Scalp kerion  Guinea pig  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885211  1012m200864  Our department  10  F  15  2011  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885212  1102m170903  External source  11  F  12  2011  Shoulder  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885213  1102m250627  External source  12  F  5  2011  Thorax (sternum)  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885214  1102m280391  External source  13  F  5  2011  Pubis, groin  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885215  1104m210816  External source  14  F  20  2011  Arm  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885216  1105m260453  External source  15  F  10  2011  Face, thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885217  1105m270378  External source  16  F  11  2011  Thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1108m030060  Our department  17  F  12  2011  Thorax  Guinea pig, hamster, gerbil, kitten  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885218  1110m250836  External source  18  M  12  2011  Abdomen  Rabbit  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885219  1111m220427  External source  19  F  7  2012  Scalp kerion  Guinea pig, rabbit, cow, dog, horse  NI  White  μ and macro  +  A. benhamiae  T. benhamiae  KY885220  1204m110484  Our department  20  F  36  2012  Thorax (nipple)  NI  NI  Yellow  some μ  ++  T. m. var porcellae  T. benhamiae  KY885221  1205m290936  External source  21  F  33  2012  Calf, knee  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885222  1206m110884  Our department  22  F  22  2012  Arm  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  NP  NA  1208m080872  Our department  23  F  26  2012  Arm  Guinea pig  Yes  Yellow  few μ  −  T. m. var porcellae  T. benhamiae  KY885223  1208m170694  Our department  24  M  60  2012  Forearm  NI  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885224  1211m220440  External source  25  M  4  2013  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885225  1308m120592  Our department  26  F  43  2013  Leg  NI  NI  Yellow  few μ  +  T. m. var porcellae  NP  NA  1310m090513  External source  27  A  NA  2013  NI  NA  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885226  1310m160506  External source  28  A  NA  2014  NI  NA  Yes  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885227  1401m280504  External source  29  F  12  2014  Thorax (back)  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885228  1403m070728  Our department  30  F  49  2014  Face, neck  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885229  1405m100253  Our department  31  F  8  2014  Scalp, face  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885230  1405m170464  Our department  32  F  6  2014  Thorax  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885231  1405m191015  Our department  33  F  15  2014  NI  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885232  1410m030306  External source  34  F  2  2014  Scalp  NI  NI  Yellow  few μ  −  T. m. var porcellae  NP  NA  1410m160928  Our department  35  F  15  2014  Thigh  NI  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885233  1411m170810  Our department  36  A  NA  2014  NI  NA  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  GRA Sév Spik  Our department  37  M  9  2015  NI  Guinea pig  NI  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1506m020295  External source  38  F  54  2015  Arm  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885234  1507m100694  Our department  39  F  11  2016  Eyelid  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885235  1607m060843  Our department  40  F  52  2016  Lip  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885236  1608m220245  Our department  41  M  43  2016  Elbow, knee  NI  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885237  1611m220253  Our department  No.  Sex  Age  Year  Lesion localization  Contact with animals  Animal lesion  phenotype  Microscopy  Urea broth  Initial identification  ITS sequencing  GenBank Accession no.  Culture collection ID  Source  1  F  9  2008  Thorax (back)  NI  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885203  0801m210208  Our department  2  F  8  2008  NI  Rabbit  NI  White  abundant μ  +  A. benhamiae  T. benhamiae  KY885204  0802m180906  Our department  3  F  15  2008  Thigh  NI  NI  White  sterile  NP  A. benhamiae  T. bullosum  KY885205  0805m150877  External source  4  M  9  2008  Cheek  Cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885206  0810m040057  Our department  5  F  12  2009  Arm  NI  NI  Yellow  few μ  NP  A benhamiae  T. benhamiae  KY885207  0906m180356  External source  6  M  24  2009  Beard sycosis  NI  NI  White  μ and macro  +  A. benhamiae  T. eriotrephon  KY885208  0912m230081  Our department  7  M  8  2010  NI  NI  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885209  1001m210429  External source  8  F  4  2010  Face  Guinea pig, pony, cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885210  1005m220254  Our department  9  F  8  2010  Scalp kerion  Guinea pig  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885211  1012m200864  Our department  10  F  15  2011  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885212  1102m170903  External source  11  F  12  2011  Shoulder  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885213  1102m250627  External source  12  F  5  2011  Thorax (sternum)  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885214  1102m280391  External source  13  F  5  2011  Pubis, groin  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885215  1104m210816  External source  14  F  20  2011  Arm  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885216  1105m260453  External source  15  F  10  2011  Face, thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885217  1105m270378  External source  16  F  11  2011  Thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1108m030060  Our department  17  F  12  2011  Thorax  Guinea pig, hamster, gerbil, kitten  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885218  1110m250836  External source  18  M  12  2011  Abdomen  Rabbit  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885219  1111m220427  External source  19  F  7  2012  Scalp kerion  Guinea pig, rabbit, cow, dog, horse  NI  White  μ and macro  +  A. benhamiae  T. benhamiae  KY885220  1204m110484  Our department  20  F  36  2012  Thorax (nipple)  NI  NI  Yellow  some μ  ++  T. m. var porcellae  T. benhamiae  KY885221  1205m290936  External source  21  F  33  2012  Calf, knee  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885222  1206m110884  Our department  22  F  22  2012  Arm  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  NP  NA  1208m080872  Our department  23  F  26  2012  Arm  Guinea pig  Yes  Yellow  few μ  −  T. m. var porcellae  T. benhamiae  KY885223  1208m170694  Our department  24  M  60  2012  Forearm  NI  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885224  1211m220440  External source  25  M  4  2013  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885225  1308m120592  Our department  26  F  43  2013  Leg  NI  NI  Yellow  few μ  +  T. m. var porcellae  NP  NA  1310m090513  External source  27  A  NA  2013  NI  NA  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885226  1310m160506  External source  28  A  NA  2014  NI  NA  Yes  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885227  1401m280504  External source  29  F  12  2014  Thorax (back)  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885228  1403m070728  Our department  30  F  49  2014  Face, neck  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885229  1405m100253  Our department  31  F  8  2014  Scalp, face  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885230  1405m170464  Our department  32  F  6  2014  Thorax  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885231  1405m191015  Our department  33  F  15  2014  NI  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885232  1410m030306  External source  34  F  2  2014  Scalp  NI  NI  Yellow  few μ  −  T. m. var porcellae  NP  NA  1410m160928  Our department  35  F  15  2014  Thigh  NI  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885233  1411m170810  Our department  36  A  NA  2014  NI  NA  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  GRA Sév Spik  Our department  37  M  9  2015  NI  Guinea pig  NI  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1506m020295  External source  38  F  54  2015  Arm  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885234  1507m100694  Our department  39  F  11  2016  Eyelid  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885235  1607m060843  Our department  40  F  52  2016  Lip  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885236  1608m220245  Our department  41  M  43  2016  Elbow, knee  NI  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885237  1611m220253  Our department  A, animal; F, female; M, male; NA, not applicable; NI, no information; NP, not performed; μ, microconidia; macro, macroconidia; −, negative; ±, equivocal; +, weakly positive; ++, positive. T. m., T. mentagrophytes. View Large Table 2. List of strains used in this study. No.  Sex  Age  Year  Lesion localization  Contact with animals  Animal lesion  phenotype  Microscopy  Urea broth  Initial identification  ITS sequencing  GenBank Accession no.  Culture collection ID  Source  1  F  9  2008  Thorax (back)  NI  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885203  0801m210208  Our department  2  F  8  2008  NI  Rabbit  NI  White  abundant μ  +  A. benhamiae  T. benhamiae  KY885204  0802m180906  Our department  3  F  15  2008  Thigh  NI  NI  White  sterile  NP  A. benhamiae  T. bullosum  KY885205  0805m150877  External source  4  M  9  2008  Cheek  Cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885206  0810m040057  Our department  5  F  12  2009  Arm  NI  NI  Yellow  few μ  NP  A benhamiae  T. benhamiae  KY885207  0906m180356  External source  6  M  24  2009  Beard sycosis  NI  NI  White  μ and macro  +  A. benhamiae  T. eriotrephon  KY885208  0912m230081  Our department  7  M  8  2010  NI  NI  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885209  1001m210429  External source  8  F  4  2010  Face  Guinea pig, pony, cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885210  1005m220254  Our department  9  F  8  2010  Scalp kerion  Guinea pig  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885211  1012m200864  Our department  10  F  15  2011  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885212  1102m170903  External source  11  F  12  2011  Shoulder  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885213  1102m250627  External source  12  F  5  2011  Thorax (sternum)  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885214  1102m280391  External source  13  F  5  2011  Pubis, groin  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885215  1104m210816  External source  14  F  20  2011  Arm  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885216  1105m260453  External source  15  F  10  2011  Face, thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885217  1105m270378  External source  16  F  11  2011  Thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1108m030060  Our department  17  F  12  2011  Thorax  Guinea pig, hamster, gerbil, kitten  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885218  1110m250836  External source  18  M  12  2011  Abdomen  Rabbit  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885219  1111m220427  External source  19  F  7  2012  Scalp kerion  Guinea pig, rabbit, cow, dog, horse  NI  White  μ and macro  +  A. benhamiae  T. benhamiae  KY885220  1204m110484  Our department  20  F  36  2012  Thorax (nipple)  NI  NI  Yellow  some μ  ++  T. m. var porcellae  T. benhamiae  KY885221  1205m290936  External source  21  F  33  2012  Calf, knee  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885222  1206m110884  Our department  22  F  22  2012  Arm  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  NP  NA  1208m080872  Our department  23  F  26  2012  Arm  Guinea pig  Yes  Yellow  few μ  −  T. m. var porcellae  T. benhamiae  KY885223  1208m170694  Our department  24  M  60  2012  Forearm  NI  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885224  1211m220440  External source  25  M  4  2013  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885225  1308m120592  Our department  26  F  43  2013  Leg  NI  NI  Yellow  few μ  +  T. m. var porcellae  NP  NA  1310m090513  External source  27  A  NA  2013  NI  NA  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885226  1310m160506  External source  28  A  NA  2014  NI  NA  Yes  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885227  1401m280504  External source  29  F  12  2014  Thorax (back)  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885228  1403m070728  Our department  30  F  49  2014  Face, neck  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885229  1405m100253  Our department  31  F  8  2014  Scalp, face  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885230  1405m170464  Our department  32  F  6  2014  Thorax  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885231  1405m191015  Our department  33  F  15  2014  NI  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885232  1410m030306  External source  34  F  2  2014  Scalp  NI  NI  Yellow  few μ  −  T. m. var porcellae  NP  NA  1410m160928  Our department  35  F  15  2014  Thigh  NI  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885233  1411m170810  Our department  36  A  NA  2014  NI  NA  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  GRA Sév Spik  Our department  37  M  9  2015  NI  Guinea pig  NI  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1506m020295  External source  38  F  54  2015  Arm  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885234  1507m100694  Our department  39  F  11  2016  Eyelid  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885235  1607m060843  Our department  40  F  52  2016  Lip  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885236  1608m220245  Our department  41  M  43  2016  Elbow, knee  NI  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885237  1611m220253  Our department  No.  Sex  Age  Year  Lesion localization  Contact with animals  Animal lesion  phenotype  Microscopy  Urea broth  Initial identification  ITS sequencing  GenBank Accession no.  Culture collection ID  Source  1  F  9  2008  Thorax (back)  NI  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885203  0801m210208  Our department  2  F  8  2008  NI  Rabbit  NI  White  abundant μ  +  A. benhamiae  T. benhamiae  KY885204  0802m180906  Our department  3  F  15  2008  Thigh  NI  NI  White  sterile  NP  A. benhamiae  T. bullosum  KY885205  0805m150877  External source  4  M  9  2008  Cheek  Cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885206  0810m040057  Our department  5  F  12  2009  Arm  NI  NI  Yellow  few μ  NP  A benhamiae  T. benhamiae  KY885207  0906m180356  External source  6  M  24  2009  Beard sycosis  NI  NI  White  μ and macro  +  A. benhamiae  T. eriotrephon  KY885208  0912m230081  Our department  7  M  8  2010  NI  NI  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885209  1001m210429  External source  8  F  4  2010  Face  Guinea pig, pony, cat  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885210  1005m220254  Our department  9  F  8  2010  Scalp kerion  Guinea pig  NI  White  μ and spirals  ++  A. benhamiae  T. benhamiae  KY885211  1012m200864  Our department  10  F  15  2011  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885212  1102m170903  External source  11  F  12  2011  Shoulder  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885213  1102m250627  External source  12  F  5  2011  Thorax (sternum)  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885214  1102m280391  External source  13  F  5  2011  Pubis, groin  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885215  1104m210816  External source  14  F  20  2011  Arm  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885216  1105m260453  External source  15  F  10  2011  Face, thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  T. benhamiae  KY885217  1105m270378  External source  16  F  11  2011  Thorax  Guinea pig  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1108m030060  Our department  17  F  12  2011  Thorax  Guinea pig, hamster, gerbil, kitten  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885218  1110m250836  External source  18  M  12  2011  Abdomen  Rabbit  NI  Yellow  few μ  +  A. benhamiae  T. benhamiae  KY885219  1111m220427  External source  19  F  7  2012  Scalp kerion  Guinea pig, rabbit, cow, dog, horse  NI  White  μ and macro  +  A. benhamiae  T. benhamiae  KY885220  1204m110484  Our department  20  F  36  2012  Thorax (nipple)  NI  NI  Yellow  some μ  ++  T. m. var porcellae  T. benhamiae  KY885221  1205m290936  External source  21  F  33  2012  Calf, knee  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885222  1206m110884  Our department  22  F  22  2012  Arm  Guinea pig  NI  Yellow  few μ  ±  T. m. var porcellae  NP  NA  1208m080872  Our department  23  F  26  2012  Arm  Guinea pig  Yes  Yellow  few μ  −  T. m. var porcellae  T. benhamiae  KY885223  1208m170694  Our department  24  M  60  2012  Forearm  NI  NI  Yellow  few μ  ±  T. m. var porcellae  T. benhamiae  KY885224  1211m220440  External source  25  M  4  2013  Scalp kerion  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885225  1308m120592  Our department  26  F  43  2013  Leg  NI  NI  Yellow  few μ  +  T. m. var porcellae  NP  NA  1310m090513  External source  27  A  NA  2013  NI  NA  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885226  1310m160506  External source  28  A  NA  2014  NI  NA  Yes  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885227  1401m280504  External source  29  F  12  2014  Thorax (back)  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885228  1403m070728  Our department  30  F  49  2014  Face, neck  Guinea pig  Yes  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885229  1405m100253  Our department  31  F  8  2014  Scalp, face  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885230  1405m170464  Our department  32  F  6  2014  Thorax  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885231  1405m191015  Our department  33  F  15  2014  NI  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885232  1410m030306  External source  34  F  2  2014  Scalp  NI  NI  Yellow  few μ  −  T. m. var porcellae  NP  NA  1410m160928  Our department  35  F  15  2014  Thigh  NI  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885233  1411m170810  Our department  36  A  NA  2014  NI  NA  Yes  Yellow  few μ  NP  T. m. var porcellae  NP  NA  GRA Sév Spik  Our department  37  M  9  2015  NI  Guinea pig  NI  Yellow  few μ  NP  T. m. var porcellae  NP  NA  1506m020295  External source  38  F  54  2015  Arm  Guinea pig  NI  Yellow  few μ  +  T. m. var porcellae  T. benhamiae  KY885234  1507m100694  Our department  39  F  11  2016  Eyelid  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885235  1607m060843  Our department  40  F  52  2016  Lip  Guinea pig  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885236  1608m220245  Our department  41  M  43  2016  Elbow, knee  NI  NI  Yellow  few μ  ++  T. m. var porcellae  T. benhamiae  KY885237  1611m220253  Our department  A, animal; F, female; M, male; NA, not applicable; NI, no information; NP, not performed; μ, microconidia; macro, macroconidia; −, negative; ±, equivocal; +, weakly positive; ++, positive. T. m., T. mentagrophytes. View Large No M. canis were included in this study because all of the strains had typical macroconidia that made them easy to differentiate from the yellow phenotype of T. benhamiae. We had no T. concentricum or T. eriotrephon in our collections, and all of our T. verrucosum isolates were already confirmed by ITS sequencing. Only 35 of 41 strains were available for retrospective study in our mycology bank; for these, the morphological identification was verified by sequencing the ITS region of the ribosomal DNA. The six remaining strains had been identified according to morphology as T. mentagrophytes var. porcellae, name used incorrectly for the yellow phenotype Trichophyton sp. of A. benhamiae (Figs 1 and 2). Figure 1. View largeDownload slide Flow chart of the 41 strains included in this study. Figure 1. View largeDownload slide Flow chart of the 41 strains included in this study. Figure 2. View largeDownload slide Macroscopy and microscopy of T. benhamiae. Up: Macroscopy of white (left) and yellow (right) phenotypes of T. benhamiae. Potato-dextrose-agar slant media, diluted Sabouraud dextrose agar (two upper Petri dishes) and Borelli's lactrimel agar (two lower Petri dishes). Low-left: Microscopy of the white phenotype of T. benhamiae (magnification 400 ×): numerous micro- and macroconidia. Low-right: Microscopy of the yellow phenotype of T. benhamiae (magnification 400 ×): absent macroconidia, few microconidia. Figure 2. View largeDownload slide Macroscopy and microscopy of T. benhamiae. Up: Macroscopy of white (left) and yellow (right) phenotypes of T. benhamiae. Potato-dextrose-agar slant media, diluted Sabouraud dextrose agar (two upper Petri dishes) and Borelli's lactrimel agar (two lower Petri dishes). Low-left: Microscopy of the white phenotype of T. benhamiae (magnification 400 ×): numerous micro- and macroconidia. Low-right: Microscopy of the yellow phenotype of T. benhamiae (magnification 400 ×): absent macroconidia, few microconidia. Strain 3 had a white phenotype and was identified by ITS sequencing as T. bullosum (Table 2, Fig. 1, Table 3). This result was confirmed by the phylogenetic analysis presented in Figure 3. Strain 3 had been isolated in 2008 from a thigh lesion of a 15-year-old girl, presented a white phenotype and a sterile microscopy; no information concerning animal contact was known. Table 3. Results of GenBank and CBS comparison for our 35 ITS sequenced strains. Strains from this study  BLASTN first hits  Score  Identity  Query cover  Organism  GenBank definition  Strain 6  GenBank EU181452.1  1230 bits (666)  100%  99%  T. erinacei  A. benhamiae NCPF 431  672 bp  GenBank KJ606083.1  1229 bits (665)  99%  99%  Trichophyton cf. erinacei ATCC 24552  Trichophyton cf. erinacei ATCC 24552  ITS sequencing: A. benhamiae / T. erinacei  GenBank JN134091.1  1203 bits (651)  99%  100%  T. erinacei  T. erinacei isolate 379  Phylogeny: T. eriotrephon  GenBank EU622882.1  1203 bits (651)  99%  100%  T. benhamiae  A. benhamiae AMC 07101    GenBank JX413539  1044.49  98.958%  100%  T. erinacei  A. benhamiae CS783  Strain 3  CBS 131645  1041.32  100%  100%  T. bullosum BCCM/IHEM 24321    656 bp  CBS 363.35 GenBank FM992675.1  1038.32  100%  99.695%  T. bullosum LP 770    ITS sequencing and phylogeny: T. bullosum  GenBank LN589975.1  1197 bits (648)  99%  99%  T. bullosum CCF 4831      CBS 557.50 GenBank FM992676.1  958.02  100%  92%  T. bullosum No. 493      GenBank KU257463.1    99%  100%  T. benhamiae SU901378    White phenotype  GenBank KJ606075.1    99%  100%  A. benhamiae ATCC MYA-4681    Strains: 1, 2, 9, 19  GenBank JN134088.1    99%  100%  T. benhamiae 269    T. benhamiae  CBS 112370    99.201%  100%  A. benhamiae CHUV 2352/02    Yellow phenotype T. benhamiae  GenBank KU257464.1    99%  100%  T. benhamiae SU209346      GenBank AB458216.1    99%  100%  A. benhamiae IFM 54424    Strains: 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 28, 29, 30, 31, 32, 33, 35, 38, 39, 40, 41  GenBank AF170457.1    99%  100%  A. benhamiae RV 26680      CBS 624.66 GenBank JX122282.1    99%  100%  A. benhamiae ATCC 16782      CBS 623.66 GenBank JX122281.1    99%  100%  A. benhamiae ATCC 16781    Yellow phenotype T. benhamiae  GenBank KU257464.1    97%  99%  T. benhamiae SU209346      GenBank KX092365.1    97%  99%  T. benhamiae ATCC 42873    Strain 27  CBS 624.66    97%  99%  A. benhamiae ATCC 16782    Strains from this study  BLASTN first hits  Score  Identity  Query cover  Organism  GenBank definition  Strain 6  GenBank EU181452.1  1230 bits (666)  100%  99%  T. erinacei  A. benhamiae NCPF 431  672 bp  GenBank KJ606083.1  1229 bits (665)  99%  99%  Trichophyton cf. erinacei ATCC 24552  Trichophyton cf. erinacei ATCC 24552  ITS sequencing: A. benhamiae / T. erinacei  GenBank JN134091.1  1203 bits (651)  99%  100%  T. erinacei  T. erinacei isolate 379  Phylogeny: T. eriotrephon  GenBank EU622882.1  1203 bits (651)  99%  100%  T. benhamiae  A. benhamiae AMC 07101    GenBank JX413539  1044.49  98.958%  100%  T. erinacei  A. benhamiae CS783  Strain 3  CBS 131645  1041.32  100%  100%  T. bullosum BCCM/IHEM 24321    656 bp  CBS 363.35 GenBank FM992675.1  1038.32  100%  99.695%  T. bullosum LP 770    ITS sequencing and phylogeny: T. bullosum  GenBank LN589975.1  1197 bits (648)  99%  99%  T. bullosum CCF 4831      CBS 557.50 GenBank FM992676.1  958.02  100%  92%  T. bullosum No. 493      GenBank KU257463.1    99%  100%  T. benhamiae SU901378    White phenotype  GenBank KJ606075.1    99%  100%  A. benhamiae ATCC MYA-4681    Strains: 1, 2, 9, 19  GenBank JN134088.1    99%  100%  T. benhamiae 269    T. benhamiae  CBS 112370    99.201%  100%  A. benhamiae CHUV 2352/02    Yellow phenotype T. benhamiae  GenBank KU257464.1    99%  100%  T. benhamiae SU209346      GenBank AB458216.1    99%  100%  A. benhamiae IFM 54424    Strains: 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 28, 29, 30, 31, 32, 33, 35, 38, 39, 40, 41  GenBank AF170457.1    99%  100%  A. benhamiae RV 26680      CBS 624.66 GenBank JX122282.1    99%  100%  A. benhamiae ATCC 16782      CBS 623.66 GenBank JX122281.1    99%  100%  A. benhamiae ATCC 16781    Yellow phenotype T. benhamiae  GenBank KU257464.1    97%  99%  T. benhamiae SU209346      GenBank KX092365.1    97%  99%  T. benhamiae ATCC 42873    Strain 27  CBS 624.66    97%  99%  A. benhamiae ATCC 16782    View Large Table 3. Results of GenBank and CBS comparison for our 35 ITS sequenced strains. Strains from this study  BLASTN first hits  Score  Identity  Query cover  Organism  GenBank definition  Strain 6  GenBank EU181452.1  1230 bits (666)  100%  99%  T. erinacei  A. benhamiae NCPF 431  672 bp  GenBank KJ606083.1  1229 bits (665)  99%  99%  Trichophyton cf. erinacei ATCC 24552  Trichophyton cf. erinacei ATCC 24552  ITS sequencing: A. benhamiae / T. erinacei  GenBank JN134091.1  1203 bits (651)  99%  100%  T. erinacei  T. erinacei isolate 379  Phylogeny: T. eriotrephon  GenBank EU622882.1  1203 bits (651)  99%  100%  T. benhamiae  A. benhamiae AMC 07101    GenBank JX413539  1044.49  98.958%  100%  T. erinacei  A. benhamiae CS783  Strain 3  CBS 131645  1041.32  100%  100%  T. bullosum BCCM/IHEM 24321    656 bp  CBS 363.35 GenBank FM992675.1  1038.32  100%  99.695%  T. bullosum LP 770    ITS sequencing and phylogeny: T. bullosum  GenBank LN589975.1  1197 bits (648)  99%  99%  T. bullosum CCF 4831      CBS 557.50 GenBank FM992676.1  958.02  100%  92%  T. bullosum No. 493      GenBank KU257463.1    99%  100%  T. benhamiae SU901378    White phenotype  GenBank KJ606075.1    99%  100%  A. benhamiae ATCC MYA-4681    Strains: 1, 2, 9, 19  GenBank JN134088.1    99%  100%  T. benhamiae 269    T. benhamiae  CBS 112370    99.201%  100%  A. benhamiae CHUV 2352/02    Yellow phenotype T. benhamiae  GenBank KU257464.1    99%  100%  T. benhamiae SU209346      GenBank AB458216.1    99%  100%  A. benhamiae IFM 54424    Strains: 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 28, 29, 30, 31, 32, 33, 35, 38, 39, 40, 41  GenBank AF170457.1    99%  100%  A. benhamiae RV 26680      CBS 624.66 GenBank JX122282.1    99%  100%  A. benhamiae ATCC 16782      CBS 623.66 GenBank JX122281.1    99%  100%  A. benhamiae ATCC 16781    Yellow phenotype T. benhamiae  GenBank KU257464.1    97%  99%  T. benhamiae SU209346      GenBank KX092365.1    97%  99%  T. benhamiae ATCC 42873    Strain 27  CBS 624.66    97%  99%  A. benhamiae ATCC 16782    Strains from this study  BLASTN first hits  Score  Identity  Query cover  Organism  GenBank definition  Strain 6  GenBank EU181452.1  1230 bits (666)  100%  99%  T. erinacei  A. benhamiae NCPF 431  672 bp  GenBank KJ606083.1  1229 bits (665)  99%  99%  Trichophyton cf. erinacei ATCC 24552  Trichophyton cf. erinacei ATCC 24552  ITS sequencing: A. benhamiae / T. erinacei  GenBank JN134091.1  1203 bits (651)  99%  100%  T. erinacei  T. erinacei isolate 379  Phylogeny: T. eriotrephon  GenBank EU622882.1  1203 bits (651)  99%  100%  T. benhamiae  A. benhamiae AMC 07101    GenBank JX413539  1044.49  98.958%  100%  T. erinacei  A. benhamiae CS783  Strain 3  CBS 131645  1041.32  100%  100%  T. bullosum BCCM/IHEM 24321    656 bp  CBS 363.35 GenBank FM992675.1  1038.32  100%  99.695%  T. bullosum LP 770    ITS sequencing and phylogeny: T. bullosum  GenBank LN589975.1  1197 bits (648)  99%  99%  T. bullosum CCF 4831      CBS 557.50 GenBank FM992676.1  958.02  100%  92%  T. bullosum No. 493      GenBank KU257463.1    99%  100%  T. benhamiae SU901378    White phenotype  GenBank KJ606075.1    99%  100%  A. benhamiae ATCC MYA-4681    Strains: 1, 2, 9, 19  GenBank JN134088.1    99%  100%  T. benhamiae 269    T. benhamiae  CBS 112370    99.201%  100%  A. benhamiae CHUV 2352/02    Yellow phenotype T. benhamiae  GenBank KU257464.1    99%  100%  T. benhamiae SU209346      GenBank AB458216.1    99%  100%  A. benhamiae IFM 54424    Strains: 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21, 23, 24, 25, 28, 29, 30, 31, 32, 33, 35, 38, 39, 40, 41  GenBank AF170457.1    99%  100%  A. benhamiae RV 26680      CBS 624.66 GenBank JX122282.1    99%  100%  A. benhamiae ATCC 16782      CBS 623.66 GenBank JX122281.1    99%  100%  A. benhamiae ATCC 16781    Yellow phenotype T. benhamiae  GenBank KU257464.1    97%  99%  T. benhamiae SU209346      GenBank KX092365.1    97%  99%  T. benhamiae ATCC 42873    Strain 27  CBS 624.66    97%  99%  A. benhamiae ATCC 16782    View Large Figure 3. View largeDownload slide Maximum likelihood phylogenetic tree (MEGA® 6.0 software) based on ITS sequences of Arthroderma benhamiae complex using T92+G+I model, with 10,000 bootstrap replications. Bootstrap values above 70% are shown, Trichophyton rubrum was used as outgroup. A: animal strain (2); *: type strains. Figure 3. View largeDownload slide Maximum likelihood phylogenetic tree (MEGA® 6.0 software) based on ITS sequences of Arthroderma benhamiae complex using T92+G+I model, with 10,000 bootstrap replications. Bootstrap values above 70% are shown, Trichophyton rubrum was used as outgroup. A: animal strain (2); *: type strains. Strain 6 had a white phenotype, and ITS sequencing showed 99–100% identity with A. benhamiae / T. erinacei strains (Table 3). However, the phylogenetic analysis with high bootstrap values presented in Figure 3 showed that it belongs to the T. eriotrephon clade. This strain had been isolated from a beard sycosis of a 24-year-old man in 2009 and no information concerning animal contact was given. Microscopically, it presented numerous microconidia and some macroconidia; urease activity was weekly positive (Table 2). The remaining 33 sequenced strains were confirmed as A. benhamiae or T. benhamiae. One animal strain (strain 27) only had 97% identity with three strains of T. benhamiae (Table 3). The other 32 strains all have 99–100% identity with strains of T. benhamiae, including the type strain. In sum, 36 of the total 39 T. benhamiae/T. mentagrophytes var. porcellae strains were isolated from humans and three from guinea pig samples (Fig. 1). And 35 of the 39 T. benhamiae/T. mentagrophytes var. porcellae strains (89.7%) belong to the yellow phenotype and four (10.3%) to the white phenotype. All white phenotype strains were isolated from human samples (strains 1, 2, 9, and 19); they all clustered together and separately from the yellow ones in the phylogenetic analysis presented in Figure 3. With regards to the 36 human T. benhamiae strains, 25 (69.4%) were from children with ages ranging from 2 to 15 years; the lesions were mostly inflammatory and located on the thorax or abdomen (seven), scalp (six), face (five), arms (two), leg (one), and groin (one). At least two children had more than one lesion (face and thorax, face and scalp). For four children, the clinical data were not precise (only “skin lesion” was specified). The 11 adults (30.6% of T. benhamiae strains) had ages ranging from 20 to 89 and presented lesions on arms (six), legs (four), thorax (one), lip (one), scalp (one), face and neck (one). Only two human strains come from two members (children) of the same family (strains 31 and 32). Both strains were of the T. benhamiae yellow phenotype, presented weakly positive urease activity but did not cluster next to one another in the dendrogramm presented in Figure 3. In nine of the 36 human T. benhamiae infections (25%), contact with an animal could not be established at the time of diagnosis, mostly because the interrogatory had been performed out of our department and no detailed information was transmitted to our laboratory. In sum, 27 out of 36 T. benhamiae patients (75%) had been in contact with an animal. And 21 of these 27 patients had been in contact only with guinea pigs; three patients had been in contact with other animals besides guinea pigs (guinea pig, pony, and cat for patient 8; guinea pig, hamster, gerbil, and kitten for patient 17; and guinea pig, rabbit, cow, dog, and horse for patient 19). Seven of the 24 guinea pigs were symptomatic, but we did not receive samples from these animals. Three patients with no guinea pig contact had been in contact with rabbits (patient 2 and patient 18) and patient 4 with a cat (Table 2). Three T. benhamiae strains isolated from guinea pigs were sent to us for identification. These animals were symptomatic but were not pets of any of the patients included in this article. They were all yellow type T. benhamiae. Only 32 T. benhamiae strains of the total of 39 were tested for urease activity: eight were positive, 19 were weakly positive, five were equivocal, and two were negative (Table 2 and Fig. 4). Figure 4. View largeDownload slide Christensen's urea broth test. From left to right: negative control, equivocal reaction (strain 11), weakly positive reaction (strain 10), positive reaction (strain 40). Figure 4. View largeDownload slide Christensen's urea broth test. From left to right: negative control, equivocal reaction (strain 11), weakly positive reaction (strain 10), positive reaction (strain 40). Regarding the incidence of T. benhamiae and M. canis, the former was more frequent than the latter in only 3 of the 9-year survey (Table 1): 2011 (1.06% vs 0.82%), 2012 (0.85% vs 0.57%), and 2014 (1.18% vs 0.4%). Discussion Most of the dermatophytes are cosmopolite, but some are confined to specific regions or areas of the globe or are associated with certain animals. Population migrations, changes in lifestyle, improvement of hygiene, and practice of more physical activity are changing the geographical distribution of dermatophytosis. Another important factor influencing the epidemiology of these infections is the increasing number and variety of pets. Lately, under the influence of fashion and mediatisation, these are no longer limited to cats and dogs. Reptiles, mice, rabbits, guinea pigs or ferrets have become equally common9,25,32. Children and staff working in pet shops are populations especially at risk of contracting an infection transmitted by these no longer exotic animals.32,33,34,35,36 More than adults, children are at risk of developing an infection due to zoophilic dermatophytes because of their increased outdoors activity and preferred close contact with pets and other animals.33 This was also evidenced by our cases, with infections concerning mostly children (25 out of 38 patients = 65.78%). The adults’ lesions were mostly situated on the exposed parts of the skin (arms and legs), whereas lesions on thorax, abdomen, scalp, face, groin, thigh, or shoulder, were predominant in children, probably due to petting and playing with their animals. T. benhamiae is transmitted mainly by guinea pigs but also occasionally by other animals.4,18,21,25,37 In our survey, three patients had been in contact with other animals besides guinea pigs (pony, cat, rabbit, cow, dog, and horse) and three patients had been in contact only with animals other than guinea pigs (rabbit and cat). These data correspond to those of the literature, since other hosts have recently been identified for T. benhamiae, such as cats, dogs, rabbits, mice, rats, foxes, and more rarely degus or porcupines.6,10,13,38,39 Lesions caused by T. benhamiae tend to be highly inflammatory. The animals are usually asymptomatic; when apparent, typical lesions are circumscribed areas of alopecia with erythema, scaling and crusting.4 In 27 of our 36 T. benhamiae human cases (75%), contact with guinea pigs has been established. Out of these 27 cases, the animals were confirmed symptomatic in only seven cases (25.92%). Khettar et al. in 20129 and Bloch et al. in 201640 have investigated pet shops in the city of Nancy (Eastern France) and found that 1/2 of the guinea pigs in 2012 and 2/3 in 2016 were carriers of T. benhamiae (morphological identification), most of them asymptomatic. The percentages of carriers for hosts other than guinea pigs have not been investigated. Transmission can take place directly by contact with the animals (even if asymptomatic) but also via soil, animal hairs, and scales. The infectivity could be as long as 2 years.8 These are elements that add to the spreading of the infection, since the animal is neither identified as a potential source of infection for its entourage, nor isolated or treated. In terms of incidence, in most regions with a temperate climate, M. canis remains the second most frequently isolated zoophilic dermatophyte37 after T. mentagrophytes. It is mainly transmitted by cats and dogs33,41 and determines lesions that are not very inflammatory, unlike most of the other zoophilic dermatophytes. The observed frequency of T. benhamiae infection has been increasing recently in Japan,11,16 Switzerland,4,10,17 Germany,5,14,18,42,43,44 France,8 Belgium,9 the Netherlands,37 and Chile36. In recent years in Japan11,16T. benhamiae has become the second most frequent dermatophyte after M. canis, and a study performed in Germany between March 2010 and March 2013 showed that T. benhamiae had already become the most frequent zoophilic dermatophyte responsible of human infections with a prevalence of 2.9%.13,14 In the past 9 years in our laboratory we diagnosed a total of 658 strains of T. mentagrophytes, 65 strains of M. canis and 39 strains of T. benhamiae/T. mentagrophytes var. porcellae (Tables 1 and 2). T. mentagrophytes remains the most frequent zoophilic dermatophyte (incidence around 9%). M. canis comes in second (incidence around 1%) and T. benhamiae third (incidence around 0.5%, varying between 0.25% and 1.18%). From our 9-year survey we can conclude (Table 1) that T. benhamiae was the third most frequent zoophilic dermatophyte after T. mentagrophytes and M. canis in our laboratory, except for 2011 (1.06% vs 0.82%), 2012 (0.85% vs 0.57%), and 2014 (1.18% vs 0.4%), when it came second, surpassing M. canis. This seems to be mostly due to the increased number of guinea pigs as popular pets and to the fact that up to 2/3 of these animals can be carriers of T. benhamiae, even if asymptomatic.9,40 Microscopical differential diagnosis between M. canis and the yellow phenotype of T. benhamiae can be straightforward when rough-walled spindle-like macroconidia are present for the former and only few microconidia for the latter. Recently, Brasch and Wodarg14 described loop or circuit-like mycelial junctions for T. benhamiae, which could make morphological identification easier. Easy and fast methods, like the one proposed by Mayser et al. in 201345 can be very useful. The authors used chromogenic media CandiSelect™ 4 (Bio Rad, France), which allowed differentiation of the two dermatophytes after a few hours of incubation: the medium turned pink or purple for M. canis and turquoise-green for T. benhamiae.45 The chromogenic medium used routinely in our laboratory is ChromID Candida™ (bioMérieux, France), but it does not allow the differentiation of M. canis and T. benhamiae described by Mayser et al. for CandiSelect™ 4. When both M. canis and the yellow phenotype of T. benhamiae present as sterile mycelia, they are difficult to differentiate and ITS sequencing is recommended. Differential diagnosis between T. mentagrophytes and the white phenotype of T. benhamiae can be problematic, considering their similar macroscopy and microscopy.15 Since these two species also show overlapping host specificity, ITS sequencing is the only method that can provide reliable identification. Christensen urea broth test for T. benhamiae has been described as negative,8 weakly positive,45 positive,13 or variable.14 Our results confirm the variability observed by Brasch and Wodarg14; 32 strains out of the 39 T. benhamiae strains were tested for urease activity: eight were positive, 19 were weakly positive, five were equivocal, and two were negative. Considering the subjective interpretation of this test, the different types of tests available and especially the strain variability of the urea hydrolysis activity, this test is less and less used and/or reported in the literature. Recently, mass spectrometry has been used for the identification of T. benhamiae, with great correlations when compared to PCR.28,46 This technique is now widely available in routine and hospital laboratories but some problems persist: sporulation of the mould is needed, extraction protocols are not standardized, databases need to be regularly updated, and most of them are expensive and/or contain a limited number of dermatophytes in general and of T. benhamiae isolates in particular. Dermatophyte species identification can be performed or confirmed by DNA sequencing, most frequently targeting the ITS region of the ribosomal DNA.2,21,25,27 Multilocus DNA sequencing has recently been used to revise the classification and taxonomy of dermatophytes.2 De Hoog et al. showed that dermatophyte taxonomy has reached an acceptable level of stability. ITS sequencing can also reveal unexpected results: in our study, one of the strains we suspected of being a white phenotype of T. benhamiae proved to be T. bullosum, a zoophilic dermatophyte rarely isolated from the coat of horses and possibly donkeys. Only two cases have been described in the literature: one from a forearm lesion of a 21-year-old male in rural France47 and one from a saddle-area lesion of a 6-year-old male horse in the Czech Republic.48 As shown by the dendrogram presented in Figure 3, the ITS sequence of our T. bullosum strain clustered with the other two available strains. This newly described species is closely related to T. verrucosum and T. eriotrephon and systematic molecular identification of the dermatophytes comprised in these genera could give us more insight into the real prevalence of this species. Another strain we suspected of being a white phenotype of T. benhamiae was strain 6, isolated from a beard sycosis of a 24-year-old man in 2009 (no information concerning animal contact was available). ITS sequencing showed a 99–100% identity with A. benhamiae and T. erinacei strains (Table 3), but the phylogenetic analysis with high bootstrap value presented in Figure 3 places this strain in the T. eriotrephon clade, next to the type strain. There are at least two limiting factors in our case: the sequence length (672 bp) and having used only one marker for the phylogenetic analysis presented in Figure 3. All four white phenotype T. benhamiae strains were isolated from human samples (strains 1, 2, 9, and 19); they all cluster together and separately from the yellow ones in the phylogenetic analysis presented in Figure 3. The phylogenetic analysis shows clusters of A. benhamiae as well as T. benhamiae. We think this is due to ongoing updates taking place in the different databases following the new taxonomy of dermatophytes.2 These efforts of the scientific community will be useful in deepening the knowledge of local epidemiologies and will improve communication between scientists. T. benhamiae is a zoophilic dermatophyte diagnosed as an agent of human infection with increased frequency in the past years on three continents (Asia, Europe, South America). Our study shows that ITS sequencing is necessary for accurate identification of both phenotypes (white or yellow) of the species. The new taxonomy2 should simplify identifying T. benhamiae and monitoring the epidemiology of this zoophilic dermatophyte. Acknowledgments We thank private laboratories and private dermatologists in the Strasbourg area for contributing patient samples and fungal strains, and the technicians from the Medical Mycology Laboratory of the Strasbourg University Hospital for their technical assistance. 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Medical MycologyOxford University Press

Published: Nov 17, 2017

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