An Outbreak of Trichophyton quinckeanum Zoonotic Infections in the Czech Republic Transmitted from Cats and Dogs

Trichophyton quinckeanum, a zoophilic dermatophyte mostly known as the causative agent of rodent favus, is relatively rarely reported to cause human infections. Indeed, no infections were detected in Czechia between 2012 and 2015 despite routine verification of species identification by ITS rDNA sequencing. By contrast, 25 human and 11 animal cases of infection were documented from December 2016 to December 2020 and the rates tended to grow every following year. Interestingly, most of the cases were reported in the Olomouc region, suggesting a local outbreak. We bring the evidence that human T. quinckeanum infections are most commonly contracted from infected cats or, less frequently, dogs. Although rodents or contaminated soil and environment could be the source of infection to cats and dogs, the occurrence of infections in multiple animals in the same household suggests direct transmission among animals. Confirmation of the identification by molecular methods is highly recommended due to morphological similarity with T. mentagrophytes/T. interdigitale. Antifungal susceptibility testing of isolates to eight antifungals was performed using EUCAST methodology (E.Def 11.0). Among the tested antifungals, terbinafine, amorolfine, ciclopirox and efinaconazole were most potent in vitro and elevated minimum inhibitory concentrations were obtained for fluconazole and ketoconazole.


Introduction
The zoophilic species Trichophyton quinckeanum was earlier known as a variety of T. mentagrophytes, that is, T. mentagrophytes var. quinckeanum [1,2]. In the past, the taxonomic status of this pathogen was the subject of many controversies which have been reviewed in detail by Beguin et al. [3]. Based on the recently published taxonomy, which is predominantly based on molecular biology criteria, T. quinckeanum again constitutes a distinct species closely related to anthropophilic T. schoenleinii [4]. Rodents and camels are considered the main natural reservoirs of T. quinckeanum which preferably occurs in the Middle East [5]. In Europe, this pathogen has been only sporadically isolated from humans, and there have been no or only sporadic reports of T. quinckeanum in Czechia [1,6,7].
Thanks to a large epidemiological study on dermatophytosis ongoing in Czechia since 2011, exact prospective data substantiated by molecularly identified isolates are available [7,8]. This has led to the detection of an increasing number of cases of infections in recent years which are summarized in this study. In addition to an analysis of epidemiological data, we searched for possible sources of infections and characterized antifungal susceptibility patterns, morphology and MALDI-TOF mass spectrometry (MS) in obtained isolates to offer appropriate treatment options and diagnostic tools.

Dermatophyte Isolates, Specimen Collection and Examination
Dermatophytes from human tinea infections were collected prospectively from eight clinical laboratories across Czechia since 2012 as described previously [7,8]. During this period, more than 15,000 dermatophytosis cases were recorded, representing all clinical types of tinea, with approximately 1500-1700 cases registered per year. Identification of all dermatophytes except T. rubrum was confirmed by internal transcribed spacer (ITS) rDNA sequencing (see below). A total of 36 isolates of T. quinckeanum were examined in this study. The information on the isolation source of all isolates is provided in Tables 1 and 2. Skin lesions were scraped from the edge or entirely (small lesions) using scalpel blades or dermal curette; hairs were plucked and crusts picked from scalp lesions. The collected material was divided for cultivation (Sabouraud's dextrose agar with cycloheximide and chloramphenicol, and Sabouraud's dextrose agar without antibiotics, 25 • C) and direct microscopic examination. Skin squames and hairs were mounted between the glass slide and coverslip in a drop of 20% KOH with Parker ink or Blankophor (Bayer, Leverkusen, Germany) [9], depending on the laboratory. Microscopic examination was performed under a light or fluorescence microscope.

Molecular Methods
Genomic DNA was extracted from seven-day-old colonies using Fungal/Bacterial Miniprep Kit (Zymo research, Irvine, CA, USA). The quality of the extracted DNA was evaluated with NanoDrop 1000 Spectrophotometer. The ITS rDNA region (ITS1-5.8S-ITS2 cluster) was amplified using the forward primer ITS1F and reverse primers ITS4 or NL4 [14,15]. Reaction volume of 20 µL contained 1 µL (50 ng mL −1 ) of DNA, 0.3 µL of both primers (25 pM mL −1 ), 0.2 µL of My Taq Polymerase and 4 µL of 5 × My Taq PCR buffer (Bioline, London, UK). PCR conditions followed the protocol described by Hubka et al. [16]. PCR product purification followed the previously described protocol [17]. Automated sequencing was performed at Macrogen Sequencing Service (Amsterdam, The Netherlands) using both terminal primers.

MALDI-TOF Mass Spectrometry
Thirty-six isolates of T. quinckeanum were analyzed by MALDI-TOF-MS and compared with spectra of other common zoophilic Trichophyton species occurring in Czechia, namely the T. mentagrophytes/T. interdigitale complex (n = 20), T. benhamiae var. luteum (n = 20) and T. erinacei (n = 20). Culture and protein mass extraction for MALDI analysis was carried out based on recommendations of the manufacturer. Following complete washing and drying of sample pellets, each sample was coated with 1 µL of the matrix (HCCA portioned; Bruker Daltonics GmbH, Bremen, Germany), and again dried at room temperature. MALDI-TOF MS measurements were performed with the Microflex LT mass spectrometer (Bruker Daltonics GmbH, manufacturer). The MALDI Biotyper 3.1 software was used to analyze spectra. FlexAnalysis ver. 3.4 (Bruker Daltonics GmbH) was used for comparison of T. quinckeanum mass spectra with other closely related zoophilic dermatophytes. The data generated in the present study were compared with previously published reference spectra from Germany [5].

Antifungal Susceptibility Testing
Fluconazole in a powder form was obtained from Pfizer Pharmaceutical Group (New York, NY, USA); terbinafine, itraconazole, ketoconazole, clotrimazole, efinaconazole, amorolfine and ciclopirox were obtained from Merck (Prague, Czech Republic).
Broth microdilution was performed according to the EUCAST document E.Def 11.0 for dermatophyte testing [18]. Briefly, the isolates were incubated at 25 • C on SDA (Trios, Prague, Czech Republic). Inoculum suspensions were created from one-to two-weekold colonies (to achieve acceptable sporulation). The suspension was prepared in sterile distilled water supplemented with 0.1% Tween 20 and filtered using sterile nylon filters with 11 µm pore size (Merck). The suspension was adjusted to a concentration of 2-5 × 10 6 conidia per mL by counting the conidia in a hemocytometer chamber. The suspension was then diluted 1:10 with sterile distilled water to obtain the final working inoculum 2-5 × 10 5 colony-forming units per mL. The inoculum suspension was supplemented with chloramphenicol (Merck; final concentration 50 mg/L) and cycloheximide (Merck; final concentration 300 mg/L). Microplates were incubated at 25 • C in ambient air for 5 days. The endpoints, minimum inhibitory concentrations (MICs), were read visually (the drug concentration yielding no visible growth by eye was scored as the MIC value) and spectrophotometrically (50% inhibition) [18]. Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as quality control strains.
MIC ranges, corresponding geometric mean (GM) values, MIC50 (MIC causing inhibition of 50% of the isolates) and MIC90 (MIC causing inhibition of 90% of the isolates) were determined for the antifungal drug and reading method used. For GM calculation, MIC values of ≤0.004 mg/L were set at 0.004 mg/L and MIC values of ≤0.008 mg/L were set at 0.008 mg/L; similarly, MIC values of >64 mg/L were set at 128 mg/L.

Epidemiological Summary
A total of 36 T. quinckeanum strains were isolated from Czech human patients (n = 25) and animals (n = 11; 7 cats and 4 dogs). Microscopic hyphae presented in skin lesion samples are illustrated in Figure 1. All isolates are listed in Tables 1 and 2. No human cases were detected in Czechia between 2012 and 2015 despite routine identification of dermatophytes by DNA sequencing. The first T. quinckeanum strain was isolated in late 2016 and since then, its incidence in Czechia has been noticeably growing ( Figure 2). Two human cases were recorded in 2017, four in 2018, eight in 2019, and ten in 2020. Twenty-five strains were isolated in humans and eleven from animals. Almost all cases were detected in the Olomouc region ( Figure 2) and predominantly in spring and winter months (Tables 1 and 2).  Trichophyton quinckeanum was more frequently isolated in females (72%). The median age of patients was 23 years (range, 4-77 years); adults predominated (56%) over children and adolescents (≤18 years of age). The infections were mainly manifested as tinea corporis and tinea cruris (n = 18; 69.2%), less commonly as tinea capitis (n = 4) and tinea faciei (n = 4). The patients most commonly reported contacts with cats and dogs in their history (Table 1).

DNA Sequence-Based Identification
The DNA sequences of the ITS rDNA region were identical in all examined strains of T. quinckeanum from human patients as well as from animals. The resulting sequences were deposited into the GenBank database (www.ncbi.nlm.nih.gov) under accession numbers MZ312189-MZ312224.
The sequences were 100% identical to strains associated with recent outbreak of infections in Germany (GenBank accession numbers KY680503, KU257460-KU257462, KU257469) and displayed two and one substitutions in the ITS1 and ITS2 regions, respectively, in comparison with the ex-neotype strain IHEM 13697 (GenBank: MK298745), resulting in a 99.5% similarity. The differentiation of the most closely related species, anthropophilic T. schoenleinii, could not be achieved by using ITS region sequencing. Despite the fact that all T. quinckeanum isolates showed two substitutions in the ITS1 region compared to the majority of T. schoenleinii strains deposited in the GenBank, these substitutions were not conserved for T. quinckeanum as a whole. On the other hand, the misidentification of the two species in the clinical laboratory is highly improbable due to their strikingly different morphology and ecology. The differentiation of other dermatophytes by ITS rDNA was possible thanks to many unique substitutions.

MALDI-TOF Mass Spectrometry
In the mass range of approximately 4050-4200 m/z, T. quinckeanum could be reliably distinguished from T. benhamiae var. luteum and the T. mentagrophytes/T. interdigitale complex in the specific peaks at 4110 m/z and 4160 m/z, respectively ( Figure 4A). In the mass range of approximately 7550-8250 m/z, the MALDI-TOF MS spectra of T. quinckeanum were very similar to the above species and their differentiation was not possible ( Figure 4B,C).
The differences between T. erinacei and T. quinckeanum could be found especially in the mass ranges of approximately 7650-7800 m/z and 8050-8200 m/z in two specific peaks at 7730 m/z and 8130-8140 m/z ( Figure 4B,C). Specific peaks in the range of 4110-4160 m/z could be used in the routine identification of T. quinckeanum in practice using the FlexAnalysis tool after the automatic analysis with a MALDI Biotyper.

Antifungal Susceptibility Testing
Results of the susceptibility testing of 36 T. quinckeanum strains are given in Table 4. The obtained MICs for fluconazole were elevated in all the tested isolates. MICs for other antifungals tested were low, depending on the reading method used, with generally higher values achieved using visual reading compared to spectrophotometric reading. MICs for amorolfine, ciclopirox and efinaconazole were low. MICs for ketoconazole were elevated. MICs for terbinafine were very low with both testing methods for all isolates, with MIC ≤ 0.016 mg/L. The differences between T. erinacei and T. quinckeanum could be found especially in the mass ranges of approximately 7650-7800 m/z and 8050-8200 m/z in two specific peaks at 7730 m/z and 8130-8140 m/z ( Figure 4B,C). Specific peaks in the range of 4110-4160 m/z could be used in the routine identification of T. quinckeanum in practice using the FlexAnalysis tool after the automatic analysis with a MALDI Biotyper.

Antifungal Susceptibility Testing
Results of the susceptibility testing of 36 T. quinckeanum strains are given in Table 4. The obtained MICs for fluconazole were elevated in all the tested isolates. MICs for other antifungals tested were low, depending on the reading method used, with generally higher values achieved using visual reading compared to spectrophotometric reading. MICs for amorolfine, ciclopirox and efinaconazole were low. MICs for ketoconazole were elevated. MICs for terbinafine were very low with both testing methods for all isolates, with MIC ≤ 0.016 mg/L.  Antifungals: FLU-fluconazole; TER-terbinafine; ITR-itraconazole; KET-ketoconazole; CLO-clotrimazole; AMO-amorolfine; CIC-ciclopirox; EFI-efinaconazole; OD-optical density, i.e., MIC value read spectrophotometrically as 50% inhibition of the growth; vis-MIC value read visually as complete inhibition (no growth detected by eye); GM-geometric mean; MIC50 and MIC 90-minimum concentration at which 50% and 90% of the isolates were inhibited, respectively.

Discussion
In Europe, T. quinckeanum has been only sporadically isolated from humans. It seemed logical that the pathogen was in decline with an improving standard of life in developed countries as, historically, infections were mostly connected with low social and economic levels and poor hygiene [7,22,23]. For many years, there were no or only rare reports of T. quinckeanum in Czechia [1,7,24,25]. The first isolate verified by molecular methods was detected at the end of 2016 and the incidence of cases has been considerably growing since then (Table 1, Figure 2). A similar increase in the incidence of this pathogen was recorded several years earlier in Germany [5].
The species is historically connected with mouse favus or rodent favus in general [1,26], but the occasional hosts are also cats, dogs, rabbits, camels, chickens, horses and sheep [5]. Uhrlaß et al. [5] suspected cats as the main source of human zoonotic infections, which is in agreement with our data. In addition, we showed that dogs can also be the source of infection. It is usually assumed that the infection in domestic animals occurs due to transmission from rodents (mice) and then is eventually transmitted to humans. In this study, we showed that the infection may probably spread among domestic animals independently on rodents as indicated by the occurrence of infection in several animals in the same household (Table 2). This information is valuable because dermatophytes usually spread effectively only in their main host(s) and further transmission among other occasionally infected hosts is rare.
The occurrence of this pathogen in Czechia is a new phenomenon in terms of recent decades and is in contrast to the absence of this species in previous years (2012-2015) during which we confirmed the identification of dermatophytes coming from identical laboratories using ITS rDNA sequencing. Thanks to that, we can confidently exclude the possibility that this pathogen was missed or misidentified due to its morphological similarity to T. mentagrophytes/T. interdigitale or other superficially similar species [21,27]. The emergence of T. quinckeanum in animals in recent years is also in concordance with the incidence of human cases (Figure 2).
Although we have no clear explanation for the sudden rise of T. quinckeanum infections substantiated by data, there are several possible hypotheses. The increase can be caused by an overpopulation of rodents in Czechia observed in previous years, potentially leading to an increased transmission to domestic animals and eventually humans [28,29]. This hypothesis, however, does not explain the geographic clustering of the majority of cases into the Olomouc region around Prostějov and Olomouc cities. All but four isolates came from this area, indicating a local outbreak. In addition, a study on the occurrence of dermatophytes in rodents was carried out in the Czech Republic in recent years, which did not detect the presence of this pathogen [30]. In contrast to previous sporadic infections due to T. quinckeanum, the spread of infections among domestic animals is probably the main driving force of the current outbreak similar to the German one [5]. The spread among domestic animals is a completely new phenomenon that has not been described and may be caused by a shift in the virulence and other biological properties of the pathogen.
Interestingly, the Czech and German strains show identical ITS rDNA sequences which were different from all other T. quinckeanum isolates in the GenBank database. This suggests that the current outbreaks were caused by genetically different strains with possible new features compared to T. quinckeanum strains occurring in the past. We hypothesize that these strains could be more virulent, easily transmissible from pets to humans, and able to cause a small epidemic. However, more detailed comparative studies mapping differences in the physiology, virulence and host preferences of current and historical strains are needed for conclusions to be drawn. In the last decade, several new or emerging pathogenic dermatophytes have been reported. Namely, T. benhamiae var. benhamiae is an emerging pathogen in Europe mostly transmitted to humans from guinea pig [19,31,32]. Trichophyton erinacei, a pathogen of hedgehogs, is increasingly reported from human infections worldwide due to the growing interest of people in pet hedgehogs [33][34][35]. Another member of the T. benhamiae complex, T. persicum, was discovered as one of the major causes of zoonotic infections in Iran [36]. Finally, the name T. indotineae (=T. mentagrophytes genotype VIII) was introduced for a presumed clonal offshoot of T. mentagrophytes which causes epidemic or tinea infections in India and some other countries [37]. In this respect, T. quinckeanum can be considered an emerging pathogen in Germany and the Czech Republic.
Uhrlaß et al. [5] recorded that the majority of patients were older than 50 years (40%). In contrast to the German study, the Czech patients were mostly in the age groups 19 to 49 years (48%) and 1 to 18 years (44%), with only two patients being older than 50 years. In agreement with Uhrlaß et al. [5], we observed higher infection rates in females. This can probably be explained by the fact that women more often cuddle their pets [5].
A recent study showed differences in specific parts of the mass spectrum between strains of T. quinckeanum and other common zoophilic dermatophytes such as the T. mentagrophytes and T. benhamiae [5], but it is not entirely certain that the MALDI Biotyper automatic identification mode is possible for T. quinckeanum. However, the specific peak in the 4050-4200 m/z spectrum for T. quinckeanum supports the possibility of routine differentiation from other zoophilic species in practice using the FlexAnalysis tool in combination with micro-and macromorphological features. Further comparisons are certainly required, mainly due to the intra-species variability of the T. mentagrophytes/T. interdigitale complex and similarities of mass spectra in other dermatophyte species, especially the closely related T. schoenleinii. As showed by Packeu et al. [38], one out of six strains of T. quinckeanum was incorrectly identified as T. schoenleinii using MALDI-TOF MS.
The standardized methodology E.Def 11.0 was recently updated by the EUCAST-AFST and reading of the 50% inhibition endpoint has been recommended [18]. Based on this approach, obtained results will be comparable in future studies, enabling the definition of the epidemiological cut-off values (ECOFFs) and breakpoints for the categorization of results. These days, ECOFF wild-type upper limits (WT-ULs) have been set for T. rubrum and T. interdigitale tested by the EUCAST E.Def 11.0 methodology for four antifungals (terbinafine, voriconazole, itraconazole and amorolfine). The proposed WT-ULs for terbinafine, itraconazole and amorolfine are 0.03, 0.25 and 0.125 for T. rubrum and 0.125, 0.25 and 0.5 for T. interdigitale, respectively [18]. Values obtained in all T. quinckeanum isolates were lower in comparison with both proposed cut-offs [18]. Articles on T. quinckeanum antifungal susceptibility testing are scarce. Niewerth et al. [39] tested one strain of T. quinckeanum for itraconazole (1 mg/L), terbinafine (0.001 mg/L) and ciclopirox (3 mg/L) and obtained comparable results to ours by using the broth microdilution test (results were read by eye as no visible growth).
According to the literature, T. quinckeanum infections were usually treated with various systemic and local antifungals, mainly their combinations. Griseofulvin, terbinafine, econazole, clotrimazole, bifonazole, ciclopirox and ketoconazole were successfully used in the therapy [5,6,22,25,40]. One case of bifonazole cream treatment failure has been described [25]. The affected animals in our study were successfully treated with Pythium oligandrum whole-body baths three times a week for 14 days, usually in combination with a disinfectant (Alfadin). This approach seems to be considerate to animals, welltolerated and does not support the development of resistance. The activity of P. oligandrum against dermatophytes has been demonstrated in vitro and in a small sample of patients in vivo [41,42]. Although no large-scale clinical trials have been conducted, our study provides more evidence about the effectiveness of this treatment.

Conclusions
We described a second local outbreak of T. quinckeanum infections in addition to the recent outbreak in Germany. We also brought evidence that rising human infections were mostly mediated by infected cats and dogs among which the infection spread at least partly independent of rodents. The spread of these infections underscores the need for a closer collaboration between veterinarians, dermatologists, epidemiologists and public health personnel to set up appropriate preventive measures. The confirmation of species identification by molecular methods is desired because the pathogen morphologically resembles