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Detection of emerging genotypes in Trichophyton mentagrophytes species complex: A proposal for handling biodiversity in dermatophytes

A resistant and hypervirulent dermatophyte from India has been described as a taxonomic novelty, Trichophyton indotineae, a species of the Trichophyton mentagrophytes complex. Rapid detection and correct identification of closely similar dermatophytes with different predilections are essential for efficient clinical management. We evaluated the efficacy of rapid diagnostic methods clinical and environmental strains in the T. mentagrophytes complex. The methods included Real-time-PCR, DermaGenius, LAMP, and MALDI-ToF MS, using rDNA ITS sequences as taxonomic standard. The results show that only MALDI-ToF MS can distinguish 96.97% T. indotineae from other closely related species. The complex comprises numerous clones which may differ in anonymous markers but with similar evolutionary behavior. Therefore, we recommend to distinguish species only when they show an appreciable degree of adaptation and thus are clinically significant. The distinction of remaining clonal diversity is an epidemiological query and can be solved by haplotype numbering.

A resistant and hypervirulent dermatophyte from India has been described as a taxonomic novelty, Trichophyton indotineae, a species of the Trichophyton mentagrophytes complex. Rapid detection and correct identification of closely similar dermatophytes with different predilections are essential for efficient clinical management. We evaluated the efficacy of rapid diagnostic methods clinical and environmental strains in the T. mentagrophytes complex. The methods included Real-time-PCR, DermaGenius, LAMP, and MALDI-ToF MS, using rDNA ITS sequences as taxonomic standard. The results show that only MALDI-ToF MS can distinguish 96.97% T. indotineae from other closely related species. The complex comprises numerous clones which may differ in anonymous markers but with similar evolutionary behavior. Therefore, we recommend to distinguish species only when they show an appreciable degree of adaptation and thus are clinically significant. The distinction of remaining clonal diversity is an epidemiological query and can be solved by haplotype numbering.

Introduction
Dermatophytoses are among the most common fungal infections, affecting 20-25% of the world's population (Havlickova et al., 2008;Ameen, 2010). The species spectrum of dermatophyte infections has changed dramatically over time with socioeconomic developments (Zhan et al., 2018). Most pathogenic agents of humans originated from domesticated animals such as cattle, horse or camels (Deng et al., 2008;Morrell and Stratman, 2011;Hameed et al., 2017). Infections by zoophilic dermatophytes cause highly inflammatory lesions due to immunopathogenesis (Drouot et al., 2009;Celestrino et al., 2021;Sardana et al., 2021). The spectrum of infections of urban populations has changed in favor of species with human-to-human transmission and milder clinical pictures, in addition to those by pet-associated species causing self-limiting outbreaks. It has previously been suggested that longterm alterations of human-animal relationships such as domestication leads to novel lines of evolution in dermatophytes . This is inherent to dermatophyte natural life cycles, which comprise elaborate sexual reproduction in the environment and asymptomatic carriage of spores in mammalian fur. Domestication interrupts the sexual part of this life cycle resulting in clusters of clonal offshoots (Gräser et al., 2006). The human host is exceptional in lacking a fur, and thus the fungus becomes invasive upon skin contact. This has led to repeated adaptations from zoophilic species with T. equinum originally associated with horse as an example (Kandemir et al., 2020). Several clones in the T. mentagrophytes complex, an originally zoophilic species of small mammals (de Hoog et al., 2017;Kupsch et al., 2019), are now commonly found on humans (Chowdhary et al., 2019;Nenoff et al., 2019a;Das et al., 2020). Clones with mutations in the ribosomal internal transcribed spacers have been assigned numbers (Singh et al., 2019). T. interdigitale, causing chronic, prevalently pedal infections, has been hypothesized to be one of these clones.
Since almost a decade, dermatology in India has experienced a novel driver of dermatophyte evolution due to the overuse of over-the-counter antifungal drugs by the general public (Ebert et al., 2020). An emerging novel species has been named T. indotineae . This clone shows frequent resistance to terbinafine (>1 μg/ml), which is the most commonly used antifungal to treat dermatophyte infections in India . In addition, the clone is significantly more virulent than T. interdigitale in the same species complex, causing severe outbreaks (Singh et al., 2019), and is already spreading globally through physical contact and travel Jabet et al., 2022;Posso-De Los Rios et al., 2022). Recognition of this clone is therefore clinically significant for proper patient management and public health.
Classically, laboratory diagnosis of dermatophytosis is based on microscopy of strains grown in vitro (Kobylak et al., 2015). Routine fluorescence-microscopy of KOH-digested clinical specimens demonstrating fungal elements is rapid, but unable to differentiate between dermatophytes and non-dermatophyte filamentous fungi (Haghani et al., 2013). Sequencing of rDNA internal transcribed spacer (ITS) in few-days-old cultures is currently the gold standard for dermatophyte identification, despite relatively limited polymorphism (Makimura et al., 1999;Deng et al., 2015). For limited sets of the most common species, a commercial non-culture, molecular assay, DermaGenius 2.0 multiplex real-time PCR kit (Pathonostics, Maastricht, The Netherlands) are available. This tool provides rapid detection of superficial fungal infections of nail, hair, and skin samples and readily differentiates species of Trichophyton, Microsporum and Epidermophyton in addition to Candida albicans (Ndiaye et al., 2022). The kit lacks a probe for T. indotineae, a resistant and hypervirulent dermatophytes species . The DermaGenius ® Resistance Multiplex real-time PCR, is another kit used for detection of terbinafine-resistant T. indotineae strains, but does not recognize susceptible T. indotineae strains . Other economical molecular methods are available that have not yet been applied to dermatophytes. Loop-mediated isothermal amplification (LAMP) is a rapid assay enabling DNA amplification at constant temperature (Watanabe et al., 2019). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) is another routine diagnostic technique for the identification of microorganisms in clinical microbiology laboratories, such as T. rubrum (Wattal et al., 2017;Shaw et al., 2021). Several studies have used MALDI-ToF MS to identify dermatophytes of the T. mentagrophytes complex (Packeu et al., 2013;Calderaro et al., 2014;Shaw et al., 2021). However, recognition of T. indotineae has not been enabled; the reference spectra has not been linked to the species in the database (Calderaro et al., 2014). In this study we determined the performance of AUToF MS 1000 in differentiating the various subspecies by using the profiles of MALDI-ToF MS for hierarchical cluster analysis (HCA) and single-peak analysis.
The expansion of the T. mentagrophytes complex with T. indotineae as a clonal species is of recent date . T. mentagrophytes, T. interdigitale and T. indotineae are very similar, differing by only a few SNPs in ITS region (Singh et al., 2019;Kano et al., 2020). In order to rapidly and accurately identify the classical species T. mentagrophytes and T. interdigitale and the novelty T. indotineae, current diagnostic assays have to be updated. The present study evaluates several the methods and discusses the taxonomic approach in anthropophilic species complexes.  Arentshorst et al. (2012); Tang et al. (2021). Sequences were blasted in GenBank and 100% identity was taken as species identification. Determination of genotypes were based on ITS sequencing according to Nenoff et al., 2019b and including IHEM 4268 (type of T. mentagrophytes) and CBS 428.63 (type of T. interdigitale) in the comparison. Clone VIII is now known to be identical to the type strain CBS 146623 of T. indotineae . Forty-nine clinical samples were collected to study DermaGenius (Table 1). Besides, a total of nineteen strains (one to three strains from each genotype) were selected to study DermaGenius, Real-time PCR, and LAMP (Table 2). 81 strains were selected to be studied with MALDI-ToF MS.

Dermagenius® 2.0
Forty-nine clinical samples (skin scrapings and hair) from symptomatic patients in India were collected by R. Thakur (Table 1). Each specimen was divided into three parts: the first one for SGA culture, the second for culture on Taplin agar (Oxoid, Munich, Germany), and the third for direct non-culture diagnostics with DermaGenius. Materials were cultured on agar for 1-2 weeks at 28°C . For DNA extraction, hair and skin scrapings were added to sterile 1.5 mL tubes and extracted with glass beads (Sigma G9143, St. Louis, USA) using the PathoNostics Extraction Kit according to the manufacturer's instructions. Quality of DNA was tested based on methods described by Arentshorst et al. (2012).
Besides, nineteen strains were selected out of 88 strains to perform DermaGenius testing ( Table 2). The DermaGenius ® 2.0 complete multiplex real-time PCR (PathoNostics, Maastricht, The Netherlands) was performed according to manufacturer's instructions. Five μL of DNA extract was added to the PCR mix and a LightCycler 480 II (Roche, Mannheim, Germany) was used for amplification and melting curve analysis. Positive and negative controls were included in each PCR run. Data analysis was performed using the 2nd-derivative and Tm-calling function of the LightCycler 480 II software (v1.5.1.62 SP2).

Real-time PCR
Nineteen strains were selected to represent the described genotypes and species based on the ITS gene region; this marker was used to design the primers and probes shown in Supplementary Table S2. 1 DNA of reference strains representing all genotypes of the T. mentagrophytes complex was purified, and amplified by real-time PCR using a T. indotineae-specific probe. Real-time PCR reactions were carried out in 20 μL volumes containing 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM 1 https://benchling.com/ reverse primer, 0.5 μL probe, 0.5 μL of 40 to 100 ng/μL DNA, and 10 μL LightCycler ® 480 Probe Master. The instrument used was a LightCycler ® 480 II (Kobylak et al., 2015). Reaction conditions were as follows: 95°C for 10 min, followed by 50 cycles at 95°C for 15 s, 68°C or 60°C for 1 min, with an extension cycle of 40°C for 15 s. The curves indicate positive and the straight lines indicate negative samples (Supplementary Figure S3). Water was used as negative control. CBS 146623 was used as positive control.

Lamp
WarmStart colorimetric Loop-mediated isothermal amplification (LAMP) was applied to detect T. interdigitale, T. mentagrophytes and T. indotineae, with T. benhamiae and T. rubrum as negative controls, through isothermal amplification of the ITS gene. The same nineteen strains as for Real-time PCR were used to represent genotypes ( Table 1). The ITS type-specific LAMP primer sets were designed by using the software NEB LAMP Primer Design Tool, 2 consisting of two outer (F3, B3), two inner (FIP, BIP) primers, and two loop primers (LF, LB). Used primers are shown in Supplementary Table S2. Reactions were performed according to WarmStart Colorimetric LAMP 2× Master Mix (New England Biolabs, Ipswich, UK) protocol (Dao Thi et al., 2020;Daskou et al., 2021). Samples were incubated for 15 min and 30 min at 65°C in a heating block. Color change was visible by visual observation directly upon removal from the incubation temperature. As additional verification, amplification products were analyzed by 2% agarose gel electrophoresis and visualized under an UV transilluminator. Water was used as negative control. CBS 146623 was used as positive control. Red and yellow indicate negative and positive results, respectively.

MALDI-ToF MS
The data include T. mentagrophytes (n = 23), T. interdigitale (n = 19), T. indotineae (n = 33), T. benhamiae (n = 3), T. quinckeanum (n = 3). MALDI-ToF MS was performed by the formic acid extraction method according to the manufacturer's instruction (AUToF MS1000, Autobio, Zheng Zhou, China) with minor modifications. All chemical reagents used were of LC-MS grade. Briefly, dermatophytes isolates were cultured on SGA for 7 days at 28°C. After growth, the sample was collected in a 1.5 mL centrifuge tube containing 0.5 mL 75% ethanol. After mixing, the sample was centrifuged at 12,000 × g for 3 min, and the supernatant was discarded. After drying of the residue at 37°C, 40 μL of lysis solution 1 (containing formic acid) was added. Subsequently, 1 μL of supernatant was transferred on a target plate (Autobio, Zheng Zhou, China) after mixing, and dried naturally in a bio-safety cabinet. Afterward, 1 μL of matrix solution was added on the Frontiers in Microbiology 04 frontiersin.org above dried supernatant, and dried again at room temperature. Each strain was prepared on eight MALDI target positions in parallel. For each strain, a mass spectrum was generated and integrated to a sum spectrum using AUToF MS1000. Finally, five spectra were selected from each species for better spectra handling and visualization. MALDI-tree was built up by software inside of AUTO MS1000 with hierarchical cluster analysis.

Clinical samples
DermaGenius 2.0 found the majority of clinical samples from India to be positive for dermatophytes (n = 40/49, 81.63%). Of these, 39 were identified as T. interdigitale, and one as T. rubrum (Table 1; Supplementary Figure S1). Fungal cultures on SGA and Taplin agar yielded 31 (63.26%) samples positive for dermatophytes (Table 1). DermaGenius identified the India samples as T. interdigitale. Comparison of ITS sequences generated from the strains (Table 1; Supplementary Figure S1), 30 isolates matched with T. mentagrophytes ITS-genotype VIII according to Nenoff et al. (2019a) and with CBS 146623, the type strain of T. indotineae.
Nineteen strains from the culture collection representing each genotype using the ITS classification of Nenoff et al. (2019a) were tested by DermaGenius (Supplementary Figure S2). DermaGenius correctly recognized genotypes IV, VII, and IX as belonging to T. mentagrophytes, and all strains of T. interdigitale. However, genotype III and III* of T. mentagrophytes were recognized as T. interdigitale (Table 2). T. indotineae, not present in the DermaGenius database, was identified as T. interdigitale.

Real-time PCR
The assays were positive for all genotypes, while controls (T. rubrum and T. benhamiae) remained negative (Table 2; Supplementary Figure S3). The ITS variable positions used for primer and probe design did not allow distinction between genotypes, which was maximally 1 bp difference between entities.

Warmstart colorimetric RT-LAMP
WarmStart Colorimetric RT-LAMP assay was first executed to determine the incubation time range. The experiment result shows the optimal incubation time is 10 to 20 min. Moreover, an incubation period of 30 min leads to false-positive results with non-templatecontaining samples as well as with non-T. mentagrophytes complex samples. When samples were incubated at 65°C for 15 min, those matching with T. indotineae, T. mentagrophytes III*, III, and IV, and T. interdigitale were positive (Table 2; Supplementary Figure S4). T. benhamiae, T. rubrum, T. mentagrophytes genotype VII, one strain of T. mentagrophytes genotype IX, and water were negative (Table 2; Supplementary Figure S4). The colorimetric assay evaluated by visual observation was confirmed on the gel (Supplementary Figure S4). However, the protocol did not allow to separate all genotypes unambiguously.

MALDI-ToF mass spectrometry
Representative isolates of each species from the T. mentagrophytes clade were analyzed using MALDI-ToF MS. High-quality (peak rich) MALDI spectra samples were selected to build the MALDI-ToF MS tree. A clustering MALDI-ToF MS analysis results for 81 isolates are shown in Supplementary Table S1; Figure 1. All T. benhamiae and T. quinckeanum strains clustered in their clades and 96.97% (32/33) of T. indotineae strains clustered also together. Six T. interdigitale strains, in the upper single clade, were almost isolated from cases with onychomycosis. The remaining strains, mostly T. mentagrophytes, did not form regularly clusters. CBS 146623, CBS 428.63, CBS 124421 were selected to represent the spectra of T. indotineae, T. interdigitale, and T. mentagrophytes, respectively. In the mass range between approximately 4,000 to 5,000 m/z (as a representative example), the MALDI-ToF MS of CBS 146623, CBS 428.63, and CBS 124421 were very similar, and cannot be differentiated. However, several specific peaks could be found for analyzed taxa in the mass range of approximately between 2,000 to 4,000 and 6,000 to 8,000 m/z (Supplementary Figure S5). The most variable mass range of approximately 2,000 to 4,000 m/z is shown in Supplementary Figure S5. T. indotineae and T. mentagrophytes showed peaks at 2,206 m/z. T. interdigitale and T. indotineae showed peaks at 2,610 to 2,680 m/z. T. interdigitale and T. mentagrophytes showed high peaks at 3,810 to 3,830 and 7,800 to 8,000 m/z, but T. indotineae showed very low peaks at these ranges ( Figure 2).

Discussion
Dermatophytes infections have long been regarded as curable, relatively insignificant esthetic problems. The severe infections by, e.g., T. schoenleinii have practically disappeared by changes in life style and hygienic measures (Prasanna et al., 2016), and today a wide panel of effective antifungals is available without prescription. However, a new problem has emerged in South Asia and spread to other continents with outbreaks of dermatophyte skin diseases showing a remarkably virulence and resistance to the most commonly used antifungal, terbinafine (Singh et al., 2019;Kong et al., 2021). Several species of dermatophytes are involved in this new problem, including T. rubrum (Shankarnarayan et al., 2020) and particularly species of the T. mentagrophytes complex (Singh et al., 2019). Inappropriate use of antifungal creams with corticosteroids has been hypothesized to be a main reason for this emergence (Verma et al., 2021).
T. indotineae, an emerging dermatophyte in India, is a multidrug-resistant taxonomic novelty, regarded as a clonal offshoot in T. mentagrophytes complex (Singh et al., 2019;Kong et al., 2021;Tang et al., 2021). The main objective of the present study was to evaluate the efficacy of diagnostic methods for rapid detection of this clinically relevant dermatophyte. The DermaGenius ® 2.0 multiplex real-time PCR assay is a fast molecular diagnostic method that identifies several dermatophytes directly in nail, hair and skin samples within 3 h . Compared to traditional methods, such as culture, the DermaGenius kit proved to have a higher detection rate (DermaGenius vs. culture; Table 1). Several samples that did not show growth or yielded contaminants on Taplin agar / SGA were positive for dermatophytes with DermaGenius. However, T. indotineae has not yet been included in the kit. The T. mentagrophytes complex comprises three entities, which are in our dataset, in addition to the ancestral sexually interacting species T. mentagrophytes (comprising ITS genotypes III, III*, IV, VII and IX), two clonal offshoots which have been denominated T. interdigitale (also comprising ITS genotype II) and T. indotineae (ITS genotype VIII) (Nenoff et al., 2019b;Tang et al., 2021); the denominations are after Nenoff et al., 2019a. Since T. indotineae is a new name for T. mentagrophytes genotype VIII, existing databases require updating. DermaGenius made a bipartition in identifying most genotypes (including the T. mentagrophytes type strain IHEM 4268), as T. interdigitale (type strain CBS 428.63), while IV, VII and IX were identified as T. mentagrophytes. The tripartition T. indotineae / T. interdigitale / T. mentagrophytes of Tang et al., 2021 was primarily based on multilocus sequence data (TEF1 and HMG) supplemented with phenotypic markers, matching with minute ITS differences. The DermaGenius probes are based on ITS, but their exact sequence is unknown. Using MEGA v7.0 to align the sequences, we found potential primer sites to separate T. mentagrophytes genotypes IV, VII, and IX from T. interdigitale, T. indotineae, and T. mentagrophytes genotypes III and III* (Supplementary Figure S6). Most doubtful group is III/III*, which variously is classified in T. interdigitale or T. mentagrophytes. Since the latter is the latest a valid species name for this entity, the other databases require updating.
A potential area within ITS for primers design for the simultaneous distinction of all three species sensu stricto (Singh et al., 2019;Tang et al., 2021) just a single SNP of T. indotineae is different from T. mentagrophytes and T. interdigitale (Supplementary Table S2; Supplementary Figure S6). Unfortunately, the number of characteristic SNPs proved insufficient for RT-PCR; in addition, this site was invariable between outgroups T. rubrum and T. benhamiae. A more consistent distinction was observed in the HMG gene, which is the prevalent mating type of T. indotineae . However, the alpha-box mating type did not reveal a usable difference, and some strains only have one mating type gene (Singh et al., 2019). The ITS region in general did not exhibit difference to design probes and primers.
A similar problem was encountered with other molecular methods tested. In comparison to conventional PCR and realtime PCR, WarmStart ® Colorimetric LAMP assay is faster and simpler. Published data showed that the assay does not require expensive special equipment such as a thermal cycler, positive Frontiers in Microbiology 07 frontiersin.org samples being determined by a color change from pink to yellow within 30 min of incubation at 65°C. For recording simple mobile phone cameras can be used (Dao Thi et al., 2020;Reynés et al., 2021). It utilizes four oligonucleotide primers to recognize six different regions of the target gene, while two additional primers, LF and LB, are also incorporated in order to accelerate the amplification reaction and enhance the specificity (Tumino et al., 2020). In this study, colorimetric LAMP was positive with T. indotineae, T. mentagrophytes ITS genotype III*, III, IV and T. interdigitale, but is unable to detect all genotypes in the T. mentagrophytes species complex. T. indotineae is a member of the T. mentagrophytes complex, an originally zoophilic species which loss ability for sexual reproduction due to domestication of host animals . The species now mainly consists of a cluster of clonal offshoots, and has a skewed mating type distribution with a preponderance of MAT1-2 (HMG gene). Only two of these clones are as yet clinically significant: the classical species T. interdigitale, mostly causing human pedal infections, and the novel taxon T. indotineae. The latter species shows reasonable specificity (96.97%) with MALDI-ToF MS, but T. interdigitale, which possibly resides on the human host already for prolonged periods, is barely different from other clones, the most reliable approach being ITS sequencing. It may be questioned whether taxonomic distinction of every clone is meaningful for reasons other than epidemiology. For clinical practice, direct analysis of genes which confer resistance may be a good way forward (Burmester et al., 2022).
In the course of evolution, Dermatophytes (family Arthrodermataceae in the order Onygenales) show a trend of adaptation to vertebrate hosts. Ancestral life cycles involve production of elaborate sexual fruiting bodies in the natural environmental and distribution of clones via the fur of terrestrial animals. These species, known as geophiles, have low infective abilities. Zoophilic species are prevalently associated with domesticated animals, have less soil contact and are carried longer in animal fur, as 'clonal offshoots' (Gräser et al., 2006). A last and most recent adaptation is to the human host who is devoid of fur, and thus superficial infection of the skin takes place rather than asymptomatic colonization of fur. These species tend to adapt to particular body sites and lose sexual reproduction (Metin and Heitman, 2017;Kosanke et al., 2018). The evolution of the family Arthrodermataceae has been estimated to have taken about 37 million year (Kandemir et al., 2021), but Tang et al., 2021 suggested that similar adaptations Distance dendrogram of MALDI-ToF MS analysis of T. mentagrophytes species complex. T. benhamiae is outgroup.
Frontiers in Microbiology 08 frontiersin.org may proceed rapidly after domestication. Indeed, we observe anthropophilic dermatophytes in several species such as: T. concentricum close to T. benhamiae (Čmoková et al., 2020), T. tonsurans close to T. equinum (Kandemir et al., 2020), and Microsporum ferrugineum close to M. canis (de Hoog et al., 2017;Kosanke et al., 2018). As a result, the human host carries a larger number of specifically adapted dermatophytes than any other mammal. In the T. rubrum complex, no sexual reproduction has been observed. It may be hypothesized that this complex is associated with humans for longer periods, which has led to complete loss of sexuality and divergently adapted clones, such as T. rubrum on glabrous skin and T. violaceum, T. soudanense on the scalp. The evolutionary trend over the entire family can thus be summarized as sexuality with clonal offshoots in terrestrial species ( Figure 3A), gradual loss of sexuality with longer transmission periods of clonal offshoots in zoophilic species ( Figure 3B), and complete loss of sexuality with specialization of some clones surviving on the human host ( Figure 3C). Unisexual reproduction is considered a good strategy for short-term survival and population expansion. There is only limited genetic diversity that might be generated by aneuploidy or chromosomal translocations, which might improve the fitness of the progeny without disturbing a welladapted genotype and phenotype, while the occurrence of sexual reproduction may enhance its fitness by the introduced limited genetic diversity (Feretzaki and Heitman, 2013;Metin and Heitman, 2017;Kosanke et al., 2018). Sex is more suitable for long-term survival and adaptability to an ever-changing environment (Drenth et al., 2019).
In the present paper, we described the intermediate situation ( Figure 3B), with decreased sexual reproduction and the presence of numerous clones. Various authors (Nenoff et al., 2019a;Taghipour et al., 2019;Pashootan et al., 2022) distinguished 19 genotypes within the T. mentagrophytes complex, based on ITS sequence diversity. The zoophilic species tend to comprise a plethora of genotypes, as is also observed, e.g., in the T. benhamiae group (Čmoková et al., 2020). With the identification of geophilic dermatophytes, which interact sexually, the genotypes are usually disregarded as just causing some intraspecific variability (Čmoková et al., 2020). In the strictly anthropophilic species of the T. rubrum complex, clones are distinguished because of their ecological adaptation and clinical significance (Su et al., 2019), rather than based on their molecular distance. We recommend to distinguish species of the intermediate group ( Figure 3B) only when they show an appreciable degree of adaptation and thus are clinically significant. Clinical relevance is effectuated evolution and is thus also biologically relevant. We advocate to regard the molecular diversity of the T. mentagrophytes complex other than the clones T. indotineae and T. interdigitale as variation within a single species. The lineages within these entities may be epidemiological relevant and can be numbered as haplotypes, rather than attributing formal taxonomic species names.