Epidemiology and Molecular Identification of Dermatophytes: Focus on the Detection of the Emerging Species Trichophyton indotineae in Northern Italy

Abstract

Background: Trichophyton indotineae, a new emerging pathogen according to the WHO, is known to cause severe forms of tinea. Given that traditional identification methods rely on morphological characteristics, and the morphological distinctions among T. indotineae, T. mentagrophytes, and T. interdigitale are minimal, the adoption of alternative diagnostic techniques, such as RT-PCR or gene sequencing, has become critically important to prevent misidentification. The purpose of this study was firstly to analyze the local epidemiology of dermatophytes isolated and secondly to investigate the presence of T. indotineae among the isolated strains. Methods: Between January 2021 and June 2024, 1096 samples of skin adnexa were analysed. The isolated strains belonging to the genus Trichophyton were submitted to molecular identification by ITS sequencing, and T. indotineae strains were subjected to squalene epoxidase (SQLE) sequencing analysis. Results: Trichophyton rubrum and Trichophyton interdigitale appear to be the most prevalent pathogenic species. Molecular identification reveals four T. indotineae strains (4/87; 4.61%) from Asian patients, which were also characterized by gene mutations associated with terbinafine resistance. Conclusions: This study has made it clear that there is a need to implement basic mycological diagnostics with molecular methods to avoid misidentifications, ensure the correct identification, and evaluate the presence of mutations associated with antifungal drug resistance.

1. Introduction

Dermatophytosis is the most common fungal infection in the world, and includes infections of the skin, nails, and hair affecting approximately 25% of the general population [1]. The etiological agents of these infections are keratinolytic filamentous fungi, categorized as dermatophytes, which were originally classified into three different genera, namely, Epidermophyton, Microsporum, and Trichophyton. However, with the advancement of diagnostic techniques, these dermatophytes were classified into a further six genera: Arthroderma, Ctenomyces, Guarromyces, Lophophyton, Nannizzia, and Paraphyton [2]. These fungi are generally categorized into three groups according to their primary habitat: zoophilic species associated with animals, anthropophilic species with humans, and geophilic species related to the environment [3].

Among the fungi causing human dermatophytosis in many European countries, Trichophyton rubrum is the most frequently isolated species, followed by Trichophyton mentagrophytes/T. interdigitale, at all sampling sites [4,5,6,7,8].

The increased frequency of dermatophytosis is due to several factors, such as poor personal hygiene, occupation, occlusive footwear, climate change, socioeconomic problems, large-scale international travel, immigration from tropical countries, use of immunosuppressive drugs by many patients, and improvement in diagnostic techniques [5,6].

In recent years, dermatologists in India have observed a wide spread of recalcitrant extensive dermatophytosis across the country. The emerging pathogen associated with these infections was identified in 2019 as the novel species T. indotineae, a distinct clonal offshoot of the T. mentagrophytes series formerly described as T. mentagrophytes genotype VIII. Most cases of T. indotineae infection present with highly inflammatory lesions accompanied by itching and a burning sensation, often involving the lower part of the body and inguinal region, causing tinea corporis, cruris, and less frequently, faciei and pedis [9,10,11,12,13,14,15,16].

Resistance mechanisms in dermatophytes can be attributed to various factors, including the overexpression of drug efflux pumps and biofilm formation. However, one of the most commonly reported mechanisms in T. indotineae involves point mutations in the squalene epoxidase (SQLE) gene [11,12,17,18].

The rapid identification of etiological agents is fundamental for establishing appropriate antifungal therapy, prognosis, and prevention of further dissemination.

Since conventional methods are often based on morphological differences between T. indotineae, T. mentagrophytes, and T. interdigitale, other identification tools are fundamental for good diagnostic practice.

The first objective of this study was to examine the epidemiology of two hospitals in northern Italy: Piacenza and Bergamo. Based on the data obtained from conventional methods, we focused on the most widespread dermatophyte species and the age groups most affected by these infections.

Second, we wanted to investigate, using molecular methods, the presence of T. indotineae among the isolated strains and the possible presence of squalene epoxidase gene mutations associated with terbinafine resistance.

2. Materials and Methods

2.1. Samples

In this study, a total of 1096 skin adnexa samples (nails, hair, and skin scrapings) collected over the period January 2021–June 2024 from patients referred to microbiology laboratories of two hospitals in northern Italy, AUSL Piacenza (844 samples) and Papa Giovanni XXIII of Bergamo (256 samples), were analyzed.

As a part of the diagnostic procedure, samples were subjected to microscopic examination with Calcofluor fluorescence (Becton Dickinson, Sparks, MD, USA) staining following treatment with 10% Potassium Hydroxide (Becton Dickinson, Sparks, MD, USA).

The culture examination was set up by inoculating a Sabouraud glucose agar with gentamicin and chloramphenicol (Oxoid Ltd., Wesel, Germany) and a Dermasel selective medium (Oxoid Ltd., Wesel, Germany), both then incubated at 30 °C for 21 days, with readings every 48 h.

For reporting purposes, microorganisms were identified based on their morphology using microscopy and Lactophenol blue staining (Becton Dickinson, Sparks, MD, USA) or, alternatively, using MALDI-ToF (VITEK^® MS, BioMérieux, Marcy l’Etoile, France).

2.2. Molecular Identification of the Isolates

Among the isolated strains, 87 isolates from 87 patients, all belonging to the genus Trichophyton, were selected for molecular identification to preliminarily assess the accuracy of morphological identification. Specifically, the isolates included 7 T. mentagrophytes, 40 T. interdigitale, 1 T. violaceum, and 39 T. rubrum, which were identified through Sanger sequencing of the ITS region.

DNA was extracted by preparing a 2.0 McFarland suspension of fungal material obtained from plates cultured for a duration exceeding seven days. This suspension was subsequently processed and eluted in 50 µL using the ELITe InGenius SP 1000 cartridge on the automated Elite InGenius platform (ELITechGroup, Inc., Bothell, WA, USA).

ITS PCR was performed using 2.5 µL DNA in a 25 µL reaction containing 0.25 µL (0.4 µM) primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′).

PCR products were enzymatically purified using the ExoSAP-IT PCR cleanup reagent (Thermo Fisher, Waltham, MA, USA) prior to Sanger sequencing. Purified amplicons were sequenced using the BigDye v1.1 Sanger sequencing reagent (Thermo Fisher). Sanger reaction products were run on a 3500xL genetic analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were processed using Sequencing Analysis Software 6 (Applied Biosystems).

BLASTn searches were then performed in GenBank, and results were considered significant with an E value of 0.0 at 98 to 100% identity and with at least 90% query coverage. Species identification was based on the following well-defined reference strains described in the literature (GenBank ID): KJ606115 for T. interdigitale, KT155896 for T. mentagrophytes, NR_144901 for T. violaceum, and ON182016 for T. indotineae.

2.3. Squalene Epoxidase Gene Sequencing

Dermatophytes exhibit resistance through several mechanisms, such as the overproduction of drug efflux pumps and the development of biofilms. Nonetheless, in T. indotineae, one of the most frequently observed resistance mechanisms involves point mutations in the squalene epoxidase (SQLE) gene, ERG1. These mutations are linked to terbinafine resistance by modifying the binding of terbinafine to the SQLE enzyme.

The squalene epoxidase gene was sequenced in all T. indotineae Piacenza isolates. DNA was extracted as described above, and the entire gene encoding squalene epoxidase was amplified by PCR using primers SQLE-F0 (5′-TGTAAAACGACGGCCAGTTGACAGCGACAAGTGCCA-3′) and SQLE-R0 (5′-CAGGAAACAGCTATGACCAAAGAGCTAGAGATAAGCCTATCTG-3′), and sequenced using internal primers SQLE-F1 (5′-TGTAAAACGACGGCCAGTGGAATATCTCCCCATACAACCAG-3′) and SQLE-R1 (5′-CAGGAAACAGCTATGACCCCTCCCTTCTCCAACGCAG-3′) [8,19].

PCR products were purified, sequenced, and run as above. Sequences were assembled using SeqScape^® Software 4 (Applied Biosystems) and aligned to the wild-type SQLE reference mRNA sequence for T. indotineae (GenBank accession number MW187977).

2.4. Data Analysis

Descriptive statistics were used to describe species distributions and their allocation in age groups.

Microsoft Excel (Microsoft, Redmond, WA, USA) was used to carry out the statistical analysis.

In order to evaluate the level of agreement between methods, contingency tables were constructed, followed by chi-square test and Cramer’s V coefficient values.

3. Results

There were a total of 1096 cultures during the period January 2021–June 2024. Of the samples, 237 (21.6%) from 236 patients were positive for dermatophytes. One patient had a co-infection with Epidermophyton floccosum and Trichophyton rubrum.

Of the 237 dermatophytes, identified on a morphological basis, there were 223 (94.1%) Trichophyton species, 13 (5.5%) Microsporum species, and 1 (0.4%) Epidermophyton species. The species distributions of the isolates are shown in

Table 1. Species distribution of the 237 dermatophyte isolates.

Species	Number of Isolates (%)
Trichophyton rubrum	138 (58.2)
Trichophyton interdigitale	48 (20.3)
Trichophyton mentagrophytes	25 (10.6)
Microsporum canis	8 (3.4)
Trichophyton tonsurans	6 (2.5)
Trichophyton spp.	3 (1.3)
Microsporum gypseum	3 (1.3)
Trichophyton verrucosum	2 (0.8)
Microsporum audouinii	2 (0.8)
Trichophyton violaceum	1 (0.4)
Epidermophyton floccosum	1 (0.4)
Total	237

.

Table 2. Positivity rate of isolates according to the age groups (* one patient had a co-infection with Epidermophyton floccosum and Trichophyton rubrum).

Age	E. floccosum	M. audouinii	M. gypseum	M. canis	T. interdigitale	T. verrucosum	T. rubrum	T. mentagrophytes	Trichophyton spp.	T. tonsurans	T. violaceum	Total
<10		1		3		2	5	1		2		14
10–19			1				7	1	1	1		11
20–29				3	2		11	3				19
30–39		1		1	4		15	2		1		24
40–49				1	4		29	6	1			41
50–59					11		23	3	1	1		39
60–69					11		27	5		1		44
70–79	1		1		11		15	4			1	33 *
>80			1		5		6					12
Total	1	2	3	8	48	2	138	25	3	6	1	237

shows the stratification by age of the isolated dermatophyte species identified on a morphological basis. Culture positivity relative to the age groups reveals that the population most affected by this type of infection is between the ages of 60 and 69, followed by 40–49 and 50–59 years.

Across all age groups, T. rubrum and T. interdigitale remained the most prevalent pathogens, consistent with the overall study population.

Table 3. Morphological-based identification compared to ITS identification.

Morphological-Based Identification	Molecular Identification (n.)	Disagreement (%)
T. violaceum (n = 1)	T. violaceum (1)	0/1 (0%)
T. interdigitale (n = 40)	T. interdigitale (37)	3/40 (7.5%)
	T. indotineae (2)
	T. mentagrophytes (1)
T. mentagrophytes (n = 7)	T. mentagrophytes (2)	5/7 (71.4%)
	T. interdigitale (3)
	T. indotineae (2)
T. rubrum (n = 39)	T. rubrum (24)	15/39 (38.5%)
	T. interdigitale (15)

shows the results obtained by the ITS sequencing of the 87 strains belonging to the Trichophyton genus and the differences in identification based on the morphological characteristics method.

As reported in Table 3, for 23 (26.4%) of the strains submitted to molecular analysis, the identification on a morphological basis was not confirmed.

Differences in identification among the tests were examined using chi-square and Cramer’s V analyses, with a 95% confidence interval. We observed a moderate association (Cramer’s V = 0.59) between identification by the morphological method and identification through sequencing.

In particular, the Trichophyton violaceum strain was correctly identified; of the 40 strains morphologically identified as T. interdigitale, 37 (92.5%) were confirmed, 1 (2.5%) was T. mentagrophytes, and 2 (5%) were identified as T. indotineae.

Of the seven strains morphologically identified as T. mentagrophytes, two (28.6%) were confirmed, three (42.8%) were T. interdigitale, and two (28.6%) were identified as T. indotineae.

Strikingly, among the Trichophyton rubrum strains, 24 (61.5%) were confirmed and 15 (38.5%) were identified as T. interdigitale.

In order to investigate the T. rubrum strains identified as T. interdigitale by molecular analysis, some of these were plated on Sabouraud dextrose agar and then incubated for 7 days at 30 °C. As can be observed from the images (Figure 1), none of the typical characteristics of T. interdigitale could be observed; there were no spiral hyphae and no numerous microconidia arranged in clusters.

Molecular examination of mutations in the SQLE gene across four T. indotineae strains identified mutations linked to terbinafine resistance in some strains that were also present in our country. Sequencing of the SQLE gene revealed nucleotide changes (1189 T>C and 1342 G>A) that resulted in the missense mutations F397L and A448T in one strain from the Piacenza hospital. A different nucleotide change (1178 T>C) led to the L393S mutation in the strain from Bergamo hospital. No mutations were found in the other two T. indotineae strains from Piacenza.

Table 4. Information on the Trichophyton indotineae affected patients and SQLE gene mutations detected. (BG, Papa Giovanni XXIII of Bergamo; PC, AUSL Piacenza)

Case Number	Sex	Age	Native Country	SQLE Mutation
1 PC	F	35	Bangladesh	No mutation
2 PC	F	34	India	F397L + A448T
3 PC	M	31	Bangladesh	No mutation
4 BG	F	33	Sri Lanka	L393S

shows the T. indotineae strains identified by gene sequencing, the origin of the patients, and the type of mutation found. The patients were from Bangladesh (n = 2), India (n = 1), and Sri Lanka (n = 1), suggesting recent travel to endemic areas. Most of them were female (3/4, 75%), and their mean age was 33 years (range 31–35 years).

4. Discussion

To the best of our knowledge, this study presents the first comprehensive analysis of dermatophytes in northern Italy. In particular, this study investigated the epidemiology of dermatophytosis in two Italian cities: Bergamo and Piacenza.

The dermatophyte isolates examined in this study were initially screened using both macroscopic and microscopic characteristics to verify their classification as dermatophytes. This was followed by ITS sequencing, which facilitated the definitive identification of 87 strains belonging to the Trichophyton genus.

We observed a dermatophyte positivity rate of 21.6%. Similar data has also been evaluated in other epidemiological studies in different areas of the world [1].

Morphological identification revealed that T. rubrum was the most frequently identified species (n = 138, 58.2%), followed by T. interdigitale (n = 48, 20.3%), and T. mentagrophytes (n = 25, 10.6%). These data were confirmed by reports from European countries in the literature [4,8].

We observed a moderate discrepancy between the data obtained through morphological identification and that obtained with the molecular method. We specifically observed that 26% of the strains submitted for molecular confirmation of identification were incorrectly assigned to a species. Although our epidemiology remained unchanged (Table 3), with T. rubrum as the most frequently identified species, this finding strongly suggests that the epidemiology of this type of filamentous fungi may be distorted by inaccurate data, as morphologically based identification remains the most prevalent routine technique in this field.

Secondly, we focused on the distribution of different species of dermatophytes and their relationship with basic demographic characteristics. Our analysis showed that age could be a significant factor influencing the occurrence of dermatomycosis, with a notable predominance of patients aged 40–69 years, which is remarkable regarding other epidemiological surveys [7,8]. A low prevalence was observed in pediatric patients.

The primary objective of this study was to assess the presence of T. indotineae in our geographic region, given its growing contribution to the global prevalence of tinea infections and the difficulty in distinguishing it from T. mentagrophytes and T. interdigitale through routine morphological analysis [11,20]. This newly emerged fungus frequently causes inflammatory and pruritic forms of tinea cruris, tinea corporis, and tinea faciei, which are often difficult to treat [21].

Due to globalization, this emerging pathogen has been isolated in numerous countries outside of Asia. T. indotineae appears to be spreading across Europe, with infections reported in France [22,23], Belgium [24], Switzerland [25], Greece [26], and Denmark [27]. Most of these European cases involved patients who either originated from or had traveled to endemic regions—similar to the cases in our study. Our patients were from Bangladesh (n = 2), India (n = 1), and Sri Lanka (n = 1).

This highlights two key points: First, globalization and migratory flows have facilitated the global spread of this species. Second, to the best of our knowledge, there have been no cases of infection found among patients in northern Italy. This could suggest, as has been assumed in the literature, that individuals from the most affected countries are more susceptible to infection by T. indotineae [10].

Our study highlights the presence of T. indotineae in our country, with four strains previously misidentified as T. interdigitale and T. mentagrophytes. This misclassification may indicate that the actual prevalence of this species in Italy has been underestimated.

Infections caused by dermatophytes that are predominantly resistant to terbinafine are now being reported worldwide. Over the past decade, outbreaks of antifungal-resistant dermatophytosis caused by T. indotineae have been documented in India [28,29,30].

Compared to wild-type strains, T. indotineae isolates carrying the F397L and L393S mutations in the SQLE gene exhibit reduced sensitivity to terbinafine in vitro [31]. Conversely, the A448T mutation is associated with increased susceptibility to terbinafine, although there is ongoing debate regarding its potential reduced responsiveness to azoles [32,33]. The presence of the double mutation F397L/A448T is linked to decreased sensitivity to both terbinafine and azoles [34]. Similarly, the L393S/A448T double mutant also shows reduced susceptibility to terbinafine [31].

In this study, we confirmed the presence of T. indotineae variants in our country, characterized by SQLE mutations (F397L, A448T, and L393S) associated with resistance to both terbinafine and azoles.

The main limitation of this study is that we did not sequence the entirety of the isolated dermatophyte strains; our conclusions were based on a randomly selected pool of isolates. Additionally, the study was constrained by its small sample size and the inclusion of patients from only one region of the country.

Another limitation was the lack of clinical information and treatment details for the patients. No data were available regarding the antifungal susceptibility of the isolated strains, as such testing is not routinely performed. However, this information would have provided valuable insights into the correlation between molecular findings (e.g., squalene epoxidase gene mutations) and in vitro resistance.

Further research is needed to include other geographic regions in Italy and to expand the clinical, epidemiological, molecular, and mycological understanding of drug resistance in T. indotineae strains.

5. Conclusions

This study underscores the limitations of traditional diagnostic tools in accurately differentiating between dermatophyte species. We emphasize the need for medical mycology laboratories to adopt advanced technologies and methodologies to ensure precise species identification. Such improvements are essential for accurately estimating species distribution and detecting emerging pathogens like T. indotineae.

The data presented highlight the importance of elucidating the epidemiology of fungal species responsible for dermatological mycoses. A deeper understanding of species distribution is crucial for developing effective surveillance strategies and establishing a robust foundation for routine monitoring within our country.

T. indotineae has emerged as a significant global public health concern due to its resistance to standard antifungal therapies and its potential to cause widespread infections. Therefore, early identification and detection of factors contributing to poor response to the first-line antifungal agent, terbinafine, are essential to controlling the spread of this pathogen—not only in Europe but globally.