Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of ...patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all
clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor
gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu
, Phe
, Phe
, and His
) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous
gene of a terbinafine-sensitive
(formerly
) strain. All of the generated
transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in
transformants expressing mutant forms of
SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.
The mechanisms of terbinafine resistance in a set of clinical isolates of
have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of ...TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of
to develop resistance through multidrug efflux transporters. The introduction of a
cDNA library into
allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the
genome, we performed a BLASTp analysis of
,
, and
on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in
Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both
and
were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of
in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in
, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.
Dermatophytes are the most common pathogenic agents of superficial mycoses in humans and animals. Knowledge of their epidemiology can facilitate the prevention of dermatophytosis and improve ...prophylactic measures. We sought to determine the incidence of the different dermatophyte species diagnosed in Lausanne (Switzerland) from 2001 to 2018. In total, 10,958 dermatophytes were isolated from patients and 459 from pets. Overall, 99% of tinea unguium and tinea pedis were caused by Trichophyton rubrum and Trichophyton interdigitale with a prevalence ratio of 3:1. Trichophyton violaceum and Trichophyton soudanense were mainly found in tinea capitis in patients of African and Mediterranean origin. Interestingly, while Epidermophyton floccosum and Trichophyton verrucosum were prevalent 50 years ago in an epidemiological analysis carried out in the same laboratory from 1967 to 1970, these two species were rarely detected from 2001 to 2018. Trichophyton mentagrophytes, Trichophyton benhamiae and Microsporum canis were the prevalent zoophilic pathogenic species in children and young adults. Our investigation of animal samples revealed the main reservoirs of these zoophilic species to be cats and dogs for T. mentagrophytes and M. canis, and Guinea pigs for T. benhamiae. This study provides an epidemiological overview of dermatophytoses in Switzerland to improve their surveillance.
Most inflammatory dermatophytoses in humans are caused by zoophilic and geophilic dermatophytes. Knowledge of the epidemiology of these fungi in animals facilitates the prevention of dermatophytosis ...of animal origin in humans. We studied the prevalence of dermatophyte species in domestic animals in Switzerland and examined the effectiveness of direct mycological examination (DME) for their detection compared to mycological cultures. In total, 3515 hair and skin samples, collected between 2008 and 2022 by practicing veterinarians, were subjected to direct fluorescence microscopy and fungal culture. Overall, 611 dermatophytes were isolated, of which 547 (89.5%) were from DME-positive samples. Cats and dogs were the main reservoirs of
and
, whereas
was predominantly found in guinea pigs. Cultures with
significantly (
< 0.001) outnumbered those with
in DME-negative samples (19.3% versus 6.8%), possibly because
can be asymptomatic in cats and dogs, unlike
, which is always infectious. Our data confirm DME as a reliable, quick, and easy method to identify the presence of dermatophytes in animals. A positive DME in an animal hair or skin sample should alert people in contact with the animal to the risk of contracting dermatophytosis.
Several dermatophytes producing numerous pyriform or round microconidia were called
Trichophyton mentagrophytes
. Among these dermatophytes are the teleomorph species
Arthroderma benhamiae
,
...Arthroderma vanbreuseghemii
and
Arthroderma simii
, and other species such as
Trichophyton interdigitale
,
Trichophyton erinacei
and
Trichophyton quinckeanum
for which only the anamorph is known. Confusion exists about which fungus should be really called
T. mentagrophytes
and about the rational use of this name in practice. We report a case of beard ringworm (tinea barbae) with
A. vanbreuseghemii
. According to both clinical signs and the type of hair parasitism, this case was exactly compatible to the first description of a non-favic dermatophytosis by Gruby under the name of “mentagrophyte” from which was derived the dermatophyte epithet
mentagrophytes
. In addition, the phenotypic characters of the isolated fungus in cultures perfectly matched with those of the first description of a dermatophyte under
T. mentagrophytes
by Blanchard (Parasites animaux et parasites végétaux à l’exclusion des Bactéries, Masson, Paris,
1896
). In conclusion,
T. mentagrophytes
corresponds to the fungus later named
A. vanbreuseghemii
. However, because the neotype of
T. mentagrophytes
was not adequately designated in regard to the ancient literature, we would privilege the use of
A. vanbreuseghemii
and abandon the name of
T. mentagrophytes
.
In cases of highly inflammatory dermatophytosis in humans, it is important to identify the possible source of animal transmission in order to prevent recurrence, family outbreaks or rapidly ...progressing epidemics. A survey of dermatophytes in pets during a 14‐month period in Switzerland revealed, in addition to Microsporum canis, two different species of the Trichophyton mentagrophytes complex, Arthroderma benhamiae and Arthroderma vanbreuseghemii, all causing inflammatory dermatophytoses. Arthroderma benhamiae was only and frequently isolated from guinea pigs. Arthroderma vanbreuseghemii was isolated mainly from European short hair cats, but also from dogs and in one case from a pure‐bred cat. Ninety‐three percent of the cats carrying A. vanbreuseghemii were hunters and all had skin lesions. In contrast, cats with skin lesions that were strictly indoors were found to be almost exclusively infected by M. canis. Therefore, it can be suspected that infection with A. vanbreuseghemii occurred during hunting and that the natural source of this dermatophyte is either soil or an animal other than the cat, most probably a rodent.
Rapid and reliable fungal identification is crucial to delineate infectious diseases, and to establish appropriate treatment for onychomycosis. Compared to conventional diagnostic methods, molecular ...techniques are faster and feature higher accuracy in fungal identification. However, in current clinical practice, molecular mycology is not widely available, and its practical applicability is still under discussion. This study summarizes the results of 16,094 consecutive nail specimens with clinical suspicion of onychomycosis. We performed PCR/sequencing on all primary nail specimens for which conventional mycological diagnostics remained inconclusive. In specimens with a positive direct microscopy but negative or contaminated culture, molecular mycology proved superior and specified a fungal agent in 65% (587/898). In 75% (443/587), the identified pathogen was a dermatophyte. Positive cultures for dermatophytes, yeasts and non-dermatophyte molds (NDMs) were concordant with primary-specimen-DNA PCR/sequencing in 83% (10/12), 34% (22/65) and 45% (76/169), respectively. Among NDMs, agreement was high for Fusarium spp. (32/40; 80%), but low for Penicillium spp. (5/25; 20%) and Alternaria spp. (1/20; 5%). This study underlines the improvement in diagnostic yield by fungal primary-specimen-DNA PCR/sequencing in the event of a negative or contaminated culture, as well as its significance for the diagnosis of dermatophyte and non-dermatophyte onychomycosis. Molecular mycology methods like PCR and DNA sequencing should complement conventional diagnostics in cases of equivocal findings, suspected NDM onychomycosis or treatment-resistant nail pathologies.
Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient ...contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10% of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1-3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.
In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a ...novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.
PDF
