Terbinafine resistance in Trichophyton mentagrophytes and Trichophyton rubrum in the Czech Republic: A prospective multicentric study.

BACKGROUND: Terbinafine, an allylamine antifungal, is crucial for treating dermatophytosis by inhibiting squalene epoxidase (SQLE) in the ergosterol biosynthetic pathway. However, resistance is emerging, particularly in India and Southeast Asia, but reports of resistance spread worldwide. Despite this, comprehensive studies on terbinafine resistance in Trichophyton are still limited. OBJECTIVES: This research aimed to determine the prevalence of terbinafine resistance in the Czech Republic, with a focus on Trichophyton rubrum and Trichophyton mentagrophytes, and investigate the underlying molecular mechanisms. PATIENTS/METHODS: A total of 514 clinical strains of T. rubrum and 240 T. mentagrophytes collected from four Czech clinical institutions were screened for terbinafine resistance. Molecular investigations included DNA sequencing, specifically the ITS rDNA region and SQLE gene, as well as antifungal susceptibility testing following EUCAST guidelines. RESULTS: While no resistance was observed in T. rubrum, 2.5% of T. mentagrophytes strains exhibited resistance, marked by the F397L mutation in SQLE. Notably, resistance surged from 1.2% in 2019 to 9.3% in 2020 but reverted to 0% in 2021. All resistant strains were identified as T. mentagrophytes var. indotineae. Resistant strains exhibited high MICs for terbinafine (≥4 mg L-1 ) but low MICs to the other seven antifungals tested except for fluconazole. CONCLUSIONS: This study highlights the emergence of terbinafine-resistant T. mentagrophytes strains in the Czech Republic, with the F397L mutation being pivotal. Due to the relatively low resistance level, the current guidelines for dermatomycosis treatment in the Czech Republic remain effective, but ongoing surveillance is essential for timely adaptations if resistance patterns change.

Distinguishing Invasive from Chronic Pulmonary Infections: Host Pentraxin 3 and Fungal Siderophores in Bronchoalveolar Lavage Fluids.

The multiple forms of pulmonary aspergillosis caused by Aspergillus species are the most common respiratory mycoses. Although invasive, the analysis of diagnostic biomarkers in bronchoalveolar lavage fluid (BALF) is a clinical standard for diagnosing these conditions. The BALF samples from 22 patients with proven or probable aspergillosis were assayed for human pentraxin 3 (Ptx3), fungal ferricrocin (Fc), and triacetylfusarinine C (TafC) in a retrospective study. The infected group included patients with invasive pulmonary aspergillosis (IPA) and chronic aspergillosis (CPA). The BALF data were compared to a control cohort of 67 patients with invasive pulmonary mucormycosis (IPM), non-Aspergillus colonization, or bacterial infections. The median Ptx3 concentrations in patients with and without aspergillosis were 4545.5 and 242.0 pg/mL, respectively (95% CI, p < 0.05). The optimum Ptx3 cutoff for IPA was 2545 pg/mL, giving a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100, 98, 95, and 100%, respectively. The median Ptx3 concentration for IPM was high at 4326 pg/mL. Pentraxin 3 assay alone can distinguish IPA from CPA and invasive fungal disease from colonization. Combining Ptx3 and TafC assays enabled the diagnostic discrimination of IPM and IPA, giving a specificity and PPV of 100%.

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.

Successful surgical excision of cerebral abscess caused by Fonsecaea monophora in an immunocompetent patient and review of literature.

Cerebral abscesses caused by dark-pigmented Fonsecaea fungi are rare, especially in otherwise healthy individuals. In this case report, we present a 61-year-old man from Moldova, living in the Czech Republic, who had worked as a locksmith on oil platforms in Turkmenistan, Kazakhstan, Sudan, and Iraq since 1999, and was admitted to a neurology ward for a sudden motion disorder of the right leg, dysarthria, and hypomimia. Imaging revealed presence of expansive focus around the left lateral ventricle of the brain and a pronounced peripheral edema. The intracranial infectious focus was excised under intraoperative SonoWand guidance. Tissue samples were histologically positive for dark-pigmented hyphae, suggesting dematiaceous fungi. Therefore, liposomal amphotericin B therapy was initiated immediately. Fonsecaea monophora was provisionally identified using ITS rDNA region sequencing directly from brain tissue. The identification was subsequently confirmed by cultivation and DNA sequencing from culture. The strain exhibited in vitro sensitive to voriconazole (MIC = 0.016 µg/mL) and resistance to amphotericin B (MIC = 4 µg/mL); therefore, the amphotericin B was replaced with voriconazole. Postoperatively, a significant clinical improvement was observed and no additional surgery was required. Based on the literature review, this is the third documented case of cerebral infection due to this pathogen in patients without underlying conditions and the first such case in Europe.

[Cryptic Aspergillus species - a case report of chronic cavitary pulmonary Aspergillus lentulus infection in a heart transplant recipient].

Cryptic species within the section Fumigati, that is Aspergillus fumigatus-like species, are increasingly reported in the literature as causative agents of invasive aspergillosis (IA) in both humans and animals. Their detection and proper identification are important, but even more important is to determine the susceptibility profile (minimum inhibitory concentrations, MICs) of the isolate to antifungals using appropriate methods. Cryptic species often demonstrate elevated MICs to drugs recommended for IA therapy such as voriconazole or amphotericin B. Presented is a case of pulmonary aspergillosis in a 63-year-old male heart transplant recipient. Aspergillus lentulus with reduced susceptibility to voriconazole and amphotericin B was identified as the causative agent of the infection using culture and DNA sequencing. Susceptibility to antifungals was confirmed by the standard EUCAST-AFST methods. Based on MIC values obtained in vitro, therapy was switched from voriconazole to posaconazole with excellent clinical effects. To the best of our knowledge, this is the first reported case of A. lentulus infection treated with posaconazole and, moreover, a successful one.

Antifungal Susceptibility of the Aspergillus viridinutans Complex: Comparison of Two In Vitro Methods.

Cryptic species of Aspergillus fumigatus, including the Aspergillus viridinutans species complex, are increasingly reported to be causes of invasive aspergillosis. Their identification is clinically relevant, as these species frequently have intrinsic resistance to common antifungals. We evaluated the susceptibilities of 90 environmental and clinical isolates from the A. viridinutans species complex, identified by DNA sequencing of the calmodulin gene, to seven antifungals (voriconazole, posaconazole, itraconazole, amphotericin B, anidulafungin, micafungin, and caspofungin) using the reference European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. The majority of species demonstrated elevated MICs of voriconazole (geometric mean [GM] MIC, 4.46 mg/liter) and itraconazole (GM MIC, 9.85 mg/liter) and had variable susceptibility to amphotericin B (GM MIC, 2.5 mg/liter). Overall, the MICs of posaconazole and the minimum effective concentrations of echinocandins were low. The results obtained by the EUCAST method were compared with the results obtained with Sensititre YeastOne (YO) panels. Overall, there was 67% agreement (95% confidence interval [CI], 62 to 72%) between the results obtained by the EUCAST method and those obtained with YO panels when the results were read at 48 h and 82% agreement (95% CI, 78 to 86%) when the results were read at 72 h. There was a significant difference in agreement between antifungals; agreement was high for amphotericin B, voriconazole, and posaconazole (70 to 86% at 48 h and 88 to 93% at 72 h) but was very low for itraconazole (37% at 48 h and 57% at 72 h). The agreement was also variable between species, with the maximum agreement being observed for A. felis isolates (85 and 93% at 48 and 72 h, respectively). Elevated MICs of voriconazole and itraconazole were cross-correlated, but there was no correlation between the other azoles tested.

Proven Invasive Pulmonary Aspergillosis in Stem Cell Transplant Recipient Due to Aspergillus sublatus, a Cryptic Species of A. nidulans.

Invasive fungal disease represents one of the severe complications in haematopoietic stem cell transplant recipients. We describe a case of a patient treated for relapse of chronic lymphoblastic leukaemia 6 years after HSCT. The patient was treated for invasive pulmonary aspergillosis but died 3 months later from multiple organ failures consisting of haemorrhagic necrotizing fungal pneumonia, refractory chronic hepatic graft versus host disease and cytomegalovirus hepatitis. Autopsy samples revealed histopathological evidence of fungal hyphae and an unusual Aspergillus nidulans-like species was isolated in pure culture. More precise identification was achieved by using scanning electron microscopy of ascospores and sequencing of calmodulin gene, and the isolate was subsequently re-identified as A. sublatus (section Nidulantes) and showed good in vitro susceptibility against all classes of antifungals. Commonly used ITS rDNA region and ß-tubulin gene fail to discriminate A. sublatus from related pathogenic species, especially A. quadrilineatus and A. nidulans. Although this is the first case of proven IPA attributed to A. sublatus, we demonstrated that at least some previously reported infections due to A. quadrilineatus were probably caused by this cryptic species.

Successful Posaconazole Therapy of Disseminated Alternariosis due to Alternaria infectoria in a Heart Transplant Recipient.

We report a case of phaeohyphomycosis caused by Alternaria infectoria in a 61-year-old heart transplant recipient with multiple skin lesions and pulmonary infiltrates. The infection spread via the haematogenous route from the primary cutaneous lesions into the lungs. The diagnosis was based on the histopathological examination, direct microscopy, skin lesion cultures and detection of Alternaria DNA in the bronchoalveolar lavage fluid using molecular methods. The treatment consisted of a combination of surgical excision and systemic antifungal therapy. Voriconazole was the first agent used but had a weak effect. Posaconazole was subsequently used to achieve a successful response. The isolate was identified as A. infectoria by sequencing of the rDNA ITS region and the partial ß-tubulin gene.

Early Diagnosis of Cutaneous Mucormycosis Due to Lichtheimia corymbifera After a Traffic Accident.

A case report of cutaneous mucormycosis and obstacles to early diagnosis is presented. A 38-year-old male was involved in a car accident that led to amputation of both lower limbs. Subsequently, he developed fungal wound infection of the left lower limb stump. The infection was detected very early, although the diagnosis was difficult because only a small area was affected and histopathological examination was initially negative. The infection was proven by microscopy, culture and histopathology. The isolate was identified by sequencing of the rDNA ITS region gene (internal transcribed spacer region of ribosomal DNA) as Lichtheimia corymbifera. Liposomal amphotericin B and surgery were successful in management of the disease.

[Vulvovaginal candidiasis]. / Kandidovvá vulvovaginitida.

Vulvovaginal candidiasis is the second most common vaginal infection after bacterial vaginosis. It is caused by yeasts, the vast majority of which belong to the genus Candida. Vulvovaginal candidiasis affects as many as 75 % of women during their childbearing years and 40 % of them experience recurrences. The most common etiological agent is Candida albicans, which is responsible for nearly 90 % of cases. Vulvovaginal candidiasis can be treated with topical and/or systemic antifungals while risk factors for the infection must be eliminated.

Equine Dermatophytosis due to Trichophyton bullosum, a Poorly Known Zoophilic Dermatophyte Masquerading as T. verrucosum.

Trichophyton bullosum is a zoophilic dermatophyte from the Arthroderma benhamiae complex with a poorly known distribution. In this study, we report a case of dermatophytosis caused by T. bullosum in a 6-year-old male horse who had a skin lesion located in a saddle area. The infection spread rapidly to the upper chest and to both sides of the trunk. The dermatophyte was isolated in culture and identified by sequence analysis of the internal transcribed spacer regions (ITS rDNA). To date, this is the first verified case of animal infection due to T. bullosum in Europe following the 2012 report of human infection in France. We hypothesize that this species can be relatively common in horses and donkeys, but it is confused with other zoophilic species responsible for infections with similar clinical manifestations, and when isolated in culture, it is misidentified as the phenotypically similar T. verrucosum. Previous cases of dermatophytosis caused by T. verrucosum-like dermatophytes in horses and donkeys were reviewed together with human infections transmitted from these animals. This summary estimates possible distribution width of T. bullosum. The taxonomy of T. verrucosum-like dermatophytes is extremely difficult due to lack of original material and poor morphology of species. Molecular genetic methods are necessary to verify the identification of these fungi. ITS1 or ITS2 region of rDNA alone is sufficient for correct identification.

Aspergillus pragensis sp. nov. discovered during molecular reidentification of clinical isolates belonging to Aspergillus section Candidi.

The identity of nine clinical isolates recovered from Czech patients and presumptively identified as Aspergillus sp. section Candidi based on colony morphology was revised using sequences of ß-tubulin, calmodulin gene sequence, and internal transcribed spacer rDNA. Six isolates were from suspected and proven onychomycosis, one from otitis externa, and two associated with probable invasive aspergillosis. The results showed that one Aspergillus candidus isolate was the cause of otitis externa, and both isolates obtained from sputa of patients with probable invasive aspergillosis were reidentified as A. carneus (sect. Terrei) and A. flavus (sect. Flavi). Three isolates from nail scrapings were identified as A. tritici, a verified agent of nondermatophyte onychomycosis. One isolate from toenail was determined to be A. candidus and the two isolates belonged to a hitherto undescribed species, Aspergillus pragensis sp. nov. This species is well supported by phylogenetic analysis based on ß-tubulin and calmodulin gene and is distinguishable from other members of sect. Candidi by red-brown reverse on malt extract agar, slow growth on Czapek-Dox agar and inability to grow at 37°C. A secondary metabolite analysis was also provided with comparison of metabolite spectrum to other species. Section Candidi now encompasses five species for which a dichotomous key based on colony characteristics is provided. All clinical isolates were tested for susceptibilities to selected antifungal agents using the Etest and disc diffusion method. Overall sect. Candidi members are highly susceptible to common antifungals.

Auxarthron ostraviense sp. nov., and A. umbrinum associated with non-dermatophytic onychomycosis.

Auxarthron is a genus within the Onygenales encompassing keratinophilic species with typical ascomata (gymnothecia) consisting of anastomosing network of thick-walled hyphae and small globose or oblate ascospores. No association of this genus with clinically relevant cases of human or animal infection has been reported. This paper describes the isolation of an undescribed Auxarthron species as an agent of proven onychomycosis affecting almost all fingernails in a man with psoriasis. The causality of the isolated fungus was verified by repeated sampling and direct microscopy revealing irregular septate hyphae. Based on micro- and macromorphological features and unique sequence data (ITS region, benA and RPB2 gene), the isolated fungus is proposed as the new species A. ostraviense. The sibling species of A. ostraviense, A. umbrinum, was isolated from three patients with suspected onychomycosis and a detailed clinical history is provided for one of these patients. All four isolates were tested for susceptibility to selected antifungal agents. Terbinafine and clotrimazole appear to be effective in vitro. The morphological identification of Auxarthron spp. is non-trivial, time-consuming and requires cultivation media other than Sabouraud glucose agar which is routinely used in dermatomycology.

Rare and new etiological agents revealed among 178 clinical Aspergillus strains obtained from Czech patients and characterized by molecular sequencing.

A collection of 178 Aspergillus isolates, recovered from Czech patients, mostly from 2007-2011, was subjected to multilocus DNA sequence typing using the ITS region, ß-tubulin, and calmodulin genes. An unusually wide spectrum of etiologic agents that included 36 species of Aspergillus is discussed in the context of recent taxonomic and clinical reports. Invasive aspergillosis (IA), onychomycosis, and otitis externa were the predominant clinical entities. Five cases due to species newly proven as etiologic agents of human mycoses, as well as cases with unique clinical manifestations caused by unusual agents are discussed in more detail. Three species (i.e., A. insulicola, A. westerdijkiae and A. tritici) were identified as the confirmed etiologic agents of non-dermatophytic onychomycosis. Emericella rugulosa was recovered from a premature newborn with a fatal necrotising disseminated infection and is reported for only the second time as the cause of IA. Furthermore, we document the first infection due to A. calidoustus in a patient with chronic granulomatous disease. The infection manifested as a latent brain aspergilloma with an unusual clinical-laboratory finding. In addition to the well-known agents of human mycosis, several rarely isolated or poorly documented species were identified. An undescribed cryptic species related to A. versicolor was found to be common among isolates linked to proven and probable onychomycosis. An isolate representing A. fresenii, or an unnamed sister species, were causal agents of otomycosis. Three well defined, and tentative new species belonging to section Cervini, Candidi and Aspergillus (Eurotium spp.), were associated with cases of probable onychomycosis.

[Fusarium as a cause of onychomycosis resistant to common antifungal therapy]. / Fusarium jako puvodce onychomykozy rezistentni k bezne antimykoticke terapii.

INTRODUCTION: Fusarium species are common soil saprophytes and plant pathogens. Members of the genus have been frequently reported as etiologic agents of opportunistic infections in humans and animals. We report six cases of confirmed or suspected onychomycosis caused by members of the genus Fusarium (F. solani and F. oxysporum species complexes). MATERIAL AND METHODS: The isolates were identified by rDNA ITS sequencing analysis. The EMBL accession numbers for the ITS are HE974453-HE974458. A disk diffusion method was used for in vitro susceptibility testing. Comparison of disks (ITEST) and Neo-Sensitabs tablets (Rosco) on a different media at two different temperatures (25 °C and 35 °C) was made. RESULTS: Six strains of Fusarium spp. (4 strains of F. solani and 2 strains of F. oxysporum) were isolated from patients with confirmed or suspected onychomycosis. Natamycin (pimaricin) was the only antifungal effective in vitro in all isolates tested. Variable susceptibility of the isolates was detected in amphotericin B, econazole and terbinafine. The remaining antifungals tested were not effective. The results varied depending on the culture medium and temperature for nystatin and econazole disks and amphotericin B and terbinafin tablets. CONCLUSION: It is important to adhere to recommended methods when testing in vitro susceptibility to antifungals in moulds. An incubation temperature of 35 °C is important for obtaining valid results in amphotericin B tablets (and probably also terbinafine ones). Determination of multidrug-resistant Fusarium spp. in onychomycosis make the choice of therapy difficult. Good clinical effect was recorded with nail plate ablation and subsequent local econazole therapy.

Phaeohyphomycosis and onychomycosis due to Chaetomium spp., including the first report of Chaetomium brasiliense infection.

Chaetomium species have been rarely described as aetiological agents of invasive and dermatomycotic infections in humans. The majority of cases have been reported within the last two decades. Treatment failed in most of these cases. In this paper we present two cases in which Chaetomium spp. can be clearly identified as an aetiological agent in pathological conditions. In the first report, we describe a new aetiological agent, Chaetomium brasiliense, which was implicated in a case of otitis externa in a patient with spinocellular carcinoma basis cranii. The patient had been repeatedly treated for relapsing otitis externa and had previously undergone surgery several times for otitis media. The fungal aetiology was confirmed by repeated positive culture and histologic studies. The second case involved onychomycosis with strikingly brown nail discoloration due to Chaetomium globosum in an otherwise healthy patient. The nail lesion was successfully cured by oral terbinafine. The determination of both species was supported by sequencing of rDNA regions. The morphological aspect of Chaetomium spp. identification is also discussed. In vitro antifungal susceptibility tests demonstrated that both isolates were susceptible to terbinafine and azole derivates except fluconazole. Amphotericin B was effective only against the C. brasiliense strain. We review the literature to summarize clinical presentations, histologic findings, and treatment strategies.