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.

Current and Future Pathways in Aspergillus Diagnosis.

Aspergillus fumigatus has been designated by the World Health Organization as a critical priority fungal pathogen. Some commercially available diagnostics for many forms of aspergillosis rely on fungal metabolites. These encompass intracellular molecules, cell wall components, and extracellular secretomes. This review summarizes the shortcomings of antibody tests compared to tests of fungal products in body fluids and highlights the application of ß-d-glucan, galactomannan, and pentraxin 3 in bronchoalveolar lavage fluids. We also discuss the detection of nucleic acids and next-generation sequencing, along with newer studies on Aspergillus metallophores.

Diagnosis of Aspergillosis in Horses.

Invasive pulmonary aspergillosis (IPA) may be a rare cause of granulomatous pneumonia in horses. The mortality of IPA is almost 100%; direct diagnostic tools in horses are needed. Bronchoalveolar lavage fluid (BALF) and serum samples were collected from 18 horses, including individuals suffering from IPA (n = 1), equine asthma (EA, n = 12), and 5 healthy controls. Serum samples were collected from another 6 healthy controls. Samples of BALF (n = 18) were analyzed for Aspergillus spp. DNA, fungal galactomannan (GM), ferricrocin (Fc), triacetylfusarinin C (TafC), and gliotoxin (Gtx). Analysis of 24 serum samples for (1,3)-ß-D-glucan (BDG) and GM was performed. Median serum BDG levels were 131 pg/mL in controls and 1142 pg/mL in IPA. Similar trends were observed in BALF samples for GM (Area under the Curve (AUC) = 0.941) and DNA (AUC = 0.941). The fungal secondary metabolite Gtx was detected in IPA BALF and lung tissue samples (86 ng/mL and 2.17 ng/mg, AUC = 1).

Siderophore-Based Noninvasive Differentiation of Aspergillus fumigatus Colonization and Invasion in Pulmonary Aspergillosis.

Germination from conidia to hyphae and hyphal propagation of Aspergillus fumigatus are the key pathogenic steps in the development of invasive pulmonary aspergillosis (IPA). By applying in vitro observations in a clinical study of 13 patients diagnosed with probable IPA, here, we show that the transition from colonization to the A. fumigatus invasive stage is accompanied by the secretion of triacetylfusarinine C (TafC), triacetylfusarinine B (TafB), and ferricrocin (Fc) siderophores into urine, with strikingly better sensitivity performance than serum sampling. The best-performing index, the TafC/creatinine index, with a median value of 17.2, provided 92.3% detection sensitivity (95% confidence interval [CI], 64.0 to 99.8%) and 100% specificity (95% CI, 84.6 to 100%), i.e., substantially better than the corresponding indications provided by galactomannan (GM) and ß-d-glucan (BDG) serology. For the same patient cohort, the serum GM and BDG sensitivities were 46.2 and 76.9%, respectively, and their specificities were 86.4 and 63.6%, respectively. The time-dependent specific appearance of siderophores in the host's urine represents an impactful clinical diagnostic advantage in the early discrimination of invasive aspergillosis from colonization. A favorable concentration of TafC in a clinical specimen distant from a deep infection site enables the noninvasive sampling of patients suffering from IPA. IMPORTANCE The importance of this research lies in the demonstration that siderophore analysis can distinguish between asymptomatic colonization and invasive pulmonary aspergillosis. We found clear associations between phases of fungal development, from conidial germination to the proliferative stage of invasive aspergillosis, and changes in secondary metabolite secretion. The critical extracellular fungal metabolites triacetylfusarinines C and B are produced during the polarized germination or postpolarized growth phase and reflect the morphological status of the proliferating pathogen. False positivity in Aspergillus diagnostics is minimized as mammalian cells do not synthesize Aspergillus siderophore or mycotoxin molecules.

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%.

Combined Use of Presepsin and (1,3)-ß-D-glucan as Biomarkers for Diagnosing Candida Sepsis and Monitoring the Effectiveness of Treatment in Critically Ill Patients.

New biomarker panel was developed and validated on 165 critically ill adult patients to enable a more accurate invasive candidiasis (IC) diagnosis. Serum levels of the panfungal biomarker (1,3)-ß-D-glucan (BDG) and the inflammatory biomarkers C-reactive protein, presepsin (PSEP), and procalcitonin (PCT) were correlated with culture-confirmed candidemia or bacteremia in 58 and 107 patients, respectively. The diagnostic utility was evaluated in sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). BDG was the best marker for IC, achieving 96.6% sensitivity, 97.2% specificity, 94.9% PPV, and 98.1% NPV at a cut-off of 200 pg/mL (p ≤ 0.001). PSEP exhibited 100% sensitivity and 100% NPV at a cut-off of 700 pg/mL but had a lower PPV (36.5%) and low specificity (5.6%). Combined use of PSEP and BDG, thus, seems to be the most powerful laboratory approach for diagnosing IC. Furthermore, PSEP was more accurate for 28-day mortality prediction the area under the receiver operating characteristic curve (AUC = 0.74) than PCT (AUC = 0.31; PCT cut-off = 0.5 ng/mL). Finally, serum PSEP levels decreased significantly after only 14 days of echinocandin therapy (p = 0.0012). The probability of IC is almost 100% in critically ill adults with serum BDG and PSEP concentrations > 200 pg/mL and >700 pg/mL, respectively, defining a borderline between non-invasive superficial Candida colonization and IC.

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.

Inactivation of Dermatophytes Causing Onychomycosis Using Non-Thermal Plasma as a Prerequisite for Therapy.

Following our previous study of the therapy of onychomycosis by non-thermal plasma (NTP) and nail hygiene and to obtain some prerequisite data of dermatophytes sensitivity, the dynamics of those inactivation by NTP plasma was monitored for various strains of Trichophyton iterdigitale, Trichophyton benhamiae, Trichophyton rubrum, and Microsporum canis. Three strains of each species on agar plates were exposed with plasma produced by a DC corona discharge in the point-to-ring arrangement in various time intervals. Although all strains were sufficiently sensitive to plasma action, significant differences were observed in their sensitivity and inactivation dynamics. These differences did not correlate with the species classification of individual strains, but could be assigned to four arbitrarily created types of strain response to NTP according to their sensitivity. These results indicate that the sensitivity to plasma is not an inherent property of the fungal species, but varies from strain to strain.

Noninvasive Combined Diagnosis and Monitoring of Aspergillus and Pseudomonas Infections: Proof of Concept.

In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient's course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections.

Inactivation of Dermatophytes Causing Onychomycosis and Its Therapy Using Non-Thermal Plasma.

Onychomycosis is one of the most common nail disorders. Its current treatment is not satisfactorily effective and often causes adverse side effects. This study aims to determine the optimal conditions for non-thermal plasma (NTP) inactivation of the most common dermatophytes in vitro and to apply it in patient`s therapy. The in vitro exposure to NTP produced by negative DC corona discharge caused full inactivation of Trichophyton spp. if applied during the early growth phases. This effect decreased to negligible inactivation with the exposure applied six days after inoculation. In a group of 40 patients with onychomycosis, NTP therapy was combined with nail plate abrasion and refreshment (NPAR) or treatment with antimycotics. The cohort included 17 patients treated with NPAR combined with NTP, 11 patients treated with antimycotics and NTP, and 12 patients treated with NPAR alone. The combination of NPAR and NTP resulted in clinical cure in more than 70% of patients. The synergistic effect of NPAR and NTP caused 85.7% improvement of mycological cure confirmed by negative microscopy and culture of the affected nail plate. We conclude that NTP can significantly improve the treatment of onychomycosis.

Rhizoferrin Glycosylation in Rhizopus microsporus.

Rhizopus spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a Rhizopus microsporus clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same R. microsporus strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.

[Chromoblastomycosis and phaeohyphomycosis, overlooked fungal diseases].

Dark-pigmented microscopic fungi are worldwide-spread soil saprophytes often found on plant remnants. In chromoblastomycosis, infectious particles of these fungi enter the human body at the site of injury and may cause chronic infection, mainly in tropical and subtropical endemic areas. Chromoblastomycosis is almost exclusively diagnosed in patients with fully functioning immunity, with typically muriform cells present in infected tissue distinguishing this condition from phaeohyphomycosis. Phaeohyphomycosis, a less specific disease caused by dark-pigmented fungi, usually makes tissue necrotize rather than proliferate, involves a broader range of pathogens of the kingdom Fungi and is mainly associated with immune disorders. Chromoblastomycosis is usually a threat to male adults, globally considered an occupational disease affecting farmers, gardeners, loggers, agricultural commodity traders and other workers exposed to contaminated soil or handling materials of plant origin. In the Czech Republic, immunocompetent patients may be at risk of chromoblastomycosis as imported infection. In the past, however, the infection was also rarely documented as autochthonous in the country.

[Treatment of chromoblastomycosis and phaeohyphomycosis].

Cases of chromoblastomycosis are frequent in certain parts of the world, especially in some developing countries. Clinical manifestations of chromoblastomycosis are typical. To a certain extent, pathogens causing chromoblastomycosis overlap with those causing phaeohyphomycosis. Although cases of phaeohyphomycosis are not very common, they may end fatally. Therefore early management of these life-threatening infections is rather important. Targeted antifungal therapy and surgery are effective in combating these infections. Recently, several triazole antifungals such as posaconazole and isavuconazole have been available to treat even the most severe cases. Prevention of the infection should be aimed at reducing the risk of subcutaneous trauma, particularly in persons in contact with potential sources of infection such as wood materials important from endemic areas.

[Chromoblastomycosis and phaeohyphomycosis - pathogenesis and laboratory diagnosis].

Chromoblastomycosis and phaeohyphomycosis are less common fungal infections caused by dark-pigmented fungi. Virulence factors play an important role in the pathogenesis of these diseases. One of these factors, muriform cells, are the most important element for differential diagnosis of chromoblastomycosis and phaeohyphomycosis using clinical samples and various staining techniques. Accurate identification of pathogens causing chromoblastomycosis and phaeohyphomycosis is very important for correct and early antifungal therapy. Therefore, species identification of the etiological agent should be confirmed by sequencing of DNA from the culture. Early diagnosis may be crucial, especially in case of invasive forms of these infections. The diagnosis may be guided by some immunohistochemistry methods and DNA detection using polymerase chain reaction directly from clinical samples seems to be useful for identification of pathogens causing these severe and life-threatening infections.

Presepsin in the diagnostics of sepsis.

Sepsis remains one of the most common causes of death worldwide. It is caused by a complex of inadequate host responses to infection. It is also often difficult to distinguish sepsis from a non-infectious cause of systemic inflammatory response syndrome. Early identification of an infectious origin may dramatically help to improve the outcome and reduce mortality. That is the main reason why clinicians need fast, reliable and specific biomarkers for recognition of sepsis. Presepsin (sCD-14ST) is one of promising biomarkers, the level of which increases in response to a microbial infection in the host. As a glycoprotein expressed in the membranes of monocytes and macrophages, CD14 (cluster of differentiation 14) serves especially as a co-receptor of the lipopolysaccharide-lipopolysaccharide binding protein complexes, and activates the inflammatory cascade. Consequently, during the inflammatory reaction, sCD14-ST, known as presepsin, is cleaved away from plasma. The objective of this article is to determine the diagnostic value of presepsin in the diagnostics of sepsis, assessing its severity, and monitoring the effectiveness of therapeutic interventions, and to establish the prognostic value of this biomarker.

[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.

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.

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.

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.

Microsporum aenigmaticum sp. nov. from M. gypseum complex, isolated as a cause of tinea corporis.

An undescribed Microsporum species was isolated from skin scales recovered from a 40-mm large, annular, scaling lesion on the wrist of a 46-year-old woman. The risk factors for dermatophyte infection in the patient were frequent work in the garden, hunting, and contact with dogs and horses. Direct microscopic examination of the scales revealed the presence of dermatophyte hyphae; when the samples were cultured, a morphologically similar fungus grew on all slants in pure culture. Both of these findings strongly suggested that the isolate was the true causal agent of infection. The possible geophilic nature of the species was based on phylogenetic analysis (internal transcribed spacer region of rDNA and ß-tubulin gene) that placed it in between species of the M. gypseum complex. However, its divergencies from all other Microsporum species exceeded 4% base pairs. Based on ß-tubulin phylogeny, the isolated species is a sister to M. gypseum. The species produces abundant chlamydospores and clumps of hyphae similar to those of ascomatal primordia but no conidia and ascospores. The species was unable to grow at 37°C and does not grow on T6 basal medium, which is unlike other Microsporum species; hair perforation and urease tests were positive. The addition of histidine to the T6 medium resulted in rapid growth of the fungus. The phylogenetic evidence, morphology, growth parameters, and physiology justified the proposal that the isolate is a new species, M. aenigmaticum, sp. nov.