A deletion mutation in the amino acid sequence of squalene epoxidase in terbinafine-resistant Trichophytonrubrum.

INTRODUCTION: Trichophyton rubrum is an anthropophilic dermatophyte that is most frequently isolated from onychomycosis (tinea unguium) worldwide. T. rubrum strains showing resistance to the anti-fungal drug terbinafine (TRF) have also been isolated from human patients worldwide. METHODS: In this study, we isolated a TRF-resistant strain (N99-2) of T. rubrum from a patient with recurrent tinea unguium. In vitro susceptibility of the clinical isolate to TRF, itraconazole (ITZ), ravuconazole (RVZ), and luliconazole (LCZ) was investigated using the Clinical & Laboratory Standards Institute M38-A2 test. To identify mutations, we compared the gene sequence of N99-2 to that of a TRF-susceptible strain of T. rubrum. Results; In N99-2, the minimum inhibitory concentrations were 32 mg/L for TRF, <0.03 mg/L for ITZ, <0.03 mg/L for RVZ, and <0.03 mg/L for LCZ. The squalene epoxidase (SQLE) gene sequence in N99-2 was determined to be 1467 bp in length, and it encoded a protein of 488 amino acids, beginning with a putative initiating methionine (ATG). The following mutations were identified from the SQLE of N99-2: L393F and Y394del. CONCLUSIONS: This is the first report of the detection of a deletion mutation in SQLE in a TRF-resistant strain. The protein of SQLE is the target of TRF, and it is essential for cell membrane synthesis in dermatophytes. However, dermatophyte cells were found to undergo gene mutations to escape the effects of antifungal agents.

Onychomycosis, the Active Invasion of a Normal Nail Unit by a Dermatophytic Versus the Colonization of an Existing Abnormal Nail Unit by Environmental Fungus.

Onychomycosis was described by early investigators as the presence of an abnormal nail unit and a member of the order Mycota, producing the abnormality. This interpretation has caused more than 50 years of confusion in the dermatologic literature. Unquestionably, the clinician sees more abnormal toenails than fingernails, and investigators have described a multitude of fungi as the cause of the clinically abnormal toenail. In 2010, developmental scientists proved, what we have long recognized, that there is no bilateral symmetry in living organisms and, therefore, one sole is different from the other. This causes a gait asymmetry, coupled with the pressure the closed shoe exerts on toenails while walking. This produces a series of abnormalities, which are clinically identical to what has been described for dermatophytic onychomycosis. These are fungus free and result in toenail niches. These toenail abnormalities were recently described as the asymmetric gait nail unit syndrome (AGNUS). It is possible that environmental fungi can colonize these toenail niches and, therefore, were described by investigators as a new onychomycosis entity In the normal host, onychomycosis should be only used to describe the active invasion of the nail bed (NB) corneocytes by a dermatophyte, as seen in dermatophytic onychomycosis. Dermatophytes only affect those hosts who have inherited the dermatophytosis susceptibility gene, transmitted as an autosomal dominant trait. In studies encompassing 3,000 abnormal toenails, only 27%-30% were found as dermatophyte culture positive, 25% were negative and the rest environmental fungi were recovered.

Molecular identification of isolates of the Trichophyton mentagrophytes complex.

Background: The Trichophyton mentagrophytes complex is the second most common causal agent of dermatophytosis. It comprises five species-T. mentagrophytes, T. interdigitale, T. erinacei, T quinckeanum, and T. benhamie, as well as nine different genotypes of T. mentagrophytes / T. interdigitale-which are morphologically similar; however, their susceptibility to antifungal agents may differ. For targeted therapy and better prognosis, it is important to identify these species at a molecular level. However, since many hospitals lack molecular methods, the actual aetiology of dermatophytosis caused by this complex remains unknown. Objective: To characterize 55 anthropophilic isolates of the T. mentagrophytes complex recovered from a dermatological centre in Yucatán, Mexico. Material and methods: Fifty-five isolates of the T. mentagrophytes complex were obtained from patients with tinea capitis, tinea pedis, tinea corporis, tinea barbae, and tinea unguium. They were characterized by their colonial and microscopic morphology on Sabouraud dextrose agar (SDA) and through the sequencing of a fragment from the region ITS1-5.8S-ITS2. Results: All colonies grown on SDA were white. Forty-six isolates formed colonies with a powdery texture, while nine isolates formed colonies with a velvety texture. The micromorphological features were typical of the T. mentagrophytes complex. The molecular analysis revealed that 55 isolates were microorganisms that belonged to the T. mentagrophytes complex, that 46 formed powdery colonies representing T. mentagrophytes, and that the other nine isolates that formed velvety colonies represented T. interdigitale. The latter nine isolates were obtained from patients with tinea pedis, tinea corporis, and tinea unguium. Conclusions: The colony morphology on SDA led to the identification of 46 isolates as T. mentagrophytes and nine isolates as T. interdigitale. At a molecular level, the species identified by their morphology were identified only as T. mentagrophytes complex.

Tuberculosis, onychomycosis and immune deficiency in complicated Crohn's disease.

The immune system is composed of innate humoral defence and adaptive immunity. One of the key mechanisms of the innate humoral defence is through complement activation. Mutations of certain enzyme may affect the complement activation and result in decreased defence against microorganisms. Mannan-binding lectin serine protease 2 (MASP-2) mutation was associated with recurrent infections and autoimmune diseases. Tuberculosis (TB) has been linked with mannose-binding lectin and MASP-2 gene polymorphism. We report a case of a paediatric patient with MASP-2 deficiency with classical and atypical features associated with Crohn's, onychomycosis and severe cutaneous infections including TB. We also report the presence of a new mutation variant in MASP-2 reported in whole exome sequencing of our patient.

Causative Agents of Onychomycosis: A 7-Year Study.

BACKGROUND: Onychomycosis is principally caused by dermatophyte species, but nondermatophyte molds and yeasts have also been involved, causing different clinical manifestations. The aim of this investigation is to determine the clinicomycological and epidemiological profile of the etiologic agents of onychomycosis. METHODS: The study population included 9,785 suspected cases of onychomycosis referred to the Medical Mycology Reference Laboratory in Isfahan, Iran, during 2007-2014. Nail clipping was collected in sterile Petri dishes for direct microscopic examination and culture. Clinical isolates were identified by using phenotypic tests and molecular techniques. RESULTS: Of total 9,785 cases with clinical suspicion of onychomycosis comprised in the present study, 1,284 patients (13.1%) were positive by direct microscopy. Age range of patients was between 1 and 82 years. Housewives were the commonest infected population. Candida albicans was the most prevalent species isolated from patients in this study (34.9%) followed by Trichophyton interdigitale (11.7%) and Aspergillus flavus (9.1%). CONCLUSION: The pattern of causative agents and clinical signs of onychomycosis is altering region to region, so repeated epidemiological surveys of onychomycosis seems to be fundamental. The present study provides novel and appropriate epidemiologic data of onychomycosis for the better prevention and treatment of this fungal infection.

Trichophyton rubrum DNA strain switching increases in patients with onychomycosis failing antifungal treatments.

BACKGROUND: The dermatophyte Trichophyton rubrum is responsible for approximately 80% of onychomycosis cases. Genetic strain typing was developed to help elucidate its epidemiology and pathogenicity. OBJECTIVES: To determine T. rubrum DNA strain types in North American patients with onychomycosis and to track the patients before and after their course of treatments. METHODS: T. rubrum DNA strain types were determined by restriction fragment length polymorphisms in ribosomal DNA and Southern blotting from toenails that were cultured from 50 North American patients with onychomycosis prior to treatment. Some of the patients were subsequently typed from oral terbinafine (n = 6), laser (n = 9) or placebo (n = 8) treatment groups. Three European DNA strains were obtained for comparison. DNA strains from the terbinafine group were tested for in vitro susceptibility to terbinafine. RESULTS: Six DNA strain types (A-F) accounted for 94% of T. rubrum DNA strains and corresponded to European isolates. Three DNA strains (6%) novel to North America were detected. DNA strain type switching occurred in all treatment groups: terbinafine (83%), laser (56%) and placebo (25%). Most of the switches (50%) observed in the terbinafine group coincided with mycological cures followed by relapse. Patients treated with laser therapy or placebo exhibited no intermittent cures. DNA strains from the terbinafine group were all susceptible to terbinafine in vitro. CONCLUSIONS: Nine T. rubrum DNA strains were identified in a North American population: three novel and six predominant to a European population. Although DNA strain type switching in onychomycosis is a natural phenomenon, with presence in the placebo group, increases following the course of failed onychomycosis treatment suggest an antifungal-induced response.

Do genetic mutations and genotypes contribute to onychomycosis?

BACKGROUND: The variability in susceptibility to onychomycosis for individuals exposed to the same environmental risk factors raises the possibility that there may be individuals with a genetic predisposition to dermatophyte infection. OBJECTIVE: To determine whether there are genetic mutations or genotypes which contribute to onychomycosis. METHODS: The PubMed database was searched for examples of immune deficiencies resulting in dermatophyte infections. RESULTS: There are mutations in the innate immune receptors Dectin-1 and its adaptor protein CARD9 which result in familial mucocutaneous infections. There are also specific human leukocyte antigen genotypes that are more common in individuals and families with a high prevalence of onychomycosis. In addition, some patients have been reported with insufficient levels of CD4+CD25+ regulatory T cells. These deficits impair a full innate and adaptive immune response and may result in chronic or recurrent infections. CONCLUSIONS: There are documented mutations and genotypes that contribute to familial and individual susceptibility to onychomycosis.

Diagnostic PCR of dermatophytes--an overview.

The prevalence of onychomycosis is increasing steadily, sevenfold alone in the US within the last twenty years. An important aspect in this development is the demographic development of the human population of the industrial countries like Germany. A fast and accurate laboratory diagnosis is essential for successful treatment because 50% of the cases are misdiagnosed when relying on the clinical appearance only. The current diagnosis of dermatophytosis, based on direct microscopy and culture of the clinical specimen, is problematic given the lacking specificity of the former and the length of time needed for the latter. Molecular techniques can help to solve these problems. In recent years, a number of in-house PCR assays have been developed to identify dermatophytes directly from clinical specimens. Based on the "Mikrobiologisch-infektiologischen Qualitätsstandards (MIQ) für Nukleinsäure-Amplifikationstechniken" and the MIQE guideline (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 11 studies are reviewed which were published between 2007 and 2010. The present article evaluates the quality of the PCR assays regarding false positive and false negative results due to contamination, PCR format, statistical analysis, and diagnostic performance of the studies. It shows that we are only at the beginning of providing high quality PCR diagnosis of dermatophytes.

Analysis of genetic polymorphism of the HLA-B and HLA-DR loci in patients with dermatophytic onychomycosis and in their first-degree relatives.

Onychomycosis is known to have predisposing factors and a high prevalence within families that cannot be explained by within-family transmission. We determined the frequency of HLA-B and HLA-DR haplotypes in 25 families of Mexican patients with onychomycosis in order to define the role of the class II major histocompatibility complex (MHC) in genetic susceptibility to this infection. Seventy-eight subjects participated in the study, 47 with onychomycosis and 31 healthy individuals. The frequencies of the HLA-B and HLA-DR haplotypes were compared with those found in first-degree relatives without onychomycosis and in a historic control group of healthy individuals. The frequencies in the controls were similar to those of the healthy relatives of the patients. However, on comparison of the patients with historic controls, we detected a higher frequency of the HLA-DR8 haplotype (P=.03; odds ratio, 1.89; 95% confidence interval, 0.98-36). These findings suggest that there are polymorphisms in genes of the MHC that increase susceptibility to onychomycosis, particularly haplotype HLA-DR8.

Análisis del polimorfismo genético de los loci HLA-B y HLA-DR en pacientes con onicomicosis dermatofítica y familiares en primer grado / Analysis of Genetic Polymorphism of the HLA-B and HLA-DR Loci in Patients with Dermatophytic Onychomycosis and in Their First-Degree Relatives

En la onicomicosis hay factores predisponentes conocidos y una alta prevalencia en familiares no explicada por transmisión intrafamiliar. Analizamos la frecuencia génica de alelos de los genes HLA-B y HLA-DR en 25 familias de pacientes mexicanos con onicomicosis para conocer el papel del complejo mayor de histocompatibilidad (MHC) clase II en la susceptibilidad genética a esta enfermedad. Se estudiaron 78 individuos, 47 de ellos con onicomicosis y 31 sanos. Se determinaron las frecuencias génicas de los alelos de los loci HLA-B y HLA-DR y se compararon con las presentes en familiares de primer grado sin onicomicosis y con un grupo de individuos sanos. Estas fueron semejantes a las de familiares sanos; sin embargo, al comparar pacientes con controles históricos se encontró un aumento de la frecuencia del alelo HLA-DR8 (p=0,03; OR=1,89; IC 95%: 0,98-36). Estos datos sugieren que dentro del MHC existen genes de susceptibilidad al desarrollo de onicomicosis; en particular con el alelo HLA-DR8 (AU)

Onychomycosis is known to have predisposing factors and a high prevalence within families that cannot be explained by within-family transmission. We determined the frequency of HLA-B and HLA-DR haplotypes in 25 families of Mexican patients with onychomycosis in order to define the role of the class II major histocompatibility complex (MHC) in genetic susceptibility to this infection. Seventy-eight subjects participated in the study, 47 with onychomycosis and 31 healthy individuals. The frequencies of the HLA-B and HLA-DR haplotypes were compared with those found in first-degree relatives without onychomycosis and in a historic control group of healthy individuals. The frequencies in the controls were similar to those of the healthy relatives of the patients. However, on comparison of the patients with historic controls, we detected a higher frequency of the HLA-DR8 haplotype (P=0.03; odds ratio, 1.89; 95% confidence interval, 0.98-36). These findings suggest that there are polymorphisms in genes of the MHC that increase susceptibility to onychomycosis, particularly haplotype HLA-DR8 (AU)

[Analysis of genetic polymorphism of the HLA-B and HLA-DR loci in patients with dermatophytic onychomycosis and in their first-degree relatives]. / Análisis del polimorfismo genético de los loci HLA-B y HLA-DR en pacientes con onicomicosis dermatofítica y familiares en primer grado.

Onychomycosis is known to have predisposing factors and a high prevalence within families that cannot be explained by within-family transmission. We determined the frequency of HLA-B and HLA-DR haplotypes in 25 families of Mexican patients with onychomycosis in order to define the role of the class II major histocompatibility complex (MHC) in genetic susceptibility to this infection. Seventy-eight subjects participated in the study, 47 with onychomycosis and 31 healthy individuals. The frequencies of the HLA-B and HLA-DR haplotypes were compared with those found in first-degree relatives without onychomycosis and in a historic control group of healthy individuals. The frequencies in the controls were similar to those of the healthy relatives of the patients. However, on comparison of the patients with historic controls, we detected a higher frequency of the HLA-DR8 haplotype (P=.03; odds ratio, 1.89; 95% confidence interval, 0.98-36). These findings suggest that there are polymorphisms in genes of the MHC that increase susceptibility to onychomycosis, particularly haplotype HLA-DR8.

HLA-DR6 association confers increased resistance to T. rubrum onychomycosis in Mexican Mestizos.

BACKGROUND: Onychomycosis is multifactorial in origin. Studies have suggested an autosomal dominant pattern of inheritance and human leukocyte antigen DR4 (HLA-DR4) has been shown to protect against onychomycosis in an Ashkenazi Jewish population. AIM: This study investigates HLA class II association in a Mexican Mestizo population with Trichophyton rubrum onychomycosis. METHODS: This was a prospective case-control study. Mexican Mestizos with a clinical diagnosis of onychomycosis and culture positive for T. rubrum were recruited, together with age- and sex-matched controls. First-degree relatives were also investigated for onychomycosis. Case-control samples were HLA typed by polymerase chain reaction sequence-specific primer based analysis. RESULTS: Twenty-one cases and 42 controls were recruited with a mean age of 40 years (range: 18-58 years). HLA-DR6 was found in seven (33%) cases and 19 (45%) controls [P < 0.023, odds ratio (OR) = 0.088, 95% confidence interval (CI): 0.01-0.71]. Six (29%) cases and three (7%) controls had an affected child (P < 0.043, OR = 9.15, 95% CI: 1.07-78.31), and 13 (62%) cases and 12 (29%) controls had an affected first-degree relative (P < 0.02, OR = 4.0, 95% CI: 1.1-14.3). CONCLUSIONS: These results suggest that HLA-DR6 confers protection against the development of onychomycosis in a Mexican Mestizo population. Having an affected first-degree relative significantly increases the risk of onychomycosis, suggesting genetic susceptibility.

Human dectin-1 deficiency and mucocutaneous fungal infections.

Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the beta-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate beta-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with beta-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense.

Genetic predisposition--understanding underlying mechanisms of onychomycosis.

BACKGROUND: There are numerous factors that predispose to onychomycosis including genetic predisposition, diabetes mellitus, immunosuppression, vascular disease and psoriasis. OBJECTIVES: The aims of this workshop were to discuss current knowledge of genetic risk factors and the approaches that should be used to investigate underlying mechanisms. RESULTS: The high prevalence of onychomycosis within certain families was initially attributed to intrafamilial transmission. However, the low prevalence of infection in people marrying into infected families together with the high prevalence among their offspring suggested a genetic basis. The state-of-the-art pedigree study by Zaias et al. suggested that Trichophyton rubrum infection shows an autosomal dominant pattern of inheritance. A consensus was reached that epidemiological and genetic studies are required to investigate this issue further. For epidemiological studies, families in which two or three generations are infected with T. rubrum should be selected. Patients with T. rubrum on different body sites should be included, and the presence of associated diseases or other common features in these individuals should be investigated to identify trigger factors. CONCLUSION: Genetic studies should explore the mode of inheritance of onychomycosis and look for the disease gene(s). Serum samples from patients and age-sex matched controls must be analysed centrally. The results of these studies will make it possible to develop therapeutic, preventive and prophylatic measures and to provide patients and their families with information.

Onychomycosis: predisposed populations and some predictors of suboptimal response to oral antifungal agents.

The population groups predisposed to onychomycosis and factors associated with a poor response to antifungal therapy may be subdivided into (a) genetic, (b) environmental, (c) systemic conditions, (d) local nail characteristics, and (e) other miscellaneous items. By paying attention to the scenarios that may lead to a suboptimal response to the therapy and a higher probability of relapse of the onychomycosis, it may be possible to improve the overall cost-effectiveness of treatments for onychomycosis. Besides attempting to achieve a cure when treating onychomycosis it is important to take steps to prevent reinfection with fungal organisms.

Detection and differentiation of causative fungi of onychomycosis using PCR amplification and restriction enzyme analysis.

BACKGROUND: Onychomycosis, a fungal nail infection, has become one of the most important dermatophytoses. Unfortunately, a predictably successful diagnostic approach to onychomycosis does not yet exist. OBJECTIVE: The purpose of this study was to develop a deoxyribonucleic acid (DNA)-based diagnostic method to improve the sensitivity and specificity of the detection and differentiation of the pathogenic fungi of onychomycosis. METHODS: We attempted to detect fungi in the nail using polymerase chain reaction (PCR) primer systems that were designed in conserved sequences of the small ribosomal subunit 18S-rRNA genes shared by most fungi, and differentiated between species by restriction enzyme analysis of the amplified product. RESULTS: Fragments of the gene coding for 18S-rRNA were amplified successfully from medically important fungi species, but not from normal nails. Restriction fragment length polymorphism patterns using HaeIII endonuclease were sufficiently different to allow the recognition of individual species. CONCLUSIONS: The PCR-restriction enzyme analysis method appears to be a more sensitive detection and identification technique for onychomycosis than conventional methods, and has considerable diagnostic value.