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.

Novel CARD9 mutation in a patient with chronic invasive dermatophyte infection (tinea profunda).

Caspase Recruitment Domain Family Member 9 (CARD9) is an adaptor molecule that drives antifungal activity of macrophages and neutrophils in the skin. Autosomal recessive loss-of-function mutations in CARD9 confer increased susceptibility to invasive disease with select fungi in non-immunosuppressed patients. We report on a patient with X-linked ichthyosis complicated by chronic cutaneous invasive dermatophyte infection. We identified a previously reported c.271T>C (p.Y91H) mutation and a novel intronic c.1269+18G>A mutation in CARD9 underlying recurrent deep dermatophytosis in this patient despite various antifungals for over three decades. Our case highlights susceptibility to invasive dermatophytosis related to autosomal recessive CARD9 deficiency and illustrates the range of CARD9 mutations to be pursued in immunocompetent patients with unexplained deep dermatophyte infections. Further studies are needed to define the best therapeutic regimen.

FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis.

Microsporum canis (M. canis) is a common pathogen that causes tinea capitis and is present worldwide. The incidence of M. canis infection, particularly tinea capitis, has been increasing in China. In our previous studies, family of serine hydrolases 1 (FSH1) was identified as a potential virulence factor in tinea capitis infection caused by M. canis. To determine the function of this gene in M. canis, FSH1 was knocked down using double­stranded RNA interference mediated by Agrobacterium tumefaciens. Reverse transcription­quantitative PCR analysis was used to confirm gene knockdown. Loss of FSH1 expression by RNAi resulted in a minor phenotype alteration, but M. canis pathogenicity in guinea pig cutaneous infection was decreased compared with the wild­type strain. To the best of our knowledge, the present study is the first to demonstrate that FSH1 is associated with macroconidia septa formation and is an important contributor to M. canis virulence. These findings may advance the understanding of the function of the FSH1 gene and provide a foundation for future studies on macroconidia septa formation and pathogenicity of M. canis.

Trichophyton tonsurans strains from Brazil: phenotypic heterogeneity, genetic homology, and detection of virulence genes.

The objective of this study was to establish the phenotypical and molecular patterns of clinical isolates of Trichophyton tonsurans circulating in the state of Ceará, northeastern Brazil. For this purpose, 25 T. tonsurans strains isolated from independent cases of tinea capitis in children were phenotypically evaluated regarding their macro- and micro-morphological characteristics, vitamin requirements, urease production, and antifungal susceptibility. The molecular characterization was carried out with random amplified polymorphic DNA molecular markers and M13 fingerprinting. The presence of the genes CarbM14, Sub2, CER, URE, ASP, PBL, and LAC, which encode enzymes related to fungal virulence, was also evaluated. Finally, melanin production was assessed through specific staining. The data obtained demonstrated that these T. tonsurans strains have considerable phenotypical variation, although they showed a low degree of genetic polymorphism according to the markers used. The genes CarbM14, Sub2, CER, and URE were detected in all the analyzed strains. The gene LAC was also identified in all the strains, and melanin synthesis was phenotypically confirmed. The strains were susceptible to antifungals, especially itraconazole (GM = 0.06 µg/mL) and ketoconazole (GM = 0.24 µg/mL). Therefore, T. tonsurans strains can present great phenotypical heterogeneity, even in genetically similar isolates. Moreover, the presence of the LAC gene indicates the possible participation of melanin in the pathogenesis of these dermatophytes.

Genetic predictors of susceptibility to cutaneous fungal infections: a pilot genome wide association study to refine a candidate gene search.

BACKGROUND: Trichophyton tonsurans is the foremost fungal pathogen of minority children in the U.S. Despite overwhelming infection rates, it does not appear that this fungus infects children in a non-specific manner. OBJECTIVE: This study was designed to identify genes that may predispose or protect a child from T. tonsurans infection. METHODS: Children participating in an earlier longitudinal study wherein infection rates could be reliably determined were eligible for inclusion. DNA from a subset (n=40) of these children at the population extremes underwent whole genome genotyping (WGG). Allele frequencies between cases and controls were examined and significant SNPs were used to develop a candidate gene list for which the remainder of the cohort (n=115) were genotyped. Cumulative infection rate was examined by genotype and the ability of selected genotypes to predict the likelihood of infection explored by multivariable analysis. RESULTS: 23 genes with a putative mechanistic role in cutaneous infection were selected for evaluation. Of these, 21 demonstrated significant differences in infection rate between genotypes. A risk index assigned to genotypes in the 21 genes accounted for over 60% of the variability observed in infection rate (adjusted r(2)=0.665, p<0.001). Among these, 8 appeared to account for the majority of variability that was observed (r(2)=0.603, p<0.001). These included genes involved in: leukocyte activation and migration, extracellular matrix integrity and remodeling, epidermal maintenance and wound repair, and cutaneous permeability. CONCLUSIONS: Applying WGG to individuals at the extremes of phenotype can help to guide the selection of candidate genes in populations of small cohorts where disease etiology is likely polygenic in nature.

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.

Analysis of Trichophyton antigen-induced contact hypersensitivity in mouse.

BACKGROUND: Trichophyton-induced superficial skin mycosis is a common infectious human disease, but the immunological mechanism against Trichophyton infection is unclear with regard to many points. Since Trichophyton cannot colonize mice, guinea pigs were used in previous experiments on Trichophyton infection. However, it is difficult to perform immunological and genetic analyses in guinea pigs. OBJECTIVE: The objective of this study was to establish a mouse Trichophytin-associated inflammation model of superficial skin mycosis in which immunological and genetic analyses can be performed. METHODS: We established a mouse Trichophyton-induced contact hypersensitivity model by applying Trichophytin, the Trichophyton antigen, extracted from Trichophyton mentagrophytes, to mice. Using a Th1-dominant strain, C57BL/6, and a Th2-dominant strain, BALB/c, we investigated the expression of inflammatory cytokines and receptors of the innate immune system for fungi, TLR4, TLR2, and dectin-1, and their influences on responses of the acquired immune system. RESULTS: In C57BL/6 mice, expressions of IFN-γ and IL-17 A in regional lymph nodes and IL-1ß, IFN-γ, IL-6, and IL-23 in the inflammatory auricular skin were enhanced by Trichophytin challenge, suggesting that not only Th1 cells but also Th17 cells were induced. In BALB/c mice, expressions of IL-4 in regional lymph nodes, and TSLP and IL-4 in the auricular skin were enhanced by Trichophytin challenge. Interestingly, dectin-1-neutralizing antibody inhibited the promotion of IFN-γ production in C57BL/6 mice, and dectin-1-expressing immune cells had crucial actions in Trichophyton-induced IFN-γ production. CONCLUSION: These results suggest that inflammatory mediators differently regulate Trichophytin-induced contact hypersensitivity on the basis of the status of host immunity.

Epidemiology of sporadic (non-epidemic) cases of Trichophyton tonsurans infection in Japan based on PCR-RFLP analysis of non-transcribed spacer region of ribosomal RNA gene.

A number of cases of Trichophyton tonsurans infection have been reported among sportsmen and women participating in wrestling, judo, and sumo wrestling in Japan, but there have also been sporadic reports of cases with no history of contact with these sports. A molecular method using restriction enzyme analysis of PCR-amplified fragments targeting the non-transcribed spacer region (NTS) of ribosomal RNA gene in fungal nuclei was applied to T. tonsurans strains isolated from sporadic cases in Japan. Five of 6 molecular types recorded in Japan, i.e., NTS types I, II, IV, V, and VI, and two new types, designated NTS VII and NTS VIII, were observed among 10 strains isolated from sporadic cases. The NTS IV strains, considered not to be related to the present epidemic, were found to be the most prevalent molecular type accounting for 4 of the 10 strains isolated. NTS I was the most prevalent type in the current epidemic in Japan, but it was cultured from only one patient who was later noted to be the daughter of a retired judo practitioner. Four subjects had histories of living abroad and were considered to have been infected outside Japan. The strains in these cases were NTS II, V, VI, and VII. The results of this study suggested that the NTS IV strains were originally present in Japan at a low incidence, but that there has been a recent influx of NTS I, II, V, VI, and VII from abroad, which has been accompanied by the secondary spread of strains from wrestlers and practitioners of martial arts to the general community.

Periodontal therapy in siblings with Papillon-Lefèvre syndrome and tinea capitis: a report of two cases.

OBJECTIVE: Report of clinical and microbiological periodontal findings before and 6 months after treatment of two siblings with Papillon-Lefèvre syndrome (PLS) and tinea capitis. METHODS: Two brothers, RG 3 years and NG 5 years of age, were referred for treatment due to premature mobility of their deciduous teeth. Probing depths (PPD), attachment levels (PAL-V), and furcation involvements were examined clinically. Panoramic radiographs were taken. Subgingival plaque samples within the deepest pocket of each tooth were taken and analysed by real-time polymerase chain reaction (PCR) for Actinobacillus actinomycetemcomitans (AA), Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, Fusobacterium nucleatum, and Prevotella intermedia. One-stage full-mouth scaling and extraction of hopeless teeth were performed under general anaesthesia, followed by systemic amoxicillin and metronidazole for 7 days. Clinical and microbiological analyses were performed 6 months after treatment. RESULTS: Before treatment, both siblings had exhibited PPD of up to 13 mm, Class III furcation defects at four teeth, and marginal suppuration. AA was detected in both patients and at all teeth at levels ranging from 3.0 x 10(2) to 5.1 x 10(6). Both patients exhibited palmar and plantar hyperkeratosis. Seven teeth were extracted from RG, and nine from NG. Six months after treatment, PPD had been reduced to

[Questionnaire investigation of incidence of Trichophyton tonsurans infection in dermatology clinics in the Kanto area].

We conducted a questionnaire investigation to learn the incidence of T. tonsurans infection. Subjects of this investigation were 1,060 dermatologists in 1,060 dermatology clinics in the Kanto area to whom questionnaires were mailed. We asked each dermatologist whether he/she had experienced T. tonsurans infection cases (including suspected cases) and if so, we further asked; a. time of onset, b. number of cases, c. sexuality of the patient, d. club that the subject had joined (judo club, wrestling club or other), e. age of the subject, and f. number of cases suspected of having familial infection, the response rate was 47.5% (504 of the 1060 doctors), and 25.8% (130 of the 504) had handled T. tonsurans infection cases. The total number of patients was 707 (657 males and 50 females), with 400 (56.6%) of those in the 18 high-ranking clinics. The number of cases had increased rapidly from around 2002; 72.9% of the patients were students in high schools and universities and in 8 cases familial infection was suspected. 96.5% of the patients were in a judo or wrestling club. Our investigation revealed that this infectious disease had spread more than we had expected. It is important to develop more reliable infection control measures and to determine the actual conditions of this infection using mycological examinations.

[Survey of Trichophyton tonsurans infection in the Hokuriku and Kinki regions of Japan].

In June 2004, information was gathered on Trichophyton tonsurans infections, both past and current, in the Hokuriku and Kinki regions of central-western Honshu island, Japan, by questionnaires sent to 185 dermatologists who were members of the local medical mycologist associations Hokuriku Shinkin Kondan-kai and Kansai Shinkin Kondan-kai. Of the 111 (59.4%) who returned the completed questionnaire, 32 (28.8%) had seen patients infected with T. tonsurans including suspicious cases. The earliest recorded cases were linked to an endemic that occurred in 1994 or 1995 among a high school wrestling team in Toyama. The majority of the dermatologists saw their first case between 2001 and 2003. When the patients were grouped according to contact sports, judo players formed the largest group, followed by wrestlers. When grouped according to age, high school students formed the largest group, but the endemic had also expanded among junior high school students and adults, and there was one nursery school child who was a member of a judo club. Seventy-four of the dermatologists were sent sterilized hairbrushes to collect samples from patients suspected as having tinea capitis during July and September 2004. Trichophyton tonsurans was detected in samples from 6 patients. To investigate the molecular epidemiology, 71 of the clinical strains of T. tonsurans isolated from the Hokuriku and Kinki regions were analyzed using restriction fragment length polymorphisms of the non-transcribed spacer regions of ribosomal RNA genes. With the restriction enzyme Mva I, two molecular types were detected among the strains, indicating that the causative agents of the endemic were derived from different origins.

[Trichophyton violaceum infection in three black brothers].

Case 1 was a 3-year-old with tinea capitis, while Case 2 was a 5-year-old with kerion celsi. These conditions developed between December 1997 and January 1998 when the patients were living in Canada. Case 3 was a 7-year-old who developed kerion celsi in March 1998 in Japan. For Cases 1 and 2, the referring doctor had detected a fungal infection a few weeks previously, and griseofulvin was administered orally to Case 1 for three weeks and Case 2 for one week prior to consultation at our department. Both Cases 1 and 2 visited our department initially on May 7, 1998 with the causative agent separated from their lesions by the referring doctor respectively. The causative agent was identified as Trycophyton violaceum by our department. KOH-prepared direct microscopy and culture were positive for Case 1, but negative for Case 2. Infected hairs of Case 1 showed a chain of large arthrospores arranged in parallel rows inside the hair. Case 3 visited our department initially on May 21, and T. violaceum was also isolated from this patient's lesion. A reddish purple isolate that was stored for five months after isolation and culture was subcultured using a Sabouraud agar containing thiamin. After two months, brown colonies without red pigment were seen in the reddish purple colonies. Slide culture performed for both colonies showed intercalary and terminal chlamydospores, thus confirming that they were the same fungus. These findings appear to represent the conversion of T. violaceum into T. glabrum.