Evaluation of trichophyton agars for identification of Trichophyton soudanense

Studies of three monoconidial subcultures of each of 10 isolates of Trichophyton soudanense on seven trichophyton agars revealed variations in growth among the subcultures of each isolate and among the isolates themselves on six agars. In contrast, greater consistency and generally good to excellent growth were noted with all isolates on trichophyton agar 1 (basal medium). These results are contrary to those found with other Trichophyton species and suggest that growth on the trichophyton agars is not a suitable test for the identification of T. soudanense.     

Rapid method for differentiation of Trichophyton rubrum, Trichophyton mentagrophytes, and related dermatophyte species.

Bromocresol purple-milk solids-glucose medium, proposed by Fischer and Kane in 1971 (Mycopathol. Mycol. Appl. 43:169-180, 1971) as an aid in the rapid determination of Trichophyton rubrum and Trichophyton mentagrophytes, was evaluated across a wide range of isolates to determine its accuracy and efficacy in the clinical laboratory. Results showed that it facilitated accurate determination of typical and atypical isolates of both species and also permitted the rapid identification of other closely related and similar species. Identification of all dermatophyte species tested was possible within 7 to 10 days. Occult contamination of isolates by antibiotic-resistant bacteria did not hinder identification.     

Trichophyton eboreum sp. nov. isolated from human skin

An unusual dermatophyte was isolated from the plantar scales of a human immunodeficiency virus-positive man with tinea pedis. Morphology, physiology, and molecular data provided evidence to support the new species Trichophyton eboreum. This dermatophyte is characterized by rapid growth on common mycological media, a flat powdery off-white colony, formation of clavate microconidia, smooth- and thin-walled cylindrical or club-shaped macroconidia with two to nine cells, the presence of hook-shaped hyphae, the production of cleistothecium-like structures and spiral hyphae in older cultures, positive hair perforation, the absence of pigmentation on potato glucose agar, the absence of a requirement for vitamins, a weak positive urease reaction, no growth at 37 degrees C, resistance to 5% NaCl, resistance to fluconazole, good growth on human epidermal keratin, and the production of various enzymes on different media by the API-ZYM test. More than 5% divergence from any known species of dermatophyte was revealed by sequence analysis of the internal transcribed spacer of the rRNA gene.     

A case of black dot ringworm caused by Trichophyton tonsurans in Chiba Prefecture]

A 74-year-old woman visited a clinic in Kisarazu, Chiba Prefecture in December 2002 complaining of itching, scale and alopecia. She had been diagnosed as having tinea capitis by a direct microscopic examination of scales, and been treated with an antifungal cream and steroid lotion since 1999. The bald area spread from frontal to occipital in which multiple black dots and red papules were scattered. Abundant endothrix spores were observed in the hair shaft. A mycelial colony was isolated from the black dots. A giant colony on Sabouraud's agar was white, powdery and flattened with cottony elevation at the center in the obverse, and a reddish-brown pigmentation in the reverse. The isolate produced abundant microconidia that were round to club-and balloon-shaped with extreme swelling, while macroconidia and spiral bodies were few. Hair perforation test was negative and urease activity test was positive. ITS1-5.8S-ITS2 rDNA sequencing revealed 100% homology with T. tonsurans isolated from two old women in Niigata Prefecture. On the other hand, 3 bases were different from those of the outbreak isolates from judo and wrestling players infected through international matches. T. tonsurans has polymorphism and the present isolate might be an autochthonous genotype in Japan. This is the first time T. tonsurans was isolated in Chiba Prefecture. But this prefecture had been known as an endemic area of Trichophyton coccineum, which was very similar in morphological and physiological characteristics to those of T. tonsurans before World War II. These facts raise the question of whether T. tonsurans has existed in this prefecture before.     

A case of Tinea capitis caused by Trichophyton tonsurans]

A 10-year-old Peruvian girl, living in Japan since 1996, visited our hospital in August 2000 complaining of alopecia which had been present on her scalp for one year. The bald areas appeared as multiple small, scattered, angular patches with indistinct margins. Follicular pustules, erythemic nodules and lymphadenopathy were also seen. In the culture of the affected hair, a tan surface with wiry undulations grew on Sabouraud's media. The colony reverse had reddish-brown central pigmentation. Slide cultured fungi produced great numbers of round and short club-shaped microconidia, hyphae and intercalary chlamydospores. These fungi showed the following characteristics: positive urease test, no pigment production on cornmeal agar and positive thiamine dependency. The restriction fragment length polymorphism pattern and the nucleotide sequences of ribosomal-DNA internal transcribed spacer region of the causative fungus was compatible with Trichophyton tonsurans. Daily administration of 125 mg of terbinafine resulted in a satisfactory response and the lesion healed almost completely.     

Trichophyton violaceum infection occurring in a nursing home]

Six cases of Trichophyton (T.) violaceum infection seen in a nursing home are reported. A 66-year-old female (case 1) was found with tinea corporis on her face, chest and shoulder, associated with black dot ringworm. A KOH examination of hair showed endothrix parasitism. Reddish purple colonies were isolated from the patient on Sabouraud's dextrose agar, and intercalary and terminal chlamydospores were observed on slide culture. PCR-RFLP analysis of the microorganism showed a pattern of T. violaceum type. Therefore, the isolated fungus was identified as T. violaceum, a typical anthropophilic dermatophyte which had spread among residents and staffs easily. Using a mycological method, we examined 59 persons (21 residents and 38 staff members) who had had contact with case 1. The results were as follows. An 85-year-old female (case 2) and an 83-year-old female (case 3) were carriers of T. violaceum. A 23-year-old male (case 4) had tinea corporis on his right forearm due to T. violaceum. A 24-year-old male (case 5) probably had tinea corporis on his right forearm due to T. violaceum. One year after case 1's first visit to our clinic, we observed an 88-year-old female (case 6) of tinea capitis by T. violaceum. It seems that the organism was preserved in surroundings and members of the nursing home. The contagion in our cases could either have been caused by directly touching the person or by sharing their comb. PCR-RFLP analysis was performed within a short time, so that we managed effectively to select a way of treatment and to prevent the infection from spreading.     

Five-hour diagnosis of dermatophyte nail infections with specific detection of Trichophyton rubrum

A rapid two-step DNA extraction method and a multiplex PCR for the detection of dermatophytes in general and Trichophyton rubrum specifically were developed and evaluated with DNA extracted from pure cultures and from clinically diseased nails. DNA from the following dermatophytes was used: Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum gypseum, Microsporum nanum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton soudanense, Trichophyton terrestre, Trichophyton tonsurans, Trichophyton verrucosum, and Trichophyton violaceum. Human DNA and DNA from the following nondermatophyte fungi were included as controls: Alternaria, Aspergillus niger, Candida albicans, Candida glabrata, Candida krusei, Malassezia furfur, Saccharomyces cerevisiae, and Scopulariopsis brevicaulis. A total of 118 nail samples received for routine microscopy and culture for dermatophytes were subsequently tested by the two PCRs separately and in a multiplex format. Using DNA extracted from pure cultures and the pan-dermatophyte PCR, the T. rubrum-specific PCR sequentially and in a multiplex format correctly detected all dermatophytes and additionally correctly identified T. rubrum. Comparison of the traditional diagnostic evaluation (microscopy and culture) of nail samples with PCR on DNA directly extracted from the nails showed excellent agreement between PCR and microscopy, but the number of samples with dermatophyte species identification was increased considerably from 22.9% to 41.5%, mainly due to the identification of T. rubrum by PCR in microscopy-positive but culture-negative samples. In conclusion, this 5-hour diagnostic test was shown to increase not only the speed but also the sensitivity of investigation for nail dermatophytosis.     

A comparison of the performance of cystine lactose electrolyte deficient (CLED) agar with Oxoid chromogenic urinary tract infection (CUTI) medium for the isolation and presumptive identification of organisms from urine

AIMS: As part of the UK antimicrobial resistance strategy and action plan, the Public Health Laboratory Service (PHLS) is required to collect antibiotic susceptibility data so that resistance trends and patterns can be monitored. Most laboratories report urine Gram negative isolates, as "coliforms" according to morphological appearance, but without an acceptable identification system the antimicrobial surveillance data will be meaningless. Commercially available identification systems tend to be expensive and time consuming. Chromogenic agars, which claim to improve the detection of mixed cultures and identification of organisms from urine, have now become available and may provide a cost effective alternative. The primary aim of this study was to compare the performance of cystine lactose electrolyte deficient (CLED) agar with a chromogenic agar (Oxoid urinary tract infection medium; CUTI) in terms of isolation rates and ability to detect mixed cultures. Secondary aims were to evaluate the correlation of "presumptive" identification of isolates from chromogenic media with that of two commercial identification systems and to appraise the sensitivity of the semiquantitative loop and filter paper strip culture techniques. METHOD: One thousand, four hundred and sixty six urine samples were examined in four laboratories using the semiquantitative culture methods of 1 microl loop and filter paper strip. The degree of accuracy of organism identification was measured by comparing the presumptive identification using colony colour supplemented with simple bench tests, with identification obtained from two more complex commercial systems. RESULTS: There was no significant difference between the performance of the loop and filter paper strip methods on the CLED agar, but the CUTI agar performed significantly better than the CLED agar for the detection of significant isolates and mixed cultures. This difference was greater using the loop method. Identification of the organisms using the commercial systems gave > 99% agreement and was therefore considered suitable as a standard against which to compare the presumptive CUTI identification. Using the manufacturer's colony colour criteria in combination with a bench indole test, the CUTI medium was 99% specific for Escherichia coli, although this was reduced to 97% if the indole test was omitted. Citrobacter spp were the most commonly misidentified organisms, giving false presumptive identification as E coli. By testing oxidase activity to differentiate Pseudomonas spp and the absence of indole production to support the identification of Proteus mirabilis, the CUTI medium provided a suitable identification for 86.8% of Gram negative isolates. The remaining 13.2% would require further identification. CONCLUSION: CUTI medium improves the detection of mixed cultures, thereby improving the reliability of reporting of significant isolates when compared with CLED agar. When supplemented with simple bench tests it provides an identification system capable of speciating 86.8% of Gram negative isolates and providing a valuable cost effective mechanism for antimicrobial resistance surveillance.     

Validation of PCR–reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples

A dermatophyte-specific PCR–reverse line blot (PCR-RLB) assay based on internal transcribed sequences was developed. This assay allows the rapid detection and identification of nine clinically relevant species within the three dermatophyte genera Trichophyton , Microsporum and Epidermophyton in nail, skin and hair samples within 1 day. Analysis of 819 clinical samples (596 nail, 203 skin and 20 hair) revealed a positive PCR-RLB result in 93.6% of 172 culture-positive and microscopy-positive samples. PCR-RLB was superior to culture and direct microscopy, in both detection and species identification. Comparison of identification results of 208 PCR-positive and culture-positive clinical samples showed five discrepancies (2.4%) between PCR-RLB identification and classical microscopic/biochemical identification of isolates. Comparison of PCR-RLB identification and classical identification of 98 other isolates (dermatophytes and non-dermatophytes) revealed 13 discrepancies (13.3%) and five incomplete identifications of Trichophyton spp. Sequence analysis of ITS1 regions of 23 samples with discrepant or incomplete identification results (four Centraalbureau voor Schimmelcultures dermatophyte strains, four clinical samples and 15 clinical isolates) confirmed identification results of PCR-RLB in 21 of the 23 analyzed samples. PCR-RLB proved to be extremely suitable for routine detection and identification of dermatophytes directly in nail, skin and hair samples because it is rapid, sensitive, specific and accurate.     

The identification of pseudomonads and related bacteria in a clinical laboratory.

Non-fermenting, catalase-positive Gram-negative bacilli that grow on nutrient agar are often isolated in clinical laboratories. We have applied biochemical techniques appropriate to a typical clinical microbiology laboratory, and for the most part described in Cowan and Steel's Manual for the identification of medical bacteria (Cowan, 1974), to 428 clinical isolates and have evolved a scheme for their identification. Organisms were subdivided into groups on the basis of three tests, namely the glucose oxidation-fermentation test and tests for oxidase activity and motility. A choice was then made among other tests to produce indentification tables, containing only the most useful tests, for the various groups. The most complicated table has only 16 tests. This simple system identified 96.5% of the 428 organisms, as well as many subsequent isolates of the more common organisms.     

The association of perennial rhinitis with Trichophyton infection

Trichophyton was recently reported to be the cause of respiratory allergy in patients with severe bronchial asthma. We describe eight patients with perennial rhinitis in combination with skin or toe-nail infection, in whom Type I hypersensitivity reaction to Trichophyton was confirmed by skin test, RAST and nasal provocation. When treated with oral fungicidal therapy, there was significant improvement in both their skin and nasal symptoms. Local therapy produced only a mild, transient improvement. We emphasize the need for thorough evaluations in patients with respiratory allergies and negative routine skin tests.     

Trichophyton sensitivity in allergic and nonallergic asthma

BACKGROUND: Although the role of inhaled fungi in inducing asthma has been repeatedly confirmed, there are few reports about the association of asthma with dermatophyte sensitivity and the causal role of Trichophyton allergy in asthma. The objective was to investigate the presence of Trichophyton sensitivity among patients with allergic and nonallergic asthma in combination with tinea, and to compare the situation with several control groups in order to evaluate the factors determining Trichophyton sensitivity. METHODS: A total of 86 subjects (55 female, 31 male) with a mean age of 38.6 +/- 11.1 years were included in the study. The patients were divided into five groups: 1) nonallergic asthma plus tinea (n = 19) 2) allergic asthma plus tinea (n = 15) 3) asthma without tinea (n = 22) 4) tinea without asthma (n = 17) 5) healthy controls (n = 13). Skin tests with standardized extracts of T. rubrum and specific IgE measurements were performed in all subjects. All patients were also subjected to microscopic evaluation and fungal culture for dermatophyte infection. RESULTS: The skin test positivity rate to Trichophyton extract of groups 1 (63.1%), 2 (46.7%), and 4 (47.1%) was higher than that in groups 3 (4.4%) and 5 (7.7%) (P < 0.05). Although not significant, the rates of sensitivity to T. rubrum (63.1%) and of severe asthma (31.6%) were higher in the group with nonallergic asthma with tinea (group 1) than in other groups. Among 51 patients in whom direct microscopic evaluation revealed dermatophyte infection, 60.8% had positive fungal cultures for T. rubrum (58.1%), T. mentagrophytes (35.5%), and Candida (6.4%). CONCLUSION: According to our data, the presence of fungal infection seems to be an important determinant in hypersensitivity to Trichophyton whether or not the subject is asthmatic and/or allergic. Since a greater proportion of patients with nonallergic asthma--in whom the rate of severe asthma was also higher - showed positive skin tests to Trichophyton extracts in this study, we believe that patients with severe, intrinsic asthma should be examined for signs of fungal infection and tested to determine immediate hypersensitivity to dermatophyte antigens.     

Two arthroderma benhamiae isolates showing mitochondrial DNA type of Trichophyton verrucosum.

Two Trichophyton mentagrophytes isolates from skin lesions on a girl and her rabbit, which were identified as Arthroderma benhamiae by mating tests, were studied by mtDNA (mitochondrial DNA) analysis and sequencing of ITS (internal transcribed spacer) regions of nuclear ribosomal RNA genes. The mtDNA-RFLP (restriction fragment length polymorphism) patterns showed the two isolates to be T. verrucosum rather than A. benhamiae. The ITS sequence showed the isolates to be closer to A. benhamiae Americano-European race than to T. verrucosum and closer to T. verrucosum than to their mating partner A. benhamiae African race. These results suggest that there are three genetic types of A. benhamiae - an Americano-European type, an African type and a T. verrucosum type - and that T. verrucosum is an anamorph of A. benhamiae.     

Molecular and phenotypic identification of the yeast pathogen Candida dubliniensis

Candida dubliniensis is an emerging yeast pathogen generally misclassified as Candida albicans by standard diagnostic procedures. This study examined the efficiency of molecular identification, based on a discriminative PCR test, in a prospective study on the prevalence of C. dubliniensis among 103 oropharyngeal isolates from HIV-infected individuals or transplant recipients, and 30 vaginal isolates. All of the isolates had been classified as C. albicans by standard laboratory procedures. The PCR was evaluated in a blinded fashion against classification achieved by sequencing rDNA. Sequencing results corresponded 100% to the results of the discriminative PCR, indicating the validity of this rapid test. Twenty-one C. dubliniensis isolates were identified, all of them from HIV-infected individuals (prevalence 30%). The internal transcribed spacer regions of the C. dubliniensis isolates were sequenced. Phenotypic features of C. dubliniensis, namely abundant chlamydospore formation, atypical color on CHROMagar, growth defect at 45 degrees C, and colony morphology on Staib agar, were evaluated in a blinded fashion with respect to their discriminative potential, facilitating the design of further epidemiological studies. Carbohydrate assimilation patterns were determined for C. dubliniensis with a novel automated system showing that, in contrast to previous reports, C. dubliniensis is able to utilize D-xylose and trehalose. In evaluating these tests we present a rational approach to identification of the new species and characterization of C. dubliniensis isolates.     

An inexpensive and reliable method for routine identification of staphylococcal species

The aim of this study was to develop a simple, reliable, and inexpensive in-house system for routine species identification of staphylococci in clinical practice. The system combines 15 key tests (including carbohydrate fermentation) performed in micro-well strips and antimicrobial disk diffusion susceptibility tests performed on standardised paper disk method antibiotic sensitivity medium agar. Twenty-eight Staphylococcal reference strains belonging to 18 different species were correctly identified using this in-house system. A total of 291 clinical staphylococci isolates were evaluated with the in-house system and a conventional identification scheme. The in-house system identified 281 (96.6%) of these 291 isolates. Eleven different species were recognised. The five species most frequently identified wereStaphylococcus epidermidis (48.6%),Staphylococcus aureus (27.8%),Staphylococcus haemolyticus (8.2%),Staphylococcus hominis (5.7%), andStaphylococcus warneri (5.3%). There was an agreement of 86.3% between the species identification obtained with the in-house system and the conventional identification scheme. All coagulase-negative isolates initially identified as species other thanStaphylococcus epidermidis as well as indistinctly identified isolates were also evaluated with a commercial identification system. The agreement between species identification obtained with the inhouse system and the commercial system for 101 identified isolates was 73%. Several isolates that were difficult to distinguish with the conventional scheme and/or the commercial system were identified with the aid of the antimicrobial susceptibility test included in the in-house system. The described test scheme should be of value for identification of clinically significant staphylococci species.     

Multiple gene analyses are necessary to understand accurate phylogenetic relationships among Trichophyton species

Phylogenetic relationships among 34 isolates from 11 Trichophyton and 3 Arthroderma species were investigated using the nucleotide sequences from 4 DNA regions: internal transcribed spacers (ITS) 1 and 2 including the 5.8S rRNA gene, and the actin (ACT), DNA topoisomerase (TOP) 2 and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes. All four phylogenetic trees showed that the 34 isolates can be divided into 3 clades, the Arthroderma simii, A. benhamiae and Trichophyton rubrum clades. The Shimodaira-Hasegawa test (SH test) revealed significant topological incongruities within the A. benhamiae and A. simii clades. Although branching patterns of the 3 clades were inconsistent among the four trees, the SH test did not support these differences except that the best tree topology according to ACT sequences was significantly rejected by the TOP data set. These results show that multiple gene analyses are necessary to more precisely understand the phylogenetic relationships among these fungi.     

In vitro antifungal oral drug and drug-combination activity against onychomycosis causative dermatophytes.

We present the results of studies of the in vitro susceptibility of 52 isolates of Trichophyton rubrum and 40 of Trichophyton mentagrophytes to griseofulvin, terbinafine, itraconazole, ketoconazole, fluconazole and cyclopiroxolamine. All test strains were recovered from patients with toe nail onychomycosis and the minimum inhibitory concentration (MIC) of each antifungal against both species was individually assessed. In addition, we investigated the MIC of the combination of cyclopiroxolamine and itraconazole and cyclopiroxolamine and ketoconazole. The NCCLS approved procedure M38-A as modified by Santos and Hamdan was employed. The studies of the two drug combinations were conducted with a checkerboard design. Analysis of the data revealed that terbinafine was the most effective in vitro against all isolates, followed in order by itraconazole, cyclopiroxolamine, ketoconazole and fluconazole. We observed no significant difference in the in vitro susceptibility profiles between either species to any of the antifungals (P<0.05). Our in vitro results confirm that terbinafine is the most effective of the antifungals included in this study. Furthermore, synergistic interactions were found in the two drug combinations with all of the dermatophyte test isolates. The latter results are in agreement with clinical data that show synergism between oral and topical antifungals in the treatment of onychomycosis.     

Antifungal susceptibility testing of dermatophytes: establishing a medium for inducing conidial growth and evaluation of susceptibility of clinical isolates

A standardized reference method for dermatophyte in vitro susceptibility testing is lacking. In a previous study, Norris et al. (H. A. Norris, B. E. Elewski, and M. A. Ghannoum, J. Am. Acad. Dermatol. 40(6, part 2):S9-S13) established the optimal medium and other growth variables. However, the earlier study did not address two issues: (i) selection of an optimal medium for conidial formation by dermatophytes and (ii) validation of the method with a large number of dermatophytes. The present study addresses these two points. To select which agar medium best supported conidial growth, representative isolates of dermatophytes were grown on different agars. Preliminary experiments showed that only oatmeal cereal agar supported the production of conidia by Trichophyton rubrum. We tested the abilities of 251 T. rubrum isolates to form conidia using three different cereal agars and potato dextrose agar. Overall, oatmeal cereal and rice agar media were comparable in their abilities to support T. rubrum conidial growth. Next, we used the oatmeal cereal agar for conidial formation along with the optimal conditions for dermatophyte susceptibility testing proposed by Norris et al. and determined the antifungal susceptibilities of 217 dermatophytes to fluconazole, griseofulvin, itraconazole, and terbinafine. Relative to the other agents tested, terbinafine possessed the highest antifungal activity against all of the dermatophytes. The mean +/- standard error of the mean MICs of fluconazole, itraconazole, terbinafine, and griseofulvin were 2.07 +/- 0.29, 0.13 +/- 0.01, 0.002 +/- 0.0003, and 0.71 +/- 0.05 microgram/ml, respectively. This study is the first step in the identification of optimal conditions that could be used for the standardization of the antifungal susceptibility testing method for dermatophytes. Inter- and intralaboratory agreement as well as clinical correlations need to be established.     

White variants of Trichophyton violaceum isolated in Ethiopia

Certain dermatophytes are geographically restricted and endemic in particular parts of the world, while other species may have a sporadic but worldwide distribution. Trichophyton violaceum is one of the most common dermatophytes causing tinea capitis, and is the predominant cause of tinea in Africa, South America and the Indian subcontinent. Among 1187 dermatophyte isolates collected from Ethiopian patients with various types of tinea, 32 isolates had uncharacteristic phenotypic features. Based on conventional methods complemented by sequence analysis of the rDNA ITS2 region, these isolates were identified as white variants of T. violaceum. This is the first time that white isolates of T. violaceum have been identified in Ethiopia.