Treatment of nail clippings with ethyl alcohol improves the efficacy of Raman spectroscopy in the diagnosis of Trichophyton rubrum onychomycosis.

The purpose of this work was to enhance the diagnostic accuracy of nail Raman spectroscopy for fungal nail infections, specifically onychomycosis caused by Trichophyton rubrum. The study assessed the different ethyl alcohol retention rates between control and infected nails after soaking nail clippings in ethanolic solutions and drying. Results revealed that ethyl alcohol completely evaporated from infected nail samples, while significant amounts were still present in control samples. Principal component analysis (PCA) was applied to discriminate control from infected nails and showed superior group separation when nails were treated with ethyl alcohol. PCA loadings plot attributed the efficient classification to the νs (CCO) Raman vibrational mode of ethyl alcohol. As Raman spectroscopy can detect minute concentration changes of ethyl alcohol in nails and the deterioration caused by onychomycosis accelerates its evaporation, a simple and rapid method for detecting T. rubrum onychomycosis is proposed.

The effect of cytoplasmic crude extracts of Trichophyton verrucosum on cell mediated immunity.

OBJECTIVE: Trichophyton verrucosum is a slow growing dermatophyte responsible for a number of skin diseases such as ringworm, and is characterized by patches of hair loss and thick crusts on the host skin in domestic animals. In this study, we examined the immunomodulatory effects of crude extract of Trichophyton verrucosum (TV)cytoplasm in a mouse model. METHODS: The TV variate was cultured on Sabouraud dextrose agar and the mycelium was grinded by mechanical force. The purified protein was obtained from crude extract of the fungus, and protein concentration was measured by BradFord assay. Six to eight week-female BALB/c mice were divided into three groups: test group, receiving cytoplasmic crude extract plus defibrinated sheep blood; control group, receiving defibrinated sheep blood; and normal group, receiving normal saline. Injections were performed on days 0, 3, 5, 7 and 9 and the mice were sacrificed four days after the last injection. T lymphocyte metabolic activity was examined by methyl thiazol tetrazolium (MTT) assay, and also interleukin-4 (IL-4) and interferon-γ (IFNγ) levels were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: MTT assay showed that the TV extract stimulated lymphocyte metabolic activity. ELISA results showed that despite increase in the level of IFNγ, no changes were observed in IL-4 level. CONCLUSIONS: Results indicated that crude extract of TV cytoplasm may probably act as an immune modulator, which affects Th1 responses. The TV crude extract may be an appropriate agent to induce cellular immunity for combating dermatophytosis infection in animals; and therefore, TV extract may have some potential applications in vaccine/adjuvant technology.

Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 µg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and ß(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of ß(1→3) glucans and ß(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses.

Triintsin, a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity.

Since there is a symbiotic and competitive relationship between microorganisms in the same ecological niche, fungal defensins have been found to be important resources for antimicrobial peptides. Here, a fungal defensin, triintsin, was characterized in a clinical isolate of Trichophyton interdigitale from a patient with onychomycosis. The comparison of its genomic and mRNA sequences showed the gene organization and structure of three coding exons separated by two introns. The precursor peptide of triintsin contained 85 amino acid residues, which were composed of three parts including an N-terminal signal domain of 21 residues, a pro-peptide of 47 residues that ended at lysine-arginine and a mature peptide of 38 residues at the C-terminus. The 3D-structure established by homology modeling revealed that triintsin presented a representative typical cysteine-stabilized α-helical and ß-sheet fold. The reductive linear peptide of triintsin was obtained by chemical synthesis. After cyclization to form three pairs of disulfide bonds, the oxidative-type peptide displayed broad-spectrum antimicrobial activity against both gram-positive and gram-negative bacteria but also showed anti-fungal activity. Moreover, triintsin can effectively inhibit the growth of clinical strains. Altogether, the peptide is a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity.

Ultra-High-Resolution Mass Spectrometry for Identification of Closely Related Dermatophytes with Different Clinical Predilections.

In the present study, an innovative top-down liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification of clinically relevant fungi is tested using a model set of dermatophyte strains. The methodology characterizes intact proteins derived from Trichophyton species, which are used as parameters of differentiation. To test its resolving power compared to that of traditional Sanger sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), 24 strains of closely related dermatophytes, Trichophyton rubrum, T. violaceum, T. tonsurans, T. equinum, and T. interdigitale, were subjected to this new approach. Using MS/MS and different deconvolution algorithms, we identified hundreds of individual proteins, with a subpopulation of these used as strain- or species-specific markers. Three species, i.e., T. rubrum, T. violaceum, and T. interdigitale, were identified correctly down to the species level. Moreover, all isolates associated with these three species were identified correctly down to the strain level. In the T. tonsurans-equinum complex, eight out of 12 strains showed nearly identical proteomes, indicating an unresolved taxonomic conflict already apparent from previous phylogenetic data. In this case, it was determined with high probability that only a single species can be present. Our study successfully demonstrates applicability of the mass spectrometric approach to identify clinically relevant filamentous fungi. Here, we present the first proof-of-principle study employing the mentioned technology to differentiate microbial pathogens. The ability to differentiate fungi at the strain level sets the stage to improve patient outcomes, such as early detection of strains that carry resistance to antifungals.

The First Whole-Cell Proteome- and Lysine-Acetylome-Based Comparison between Trichophyton rubrum Conidial and Mycelial Stages.

Trichophyton rubrum is the most common fungal pathogen in the world, which has been studied as an important dermatophyte model organism. Despite the prevalence of T. rubrum, the available antifungal therapies are not sufficiently efficient. In this study, we performed the first comparison between the two major growth stages of T. rubrum: conidial and mycelial stages, based on their whole-cell proteomes and lysine acetylomes. In total, 4343 proteins were identified in both stages, and 1879 proteins were identified as differentially expressed between the two stages. The results showed that secretory proteases were more abundant in conidia, while aerobic metabolism and protein synthesis were significantly activated in the mycelial stage. In addition, 386 acetylated sites on 285 proteins and 5414 acetylated sites on 2335 proteins were identified in conidia and mycelia, respectively. The acetylation modifications were highly involved in metabolism and protein synthesis in both stages but differentially involved in Kyoto Encyclopedia of Genes and Genomes pathways and in epigenetic regulation between the two stages. Furthermore, inhibition of acetyltransferases or deacetylases significantly inhibited fungal growth and induced apoptosis. These results will enhance our understanding of the biological and physiological characteristics of T. rubrum and facilitate the development of improved therapies targeting these medically important pathogenic fungi.

Nail Raman spectroscopy: A promising method for the diagnosis of onychomycosis. An ex vivo pilot study.

Trichophyton rubrum and Candida species comprise the majority of onychomycosis pathogens. The aim of this study was to evaluate Raman spectroscopy for the differentiation between healthy and either T. rubrum or Candida infected nails. Raman measurements were performed on clippings (N = 52) infected either by T. rubrum (N = 12) or Candida species (N = 14; C. parapsilosis (sensu lato): N = 11, C. glabrata: N = 1, C. albicans: N = 2) with healthy nails (N = 26) used as controls. Systematic spectral differences were observed in the 500-520 cm-1 band region, attributable to a diverting imprint of the disulfide stretching of cystine and cysteine residues among samples. Particularly, Candida infected nails demonstrated a shoulder at 519 cm-1, corresponding to the signal of the less stable gauche-gauche-trans conformation of the disulfide bond. Two additional bands at 619 and 648 cm-1, corresponding to the C-S stretching vibration, were more evident in the T. rubrum infected nails. Finally, a Raman band at 1550 cm-1, attributable to amide II and tryptophan (Trp) content, was undetectable in Candida infected nails. Using principal component analysis (PCA), efficient differentiation of healthy, T. rubrum and Candida species infected nails was achieved. Soft independent modeling of class analogy (SIMCA) and partial least squares-discriminant analysis (PLS-DA) were further applied to generate diagnostic algorithms for the classification of Raman spectra. Both techniques succeeded in modeling clinical nail samples in three groups according to their mycological categories. Raman spectroscopy is a promising method for the differentiation of healthy vs. diseased nails, including efficient differentiation between onychomycosis caused by T. rubrum and Candida species.

MALDI-TOF MS analysis of bovine and zoonotic Trichophyton verrucosum isolates reveals a distinct peak and cluster formation of a subgroup with Trichophyton benhamiae.

The zoophilic dermatophyte Trichophyton verrucosum is the most important causative agent of bovine dermatophytosis. Additionally, it causes profound and poorly healing skin infections in humans indicating the high zoonotic potential. The objective of this study was to establish differentiation of T. verrucosum from other dermatophytes by mass spectrometry and to identify distinct features of the mass spectra. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successful for identification of this pathogen only after extension of the database of the manufacturer with spectra from T. verrucosum strains, which were identified as such by sequencing of the internal transcribed spacer (ITS) region. MALDI-TOF MS analysis was conducted with 46 field isolates from cattle, two live vaccine strains, and 10 isolates from humans identified as T. verrucosum by sequence analysis of the ITS region. The results suggest a very good agreement of both methods. Comparison with the mass spectra of 68 strains of other keratinophilic fungi revealed that most T. verrucosum wild-type isolates showed a characteristic peak at 7950-7954 m/z, which was missing in the spectra of other keratinophilic fungi and the live vaccine strains. The spectra of T. verrucosum were most similar to the spectra of T. benhamiae, an emerging zoophilic dermatophyte. In summary, MALDI-TOF MS is a powerful and reliable tool to identify T. verrucosum.

Proteogenomic Analysis of Trichophyton rubrum Aided by RNA Sequencing.

Infections caused by dermatophytes, Trichophyton rubrum in particular, are among the most common diseases in humans. In this study, we present a proteogenomic analysis of T. rubrum based on whole-genome proteomics and RNA-Seq studies. We confirmed 4291 expressed proteins in T. rubrum and validated their annotated gene structures based on 35 874 supporting peptides. In addition, we identified 323 novel peptides (not present in the current annotated protein database of T. rubrum) that can be used to enhance current T. rubrum annotations. A total of 104 predicted genes supported by novel peptides were identified, and 127 gene models suggested by the novel peptides that conflicted with existing annotations were manually assigned based on transcriptomic evidence. RNA-Seq confirmed the validity of 95% of the total peptides. Our study provides evidence that confirms and improves the genome annotation of T. rubrum and represents the first survey of T. rubrum genome annotations based on experimental evidence. Additionally, our integrated proteomics and multisourced transcriptomics approach provides stronger evidence for annotation refinement than proteomic data alone, which helps to address the dilemma of one-hit wonders (uncertainties supported by only one peptide).

Identification of Dermatophyte species after implementation of the in-house MALDI-TOF MS database.

Despite that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool in the clinical microbiology setting, few studies have till now focused on MALDI-TOF MS-based identification of dermatophytes. In this study, we analyze dermatophytes strains isolated from clinical samples by MALDI-TOF MS to supplement the reference database available in our laboratory. Twenty four dermatophytes (13 reference strains and 11 field isolated strains), identified by both conventional and molecular standard procedures, were analyzed by MALDI-TOF MS, and the spectra obtained were used to supplement the available database, limited to a few species. To verify the robustness of the implemented database, 64 clinical isolates other than those used for the implementation were identified by MALDI-TOF MS. The implementation allowed the identification of the species not included in the original database, reinforced the identification of the species already present and correctly identified those within the Trichophyton mentagrophytes complex previously classified as Trichophyton. tonsurans by MALDI-TOF MS. The dendrogram obtained by analyzing the proteic profiles of the different species of dermatophytes reflected their taxonomy, showing moreover, in some cases, a different clusterization between the spectra already present in the database and those newly added. In this study, MALDI-TOF MS proved to be a useful tool suitable for the identification of dermatophytes for diagnostic purpose.

Hippolachnin A, a new antifungal polyketide from the South China Sea sponge Hippospongia lachne.

Hippolachnin A (1), a polyketide possessing an unprecedented carbon skeleton with a four-membered ring, was isolated from the South China Sea sponge Hippospongia lachne. The structure was elucidated using MS and NMR spectroscopic analyses, and the absolute configuration was determined using a calculated ECD method. Hippolachnin A demonstrated potent antifungal activity against three pathogenic fungi, Cryptococcus neoformans, Trichophyton rubrum, and Microsporum gypseum, with a MIC value of 0.41 µM for each fungus.

De novo production of metabolites by fungal co-culture of Trichophyton rubrum and Bionectria ochroleuca.

The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″-dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca.

Identification of the Trichophyton mentagrophytes complex species using MALDI-TOF mass spectrometry.

Dermatophytes are fungi capable of invading keratinized tissues and are responsible for the most common fungal infection worldwide: dermatophytosis. Identification of these organisms to the species level is often necessary for the correct treatment of these infections, and is always recommended from an epidemiological point of view. Since the identification of dermatophytes is sometimes problematic, we assessed whether Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) could provide a useful tool to identify dermatophytes of the Trichophyton mentagrophytes complex. A reference database was constructed with 17 strains of six different species belonging to this complex. A total of 54 dermatophyte strains of the Belgian co-ordinated collections of micro-organisms, Scientific Institute of Public Health, Brussels, Belgium (BCCM™/IHEM) collection were used to challenge this database; 89% of the tested strains (not used as reference strains in the database) could readily be identified. When incorrect identifications were encountered, the confusion was always between phylogenetically closely related taxa which indicates that observations made by MALDI-TOF MS correlate with phylogenetic data. To assess this observation, a dendrogram outlining the similarities between the obtained spectra was constructed. Strikingly, the relationships found in this dendrogram were highly similar to the ones observed in the phylogenetic tree recently reported by Beguin and co-workers. In conclusion, MALDI-TOF MS is a fast and reliable tool for the identification of dermatophytes, since it can even discriminate between the closely related species of the T. mentagrophytes complex. Moreover, our data indicate that the data obtained by MALDI-TOF MS correlate with phylogenetic data.

Fourier transform infrared spectral evaluation for the differentiation of clinically relevant Trichophyton species.

Routine mold identification methods have been established to provide actual data to facilitate reliable diagnoses in clinical laboratories, as well as the management of infection and health practice planning, particularly for dermatophytes. Some species of the Trichophyton genera, particularly T. rubrum and T. mentagrophytes complexes, exhibit more complexity in species recognition. In this study, the intriguing technique of Fourier-transform infrared (FT-IR) spectroscopy is evaluated for species recognition of Trichophyton spp. A total of 32 reference isolates, belonging to T. mentagrophytes (n=7), T. rubrum (n=21) complexes and Arthroderma spp. (n=4), were included in the study. Numerous spectral window FTIR spectroscopy data were analyzed by principal component analysis and hierarchical clustering was performed. There were not any spectral ranges presenting clusters at the main Trichophyton species (e.g. T. rubrum, T. mentagrophytes and Arthroderma spp.). Notably, only T. violaceum (including T. yaoundei and T. soudanense) was clustered in several ranges. In intra-species evaluation, T. erinacei, belonging to the T. mentagrophytes complex, was distinguishable by FT-IR spectroscopy with different spectral range calculations. We suggested that further research with several reference and clinical isolates of Trichophyton species will be crucial to accurately identify intra-species of T. rubrum and T. mentagrophytes complexes.

Efficacy of the Trichophyton ajelloi and Lagenidium giganteum metabolites against mosquitoes after flash chromatography.

Entomopathogens are significant natural enemies for mosquitoes. We have investigated the adulticidal efficacies of metabolites of Trichophyton ajelloi and Lagenidium giganteum against Culex quinquefasciatus and Aedes aegypti simultaneously. The T. ajelloi was grown on Sabouraud's dextrose broth medium at 25 ± 2°C and relative humidity at 75 ± 5% for 15 days. L. giganteum was grown in peptone yeast extract glucose broth at 25 ± 2°C and relative humidity 75 ± 5% for 15 days. The filtrations of metabolites have been made by using Whatman-1 filter paper then with the flash chromatograph. The bioassays were conducted as per the World Health Organization's methods and protocols (2006). In this significant investigations, the metabolites of T. ajelloi have been found highly susceptible against A. aegypti with LC(99)-7.24 ml after an exposure time of 24 h with a comparison, the LC(99)-66 ml was observed against C. quinquefasciatus after exposure of 24 h. Moreover, the L. giganteum metabolites have shown higher pathogenicity against C. quinquefasciatus with LC(99)-11.3 ml and A. aegypti with LC(99)-15.49 ml. Although, the efficacy in adults could be achieved with higher concentration can be significant also. Their adulticidal activities in different climatic zones are plausible with metabolites which have better LT values of T. ajelloi.

Mating genes of the Trichophyton mentagrophytes complex.

The mating type (-)-specific gene of the alpha-box and the mating type (+)-specific gene of the high-mobility group (HMG) DNA-binding domain were confirmed in zoophilic dematophytes of Arthroderma simii and A. vanbreuseghemii. The sequence of the alpha-box gene was 1,375 bp, containing 2 exons (from 172 to 463 bp and from 513 to 1,375 bp) in the A. simii (-) mating type strain and 1,380 bp, containing 2 exons (from 177 to 468 bp and from 518 to 1,380 bp) in the A. vanbreuseghemii (-) mating type strain. The sequence of the HMG gene was 1,871 bp, containing 2 exons (from 181 to 362 bp and from 426 to 1,440 bp, coding a protein of 398 amino acids) in the A. simii (+) mating type strain and 1,811 bp containing 2 exons (from 158 to 339 bp and from 403 to 1,381 bp, coding a protein of 386 amino acids) in the A. vanbreuseghemii (+) mating type strain. Of 15 animal isolates and 72 human isolates examined, the alpha-box gene was detected in five of the animal isolates and in none of the human isolates, while the HMG gene was detected in the other 10 of the animal isolates and in all of the human isolates. Phylogenetic analysis of the alpha-box and HMG genes of Trichophyton mentagrophytes complex strains and the Microsporum gypseum strain revealed that these strains were divided into 4 clusters; the first cluster consisting of A. vanbreuseghemii and the isolates from animals and humans, the second cluster consisting of A. simii, the third cluster consisting of A. benhamiae and the fourth cluster consisting of M. gypseum. These results indicate that anthropophilic T. mentagrophytes evolved from the A. vanbreuseghemii (+) mating strain.

Melanogenesis in dermatophyte species in vitro and during infection.

Dermatophytes are keratinophilic fungi that are the most common cause of fungal skin infections worldwide. Melanin has been isolated from several important human fungal pathogens, and the polymeric pigment is now recognized as an important virulence determinant. This study investigated whether dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum gypseum, produce melanin or melanin-like compounds in vitro and during infection. Digestion of the pigmented microconidia and macroconidia of dermatophytes with proteolytic enzymes, denaturant and hot concentrated acid yielded dark particles that retained the size and shape of the original fungal cells. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical signal, consistent with the pigments being a melanin. Immunofluorescence analysis demonstrated reactivity of a melanin-binding mAb with the pigmented conidia and hyphae, as well as the isolate particles. Laccase, an enzyme involved in melanization, was detected in the dermatophytes by an agar plate assay using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Skin scrapings from patients with dermatophytoses contained septate hyphae and arthrospores that were reactive with the melanin-binding mAb. These findings indicate that dermatophytes can produce melanin or melanin-like compounds in vitro and during infection. Based on what is known about the function of melanin as a virulence factor of other pathogenic fungi, this pigment may have a similar role in the pathogenesis of dermatophytic diseases.

Arthroderma benhamiae (the teleomorph of Trichophyton mentagrophytes) mating type-specific genes.

This study first report to identify the mating type (-)-specific gene of alpha-box and the mating type (+)-specific gene of the high-mobility-group (HMG) DNA-binding domain in zoophilic dermatophytes of Arthroderma benhamiae in an effort to understand the epidemiological characteristics of Trichophyton mentagrophytes. The sequence of the alpha-box gene (1,387 bp) was found to contain two exons, from 184 to 475 bp and from 525 to 1,387 bp, coding a protein of 384 amino acids, beginning with a putative initiating methionine (ATG). The sequence of the HMG gene (1,910 bp) contained two exons, from 234 to 415 bp and from 479 to 1,457 bp, coding a protein of 386 amino acids, beginning with a putative initiating methionine (ATG). PCR analysis detected the alpha-box gene in A. benhamiae (-) mating type strains but not in (+) mating type strains. On the other hand, the HMG gene was detected in A. benhamiae (+) mating type strains but not in (-) mating type strains. These findings suggest that the HMG and alpha-box genes could be specific to the (+) and (-) mating types, respectively.

Biolarvicidal activity of extracellular metabolites of the keratinophilic fungus Trichophyton mentagrophytes against larvae of Aedes aegypti - a major vector for chikungunya and dengue.

The larvicidal activity of extracellular metabolites of keratinophilic fungus Trichophyton mentagrophytes against Aedes aegypti larvae was determined. T. mentagrophytes was isolated from soil by the feather baiting technique. Culture filtrates (10-100 microL/mL) were found to be entomotoxic to 3rd instars larvae of A. aegypti (L3), LC(50) and LC(90) being 110 +/- 11.5 and 200 +/- 20.7 microL/mL, respectively, after 2 d. Extracellular metabolites are proteinaceous in nature and more specific to chitin of mosquito larvae. They degraded cock feather causing an average of 20.0 +/- 2.6 % loss in feather mass. Culture filtrate at 100 microL/mL produced 90 % mortality against L3 after 3 d; mortality was increased in dose- and time-dependent manner. These extracellular metabolites of T. mentagrophytes could be regarded as alternatives to synthetic insecticides.

Identification of 30-kDa heat shock protein gene in Trichophyton rubrum.

Small heat shock proteins (sHSPs) are chaperones that are crucial in the heat shock response but also have important non-stress roles within the cell. HSP70 in Trichophyton rubrum is already detected and carefully characterised; however, no study was carried out for HSP30 in this pathogenic fungus. In the present study, T. rubrum was obtained from patients with dermatophytosis and cultured in appropriate conditions. High-molecular-weight DNA was extracted using standard extraction methods. Pairs of 21 nt primers were designed from highly conserved regions of the similar genes in other eukaryotic cells. Mentioned primers were utilised in PCR using isolated genomic DNA and extracted RNA templates of T. rubrum. The PCR fragments were then sequenced and 415 nucleotides of HSP30 in this pathogenic fungus were detected; the open reading frame had 156 nucleotides and was coding 51 amino acids. This gene (called TrHSP30) is registered in GenBank at National Center for Biotechnology Information (NIH, USA) database. Detection of TrHSP30 gene may open the way to determination of its possible role in the pathogenesis of dermatophyte infections due to T. rubrum.