The effect of isoflavaspidic acid PB extracted from Dryopteris fragrans (L.) Schott on planktonic and biofilm growth of dermatophytes and the possible mechanism of antibiofilm.

ETHNOPHARMACOLOGICAL RELEVANCE: Dryopteris fragrans (L.) Schott (D. fragrans), a deciduous perennial herb, has been traditionally used for treatment of various skin diseases in Heilongjiang province of China for many years. Phloroglucinol derivatives extracted from D. fragrans were the most effective fraction against dermatophytes. Isoflavaspidic acid PB is a typically phloroglucinol derivative which extracted from D. fragrans and has been reported to exert anti-fungal activities against several dermatophytes. AIM OF THE STUDY: This study aimed to evaluate anti-fungal and anti-biofilm activity of isoflavaspidic acid PB on planktonic and biofilm growth of dermatophytes and explore possible mechanisms of anti-biofilm. MATERIALS AND METHODS: Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of isoflavaspidic acid PB against 25 isolates of dermatophytes were determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method. The effects of isoflavaspidic acid PB on dermatophytes biofilm formation and pre-formed biofilm were assessed by 2.3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl (phenylamino)]-2H-tetrazolium hydroxide (XTT) assay. Morphology of mature biofilm were observed by Scanning Electron Microscope (SEM). Biomass, exopolysaccharide and ergosterol content of mature biofilm were analyzed by gravimetric analysis, anthranone sulfuric acid method and Ultra Performance Liquid Chromatography (UPLC) assay respectively. RESULT: The MIC and MFC ranges of isoflavaspidic acid PB against 25 isolates of dermatophytes were 20-80 µg/mL and 40-80 µg/mL respectively. Isoflavaspidic acid PB (2 MIC) inhibited not only Trichophyton biofilm formation (54.8% ∼ 81.2%) but also the metabolic activity of mature biofilm (20.7% ∼ 44.2%). The result of SEM showed that isoflavaspidic acid PB (8 MIC) could destroy the morphology of hyphae seriously. Comparing with control group, biomass, exopolysaccharide and ergosterol content of the mature biofilm under isoflavaspidic acid PB (8 MIC) were significantly decreased (P < 0.01). CONCLUSION: Isoflavaspidic acid PB had anti-fungal and fungicidal activities against dermatophytes. Isoflavaspidic acid PB could inhibit the biofilm of Trichophyton. The mechanism might be related to the decline of the biofilm biomass, exopolysaccharide and ergosterol content. These results showed that isoflavaspidic acid PB could be explored for promising anti-biofilm drugs.

Clinico-mycological study of dermatophytic infections and their sensitivity to antifungal drugs in a tertiary care center.

BACKGROUND: Worldwide, dermatophytic infections are running a chronic course either due to ineffective treatment or emerging drug resistance. In the past three decades, there has been an increase in incidence and non-responsiveness to conventional antifungals, which suggests that there is a need of antifungal sensitivity testing. AIMS: This study was aimed at identifying clinico-mycological pattern of dermatophytic infections in patients attending thedermatology outpatient department of a tertiary care hospital, and to obtain the sensitivity pattern of isolates against six commonly used oral antifungals (fluconazole, terbinafine, itraconazole, ketoconazole, griseofulvin and voriconazole). METHODS: Patients with suspected dermatophytoses attending the outpatient department of Sir Sunderlal Hospital, Varanasi, were enrolled in the study. A detailed history, clinical examination and sample collection for mycological examinations was done. In vitro antifungal sensitivity testing was done on species isolated from culture as per the Clinical and Laboratory Standard Institute M38-A standards, with broth microdilution method. RESULTS: There were 256 patients recruited in the study, with a male: female ratio of 3:1. The most commonly affected age group was 20-40 years (52.4%). Tinea corporis et cruris was the most common type observed (27.2%). Potassium hydroxide positivity was seen in 211 samples (79.6%) and culture positivity was found in 139 samples (52.4%). The most common species identified was Trichophyton mentagrophytes (75.9%). Sensitivity testing was done on fifty isolates of T. mentagrophytes. Minimum inhibitory concentrations of itraconazole, ketoconazole, terbinafine and voriconazole were comparable, while griseofulvin showed the highest minimum inhibitory concentration. Itraconazole was found to be the most effective drug, followed by ketoconazole, terbinafine and fluconazole. Griseofulvin was the least effective drug among the tested antifungals. LIMITATIONS: This is a hospital-based study, and may not reflect the true pattern in the community. Sensitivity pattern of only one species T. mentagrophytes was carried out. CONCLUSION: Inadequate and irregular use of antifungal drugs has led to the emergence of resistant strains, which cause poor treatment outcomes. Thus, it is very important to test for antifungal sensitivity to check for resistance to antifungals.

Antifungal and antioxidant activities of Coleonema album and C. pulchellum against skin diseases.

CONTEXT: Coleonema album (Thunb) Bart. & H. L. Wendl (Rutaceae) has been used in the formulation of skincare products, and the Khoisan people rub it on their skin to add luster. Coleonema pulchellum I. Williams has received less attention in the South African traditional medicine. OBJECTIVE: This study investigates the antifungal and antioxidant activities of C. album and C. pulchellum essential oil (EO) and leaf extracts; and analyzes the chemical components of their EOs. MATERIALS AND METHODS: Antifungal activity of leaf extracts was determined using the microdilution method with griseofulvin and ketoconazole as controls. Antifungal capacity of EO was investigated using the 'Volatile release plate method'. Trichophyton rubrum (ATCC 28188) and T. mentagrophytes (ATCC 9533) mycelia (0.3 cm diameter) were placed on fresh yeast malt agar in Petri dishes with filter paper (impregnated with 20 µL of EO) on the lid for direct exposure to EO volatiles while plates without EO were used as controls. The incubation time was seven days. Antioxidant activities of the leaf extracts were determined. RESULTS: Methanol leaf extract of C. pulchellum inhibited the growth of three fungi tested with MIC values of 195, 391 and 49 µg/mL for Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum gypseum, respectively. Terpenes formed the major components of the EO. The EO from both plants inhibited the growth of T. rubrum in vitro. DISCUSSION AND CONCLUSION: This study revealed the therapeutic value of C. pulchellum. Coleonema album and C. pulchellum should be considered as potential plants for skin ointment from natural origin.

BIOLOGICAL PROPERTIES AND CHEMICAL COMPOSITION OF JATROPHA NEOPAUCIFLORA PAX.

BACKGROUND: Ethnopharmacological relevance. Jatropha neopauciflora (Pax) is an endemic species of the Tehuacan- Cuicatlan Valley, Mexico. This species has long been used as a remedy to alleviate illnesses of bacterial, fungal and viral origin. Aim of the study. Experimentally test the traditional use of Jatropha neopauciflora in Mexican traditional medicine. MATERIALS AND METHODS: The methanol extract (MeOH1), of Jatropha neopauciflora (Euphorbiaceae) was obtained by maceration. Next, the methanol (MeOH2) and hexane (H) fractions were obtained. The essential oil was obtained by hydro- distillation. The extract, fractions and essential oil were analyzed by GC-MS. The antimicrobial activity was measured by the disc diffusion agar and radial inhibition growth methods. RESULTS: The extract and fractions showed antibacterial activity against eleven strains (five Gram-positive and six Gram- negative) and a bacteriostatic effect in the survival curves for Staphylococcus aureus and Vibrio cholerae. The extract and fractions were also shown to have antifungal activity, particularly against Trichophyton mentagrophytes (CF50 = MeOH1: 1.07 mg/mL, MeOH2: 1.32 mg/mL and H: 1.08 mg/mL). The antioxidant activity of MeOH1 (68.6 µg/mL) was higher than for MeOH2 (108.1 µg/mL). The main compounds of the essential oil were ß-pinene, 1,3,8-p-menthatriene, ledene, m- menthane, linalyl acetate and 3-carene. The main compounds of MeOH1 were ß-sitosterol, lupeol and pyrogallol; the main compounds of MeOH2 were ß-sitosterol, spathulenol, coniferyl alcohol and lupeol; and the main compounds of H were ß-sitostenone, γ-sitosterol and stigmasterol. CONCLUSIONS: This study indicates that Jatropha neopauciflora is a potential antibacterial and antifungal agent.

Synergistic antimicrobial activity of Boswellia serrata Roxb. ex Colebr. (Burseraceae) essential oil with various azoles against pathogens associated with skin, scalp and nail infections.

Antimicrobials from natural sources have gained immense importance in recent times to combat the global challenge of antibiotic resistance. Essential oils are implicated in antimicrobial action against several species. Here, we have screened nine commercially available essential oils for their antimicrobial activity against organisms associated with skin, scalp and nail infections mainly Propionibacterium acnes, Malassezia spp., Candida albicans and Trichophyton spp. Among nine essential oils, Boswellia serrata essential oil demonstrated superior antimicrobial activity against all the micro-organisms and surprisingly it showed maximum activity against Trichophyton spp. The gas chromatography-mass spectrometry analysis of B. serrata oil indicates a major composition of α thujene, ρ cymene and sabinene. Additionally, B. serrata oil was found to inhibit Staphylococcus epidermidis biofilm, and its combination with azoles has shown synergistic activity against azole-resistant strain of C. albicans. These broad-spectrum antimicrobial activities of B. serrata oil will make it an ideal candidate for topical use. SIGNIFICANCE AND IMPACT OF THE STUDY: Eradication of skin and nail infections still remain a challenge and there are serious concerns regarding the recurrence of the diseases associated with these infections. Antimicrobials from plant sources are gaining importance in therapeutics because they encounter minimal challenges of emergence of resistance. We have demonstrated the antimicrobial activity of Boswellia serrata essential oil against micro-organisms involved in skin, scalp and nail infections, especially if it has shown favourable synergistic antifungal activity in combination with azoles against the azole-resistant Candida albicans strain. Thus, B. serrata oil can be one of the plausible therapeutic agents for management of skin, scalp and nail infections.

Antifungal properties of hypericin, hypericin tetrasulphonic acid and fagopyrin on pathogenic fungi and spoilage yeasts.

CONTEXT: The role of hypericin-mediated photodynamic antimicrobial properties on pathogenic fungi and photodynamic therapy for human cancer cells is known. Antifungal properties of Hypericum perforatum L. (Hypericaceae) and Fagopyrum esculentum Moench. (Polygonaceae) extracts were also studied. The different polarities of solvents can cause complication in the identification of antifungal effects of separate biologically active compounds. In recent experimental work, we compared antifungal properties of purified hypericin, hypericin tetrasulphonic acid (hypericin + S) and fagopyrin, which is analogue of hypericin. OBJECTIVE: The antifungal properties of aromatic polyketide derivatives such as hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts have been studied. MATERIALS AND METHODS: The antifungal properties of hypericin, hypericin + S and fagopyrin were determined using the broth microdilution method against a set of pathogenic fungi and spoilage yeasts including: Microsporum canis, Trichophyton rubrum, Fusarium oxysporum, Exophiala dermatitidis, Candida albicans, Kluyveromyces marxianus, Pichia fermentans and Saccharomyces cerevisiae. The tested concentrations of hypericin, hypericin + S and fagopyrin ranged from 750 to 0.011 µg/mL and MIC values were evaluated after 48 h incubation at 30 °C. RESULTS: The results confirm different antifungal properties of hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts. For pathogenic fungi, the minimum inhibitory concentrations of hypericin ranged 0.18-46.9 µg/mL, hypericin + S 0.18-750 µg/mL and fagopyrin 11.7-46.9 µg/mL. For spoilage yeasts, the MICs of hypericin and hypericin + S ranged 0.18-46.9 and 0.011-0.73 µg/mL, respectively. DISCUSSION AND CONCLUSION: The results obtained herein indicate that various chemical structures of hypericin, hypericin + S and fagopyrin can develop different antifungal properties.

Intravenous Iron Sucrose Therapy in Iron Deficiency Anemia in Antenatal and Postnatal Patients.

INTRODUCTION: Dermatophytosis is a common superficial fungal infection of the skin, hair and nails caused by Trichophyton, Microsporum and Epidermophyton dermatophytic species. Identification of causative dermatophytic species tend to vary with time and place and antifungal sensitivity is of epidemiological concern as well as significant for the treatment with precision at the current scenario of increasing antifungal resistance. The objectives was to study clinicomycological profile and antifungal sensitivity pattern of commonly used azoles in dermatophytosis. METHODS: The prospective analysis of 145 clinically suspected cases of dermatophytosis was conducted from January 2014 to January 2015 at Department of Dermatology of COMS, Bharatpur, Chitwan, Nepal. Cases were evaluated and data recorded as per proforma. Samples were collected for microscopy and culture from skin, hair and nail. Antifungal sensitivity pattern was evaluated by standard disk diffusion technique. RESULTS: Maximum numbers of cases with dermatophytosis were observed between June to September with male to female ratio of 1.4:1. The youngest patient was 3 years and the oldest was 76 years. Tinea corporis (25.5%) was the dominant clnical type observed. Overall direct microscopy (KOH) positivity and culture positivity was 64.8% and 57.2% respectively. Predominant species of dermatophyte isolated was T. mentagrophyte (23.4%). Out of five antifungals used in our study, fluconazole and ketoconazole were found 100% resistant. CONCLUSIONS: This study highlighted the increasing resistance of the antifungals, which is responsible for the treatment failure in dermatophye infections.

In vitro combination therapy using low dose clotrimazole and photodynamic therapy leads to enhanced killing of the dermatophyte Trichophyton rubrum.

BACKGROUND: Superficial infections of the skin and mucous membranes caused by dermatophyte fungi are amongst the most common and challenging infections to treat. Previously we demonstrated the phototoxic effects of photodynamic therapy (PDT) towards Trichophyton rubrum, using a green laser to photoactivate Rose Bengal (RB). The aim of this study was to evaluate whether we could; (1) achieve a similar effect using an inexpensive light-emitting diode (LED) to photoactivate RB and (2) to evaluate whether our PDT regime could be combined with standard antifungal drug therapy and increase its effectiveness. METHODS: We designed and built our own inexpensive green (530 nm) LED source and tested its efficacy as part our RB-PDT regime in vitro against T. rubrum. We also examined the potential benefits of incorporating PDT as part of combination therapy and whether the order in which this was done had an impact. First we subjected spore suspensions to sub-inhibitory concentrations of a number of antifungal agents (CLT, MCZ and TRB) for 72 hours followed by RB-PDT. Secondly we subjected spore suspensions to sub-inhibitory PDT followed by drug treatment and evaluated if there were any changes to the minimum inhibitory concentrations (MICs) of the drugs tested. RESULTS: The optimal conditions for photoinactivation of T. rubrum using RB-PDT alone were 140 µM of RB and 24 J/cm2 of LED (equating to a 30-minute exposure). These parameters also caused a 100% reduction in the viability of the pathogenic yeast Candida albicans and the model fungus Saccharomyces cerevisiae. By combining our RB-PDT regime as an adjunct to antifungal drugs we were able to dramatically reduce the exposure times. Treatment of spore suspensions using a sub-inhibitory dose of clotrimazole (CLT) followed by RB-PDT, this order was critical, significantly reduced the exposure times required to achieve 100% inhibition of T. rubrum to 15 minutes as compared to RB-PDT alone. CONCLUSIONS: The combination of antifungal drug and RB-PDT represents an attractive alternative to the current antifungal therapies used to treat superficial fungal diseases. Our approach has the potential to reduce treatment times and drug dosages which can also reduce drug toxicity and improve patient compliance.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp.

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Isolation of flavonoids from Anemopaegma arvense (Vell) Stellf. ex de Souza and their antifungal activity against Trichophyton rubrum

Anemopaegma arvense (Vell) Stellf. ex de Souza belongs to the family Bignoniaceae, and is popularly known as catuaba. To evaluate the cytotoxic and antimicrobial activity of A. arvense, fraction F3 and flavonoids 1 (quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside) (rutin) and flavonoid 2 (quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside) were isolated from the leaves of this plant. Fraction F3 and flavonoids 1 and 2 exhibited no antibacterial activity. Furthermore, no cytotoxic activity of fraction 3 or flavonoids 1 and 2 was observed against the tumor cells tested. However, analysis of the antifungal activity of flavonoids 1 and 2 revealed minimum inhibitory concentrations of 0.5 and 0.25 mg/mL, respectively, against the Trichophyton rubrum strains tested (wild type and mutant). This study demonstrates for the first time the antifungal activity of isolated flavonoids, validating the same activity for A. arvense.

Anemopaegma arvense pertence à família Bignoniaceae, sendo conhecida popularmente como Catuaba. Para avaliação de sua atividade citotóxica e antimicrobiana, a fração cromatográfica F3 e os flavonoides 1 (quercetina 3-O-α-L-ramnopiranosil-(1→6)-β-D-glucopiranosídeo) (rutina) e flavonoide 2 (quercetina 3-O-α-L-ramnopiranosil-(1→6)-β-D-galactopiranosídeo) foram isolados das folhas de A. arvense. A fração 3 e os flavonoides não apresentaram atividade antibacteriana. Nenhuma atividade citotóxica foi observada para a fração F3 e para os flavonoides, quando avaliados contra as células tumorais em teste. Entretanto, e considerando a atividade antifúngica, o flavonóide 1 apresentou valor de concentração inibitória mínima (CIM) de 0,5 mg/mL, enquanto o flavonóide 2, CIM de 0,25 mg/mL contra as cepas selvagem e mutante de Trichophyton rubrum, demonstrando, pela primeira vez, que os flavonoides isolados possuem atividade antifúngica, o que valida a mesma atividade para A. arvense.

Commonly affected body sites in 92 Japanese combat sports participants with Trichophyton tonsurans infection.

Outbreaks of Trichophyton tonsurans infection constitute one of the serious problems among combat sports practitioners in Japan. To facilitate the diagnosis of individuals at risk, we undertook a study to determine which body sites are most commonly infected. We reviewed medical data, hairbrush culture results and questionnaire information from patients with T. tonsurans infection who were admitted to the dermatology clinic of Juntendo University hospital from 2000 to 2004. The study included 92 patients (87 males), aged 6-38 years (mean age: 18.4 years old). Eighty-nine patients were judo practitioners and three were wrestlers. Twenty-eight patients (30.4%) were asymptomatic carriers. In 64 patients, 51 patients (55.4%) with tinea corporis, 27 patients (29.3%) with tinea capitis, and/or one patient (1.1%) with tinea manuum were seen. Tinea corporis was observed on the forehead, auricles, nape of the neck, bilateral shoulders, left side of the upper chest, both elbows, back of the left hand to the wrist and both knees. Tinea capitis was most common in the occipitonuchal region at the hairline and in the temporal and frontal regions, at both auricles. Initial screening of these sites might facilitate the identification of the infection especially in judo practitioners.

The boosted antifungal topical treatment (BATT) for onychomycosis.

Despite promising pharmacodynamic and pharmacokinetic profiles, modern antifungals exhibit limited efficacy in the treatment of onychomycoses. The clinical and mycological data are far from reaching a complete cure even after long-course treatments extending over many months. After reviewing the histological presentation of onychomycoses, one of the main reasons for treatment failure and disease recurrence appears to be the presence of dormant chlamydospores and arthroconidia inside the infected nail plate. Accordingly, we present a novel concept in onychomycosis treatment. It consists of the combination of an antifungal with a device boosting germination of fungal propagules in order to render these cells more responsive to treatment. Preliminary results of a pilot study support such a concept.

Effect of glucose and thiamine concentrations on the formation of macroconidia in dermatophytes. Occurrence of dysgonic Microsporum canis strains in Athens, Greece.

Data collected from multiple trials with 110 fresh and preserved clinical isolates of Trichophyton mentagrophytes var. mentagrophytes, T. violaceum, T. rubrum, T. verrucosum, Microsporum canis and Epidermophyton floccosum revealed that production of macroconidia depends on glucose and thiamine concentrations in the medium. Optimal macroconidia production was obtained at the critical concentrations of 5 g l-1 glucose and 0.6 g l-1 thiamine when the two compounds were used in combination. The same conditions also encouraged macroconidia production in aconidial strains of T. verrucosum. Cutaneous inoculation in immunocompetent laboratory rabbits further enhanced the macroconidia producing capacity of the tested strains. Emphasis was placed on the occurrence of dysgonic/atypical strains of M. canis, which readily reverted to their typical phenotypes after growth on medium supplemented with 0.6 g l-1 thiamine, a process greatly augmented after cutaneous animal inoculation. It was verified that selective exogenous factors affect macroconidial production and that the dysgonic group of M. canis constitutes an epidemiologically significant group in the Greater Athens area. This is the first report of the occurrence of such M. canis strains from Greece.