Identification of Dermatophytes | Experiment

Identification of Derma acmehytes ExperimentAbstractDermatophytosis constitutes a group of superficial fungal contagions of keratinized structures like stratum corneum of the skin, digs and hair. Despite the availability of utile antifungal agents, dermatophytic infections continue to be one of the principal infections throughout the world, especially in tropical countries like India. Identification of causative dermatophytic species in clinical settings are not only of epidemiological concerns but also important for the interference of rather common dermatomycosis with great precision.To study the clinical variants, species of fungus kingdom create dermatophytic infections, epidemiological factors responsible for the disease in central Mysore, molecular characterization and antifungal expertness tests by MIC.The prospective observational analyses of clinically suspected 600 dermatophytic give cases were studied at the Dermatology department of K.R. Hospital and C.S.I. H gag asworth Memorial Hospital, Mysore, India. The study was conducted from November 2010 to October 2012. All the cases were evaluated and patients data were recorded as per pre-designed proforma. Skin scraping, hair and nail samples were amass, processed, molecular characterization and antifungal susceptibility test were done according to standard mycological protocol. Patients on topical and systemic antifungal treatment were excluded from the present study.clinically suspected 600 patients samples were collected, among them 389 (64.8%) were skin scraping, 113 (18.8%) were hair plucking and 98 (16.3%) were nail clipping. They were processed for isolation and identification of dermatophytes from different clinical types. The specific and non specific variables effecting dermatophytosis were also analyzed.Samples were collected throughout the year, of which samples collected from July to October showed highest incidence of dermatophyte infection among patients. The number of specimen c ollected under the age group 21-30 years were 333 (55.5%), which was statistically significant (PMicrosporum genus Canis and 12 were infected by Trichophyton verrucosum.Among 600 clinically suspected dermatophytes infected patients, 433 (72.2%) samples were collected from K.R. Hospital and 167 (27.8%) samples were collected from C.S.I. Holdsworth Memorial Hospital, Mysore. The various factors which were found to increase chances of acquiring dermatophytosis were by sharing bed, 343 (57.2%) not bathing daily, 362 (60.3%) and domestic animals in their house, 102 (17.0%). Partial treatment and over the counter medication was also considered as major factors influencing dermatophytic infection among patients. Less number of patients 86 (14.3%) showed family history of dermatophytosis. Most of the patients visited to the Doctor in 5th-8th week after infection, 391 (53.2%). During that period of infection, they were either taking home made music or over the counter medicine.Most of the c ases found predominantly were roundworm corporis and tinea capitis. Tinea corporis 282 (47.0%) and tinea capitis 75 (12.5%) were seen among 26-30 year old patients and 6-10 year old patients respectively. Most of the isolates belonged to three genera and eight species, where T. mentagrophytes was predominantly isolated 105 (29.7%), T. rubrum 84 (23.8%) was isolated from tinea corporis cases, T. violacium 4 (12.2%), T. verrucosum 54 (15.3%), Epidermophyton floccosum 11 (3.1%), M. canis 13 (3.6%), T. tonsurans 38 (10.8%) and T. schoenleinii 5 (1.4%) were also isolated from different cases of tinea.Dermatophytes infected horticulture positive, 132 patient samples (skin scrapings, hair plucking and nail clipping), were obtained for molecular characterization in the study. Nearly 87 males and 45 females were referred by Department of Dermatology, K.R. Hospital and C.S.I. Holdsworth Memorial Hospital, Mysore during two years of study period. The collected samples were analyzed by two d irect microscopic examination and culture assays. Of the 132 culture positive cases, the organisms identified by phenotypic characterization were belonging to three genera and eight species viz., T. mentagrophytes 52 (39.4%), T. rubrum 30 (22.7%), T. violacium 18 (13.6%), T. verrucosum 11 (8.3%), E. floccosum 10 (7.6%), M. canis 6 (4.5%), T. tonsurans 3 (2.3%) and T. schoenleinii 2 (1.5%). The culture isolates of these organisms were further characterized by PCR. The present study showed positive result for phenotypically analyzed culture positive with 100% aesthesia and specificity by PCR.PCR targeting internal transcribed spacer (ITS) and Chitin synthase 1 was sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA respectively. From the same source (houses) 10 isolates from patients and 10 domestic animals were subjected to both PCR and RFLP. The product after agarosegel electrophoresis, breastwork enzyme burn upting patern was found to be infected by same T. verruc osum with 100% sensitivity and specificity. This indicates that T. verrucosum infection is from animal source.The dermatophyte specific terra firma based PCR-RFLP which targets the internal transcribed spacer and chitin synthase 1 region are useful in the direct identification of dermatophytosis from clinical specimens. The application of the Hae III, Hinf I and Mva I restriction enzymes by using the ITS amplicons and chitin synthase 1 region are constant, stable and reproducible show variation within the species, so these sequences have been widely use to pause rapid procedures for the identification of fungal species by PCR-RFLP analysis. The PCR-RFLP method, on using the dermatophyte specific primer with restriction enzymes Mva I, Hae III and Hinf I, showed species differentiation among the T. rubrum and T. mentagrophytes isolates. Since direct microscopy and culture have limitations, execute a direct PCR on the clinical specimens can augment the diagnosis of more dermatophy tic cases.PCR confirmed 132 dermatophytic samples were taken for antifungal sensitivity test. In the current study, among 132 isolates of dermatophytes or so are sensitive and some are moving towards resistance, but high MIC value indicated that it has slowly acquired adaptation towards the medicine. This indicates in near future it will contrive drug resistance against the antifungal agents. Twenty three isolates (14.4%) were showing high MIC value for fluconazole of which T. mentagrophytes (8), T. rubrum (5) and T. verrucosum (10) and M. canis (3) had MIC50 of 16 g ml-1. Second most frequently utilise drug next to fluconazole is ketoconazole, which had MIC50 of 0.125 g ml-1 for most of the isolates. Griseofulvin, itraconazole and terbinafine showed similar results of 0.03-0.06 g ml-1.The present study showed drug concentration inhibition of some fungi against 50% of isolates and 90% of isolates and their geometric mean of five drugs against 132 isolates. MIC50 and MIC90 were n ot determined because of small number of samples i.e., C. parapsilosis ATCC-22019 were within the value standardized by CLSI guidelines.The study high uncloudeded tinea corporis as the most common clinical pattern of dermatophytosis followed by tinea capitis in Mysore. The predominant causative fungal species isolated were T. mentagrophytes, T. rubrum, T. violacium, T. verrucosum, E. floccosum, M. canis, T. tonsurans and T. schoenleinii confirmed by PCR with 100% sensitivity and specificity. Trichophyton verrucosum was isolated and confirmed by PCR and RFLP from world and their domestic animals (from animals to human). PCR-RFLP provides the best technique for dermatophytic identification at species level. MIC was done to see susceptibility and resistance pattern for clinician as well as for lab workers where terbinafine was 100% sensitive drug against dermatophytes. Clinical evaluation and fungal species identification are mandatory for proper management of dermatophytosis rather t han empirical treatment.Need for the studyIn the medical science dermatophytes are constant parasites of human beings, and has survived some(prenominal) generations of therapeutic regimens, ranging from single dose antifungal to several dose drugs like griseofulvin, tolnaftate and early imidazole. There are some dermatophytes which were abundant once upon a period but now it is rare, it may be due to geographical restrictions, indicate that economy, geography, type of the species and environmental factors also interferes with the dermatophytic survival and distribution. It is equally important to review anthropophilic dermatophyte as well as zoophilic dermatophytes, which may increase chances of infection in the near future, as immunocompromised diseases are increasing day by day.The dermatophytes are oldest parasites of human and animals. Good and specified knowledge about these agents are still far from human understanding. The medical mycologists have done considerable work, bu t the studies were mainly utilize to the reactions of the host rather than the study of the parasites. The rates of publication on dermatophytes are limited as compared to the extensive work being done in the study of bacteriology and virology. As we know, these dermatophytic infections are not usually life threatening, but offer an interesting approach to the variety of fundamental problems in human, animals and fungal biology.The obvious reasons manifestly disregard to the study of Medial MycologyMost of the fungal diseases are not fatal and hence do not attract much attention of the investigators.The phylogeny of the infection is very slow and persists for years without any serious damage to the host.Carrier among human and animals is very common and survives for decades without harming the host.Methods of isolation and identification are relatively very complicated and require long time to obtain positive results.Most of them are sensitive to the limited antifungal drugs ex cept few and drug resistance is very slow and limited.Sources of infection are mostly human, animals and soil.Factors that attract to do research on dermatophytesIncreasing incidence of immunocompromised diseases in and somewhat the world.Changing patterns of dermatophytic infections with change in environment.Development of resistant species among the dermatophytes.Carrier harbours morbific species for many years to produce disease under favourable condition.Formulate treatment of choice according to the new changing pattern of drug resistant dermatophytes.Fungal infections of skin, hair and nail are common in hot and wet climate. Humans acquire various dermatophytic infections due to poor health hygienic conditions. The present investigation was aimed to study molecular level identification of tinea by PCR and its antifungal susceptibility test.Objectives of the studyTo study the incidence of dermatophytic infections and their clinical variantsTo identify the sources of infecti on by PCR for dermatophytesTo determine the in vitro antifungal susceptibility test lantern sliding board cultureFor visualization of the normal colonial characteristics, arrangement and morphology of spores and mycelial appendages, slide culture was done. SDA media without antibiotics or cornmeal agar with 1% glucose was utilise. Lactophenol cotton blue was used as the mounting fluid for easy visualization of hyphae and spores (WHO, 1986).AdvantagesIt is arapid method of preparing fungal colonies for examination and identification.Permits fungi to be studied virtually in situ with as elfin disturbance as possible fungi were identified mostly by close examination of its morphology and the characteristics it possess.We were growing the fungi directly on the slide on a thin film of agar. By doing this, there was no need to remove a portion of the fungus from a culture plate and transit it to the slide. So there wasless chances for the features that were key to identification, no tably the spore-bearing structures, without any damage.A modified method adapted from Roberts et al. (2003) and described here fulfills those requirements. Sterile 1.5% water agar (7 to 8 ml) was poured into sterile 60-mm plastic petri plates and allowed to solidify. A sterile 22-mm2 cover glass was centered on the agar. The desired nutrient agar moderate (10 ml) was poured into a second 60-mm petri dish, allowed to solidify, and cut with a sterile stainless steel spatula into blocks approximately 5 to 8 mm2. One block was aseptically removed and placed on the cover glass. Inoculation of the agar block on one or more sides with fungal hyphae or conidia was followed by placement of a second sterile cover glass on top of it. After the petridish lid was replaced, the completed modified slide culture was incubated at the desired temperature until adequate growth and conidiogenesis had occurred. Each cover glass was used to prepare a semi permanent mount on a standard microscope slide 3 by 1 inch (7.62 by 2.54 cm). The top cover glass was lifted off with forceps and wetted on the specimen side with a drop of ethanol (70 to 90%). One drop of fungus mounting medium (e.g., lactophenol cotton blue) was applied to the specimen, and the cover glass was lowered gently onto the slide, specimen side down. The bottom cover glass was lifted from the water agar and as well mounted on a second slide. The nutrient agar block adhered to one of the cover glasses during the cover glass mounting procedure and was carefully lifted off with a sterile dissecting needle before the application of alcohol and mounting medium. Heat fixation of the cover glasses before mounting as described by McGinnis may improve the stability of the conidium-bearing structures (James, 2011).Lactophenol Cotton Blue (LPCB)The Lactophenol Cotton Blue wet mount preparation was the most widely used method of staining and observing fungi and was simple to prepare. The preparation has three components phenol, which will kill any live organisms lactic acid which keep fungal structures, and cotton blue which stains the chitin in the fungal cell walls. It was used constantly as a mounting fluid when examining culture of fungi microscopically (Leck, 2012).MaterialsLactic acid .. 20 mlPhenol (crystals.. 20 gmGlycerine.. 40 mlWater (distilled) 20 mlMixed the supra gently and added 50 mg the dye,cotton blue.ProcedurePlaced a drop of 70% alcohol on a microscope slide.Immersed the specimen / material in the drop of alcohol.Added one or at most two drops of the lactophenol cotton blue mount and stained before the alcohol dries out.Holding the cover curve between forefinger and thumb, touched one edge of the drop of mount with the cover slip edge, and lowered gently, avoiding air bubbles. The preparation was ready for examination.Hair discernment testMany isolates of T. rubrum and T. mentagrophytes are difficult to distinguish between on the basis of colony morphology and microscopic appearance . This in vitro test is the best method to distinguish between these two dermatophytesProcedureObtained a small amount of juvenile human hair and washed off fats and oils (kept hair in flaskful or beaker with 50 to 100 ml of ether, acetone or alcohol, shacked for 2 to 5 minutes and pour off the liquid).With scissors, a scalpel or a shave blade cut the hair into segments approximately 1 cm long.Put the hair in a glass petri plates, place on the lid and autoclaved for 10 to 15 minutes. This maintained sterility in the petri plates and can be used for 10 years.Calcofluor White malignment (CFW)It is a fluorescent stain for rapid spying of yeasts, fungi and parasitic organisms. Calcofluor White stain is a non-specific fluorochrome that binds to cellucose and chitin in cell walls (Chattaway and Barlow, 1954 Green et al., 1983 Hageage and Harrington, 2005).CompositionCalcofluor White M2R 1g/l and Evans blue 0.5g/l.DirectionsPut the sample to be examined onto a clean glass slide.Add one drop of Calcofluor White Stain and one drop of 10% Potassium HydroxidePlace a cover slip over the specimen and let stand for 1 minute.Examine the slide under UV light at 100X to 400X magnification.Calcofluor White Stain was a non-specific fluorochrome that binds with cellulose and chitin contained in the cell walls of fungi and other organisms. The staining procedure with Calcofluor White Stain is a rapid method for the detection of many yeasts and pathogenic fungi like Microsporidium, Acanthamoeba, Pneumocystis, Naegleria, and Balamuthia species. Evans blue present in the stain act as a counter stain and diminishes background fluorescence of tissues and cells when using blue light temper (not UV). A range of 300 to 440 nm (Emmax 433nm 0.1 M phosphate pH 7.0 cellulose) can be taken for emission wave length and the excitation occurs around 355nm. Fungal or parasitic organisms appear fluorescent bright green to blue, while other material is reddish-orange fluorescent. Attentions to b e taken as cotton fibers will fluoresce strongly as well amoebic cysts are fluorescent. One drop of 10% potassium hydroxide solution can be added for better visualization of fungal elements.