Identification of Dermatophytes | Experiment

Abstract

Dermatophytosis constitutes a group of superficial fungal infections of keratinized structures like stratum corneum of the skin, nails and hair. Despite the availability of effective 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 treatment of rather common dermatophytosis with great precision.

To study the clinical variants, species of fungi causing dermatophytic infections, epidemiological factors responsible for the disease in central Mysore, molecular characterization and antifungal susceptibility tests by MIC.

The prospective observational analyses of clinically suspected 600 dermatophytic infected cases were studied at the Dermatology department of K.R. Hospital and C.S.I. Holdsworth Memorial Hospital, Mysore, India. The study was conducted from November 2010 to October 2012. All the cases were evaluated and patient’s data were recorded as per pre-designed proforma. Skin scraping, hair and nail samples were collected, 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.

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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 collected under the age group 21-30 years were 333 (55.5%), which was statistically significant (P<0.05). Among them, 231 (38.5%) were farmers and 153 (25.5%) were students who acquired highest number of infection. The next group was housewives 113 (18.8%) having third highest number of infection by dermatophytes. Most of the samples were collected from the patients of Mysore 172 (28.7%). Samples from domestic animals in the homes of infected patients were analyzed. Out of 15 domestic animals analyzed three were found to be infected by Microsporum 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 5^th-8^th week after infection, 391 (53.2%). During that period of infection, they were either taking home made medicine or over the counter medicine.

Most of the cases found predominantly were tinea 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 culture 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 both direct 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% sensitivity 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, restriction enzyme cutting patern was found to be infected by same T. verrucosum with 100% sensitivity and specificity. This indicates that T. verrucosum infection is from animal source.

The dermatophyte specific primer 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 used to develop 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, performing a direct PCR on the clinical speci­mens can augment the diagnosis of more dermatophytic cases.

PCR confirmed 132 dermatophytic samples were taken for antifungal sensitivity test. In the current study, among 132 isolates of dermatophytes some are sensitive and some are moving towards resistance, but high MIC value indicated that it has slowly acquired adaptation towards the drug. This indicates in near future it will develop 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 µg ml^-1. Second most frequently used drug next to fluconazole is ketoconazole, which had MIC₅₀ of 0.125 µg ml^-1 for most of the isolates. Griseofulvin, itraconazole and terbinafine showed similar results of 0.03-0.06 µg 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 not determined because of small number of samples i.e., <10 samples. The MIC ranges of fluconazole, itraconazole and ketoconazole for C. parapsilosis ATCC-22019 were within the value standardized by CLSI guidelines.

The study highlighted 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 human 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 than empirical treatment.

Need for the study

In the medical science dermatophytes are constant parasites of human beings, and has survived several 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 time 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, but the studies were mainly devoted 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 field 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 apparently disregard to the study of Medial Mycology

• Most of the fungal diseases are not fatal and hence do not attract much attention of the investigators.
• The development 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 except 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 dermatophytes

• Increasing incidence of immunocompromised diseases in and around the world.
• Changing patterns of dermatophytic infections with change in environment.
• Development of resistant species among the dermatophytes.
• Carrier harbours pathogenic 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 humid 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 study

1. To study the incidence of dermatophytic infections and their clinical variants
2. To identify the sources of infection by PCR for dermatophytes
3. To determine the in vitro antifungal susceptibility test

Slide culture

For 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 used. Lactophenol cotton blue was used as the mounting fluid for easy visualization of hyphae and spores (WHO, 1986).

Advantages

• It is arapid method of preparing fungal colonies for examination and identification.
• Permits fungi to be studied virtually in situ with as little 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 transfer it to the slide. So there wasless chances for the features that were key to identification, notably 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 medium (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 similarly 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 preserves 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).

Materials

• Lactic acid ……………………….. 20 ml
• Phenol (crystals………………….. 20 gm
• Glycerine….………………………. 40 ml
• Water (distilled)…………………… 20 ml

Mixed the above gently and added 50 mg the dye, cotton blue.

Procedure

• Placed 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 slip 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 penetration test

Many 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 dermatophytes:

Procedure

• Obtained a small amount of juvenile human hair and washed off fats and oils (kept hair in flask 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 razor 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 Stain (CFW)

It is a fluorescent stain for rapid detection 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).

Composition:

Calcofluor White M2R 1g/l and Evans blue 0.5g/l.

Directions:

• Put the sample to be examined onto a clean glass slide.
• Add one drop of Calcofluor White Stain and one drop of 10% Potassium Hydroxide
• Place 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 excitation (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 be 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.