a) Acquired resistance
The efficient and protective response against dermatophytosis is a cell-mediated response of the DTH, characterized namely by the action of macrophages as effector cells, interferon-α secretion from type 1 T-helper lymphocytes and by some key cytokines like interferon-γ (IFN-γ). Immune detection and chemotaxis occur via low-molecular weight chemotactic factors or alternative complement pathway activation. However, the immune response that is raised, and especially the degree of inflammation, varies according to the dermatophyte species, the host species and the pathophysiological status of the host.26
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In general, the zoophilic species cause more inflammatory infections which may heal spontaneously and result in relative resistance to reinfection. The anthropophilic species usually cause more chronic, less circumscribed infections which result in less resistance to reinfection.26
b) Hypersensitivity (“Trichophytin” Reaction)
The “trichophytin” reaction is the term used for cutaneous hypersensitivity to dermatophyte antigens injected intradermally in humans. Both immediate- and delayed-type reactions occur, but the latter is most often associated with infection.26
Trichophyton species can be isolated from patients with deep-seated trichophytosis in a liquid medium consisting of beef extract, peptone, and maltose. After 2 to 3 months at room temperature, the growth is ground and filtered.26
In patients with deep-seated trichophytosis, parenteral injection of “trichophytin” caused signs and symptoms analogous to those induced in tuberculous patients by injection of tuberculin: general toxic reactions including elevated temperature, perspiration, loss of appetite, headache, and pain in the joints. There was inflammation, formation of pustules, and burning at the injection site.26
Dermatophytid reactions (4–5% of patients) are inflammatory eczematous allergic skin reactions at sites distant from primary fungal infection. Being KOH and culture negative, it is associated with a DTH response to trichophytin test and may involve a local DTH response to systemically absorbedfungal antigen.26
Antibody formation does not seem to be protective. The dermatophyteantigen is thought to be processed by epidermal Langerhans cells and presented in local lymph nodes to T lymphocytes which proliferate, migrate to the infected site, and produce inflammation. The epidermal barrier becomes permeable to transferring and migrating cells leading to spontaneous resolution of lesions. Trichophytin skin test is now positive and clearing of second infection will be more rapid. Rivalier showed that a dermatophytic infection in humans results in a relative resistance to subsequent infection called‘le phenomene de la reaction acceleree’or‘le phenomene de Bruno Bloch’, mainly by the inflammatory forms (kerion), caused by zoophilic species, but not always follow the more chronic anthropophilic infections. Fungi which do not invade the hair follicle do not seem to give rise to an equivalent immunity when growing in the horny layer of the smooth skin. In contrast, a study could not demonstrate such acquired immunity in experimentalT. rubruminfection of smooth skin.26
d) Non-Specific Resistance
Natural defenses against dermatophytes depend on immunological and nonimmunological mechanisms. Many nonspecific factors may account for natural resistance to infection. It is mainly related to the “serum factor,” a fungistatic substance in serum of normal individuals and animals. This factor is believed to limit the growth of the dermatophytes to the keratinized layers, i.e., prevent their invasion of living tissues.26
Host factors that help limiting the infection to keratinized tissue include their preference for cooler skin temperatures than the normal body temperature, serum inhibitory factors(beta-globulins, ferritin and other metal chelators) binding to iron essential for growth of dermatophytes. Unsaturated transferrin inhibits the growth of dermatophytes by binding to the hyphae. A growth modifying, α2 macroglobulin keratin inhibitor, has also been identified in serum. The natural resistance of scalp to
1. Direct Microscopic Examination
Direct microscopy provides an early and reasonably reliable method of diagnosing or excluding fungal infections.
Potassium Hydroxide (KOH) preparation
Direct mounts are made by mixing a small portion of the material in 2-3 drops of 10% KOH on a microscope slide. A cover slip is placed over KOH specimen and the slide is gently heated. The slide is allowed to cool and ‘ripen’ for few minutes before examination. The KOH ‘cleans’ the specimen by digesting proteinaceous debris, bleaching pigments and loosening sclerotic material without damaging fungus, making hyphal forms easier to see. The slides are examined under bright field microscope with low condenser, first under 10x and then under 40x. The hyphae stand out as highly refractile long undulating branched septate threads. At times these hyphae fragment into rounded or barrel shaped arthrospores. The arthrospores are outside the hair shaft in chains in a mosaic pattern or intrapilar depending on the species involved and whether it is endothrix or ectothrix. 20% KOH are used for nail samples. In case the nails do not soften satisfactorily, the slide may be kept in an incubator at 370C for 1 hour. Hair should be examined as soon as possible after mounting.
Some modifications of KOH preparation
- Addition of 5% glycerin to 25% KOH or NaOH prevents desiccation.
- 20% KOH dissolved in 40% DMSO helps in rapid penetration and maceration of tissue without resorting to heating.36
- Addition of Parker Superchrome Blue-black ink to KOH solution selectively colors the hyphae making them more prominent.
- Sodium sulphide may also be used as a clearing agent.
- Eosin 1% may be added to KOH to stain the keratin. It lends a pinkish background while fungal elements remain unstained.37
- Modified Parker’s ink and 1% Eosin method: Eosin 1% is added to Parker’s ink in 2:1 proportion. The mixture is painted over the affected site and allowed to dry. Apply cellophane tape, gently press, remove it, stick over glass slide and observe under microscope. Background stains pink and fungal elements stain blue.
2. Calcofluor white stain
Calcofluor white is a fluorescent brightener which selectively binds to chitin and cellulose in the fungal cell wall. It fluoresces light blue color when exposed to ultraviolet light (346-365nm).
3. Acridine Orange38
A drop of 0.01% acridine orange may be added to KOH and observed under fluorescent microscope.
4. PAS (Periodic Acid Schiff)38
Nail clipping stained with PAS is more rewarding as compared to KOH wet mount. The polysaccharides of fungi are oxidized by periodic acid to form aldehyde groups that yield magenta coloured compound with Schiff’s fuchsin sulfide.
5. Gomori Methenamine Silver Stain38
This stain works on the principle of liberation of aldehyde groups and their subsequent identification by reduced silver method. The aldehyde reduces methenamine silver nitrate complex resulting in brown black staining fungal cell wall due to deposition of reduced silver wherever aldehydes are located.
The most common media used for the isolation of dermatophytes is Sabourauds Dextrose agar with chloramphenicol and cycloheximide to inhibit bacterial and saprobic fungal contamination, incubated at three temperatures i.e., 250 C, 300C and 370C
Dermatophyte test medium (DTM) is used for the presumptive identification of dermatophytes. On incubation at 250C, the dermatophyte test media turns red due to change in color of the indicator phenol red by increased pH through their metabolic activity while most fungi do not.38 Potato flakes agar amended with cycloheximide and chloramphenicol is available as Rapid Sporulating Medium to promote rapid conidiation and colony pigmentation.39
SDA with 1% thiamine can be used for sporulation. The media should be inoculated and kept at room temperature for minimum of weeks. Sporulation usually occurs in 7 – 10 days. Some stains like T.verrucosum may take longer and some stains of T.tonsurans grow better when incubated at 370 C.
Identification is based on
- Colony characteristics in pure culture on SDA
- Microscopic morphology
1. Colony characteristics39
In observing gross colony morphology, note the color of the surface and the reverse of the colony, the texture of the surface (powdery, granular, wooly, cottony, velvety or glabrous) the topography (elevation, folding, margins, etc.,) and the rate of growth.
2. Microscopic morphology
The appearance and arrangement of the conidia and other structures may be determined by tease mounts or slide culture preparation mounted on lactophenol cotton blue. Sometimes special media like corn meal agar, potato glucose agar, lactrimel agar, rapid sporulation medium may be required to stimulate sporulation.
a. Tease mount (Lactophenol Cotton Blue)
For preparing a mount, a portion of fungal fragment is removed with a spud and is teased on a glass slide in a drop of LCB stain using 2 teasing needles. A cover slip is placed and examined under the microscope.
b. Slide culture
Microscopic structures are beautifully preserved for study in fine details. A microscopic slide is placed on a bent glass rod at the bottom of a petri dish along with 1-2 cover slips and a filter paper. Petri dishes are closed with their lid, wrapped with craft paper and sterilized using hot air oven. Block of 1x2cm of Sabourauds agar poured into petri dishes up to a depth of 4mm is cut using sterile scalpel blade. The block is transferred to the surface of the glass slide. The agar block is inoculated at four sides using the fungal strain to be identified. The inoculated block is covered with sterile cover slip and incubated at 250 C. A little sterile distilled water is added on the filter paper to avoid drying of agar. When growth appears, a drop of LCB is placed on a slide and cover slip from block is placed over it. Likewise drop of stain is placed on glass slide of the slide culture after removing agar block; fresh cover slip is applied over it and is examined under the microscope.38,40
c. Scotch Tape Technique
A 4 cm strip of scotch tape No. 800 is looped back on itself with the adhesive side out and held between the thumb and index finger. The adhesive side is pressed firmly to the surface of the fungal colony. It is gently pulled and is placed in a small drop of LCB on a microscopic slide.
1. In vitro hair perforation test
This is performed to differentiate between T.mentagrophytes and T.rubrum as well as M.canis and M.equinum. This test is taken positive when dermatophyte species show wedge shaped perforation in hair. It is positive in T.mentagrophytes and M.canis.
2. Urease Test
Is done on Christensen’s medium. T.mentagrophytes hydrolyze urea thereby turns medium red while T.rubrum shows negative result.
3. Special nutritional requirements
The method employs a casamino acids basal medium i.e., vitamin free [Trichophyton agar, (T1)] and to which various vitamins are added i.e., inositol (T2), thiamine + inositol (T3), thiamine (T4), and nicotinic acid (T5). In addition, the series includes an ammonium nitrate basal medium (T6) to which histidine is added (T7). After inoculation, incubated at room temperature or 370C (if T.verrucosum is suspected) and read after 7 and 14 days. The amount of growth is graded from 0-4+.
4. Growth on Polished Rice Grains
This is a useful test for differentiating M.audouinii from M.canis and from other dermatophytes that typically grow and sporulate on rice grains.
5. Temperature tolerance and temperance enhancement
Used for distinguishing T.mentagrophytes complex from T.terrestre, T.mentagrophytes from M.persicolor and T.verrucosum from T.schoenleinii. At 370 C, members of T.mentagrophytes complex show good result whereas T.terrestre does not grow and M.persicolor generally grows poorly or not at all; growth of T.verrucosum and T.soudanense is enhanced but that of T.schoenleinii and M.ferruginum is not.
6. Hair bait technique
This technique is used for the isolation of geophilic species like M.gypseum from soil.
- Skin tests with dermatophyte antigen trichophytin are used for the diagnosis of dermatophytosis.
- Trichophytin is a crude extract from dermatophytes producing positive delayed type hypersensitivity in most of the adults.
- The patients without delayed type reaction are more susceptible to chronic dermatophytosis.
Various serological tests like immunodiffusion are done to establish the diagnosis of dermatophytosis.
It is done on guinea pigs. M.canis, M.gypseum and T.mentagrophytes may be established more readily in laboratory animals as compared to other species. It is done for studying nature of lesions and immunity produced by the organism.
Molecular Identification Techniques
Methods used are largely based on arbitrarily primed PCR or restriction fragment length polymorphism generated from PCR products. An oligonucleotide probe for T.rubrum has been developed. Nested PCR targeting Chitin Synthase I (CHSI) gene in skin and hair specimen of patients clinically suspected with dermatophytosis is used.41
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