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=== Laboratory investigations === | |||
For a laboratory to provide optimal results, quantity and quality of material examined is critical. Scraping should be collected from active margin and transported in a presterilized black chart paper which keeps the specimen dry thus, preventing over growth of bacteria contaminants. Following are the various laboratory tests that can be used for confirming a diagnosis of dermatophytosis. | |||
# Direct microscopic examination:[20] Treatment of skin specimen with 10–20% potassium hydroxide (KOH) is a quick and inexpensive bedside tool to provide evidence of dermatophytic infection. Positive scrapings are characterized by presence of refractile, long, smooth, undulating, branching, and septate hyphal filaments with or without arthroconidiospores. False negative results are seen in 15% cases. Fluorescent staining with optical brighteners (diaminostilbene) is the most sensitive method to microscopically detect fungi in skin scales as well as in specimens from nails and hair.[21] These substances bind to chitin, the main cell wall component of fungi | |||
# Culture and antifungal sensitivity:[22] Sabouraud dextrose agar (SDA, 4% peptone, 1% glucose, agar, water) is the most commonly used isolation media for dermatophytosis and serves as the medium on which most morphologic descriptions are based. Development of colony takes 7–14 days. Modified SDA, with addition of gentamicin, chloramphenicol and cycloheximide is more selective for dermatophytes as chroramphenicol inhibits the growth of saprophytic fungus. Dermatophyte test medium is an alternative to isolation media that contain pH indicator phenol red. It is incubated at room temperature for 5–14 days. Dermatophytes utilize the protein resulting in excess ammonium ion and alkaline environment which turn the medium from yellow to bright red. | |||
==== Antifungal susceptibility testing ==== | |||
# Microdilution method: The broth microdilution assay for antifungal susceptibility testing of dermatophytes has been previously developed as a modification of the Clinical and Laboratory Standards Institute M38-A2 standard method. The final concentrations of terbinafine and itraconazole used is 0.06–32.0 μg/ml and for fluconazole, 0.13–64.0 μg/ml.[23] A standardized inoculum is prepared by counting the microconidia microscopically. Cultures are grown on SDA slants for 7 days at 35°C to produce conidia. Sterile normal saline (85%) is added to the agar slant, and the cultures are gently swabbed with a cotton-tipped applicator to dislodge the conidia from the hyphal mat. The suspension is transferred to a sterile centrifuge tube, and the volume is adjusted to 5 ml with sterile normal saline. The resulting suspension is counted on a hemacytometer and is diluted in RPMI 1640 medium to the desired concentration. Microdilution plates are set up in accordance with the reference method. The microdilution plates are incubated at 35°C and read visually after 4 days of incubation. The minimum inhibitory concentration is defined as the concentration at which the growth of the organism will be inhibited by 80% compared with the growth in the control well | |||
# Minimum fungicidal concentration (MFC) determination: For determination of the MFC, 100-μl aliquots are removed from the assay wells showing no visible growth at the end of incubation and streaked onto SDA plates. The plates are incubated at 30°C for 7 days. The MFC is defined as the lowest drug concentration at which no visible fungal growth or colonies developed | |||
* 3. Dermatophyte identification: This can be based on colony characteristics, microscopic morphology, and physiologic tests. Dermatophytes can be distinguished based upon their morphology of the macroconidia. Few physiological tests are available which help in confirmation of certain species. In addition, special amino acid and vitamin requirements can differentiate ''Trichohyton'' species from others. Ability to hydrolyse urea differentiates ''T. mentagrophytes'' (urease positive) from ''T. rubrum'' (urease negative). | |||
=== Histopathology === | |||
Histology may be used in diagnosis of Majocchi's granuloma in which KOH examination of scale on the surface may more often be negative. When present, hyphae may be appreciated in stratum corneum on hematoxylin and eosin staining. Special stains most commonly used are periodic acid-Schiff and Gomori methanamine silver which helps to highlight hyphae. | |||
=== Dermoscopy === | |||
The comma hairs, which are slightly curved, fractured hair shafts, and corkscrew hair shave been described as the dermoscopic marker of tinea capitis. Broken and dystrophic hairs are also seen. However, in tinea corporis, the involvement of vellus hair as seen on dermoscopy is an indicator of systemic therapy.[24] | |||
=== Polymerase chain reaction and nucleic acid sequence based amplification === | |||
These tests not only help in the rapid and early diagnosis of infection but also help in determining drug resistance,[25] and include: | |||
* Uniplex PCR for direct dermatophyte detection in clinical samples: A PCR for the direct detection of dermatophytes in skin scales is available as in-house PCR-ELISA assay which separately identifies numerous dermatophyte species. In a pilot study, the sensitivity and specificity of the test compared to cultures was 80.1% and 80.6% | |||
* Multiplex PCR for fungal detection in dermatophytes: Commercially available multiplex PCR tests enable simultaneous amplification of 21 dermatomycotic pathogens with subsequent DNA detection by means of agarose gel electrophoresis. | |||
==References== | ==References== | ||
{{Reflist|2}} | {{Reflist|2}} | ||
Revision as of 20:20, 28 June 2017
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Laboratory investigations
For a laboratory to provide optimal results, quantity and quality of material examined is critical. Scraping should be collected from active margin and transported in a presterilized black chart paper which keeps the specimen dry thus, preventing over growth of bacteria contaminants. Following are the various laboratory tests that can be used for confirming a diagnosis of dermatophytosis.
- Direct microscopic examination:[20] Treatment of skin specimen with 10–20% potassium hydroxide (KOH) is a quick and inexpensive bedside tool to provide evidence of dermatophytic infection. Positive scrapings are characterized by presence of refractile, long, smooth, undulating, branching, and septate hyphal filaments with or without arthroconidiospores. False negative results are seen in 15% cases. Fluorescent staining with optical brighteners (diaminostilbene) is the most sensitive method to microscopically detect fungi in skin scales as well as in specimens from nails and hair.[21] These substances bind to chitin, the main cell wall component of fungi
- Culture and antifungal sensitivity:[22] Sabouraud dextrose agar (SDA, 4% peptone, 1% glucose, agar, water) is the most commonly used isolation media for dermatophytosis and serves as the medium on which most morphologic descriptions are based. Development of colony takes 7–14 days. Modified SDA, with addition of gentamicin, chloramphenicol and cycloheximide is more selective for dermatophytes as chroramphenicol inhibits the growth of saprophytic fungus. Dermatophyte test medium is an alternative to isolation media that contain pH indicator phenol red. It is incubated at room temperature for 5–14 days. Dermatophytes utilize the protein resulting in excess ammonium ion and alkaline environment which turn the medium from yellow to bright red.
Antifungal susceptibility testing
- Microdilution method: The broth microdilution assay for antifungal susceptibility testing of dermatophytes has been previously developed as a modification of the Clinical and Laboratory Standards Institute M38-A2 standard method. The final concentrations of terbinafine and itraconazole used is 0.06–32.0 μg/ml and for fluconazole, 0.13–64.0 μg/ml.[23] A standardized inoculum is prepared by counting the microconidia microscopically. Cultures are grown on SDA slants for 7 days at 35°C to produce conidia. Sterile normal saline (85%) is added to the agar slant, and the cultures are gently swabbed with a cotton-tipped applicator to dislodge the conidia from the hyphal mat. The suspension is transferred to a sterile centrifuge tube, and the volume is adjusted to 5 ml with sterile normal saline. The resulting suspension is counted on a hemacytometer and is diluted in RPMI 1640 medium to the desired concentration. Microdilution plates are set up in accordance with the reference method. The microdilution plates are incubated at 35°C and read visually after 4 days of incubation. The minimum inhibitory concentration is defined as the concentration at which the growth of the organism will be inhibited by 80% compared with the growth in the control well
- Minimum fungicidal concentration (MFC) determination: For determination of the MFC, 100-μl aliquots are removed from the assay wells showing no visible growth at the end of incubation and streaked onto SDA plates. The plates are incubated at 30°C for 7 days. The MFC is defined as the lowest drug concentration at which no visible fungal growth or colonies developed
- 3. Dermatophyte identification: This can be based on colony characteristics, microscopic morphology, and physiologic tests. Dermatophytes can be distinguished based upon their morphology of the macroconidia. Few physiological tests are available which help in confirmation of certain species. In addition, special amino acid and vitamin requirements can differentiate Trichohyton species from others. Ability to hydrolyse urea differentiates T. mentagrophytes (urease positive) from T. rubrum (urease negative).
Histopathology
Histology may be used in diagnosis of Majocchi's granuloma in which KOH examination of scale on the surface may more often be negative. When present, hyphae may be appreciated in stratum corneum on hematoxylin and eosin staining. Special stains most commonly used are periodic acid-Schiff and Gomori methanamine silver which helps to highlight hyphae.
Dermoscopy
The comma hairs, which are slightly curved, fractured hair shafts, and corkscrew hair shave been described as the dermoscopic marker of tinea capitis. Broken and dystrophic hairs are also seen. However, in tinea corporis, the involvement of vellus hair as seen on dermoscopy is an indicator of systemic therapy.[24]
Polymerase chain reaction and nucleic acid sequence based amplification
These tests not only help in the rapid and early diagnosis of infection but also help in determining drug resistance,[25] and include:
- Uniplex PCR for direct dermatophyte detection in clinical samples: A PCR for the direct detection of dermatophytes in skin scales is available as in-house PCR-ELISA assay which separately identifies numerous dermatophyte species. In a pilot study, the sensitivity and specificity of the test compared to cultures was 80.1% and 80.6%
- Multiplex PCR for fungal detection in dermatophytes: Commercially available multiplex PCR tests enable simultaneous amplification of 21 dermatomycotic pathogens with subsequent DNA detection by means of agarose gel electrophoresis.