Superficial

Superficial fungal infections of the skin, hair, and nails are incredibly common, making the proper collection and analysis of these specimens vital for accurate diagnosis and effective treatment. The ease of access to these sites makes them primary targets for initial investigation. This section will delve into the specific techniques for collecting skin scrapings, hair samples, and nail clippings, emphasizing the importance of site selection, sterile technique, and appropriate storage to ensure reliable and informative mycological analysis

Importance of Superficial Samples

• Prevalence: Superficial fungal infections are incredibly common. Think athlete’s foot, ringworm, and nail infections
• Diagnostic Value: Proper collection and processing of these samples are crucial for accurate diagnosis and guiding appropriate treatment. Misdiagnosis or delayed treatment can lead to chronic infections, spread to other body sites, or transmission to other individuals
• Accessibility: These specimens are generally easy to obtain, making them a primary target for initial investigation

Types of Specimens and Collection Techniques

Here’s a breakdown of each specimen type, along with the recommended collection methods:

Skin Scrapings

• Purpose: To detect dermatophytes (like Trichophyton, Microsporum, Epidermophyton) and Malassezia furfur (causing pityriasis versicolor)
• Collection
  • Site Selection: Clean the area with 70% alcohol to remove surface debris. Scrape from the active edge of the lesion (where the fungus is most likely to be thriving). Avoid the center, which might be healing
  • Tools: Use a sterile scalpel blade (size 15), the edge of a glass slide, or a curette
  • Technique: Gently scrape the skin, collecting scales and debris. Aim for a representative sample, not just a superficial dusting
  • Storage/Transport: Place the scrapings in a sterile, dry container (e.g., a Petri dish, a folded piece of black paper, or a small, screw-capped vial). Black paper is great because it provides contrast for visualizing the scales

Hair Samples

• Purpose: To diagnose tinea capitis (ringworm of the scalp) and other hair shaft infections
• Collection
  • Plucked Hairs: Ideal for detecting endothrix infections (fungus inside the hair shaft). Pluck several infected hairs including the root (where the fungus is concentrated). Use sterile forceps
  • Cut Hairs: Can be used if plucking is difficult or painful. Cut hairs close to the scalp
  • Scrapings: Collect scales from the scalp around the infected hairs
  • Wood’s Lamp Examination: Use a Wood’s lamp (UV light) to identify infected hairs that fluoresce (e.g., some Microsporum species). Collect only the fluorescent hairs
  • Storage/Transport: Place hair samples in a sterile, dry container

Nail Clippings and Scrapings

• Purpose: To diagnose onychomycosis (nail fungus)
• Collection
  • Cleaning: Clean the nail with 70% alcohol
  • Clippings: Clip the affected part of the nail, as far proximally as possible. Discard the very tip of the nail, as it’s often contaminated
  • Scrapings: Scrape debris from under the nail plate, especially from areas that appear discolored or thickened. A small curette or a nail file can be helpful
  • Subungual Debris: This material found under the nail is often the most productive source of fungi
  • Storage/Transport: Place nail clippings and scrapings in a sterile, dry container

Important Considerations

• Proper Labeling: Label all specimens clearly with patient information (name, date of birth, medical record number), the date and time of collection, and the specific body site
• Avoid Contamination: Use sterile equipment and avoid touching the inside of containers
• Quantity: Collect an adequate amount of material. A small, barely visible sample may not be sufficient for accurate diagnosis
• Timely Processing: Process specimens as soon as possible after collection. If there’s a delay, store them properly (usually at room temperature) and consult your lab’s specific protocols
• Communication with the Clinician: If the specimen seems inadequate or inappropriate, communicate with the requesting physician to determine the best course of action

Direct Microscopic Examination

• KOH Preparation
  • A drop of 10-20% potassium hydroxide (KOH) is added to the specimen on a slide
  • KOH dissolves keratin, making fungal elements (hyphae, spores) easier to see
  • Gentle heating may speed up the clearing process
  • Examine under a microscope for fungal elements
• Calcofluor White Stain
  • Calcofluor white is a fluorescent dye that binds to chitin in fungal cell walls
  • Specimens are stained with calcofluor white and examined under a fluorescent microscope
  • Fungal elements appear bright white or blue against a dark background
  • This stain is more sensitive than KOH preparation
• India Ink
  • Used to detect capsules around Cryptococcus neoformans in CSF samples
  • India ink particles are excluded from the capsule, creating a halo effect around the yeast cell

Culture

• Media: Sabouraud Dextrose Agar (SDA) is a common general-purpose fungal culture medium. SDA with antibiotics (e.g., chloramphenicol, gentamicin) can inhibit bacterial growth. Dermatophyte Test Medium (DTM) is used to presumptively identify dermatophytes based on color change
• Inoculation: Inoculate the specimen onto the appropriate media
• Incubation: Incubate cultures at appropriate temperature (typically 25-30°C) for up to 4 weeks, examining regularly for growth
• Identification: Identify fungal isolates based on macroscopic (colony morphology) and microscopic characteristics (e.g., hyphal structure, conidia)

Reporting

• Report all positive and negative results clearly and accurately
• Include the type of specimen, the methods used, and the identification of any fungi isolated
• Report any relevant information about the specimen quality or the presence of interfering substances

Key Takeaways

• Accurate diagnosis of superficial fungal infections relies on proper specimen collection and processing
• Select the appropriate collection technique based on the type of specimen and the suspected pathogen
• Use sterile equipment and avoid contamination
• Process specimens promptly and store them properly if there’s a delay
• Perform direct microscopic examination and culture to identify fungal elements
• Report all results clearly and accurately

Key Terms

• Dermatophytes: A group of fungi that infect the skin, hair, and nails because they can utilize keratin as a nutrient source. Common genera include Trichophyton, Microsporum, and Epidermophyton
• Hyphae: The thread-like, branching filaments that form the mycelium of a fungus. They are a key microscopic feature used in fungal identification
• Conidia: Asexual spores produced by fungi, often used in identification due to their varying shapes, sizes, and arrangements
• Keratin: A fibrous structural protein that is the main component of skin, hair, and nails. Dermatophytes produce enzymes (keratinases) to break down keratin
• KOH Preparation: A microscopic technique where a sample is mixed with potassium hydroxide (KOH) to dissolve keratin, making fungal elements (hyphae, spores) more visible
• Calcofluor White Stain: A fluorescent dye that binds to chitin in fungal cell walls, causing them to fluoresce under UV light, enhancing their visibility during microscopy
• Wood’s Lamp: A UV light used to examine skin and hair for fungal infections. Certain dermatophytes fluoresce under Wood’s lamp illumination, aiding in diagnosis
• Onychomycosis: A fungal infection of the nail, typically caused by dermatophytes, yeasts, or molds. It can result in thickened, discolored, and deformed nails
• Sabouraud Dextrose Agar (SDA): A common fungal culture medium containing peptones and a high concentration of glucose. It is often used to cultivate fungi from clinical specimens
• Subungual Debris: The material found under the nail plate, often a rich source of fungi in cases of onychomycosis. Scraping this debris is crucial for accurate diagnosis