Investigations For Diagnosis Of Cutaneous Fungal Infections

Investigations for diagnosis of cutaneous fungal infections

Wood’s light examination Dermoscopic examination Biopsy and histopathological

examination Direct microscopic examination Culture PCR examination

Wood’s Light

Wood’s light examination An ultraviolet light Wave length 365 nm It is a UVR filtered by Wood’s glass (consist of barium silicate

containing 9% Nickel oxide) Some of dermatophytes causing tinea capitis will induce

flouresence, those are: The fungus is capable of invading hair Infected hairs are living and growing (anagen hair) These fungi flourescent due to petridine produced as a

metabolite of the fungus However the most common fungi producing T.capitis don’t

fluorescein.

http://www.dermnetnz.org/topics/wood-lamp-skin-examination/

Wood’s light examination T. schoenleinii: pale green

What this fungus cause?

Wood’s light examination M.canis and M.audouni: brilliant green

Fungi produce fluorescence: Cats And Dogs Fight and Growl Sometimes M.canis M.audouninii M.distortum all produce small spore ectothrix M.ferrugineum M.gypseum

T.schoenleinii

Dermoscopic Examination

Dermoscopic features of cutaneous fungal infection

Using low magnification (×30): typical comma hairs, "Morse code-like" hairs, "zigzag" hairs When using high magnification (×150): horizontal white bands that appear as empty bands that

are likely related to localized areas of fungal infection. These horizontal white bands are usually multiple and may

cause the hair to bend and break. a new dermoscopic feature identified consist of

translucent, easily deformable hairs that look weakened and transparent and show unusual bends; they are likely the result of a massive fungal invasion involving the whole hair shaft.

Dermoscopic Examination The association of clinical and

dermatoscopic findings in suspected Tinea capitis cases may help with the differential diagnosis of the etiological agent, making feasible the precocious, specific treatment.

KOH examination

KOH examination value KOH examination can be performed rapidly

at the “bed-side” Done with or without staining (e.g. by

Parker’s blue black ink, chlorazole black) Quick and inexpensive Could differentiate dermatophytes or yeast

infection from other diagnoses as psoriasis and eczema.

It is useful in diagnosis, treatment and control of epidemics of T.capitis

KOH Specimens Scraping (to obtain scales): Scraping of

diseased scalp skin for fungal study is the recommended approach of the British Association of Dermatologists.

A scalpel or edge of a glass slide is used to gently scrape skin scales from the infected area.

Specimen collection by cytobrush or toothbrush are alternative methods of sample collection especially in the context of outbreak investigation.

KOH

KOH Specimens Nail scraping/clipping/subungual debris:

Subungual hyperkeratotic material should be collected with a curette in those cases of suspected onychomycosis. Sampling should also be collected as proximal as possible in those cases of clinical distal lateral subungual onychomycosis.

Simple nail clipping of the distal diseased nails may not give the maximum yield.

Repeated sampling is sometimes required to isolate the causative fungi.

KOH Specimens Hair: Diseased hairs should be plucked

(not cut) in those cases of suspected tinea capitis. A forceps is used to remove hair shafts and follicles from the infected site.

Swabs from moist areas

Scotch tape or cellotape KOH technique

KOH procedure Collection: Skin, nail, or hair samples are collected from the

infected area on the patient. If the test is being sent to a laboratory, the scrapings are placed in a sterile covered container (folded slips of paper)

The scrapings are placed directly onto a microscope slide and are covered with 1-2 drops of 10% or 20% potassium hydroxide.

The slide is left to stand until clear, normally between five and fifteen minutes, in order to dissolve skin cells, hair, and debris.

To enhance clearing dimethyl sulfoxide (DMSO) can be added to the slide. To make the fungi easier to see. Mainly used to dissolve nails.

The slide is gently heated to speed up the action of the KOH. Place the slide under a microscope to read

KOH procedure Calcofluor-white is a

special fluorescent stain that binds to the chitin in the cell walls of the fungi.

Adding calcofluor-white stain to the slide will cause the fungi to become fluorescent, making them easier to identify under a fluorescent microscope.

KOH evaluation Dermatophytes are easily recognized under the

microscope by their long branch-like tubular structures (hyphae).

Fungi causing ringworm infections produce septate (segmented) hyphae. Some show the presence of spores formed directly from the hyphae (arthroconidia).

Under the microscope Tinea versicolor is recognized by curved hyphae and round yeast forms that give it a spaghetti-and-meatball appearance.

Yeast cells appear round or oval and budding forms may be seen.

KOH test of candida

KOH test of T.V

KOH test:

KOH test

KOH test limitations The KOH prep cannot identify the

specific organism; the specimen can be submitted for fungal culture to identify the organism.

Crystals , oil droplets, threads can produce artifacts

Culture

Culture On Sabouraud’s agar medium Incubated at room temperature For 21 days: Fast growers e.g, candida, M.canis: 2-3 days Slow growers e.g, T.violaceum, T.schoneleinii: 14

days T.rubrum:21 days Macroscopic examination of colonies that grow on

culture media and microscopic examination of slide mounts from culture often allow the identification of fungus.

Culture For optimal recovery of fungal pathogen, the followings are added: Cycloheximide is added to inhibit the

growth of rapidly growing contaminating molds.

An antibacterial agent (Chloramphenicol) is commonly added to control bacterial contamination.

Biopsy and histopathological examination

Biopsy Could be stained with: Periodic acid-Schiff (PAS) Fontana-Masson stain Gomori methamine silver stain (GMS) Mucicarmine

Periodic acid-Schiff (PAS) Outline fungal elements magenta with

green background

Gomori methamine silver stain (GMS)

Outlines fungal elements black

Mucicarmine Stains capsule of

Cryptococcus neoformans

PCR & other molecular diagnostic methods

PCR need The incidence of human infections caused by fungi

has increased dramatically over the past two to three decades, owing to an enlarging immunocompromised patient population.

Recent epidemiology data indicate that fungal infection has become one of the most common infection-associated mortalities in the USA.

The laboratory diagnosis of fungal infections is still largely dependent on microscopic and culture-based methods, and these methods do not always meet clinical needs owing to poor sensitivity and lengthy incubation time. 

PCR advantages Molecular tests can improve the diagnosis of

fungal infections The development of nucleic acid amplification

and detection platforms offers ultrasensitive and rapid detection of fungal pathogens directly in clinical samples

Also for the identification of antifungal drug resistance directly in blood, bronchoalveolar lavage fluid, cerebrospinal fluid, and formalin-fixed and paraffin-embedded tissues.

PCR current use PCR assays could be used for the

diagnosis of invasive aspergillosis, candidiasis and Pneumocystis pneumonia.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845394/

PCR limitations Despite almost two decades of

experience and a recent rapid growth of using molecular assays for fungal detection, they are still not included in EORTC/MSG criteria to define fungal disease, and none of them have been implemented by clinical laboratories for clinical use, primarily due to a lack of method standardization and adequate clinical outcome data.

PCR future With new emerging technologies, commercially developed molecular

assays will drive the development and implementation of molecular tests for early and rapid diagnosis of invasive fungal infections. A sensitive and multiplex PCR platform capable of reliably detecting multiple fungi simultaneously in a single reaction will represent an attractive platform to enhance fungal detection.

More data on clinical outcome and cost–effectiveness are needed to understand the clinical utility of the molecular tests.

Owing to the ubiquitous nature of fungi and ultrasensitivity of molecular assays, environmental contamination is a big challenge in employing these molecular assays for patient care testing. Harrison et al. found up to 18% of blood collection tubes were contaminated with fungal DNA.

It is therefore paramount to implement negative controls to rigorously monitor each step during the molecular testing that could cause false positives due to environmental contamination, and to avoid bringing any negative impact on patient care.

Conclusion

Conclusion Clinical diagnosis is usually good enough for

the routine management of patients. The laboratory approach to these cutaneous

conditions may involve answering the following 3 questions:

1) what is the purpose of performing the laboratory tests under consideration? 2) which is the most appropriate laboratory test? 3) how to interpret the laboratory results in the concerned clinical context?

Conclusion The laboratory diagnostic approach will

involve:1) wet mount KOH examination 2) culture for proper species identification

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