Investigations for diagnosis of cutaneous fungal infections

1. Investigations For
Diagnosis
Of Cutaneous Fungal
Infections

2. Investigations for diagnosis
of cutaneous fungal infections
 Wood’s light examination
 Dermoscopic examination
 Biopsy and histopathological examination
 Direct microscopic examination
 Culture
 PCR examination

3. Wood’s Light

4. 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/

5. Wood’s light examination
 T. schoenleinii: pale green What this
fungus
cause?

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

7.  Fungi produce fluorescence:
 M.canis
 M.audouninii
 M.distortum all produce small spore
ectothrix
 M.ferrugineum
 M.gypseum
 T.schoenleinii

8. Dermoscopic Examination

9. 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.

10. 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.

15. KOH examination

16. 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

17. 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.

18. KOH

19. 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.

20. 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

21. 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

23. 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 microscop
e.

24. 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.

25. KOH test of candida

26. KOH test of T.V

27. KOH test:

28. KOH test

29. 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

30. Culture

31. 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.

32. 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.

34. Biopsy and histopathological
examination

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

36. Periodic acid-Schiff (PAS)
 Outline fungal elements magenta with green
background

37. Gomori methamine silver stain
(GMS)
 Outlines fungal elements black

38. Mucicarmine
 Stains capsule of
Cryptococcus
neoformans

40. PCR & other molecular
diagnostic methods

41. 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.

42. 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.

43. 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/

44. 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.

45. 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.

46. Conclusion

47. 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?

48. Conclusion
 The laboratory diagnostic approach will
involve:
1) wet mount KOH examination
2) culture for proper species identification

49. THANK YOU