@David lufafa

Lab Diagnosis and Treatment of

mycoses

Wasswa Fredrickson B
2021/PhD/047/PS

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 LEARNING OBJECTIVES

• Laboratory diagnosis of fungal diseases (Myco

ses); Direct, isolation, Biochemicals, serology,
staining, cutenous delayed hypersenstivity te
sts and Molecular Techniques.
• Chemotherapy; commonly used drugs; azoles.
Polynes e.t.c.

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 Laboratory diagnosis of mycoses
Specimen collection: specimen collection depends on the
site affected. Different specimens include hair, skin scra
pings, nail clippings, sputum, blood, CSF, urine, cornea
l scraping, discharge or pus from lesions and biopsy.
• All specimens must be transported to the laboratory wi
thout any delay to prevent bacterial overgrowth.
• In case of delay specimens except skin specimen, blood
and CSF may be refrigerated for a short period.
• Infected hairs may be plucked using forceps. Those hair
s that fluoresce under Wood’s lamp may be selectively
plucked. Hairs may be collected in sterilized paper envel
opes.
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 Laboratory diagnosis of mycoses
• Surface of the skin must be disinfected with 70% alcohol before speci
men collection. The advancing edge of the lesion is scraped with the
help of a blunt forceps /scarpel and collected in sterilized paper envel
opes.

• Discoloured or hyperkeratotic areas of nail may be scraped. For disea

sed nail clipping may be collected in sterilized paper envelopes.

• Specimens from mucus membranes (oral) must be collected by g

entle scraping and transported to laboratory in sterile tube containi
ng saline.

• Swabs may be collected from vagina (High vaginal swabs or intra-vagi

nal swabs). 4
 Laboratory diagnosis of mycoses
• Corneal scrapings may be collected using a fine nee
dle and inoculated at bedside.
• Pus may be collected by aspiration; use of cotton sw
abs may give false positive microscopic results.
• Clean catch urine may be collected in a sterile wide-
mouthed container, MSU
• Biopsy specimens must be transported in formal-sal
ine.

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 Laboratory diagnosis of mycoses
Laboratory diagnosis
• Microscopy: Microscopy is used to observe clinical s
pecimens for the presence of fungal elements or to
identify the fungus following culture.
• In the latter case, lactophenol cotton blue is stain of
choice, which stains the fungal elements blue.
• Direct examination of clinical specimens could be st
ained or unstained.
• Wet mount: Candida may be observed in urine or H
VS wet mounts.
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 Laboratory diagnosis
• 10-20% KOH mount: Several specimens are subject
ed to KOH mount for direct examination.
• The material is mixed with 20% KOH on a slide and a
cover slip is placed. The slide is then gently heated b
y passing through the flame 2-3 times. The slide is o
bserved on cooling.
• KOH serves to digest the protein debris and clears k
eratinized tissue and increases the visibility.
• Addition of Dimethyl sulphoxide (DMSO) permits ra
pid clearing in the absence of heat.
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 Laboratory diagnosis
• Calcofluor white: This is a fluorescent dye, whi
ch binds selectively to chitin of the fungal cell
wall. The specimen then can be observed und
er fluorescent microscope.
• India Ink: Capsules of Cryptococcus neoforma
ns can be demonstrated by this negative staini
ng technique.

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 Laboratory diagnosis
• Haematoxylin and Eosin (H&E) stain: Useful for
staining tissue sections.
• Gomori’s methenamine silver nitrate (GMS) sta
in: Outlines of the fungi are black, internal par
ts stain pink- black while the background stain
s light green. Candida and Aspergillus may be
missed in H&E stained sections, therefore GM
S stained sections are essential for tissue path
ology.
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 Laboratory diagnosis
• Periodic Acid-Schiff (PAS) stain: On staining by this s
tain, fungal elements appear bright magenta colour
ed while the background stains green. It is useful in
staining tissue specimens.
• Giemsa’s stain: It is particularly useful in the detecti
on of Histoplamsa capsulatum in the bone marrow s
mears.

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 Laboratory diagnosis
• Gridley’s stain: It stains hyphae and yeasts dark blue
-pink, tissues deep blue and background yellow.
• Meyer mucicarmine stain: Capsules of C. neofor
mans and inner walls of Rhinosporidium seeberi’s s
porangium are stained pink.
• Gram stain: Candida is best demonstrated in clinical
specimen by Gram stain.
• Masson-Fontana stain is helpful in staining phaeoid
(dermatiaceous) fungi in tissue.
• Immunofluorescence: Monoclonal antibody labelled
with fluorescent dyes can be used to detect several
fungi in the clinical specimens. 11
 Laboratory diagnosis
Culture
• One of the most common media used to culture fungi i
n laboratory is Sabouraud’s Dextrose Agar (SDA). It cons
ists of peptone, dextrose and agar. High concentration
of sugar and a low pH (4.5-5.5) prevents growth of mos
t bacteria and makes it selective for fungi. Emmon’s mo
dification of SDA contains 2% dextrose and has pH of 6.
8.

• Other basal media to grow fungi include Potato Dextros

e Agar, Malt Extract Agar, Mycosel etc. Most fungi are a
ble to grow at room temperature while few pathogenic
fungi (e.g, Cryptococcus, dimorphic fungi) can grow at 3
7oC. Saprophytic fungi grow much quickly than pathoge
nic fungi (e.g, dermatophytes). 12
 Laboratory diagnosis
Culture
• In such situations the saprophytic fungi can be inhib
ited by the addition of cycloheximide (actidione) to
the SDA.
• Addition of antibiotics such as Chloramphenicol, Ge
ntamicin or Streptomycin to SDA serves to inhibit ba
cterial growth or multiplication.
• An example of SDA with cycloheximide and Chloram
phenicol is Mycosel agar.

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 Culture
Other specialized media used for different fungi inc
lude:
• Brain Heart Infusion Agar general isolation of fungi
and conversion of dimorphic fungi. (broth used for
blood culture)
• Inhibitory Mould Agar, an isolation medium with Chl
oramphenicol to suppress most bacteria.
• Caffeic Acid Agar and Birdseed Agar for isolation of
Cryptococcus neoformans.
• Corn Meal Agar: Enhances production of chlamydos
pores in Candida albicans and formation of conidia i
n fungi.
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 Culture
• Trichophyton Agars: Used for selective identifi
cation of Trichophyton species.
• Dermatophyte Test Medium: Used for recover
y of dermatophytes from clinical specimens.
• Sabhi Medium: Isolation of Histoplasma capsu
latum.
• ‘CHROM agar Candida’ is useful in identificatio
n of Candida species.

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 Culture
• Conversion of mould to yeast phase must be demonstrated in vitro
for identification of dimorphic fungi.

• Since some fungi grow slowly cultures should not be discarded for
4-6 weeks. Fungi are identified on the basis of colony morphology
(including pigmentation) and microscopic observation by tease-mo
unt preparation or slide culture technique.

Serology
• Detection of anti-fungal antibody is helpful in diagnosis of sub
-cutaneous and systemic mycoses, prognosis and response to anti
-fungal drugs.

• Different serologic techniques that are used include agglutination,

immunodiffusion, counter-immunoelectrophoresis, complement fi
xation test, immunofluorescence, RIA and ELISA.
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 Antigen detection
• It is particularly useful in the diagnosis of cryptococcal meningitis from CSF
specimens (CRAG test). The test is performed by Latex Agglutination or imm
unodiffusion tests. It is also helpful in the detection of Aspergillus and Candi
da antigens in systemic infections.
•
Skin tests (mycotic antigen -sporotrichin, blastomycin and coccidioi
din)

• Delayed hypersensitivity reactions to fungal antigens can be demonstrated

by skin tests. A positive skin does not necessarily indicate an active infection
; it only indicates sensitization of the individual. Hence, its value is in epide
miological studies than diagnosis. These tests may be performed in Histopla
smosis, Candidiasis, Sporotrichosis, Coccidioidomycosis, Blastomycosis, Para
coccidiodomycosis and dermatophytosis.

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 Molecular techniques

• Newer techniques such as DNA hybridization,

PCR are useful in diagnosis of mycoses in a sh
orter period as well as detect those fungi that
are difficult or dangerous to cultivate in vitro.

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 Treatment of Mycoses
An antifungal agent is a drug that selectively eliminates fungal
pathogens from a host with minimal toxicity to the host.
Polyene Antifungal Drugs
• Amphotericin B, nystatin, and pimaricin interact with sterols i
n the cell membrane (ergosterol in fungi, cholesterol in huma
ns) to form channels through which small molecules leak from
the inside of the fungal cell to the outside.
Azole Antifungal Drugs
• Fluconazole, itraconazole, and ketoconazole inhibit cytochro
me P450-dependent enzymes (particularly C14-demethylase) i
nvolved in the biosynthesis of ergosterol, which is required fo
r fungal cell membrane structure and function.

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 Treatment of Mycoses

Allylamine and Morpholine Antifungal Drugs

• Allylamines (naftifine, terbinafine) inhibit ergo
sterol biosynthesis at the level of squalene epo
xidase. The morpholine drug, amorolfine, inhi
bits the same pathway at a later step.
Antimetabolite Antifungal Drugs
• 5-Fluorocytosine acts as an inhibitor of both D
NA and RNA synthesis via the intracytoplasmic
conversion of 5-fluorocytosine to 5-fluorouraci
l. 20
 Treatment of Mycoses
• Griseofulvin is an antifungal drug produced by
Penicillium griseofulvum.
• Echinocandins e.g. Caspofungin. This is a new
class of antifungal drug.

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QUESTIONS???

Thank You!

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