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Diagnostic and treatment of Fungal
infection
Muh. Nasrum Massi, MD., Ph.D
Makassar, Indonesia
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DIAGNOSIS
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Introduction
Fungal Infection superficial, mucose,skin --- local destruction ---invasive----------
systemic/ pathogen opportunistic
Diagnosed by:
Doctor/Microbiologist/Pathologist
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LABORATORY DIAGNOSTIC OF
FUNGAL INFECTION
Examinations:
Physical examination:
predisposition
appropriate features of fungalinfection
clinical history, occupation, travelling
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Specimens for fungal
investigation:
swab
sputum
scrapings of skin
A small segment of infected tissues
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Collection, transport and specimens
processing base on:
a. Correlation between clinicaldiagnostic and specimens
b. Transport facility/rapidity
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Specimen collection should be:
a. Aseptic
b. Quantityc. Sterilize container, closing and
label.
d. Clinical information
e. Universal precaution
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CUTANEOUS SPECIMEN
- Scalp Woods Lamp luminescent
hair, distortion, breakable and easy totweez
- Skin : epidermal flakes at activelesions
- Nail : deep scratches & nail cuts - Collected in petri dish / sterile
envelope
- Cultured in Sabouraud dextrose agar+ chloramphenicol & cyclohexamide ; +gentamycin if there is bacterialcontamination
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EYE SPECIMEN - Scratch from ulceration and supuration
area of the cornea. - Aspiration or ocular sucking - Culture media is transported to
operation room URINE SPECIMEN - Urine catheter, suprapubic punction - Shipment as soon as possible or at
4oC (12 15 h) Quantitation & centrifugation
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VAGINAL SECRET
- Specimen is taken with a sterile swab.
- Cultured in Sabouroud dextrose agar,Inhibitory mold agar or BHI
EAR, NOSE, AND MOUTH SPECIMEN
- Sent with sterile
- Cultured in enriched media
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RESPIRATORY SECRET
- Morning Sputum 10-15 ml
- Collected inside a sterile bottle with wideopening.
- Direct shipment or at 4oC
- Cultured in yeast extract phosphate + NH4OHmedium
- Culture tubes are put in horizontal position for12-24 h
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BODY FLUID SPECIMEN
- Aseptic collection
- Shipment as soon as possible in a sterile container
- Could be kept at 4oC for one night
- Centrifugation previous to culture
BONE MARROW SPECIMEN
- Dikirim dalam semprit steril atau tabung mgd heparin
- Simpan dlm 4oC selama 12 jam
- Inokulasi lebih dari satu medium
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SPESIMEN CAIRAN SEREBRO SPINAL
- Pengambilan CSS minimal 2 ml
- Dalam tabung steril tertutup- Difilter, filter yg ada organisme ikut dikultur
- Filter dapat dipotong2 & diletakkan pd
permukaan medium- Alternatif : Sentrifugasi 15 menit, 1000 rpm
sedimen diteteskan pd media kultur
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SPESIMEN DARAH
- Sampel diambil sebanyak 10 ml
- Dikultur dalam botol kultur
- Diinkubasi minimal 30 hari
- Isolat yeast terdeteksi dalam 4 hari pertama
- Fungi dimorfik, terdeteksi selama 2 minggu
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Identifying Fungi
Yeast : biochemical tests
Filamentous :
physical appearance spore structure
colony characteristics
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Fungal investigations involves:
Microscopic examination for evidenceof hyphae or spores
Skin scrapings in 10% KOH directunstained smear
Sputum or ulcer swab Gram staining
Infected tissues: Direct
Immunofluorescense Infected tissues: Gomori Methenamine
silver (GMS) staining
Fungal Antigen detection
Latex agglutination test Cryptococcus as antigen
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Culture media
On Sabouraud dextrose agar (acidic pH +5,6 and contains antibiotic)
One incubate at 37oC and another at
room temperature to see dimorfismeObserve:colony appearance: pigment, size of mycelium
(varies greatly), spore or conidia
microscopic morphology: hyphae,pseudohyphae, mycelium. Spore, conidia,septum
Chemical reaction (carbohydrate fermentation)
Nucleic acid probe
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Host response tests:
Skin tests common test, frequently falsepos, useful to evaluate host response anddetermine exposure index forepidemiological purposes
Serology
Latex agglutination (detect IgM)
complement fixation (detect IgG),frequently false pos due to crossreactions, Antibody detection 2-3months after onset of disease
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Diagnosis
1. Wet Mount2. Skin test
3. Serology4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
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Diagnosis
1. Wet Mount2. Skin test
3. Serology4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
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DIRECT MICROSCOPIC
OBSERVATION
10 % KOH
Gentle Heat
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KOH Wet Mount
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Diagnosis
1. Wet Mount2. Skin test
3. Serology4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
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SKIN TESTING
(DERMAL HYPERSENSTIVITY)
Use is limited to :
Determine cellular defense mechanisms
Epidemiologic studies
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Diagnosis
1. Wet Mount2. Skin test
3. Serology4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
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Most serological tests for fungi measureantibody. Newer tests to measure
antigen are now being developed
ANTIGEN DETECTION PRESENTLYAVAILABLE
Cryptococcosis
Histoplasmosis
Di i
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Diagnosis
1. Wet Mount2. Skin test
3. Serology4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
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DIRECT FLUORESCENT
ANTIBODYCAN BE APPLIED TO
1. TISSUE
2. CULTURE
Viable
Non-viable
Di i
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Diagnosis
1. Wet Mount2. Skin test
3. Serology4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
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INFLAMMATORY REACTION
Normal host
Pyogenic
Granulomatous
Immunodeficient host
Necrosis
Di i
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Diagnosis
1. Wet Mount2. Skin test
3. Serology4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
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ISOLATION MEDIA
SABOURAUD DEXTROSEAGAR
(pH ~ 5.6)
PlainWith antibiotics
With cycloheximide
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INCUBATION TEMPERATURE
37 C - Body temperature
25 C - Room temperature
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TREATMENT
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History of anti-fungal drug
GILCHRIST (1888) : carbolic acid, methyl
violet, bromine, KMnO4, minyak terpentin &
olive oil
SSKI (Saturated solution of Kalium Iodide)
cutaneous sporotrichosis
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Brown & Hazen : polyenes (Amphotericin B) toxic
Smith (1961) : Amphotericin B parenteral use
coccidioidomycosis ----------> nephrotoxic
Nystatin, Hamycin topical use
1970 : Fluocytosin myelotoxicity, GITtoxicity; spectrum
limited forCandida spp & Cryptococcus neoformans
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Imidazol : broad spectrum activity againstdermatophyte, Candida & others
Contoh : Clotrimazol, Mikonazol, Ketokonazol
1980Epidemic AIDS 90% causedCandidiasis ; combination with imunomodulator
USA : Terbinafine & itrakonazol infectiondermatophyte; fluconazol vaginal candidiasis
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ALL EUKARYOTIC CELLS CONTAIN
STEROLS
Mammalian cells cholesterol
Fungal cells - ergosterol
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Acetyl-CoA
Acetoacetyl-CoA
HMG-CoA
Mevalonic Acid
Squalene
Squalene-2,3-Epoxide
Lanosterol
ErgosterolPolyenes
Azoles
Morpholines
Allylamines
JB
Ergosterol Synthesis
PRIMARY ANTI FUNGAL
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PRIMARY ANTI-FUNGAL
AGENTS1. Polyene derivatives
Amphotericin B
Nystatin
2. Azoles Ketoconazole
Fluconazole
Itraconazole Voriconazole
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Azoles
There are a few rare serious
side effects from Itraconazoleand Fluconazole
PRIMARY ANTI FUNGAL
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3. Griseofulvin
4. 5-fluorcytosine
5.
Allylamines Terbinafine (Lamisil)
6. Echinocandins
caspofungin
PRIMARY ANTI-FUNGAL
AGENTS
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GriseofulvinA slow acting drug used for
skin and nail infections. Itaccumulates in the stratumcorneum and prevent hyphal
penetration through theselayers
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5-fluorocytosine
(5-FC)Interferes With RNA Synthesis
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MECHANISMS OF ACTION
Polyenes
Azoles
Griseofulvin
5 - FC
Ergosterol in cellmembrane
Interfere with
ergosterolsynthesis
Forms a barrier tofungal growth
Inhibits RNAsynthesis
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Medical Mycology Iceberg