diag jamur ddt

7/28/2019 Diag Jamur DDT

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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



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



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



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



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



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



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

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