Diagnostic and treatment of Fungal infection

Muh. Nasrum Massi, MD., Ph.DMakassar, Indonesia

DIAGNOSISDIAGNOSIS

IntroductionIntroduction

Fungal InfectionFungal Infection superficial, superficial, mucose, skin --- local destruction ---mucose, skin --- local destruction ---invasive---------- systemic/ pathogen invasive---------- systemic/ pathogen opportunisticopportunistic

Diagnosed by:Diagnosed by:

Doctor/Microbiologist/PathologistDoctor/Microbiologist/Pathologist

LABORATORY DIAGNOSTIC OF LABORATORY DIAGNOSTIC OF FUNGAL INFECTIONFUNGAL INFECTION

Examinations:Examinations: Physical examination: Physical examination:

predispositionpredisposition appropriate features of fungal appropriate features of fungal

infectioninfection clinical history, occupation, travellingclinical history, occupation, travelling

Specimens for fungal Specimens for fungal investigation:investigation:

swabswab sputumsputum scrapings of skinscrapings of skin A small segment of infected tissuesA small segment of infected tissues

Collection, transport and Collection, transport and specimens processing base on:specimens processing base on:

a. Correlation between clinical a. Correlation between clinical diagnostic and specimensdiagnostic and specimens

b. Transport facility/rapidityb. Transport facility/rapidity

Specimen collection should be:Specimen collection should be:

a. Aseptica. Aseptic

b. Quantityb. Quantity

c. Sterilize container, closing and c. Sterilize container, closing and label.label.

d. Clinical informationd. Clinical information

e. Universal precaution e. Universal precaution

CUTANEOUS SPECIMEN - Scalp Wood’s Lamp

luminescent hair, distortion, breakable and easy to tweez

- Skin : epidermal flakes at active lesions

- Nail : deep scratches & nail cuts - Collected in petri dish / sterile

envelope - Cultured in Sabouraud dextrose

agar + chloramphenicol & cyclohexamide ; + gentamycin if there is bacterial contamination

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

VAGINAL SECRETVAGINAL SECRET- Specimen is taken with a sterile - Specimen is taken with a sterile swab. swab. - Cultured in Sabouroud dextrose - Cultured in Sabouroud dextrose agar, Inhibitory mold agar or BHIagar, Inhibitory mold agar or BHI

EAR, NOSE, AND MOUTH SPECIMEN EAR, NOSE, AND MOUTH SPECIMEN - Sent with sterile - Sent with sterile - Cultured in enriched media- Cultured in enriched media

RESPIRATORY SECRET - Morning Sputum 10-15 ml - Collected inside a sterile bottle with wide

opening. - Direct shipment or at 4oC - Cultured in yeast extract phosphate + NH4OH

medium - Culture tubes are put in horizontal position

for 12-24 h

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

SPESIMEN CAIRAN SEREBRO SPINALSPESIMEN CAIRAN SEREBRO SPINAL

- Pengambilan CSS minimal 2 ml- Pengambilan CSS minimal 2 ml

- Dalam tabung steril tertutup- Dalam tabung steril tertutup

- Difilter, filter yg ada organisme ikut dikultur- Difilter, filter yg ada organisme ikut dikultur

- Filter dapat dipotong- Filter dapat dipotong22 & diletakkan pd & diletakkan pd permukaan mediumpermukaan medium

- Alternatif : Sentrifugasi 15 menit, 1000 rpm - Alternatif : Sentrifugasi 15 menit, 1000 rpm sedimen diteteskan pd media kultur sedimen diteteskan pd media kultur

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

Identifying FungiIdentifying Fungi

Yeast : biochemical testsYeast : biochemical tests Filamentous : Filamentous :

– physical appearancephysical appearance– spore structurespore structure– colony characteristicscolony characteristics

Fungal investigations involves:Fungal investigations involves:

• Microscopic examinationMicroscopic examination for evidence of for evidence of hyphae or spores hyphae or spores Skin scrapings in 10% KOH – direct unstained Skin scrapings in 10% KOH – direct unstained

smearsmear Sputum or ulcer swab – Gram stainingSputum or ulcer swab – Gram staining Infected tissues: Direct ImmunofluorescenseInfected tissues: Direct Immunofluorescense Infected tissues: Gomori Methenamine silver Infected tissues: Gomori Methenamine silver

(GMS) staining(GMS) staining Fungal Antigen detectionFungal Antigen detection

Latex agglutination test – Latex agglutination test – CryptococcusCryptococcus as antigen as antigen

Culture mediaCulture media

On Sabouraud dextrose agar (acidic pH On Sabouraud dextrose agar (acidic pH ++ 5,6 and contains antibiotic) 5,6 and contains antibiotic)

One incubate at 37One incubate at 37ooC and another at room C and another at room temperature to see dimorfismetemperature to see dimorfisme

Observe:Observe: colony appearance: pigment, size of mycelium colony appearance: pigment, size of mycelium

(varies greatly), spore or conidia(varies greatly), spore or conidia• microscopic morphology: hyphae, pseudohyphae, microscopic morphology: hyphae, pseudohyphae,

mycelium. Spore, conidia, septummycelium. Spore, conidia, septum• Chemical reaction (carbohydrate fermentation)Chemical reaction (carbohydrate fermentation)• Nucleic acid probeNucleic acid probe

Host response tests:Host response tests:

Skin tests – common test, frequently false Skin tests – common test, frequently false pos, useful to evaluate host response and pos, useful to evaluate host response and determine exposure index for determine exposure index for epidemiological purposesepidemiological purposes

Serology Serology Latex agglutinationLatex agglutination (detect IgM) (detect IgM) complement fixationcomplement fixation (detect IgG), (detect IgG),

frequently false pos due to cross frequently false pos due to cross reactions, Antibody detection 2-3 reactions, Antibody detection 2-3 months after onset of diseasemonths after onset of disease

DiagnosisDiagnosis

1. Wet Mount1. Wet Mount2. Skin test2. Skin test3. Serology3. Serology4. Fluorescent antibody4. Fluorescent antibody5. Biopsy and 5. Biopsy and histopathologyhistopathology6. Culture6. Culture

DiagnosisDiagnosis

1. Wet Mount1. Wet Mount2. Skin test2. Skin test3. Serology3. Serology4. Fluorescent antibody4. Fluorescent antibody5. Biopsy and 5. Biopsy and histopathologyhistopathology6. Culture6. Culture

DIRECT MICROSCOPIC DIRECT MICROSCOPIC OBSERVATIONOBSERVATION

10 % KOH10 % KOH

Gentle HeatGentle Heat

KOH Wet MountKOH Wet Mount

DiagnosisDiagnosis

1. Wet Mount1. Wet Mount2. Skin test2. Skin test3. Serology3. Serology4. Fluorescent antibody4. Fluorescent antibody5. Biopsy and 5. Biopsy and histopathologyhistopathology6. Culture6. Culture

SKIN TESTINGSKIN TESTING(DERMAL HYPERSENSTIVITY(DERMAL HYPERSENSTIVITY))

Use is limited to :Use is limited to :

• Determine cellular defense mechanismsDetermine cellular defense mechanisms• Epidemiologic studiesEpidemiologic studies

DiagnosisDiagnosis

1. Wet Mount1. Wet Mount2. Skin test2. Skin test3. Serology3. Serology4. Fluorescent antibody4. Fluorescent antibody5. Biopsy and 5. Biopsy and histopathologyhistopathology6. Culture6. Culture

Most serological tests for fungi measure Most serological tests for fungi measure antibody. Newer tests to measure antibody. Newer tests to measure antigen are now being developedantigen are now being developed

ANTIGEN DETECTION PRESENTLY ANTIGEN DETECTION PRESENTLY AVAILABLEAVAILABLE

CryptococcosisCryptococcosis HistoplasmosisHistoplasmosis

DiagnosisDiagnosis

1. Wet Mount1. Wet Mount2. Skin test2. Skin test3. Serology3. Serology4. Fluorescent antibody4. Fluorescent antibody5. Biopsy and 5. Biopsy and histopathologyhistopathology6. Culture6. Culture

DIRECT FLUORESCENT DIRECT FLUORESCENT ANTIBODYANTIBODY

CAN BE APPLIED TOCAN BE APPLIED TO

1.1. TISSUETISSUE

2.2. CULTURECULTURE

• ViableViable• Non-viableNon-viable

DiagnosisDiagnosis

1. Wet Mount1. Wet Mount2. Skin test2. Skin test3. Serology3. Serology4. Fluorescent antibody4. Fluorescent antibody5. Biopsy and 5. Biopsy and histopathologyhistopathology6. Culture6. Culture

INFLAMMATORY REACTIONINFLAMMATORY REACTION

Normal hostNormal host•PyogenicPyogenic•GranulomatousGranulomatous

Immunodeficient hostImmunodeficient host•NecrosisNecrosis

DiagnosisDiagnosis

1. Wet Mount1. Wet Mount2. Skin test2. Skin test3. Serology3. Serology4. Fluorescent antibody4. Fluorescent antibody5. Biopsy and 5. Biopsy and histopathologyhistopathology66. Culture. Culture

ISOLATION MEDIAISOLATION MEDIA

SABOURAUD DEXTROSE AGARSABOURAUD DEXTROSE AGAR

(pH ~ 5.6)(pH ~ 5.6)PlainPlainWith antibioticsWith antibioticsWith cycloheximideWith cycloheximide

INCUBATION TEMPERATUREINCUBATION TEMPERATURE

37 C - Body temperature37 C - Body temperature

25 C - Room temperature25 C - Room temperature

TREATMENTTREATMENT

History of anti-fungal drugHistory of anti-fungal drug

GILCHRIST (1888) : carbolic acid, methyl GILCHRIST (1888) : carbolic acid, methyl violet, bromine, KMnOviolet, bromine, KMnO44, minyak terpentin & , minyak terpentin &

olive oilolive oil

SSKI (Saturated solution of Kalium Iodide) SSKI (Saturated solution of Kalium Iodide) cutaneous sporotrichosiscutaneous sporotrichosis

Brown & Hazen : polyenes (Amphotericin B)Brown & Hazen : polyenes (Amphotericin B) toxic toxic

Smith (1961) : Amphotericin B Smith (1961) : Amphotericin B parenteral use parenteral use coccidioidomycosis ----------> nephrotoxiccoccidioidomycosis ----------> nephrotoxic

Nystatin, Hamycin Nystatin, Hamycin topical use topical use

1970 : Fluocytosin 1970 : Fluocytosin myelotoxicity, GITtoxicity; spectrum myelotoxicity, GITtoxicity; spectrum limited for limited for Candida spp Candida spp & & Cryptococcus neoformansCryptococcus neoformans

Imidazol : broad spectrum activity against Imidazol : broad spectrum activity against dermatophyte, dermatophyte, Candida Candida & others& othersContoh : Contoh : Clotrimazol, Mikonazol, KetokonazolClotrimazol, Mikonazol, Ketokonazol

1980 1980 Epidemic AIDS Epidemic AIDS 90% caused 90% caused Candidiasis ; combination with imunomodulatorCandidiasis ; combination with imunomodulator

USA USA : Terbinafine & itrakonazol : Terbinafine & itrakonazol infection infection dermatophyte; fluconazol dermatophyte; fluconazol vaginal candidiasis vaginal candidiasis

ALL EUKARYOTIC CELLS CONTAIN ALL EUKARYOTIC CELLS CONTAIN STEROLSSTEROLS

Mammalian cells – cholesterolMammalian cells – cholesterolFungal cells - ergosterolFungal cells - ergosterol

Acetyl-CoAAcetoacetyl-CoA

HMG-CoAMevalonic Acid

Squalene

Squalene-2,3-Epoxide

Lanosterol

ErgosterolPolyenes

Azoles

Morpholines

Allylamines

JB 5/00

Ergosterol Synthesis

PRIMARY ANTI-FUNGAL PRIMARY ANTI-FUNGAL AGENTSAGENTS

1.1. Polyene derivativesPolyene derivatives• Amphotericin BAmphotericin B• NystatinNystatin

2.2. AzolesAzoles• KetoconazoleKetoconazole• FluconazoleFluconazole• ItraconazoleItraconazole• VoriconazoleVoriconazole

AzolesAzoles

There are a few rare serious There are a few rare serious side effects from Itraconazole side effects from Itraconazole

and Fluconazoleand Fluconazole

3.3. GriseofulvinGriseofulvin

4.4. 5-fluorcytosine5-fluorcytosine

5.5. AllylaminesAllylamines• Terbinafine (Lamisil)Terbinafine (Lamisil)

6. Echinocandins6. Echinocandins• caspofungincaspofungin

PRIMARY ANTI-FUNGAL PRIMARY ANTI-FUNGAL AGENTSAGENTS

GriseofulvinGriseofulvinA slow acting drug used for A slow acting drug used for skin and nail infections. It skin and nail infections. It accumulates in the stratum accumulates in the stratum corneum and prevent hyphal corneum and prevent hyphal penetration through these penetration through these layerslayers

5-5-fluorocytosinefluorocytosine(5-FC)(5-FC)

Interferes With RNA SynthesisInterferes With RNA Synthesis

MECHANISMS OF ACTIONMECHANISMS OF ACTION

PolyenesPolyenes

AzolesAzoles

GriseofulvinGriseofulvin

5 - FC5 - FC

Ergosterol in cell Ergosterol in cell membranemembrane

Interfere with Interfere with ergosterol ergosterol synthesissynthesis

Forms a barrier to Forms a barrier to fungal growthfungal growth

Inhibits RNA Inhibits RNA synthesissynthesis

Medical Mycology Iceberg