who workshop on laboratory diagnosis of diptheria labkes
TRANSCRIPT
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WHO Workshop on Laboratory Diagnosis of Diptheria
Balai Besar Laboratorium Kesehatan Surabaya 13 – 15 Juli 2011
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Corynebacterium diphtheriae• Bakteri gram positif, batang lurus atau sedikit
bengkok, diujungnya membesar• Anaerob fakultatif• Sel-sel tersusun tunggal atau berpasangan,
sering membentuk formasi V, L atau palisade (spt huruf Cina )
• Non motil, spora negatif, granulametakromatik positif
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Alur Diagnosa Laboratorium
Diphteria(WHO manual)
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Media kultur yang digunakan
• Columbia blood agar plate• Tellurite blood agar plate ( CTBA ; Hoyle’s)• Media Tinsdale (tes cystinase) → screening test
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Tes Biokimia• Reduksi nitrat (Rosco)• Hidrolisis Urea (Rosco)• Tes Pyrazinamidase (Rosco)• Fermentasi gula (Glukosa, Sukrosa, Maltosa, Starch) →
Hiss Serum Water Sugar• Tes Katalase• Biotyping (API CORYNE)
Tes Toxigenitas → Elek Test
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Swab tenggorok ↓
Colombia blood agar plate + Hoyle’s agar plate(inkubasi 18 – 48 jam)
↓ CBA = abu2, kecil ↓ Hoyle’s = hitam, kering, kecil
Colombia blood agar + Hoyle’s agar plateDiambil 4 koloni tersangka
(inkubasi 18 – 48 jam) ↓ (koloni murni)
• Media Tinsdale (24 jam)• tes nitrat + tes urea + tes pyz (4 jam) → rapid test
• Hiis serum water sugar (24 jam)• tes biotyping (24 jam)• elek test (24 – 48 jam)
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Colombia blood agar plate
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Hoyle’s agar plate
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Media Tinsdale (cystinase test)
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Media Tinsdale (cystinase test)
NEGATIF POSITIFC. diphtheriae
C. ulceransC.
pseudotuberculosis
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Tes Nitrat• Dibuat suspensi kuman Mc Farland No.8• Dipipet 0,25 ml suspensi kuman ke dalam tabung steril• Ditambahkan 1 tablet Rosco Nitrate Reduction
Diagnostic• Di inkubasi selama 4 jam pada suhu 37 derajat Celcius• Ditambahkan 1 tetes dimethylnaphthylamine solution +
1 tetes sulfanilic acid solution
Positif = merah/pinkNegatif = tidak berwarna
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Tes Nitrat
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Tes Urease• Dibuat suspensi kuman Mc Farland No.8• Dipipet 0,25 ml suspensi kuman ke dalam tabung
steril• Ditambahkan 1 tablet Rosco Urease Reduction
Diagnostic• Di inkubasi selama 4 jam pada suhu 37 derajat
Celcius
Positif = merah/unguNegatif = kuning/oranye
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Tes Urease
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Tes Pyrazinamidase
• Dipipet 0,25 ml aquades steril ke dalam tabung steril• Ditambahkan koloni kuman hingga tersuspensi (Mc
Farland No.8)• Ditambahkan 1 tablet PYZ• Di inkubasi selama 4 jam pada suhu 37 derajat
Celcius
Positif = merah/oranyeNegatif = tidak berwarna
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Tes Pyrazinamidase
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Hiss serum water sugar
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API Coryne kit
Tes Negatif
Tes Positif
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• Typical “gravis” 1010326
• Typical “mitis” 1010324
• Typical ”ulcerans” 0111326
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BIOCHEMICAL IDENTIFICATION OF PATHOGENIC CORYNEBACTERIA
CYS
PYZ
Alkaline Phosphatase
Nitrate
Urease
Acid produced from Gelatin Liquification
Glucose
Ribose
Maltose
Sucrose
Glycogen
Trehalose
C. diphtheriae
Var gravis + - - + - + + + - + N/A N/A
Var mitis + - - + - + + + - - N/A N/A
Var intermedius + - - + - + + + - - N/A N/A
Var belfanti + - - - - + + + - - N/A N/A
C. ulcerans + - + - + + + + - + + + at 25OC
C. pseudotuberculosis + - + - + + + + - - - - at 25OC
C. pseudodiphtheriticum - + V + + - - - - - N/A N/A
C. xerosis - + + + - + + + + - N/A N/A
C. striatum - + + + - + V - V - N/A N/A
C. amycolatum - + + V V + V V V - N/A N/A
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Elek test
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Elek Test
NCTC 10648++
test
NCTC 10356-
NCTC 3984±
NCTC 10648++
test
NCTC 10356-
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Modifikasi Elek test
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Sample preparation • Pipet 0.5 ml aquades steril ke dalam sterile Eppendorf
tubes (tutup ulir/screw tube)• Masukan koloni dari media Colombia/Hoyle (lakukan
hal yang sama pada kontrol)• Tabung2 diletakan pada dry heating block dan
dipanaskan pd suhu 100 derajat Celcius selama 15 menit
• di sentrifuge dgn kecepatan tinggi (13000 rpm) slm 1 menit
PCR for the detection of diphtheria toxin
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Persiapan PCR Reaction Mixture misalnya untuk 9 reaksi (6 template DNA, water control, internal positive control (IPC), plus 1 extra to allow for pipette
error).
• 1. 10 x Reaction buffer 45 l • 2. MgCl2 (1.5 mM) 18 l
• 3. Nucleotides:(10mM, containing, 9 l
dATP, dCTP, dGTP and dTTP) • 4. Taq polymerase 2.5 units 4.5 l
• 5. Primer 1 (15pmol/l) 9 l • 6. Primer 2 (15pmol/l) 9 l • 7. Water (PCR grade) 337.5 l
TOTAL 432 l Vortex the PCR reaction mixture and proceed to the second stage.
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Masing2 tabung ditambahkan : a. PCR reaction mixture 48 l
b. Internal control 1 l-add to all tubes except water control
c. Crude DNA sample 1 l-add to all tubes except water control
d. Water 2 -add to water control only
*Masing2 tabung ditambahkan mineral oil *Sentrifuge selama beberapa detik dalam micro centrifuge
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• Cycling times :1 cycle 96 derajat celcius 2 menit30 cycles 94 derajat celcius 15 detik
50 derajat celcius 15 detik
72 derajat celcius 30 detik1 cycle 72 derajat celcius 10 menit
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Electrophoresis
• 20 µl produk + 4 µl loading dye• Sampel di elektroforesa pada 2 % agarose gel
dalam buffer Tris Borate EDTA pH 8 (150 v)• Gel diwarnai dengan Ethidium bromide dan di
lihat hasilnya pada UV transilluminator
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• 1 Marker• 2-5 Test isolate • 6 Positive control• 7 Weak positive
control• 8 Negative control• 9 IPC control• 10 water control• 11 Marker
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buffer Tris Borate EDTA pH 8
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DR. ANDROULLA EFSTRATIOU
(Androu)
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DR. ARUNI DE ZOYSA(RU)
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SekianDan
Terima kasih