Tutor  Divisi Imunologi PEMERIKSAAN  VIRAL LOAD TEKNOLOGI NASBA Febtarini. R,dr/ Endang Retnowati,dr,MS,Sp.PK (K) Rabu, 8-...
PENDAHULUAN <ul><li>Viral load  : </li></ul><ul><li>*Jumlah turunan virus dalam darah atau cairan tubuh yang diukur melalu...
Indikasi Pemeriksaan  Viral load  : <ul><li>Infeksi akut : membantu menegakkan diagnosis </li></ul><ul><li>secara dini  </...
Metode pemeriksaan asam nukleat virus <ul><li>1. PCR  :  Polymerase chain reaction </li></ul><ul><li>(RT-PCR, rt-PCR)  </l...
NASBA ( Nucleic acid sequence-based amplification ) <ul><li>Teknologi uji amplifikasi virus RNA atau DNA </li></ul><ul><li...
5
Plasma Serum HIV-1 HTLV-1 HCV HBV HHV-6 Whole blood HIV-1 CMV Factor V Leiden Lymph nodes/skin biopsies HIV-1 Sputum/Saliv...
Menggunakan 3 enzim : <ul><li>Reverse transcriptase (Avian myeloblastosis  virus /AMV) </li></ul><ul><li>RNA atau DNA  tem...
Contoh: Pemeriksaan  viral load  HIV dengan sampel tetesan darah kering 1. persiapan sampel dan reagen 1 ml darah vena + E...
Kertas dikeluarkan Menyiapkan larutan  premix  : CAL + 550 µl CAL  diluent  + 550 µl larutan silika ( vortex ) 2. EKSTRAKS...
Magnetic  separatio n Complex  biological  sample Pure  nucleic acid 2. EKSTRAKSI 10 Proteins  and Lipids Wash buffer 3. P...
2. Lanjutan fase ekstraksi Larutan  premix  + larutan  lysis buffer  & sampel vorteks Inkubasi suhu kamar,10 menit Sentrif...
<ul><li>Pipet 1,5 ml ( washing buffer  1 + silika-RNA) </li></ul>Cuci selama 30 detik (menu STEP 1,  magnet on ) Buang sup...
<ul><li>15 µl  Eluate  + 25 µl  elution buffer </li></ul>Inkubasi selama 5 menit, suhu 60 0  C  Tempatkan tabung di rak ma...
<ul><li>15 µl ( eluate&elution buffer ) + 20 µl primer </li></ul>Menyiapkan enzim = kemasan enzim ( lyophilized enzymes ) ...
15
16
17
3.  Real time PCR oligo P2 RT RT T7 RNAP anti-sense RNA RNase H oligo P1 oligo P1 RNase H  & oligo P2 sense RNA T7 RNA pol...
Molecular beacon DNA  probe NASBA RNA  amplicon SIGNAL:  ++ “ open” NO SIGNAL “ closed” Deteksi 19 -Alat dinyalakan 2 meni...
Amplifikasi  real time PCR  & deteksi oligo P2 RT RT T7 RNAP anti-sense RNA RNase H oligo P1 oligo P1 RNase H & oligo P2 s...
Deteksi ( Molecular beacon ) 0 10 20 30 40 50 60 Normalized fluorescence Fluorescent signal (real-time) NASBA  reaction Ti...
Fluorescence Analyzer, Optics (Schematic layout) 22
23 Desktop computer Strip centrifuge Incubator Analyzer
Interpretasi hasil Pasien 1 = 840.000 kopi/ml darah  Pasien 5 = 110.000 kopi/ml darah Pasien 2 = 870.000 kopi/ml darah  Pa...
MOHON MAAF LAHIR - BATIN
 
 
 
 
Benefits <ul><ul><li>High-throughput (n=48) </li></ul></ul><ul><ul><li>Minimal hands-on-time (30 min for 48 samples) </li>...
Lysis buffer = Chaotropic agent  Gu SCN/  guanidine thiocyanate Perbedaan NASBA dan PCR : NASBA PCR Target utama RNA Targe...
RT-PCR rt-PCR bDNA NASBA *replikasi as. nukleat *deteksi  capture  amplikon,divisualisasi kan mell reaksi enzim/substrat (...
<ul><li>Performance Claims of HIV-1 viral load, product FDA-approved </li></ul><ul><li>HIV-1 viral load values In Internat...
<ul><li>Sample </li></ul><ul><li>Blood  </li></ul><ul><li>Water </li></ul><ul><li>Filterable </li></ul><ul><li>product </l...
Plasma Serum HIV-1 HTLV-1 HCV HBV HHV-6 Neisseria gonorrhoeae Neisseria meningitidis Whole blood HIV-1 CMV Factor V Leiden...
<ul><li>Main features: </li></ul><ul><ul><li>High purity of eluate  (one of the best) </li></ul></ul><ul><ul><ul><li>magne...
The EasyMag instrument <ul><li>Performances: </li></ul><ul><ul><li>Nucleic acid recovery: </li></ul></ul><ul><ul><li>As ef...
<ul><ul><li>NASBA is available as a research tool through the “basic kit” set of Nasba reagents used on the EasyQ instrume...
Nasba & sensitivity <ul><ul><li>Enterovirus: 11 copies / µl of extract (95% hit rate) targeting 5’NCR-product claim   </li...
Nasba & Quantification <ul><li>Performance Claims of HIV-1 viral load, product FDA-approved </li></ul><ul><li>HIV-1 viral ...
 
PCR
 
 
Indikasi Pemeriksaan  viral load  pada HIV Indikasi klinik Informasi Penggunaan Sindroma HIV akut Menegakkan Dx (tes serol...
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  • Een aantal toepassingen in de humane diagnostiek
  • Een aantal toepassingen in de humane diagnostiek
  • Tibaru14

    1. 1. Tutor Divisi Imunologi PEMERIKSAAN VIRAL LOAD TEKNOLOGI NASBA Febtarini. R,dr/ Endang Retnowati,dr,MS,Sp.PK (K) Rabu, 8- September- 2010
    2. 2. PENDAHULUAN <ul><li>Viral load : </li></ul><ul><li>*Jumlah turunan virus dalam darah atau cairan tubuh yang diukur melalui pemeriksaan asam nukleat virus. </li></ul><ul><li>*Petunjuk aktivitas virus dlm darah/cairan tubuh, </li></ul><ul><li>jika aktivitas virus tinggi  didapatkan viral load tinggi  semakin berat kerusakan yg ditimbulkan terhadap sistem imun , kerusakan sel </li></ul>1
    3. 3. Indikasi Pemeriksaan Viral load : <ul><li>Infeksi akut : membantu menegakkan diagnosis </li></ul><ul><li>secara dini </li></ul><ul><li>Evaluasi infeksi virus : menentukan awal terapi </li></ul><ul><li>& pemantauan terapi </li></ul><ul><li>Menentukan prognosis </li></ul>2
    4. 4. Metode pemeriksaan asam nukleat virus <ul><li>1. PCR : Polymerase chain reaction </li></ul><ul><li>(RT-PCR, rt-PCR) </li></ul><ul><li>2. bDNA : Branched chain DNA assay </li></ul><ul><li>3. NASBA : Nucleic acid sequence-based amplification </li></ul>3
    5. 5. NASBA ( Nucleic acid sequence-based amplification ) <ul><li>Teknologi uji amplifikasi virus RNA atau DNA </li></ul><ul><li>Pengembangan metode ELISA </li></ul><ul><li>Terdiri dari proses : </li></ul><ul><li>1. Pre ekstraksi </li></ul><ul><li>2. Ekstraksi </li></ul><ul><li>3. Amplifikasi dan deteksi </li></ul>4
    6. 6. 5
    7. 7. Plasma Serum HIV-1 HTLV-1 HCV HBV HHV-6 Whole blood HIV-1 CMV Factor V Leiden Lymph nodes/skin biopsies HIV-1 Sputum/Saliva HRV Influenza Virus Measles virus Cervical swabs HIV-1 HPV Urine Measles virus Faeces HIV-1 CMV astrovirus Cells HIV-1 HTLV-1 CMV CSF HIV-1 JCV LCMV mumps virus Semen HIV-1 6
    8. 8. Menggunakan 3 enzim : <ul><li>Reverse transcriptase (Avian myeloblastosis virus /AMV) </li></ul><ul><li>RNA atau DNA template  mensintesis DNA </li></ul><ul><li>2. RNAse Hybrid ( E. coli ) </li></ul><ul><li>3. T7 RNA polymerase (phage T7 ) : </li></ul><ul><li>DNA template  sintesis RNA </li></ul>7
    9. 9. Contoh: Pemeriksaan viral load HIV dengan sampel tetesan darah kering 1. persiapan sampel dan reagen 1 ml darah vena + EDTA Pipet & teteskan masing-masing 50 µl darah (ke lingkaran di kertas proteinsaver 903 Whatman ), beri identitas Px. Biarkan kering (3 s/d 24 jam, suhu kamar) Dimasukkan ke dalam kantong plastik Zip Lock yg telah diberi silika gel  ke dalam kantong Biohazard  amplop dan kirim ke Laboratorium 8
    10. 10. Kertas dikeluarkan Menyiapkan larutan premix : CAL + 550 µl CAL diluent + 550 µl larutan silika ( vortex ) 2. EKSTRAKSI 9
    11. 11. Magnetic separatio n Complex biological sample Pure nucleic acid 2. EKSTRAKSI 10 Proteins and Lipids Wash buffer 3. Purification of nucleic acid, removal of inhibitors Elution buffer 4. Recover nucleic acid in small volume Silica Low [GuSCN] pH > 8.0 Silica 2. Binding of nucleic acid High [GuSCN] neutral pH Sample + Lysis buffer Nucleic acid 1. Release of nucleic acid Stabilization High [GuSCN] neutral pH Function: Chemistry:
    12. 12. 2. Lanjutan fase ekstraksi Larutan premix + larutan lysis buffer & sampel vorteks Inkubasi suhu kamar,10 menit Sentrifus 1500g, 15 detik <ul><li>Supernatan dibuang </li></ul><ul><li>endapan : silika & RNA </li></ul>+ washing buffer 1 Diaduk dg pipet  100 µl 400 µl 2 µl 100 µl 11
    13. 13. <ul><li>Pipet 1,5 ml ( washing buffer 1 + silika-RNA) </li></ul>Cuci selama 30 detik (menu STEP 1, magnet on ) Buang supernatan, tambahkan 400 µl washing buffer 1 (mengulangi pencucian) Buang supernatan, tambahkan 500 µl washing buffer 2 (ulangi 2X) Buang supernatan, tambahkan 500 µl washing buffer 3, cuci selama 15 detik Buang supernatan = eluate 12
    14. 14. <ul><li>15 µl Eluate + 25 µl elution buffer </li></ul>Inkubasi selama 5 menit, suhu 60 0 C Tempatkan tabung di rak magnetik Buka tabung, dan ambil 15 µl extracted sample (eluate & elution buffer) ke eight tube strip di dalam area amplifikasi ,beri identitas (atau jika tidak langsung dikerjakan, eluate bisa disimpan pada suhu 4 0 C) 13
    15. 15. <ul><li>15 µl ( eluate&elution buffer ) + 20 µl primer </li></ul>Menyiapkan enzim = kemasan enzim ( lyophilized enzymes ) + 45 µl enzyme diluents  mencampurkannya dengan cara di tapping 3 detik  inkubasi suhu kamar 15 menit  sentrifus 15 detik  inkubasi (di inkubator) selama 15 menit  ambil 5 µl larutan enzim tsb  letakkan pada tutup eight tube Lyophilized primers + 180µl primerdiluents  vortex  ambil 20 µl Inkubasi (di inkubator), suhu 41 0 C selama 4 menit sentrifus 2 detik, 1500 rpm  tapping 3 detik  sentrifus 2 detik  ke Analyzer (real time PCR and detection ) 14
    16. 16. 15
    17. 17. 16
    18. 18. 17
    19. 19. 3. Real time PCR oligo P2 RT RT T7 RNAP anti-sense RNA RNase H oligo P1 oligo P1 RNase H & oligo P2 sense RNA T7 RNA polymerase Reverse Transcriptase Reverse Transcriptase Fase Linier Fase amplifikasi 18 ( 5 menit ) ( 90 menit )
    20. 20. Molecular beacon DNA probe NASBA RNA amplicon SIGNAL: ++ “ open” NO SIGNAL “ closed” Deteksi 19 -Alat dinyalakan 2 menit (saat enzim diletakkan pd tutup eight tubes - Analizer mendeteksi 10- 10 7 turunan HIV RNA / ml darah -menggunakan 1 suhu( isothermal ) 41 0 C -Alat tersambung dg komputer & printer F Q G Q F
    21. 21. Amplifikasi real time PCR & deteksi oligo P2 RT RT T7 RNAP anti-sense RNA RNase H oligo P1 oligo P1 RNase H & oligo P2 sense RNA T7 RNA polymerase Reverse Transcriptase Reverse Transcriptase Molecular beacon hybridization 20 F Q Q F Q F F Q
    22. 22. Deteksi ( Molecular beacon ) 0 10 20 30 40 50 60 Normalized fluorescence Fluorescent signal (real-time) NASBA reaction Time (minutes) 21 F Q Q F Q F F Q
    23. 23. Fluorescence Analyzer, Optics (Schematic layout) 22
    24. 24. 23 Desktop computer Strip centrifuge Incubator Analyzer
    25. 25. Interpretasi hasil Pasien 1 = 840.000 kopi/ml darah Pasien 5 = 110.000 kopi/ml darah Pasien 2 = 870.000 kopi/ml darah Pasien 6 = 620.000 kopi/ml darah Pasien 3 = 780.000 kopi/ml darah Pasien 7 = 290.000 kopi/ml darah Pasien 4 = TND Pasien 8 = 3300 kopi/ml darah 24
    26. 26. MOHON MAAF LAHIR - BATIN
    27. 31. Benefits <ul><ul><li>High-throughput (n=48) </li></ul></ul><ul><ul><li>Minimal hands-on-time (30 min for 48 samples) </li></ul></ul><ul><ul><li>Fast-time to result (30 to 60 min.) for amplification / detection steps, </li></ul></ul><ul><ul><li>sample prep. dependent on sample </li></ul></ul><ul><ul><li>Internal control (also used as calibrator for QT) due to duplex amplification </li></ul></ul><ul><ul><li>No post-amplification step, no carry-over contaminations (closed tube) </li></ul></ul><ul><ul><li>Combining various applications in one run </li></ul></ul><ul><ul><ul><li>Allows panel approach based on clinical signs e.g. pneumonia </li></ul></ul></ul><ul><ul><ul><li>on sample-related risks e.g. water, food </li></ul></ul></ul>
    28. 32. Lysis buffer = Chaotropic agent Gu SCN/ guanidine thiocyanate Perbedaan NASBA dan PCR : NASBA PCR Target utama RNA Target utama DNA Isothermal ( 1 suhu ) 3 suhu RNA 3 enzim, DNA 4 enzim 2 Primer RNA 2 enzim, DNA 1 enzim 1 primer Continuous Cyclic ssRNA amplicons dsDNA amplicons Sampel sedikit, hemat waktu Real time PCR Lebih spesifik & sensitif Satu tabung tertutup Waktu lebih lama
    29. 33. RT-PCR rt-PCR bDNA NASBA *replikasi as. nukleat *deteksi capture amplikon,divisualisasi kan mell reaksi enzim/substrat (kolorimetrik) *enzim reverse transcriptase *hasil: Kuantitatif:ELISA Kualitatif: Gel *Amplifikasi signal Probe spesifik fluoresence * deteksi amplikon pd setiap siklus amplifikasi *hasil: grafik kuantitas jumlah turunan asam nukleat virus Proteinase K  RNA keluar  probe sintetik oligonukleotida berlabel alkali fosfatase & substrat  bDNA kompleks & signal dideteksi dg teknik chemiluminescense *deteksi 50-500.000 kopi/ml *amplifikasi signal isothermal *amplifikasi transkripsi RNA satu tabung *kalibrator internal *selektif HIV1RNA (pd HIV) * one step sandwich hybridization * deteksi 10-10 7 kopi/ml
    30. 34. <ul><li>Performance Claims of HIV-1 viral load, product FDA-approved </li></ul><ul><li>HIV-1 viral load values In International Units (IU) per ml </li></ul><ul><li>Sensitivity (sample 1 and 2 ml of human plasma </li></ul><ul><ul><li>cut-off 25 IU/ml (1.0 ml) and 12.5 IU/ml (2.0 ml) </li></ul></ul><ul><ul><li>50% detection rate: 62 IU/ml (1.0 ml) and 36 IU/ml (2.0 ml) </li></ul></ul><ul><ul><li>95% detection rate: 357 IU/ml (1.0 ml) and 141 IU/ml (2.0 ml) </li></ul></ul><ul><li>Linear dynamic range from 50 – 3,000,000 IU / ml: 5 Log </li></ul><ul><li>Specificity 99.7% (95% confidence limit 98.5 – 100%) </li></ul><ul><li>HIV-1 subtype reactivity HIV-1 group M subtypes A to J </li></ul><ul><li>Accuracy < 0.2 log deviation from 1st international standard </li></ul><ul><li>Precision 0.12 log in the range 1,000 – 3,000,000 IU / ml </li></ul><ul><li>0.28 log in the range <= 1,000 IU / ml </li></ul>
    31. 35. <ul><li>Sample </li></ul><ul><li>Blood </li></ul><ul><li>Water </li></ul><ul><li>Filterable </li></ul><ul><li>product </li></ul><ul><li>Air … </li></ul><ul><li>Pre-processing </li></ul><ul><li>Inactivation </li></ul><ul><li>Viscosity </li></ul><ul><li>Concentration </li></ul><ul><li>Filtration </li></ul><ul><li>Standardization for difficult sample </li></ul><ul><li>Lysis of µ-org. </li></ul><ul><li>Disrupt target </li></ul><ul><li>µ-organism </li></ul><ul><li>for Nucleic Acid </li></ul><ul><li>extraction </li></ul><ul><li>(yield, sensitivity) </li></ul><ul><li>- Stabilization of N.A </li></ul><ul><li>N.A. Capture, purification & concentration </li></ul><ul><li>Generic or specific capture of target DNA/RNA </li></ul><ul><li>Remove of potential amplification step inhibitors </li></ul><ul><li>Concentrate to increase kinetic and decrease volume of reagents </li></ul>Results for a few targets Mono-detection Amplification & Detection Obtain a high number of copies labelled for detection (real time) Hybridization Labelling Washing on DNA Chip Results for high number of targets Multi-detection Pre ekstraksi Ekstraksi Amplifikasi (real-time PCR) & Deteksi
    32. 36. Plasma Serum HIV-1 HTLV-1 HCV HBV HHV-6 Neisseria gonorrhoeae Neisseria meningitidis Whole blood HIV-1 CMV Factor V Leiden Lymph nodes/skin biopsies HIV-1 Mycobacterium lepra Mycobacterium tuberculosis Sputum/Saliva HRV Influenza Virus Measles virus Mycobacterium tuberculosis Mycoplasma pneumoniae Legionella pneumoniae Cervical swaps HIV-1 HPV Chlamydia trachomatis Urine Measles virus Chlamydia trachomatis Neisseria gonorrhoeae Faeces HIV-1 CMV astrovirus Microspordia Campylobacter jejuni Campylobacter coli Cells HIV-1 HTLV-1 CMV CSF HIV-1 JCV LCMV mumps virus Measles virus Mycobacterium tuberculosis Borrelia burgdorferi Leptospira interrogans Semen HIV-1
    33. 37. <ul><li>Main features: </li></ul><ul><ul><li>High purity of eluate (one of the best) </li></ul></ul><ul><ul><ul><li>magnetic silica with high binding capacity </li></ul></ul></ul><ul><ul><ul><li>optimum mixing through disposable design </li></ul></ul></ul><ul><ul><li>Compatible with the use of an internal control </li></ul></ul><ul><ul><li>A single set of reagent and 2 disposables / sample: 1 protocole for all extractions </li></ul></ul><ul><ul><li>3 X 8 samples extracted in 40 or 60 min. (the fastest). Up to 240 extrac. / day. </li></ul></ul><ul><ul><li>Hands-on-time <15 min. </li></ul></ul><ul><ul><li>Input sample vol.: 10 µl up to 1 ml </li></ul></ul><ul><ul><li>Output vol.: 110 µl down to 12 µl [80X] </li></ul></ul><ul><ul><li>Flexibility (N.A. /vol. / applications / workflow) </li></ul></ul><ul><ul><li>Intuitive software with touch screen interface </li></ul></ul><ul><ul><li>Sample tracking and reagents ID via barcode scanner </li></ul></ul><ul><ul><li>Operator safety: closed environ., automated pipetting, controlled waste </li></ul></ul><ul><ul><li>CE-IVD (exclusive) </li></ul></ul>EKSTRAKSI NASBA
    34. 38. The EasyMag instrument <ul><li>Performances: </li></ul><ul><ul><li>Nucleic acid recovery: </li></ul></ul><ul><ul><li>As efficient as manual Boom </li></ul></ul><ul><ul><li>70% yield on average for 500 DNA virus particles in plasma, serum , CSF, blood, sputum (in publication) </li></ul></ul><ul><ul><li>HIV-1 RNA detected down to 10 UI /ml with excellent reproducibility (Kreuwel et al ., 2004) </li></ul></ul><ul><ul><li>«The miniMAG produced the highest quantity of nucleic acids and the best precision among the three systems Magnapure compact and biorobot EZ» for polyomavirus in urine. «…the best for specimens containing a low number of micro-organisms of interest » (Tang et al ., JCM 4830, sep 2005) </li></ul></ul><ul><ul><li>T </li></ul></ul><ul><ul><li>CV < 2% on Ct values of real-time PCR </li></ul></ul><ul><ul><li>Specimen to specimen contamination: </li></ul></ul><ul><ul><li>< 0.1 ppm alternating high positive viral specimen (> 10 8 copies/ml) and negative, using NASBA detection </li></ul></ul>EKSTRAKSI NASBA
    35. 39. <ul><ul><li>NASBA is available as a research tool through the “basic kit” set of Nasba reagents used on the EasyQ instrument </li></ul></ul><ul><ul><ul><li>Contains all the components for isolation, amplification and detection, except for the specific primers and probes </li></ul></ul></ul><ul><ul><li>Increasing number of papers describing the use of Nasba: </li></ul></ul><ul><ul><ul><li>Clinical RNA viruses (HIV-1, HCV, Influenza, Enterovirus, Poliovirus, West Nile, Dengue, Rabies, ………) </li></ul></ul></ul><ul><ul><ul><li>Food / water related viruses </li></ul></ul></ul><ul><ul><ul><li>Bacteria, fungi and parasites using various targets (rRNA, specific) that result in extremely sensitive tests </li></ul></ul></ul>
    36. 40. Nasba & sensitivity <ul><ul><li>Enterovirus: 11 copies / µl of extract (95% hit rate) targeting 5’NCR-product claim </li></ul></ul><ul><ul><li>Bacillus cereus: 0.1 eq-cell from total RNA targeting EF-tu </li></ul></ul><ul><ul><li>2 cells in Nasba after lysis + RNA purif. (internal data) </li></ul></ul><ul><ul><li>Listeria monocytogenes : 0.1 eq-cel from total RNA in 20 µl Nasba in 30 min. </li></ul></ul><ul><ul><li>estimated to be 10 copies of hlyA target (Bulteau et al ., ASM 2005) </li></ul></ul>
    37. 41. Nasba & Quantification <ul><li>Performance Claims of HIV-1 viral load, product FDA-approved </li></ul><ul><li>HIV-1 viral load values In International Units (IU) per ml </li></ul><ul><li>Sensitivity (sample 1 and 2 ml of human plasma </li></ul><ul><ul><li>cut-off 25 IU/ml (1.0 ml) and 12.5 IU/ml (2.0 ml) </li></ul></ul><ul><ul><li>50% detection rate: 62 IU/ml (1.0 ml) and 36 IU/ml (2.0 ml) </li></ul></ul><ul><ul><li>95% detection rate: 357 IU/ml (1.0 ml) and 141 IU/ml (2.0 ml) </li></ul></ul><ul><li>Linear dynamic range from 50 – 3,000,000 IU / ml: 5 Log </li></ul><ul><li>Specificity 99.7% (95% confidence limit 98.5 – 100%) </li></ul><ul><li>HIV-1 subtype reactivity HIV-1 group M subtypes A to J </li></ul><ul><li>Accuracy < 0.2 log deviation from 1st international standard </li></ul><ul><li>Precision 0.12 log in the range 1,000 – 3,000,000 IU / ml </li></ul><ul><li>0.28 log in the range <= 1,000 IU / ml </li></ul>
    38. 43. PCR
    39. 46. Indikasi Pemeriksaan viral load pada HIV Indikasi klinik Informasi Penggunaan Sindroma HIV akut Menegakkan Dx (tes serologi antibodi HIV negatif atau meragukan) Diagnosis Evaluasi awal terhadap infeksi HIV yg baru ditemukan Data dasar viral load Keputusan mulai atau menunda terapi Setiap 3-4 bulan pd ODHA yg tidak mendapat terapi Perubahan dlm viral load Keputusan utk memulai terapi 2-8 minggu setelah memulai terapi ARV Penilaian awal terhadap kemanjuran obat Keputusan utk melanjutkan atau mengubah terapi 3-4 bulan setelah permulaan terapi Efek maksimal terapi Keputusan utk melanjutkan atau mengubah terapi Setiap 3-4 bulan pd ODHA dg terapi Kesinambungan efek dari ARV Keputusan utk melanjutkan atau mengubah terapi Peristiwa klinik atau penurunan sel CD4 yg bermakna Hubungan dg viral load yg berubah atau menetap Keputusan utk melanjutkan,memulai atau mengubah terapi

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