The document discusses viral load testing using NASBA technology. NASBA is a nucleic acid amplification technique used to measure the amount of virus in a patient's blood or body fluid sample. It has several benefits over PCR including being isothermal, having less risk of contamination, and allowing for multiplex detection. The document provides details on using NASBA to test viral load in HIV patients, including the sample extraction and amplification process. NASBA testing for viral load can help diagnose acute infections, evaluate virus treatment effectiveness, and determine patient prognosis.

1. Tutor DivisiImunologiPEMERIKSAAN VIRAL LOADTEKNOLOGI NASBAFebtarini. R,dr/ EndangRetnowati,dr,MS,Sp.PK (K)Rabu, 8- September- 2010

2. PENDAHULUANViral load : *Jumlahturunan virus dalamdarahataucairantubuh yang diukurmelaluipemeriksaanasamnukleat virus. *Petunjukaktivitas virus dlmdarah/cairantubuh,jikaaktivitas virus tinggididapatkanviral loadtinggisemakinberatkerusakanygditimbulkanterhadapsistemimun, kerusakansel1

3. IndikasiPemeriksaanViral load :Infeksiakut : membantumenegakkan diagnosissecaradiniEvaluasiinfeksi virus : menentukanawalterapi & pemantauanterapiMenentukan prognosis 2

4. Metodepemeriksaanasamnukleat virus 1. PCR : Polymerase chain reaction (RT-PCR, rt-PCR) 2. bDNA : Branched chain DNA assay 3. NASBA : Nucleic acid sequence-based amplification3

5. NASBA (Nucleic acid sequence-based amplification)Teknologiujiamplifikasi virus RNA atau DNAPengembanganmetode ELISATerdiridariproses : 1. Pre ekstraksi 2. Ekstraksi 3. Amplifikasidandeteksi4

6. 5

7. Whole bloodHIV-1CMVFactor V LeidenCellsHIV-1 HTLV-1CMVPlasmaSerumHIV-1HTLV-1HCVHBVHHV-6Sputum/Saliva HRVInfluenza Virus Measles virusCSFHIV-1JCVLCMVmumps virusCervical swabsHIV-1HPVSemenHIV-1FaecesHIV-1CMVastrovirusUrineMeaslesvirusLymph nodes/skin biopsiesHIV-16

8. Menggunakan 3 enzim :Reverse transcriptase (Avian myeloblastosis virus /AMV) RNA atau DNA templatemensintesis DNA2. RNAse Hybrid ( E. coli)3. T7 RNA polymerase (phage T7) : DNA templatesintesis RNA7

9. Contoh: Pemeriksaanviral load HIVdengansampeltetesandarahkering1. persiapansampeldanreagen1 ml darah vena + EDTAPipet & 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 Laboratorium8

10. 2. EKSTRAKSIKertas dikeluarkanMenyiapkan larutan premix : CAL + 550 µl CAL diluent + 550 µl larutan silika (vortex)9

11. Function:Chemistry:Sample + Lysis buffer1.High [GuSCN]neutral pHRelease of nucleic acidStabilization Nucleic acidHigh [GuSCN]neutral pH2.Binding of nucleic acidSilicaWash bufferPurification of nucleic acid, removal of inhibitors3.Proteins and LipidsRecover nucleic acid insmall volume Low [GuSCN] pH > 8.0Elution buffer4.Silica2. EKSTRAKSIComplex biological sampleMagnetic separationPure nucleic acid10

12. 2. Lanjutan fase ekstraksiLarutan premix + larutan lysis buffer & sampel100 µl2 µl100 µlvorteksInkubasisuhu kamar,10 menitSentrifus 1500g, 15 detikSupernatandibuang

13. endapan : silika & RNA+ washing buffer 1400 µlDiaduk dg pipet11

14. Pipet 1,5 ml (washing buffer 1 + silika-RNA)Cuciselama 30 detik (menu STEP 1, magnet on)Buangsupernatan, tambahkan 400 µl washing buffer 1 (mengulangipencucian)Buangsupernatan, tambahkan 500 µl washing buffer 2 (ulangi 2X)Buangsupernatan, tambahkan 500 µl washing buffer 3, cuciselama 15 detik12Buangsupernatan = eluate

15. 15 µl Eluate + 25 µl elution bufferInkubasiselama 5 menit, suhu 600 C TempatkantabungdirakmagnetikBukatabung, danambil 15 µl extracted sample (eluate & elution buffer)keeight tube stripdidalam area amplifikasi ,beriidentitas (ataujikatidaklangsungdikerjakan, eluatebisadisimpanpadasuhu 40C)13

16. 15 µl (eluate&elution buffer) + 20 µl primerInkubasi (di inkubator), suhu 410C selama 4 menitLyophilized primers + 180µl primerdiluentsvortexambil 20 µlMenyiapkanenzim = kemasanenzim (lyophilized enzymes) + 45 µl enzyme diluentsmencampurkannyadengancaraditapping 3 detikinkubasisuhukamar 15 menitsentrifus 15 detikinkubasi (diinkubator) selama 15 menit ambil 5 µl larutanenzimtsbletakkanpadatutupeight tubesentrifus 2 detik, 1500 rpmtapping 3 detiksentrifus 2 detik keAnalyzer (real time PCR and detection)14

17. 15

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20. 3. Real time PCRFase Linier( 5 menit )Faseamplifikasi( 90 menit )sense RNARNase Holigo P1oligo P1Reverse TranscriptaseRTRTRNase H &oligo P2oligo P2Reverse TranscriptaseT7 RNAPanti-sense RNAT7 RNA polymerase18

21. FGFQQ-Alat dinyalakan 2 menit (saat enzim diletakkan pd tutup eight tubes-Analizer mendeteksi 10- 107 turunan HIV RNA / ml darah-menggunakan 1 suhu(isothermal) 410C-Alat tersambung dg komputer & printerDeteksiMolecular beaconDNA probeNASBA RNA amplicon“open”“closed”SIGNAL: ++NO SIGNAL19

22. FFFFQQQQAmplifikasi real time PCR & deteksisense RNARNase Holigo P1oligo P1Reverse TranscriptaseRTRTRNase H &oligo P2oligo P2Reverse TranscriptaseT7 RNAPanti-sense RNAT7 RNA polymeraseMolecular beacon hybridization20

23. FFFFQQQQDeteksi (Molecular beacon)Fluorescent signal (real-time)NASBA reactionNormalized fluorescence0102030405060Time (minutes)21

24. Fluorescence Analyzer, Optics (Schematic layout)22

25. IncubatorDesktop computerAnalyzerStrip centrifuge23

26. InterpretasihasilPasien 1 = 840.000 kopi/ml darah Pasien 5 = 110.000 kopi/ml darahPasien 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 darahPasien 4 = TND Pasien 8 = 3300 kopi/ml darah24

27. SELAMAT IDUL FITRI1431 HMOHON MAAF LAHIR - BATIN

32. BenefitsHigh-throughput (n=48)Minimal hands-on-time (30 min for 48 samples)Fast-time to result (30 to 60 min.) for amplification / detection steps, sample prep. dependent on sampleInternal control (also used as calibrator for QT) due to duplex amplificationNo post-amplification step, no carry-over contaminations (closed tube)Combining various applications in one runAllows panel approach based on clinical signs e.g. pneumonia on sample-related risks e.g. water, food

33. Lysis buffer = Chaotropic agent Gu SCN/ guanidine thiocyanatePerbedaan NASBA dan PCR :

35. Performance Claims of HIV-1 viral load, product FDA-approvedHIV-1 viral load values In International Units (IU) per mlSensitivity (sample 1 and 2 ml of human plasmacut-off 25 IU/ml (1.0 ml) and 12.5 IU/ml (2.0 ml)

36. 50% detection rate: 62 IU/ml (1.0 ml) and 36 IU/ml (2.0 ml)

37. 95% detection rate: 357 IU/ml (1.0 ml) and 141 IU/ml (2.0 ml)Linear dynamic range from 50 – 3,000,000 IU / ml: 5 LogSpecificity 99.7% (95% confidence limit 98.5 – 100%)HIV-1 subtype reactivity HIV-1 group M subtypes A to JAccuracy < 0.2 log deviation from 1st international standard Precision 0.12 log in the range 1,000 – 3,000,000 IU / ml 0.28 log in the range <= 1,000 IU / ml

38. Amplifikasi (real-time PCR) & DeteksiPre ekstraksiEkstraksiAmplification& DetectionObtain a high number of copies labelledfor detection(real time)N.A. Capture, purification & concentrationGeneric or specific capture of target DNA/RNA

39. Remove of potential amplification step inhibitors

40. Concentrate to increase kinetic and decrease volume of reagentsLysis of µ-org.Disrupt target µ-organism for Nucleic Acid extraction (yield, sensitivity)- Stabilization of N.AResults for a few targets Mono-detectionPre-processingInactivation

41. Viscosity

42. Concentration

43. Filtration

44. Standardization for difficult sampleSample Blood

45. Water

46. Filterable productAir…Results for high number of targets Multi-detectionHybridizationLabellingWashing on DNA Chip

47. Whole bloodHIV-1CMVFactor V LeidenSputum/Saliva HRVInfluenza Virus Measles virusMycobacterium tuberculosisMycoplasma pneumoniaeLegionella pneumoniaeCellsHIV-1 HTLV-1CMVPlasmaSerumHIV-1HTLV-1HCVHBVHHV-6Neisseria gonorrhoeaeNeisseria meningitidisCSFHIV-1JCVLCMVmumps virusMeasles virusMycobacterium tuberculosisBorrelia burgdorferiLeptospira interrogansSemenHIV-1Cervical swapsHIV-1HPVChlamydia trachomatisFaecesHIV-1CMVastrovirusMicrospordia Campylobacter jejuniCampylobacter coliLymph nodes/skin biopsiesHIV-1Mycobacterium lepraMycobacterium tuberculosisUrineMeaslesvirusChlamydia trachomatisNeisseria gonorrhoeae

48. EKSTRAKSI NASBAMain features:High purity of eluate (one of the best)

49. magnetic silica with high binding capacity

50. optimum mixing through disposable design

51. Compatible with the use of an internal control

52. A single set of reagent and 2 disposables / sample: 1 protocole for all extractions

53. 3 X 8 samples extracted in 40 or 60 min. (the fastest). Up to 240 extrac. / day.

54. Hands-on-time <15 min.

55. Input sample vol.: 10 µl up to 1 ml

56. Output vol.: 110 µl down to 12 µl [80X]

57. Flexibility (N.A. /vol. / applications / workflow)

58. Intuitive software with touch screen interface

59. Sample tracking and reagents ID via barcode scanner

60. Operator safety: closed environ., automated pipetting, controlled waste

61. CE-IVD (exclusive)EKSTRAKSI NASBAPerformances:Nucleic acid recovery:As efficient as manual Boom

62. 70% yield on average for 500 DNA virus particles in plasma, serum , CSF, blood, sputum (in publication)

63. HIV-1 RNA detected down to 10 UI /ml with excellent reproducibility (Kreuwel et al., 2004)

64. «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) TCV < 2% on Ct values of real-time PCRSpecimen to specimen contamination:< 0.1 ppm alternating high positive viral specimen (> 108 copies/ml) and negative, using NASBA detectionThe EasyMag instrument

65. NASBA is available as a research tool through the “basic kit” set of Nasba reagents used on the EasyQ instrumentContains all the components for isolation, amplification and detection, except for the specific primers and probesIncreasing number of papers describing the use of Nasba:Clinical RNA viruses (HIV-1, HCV, Influenza, Enterovirus, Poliovirus, West Nile, Dengue, Rabies, ………)Food / water related viruses Bacteria, fungi and parasites using various targets (rRNA, specific) that result in extremely sensitive tests

66. Nasba & sensitivity

67. Nasba & Quantification Performance Claims of HIV-1 viral load, product FDA-approvedHIV-1 viral load values In International Units (IU) per mlSensitivity (sample 1 and 2 ml of human plasmacut-off 25 IU/ml (1.0 ml) and 12.5 IU/ml (2.0 ml)

68. 50% detection rate: 62 IU/ml (1.0 ml) and 36 IU/ml (2.0 ml)

69. 95% detection rate: 357 IU/ml (1.0 ml) and 141 IU/ml (2.0 ml)Linear dynamic range from 50 – 3,000,000 IU / ml: 5 LogSpecificity 99.7% (95% confidence limit 98.5 – 100%)HIV-1 subtype reactivity HIV-1 group M subtypes A to JAccuracy < 0.2 log deviation from 1st international standard Precision 0.12 log in the range 1,000 – 3,000,000 IU / ml 0.28 log in the range <= 1,000 IU / ml

71. PCR

74. Indikasi Pemeriksaan viral load pada HIV