The application of molecular technology in the HBV/HCV/HIV

Transcription

1 The application of molecular technology in the development of donor screening product for HBV/HCV/HIV Shanghai Kehua Bio-engineering Co.,Ltd. Sherry M Wang

2 General Information for Donor Screening in China------current method TO PREVENT BLOOD TRANSMISSION OF INFECTIOUS DISEASES mainly by immunoassay (ELISA), such as HBV HCV HIV TP-ELISA in China; protein is the testing target, mostly are antigen(hbv) and antibody tests; for large scale screening, require high throughput and low cost;

3 General Information for Donor Screening in China------current method Disadvantage of Immunoassay: False negative for window period donor sample (can not tbe dt detected tdby antibody, tibd bt but can be dt detected tdby Nucleic Acid Testing,NAT); False negative for mutant virus; False negative for immune-silence.

4 window period of HBV infection

5 window period of HCV infection

6 window period of HIV infection

7 General Information for Donor Screening in China------new method ( NAT ) Advantage of NAT Shorten window period : by ~45% for HIV ~89% for HCV ~50% for HBV Increase sensitivity; Good specificity; Not influenced by virus subtype and mutant; High requirement for lab environment and personnel; Relative high cost than immunoassay.

8 General Information for NAT Screening around the world In England: NAT was started in 1996(48-96 pool),and the infection risk for HCV was reduced by 90% during , by 10% for HIV; In Japan:HBV/HCV/HIV NAT started in 1999 (50 pool); In US: HCV/HIV NAT started in 1999 (16-24 pool),the infection risk of HCV and dhiv1 HIV-1reduced d from 1/276,000 and 1/ to1/ ; In Hong Kong: start routine screening by NAT in 2003 in blood centers ; In Australia: National Serology Reference Laboratory, Australia (NRL) WHO Collaborating Center for HIV perform evaluation study for NAT reagents.

9 Incidence of window period around the world COUNTRY HBV HCV HIV Canada 1: : : France 1: : : Germany 1: : : Japan(50 pool) 1(~1/52,000) 1:(~1/350,000) 1:(~1/2670,000) US(24 pool) (~1/180,000) (~1/230,000) 12(~1/3090,000) H.K 1:32786 (~1/33,000) 1:5456 (~1/5000) 1:222 (~1/200)

10 General information of NAT assay for HBV/HCV/HIV (real time PCR assay) Using magnetic beads for nucleic acid extraction, and Taqman real-time PCR for nucleic acid amplification; Screening donor for HBV/HCV/HIV at the same time; An integrated system including sample scanning,pooling, nucleic acid extraction, and PCR set up; High throughput: ~750 donors in 5 hrs,and another 750 donors in another 2-3 hrs; Contain in vitro constructed positive control to QC all the steps from sample extraction to amplification; Contain internal control to control false negative; Testing procedure: testing for pooled sample (8 or 24 pool) if positive, testing for individual sample confirm the positive individual sample for plasma manufacturer: for blood center: 8 pool 24 pool

11 PRODUCT DESIGN 1 SAMPLE SCANNING 2 SAMPLE POOLING 3 NUCLEIC ACID EXTRACTION 4 NUCLEIC ACID AMPLIFICATION 5 RESULT ANALYZE 1 全自动标本管条形码 3 磁珠法核酸提取 PCR plate COOLING 全自动核酸提取 ( 初筛 拆分 确认 ) 半自动核酸提取 ( 拆分 ) 扫描 报告记录 样品跟踪表等 TIP REAGENT S Working station that integrated with magnetic platform and cooling, heating system

12 SAMPLE SCANNING

13 SAMPLE POOLING AND EXTRACTION

14 PCR AMPLIFICATION ABI 7300 /7500 MJ

15 Software Control for Hamilton Working Station

16 ANALYTICAL PERFORMANCE SENSITIVITY:(requirement from US FDA and EU Country for HCV HIV: donor:5000iu/ml,test:100iu/ml) WHO Standard (HBV;HCV;HIV= 7.0; 119.8; 93.5 IU/ml) In house panel (HBV;HCV;HIV= 9.97; 88.5; 87.8 IU/ml) National panel (HBV;HCV;HIV= 5; 50; 50 IU/ml by NICPBP) WHO-Australia NRL evaluation result for 2008,2009 (can detect HCV: 30 IU/mL;HIV: 30 c/ml) SUBTYPE DETECTION: HBV: A/B/C/D 均等效检出 ; HCV: 的 WHO HCV1-6 型均可等效检出 ; HCV: WHO HIV-1 A B C D E F G H and O subtype 均可检出 SPECIFICITY STABILITY: 14 months stability in required storage temp.

17 ANALYTICAL SPECIFICITY Interfering microorganism and nucleic acid Testing item: HPV CMV UU NG CT HSV Flu virus bird flu H5 Result:all negative Interfering substance H7 H9 NDV TB HEV HTLV MMLV Ecoli JM109 PUC18 carrier DNA yeast t RNA 16S r RNA Hela cell etc. Testing item : bilirubin up to100mg/ml TG up to1g/dl Hb 1g/dl albumin up to 6g/d Aspirin up to10mg/ml ascorbic acid to20mg/dl Result: heparin 100U/ml EDTA 至 8mg/dl all negative Healthy donor(3000 donors) Testing item : HBV HCV HIV; Result: all negative National panel from NICPBP Testing item : Negative panel Result: all negative

18 Australia NRL EQAS Participate NRL External Quality Assessment Program (NATA4315) since 2008; 3 times evaluation every year, 100% accordant with consensus result; Can detect low level of HCV and HIV (30 IU/ml).

19 Sample numbers: Clinical study sample distribution ib i Regional distribution: Coast area Undeveloped region 164,231 samples (including 163,634 donor samples) High-risk h ikregion for infectious diseases Population distribution: Healthy donor,volunteer Regular plasma donor High risk population such as druggy

20 Clinical study Summary result(immunoassay negative,nat positive) i sample source Plasma manufacturer A Plasma manufacturer B Sample numbers Donor numbers HBV-DNA Positive samples (donors) HCV-RNA Positive samples (donors) HIV-RNA Positive samples (donors) (3) (3) 0 1(1) Subtotal t 90,564 32,455 11(6) 0 1(1) Blood center A* (2) 0 0 Blood center B (3) 1(1) 0 Blood center C (11) 1(1) 0 Subtotal 73,070 73,070 16(16) 2(2) 0 Total 163, , (22) 2(2) 1(1)

21 Clinical study HIV window period sample SOUR NAT HIV NAT HIV ELISA WB Collecting SAMPLE ID. CE (KHB) (ROUCHE) Anti-HIV RESULT date N00093* / Plasma manufa -cturer B N00070* / N00064* / N / N00089 / / + GP160/P NICPBP / / / Total 6(2) 2 2 / / / *tracing sample

22 Clinical study HCV window period sample SAMPLE SOURCE SAMPLE ID. NAT HCV (KHB) NAT HCV (ROCHE) ELISA Anti-HCV COLLECTIN G DATE BLOOD CENTER B BLOOD CENTER C Ljm* Total / / *tracing sample,alt = 700 U/L

23 SOURCE PLASMA MANUFACTURER A PLASMA MANUFACTURER B BLOOD CENTER A SAMPLE ID. Clinical study HBV window period sample NAT (KHB) NAT (ROUCHE) ELISA HbsAg ELISA HbsAb ELISA HbeAg ELISA HbeAb ELISA HbcAb COLLECTI NG DATE B B * B * B * B * F F F N A A90-2* BLOOD CENTER B BLOOD CENTER C / d* + / d* / / d* / / d* + / d* + / Total (positive sample no.) / / / / *TRACING SAMPLE

24 source PLASMA MANUFACTERER A PLASMA MANUFACTURER B BLOOD CENTER A BLOOD CENTER C Total (positive sample no. Clinical study current HBsAg screening false negative Sample ID NAT NAT ELISA ELISA ELISA ELISA ELISA COLLECTING KHB ROCHE HBsAg HBsAb HBeAg HBeAb HBcAb DATE A A A A A N00059* N00018* N00021* N00041* N00030* N N H H40-2* d* / d* + / / / / / / / *TRACING SAMPLE

25 Clinical study (summary result) Risk-residual for current donor screening method in China Based on this clinical study in related region and population,the risk residual for current immunoassay screening in China is now reach to: HBV: 22: (~ 2.2/10,000); 2/10 000); HCV: 2: (~ 0.2/10,000); HIV: 1: (~0.1/10,000)

26 KHB With a proven IVD manufacturing experience for nearly 30 years; Being No.1 by revenue and profit among diagnostic reagents providers in China; Listed by WHO, UNICEF for bulk procurement program, and the only Chinese Supplier for Clinton Foundation; Has 123Production approvals(sfda) and 55 CE-marked products; Products including reagents, instrument and software;

27 KHB Reagent Diagnostic Laboratory Instruments Blood Collection System Software Rapid Tests ELISA Kits Clinical Chemistry Kits PCR Kits Micro-plate Reader Micro-plate Washer Clinical Chemistry Analyzer Blood Collection Tubes Needles LIS System PCR Amplifier Nucleic Acid Extractor

28 Thank You!