I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - Trn Võn Chi NG DNG K THUT NAT (NUCLEIC ACID) PHT HIN SM S Cể MT CA VI RT HIV, HBV, HCV NGI CHO MU LUN VN THC S KHOA HC H Ni - 2015 Trần Vân Chi Luận văn Thạc sĩ Khoa học I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - Trn Võn Chi NG DNG K THUT NAT (NUCLEIC ACID) PHT HIN SM S Cể MT CA VI RT HIV, HBV, HCV NGI CHO MU Chuyờn ngnh: Mó s: Vi sinh vt hc 60420107 LUN VN THC S KHOA HC Ngi hng dn khoa hc: PGS.TS Bch Khỏnh Hũa TS Trn Vn Tun XC NHN HC VIấN CHNH SA THEO GểP í CA HI NG Giỏo viờn hng dn Ch tch hi ng chm lun thc s khoa hc TS Trn Vn Tun PGS.TS Bựi Th Vit H H Ni - 2015 Trần Vân Chi Luận văn Thạc sĩ Khoa học LI CM N Li u tiờn, cho phộp tụi c by t lũng bit n sõu sc nht ti PGS.TS Bch Khỏnh Hũa; ngi thy ó tn tỡnh hng dn, giỳp tụi lm lun Ngi thy a tụi n vi Huyt hc Truyn mỏu, ngi thy ó truyn cho tụi nim am mờ vi Huyt hc Truyn mỏu Ngi thy ó bi dng cho tụi nhng c tớnh trung thc, cn mn ca mt ngi lm cụng tỏc nghiờn cu t nhng ngy u tiờn bc vo ngh Tụi xin by t lũng cm n sõu sc n BSCKII Phm Tun Dng ngi ó ng viờn cho tụi i hc cao hc v sut quỏ trỡnh hc Ngi ó tn tỡnh giỳp , dỡu dt tụi cụng tỏc Tụi by t lũng cm n sõu sc n TS Trn Vn Tun ngi ó hng dn ch dn, giỳp tụi lm lun Tụi cng xin chõn thnh cm n s giỳp on kt ca anh ch em ng nghip Khoa Xột nghim sng lc mỏu ó giỳp tụi hon thin s liu, mi iu kin thun li nht cho tụi sut quỏ trỡnh hc v lm lun Tụi xin gi li cm n ti: Ban lónh o Vin HHTMTW ó cp kinh phớ cho tụi i hc, cỏc khoa phũng hnh chớnh ó giỳp tụi hon thnh th tc hc tp, Khoa Di truyn v sinh hc phõn t, Khoa Thalassemia, Khoa Hemophilia, khoa HIV vin v sinh dch t ó cho phộp v giỳp tụi xột nghim nh lng virus, ton th Khi truyn mỏu ó to iu kin thun li cho tụi thc hin ti Tụi xin gi li cm n ti Phũng sau i hc, Khoa Sinh hc, B mụn Vi sinh hc ca trng i hc Khoa hc T nhiờn ó giỳp tụi hon thnh cỏc th tc lun Tụi xin by t lũng bit n ti cỏc bc b m , b m chng, chng ó giỳp ng viờn, dnh tỡnh cm v iu kin thun li cho tụi hon thnh lun Tụi xin by t lũng cm n n ngi hin mỏu ó cho tụi nhng s liu quý bỏu Hc viờn Trn Võn Chi Trần Vân Chi Luận văn Thạc sĩ Khoa học M U Vi rỳt l mt nhng nguyờn nhõn gõy mt s bnh nghiờm trng ngi núi chung Riờng vi rỳt viờm gan B gi tt l HBV (Hepatitis B virus ), vi rỳt viờm gan C gi tt l HCV (Hepatitis C virus), vi rỳt gõy suy gim dch ngi HIV (Human immunodeficiency virus) l vi rỳt gõy bnh thụng qua ng mỏu Cỏc vi rỳt ny bin i liờn tc v cu trỳc v h gen trn thoỏt cỏc loi sinh phm xột nghim hu qu l tng t l lõy nhim cng ng Phng phỏp xột nghim dựng cho sng lc HIV, HBV v HCV lõy qua ng truyn mỏu l xỏc nh tỏc nhõn gõy bnh giỏn tip da trờn kt qu ca phn ng dch khỏng nguyờn-khỏng th u im ca xột nghim l n gin, tn ớt thi gian, giỏ thnh hp lý, song phng phỏp ny cú nhc im l giai on ca s ú khỏng th hoc khỏng nguyờn cha t ngng phỏt hin, vy xột nghim s cho kt qu õm tớnh Bờn cnh ú, tng tỏc khụng c hiu ca khỏng th hoc khỏng nguyờn vi nhng bt thng protein khỏc mỏu cú cu trỳc gn ging vi khỏng nguyờn hoc khỏng th cú th dn n kt qu dng tớnh gi Cựng vi s phỏt trin ca k thut sinh hc phõn t, k thut NAT ó c a vo s dng, k thut ny cú nhy cao cho phộp phỏt hin v nhõn bn c hiu theo hm m cỏc trỡnh t ớch ca tỏc nhõn gõy bnh t mt lng nh vi rỳt, ú, cho phộp phỏt hin sm v chớnh xỏc cỏc tỏc nhõn gõy bnh Hn th na, NAT cú th c s dng phỏt hin ng thi HIV, HBV v HCV thụng qua mt xột nghim thi gian l 4-5 gi, m bo an ton cho n v mỏu truyn Trờn Th gii, cỏc nc phỏt trin, m bo sng lc mỏu Anh, Phỏp, M, c thc hin xột nghim song song gia phng phỏp giỏn tip khỏng nguyờn khỏng th v xột nghim NAT cho n v mỏu t cui nhng nm 1990 u nhng nm 2000 Nm 1997, Hi ch thp c ó s dng PCR cho mc ớch sng lc HCV RNA cho ngi hin mỏu, tip theo l Nht Bn v Scotland Sau ú vo thỏng 3/1998 ngi ta ó gii thiu NAT cho xột nghim HCV RNA[50] Trần Vân Chi Luận văn Thạc sĩ Khoa học Vit Nam, hu ht cỏc phũng xột nghim sng lc HIV, HBV v HCV u s dng phng phỏp xột nghim dch khỏng nguyờn khỏng th hay cũn gi l xột nghim huyt hc, xột nghim NAT cha c s dng Nm 2008 ti cp B v ng dng NAT cho xột nghim sng lc mỏu t kt qu tt [3] n nm 2015, Vin HHTMTW thc hin theo Thụng t 26 TT-BYT ngy 16/9/2014 Hng dn hot ng truyn mỏu, trin khai xột nghim NAT m bo an ton truyn mỏu Xut phỏt t thc t trờn, chỳng tụi tin hnh nghiờn cu ng dng k thut NAT (nucleic acid) phỏt hin sm s cú mt vi rỳt HIV, HBV, HCV ngi cho mỏu nhm hng ti cụng tỏc xột nghim sng lc sm cỏc tỏc nhõn lõy qua ng truyn mỏu ny mt cỏch chớnh xỏc, m bo an ton, cht lng cho mỏu v ch phm s dng iu tr Mc tiờu nghiờn cu ca ti - Kho sỏt t l HBsAg, anti HCV, HIVAgAb phn ng trờn n v mỏu hin c xột nghim dch bng k thut CMIA, ECLIA T l nhim HBV, HCV, HIV bnh nhõn truyn mỏu nhiu ln - T l HBV, HCV, HIV phn ng phỏt hin bng k thut NAT (nucleic acid) trờn n v mỏu hin m xột nghim dch khụng phỏt hin c, theo dừi ngi hin mỏu cú phn ng k thut NAT, phỏt hin sm h gen vi rỳt nhm b tr cho xột nghim dch khỏng nguyờn- khỏng th Trần Vân Chi Luận văn Thạc sĩ Khoa học KT LUN Xột nghim sng lc khỏng nguyờn khỏng th bng k thut CMIA, ECLIA vi t l HBsAg l 0,75%; 0,79%; anti HCV l 0,43%, 0,24%, HIV AgAb l 0,1%, 0,23% Bnh nhõn nhn mỏu nhiu ln cú t l nhim HBV l 5,9% nhim HCV l 23,89% Xột nghim NAT (nucleic acid) cú t l HBV, HCV, HIV tng ng l 1:1086 ; 1:192221; 2:192221 (HBV 0,92, HCV 0,0052, HIV 0,01) Nghiờn cu theo dừi c ngi hin mỏu mi nhim HIV giai on ca s huyt thanh, ngi mi nhim HCV giai on ca s, ngi nhim HBV giai on ca s Trần Vân Chi Luận văn Thạc sĩ Khoa học KIN NGH Thc hin xột nghim sng lc mỏu bng k thut NAT b tr cho k thut dch khỏng nguyờn- khỏng th Nghiờn cu mt s trng hp nhim HBV th n OBI Trần Vân Chi Luận văn Thạc sĩ Khoa học TI LIU THAM KHO TI LIU TING VIT V Thựy An v cng s (2012), Tỡnh hỡnh sng lc cỏc bnh nhim trựng qua ng mỏu ngi hin mỏu tỡnh nguyn khu vc ụng-Nam b ti Trung tõm truyn mỏu Ch Ry t 2009-2011, Tp Y hc Vit Nam, 396, 272-280 Phm Tun Dng v cng s (2014), Kt qu xột nghim sng lc HBV, HCV, HIV, giang mai ngi hin mỏu ti Vin Huyt hc Truyn mỏu TW nm 20122013, Tp Y hc Vit Nam, thỏng 10 s c bit, 45-50 Bch Khỏnh Hũa v cng s (2007), Xột nghim acid nucleic NAT phỏt hin sm vi rỳt HIV, viờm gan B, viờm gan C ngi cho mỏu, Tp nghiờn cu y hc, 4/51, 41-43 Nguyn Th Thu Hin v cng s (2012), Kt qu sng lc cỏc bnh nhim trựng lõy qua ng truyn mỏu ngi hin mỏu ti Hi Phũng 2008-2011, Tp Y hc Vit Nam, 396, 280-286 Lờ Th Hng v cng s (2012) Kt qu sng lc khỏng th HIV, khỏng th HCV, HBsAg, giang mai, st rột ngi hin mỏu tỡnh nguyn ti Bnh vin a khoa tnh Hũa Bỡnh 3/2007-3/2012, Tp Y hc Vit Nam, 396, 286-292 Phan Th Minh Hng v cng s (2009), Nghiờn cu tỡnh hỡnh nhim HIV, HBV, HCV bnh nhõn truyn mỏu nhiu ln ti vin Huyt hc Truyn mỏu trung ng nm 2009, Tp Y hc Vit Nam, 373; 370-374 inh Duy Khỏng, Chu Hong H, Phm Thỳy Hng, Nguyn Thanh Thy, Nụng Vn Hi, ỏi Duy Ban (1997), Phõn lp tỏch dũng v xỏc nh trỡnh t gen khỏng nguyờn b mt ca vi rỳt viờm gan B t u ca bnh nhõn viờm gan, Y hc Vit Nam, 8: 12-17 Trnh Th Minh Liờn (1998), Xỏc nh ADN ca vi rỳt viờm gan B huyt bng phn ng chui Polymerase (PCR), Y hc thc hnh, 3: 26-28 Nguyn ng Mnh, Bựi i, Nguyn Trng Chớnh, Lờ Vn Don (2001), Tỡnh hỡnh nhim vi rỳt viờm gan C (HCV) cỏc bnh nhõn viờm gan, x gan v ung th gan, Tp thụng tin Y dc, 10: 30-33 Trần Vân Chi Luận văn Thạc sĩ Khoa học 10 V Tng Võn, Nguyn Th Hnh, Nguyn Xuõn Quang, Nguyn Ngc Dip, Lờ Khỏnh Trõm (2003), Bc u ỏnh giỏ tỡnh hỡnh nhim trựng phi hp vi rỳt viờm gan B v vi rỳt viờm gan C trờn bnh nhõn HIV (+) ti bnh vin Bch Mai, Tp nghiờn cu Y hc, 23: 64-69 11 o Ngc Tuyn v cng s (2010), Tỡnh hỡnh xột nghim cỏc tỏc nhõn lõy qua ng truyn mỏu ti Bnh vin Truyn mỏu Huyt hc tp.HCM, Tp y hc Vit Nam, 373, 543-547 12 Viờm gan v cỏc bnh liờn quan n gan T trang web: www.cimsi.org.vn TI LIU TING ANH 13 Abe A., Inoue K., Tanaka T., Kato J., Kajiyama N., Kawaguchi R., Tanaka S., Yoshiba M., Kohara M (1999), Quantitation of hepatitis B virus genomic ADN by real-time detection PCR, J Clin Microbiol, 37: 2899-903 14 Barbara R., Michelina N (2005), Immunology of Hepatitis B virus and Hepatitis C virus infection, Nat Immunol, 5: 215-229 15 Bukh J., Purcell RH., Miller RH (1992), Importance of primer selection for the detection of hepatitis C virus ARN with the polymerase chain reaction assay, Proc Nat Acad Sci USA, 89: 187191 16 CDC (2008), Viral Hepatitis (http://www.cdc.gov/hepatitis/index.htm) 17 Choo QL., Richman KH., Han JH (1991), Genetic organization and diversity of the hepatitis C virus, Proc Natl Acad Sci USA, 88: 24512455 18 C.Giachetti, J.M.Linnen, et all (2002), Highly Sensitive Multiplex Assay for detection of Human Immunodeficiency Virus Type and Hepatitis C virus RNA, Journal of clinical microbiology, p 2408-2419 19 Daar ES, Little S., Pitt J., Santagelo J., Ho P, Harawa N, Kemdt P, Giogi JV, Bai J, Gaut P, Richman DA, Mandel S, Nicholas S (2001), Diagnosis of Primary HIV-1 Infection, Annul Interl Med, 134: 25-29 20 Defoort J., Martin M., Casano B.(2000), Simultaneous detection of multiplexamplified human immunodeficiency virus type ARN, hepatitis C virus ARN, and hepatitis B virus ADN using a flow cytometer microsphere-based hybridization assay, J Clin Microbiol, 38:10661071 Trần Vân Chi Luận văn Thạc sĩ Khoa học 21 Dong Hee Seo, et all (2015) Occult hepatitis B virus infection and blood transfusion, World Journal of Hepatology;7(3):600-606 22 Feinberg MB (1996), Changing the natural history of HIV disease, The Lancet, 348: 239-246 23 Glick BR., Pasternak JJ (2003), Molecular Biotechnology: Principle and Applifications of Recombinant ADN, 3rd Edition, ASM Press, 199-212 24 Guidotti LG., Chisari FV (2006), Immunology and Pathogenesis of Viral Hepatitis, Annul Rev Pathol, 1: 23-61 25 Hoffman C., Rockstroh J, Kamps BS, HIV Medicine 2006 from www HIVMedicine.com 26 Highleyman L (2008), World Health Organization, UNAIDS, and UNICEF release new report on global HIV/AIDS treatment www.hivandhepatitis.com/recent/2008/060608,1-7 27 Hu Y., Shahidi A., Park S., Guilfoyle D., Irvin Hirshfield I (2003), Detection of extrahepatic Hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction, Am J Clin Pathol, 119: 95-100 28 Hwang SJ., Lee SD (1996), Hepatitis C in Southeast Asia, Medical Progress, 23: 98-201 29 Josộ Eduardo Levi, Ricardo Antonio DAlmeida Pereira (2013), One window period donation in two years of individual donor nucleic acid test screening for hepatitis B, hepatitis C and human immunodeficiency virus, Revista Brasileira de Hematologia e Hemoterapia;35(3):167-170 30 K Malm, et all (2009), Performance of three automated fourth-generation combined HIV antigen/antibody assays in large-scale screening of blood donors and clinical samples, Transfusion Medicine, 19,78-88 31 Kao JH., Chen PJ., Lai MY., Chen DS (2002), Genotypes and Clinical Phenotypes of Hepatitis B virus in patients with Chronic Hepatitis D virus Infection, J Clin Microbiol, 40: 1207-1209 32 Kato N (2001), Molecular virology of hepatitis C virus, Acta Medica Okayama 55: 33-159 33 Khalili M (2006), Coinfection with hepatitis viruses and HIV, HIV Insite Knowledge Base Chapter.-Ngun www.hivinsite.ucsf.edu Trần Vân Chi 10 Luận văn Thạc sĩ Khoa học 34 Kidd-Ljunggren K., Miyakawa Y., Kidd AH (2002), Genetic variability in hepatitis B viruses, J Gen Virol, 83: 1267-80 35 Krekulova L., Rehak V., Riley LW (2006), Structure and function of hepatitis C virus proteins: 15 years after, Folia Microbiol, 51: 665-680 36 Kuo G., Choo QL., Alter HJ., Gitnick GL., Redeker AG, Purcell RH, Miyamura T., Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, Bonino F, Colombo WS, Lee WS, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW and Houghton M (1989), An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis, Science, 244: 362-364 37 Locarnini S, M.D, (2004), Molecular virology of Hepatitis B virus, Serminars in liver disease, 24: 3-10 38 Mahoney FJ (1999), Update on Diagnosis, Management, and Prevention of Hepatitis B virus infection, Clin MicrobioL Rev, 12: 351-36 39 Marion Vermeulen, Nico Lelie (2009), Impact of individual-donation nucleic acid testing on risk of human immunodeficiency virus, hepatitis B virus, and hepatitis C virus transmission by blood transfusion in South Africa, Transfusion; 49: 1115-1125 40 Mercier B., Burlot L., and Ferec C.(1999), Simultaneous screening for HBV ADN and HCV ARN genomes in blood donations using a novel TaqMan PCR assay, J Virol Methods, 77: 1-9 41 Michael NL, Herman SA, Kwok S, Dreyer K, Wang J, Christopherson C, Spadoro JP, Young KK, Polonis V, McCutchan FE, Carr J, Mascola JR, Jagodzinski LL, Robb ML (1999), Development of calibrated viral load standards for group M subtypes of human immunodeficienvy virus type and performance of an improved AMPLICOR HIV-1 MONITOR test with isolates of diverse subtypes, J Clin Microbiol, 37: 2557-2563 42 Michael K.Hourfar, Christine Jork, et all (2008) Experience of German Red Cross blood donor services with nucleic acid testing: results of screening more than 30 million blood donations for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus, Transfusion; 48:1558-1566 Trần Vân Chi 11 Luận văn Thạc sĩ Khoa học 43 Peter M (2004), Coinfection with HIV and HBV: Diagnosis and Therapy, The PRN notebook, 9: 14-23 44 Prasun Bhattacharya (2007), Significant increase in HBV, HCV, HIV and syphilis infections among blood donors in West Bengal, Eastern India 2004-2005: Exploratory screening reveals high frequency of occult HBV infection, World J Gastroenterol; 13(27):3730-3733 45 Roche, Cobas Taq screen MPX test 46 Raymond D., Swan T (2004), Hepatitis C virus (HCV) and HIV/HCV coinfection: A critical review of research and treatment, Treatment Action Group, New York, 16: 266-288 47 Rekha Hans and Neelam Marwaha (2014), Nucleic acid testing- benefits and constraints, Asian Journal Transfusion Science; 8(1):2-3 48 R.N.Makroo, N.Choudhury, et all (2008) Multicenter evaluation of individual donor nucleic acid testing (NAT) for simultaneous detection of human immunodeficiency virus-1&hepatitis B&C viruses in Indian blood donors, Indian Journal Medical Res 127, pp 140-147 49 Satyam Arora, Veena Doda (2014), Sensitivity of individual donor nucleic acid testing (NAT) for the detection of hepatitis B infection by studying diluted NAT yield samples, Blood transfusion; 13(2):227-232 50 Silvano Wendel et al (2007), Primary screening of blood donors by NAT testing for HCV-RNA: Development of an in-house method and results, Rev Inst.Med.trop.S.Paulo 49(3):177-185 51 Soisaang Phikulsod (2009), One-year experience of nucleic acid technology testing for human immunodeficiency virus Type 1, hepatitis C virus, and hepatitis B virus in Thai blood donations, Transfusion;49:1126-1135 52 Triques K., Coste J., Perret JL., Segarra C., Mpoudi E., Reynes J, Delaporte E, Butcher A, Dreyer K, Herman S, Spadoro J, Peeters M (1999), Efficiencies of four versions of the AMPLICOR HIV-1 MONITOR test for quantification of different subtypes of human immunodeficiency virus type 1, J Clin Microbiol, 37: 110-116 53 UNAIDS in Vietnam, from www.unaids.org.vn Trần Vân Chi 12 Luận văn Thạc sĩ Khoa học 54 Vermeulen M, Coleman C, et al (2013), Sensitivity of individual-donation and minipool nucleic acid amplification test options in detecting window period and occult hepatitis B virus infection, Transfusion 53:10, 2459-2466 55 Yaseen SG., et al (2013), Evaluation of serological transfusion-transmitted viral diseases and mutliplex nucleic acid testing in malaysian blood donors, Transfusion Apheresis Scientic, 49(3), 647-510 56 Warner C., Greene(2007), A history of AIDS: Looking back to see ahead, Eur J Immunol, 37: 94-102 57 WHO (2000), Hepatitis C virus Fact sheet, No 164 58 World Health Organization (2002), Hepatitis B Department of Communicable Disease Surveillance and Response 59 WHO (2012), The Immunological Basis for Immunization Series, Module 22: Hepatitis B 60 WHO (2014), Guidelines for the screening, care and treatment of persons with hepatitis C infection 61 WHO (2015), Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection 62 www.foundation.org.vn 63 www.hivandhepatitis.com 64 http://www.niaid.nih.gov/factsheets/howhiv.htm Trần Vân Chi 13 Luận văn Thạc sĩ Khoa học