KET QUA BAN DAU UfNG DUNG BQ KIT EXAVIR LOAD TRONG VIEC OANH GIA THAT BAI DIEU TRj ARV
VEVIRUTHpCdTRENHllMHIV
Tran Chf Thanhs Nguyen Thi Kim Chuc^ Mattias Larsson^
Bo kit ExaVir Load la cdng cu hmi hieu trong viec theo doi tai lucmg virut HIV cho nhiing benh nhan dang dieu tri ARV tai cac nuac co nguon lire han che. Tai Viet Nam, bg kit nay da dugc dung cho nghien curu thii nghiem tam soat danh gia that bai dieu tri ARV ve virut hgc o nguai lan (du an DOTARV 2007 - 2011, tai tinh Quang Ninh). Viec ung dung bp kit ExaVir Load cho tre em nhiem HIV dang dieu tr; ARV gap phai kho khan do th6 tich mau huyet tuang can dung la 1ml, trong khi do viec lay mau o tre khong the dap img lugng mau. Do do, chiing toi tien hanh thir nghiem giam the tich mau huyet tuong va buac dau danh gia ty le that bai dieu tri ARV ve virut hgc o tre em nhiem HIV tai thanh phd Hd Chi Minh.
The tich 0,5ml huyet tuong c6 the su dung dugc cho xet nghiem do tai lugng virut HIV bang bg kit ExaVir Load. Ket qua tuong dong vol ket qua lam tir 1 ml huyet tuong va phu hop vai ky thuat TaqMan PCR (hang Biocentnc, Phap). Vdi ket qua do tai lugng cua 242 tre nhiem HIV dang dieu hi ARV (thu thap tiJ 10/2013 - 12/2013), chiing toi phat hien dugc 19 mSu co tai lugrng > 1,000 ban sao/ml, chiem ty 1? 7,85%.
Tir khoa: Bg kit Exa Vir Load, th4t bai di8u tri ARV.
0ATVANE)E
Theo ket qua phan tich tong hdp tu" eac nghien cu\j, tre nhiem HIV du'dc dieu trj bang thuoe ARV co nguy cd that bai dieu tn ve mat virut hpc eao gap 2 lan so vdi ngi/di Idn. Tre nhiem HIV sau 12 thang dieu trj ARV cd ty le that bai dieu tri ve mat virut hgc la 30%, trong do 13,7% that bai dieu tri ve mat mien dich hpe'". Ngoai ra, hau qua cua that bai dieu tn d tre nghiem trong hdn nhieu so vdi ngu'di Idn khi nhutig phac do thudc ARV it sif lu'a chpn hdnt'i. Do dd, viec tam soat va tiieo doi tai lu'dng vimt HIV trong qua trinh dieu trj ARV la rat quan trpng va CO y nghia to ldn trong viee dam bao hieu qua dieu trj ARV
"'DaiA(K Y Phgm Ngi>c Thgch thanh pho Ho Chi Minh "'DqihgcYHd NQI '''Vifn Karolinska
\%k\ nY\^n\sk\: 25/12/2014 NgM phfin bifn tong: 05/01/2015 N g i y duyft dftng: 28/01/2015
NgirM ch[u trich nhifm n^i dung khoa hgc: Tran Chi Thdnh. Dgi hoc Y Phtfm Ngpt Thfich. ihdnh pho Hd Chi Mmh
Bifn thogi. 091S273164. E-mail tranchithanh(aipnl edu.vn
Bg kit ExaVir Load do tai lu'dng virut HIV tiiong qua do mii'c dp hoat dpng ciia men phien ma ngu'dc va da dddc chiitig minh ve dp chinh xae ve ket qua tu'dng du'dng vdi ky thuat chuan thiic HIV RNA^-^i. Tai Viet Nam, bp kit Ex- aVir Load da du'dc litig dung de do tai lu'dng virut HIV d ngu'di ldn dang dieu tri ARV trong dU an DOTARV tai Quang Ninh vdi do dae hieu ~ 99% va dp nhay Ifg/ml (tddng du'dng 200 ban sao/ml). So sanh vdi ky thuat Taq- Man PCR ghi nhan he so tu'dng quan r^ = 0,97 va dp chenh lech ttiap vdi miie chenh lech ve gia tn log la 0,34£^i.
Bp kit ExaVir Load theo quy trinh ehuan sii dung 1 ml huyet tu'dng ciia benh nhan de lam xet nghiem. Day chinh la trd ngai Idn khi du'a vao trien khai Uieo doi tai lu'dng virut cho tre, do the tieh mau lay d tre khong tiie lay nhieu nhd d ngu'di ldn. Chinh vi vay, ehiing tdi tien hanh nghien ciiu "U'ng dung bg kit ExaVir Load trong viec danh gia that bai dieu trj ARV ve virut hpe d tre nhiem HIV" vdi hai muc tieu sau: (1) So sanh ket qua do tai lu'dng vinjt vdi the tich huyet tu'dng 0,75 ml, 0,5 ml, va 0,25 ml so vdi ket
TRUYEN NHlilUIXfl^TNAM*S6 1(9) - 2015 - 8 7
qua do tai luUng vdi tiie ti'ch huyet tu'dng 1ml. (2) Do tai luUng vimt d tre nhiem HIV dang dieu trj ARV vdi tiie tich huyet tu'dng toi u\j va so sanh vdi ky ttiuat TaqMan PCR (hang Biocentnc, Phap).
Phu'cfng phap: Nghien ciilj du'dc diia lam hai phan:
Phan 1: Cac Oivrnghiem so sanh doi chvtng ket qua ao tai IUdng HIV tu" the tich huyit tWdng 0,75 ml, 0,5ml va 0,25ml vdi thi b'ch huyet tu'dng chuan 1ml.
Sd lu'de nguyen ly va eac buidc chinh eua xet nghiem do tai ludng HIV aia bo kit ExaVir Load: (1) Chlet xuat men phien ma nguWc ciia HIV tit 1ml mau huyet tijUng cua benh nhan; (2) Oo hoat ti'nh eua men phien ma ngu'dc tiiong qua phan iihg tong hdp cDNA trong gieng cd g§n sin cac RNA sdl ddn; (3) Phat hien sU hinh thanh mach doi cDNA trong cac gieng thong qua phan utig mau (theo nguyen ly phan ufng ELISA); (4) Ket qua xac dinh ndng dp virut thong qua nhutig mau chuan biet trudc (dudng chuan) du'dc ttii/c hien dong thdi.
Cac mau huyet tu'dng HIV od nong dp cao (500 ^ / m l
« 105 ban sao/ml), trung binh (50fg/ml ~ 10* ban sao/ml), thap (5fg/ml ~ 103 ban sao/ml) du'dc tien hanh do tai liTdng vdi the h'ch huyet tuUng 0,75ml;
0,5ml; 0,25ml du'dc tien hanh dong tiidi vdi the tich chuan 1ml (thiet ke nghien a i l i dang thii nghiem - ddi chiihg). Dung djch nude mudi sinh ly 0,9% du'dc chgn lam dung dJeh de bo sung vao m i u huyet tutfng de dam bao dii the tieh chuan 1ml theo quy trinh xet nghiem. Viec bo sung eho the b'ch mau dat 1ml nhim dam bao eac i^eu kien nong dp cac chat trong cae phan litig eua giai doan ly trich men phien ma ngu'dc.
Cac t h i i nghiem du'dc lap lai 3 lan ttong ciing lan lam va 3 fan lam kh^e nhau.
Phan 2: Nghien c&u mo ta cat ngang khao sat ty le thai bai diiu tri ARV ve virut hoc d tre nhiim HIV dang dfSu tri tai B^nh vien Nhi Dong 1 va Nhi &dng 2 tai thanh pho Ho Chi Minh.
Tieu chuan tuy€n chon doi tu'dng: Tre nhiem HIV dieu tii ARV phac do bac 1, ft nhat 6 ttiang va tdi da 3 nSm; Tre do gia dinh (bo me hoac ngudi than) cham sdc va cho uong thuoe ARV tai nha.
Cach chon m l u : Chpn mau ttiuan tien va lay het tat ca cac bojidng hdp ti^ du dieu kien. So lieu lam sang ttiu ttiap ttieo bieu mau va thu thap mau mau ciia tre de do tai lu'dng HIV. Cac mau mau eua tre tham gia nghien ciiti du'dc do tai lu'dng HIV bang bp kit ExaVir Load dutfc thiic hien tai phdng xet nghiem Vi sinh eiia Benh vien Nhi Dong 1. Nhutig mau co ket qua tai lu'dng (> 1,000 ban sao/ml, ngu9ng danh gia ttiat bai dieu tri ve virut hpc sau khi dieu trj ARV it nhat 6 thang, theo To chiie Y te The gidi va hu'dng dan Quoc gia dieu tn HIV/AIDS ciia Bp Y te) deu du'dc gui den Vien Pasteur thanh pho Ho Chi Minh lam doi chieu bang ky tiiuat TaqMan PCR Biocentnc (ciing mau huyet tu'dng).
XuT ly so lieu: Theo phutfng phap tiidng ke y hpc.
Dao du'c nghien cihi va cam ket: Chiing to! (cac tae gia cua bai bao gom: Tran Chi Thanh, Nguyen Thj Kim QiiJc, MatiJas Larsson) eiing cam ket cac dieu khoan sau:
1. So lieu du'dc nit trich mot phan trong d\i an HIVCHI hdp tae gilJa Oai hgc Y Ha Npi va Vien Karolinska, dam bao eac sd lieu trung thi/c.
2. Dii an hoan toan do to chiic SIDA (Uy ban Khoa hpc cua Thuy Dien) tai trd kinh phf va khong co bat ky moi quan he tai chinh hay xung dot ldi feh vdi eae cdng ty san xuat sinh pham xet nghiem do tai lu'dng HIV.
3. Neu cd nhutig van de phat sinh lien quan den npi dung eua bai bao thi diiing toi se chju trach nhiem giai trinh nhutig thac mac.
KET QUA
Thuf nghiem do tai lu'ctng HIV vdi cac thi tich huyet tu'dng 1ml, 0,75mlr 0,5ml va 0,25ml
Chijng tdi da tien hanh t h i i nghiem tren cac mau huyet tudng cd nong dp HIV theo kha nang phat hi§n cua bg kit ExaVir Load, b^jng dd nong dp cao (500fg/ml ~ 10^
ban sao/ml), trung binh (50fg/m! ~ 10* ban sao/ml), tiiap (5fg/ml « 10^ ban sao/ml). Cac ket qua ghi nhan d the tich huyet tutfng 0,75ml, 0,5ml cho ket qua tu'dng du'dng vdi ket qua lam tii 1ml mau huyet tu'dng, cdn d the tich 0,25 ml huyet tu'dng cho ket qua khong on djnh va hleu suat kem. Mot so ket qua thu" nghiem dUdc trinh bay d bang 1.
- T R U Y ^ N H I & « V e T N A M * s 6 1(9) - 2015
Bang 1 : Ket qua do tai lu'dng HIV vdi cac the t i d i liiiyet tu'dng Idiac nhau
cua 313n lap tai trong thii' nghiem
1ml 0,75ml
0,5ml 0,25ml
Nong do (fg/ml) cacmau huyet tu'dng thif nghiem CAO TRUNG BINH
Tliu' nghiem 1 647 657,66 647,33 555,33
Tlii^ nghiem 2 Thii' nghiem 1 620
648,33 610,66 566
58 58 59,67
48
Thii nghiem 2 54 55 61,66
49
T H A P T h u ' n g h i e m 1
5 4,33 4,66 3,66
Thljr n g h i S m 2 5 5 4 3,66
Tong hdp ket qua eua 3 lan lap lai (cung lan t h i i nghiem) va 3 lan lap lai (khac lan ttiii nghiem), chiing toi ghi nhan the tieh mau huyet tiidng co tiie giam 1/2 so vdi quy tiinh chuan, nghTa la vdi 0,5ml huyet tu'dng dUdc bo sung them 0,5 nu'dc muoi sinh ly (NaCI 0,9%) eho du 1ml va tien hanh lam mau theo quy trinh xet nghiem tht cho ket qua tu'dng txi vdi th&tieh 1ml huyet tuting va nam trong khoang dao dpng eho phep (khdng qua 20%)^l iTng dung ExaVir Load de danh gia that bai d i i u tri ARV v@ virut hpc d tre n h i l m HIV
Trong tiidi gian tii 10/2013 - 12/2013 (3 ttiang), 242 tre nhiem HIV dang iJeu tri ARV da duUe tuyen ehpn tii Benh vien Nhi dong 1 va Benh vien Nhi dong 2 tai thanh pho Ho Chi Minh. Thong tin cd ban cua dan so nghien a i d dutfc trinh bay d bang 2.
Bang 2: Mot so tfSc diem cua dan so nghien cihi D|c di€m d3n so nghien ciili
GicS t'nh Nam Gidi tinh Nif Tuoi (tmng binh)
Gia trj 124 (51,24%) 118 (48,76%) 6,39 ± 3,11 nSm JVhdmtuof
Du^ 3 hjoi TCf3-6tJ0l Tren 6 tuoi
21 (8,68%) 86 (35,54%) 135 (55,78%) PhSn b6 ndi a/tTV cua tfe
Khu v u t cac quan noi thanh Khu vuC cac quan ngoai thanh Cac tinh lan can
U l (45,87%) 105 (43,39%) 30 (10,74%) LucbatdSu
dieutii Ukvao nghien diii Phan h^l Giai doan lam sang (GBLSJ
I II
115 (47,52%) 47 (19,42%)
200 (82,64%) 32 (13,22%)
Oac di^m dan so' nghign d h i III IV Gia tri t£ bao CD4 (trung binh) So nam dieu hi ARV (phac do I - hien tai)
Giatri 65 (26,86%)
13 (5,37%) 949 ± 153
5 (2,07%) 0 1340 ± 121 2,83 ± 1,07
Ket qua do tai luUng virut cua 242 tri bang bo kft Ex- aVir Load (lam tai Benh vien Nhi dong 1 thanh phd Ho Chf Minh), ghi nhan ed 19 ti'e du'dc ghi nhan ed nong dp virut (> 1,000 ban sao/ml) chiem ty le 7,85%. Tat ca cac ket qua eiia 19 tre deu ddde lam dol ehieu viffi ky thuat Taq- Man PCR Bioeentrie (lam tai Vien Pasteur thanh phd Ho Chf Minh) va cho ket qua tu'dng dong vdi dp lech ve gia hi log la - 0,20 (xem bang 3).
Bang 3: Ket qua so sanh gia trj log ve nong do virut HIV 50 sanh giu'a 2 ky thuat
Gia tri Log (trung binh) ExaVir Load 4,35 ± 0,55
TaqMan PCR 4,55 ± 0,46
MiJTtddlidi -0,20
BAN LUAN
Bp kit ExaVir Load cd do nhay Ifg/ml ~ 200 ban sao/ml, do do khi tien hanh giam ttie tfch huyet tUdng su"
dung thi chiing toi da tinh toan va \iia ehpn cae miic dp giam theo miic ddn vj the ti'ch la 0,25 ml va duUe die the ti'ch 1ml; 0,75ml; 0,5ml va 0,25ml tu'dng du'dng vdi do nhay la 200 ban sao/ml; 266 ban sao/ml; 400 ban sao/ml va 800 ban sao/ml nham dap iing yeu cau phai ttiap hdn ngudng 1.000 ban sao/ml, ngudng danh gia ttiat bai dieu tti ARV ve mat virut hpe.
Viec pha loang huyet tu'dng ttong eac xet nghiem can lam sang nhd sinh hoa, mien djch, smh hpc phan t i i ddde
T R U Y ^ NHlilUI VIETNAM « S d 1(9) - 2015 - 8 9
xem nhu" la quy trinh can thi& tien hanh khi mau thit co nong dp vddt ngudng tiiii nghiem. Dung moi chuan nhat ddi vdi t h i i nghiem do tai lu'dng HIV ia mau huyet tu'dng am ti'nh. Tuy nhien, viec sii dung va ti'm the ti'ch Idn mau huyet tu'dng HIV am b'nh la khdng kha thi. Cae dung moi thu'dng dddc dung nhieu nhat la ndde muoi smh ly 0,9%
hay dung dieh Ringer vi chiing ft lam tiiay doi cae dieu kien khae eiia mau huyet tu'dng nhif pH, nong dp dien tichi^J. Oiling toi da \da dipn nu'dc muoi sinh ly 0,9% de bii tiie tich mau huyet tiidng dii 1ml, vi day la dung moi rat de kiem, de chuan bj va khong ton ehi phf eao.
Ket qua do tai lu'dng virut d the tfch huyet tu'dng 0,5ml eho hieu suat phan uhg tot hdn ca d the tieh 0,7ml tham ehi 1ml, ^ e u do ed the giai thich thong qua sit t a u\j ciia phan uhg ve eae dieu kien nong dp cae chat hdn la so vdi 0,7ml hay 1ml, chang han nhd d mau ed nong dp tiung binh (1ml = 58fg/ml; 0,75ml = 58fg/ml; 0,5ml = 59,67fg/ml).
LTng dung bp kit ExaVir Load vdi the tich 0,5ml huyet tu'dng bo sung 0,5ml nu'dc muoi sinh ly 0,9% da ddde ttiiic hien tten 242 mau, vdi 19 mau co nong dp (> 1.000 ban sao/ml). So sanh doi chieu ket qua gilj^ 2 ky thuat Ex- aVir Load va TaqMan PCR, ehiing tdi cung da ghi nhan ddde Sii tij'dng ddng gidng nhd eac cong trinh nghien ciUs tiVdc day da cong bo, dae biet la vdi the tieh 0,5ml huyet
tUdng, dieu do md ra huidng nghien a i d danh gia lau dai viec ling dung till tich 0,5ml huyet hTdng ttong tu'dng lai vdi so tu'dng mau nhieu hdn va so lu'dng ket qua doi chieu nhieu hdn de ed the tinh toan thdng ke.
Vdi thdi gian dieu h i ARV eiia eae tre trong nghien eii\j la 2,83 ± 1,07 nam, chiing tei ghi nhan ty le tfiat bai dieu tri ARV ve vinjt hoc trong nghien ciiu nay ghi nhan la 7,85% tiiap hdn nhieu so vdi ty le chung ve that bai dieu ttj la 30% sau 12 thang dieu trii". sir chenh lech nay cd the ly gial thdng qua ket qua danh gia GDLS vao thdi diem lay mau ttii da so tte d GOLS I (82,64%) va II (13,22%), dieu do cd nghTa tre dang dap iing tot vdi Uiuoc ARV phac do bac I nen tai lu'dng virut trong mau ddde kiem soat tot.
KET LUAN
The tieh 0,5ml huyet tu'dng dimg cho do tai lu'dng HIV bang bo kit ExaVir Load la kha thi vc^ ket qua tu'dng du'dng khi diing 1ml huyet tu'dng va co ket qua tiidng bi vdi ky tiiuat TaqMan PCR.
Ket qua ban dau vie t / le that bai dieu trj ve virut hpc d so ngu'di nghien ciiu la 7,85% ghi nhan tren 242 tie nhiem HIV dang dieu tn phac do ARV bac 1 ttong nghien ciiu nay vdi thdi gian dieu ttj ttung binh 2,83 ± 1,07 n^m.
9 0 • TIWJY&INHiMVI$ThlAM*s6 1
TAI LIEU THAM KHAO
1. Andrea L-Oaranello et al. "Effectiveness of Pediatiic Antirettoviral Therapy in Resource-Limited Settings: A Sys- tematic Review and Meta-analysis", Clinical Infectious Dis- eases 2009; 49:1915 - 27.
2. Binodh DeSilva et al., "Recommendations for tiie Bioanalytieal Method Validation of Ligand-binding Assays to Support Pharmacokinetic Assessments of Macromole- cules" Pharmaceutical Research, Vol. 20, No. 11, Novem- ber 2003.
3. Do Duy Cuong, Eva Agneskog, Nguyen Thi Kim Chuc, Miehele Santaeatterina, Anders Sdnnerborg, Mattias Larsson, "Monitoring tiie efficacy of antiretroviral therapy by a simple reverse transcriptase assay in HIV-infected adults in rural Vietnam", Future Virology. September 2012, Vol. 7, No. 9, Pages 923 - 931.
4. M.D.A.Nhu, D.TN.Diep, TH.Khanh, "Adherence to antiretroviral therapy (art) in HIV/AIDS patients at Chil- dren Hospital 1"-Journal of Medicine in Ho Chi Minh city.
No.l3,Sup.l-2009:212-218.
5. R.G.Hahn, C.Svensen, "Plasma dilution and the rate
of infusion of Ringer's solution", British Journal of Anaes- tiiesia 1997; 79: 64 - 67.
6. Rachel CVree, "A Systematic Review of Pediab-ie Adherence to Antirettoviral Therapy in Low-and Middle- Income Countties" The Pediatric Infectious Disease Jour- nal, Volume 27, Number 8, August 2008.
7. Steegen K, Luchters S, De Cabooter N, Reynaerts J, Mandallya K, Plum J, et al., et al. "Evaluation of hwo eom- mereially available alternatives for HIV-1 viral load testing in resource-limited settings". J Virol Methods 2007; 146:
178 - 87.
8. Stevens WS, Scott LE, Crowe SM. "Quantifying HIV fdr monib3ring antiretroviral therapy in resource-poor set- tings". J JnfectD/s 2010; 201: S16 - 26.
9. Sungkanuparph S., ManosuthI, W., Kiertiburanakul, S., et al., 2007, "Options for a secondl ine antiretroviral regimen for HIV type 1-infected patients whose initial reg- imen of a fixeddose combination of stavudine, lamivudine, and nevirapine feils. Clin Infect Dis 2007; 44: 447 - 452.
PRIMARY RESULTS OF APPLICATION LOAD EXAVIRLOAD I N ASSESSING ART VIROLOGIC TREATMENT FAILURE
I N HIV - INFECTED CHILDREN
Summary
ExaVir Load kit is an effective tool in monitoring HIV viral load for patients on antiretroviral tiierapy in resource- limited countries. In Vietnam, this kit has been used for a stijdy evaluating ART virologic failure in adults (DOTARV project from 2007 to 2011, in Quang Ninh). Using ExaVir Load for routine monitoring of ART effect among HIV - in- fected children could lead to eariier detection of virologi- cal failure and prevent accumulation of mutations.
However, for small children viral load monitoring using Ex- avir Load is complicated by the amount of plasma 1 ml, which may be too much blood to draw as other tests also often are needed. Therefore, we assessed if reduced Key words: ExaVirLoad kit, ART virologic feilure.
plasma volume would affect the viral load results com- pared to a standard of 1ml plasma. Virological tteatment failure rate was also assessed among HIV-infected chil- dren on ART Hochiminh city. There was a high correlation between the viral load results using 0.5ml and a standard of 1ml plasma. There was also a high correlation among results using ExaVir Load and TaqMan PCR (Bioeentrie, France). With screening results of 242 HIV-infected chil- dren under ART (enrolled from 10/2013 -12/2013), we detected 19 samples (7.85%) with the detectable viral load more than 1000 eopies/ml.
T R U Y E N N H l i M V I ^ N A M * s 6 1(9) - "^n-i^ - *^1
