Comparative study of two methods for RNA extraction prior to detection of resistance to human immunodeficiency virus type 1 with the line probe assay

JOURNAL OF CLINICAL MICROBIOLOGY, June 2002, p. 2257–2259 Vol. 40, No. 6 0095-1137/02/$04.00⫹0 DOI: 10.1128/JCM.40.6.2257–2259.2002 Copy igh © 2002, Ame ican Socie y o Mic obiology. All Righ s Rese ed. Compa a i e S udy o Two Me hods o RNA Ex ac ion P io o De ec ion o Resis ance o Human Immunode iciency Vi us Type 1 wi h he Line P obe Assay Jose Ma ía Ei os, 1 * C is ina Labay u, 2 Bea iz He nández, 1 Raúl O iz de Leja azu, 1 and An onio Rod íguez To es 1 Mic obiology Se ice, Uni e si y Hospi al o Valladolid, 1 and Mic obiology Se ice, Uni e si y Hospi al “Rio Ho ega,” 2 Valladolid, Spain Recei ed 1 No embe 2001/Re u ned o modi ica ion 5 Janua y 2002/Accep ed 26 Ma ch 2002 We e alua ed wo me hods om Roche and P omega o RNA ex ac ion p io o he geno ypic de ec ion o human immunode iciency i us ype 1 esis ance by line p obe assay (LiPA). Fi y plasma RNA ex ac s we e p ocessed in pa allel by LiPA. Resul s ob ained by he Roche me hod we e supe io in he p opo ion o ampli ied samples, he pe cen age o mu a ed samples, and band in ensi y. F om Feb ua y o May 2001, 50 plasma samples om di - e en pa ien s we e andomly selec ed om i al load (VL) de e mina ion eques s ha had ⱖ1,000 RNA copies/ml. All samples we e p ocessed o VL analysis by means o PCR a e p e ious e o ansc ip ion ( e e se ansc ip ase [RT]-PCR) (Cobas Amplico HIV-1 Moni o ; Roche Diagnos- ics, B anchbu g, N.J.) in i s ul asensi i e e sion wi h a h eshold o 50 RNA copies/ml. This e sion includes ul a- cen i uga ion a 23,600 ⫻go 500 ␮l o each plasma sample a 2 o 8°C o 60 min p io o he i al pa icle lysis. RNA is ex ac ed by adding a chao opic agen (guanidinium- hiocya- na e) ollowed by RNA p ecipi a ion wi h e hanol. A e VL de e mina ion all RNA ex ac s we e p ese ed a ⫺80°C. RNA om samples wi h a VL o ⱖ1,000 RNA copies/ml was also ex ac ed by using he SV To al RNA Isola ion Sys em (P omega Co po a ion, Madison, Wis.). This me hod is based on a lysis-cen i uga ion p ocess ollowed by a column il a ion h ough a silica memb ane in an RNase- ee en i onmen s a ing wi h 125 ␮l o plasma. RNA ex ac s ob ained by he wo me hods we e es ed in pa allel o he de ec ion o geno ypic esis ance by means o he comme cial line p obe assay (LiPA) (INNO-LiPA HIV-1 RT and INNO-LiPA HIV-1 P o ease; Innogene ics, Ghen , Belgium). B ie ly, LiPA is based on a pos -RT-PCR hyb idiza ion ha akes place on ni ocellulose s ips on o which speci ic oligo- nucleo ide p obes a e ixed in pa allel lines. RT-PCR was ca ied ou ollowing he manu ac u e ’s ins uc ions by using he RT-PCR Access ki (P omega Co po a ion), excep ha he p ime s used we e hose included in he LiPA Ampli ica- ion ki . This assay allows he s udy o wild- ype and mu an sequences a codons 41, 69, 70, 74, 184, and 215 o he RT gene (LiPA RT) and a codons 30, 46, 48, 50, 54, 82, 84, and 90 o he p o ease (P) gene (LiPA P). Mu a ions in hese posi ions ha e been epo ed as associa ed wi h esis ance o nucleoside RT inhibi o s and o p o ease inhibi o s. cDNA syn hesis and PCR wi h bio inyla ed p ime s we e pe o med as desc ibed by S uy e e al. (13). Hyb idiza ion was pe o med acco ding o he manu ac u e ’s ins uc ions. B ie ly, bio inyla ed DNA was hyb idized wi h speci ic oligonucleo ide p obes immobilized in pa allel lines on memb ane-based s ips. A e hyb idiza ion, s ep a idin labeled wi h alkaline phospha ase was added and bound o bio inyla ed hyb ids. Incuba ion wi h a ch omogen esul ed in a pu ple-b own p ecipi a e isible o he naked eye. Compa a i e eading o s ip bands was done subjec i ely. Ex ac ion by P omega was aken as e e ence. We adop ed a iple s a egy. Fi s , he human immunode iciency i us (HIV) con ol band in ensi y was checked. Second, he in ensi ies o he es o he bands we e checked, and inally, he appea ance o di e en bands by each ex ac ion me hod was e alua ed. Ou indings we e classi ied in o ou g oups: (i) equal in en- si y, when all bands we e simila ; (ii) in ensi y 1⫹, when he HIV con ol band was sligh ly da ke by he Roche me hod wi h he same numbe o bands; (iii) in ensi y 2⫹, when he HIV con ol band and he es o he bands we e ma kedly da ke , wi h he same numbe o bands, o an ex a band was obse ed by he Roche me hod; (i ) in ensi y 3⫹, when band colo ing was ma kedly supe io by he Roche me hod and mo e han one ex a band appea ed. When he Roche ex ac- ion s ip showed lowe in ensi y han he P omega s ip, a simila assessmen was done using nega i e igu es. In e p e a ion o he mu a ions ound was done ollowing he manu ac u e ’s ins uc ions and acco ding o he Medscape Guide o An i e o i al Resis ance Mu a ions (h p://hi .medscape.com/upda es/quickguide) and o he In e na ional AIDS Socie y-USA Panel (6). A desc ip i e s udy o all indings was ca ied ou using he s a is ical p og am SPSS 9.0 o Windows. “Ji-squa e” es s we e applied o s udy he possible ela ions be ween he di - e en a iables s udied. PCR ampli ica ion esul s o he ex ac s ob ained by each me hod we e di e en o LiPA RT and o LiPA P, he di - e ences being s a is ically signi ican in bo h cases (P⬍0.001). Fo LiPA RT a success ul ampli ica ion was achie ed a e P omega ex ac ion in 33 o 50 samples (66.0%; he 95% con- * Co esponding au ho . Mailing add ess: Mic obiology, Six h Floo , Facul y o Medicine, A da. Ramón y Cajal s/n, 47005 Vallado- lid, Spain. Phone: 34-983 423063. Fax: 34-983 423066. E-mail: ei os @med.u a.es. 2257 by on Decembe 3, 2007 jcm.asm.o gDownloaded om idence in e al [CI] was 51.2 o 78.8) and in 49 o 50 samples a e Roche ex ac ion (98%; 95% CI, 89.3 o 99.9). Fo LiPA P a success ul ampli ica ion was ob ained in 38 o 50 P omega ex ac s (76.0%; 95% CI, 61.8 o 86.9) and in 100.0% o Roche ex ac s (95% CI, 92.8 o 100.0). Compa a i e analysis o he esul s ob ained by he wo ex ac ion me hods led o disc epancies in indings a h ee di e en le els. Fi s , in h ee samples he inal LiPA in e p e- a ion was “wild ype” o he P omega ex ac bu “mu an ” o he co esponding Roche ex ac . This happened in wo LiPA RT de e mina ions and in one LiPA P de e mina ion. Second, 13 samples ha did no ampli y a e P omega ex ac- ion we e in e p e ed as “mu an ”a e success ul ampli ica- ion o he co esponding Roche ex ac s. This happened in ou samples o bo h LiPA RT and LiPA P, in se en samples o LiPA RT alone, and in wo samples only o LiPA P. O he 16 samples included in he p e ious wo le els, 10 (62.5%) had VL le els o ⬍50,000 RNA copies/ml, bu no s a is ical signi - icance was obse ed. Thi d, om he g oup o samples suc- cess ully ampli ied and in which esis ance mu a ions we e p esen a e ex ac ion by bo h me hodologies, disag eemen s could be documen ed bo h in he absolu e equency o ap- pea ance o indi idual mu a ions and in he pa e ns o com- bined mu a ions obse ed. Tables 1 and 2 p esen he disag ee- men s obse ed o bo h LiPA RT and LiPA P, aking in o accoun he ou come o he RNA ex ac ion me hod; indings ela ed o absolu e mu a ion equency (column sub o als) and o di e en combined mu a ion pa e ns ( o als by ows) a e shown. A ick (公) ma ks which indi idual and combined mu- a ions we e p esen in each RNA ex ac ion me hod; his is conside ed in he pe cen age calcula ion, which is shown in pa en heses. When di e ences in he band colo ing in ensi y obse ed we e analyzed o each sample acco ding o he ex ac ion me hod, o LiPA RT 11 samples (33.3%; 95% CI, 17.9 o 51.8) had he same in ensi y. In 21 cases Roche band in ensi y was supe io , being classi ied as in ensi y 1⫹in 11 cases (33.3%; 95% CI, 17.9 o 51.8), as in ensi y 2⫹in 4 cases (12.1%; 95% CI, 3.4 o 28.2), and as in ensi y 3⫹in 6 samples (18.2%; 95% CI, 6.9 o 35.4). Only once was he Roche band in ensi y lowe han ha o P omega (in ensi y 1⫺). Fo LiPA P, in 31 samples (81.6%; 95% CI, 65.7 o 92.2) he band in ensi ies o he wo ex ac ion me hods we e equal. In six samples in ensi y was supe io , he dis ibu ion being as ol- TABLE 1. Disc epancies in esul s obse ed o LiPA RT acco ding o he RNA ex ac ion me hod in he absolu e equency o each mu a ion (column sub o als) and in combined mu a ion pa e ns ( o al by ows) P esence o mu a ion and absolu e equency (%) by Roche (nex - o-las ow) and by P omega (las ow) Pa e n equency (%) L41(T) L41(C) R70 D69 D69R70 N69R70 V74 V184 Y215(18) Y215(19) F215 Roche P omega 冪 冪 1 (100) 冪冪 冪冪 冪 1 (100) 冪冪 冪冪 1 (100) 冪冪冪冪 1 (100) 冪冪冪 1 (100) 冪冪冪 1 (100) 冪冪 1 (100) 冪 冪 1 (100) 冪 1 (100) 冪冪 冪 3 (100) 2 (66.7) 冪冪 1 (50) 2 (100) 冪 1 (100) 1 (100) 2 (66.7) 3 (50) 1 (100) 4 (100) 9 (90) 7 (70) 1 (100) 1 (50) 1 (33.3) 3 (50) 2 (50) 5 (50) 7 (70) 1 (50) TABLE 2. Disc epancies in esul s obse ed o LiPA P acco ding o he RNA ex ac ion me hod in absolu e equency o each mu a ion (column sub o als) and in combined mu a ion pa e ns ( o al by ows) P esence o mu a ion and absolu e equency (%) by Roche (nex - o-las ow) and by P omega (las ow) Pa e n equency (%) N30 I46 V48 V50 V54 A54 V84 F82 F82V84 A82 T82 T82V84 M90 Roche P omega 冪冪 1 (100) 冪 冪 1 (100) 冪 冪 3 (100) 2 (66.7) 冪 3 (100) 2 (66.7) 冪冪 冪 1 (100) 冪冪 1 (100) 冪 3 (100) 2 (66.7) 冪冪 2 (100) 1 (50) 冪 1 (100) 6 (75) 1 (33.3) 1 (50) 3 (100) 2 (66.7) 6 (100) 6 (75) 2 (66.7) 1 (50) 2 (66.7) 2 (66.7) 3 (50) 2258 NOTES J. CLIN.MICROBIOL. by on Decembe 3, 2007 jcm.asm.o gDownloaded om lows: i e samples wi h in ensi y 1⫹(13.1%; 95% CI, 4.4 o 28.1) and one sample wi h in ensi y 2⫹(2.6%; 95% CI, 0.1 o 13.8). No samples wi h in ensi y 3⫹we e ound. In h ee cases he esul was classi ied as in ensi y 1⫺(7.9%; 95% CI, 1.6 o 21.3). A global compa ison o he in ensi y di e ences be ween he Roche and he P omega me hods, g ouping hem as equal o supe io ( o bo h LiPA echniques), shows s a is ical sig- ni icance (P⬍0.001). The e iden ly di e ing esul s obse ed o he wo ex ac- ion me hods compa ed in his s udy highligh he impo ance o his s ep. Ex ac ion condi ions he ou come o all PCRs (1, 2, 15, 17), which a e likewise an indispensable p e equisi e o HIV geno ypic esis ance es ing. In he li e a u e ha we ha e e iewed, he e a e some e e ences o ex ac ion ou come applied o HIV sequencing o geno ypic esis ance de ec ion, such as a compa ison o Qiagen and Boom ex ac ions ca ied ou by Niubóe al. (9) and he compa a i e s udy by Sha e e al. (11) o he silica bead me hod e sus ex ac ion by phenol- chlo o o m. Mos published s udies on he in luence o RNA ex ac ion e e o he subsequen VL de e mina ion (4, 5, 14, 15, 17). In ou expe ience, he be e esul s achie ed by Roche ex ac ion could be explained by wo easons. Fi s , i includes an ul acen i uga ion, an ope a ion ha imp o es he ex ac- ion yield due o he sample concen a ion p o ided (11, 17). Second, he s a ing sample olume is g ea e (500 e sus 125 ␮l) (10, 14, 17). An addi ional ad an age o his me hod is ha VL de e mina ion should be pe o med p io o HIV esis- ance es ing, and, as he Roche ex ac ion me hod is used o his e ec , he ex ac ob ained se es as s a ing ma e ial o bo h echniques. Ou s udy is limi ed by he absence o an objec i e sys em o band eading such as ha p o ided by a densi ome e , which o he au ho s say o e s good ou come when applied o band in ensi y eco ding (16). Assuming his limi a ion and ha ing de ined eading c i e ia in o de o compa e he esul s ob- ained by he wo ex ac ion me hods, once again he e is a clea disag eemen o LiPA RT, whe e band in ensi y was equal o he wo ex ac ion me hods in only one- hi d o he samples. The wide ange o disc epan esul s obse ed in ou s udy again highligh s he impo ance o he ex ac ion s ep. This ac is especially signi ican because o he ele an in o ma ion b ough o he clinician o he apeu ic pa ien managemen , pa icula ly o hose pa ien s who ha e al eady expe ienced any he apeu ic ailu e (7, 8, 12). The li le impo ance gi en o his echnical aspec is somewha su p ising in such a widely s udied ma e as HIV esis ance es ing is in o he aspec s. We he e o e conside ha s udies such as he p esen compa ing mo e ex ac ion me hods a e needed o op imize he ou come o geno ypic HIV esis ance es ing echniques. The wo k ca ied ou by ou g oup ai h ully e lec s ech- nical p oblems wi hin he i ology labo a o y se ing. Howe e , we do no igno e ha he c i ical poin , as ecommended by some au ho s (3, 6; see also h p://www.msc.es/sida/asis encia / esis encias.h m), is he ansc ip ion o ansla ion o such in o ma ion o he clinical ollow-up o each pa ien . This is he only way ha he echnical e o o hose de o ed o labo a- o y diagnosis can be e icien ly applied in he op imiza ion o he apeu ic esou ces. 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Imp o ed condi ions o ex ac ion and ampli ica ion o hu- man immunode iciency i us ype 1 RNA om plasma samples wi h low i al load. J. Hum. Vi ol. 3:27–34. VOL. 40, 2002 NOTES 2259 by on Decembe 3, 2007 jcm.asm.o gDownloaded om