~-240 logical Societies /94/$ chain reaction

Transcription

1 ~-240 logcal Socetes /94/$ "_ A as dagnoss ly A. Garson * ~l" Vrology. Department ~l" Medcal Mcrobology, Unt'erso' Colle,~e Col Londol :'land Street, London WIP 6DB, UK t: In the absence of tssue culture electron mcroscopy or assays for vral a eel detecton of hepatts C vm s by necessty dependent upon nuclec acd hybrdsaton methot ds. Of the ~ds, amplfcaton of HCV cl)n nerase chan reacton (PCR) commends tself by vrtue of ts extreme extr, senst sequent ablty to detect thc vc :Is of HCV-RNA that tre present n many clncal samples. In th~ ths revew the development and evoluton cvo of PCR technqu V detecton are descrbed and a number of clncal applcatons are consdered n detal. The appl )lcatons nclude dagno,, of acute e nfecton durng the seronegatve wndow perod pror to tht the appearance of HCV antbodes and dagnoss of tic nfecton n n the mmunosuppressed. PCR also enables dentfcaton of the chronc vracmc carrer state statt and t permts accura montorm -ng of the antvral effects of drugs such as nterferon. Conf ~onfrmaton of the specfcty of HCV H( antbody assays a detecton )n of ttcv contamnaton of blood donatons and blood product~ ~roducts are other mportant areas n whch PCR technques ha' proved nvaluable. In addton, PCR-based technques underle an ncreasng ncre~ number of molecular epdemologcal epden and gcnotypl studes and they are provdng nsghts nto the detals of HCV cellular tropsm and replcaton. A numbcr numbc~ of logstc problems al operatonal :real dffcultes are also dscussed. Despte these lmtatons t s concluded that PCR wll contnue to make sgnfca contrbutons atons to both clncal practce and to our understandng of the basc [ bology of HCV nfecton. KO' words." Polymerase chan reacton: m: Hepatts C vrus: Genome detecton: Dagnoss: Quantfcaton Introducton chan reacton Latent nfectons and antgen producton s lmted Snce ts ntroducton n the md 1980s [1], the cal responses are delayed or absent are also pa polymerase chan reacton has been employed for tcularly approprate for da, gnoss by PCR. Tt the detecton of many classes s of nfectous agents hepatts C vrus thus repres ~resents an deal cane ncludng a wde range of vruses 'ruses [2]. The tech- date for PCR-based detecto etecton because: () tsst nque has proved most useful ul for the dentfca- culture methods for HCV solaton., are not y culty. 9atts C ol, H~mh3'er Buhtn~. nfectons n whk or n whch serolo~ ton of those vruses whch are ether completely avalable; () practcal assays for HCV antg( non-cultvable or only cultvable wth great dff- have yet to be developed: ( ) dentfcaton HCV n clncal specmen :clmens by electron IT croscopy s not practcable: and (v) the antbo~ Correspondng author. Tel: (I)74) ; Fax: (071) 580 response to HCV s so delayed that dagno,, 5896 durng the acute phase of the 1 nfecton s fr SSDI (94)E0119-5

2 ~/ the prncples twenty to s dred base pars (bp). / Hopment, appl- three-step 1 : cyclng process s per ns of thc tech- formed: () n of the double-strandel re descrbed, target DNA ~nperaturc (95 C) to sepa rate the stra ucton of the temperatur (30-60 C) t aealng of the prmers t~ reacton the comple.'quence of each sngle stranded DI e molecule; () tempera : PCR technque s capable of hgh level tur, ture ncreas :o allow the thermostabl caton of vrtually tny segment of DNA for enzyme Ta~ lse (from the bacterun nucleotde sequence nformaton s aval- Th~ Thermus aq copy along both templat, Vhe sequence nformaton s used to desgn str~ strands n c ectons, thereby doubln tc olgonucleotde prmers (generally 15 to the amount rget DNA. The orgna rs) whch are complementary to opposte str~ trands and synthessed strands boll s of the target DNA and separated by set serve as te r the next cycle so tha rep pealed c$ rates an exponental n ere crease n th of target DNA molecule (Fg. 1). Amplfc~ ~roxmately 107 fold s ob =: larger DNA DNA + prmers =: PCR prmer + dntps + DNA polyrnera5;e ta taned by : cycles and even hghe... r:e'~ DNA lev, levels ot of amplmcanon tup (up to tc 1012 fold) can b,c~ [~ L..1 acheved by usng 'nested' pt ~rmers as descrbe~ 1 Denature and Synlheslze bel, low. Ths extraordnary d( degree of senstvt I rmts the detecton of even sngle molecule 1 B per (approxmately 10 Ls g) of th( the DNA sequence o A mterest. nu Although orgnally desgned for DNt V am J plfcaton, PCR can also be used to ampll ~ {~ Denature and Synthesze RNA sequences f they are ntally converte~ nu nto complementary DNA ( edna) by revers, l, ~, transcrpton. tral Further detals on general PCI ~ ' I U r 1 prncples, protocols and prm ~rmer desgn are gve] I v ' n the methodology text of Inns h and colleague I 1 eta: {S]. l, ;!!!:,I * eb;,,th I I IF t I Development of PCR technql ues for HCV detec ~L 1 etc lon! I[, Snce the ntal descrpton [4] of HCV detectol ', v I by PCR, the assay has been beer adapted and m etc Fg. I. Polymerasc chan reacton.dna s denatured at 95 C; proved by several groups, For t purfcaton o olgonucleotdes A and B arc annealled ded, and extenson from HCV-RNA from serum or lver tssue, protenas~ prmers of a complementary DNA strand ;trand occurs n the pres- K dgeston [5], polyethylene glycol precptatol once of 771q polymerase and the four deoxyrbonucleosde [6], slca absorpton [7], acd g uandnum sotho trphosphates (dntps). Subsequent cycles of denaturaton, cyanate-phenol-chloroform extracton e~ [8] and; anneallng and extenson result n amplfcatkm of the seg- number of other methods t ment defned by the 5' ends of the e PCR prmers. (Reprohave all been em duced from Trends n Genetcs (1989) 5(6): centrefold, wth ployed. Although most of these technques ar~ permsson). capable of producng vral RNA of adequat~

3 v _ 27 found the acd Most of 1 )ublcatons on HCV-PCI nsk and Sacch descrbed a~ ere essentally qualtatv~ t present. How- More recen we been reports of quant ntly descrbed a tatve and s atve PCR assays, the dc whch s both velopment LS been stmulated n paj antage of elm- by the reqt,r accurate montorng ( ton and alcohol vraema le~ on to nterferon therap! ance of ths at- Quantfcat ; based on lmt dluton ( e alternatve approach has yet to be fully cdna [15,~ effectve but very labou ~ted. nt, ntensve as.ts employng co-amplfc~ lowng RNA solaton all protocols nclude to~ tlon of kno s of compettve mutate ~A synthess step, prmed ether wth spe- ten templates [; rnatvc, less labour nter mtsense prmer or alternatvely wth ran- sw slve metho~ for the analyss of larg hexamers. Both methods of prmng are nm numbers ot re currently beng deve y effectve n most stuatons but random op~ md and c, ',28]. d cdna has the advantage of beng amplwth PCR prmers derved from any part of mome. For convenence, and to mnmse Ap pplcaton I HCV research and cln e manpulatons, some protocols are de- cal manage~ I to permt cdna synthess and amplfca the same tube [11]. D~ Olagnosls Da " o] acute HCV 7V mp n~ecton and q[" HC :h conventonal sngle PCR [12,13] and double ('nested' nested') PCR [5,11,14,15] protocols have been descrbed. Jescrbed. 'Nested' PCR. n whch the prodcarrage A major lmtaton of serologcal assays f( antbodes anl to HCV (ant-hc :-HCV) s ther nablty t ucts of f amplfcaton are reamplfed usng a sec- pr( )rovde a dagnoss durng the acute phase of th ond set zt of nternal prmers, has the advantage of llness, lk due to delayed serocol seroconverson [29]. We [~ ncreased specfcty and the potental to detect and an, others [30,31] have demonstrated by meat sngle molecules of cdna wthout the use of of PCR that there s frequen luently a perod of ant radoactve mtve probes. The senstvty of these assays HCV H( seronegatvty lastng from several weeks 1 s such h that approxmately 1 CIDs0 (1 CIDs0 = sex several months durng whch whcl" HCV-RNA s tldose that nfects 50% of chm hmpanzees) can be only marker of nfecton. Vraema may even I: detected [16]. Amplfcaton of HCV nuclec acd detectable before the onset of hepatc dysfun, derved from formaln-fxed effcent, however [17]. lver tssue s less ton n some cases [31]. The mplcaton of th fndng for blood donor scrcc screenng programmes Olgonucleotde prmer sets from several df- that assays for antbody alone are most unlke ferent parts of the HCV genome mne have been em- to be able to elmnate all nfectous donator ployed for PCR but many of :these proved nade- from the blood supply. Unf, lnfortunately howeve quate n the clncal context t due to marked se- due to logstc lmtatons (see below), don( quence heterogenety between dfferent HCV screenng by PCR s not currently curt a feasble Ol 'solates' [16,18,19]. Detecton problems arsng from such sequence varaton.m have now largely ton. Dagnostc problems wth HCV-antbody a been overcome by the ntroduct troducton of PCR says not only arse durng the scronegatve wj prmers based on the very hghly conserved 5' dow perod at the onset of nfecton but also noncodng regon of the genome )me [9,16,20,21]. Fur- patents who are unable to mount an mmur ther mprovements n the sens~tlvlt :nstvty of the assay response due to mmunodef anodefcency or mmure have been acheved by the use of a prmer set suppresson. The ablty of PCR to crcumvel whch amplfes a very short, approxmately 60 ths dagnostc dffculty has been demonstrat~ bp, segment of the 5' noncodn :lng regon [22]. n both organ transplant recpents [32] and p

4 py for haemato- HCV-RNA tons n chr~ gh mean ALT concentra H has been reported [41] HCV vraema Snce the many nvestgatons have aneously resolv- confrmed t "on therapy s capable o BH), a vraemc producng nent n both hstologca trs s commonly and bocher eters of lver dsease n lthough, even n proporton wth NANBH [42]. AI mnotransferase though the :rasn by whch nterferoj levels, persstent HCV vracma s usually reduces he!c maton n ths dsease ated wth hstologcal evdence of hepatc un} unknown, s~ based studes suggest tha ~e [34], recent evdence suggests that ths s the drug m drect suppressve effec varably so [35]. Intermttent vraema pat- upc ~on HCV [23,43,44]. In a coilabora have also been descrbed, both n humans tw tlve project lt and colleagues [23] w, hmpanzees [6,31,36], but the sgnfcance ha~ lave shown correlaton between th, "ognostc mplcatons of such patterns have clr clncal resp.~rferon and the declne J be establshed, ttr :Jtrc of crc V-RNA. Furthermore, w, lumber of studes on HCV carrers have [241 24] and oth e demonstrated that PCI: yed PCR to determne whether vrus s s a senstv~ ndcator of the vrologca Lt n body fluds other than blood. The rel~ relapse that occurs upon cessaton o ~s have been somewhat nconsstent; Fred therapy (F~ [37] faled to detect HCV-RNA n ether Taken l aken tc together, mese se tn fndngs ndcate tha or semen samples obtaned from 12 carrers PCR wll be an nvaluable tool for montorng th, whereas Lou [38] detected HCV-RNA n both of effl effect of antvral drugs n N/ NANBH. In addton these fluds. ]uds. Other workers [39,40] have also found qm uanttatve HCV PCR may also fnd a role l HCV g genomes n salva but not n breast mlk. In the selecton of those most sutable for therap?, addton ~n, urne and asctes have been shown to sre snce pre-treatment HCV-RN 7V-RNA ttre has recentl contan n HCV-RNA n a proporton of cases [38]. been descrbed as the stronge gest ndependent pre In vew of the mportance of establshng wth dctor of a sustaned respons~ ~onse to nterferon [261 ccrtant, lty the mechansms and routes of transmsson of f hepatts C, further studes n ths area are De~!)etectotz of HCV n blood donatons and bloo~ clearly warranted. products Followng the ntroducto ~ducton of blood dono screenng for ant-hcv t soon became apparen Vraema quantfcaton attd momtort uontorng orlll of theral~v that the relatonshp between serologcal reactv As mentoned above, varous,us types of quantta- ty n the antbody screenng assays and nfectvt rve and sem-quanttatve PCR ~R assays for HCV- of the donated blood was not a smple one RNA are now avalable and these have been used Prospectve studes n both Amsterdam AJ and Lon to assess vral load n both unto antreated and nter- don [5,45] showed that less than 20% of screej feron-treatcd patents. In untreated patents wth test reactve (ant-c100) donatons dot were nfec chronc nfecton, most reports arts suggest a wde tous. PCR for HCV-RNA has been used wtl spread of HCV-RNA ttres ran ~ngng from approx- success to dfferentate between nfectous am mately s genomes per ml of plasma non-nfectous donatons and an( thus has helpe~ [13,15,23], wth levels up to 100 fold hgher than mnmse unnecessary loss of donors and facl ths n the presence of mmunosu mosuppresson ([33]; tated donor counsellng [5]. unpublshed data). The extent t of the relatonshp Studes of blood donatol anatons n the UK am between the quantty of crculatn culatng HCV-RNA elsewhere have demonstrate( ated that postvty J and the degree of hepatocellular [lular damage s not supplementary serologcal tests, such as the scc known but a sgnfcant assocmnon.'aton between hgh ond-generaton recombnant mmunoblot assa

5 23 elates well wth atts C vral gven batch of factor VII wth both hep- and the lk that product transmttn [46]. Although NANBH tc lac recpents [51]. Fm lte the need for thermore, c that have not undergon t PCR wll stll vral nactv dures durng manufactur n of the many are sgnfca kely to contan detectabl at arse n blood HCV-RNA rally nactvated product~ ths context t s The correla ~n PCR postvty and r arthy that one of the RIBA-2 reactvty fectvty su~ the HCV sequences dc ns (5-1-1 plus c100-3 bands) regarded untl tccted tec by F ent ntact and potentall s defnte evdence of HCV nfecton has nfectous nf~ v r than genomc fragmenl ly been shown, by two ndependent PCR- get encrated ( nactvaton process. Dc studes, not apparently to be assocated sp nte ths, theoretcally possble the: tcv at all [48,49]. defectve det v or free vral nuclec act," extraordnary senstvty of PCR s well may be det( ~CR and yet be non-nfec lstrated by ts ablty to detect contamnat- tous. to~ It s e that certan blood proc CV n clottng factor concentrates. In the ucts mght c :t vrons at too low a lew raton of such concentrates, as many as to be l detect In practce, however [50 plasma donatons may be pooled pror to the.~se theort teratons do not appear t ;sng. Despte the very hgh dluton factors weaken we; the between PCR postvt ed, PCR has been shown to be capable of and an( nfectv lnfectwty. Although ;h currently avalable clo ng vral nuclec acd n both the plasma tnl g factor concentrates have an excellent safel pools [[15] and n the fnal clottng factor products record, sporadc reports of H( HCV transmsson st themselves elves [50]. A strong assocaton has been occasonally oct appear [52], tht thus emphassng th observed,ed between the detecton by PCR of hep- ne~ need for contnued vglance fance. The ncorporato + =o g o Q Interferon R x 4, 4, / OO o 400 < E 3O0-200 NEG o-o-d -g 6 s 10 Months Fg. 2. tlcv PCR montorng of nterferon therapy n a patent wth chronc NANBH. Note the rapd 'rebound' n HCV vraem that occurs wthn a month of f stoppng nterferon therapy. (Fgure adapted from Garson ct al, [24], wth permsson) ~_ upper lmt of normat

6 rates before rereased levels of e has been nmmunoglobuln erted agammather condtons. ntan antbodes V at a level hgh enough to be detectable n II :nts by ant-hcv mmunoassays. The post [] m ant-hcv seroconverson that results ve rse to anxety over the possblty of Fg. wtt ~ nfecton, although most studes ndcate rose ~e rsk of ths s extremely low [53]. In such PWl stances PCR s the only method capable of non mtatng between nnocent passvely con~ antbody and genune HCV nfecton [54]. dge 3. Demon wth hepatocell rose gel of se PTI-4 prmer noncodng reg ac- contans the dgest. (Fgun 3atts C vracma n 11 patent t. Ethdum bromde staned ag~ CR products generated by th )lles a 6(}-bp segment of tle V gcnome. The rght hand lan ss marker - 4,X 174/Haell om Garson et a]. [58], wt fsson). rrnaton' of specfcty qf' HCV antbody asand ant colleagl e been able to provde b :he blood donor screenng context, PCR s me :arts of ol Pq t't_;r, mclepenclent ndent non-serologcal ev only regarded as a 'confrmatory" test. The dence der of HCV nvolvement n type 2 autommun quotaton marks are ntended to pont out that an her )atts [57]. Smlarly, PCR has been used t~ antbod~ dy test result cannot strctly be confrmed confrm cor the role of HCV n hepatocellular carc by a test for vral nuclec acd. Although the noma nor [58] (Fg. 3), n mxed cryoglobulnaem presence of HCV-RNA n a blood donaton [59] and n porphyra cutanea tarda [60]. Involve strongly suggests that a postve ant-hcv test on ment of HCV n dopathc pulmonary fbros the same sample represents a genune antbody has also been suggested on the th~ bass of ant-hc'~ response, lse, t must be remembered that ths s only testng tesl [61], but ths has not yet been confrme~ an assum umpton. Conversely, t s mportant to be by PCR analyss. aware that the absence of HC CV-RNA n a donaton does not necessarly prove that the assoc- Molecular el)demology attd HC V genoo,png ated antbody reactvty s falsc. PCR amplfcaton has become a powerful a, The problem of false-postve ve ant-hcv results n the rapd generaton of nucleotde sequenc has not only arsen n blood donor screenng but data. Thus, PCR-based studes are defnng th also n nvestgatng the potental nta[ role of HCV n extent of sequence varaton and the mutatoj chronc actve hepatts and n a number of other rates n dfferent parts of the HCV genom dseases ncludng varous autolmmu :ommune "nune condtons [62,63]. The recognton that the I amno termnu [55,56]. Intal reports of HCV [CV nvolvement n of the putatve E2/NSI prote )roten contans a hyper autommune hepatts were doubted because of wtrable doman has mportar )ortant mplcatons wt] the potental confoundng nfluence of hypergam- regard to vaccne desgn [64]; envelope hypervar maglobulnaema and crculatn atng mmune corn- ablty may also be a sgnf nfeant factor n th, plexes on the accuracy of ant-hc,nt-hcv :ICV tests. The estab[shmcnt of chronc nfectons through th controversy s not merely academc but has sgnf- generaton of escape mutants. leant therapeutc mplcatons because nterferon, PCR-based sequencng studes st1 of varaton rather than cortcosterods, ma' may be the ratonal among solates are useful fl)r both molecular ep approach to treatment f an aetologcal role of demologcal surveys of geographcally dvers, HCV s confrmed. In collaboraton :)raton wth Banch populatons [65] and for defnng d{ routes am

7 23 local outbreaks xample, by see fragments, Inmother-to-chld wo generatons, Jt based on core Okamoto et al. ~son of HCV by :stck njury. 'logenetc analyss of PCR-generated sees from dverse solates has revealed the ce of at least sx dstnct HCV genotypes 69]. Several dfferent genotypng methods men descrbed, all are dependent on ntal Lmplfcaton and nvolve ether type-specfc ucleotde prmers [67], type-specfc hytton probes [69] or type-specfc restrcton ent length polymorphsms [70]. Such meth- Lay become mportant clncally because a,'r of observatons suggest that certan genomay be assocated wth more severe lver e [70] and wth ether resstance to or response to nterferon therapy {71]. Serologcal dagnoss s based on antbody reactvty aganst recombnant HCV antgens may also prove to be genotype-det ~pe-dependent to some extent [70]. In addt scence aplc been used clonng tect combnant munoassays PCR may a producton, cnes. Lmtatons L~ varous clncal and bas utlned above. PCR ha an adjunct to tradtom the producton of the re ns upon whch most rr ltly based. In the future o bc a useful tool n th ant protens for HCV va( Despte t and advantages of PCR a wde ranl studes, several nherer lmtatons dffcultes must be cor sdered. () I The e dtvty of PCR exacerbate the," problen~ )stvty due to contamm non tol of the Ths s a partcular prollem len n tran, watores where automate samplng san wth washable e pro{ ~robes s becomng r creasngly common. In laboratores where suc devces de~ are n use, PCR wll requre separat samplng san to avod even mnmal carry-over. Gre~ care must also be taken to avc avod contamnaton sat samples by amplfed PCR p )roducts from prev bus OUr reactons. Sample prepar~ ~araton and amplfce HCV r~ replcaton studes and recombnant proten tlon tol procedures should deally be carred out producton sef ~arate laboratores unless the uracl N-glyc{ Ver~ y lttle s known about the replcaton and syl~,lase (UNG) carry-over preve ~reventon system s use cellular tropsm of HCV but. ut, by analogy wth [76]. Strct applcaton of the contamnato flavvruses and pestvruses, HCV mght be ex- avodance measures outlned by Kwok an pected to be capable of nfectn ctng and replcatng Hguch [77] s essental. The mportance of thes n mononuclear cells as well 1 as n hepatocytes, measures has recently been dramatcally llm Ths possblty has been explored Iored n a number of trated by the fndngs of an nternatonal HC'~ studes by means of a PCR technk ",chnque desgned to PCR qualty control exercs{ xerclse n whch 9 of 3 be specfc for the mnus-strand nd (repleatve nter- laboratores were shown to have generated fals medate). Some [72,73] but not all [74] of these postve results [78], studes have found evdence of HCV replcaton () If samples contanng HCV l- are not rapdl n perpheral blood mononuclear lclear cells. The n- frozen and stored under opl ~tmal condtons (~ consstences may be due n 13art to dfferences n least - 20 C) or f they are subjectcd sut to numerot the strngency of the controls employed to freeze/thaw cycles, sgnfcan nfcant degradaton of th demonstrate strand-specfcty. Mnus-strand- lable RNA genomc may occur. Ths may resu specfc PCR has also been used to provde sup- n false negatve PCR results [79]. portve evdence for HCV re plcaton n a human () PCR for HCV-RNA remans a relatve T-cell lne [75]. Such technc ]ues are lkely to be complex, tme consumng ar and expensve assa' utlsed more frequently as many tony laboratores are Typcally, 20 samples take one person approx now attemptng to culture HCV n vtro, mately 3 days to complete so that the assay :R

8 Jtable for large kmor screenno, m ~ )tocol whch alamplfcaton to n has recently )col n conjuncproduct analyss a~-":c '~eppn t,l more,,c throughput applcatons. lsoi1.' absence of tssue culture technques, elec- 3croscopc dentfcaton methods and as- ~r vral antgen made the drect detecton of n clncal samples and blood products a hallengng problem. Fortunately, PCR profor amplfcaton of HCV cdna were 2 Kwok, S. a (1989) Applcaton of PCR t the detectk ffcctous dseases. Chapter 19 PCR Tcchn H.A, Ed.). Stockton Press. 3 lnns, M.A. I., Snnsky, J.J. and Whte, T.J (1990) PCR Gude to Methods and Applca tons. Acad ndon. 4 Wener, A.J tdley, D.W., Bonno, F., Saracc G., Let, (',.., Choo, Q.L. and Houghton, IV (19911) Delc ts C vral sequences n non-,~ non-b hepa ~5, I-3. 5 Garson. J./ S., Brggs. M., Tuke, P., Glazt_ brook, t,i.a. treras, t M. a h~rker, D., Barbara, J.A., Con ([990) Detecton of hepatts ( vral sequel lonatons by 'nested" polymeras chan ( react oron of nfectvty. Lancet 33" Garson, J./~ Makrs, M., Brggs, M., Machr S.J., PrestoJ dder, R.S. (1990) Demonstrato of vracma emophlaes treated wth hepat ts (7 vrus, factor VIII concentrates. Lance 336, 1022 I 7 Boom, I R., Salmans. M.M., Jansen. C.I. Werthcm ' M.E. and van der Noordaa, 1990) Rap lethod fl>r purfcaton of nucle acds. acres. J. I ('[ t. nn. lvncronlol. -?s, ~,8, 4"~ Chomczynsk, c P. and Sacch, N. (11987) Sngle-step metho of RNA solaton by acd gu uandnum thocyanatt I -~henol-chloroform extracton. Anal. Ar Bochem. 162, q. 9 Castllo, I I., Barlolomc, J.. Ourog,ga, J.A. and Carreno, (1992)( Comparson of several PCR procedures usng dl ferent I regons of the HCV genom enome. J. Vrol. Methods 3~ l0 Van Doorn, L.J.. wm Belkum,,~ A., Macrtens, G.. Qun W., Kos, T. and Schellekens. tl. (1992) Hepatts C vru antbody detecton by lne mmunoassay and (near) fu length genomc RNA-capture polymerase chan reacto! dcveloped once sequence nformaton be- I avalable, and the problem of HCV detec- /as thereby solved. However, the relatve complexty of thc technque, ts hgh susceptblty to contamnaton and a number of other operatonal dffcultes have largely lmted ts use to specalst research-orentated laboratores wth expertse n molecular bology. In future ths stuaton ~s s lkely to change as PCR becomcs avalable to 3 a much wder range of htboratorcs wth the ntroducton Iroducton of'user frendly' HCV-PCR kts ncorporatmg contamlnatton preventon devccs such as the UNG system. There s also a possbl- J. Med. Vroh 38, ty that alternatve non-pcr ~, methods of HCV 11 Ln, tt.j., Sh, N., Mzokam, M. a and ltollnger, F.B. (199,~ genome detecton mght bc developed, although Polymerase chan reacton assay tk for hepatts C vrus RN/ :ther or not they wll usng a sngle tube lor rcverse transcrptkm and serk at present t s uncertan whether rounds Of amplfcaton wth nest~ nested prmer pars. J. Me~ be able to match the extraordnap rdnary senstvty of Vrol ) 225. PCR [81,82]. Notwthstandng ' such developments, 12 Kato. N., Yokosuka, O.. Omata, Iv M., tosoda, K. and Oht{ t seems lkely that for several I years at least, PCR M. (1990) Detecton of hepatts C vrus nuclec acd n Itl wll contnue to make sgnfcant contrbutons to serum by amplfcaton wth polylq merase chan reacton., both clncal practce and to our )ur understandng of Cln. hwcst [3 Ulrch, P.P., Romeo, J,M., Lane, P.K., Kelly, I., Dane the basc bology of HCV nfecton. k.j. and Vyas, G.N. (1990) Detc Detecton, semquantlatol and genetc varaton n hepatts C vrus sequences fro[ the plasma of blood donors wth elevated alanne amm References transferasc..i. Cln. Invest 86, 16{ Kancko, S., Unoura, M., Koboy~ wtsh, K., Kuno, K., Mt 1 Sak, R.K., Scarf, S., Fahxma, F.. Mulls, K.B., Itorn, rakam, S. and Hanor, N. ( ) t, Detecton of serm G.T., Erlch, tt.a. and Arnhem. m, N. (I985) t{nzymatc hepatts (' vrus RNA. Lancet 335, t~76. amplfcaton of beta-globn genomlc mc sequence and restrc- 15 Smmonds, P., Zhang, L.Q., W~ Watson, H.G., Rebus, S lon ste analyss for dagnoss of sckle cell anaenlja. Ferguson, E,D., 13alfe, P., Leadt Leadbetter, G.H., Yap, P.L Scence (I Peuthcrcr, J.F. and Ludlam, C,A. {1990) Hepatts (" quac

9 23 '~)ducts, haemophl- of antbody 2 vrus n prospectvely lk~llowe transfuson acute and chronc non-a, non- ~,.t. (1992) lmpor- hepatts. N 321, cton of hepatts C 30 Shbata. M. ljyama, M., Takahash, T.. Ma eacton. Proc. Natl. sushma, M T. (199{)) F Kuzushma, K. and Morshmz al sequences n sera durng th ddleston, A.L.W.F. acute phase a,m. J. Med. 89, epatts "C' vrus n 31 Farc, P., A ng, D., Mller, R.H., Shll, J.W )lymerase chan re- Jell, B. an~ hepatts C H. (1991) A long-term stndy m n non-a, non-13 hepatts, b u. a. lvl~.~, vuul. L,I,.Jt~r-.~l,~. ano, K., D Bscegle, A.M., ttoofnagle, J.tt. and Eng. 1 J. Me( stone, S.M. (1991) ltepatts C vral RNA n serum of 32 Poterucha, I, Lumcng, L., Lcc, (7.tt., Taswcl :nts wth chronc non-a, non-b hepatts: Detecton by H.F. I and V~ 1992) Dagnoss of chronc he[ -,olymerasc chan reacton usng muhplc prmer sets. atts Cafte rotaton by the detecton of vr~ alology 14, ter, R.S., Brggs, M., Rng, C., Tuke, P.W., Jones, P., Jge, G.F., Rodgers, B. and Garson, J.A. (1991) 11epsequences v Brnk, I N., ( e chan reacton, t lepatology I~ rams, C.J., Rng, ('.J.A., (arso ; C antbody profle and vraema prevalence n adults severe haemophla. Br. J. Haematol. 79, moto, H., Okada, S. and Sugyama, Y. (1990) Detec- J. A., Brgl Tedder, R.~ undergong Jstonc, A.H.. Lnch, D. ('. an,~ hepatts C nfecton n palcn aematologcal malgnances of hepatts C vrus RNA by a two-stage polymerase 3 reacton wth two pars of prmers deduced from the m-codng regon. Jpn. J. Exp. Med. 61), clncal and 34 Alhert, A. Novcnta, 1 F dy. Br. J. ttaematol. 83, ;, ('hemello, L., Cawdlelto, I) and Ruol, A. (19t)2) Itepatts ;on, J.A., Rng, (7., Tuke, P. and Tedder, R.S. (199{I) vraema a] se n symptom-free ndvdun anced detecton by PCR of hepatts C vrus RNA. :c! 336, wth ant-tl antl-lrl('v, l,ancet 340. ~ / 698. ( 35 I Brllant, S., Fol, M., Gaan, S., Masc, ('., Mglol, b 22 Gurson, J.A., Rng, C. and Tuke, P.W. (1991) Improvement of ItCV genome detecton wth 'short" PCR prodand Barbara, L. (1993) Persstent hepatts (" vracm wthout lver dsease. Lancet 341, ~5. ucts. Lancet 338, Beach, l M.J., Meeks, E.L., Mmms, Mr L.T., Vallar, I3 23 Brllant, S., Garson, J.A., Tuke, P.W., Rng, C., Brggs, DuCharme, ] L., Spclbrng, J., Taskar, Ta! S., Schechcr,,1.I3 m., Masc, C., Mglol, M., Barbara, L. and Tedder, R.S. Krawczynsk, ] K., Bradley, D.W. (1992) Tcmporal rclatol (1991) The effect of alpha nterferon therapy on hepatts C vraema n communty acqured chronc non-a, non-b shps of hepatts C vrus RNA and antbody responst followng I expermental nfecton of chmpanzees.,1. Me~ hepatts: a quanttatve PCR study. J. Mud. Vrol. 34, Vrol. 36, Fred M.W., Shndo, M., Fong. T.L., " Fox, P.('., 11oofm 24 Garson, J.A.. Brllant, S., Rng, C., Pern, P., Mglol, M. glc, I J.H. and D Bscegle, A.M. ( 1992) Absence of hepat and Barbara, E. (1992) Hepatts C vraema rebound after ts C vral RNA from salva and semcn of patents wt 'successful' nterferon therapy n patents wth chronc chronc hepatts ('. Gastroentcro "oenturology 102, non-a, non-b hepatts. J. Med. Vrol. 37, Lou, T.C.. Chang, T.T., Young, K.C., b Ln. X.Z., Ln, ('? 25 Kaneko, S., Murakam, S., Unoura, M. and Kobayash, K. and Wu, H.L. (1992) Detecton of I ICV-RNA n sally (1992) Quanttaton of hepatts C vrus RNA by compet- urne, semnal flud, and asctes. J. Med. Vrol. 37, lve polymerase chan reacton. J. Mud. Vrol. 37, Wang, J.T., Wang, T.It., Ln, J.T., J.7 Sheu, J.C., 15n, S.N 26 ttagwara, 11., Hayash, N., Mta, E., Takehara, T., Kasa- and (hen, D.S. (1991) Hepatts C I vrus RNA n salwt hara, A.. Fusamoto, tt. and Kamada nada, T. (1993) Quantta- patents wth post-transfuson hep )atts (' nfecton, l,anc lve analyss of hepatts C vrus s RNA n serum durng nterferon alfa therapy. Gastroenterolo lterology 1(t4, , 48. 4O Ogasawara, S., Kage, M., Kosa, K.l., 1 Shmamatsu, K. all 27 hnbert, L., Caran, E., Bettnard, A.,Zonaro, A.,Alber- Kojro, M. (1993) Hepatts C vral RNA n salwt ar tn. A.. Prm., D. (1991) An mmunoassay for specfc breastmlkof hepatts(carrer mothers, n l+ancct 341, 56 amplfed I tcv sequences. J. Vrol. -ol. Methods 34, Kurosak, M., Enomoto, N., S Sato, C., Sakamolo, Ix 28 Whtby, K., Garson, J. and Baret. ~t, A. (1993) A mcrottrc Hoshno, Y., ttartan, 11. and Marumo, M~ F. (1993) Correl format quanttatve PCR assay for k)r HCV RNA employng lon of plasma hepatts C vrus RNA levels wth scru xanthne oxdase generuted chemlumne mlumnescence. nnescence. Federa- alanne amnotransfcrase m n non-a, non-? non-laj hepatts chron lon of European Mcrobologcal fl Socetes (FEMS) Sym- lver dsease J. Med. Vrol. 39, 246 2~ 251). posum on the hepatts C vrus s and ts nfecton. June 42 Davs, G.L., Balart, L.A., Schff, ", E.R., [ kndsa,v, K., Bode 29 July 1, 1993, lstanbul, Turkey, Abstract pp 137, p.76. hemer, H.C., Pcrrllo, R.P., (arty, Ca W., Jacobson, I.lV 29 Alter, H.J., Purcell, R.H., Shh, 1, J.W., Melpolder, J.C., Payne,,I., Dcnstag, J.L., Van-Thel, I).1t., Tamburro, ( Houghton, M., Choo, Q.L. and Kuo, G. (1989) Detecton Lefkowtch, J., Albrecht, J., Meschevlz. (',, Ortego, T.

10 chronc hcpatts (" 57 Garson, J.? Rng, C., Cassan, F., Ballardn nultcenter random- G., Brggs, R.S. and Banch, F.B, , Hcpatts ( aduhs wth type 2 autommun M. and Okamoto, hepatts.,i, 4, vrus RNA by nter- 58 Garson, J./ O., Schmd., Rng, ('.J.A., Joller, If., Zah ler, H. (19921 Detecton of hcf da, K., Malsumoto, atts C vra san patents wth hcpatocellula. M., Mornaga. T. carcnoma. 38, eron admnstraton 59 Agnello, V, and Kaplan, L.M A rol... t r. ltents wth.... chronc for hepatt on n type II cryoglobulnaenf: tts (', tlcpatology 13, N. 1 Engl. J Der Pool, C.L.. Reesnk, H.W., Schaasbcrg, W., 60 tlerrcro, I C Brugucra, M., Erclla, M.G tvaar-kuypers. A., Bakker, E., Exel-Ochlers, P.J. and Barrera, I J. Teres, J., Mascaro, J.M. tn ~, P.N. (1990) Infectvty of blood seropostve for Rodes, ] J. ( ts C vrus nfecton a trgger llts C vrus antbodes, l,ancet 335, I ")orphyra c Lancet 341, ;ol J.A., Clewley,,I.P., Smmonds. P., Zhang, LQ., 61 Ueda, I T., ( d, N., Yamaguch, M.. llra, K, J., Rng, C., Follet E.A.C., Dow, B.('. and Gunson, Horuch. I J., Myamoto, T. and lto, 1~ 1992) Itepatts C vracma n Unted Kngdom blood (1992)( ]do I ary' fbross and hgh prevalent ~rs: a nmltcentrc study. Vox Stng. 62, ,'t, E.A.C., Dow, B.C., McOmsh, F., Yap, ILL, of scrunl a Ds. ] 146, 2f palls (" vrus. Am. Rev. Resp hes, W., Mtchell, R., Snmonds, P. (1991). tt('v rmatoo' tcstng of blood donors, l.ancet 338, Wencr, A. Saracco, '. G. con, C., Hall, I.E., Bonno. F R., Crawfl~rd. K.. Maron, C.D ons, ('.J. and Garson, J.A. (19931 Itcpatts (' vrus ('rawfl)rd, I ttakrshna, S., Myamura, T d donor screenng: rcconlbmmt mnlnnoblot ~lssay Mct ] lutchn rs, T. and Houghton, M prctaton rc-cxamncd, Vox Stng. 64, 253. Sequence 31.Jqu~.'llCC ', VHIKItlOI v I11 llcp~ltlls atts C k vral solates. J. Hcpato,'h, M.P.. Toblcr, L., Quan, S., Wlber, J.('., Johnson. P., Polto 'olto, A.. Steane. E.. Zola, A.. Bahl C., Nellcs. M. and 13 (Suppl. 4), S Ogata, I N., Alter, tt.j., Mller, R.H. I and Purcell, R.I Lcc, S.R. (1993) A pattern of 5 I 1 {llld tll0. 3 only on ( 1991) Nuclcotde scquencc and mutaton rate of the 1 hepatts tts (' vrus (HCV) reconlbnant nuntlnoho assay does not reflect ttcv ntkecon n blood donors. Transhlstran ol hepatts (" vrus. Proc. Natl. I' Acad. Sc. USA 8f son 33, f4 Farc, ] P., Alter, t l.j., Govndaraj an, S., Wong, I).C. K 50 Makrs, M.. (;arson, J.A., Rng, ('.J.A., Tukc, P.W., Ted gle, I R., kesnewsk, R.R, Mushahwar, I.K., I)esa, S,M dcr, R.S. and Preston, F.E. (19931 l epalts (' vral RNA n clottn~ lottng factor concclltr,ltcs and the dcvelopn]cnt of Mller. R. t., Ogata, N. and Purcell, R.If ) Lack protectve I mnmnty aganst renfecton wth hepatls q hepatts tts n recpcnts. Blood Garson ~on, J.A., Preston. F.E., Makrs, M., Tuke, P., Rng, vrus. Scence 258, Smmonds, P., McOmsh, F., YaF Yap, P., Chan, S.W., L (',. Machn, S. and Teddcr R.S ) Detecton by PCR C,K, Dushcko, G., Saeed, A.A. and ttolmcs, E.(', (19% of hepatts (" vrus ll factor VIII "Ill conccntrales. Lancet Sequencc varablty n thc5' non-codng regon of hepal 335, ts (" vrus: dentfcaton of a new ne~ vrus type and reslrk 52 Schulman, S., I,ndgrcn, A-Ch.. Pelrn, P. and Allandcr, lons on scquencc dversty. J. Gen. Vrol. 74, 661-6h8. T. (1992)Tlansmsson of hepatts tts (" wth pastcurscd 66 lnouc, Y., Myamura, T., Unawlma, Unayam T., Takahash, K. an factor VIII. l,ancct 3411, Sato, I. (1991) Maternal transl'cr transfc of HCV. Nature 35~ 53 Rcznkoff-Etevant, M.F,. dc Lachaux achaux, Y., Marpcau, L and ('ouroucc, A. (1992) Intravenous mmunoglobulns (~7 Okamoto, H., Sugyama, Y.. Okada. ()M S., Kura. K.. Akt amt hepatts (' transmsson n hcalthy pregnant womcn, hanc, Y.. Suga, Y., Tanaka. T., ~[ Sam, K., Tsuda. F Ixmcet 340, 988. Myakawa, Y. and Mayum, M. (1992) Typng hepatts t 54 Lev, S., Foster, ('., Itodgson, t[.j.f...j.f., Ward, K.N., So, A., vrus by polymerasc chan reacton rca( wth lypc-specf (}arson, J.A., Waxman, J. and Swrsk wrsky, I) ) ('hronc prmcrs: applcatons to clncal surveys st and tracng nfcc lver dsease duc to hepatts C. Br. Med. J. 306, tous sources. J. Gen. Vrol. 73, McFarlane. I.G., Smth, H.M., Johnson, k)hnson, PJ. Bray, G.P., 68 ('han, S.W., McOmsh. g., t olmcs, 11~ E.C., Do'*, B Vergan. D. and Wllams, R. (1990) Hepatts (" vrus Pcntherer, J.l~'., Follctt, K., Yap, P.[.. alld Smmonds, I antbodes n chronc actve hepatts: palhogenelc factor (1992) Analyss of a new hepat mtls 17 vrus type and l or falsc-postvc result. Lancet 335. :~5, phylogenctc relatonshp to exstng wlrants. J. Ocn. V 56 Thchnann, I,., BlazeR, M., (looser, +or, T., Gmcln, K., Kom rol. 73, , mcrell, B. and Fehn W False postve anl-hepat- 6t, ~ Sttowcr, L, Rossau, P,., Wysenr, A.. l)uhamel. M., Vat ts (' vrus tests n rheumatod arthrts. "thrts. Lancet, derborght. B., tleuverswwl I].V. and Macrtens, G. 1199,:

11 23 characterzaton of (1992) Evd J. Gen. Vrol. 74, genome n 89, ~ Gllon, J., Frame, 76 Erlch, H.A tt, E.A.C. and Sm- advances r )es of hepatts vrus specfc dfferences 77 Kwok, S. a alanne amnotrans- wth PCR Zaajer, H. ;ll, I~.., l\ llku~ IVI.. kj~.{lllll.jl.l.j. In. 1~177,;~1 92) HCV Ilk-V genotypes ~-,~IIULy/)k. 3 1.N., J Gerk~ ronc hepatts C and response to nterferon. Lancet polymerase I 1543 vrus. Lanc~ ego, A.L., Maccha, D., Mont, M., Thers. V., 79 Busch, ] M.t zett, M., Fosch, M., Magg, E., Romagnan, S., Gen- Evans, ] C.S. P. and Brechot, C. (1992) Infecton of perpheral onuclear blood cells by hepatts C vrus J. Hepatol. ~ ler, H.M., Pfaff, E., Goeser, T., Kallnowsk, B., Sol-, C. and Thelmann, L. (1993) Perpheral blood leukoage on dete Young, K.I Detecton c transcrpto serve as a possble extrahepatc ste for hepatts C replcaton. J. Gen. Vrol ,'hara, T., Hayash, N., Mta, E., Hagwara, H., Ueda, Katayama, K, Kasahara. A., Fusamoto, H. and Kaa, T. (1992) Detecton of mnus strand of hepatts C RNA by reverse transcrpton and polymerase chan ton: mplcatons for hepatts C vrus replcaton n nfected tssue. Hepatology 15, Shmzl rzu, Y., Iwamoto, A., Hjkata, M. and Purcell, R.H. "o replcaton of hepatts C vrt lne. Proc. Natl. Acad. Sc. US, and Snnsky. J.J. (1991) Recel ~se chan reacton. Scence 25 (198,9) Avodng false postv~ ~ LT.M., Reesnk, tt.w., Wnke ele, P.N. (1993) Relablty 1 for the detecton of hepatts 4. 7., Johnson, l_., Tobler, L. an t of specmen handlng and sto ls C vrus RNA. Transfuson 3 R.M. and Myers, T.W. (1992 flus RNA by a combned rever~ chan reacton assay..1. Cln. M crobol. 31, 81 Hu, K.Q., d Verlng, J.M. (1992) Dre detecton o patts C vrus RNA usng proh~ from the 5' regon. J. Cln. Invest. St, ~, 21)4( Lau, J.Y.N., Davs, O.L., Knffen, Knfllel J., Qan, K.P., Urde M.S., Chan, C.S., Mzokam, M., h Neuwald, P.D. ar Wlber, J.C. (1993) Sgnfcance of o serum hepalts C vrt RNA levels n chronc hepatts C. Lance/