1.2.6.1 Liver enzym es
HCV infection classically causes elevation o f serum alanine am inotransferase (A LT) and aspartam ine am inotransferase (AST). H ow ever, the extent o f enzym e elevation may fluctuate in an individual throughout the course o f infection and although may reflect inflam m atory activity w ithin the liver, the correlation w ith the extent o f histological injury is poor. As liver injury progresses disturbances o f other liver enzym es can occur with elevation in serum bilirubin levels. R eduction in serum album in and prolongation o f the prothrom bin tim e heralds the onset o f decom pensated cirrhosis.
L2.6.2 H CV serological assays
Serological tests form the basis for screening for HCV infection in both individuals and larger populations, such as blood donors. Since the detection o f HCV, there has been considerable evolution o f the serological assays available for testing for HCV, with im provem ents in both sensitivity and specificity o f the assays [Y ounossi et al, 1996], Enzym e-linked im m unosorbent assays (ELISA or EIA) are relatively straight-forw ard, reproducible and inexpensive m eans o f detecting antibody to HCV and hence have been adopted as the m ainstay screening test. In b rief these consist o f m icrow ells coated with recom binant HCV antigens that are incubated with donor-diluted serum, follow ed by the addition o f an enzym e-linked anti-hum an conjugate that reacts with bound antibody and is detected by the addition o f a substrate m easured by optical density techniques. C urrently, three generations o f the anti-H C V ELISA exists incorporating recom binant antigen initially from a single region o f the HCV genom e (N S3/N S4) and now utilise antigens in three distinct regions o f the genom e (core, N S3/N S4 and NS5). The sensitivity o f the EIA test has im proved from 95-98% in the first generation to 99.9% in the third generation tests.
However, the specificity o f the EIA m ethods rem ains low w ith 30-50% testing falsely positive in large population screening studies [K leinm an et al, 1992] and hence supplem ental techniques w ere developed to confirm the diagnosis o f H C V infection. The most com m only utilised supplem ental technique is the recom binant im m unoblot assay (RIBA). In b rie f this technique utilises a nitrocellulose strip im pregnated w ith HCV antigens that is incubated w ith donor serum. Follow ing incubation the strip is w ashed o f non-specific antibodies, exposed to conjugated goat anti-hum an IgG bound to horseradish- peroxidase and then a solution containing hydrogen peroxidase is added resulting in the production o f colour bands, specific for the corresponding antigen and the intensity o f w hich is proportional to the am ount o f bound antibody reported on a sem i-quantitative scale. Again, three generations o f the RIBA tests exists incorporating progressively more recom binant or synthetically produced HCV antigens. W idely used throughout Europe, the
anti-H C V RIBA-3 test detects for the presence o f antibodies against the C22 (core region),
C33 (NS2 region), C 200 (incorporating the clOO region o f the N S3/N S4 region) and an
NS5 antigen. A lso included on the strips are tw o levels o f hum an IgG and the hum an super
dism utase (SO D ) antigen as internal controls. T he RIBA-3 is considered negative in the
absence o f any reactivity to HCV antigens, indetenninate w ith reactivity to one HCV
antigen and/or SOD positivity and positive w hen reactive against tw o or m ore HCV
antigens.
1.2.6.3
HCV RNA testing
T he advent o f serological testing for HCV has virtually nullified the risk o f post-transfusion
hepatitis in countries w ith routine testing. H ow ever, pitfalls still exist with respect to the
diagnosis o f HCV infection based on serology alone. Firstly, follow ing acute infection,
seroconversion m ay take up to 6 m onths to occur [M cH utchinson et al, 1991], Secondly,
there rem ains the sm all risk o f false negative serology, especially in the
im m unocom prom ised host [Lok et al, 1993]. Thirdly, up to 50% o f subjects chronically
infected will have norm al A LT values [Shakil et al, 1995]. T herefore, confirm ation o f HCV
infection is concluded by the detection o f the HCV viral RNA in the host serum by various
nucleic acid am plification techniques (N A T), most com m only RT-PCR [Gretch et al, 1996].
C urrent techniques to detect HCV in the serum by PCR allow both qualitative and
quantitative results. T he 5 ’UTR and core regions w ithin the HCV genom e show m axim um
sequence conservation am ong the various HCV genotypes and hence are the targets for
am plification. In qualitative RT-PCR assays, the target area undergoes a process o f reverse
transcription w hereby a com plim entary strand o f copy deoxyribonucleic acid (cDNA) is
generated. This is then am plified according to general PCR principals. The products are
then hybridised to oligonucleotide probes specific to the target and detected by colorim etric
determ ination.
Q uantitative PCR allow s for the estim ation o f the HCV viral load in a patien t’s serum at
one point in time. H ow ever, the natural history o f HCV is to fluctuate m ore than a m illion
fold over tim e w ithin a single person. C oupled to this was the lack o f standardisation o f the
quantitative N A T techniques am ong laboratories. To correct for this, a W H O International
standard collaborative study was undertaken in 1997 [W HO, 1998]. A sam ple o f
lypophilized m aterial containing HCV genotype 1 was accepted as the international
standard for N A T assays. T oday several m ethods exist but tw o o f the m ore w idely used
techniques include quantitative non-com petitive PCR and branched chain (bDN A) assay.
Q uantitative non-com petitive PCR includes a quantitation standard that is co-am plit'ied w ith
the target to m onitor efficiencies o f the extraction and am plification reactions. A single
p rod u cts are den atu red, transferred to a m ic r o w e ll d e te c tio n p la te and se r ia lly d ilu ted from 1:5 to 1 :625. T h e y are then h y b rid ised to H C V sp e c ific m ic r o w e lls . T h e sa m e product is again se r ia lly d ilu te d ( 1 :5 to 1 :25) b efo re h yb rid isation to Q S s p e c ific w e lls . T h e h ig h est d ilu tio n that g iv e s an A4 5 0 b e tw e e n 0 .2 and 2 .0 on th e H C V -s p e c ific w e lls and Q S -s p e c ific
w e lls are then se le c te d and u sed to c a lc u la te th e n um ber o f c o p ie s /m l in th e sam p le. T h is sy stem is u tilise d in th e R o c h e A m p lic o r H C V M o n ito r’’^^ (R o c h e D ia g n o stic S y ste m s, B ranchburg, N J ) w h e re an a ly tica l s e n sitiv ity is reported at 5 0 0 c o p ie s /m l w ith a lin ear range to 10^ c o p ie s /m l. T h is n o n -c o m p e titiv e quan titation m eth o d is e a s y to u se and h ig h ly se n sitiv e .
T h e b D N A H C V R N A a ssa y u se s a se rie s o f n u c le ic acid h y b r id isa tio n s to a c h ie v e a m p lifie d sig n a ls rather than a m p lific a tio n o f th e viral g e n o m e . In th is m eth o d , th e H C V g e n o m ic R N A is lib erated from sp e c im e n s and b ou n d to th e s o lid p h a se o f a m ic r o w e ll b y a set o f s p e c ific sy n th etic o lig o n u c le o tid e capture p rob es. V ira l R N A ’s are a lso h yb rid ised to separate target p rob es from the 5 ’U T R and co re re g io n s o f H C V . S y n th e tic branched D N A a m p lifie r m o le c u le s and m u ltip le c o p ie s o f a lk a lin e p h o sp h a ta se -lin k ed p rob e are then h y b rid ised to th e im m o b iliz e d viral n u c le ic acid c o m p le x . D e te c tio n and quan titation are a c h ie v e d by in cu b a tin g the c o m p le x w ith c h e m ilu m in e sc e n t su bstrate d io x e ta n e and m easu rin g ligh t e m iss io n . T h is m eth od is u sed in th e Quantiplex^'^ H C V R N A a ssa y (C h iron C orp oration , E m er y v ille, C A ) and is reported to d etec t virus b e tw e e n 3 5 0 ,0 0 0 and 5 8 ,0 0 0 , 0 0 0 eq u iv a le n ts/m l. T h erefo re, a lth ou gh th is sy stem is e a s ily p erform ed w ith low risk o f co n ta m in a tio n and h ig h ly rep rod u cib le, th is sy stem la c k s s e n s itiv ity to d etect p atien ts w ith lo w viral load s.
1.2.7 Treatm ent
T h e current treatm ent o p tio n s for H C V in clu d e In terfero n -a , a recom b in an t form o f the n atu rally occu rrin g c y to k in e , and ribavirin, a sy n th etic n u c le o s id e a n a lo g u e .
R ib avirin in terferes w ith viral R N A sy n th e sis, su b se q u e n tly in h ib itin g p rotein sy n th e sis and H C V r e p lica tio n p ro d u cin g a v iru s-sta tic e ffe c t [C rotty et al, 2 0 0 2 ] . In ch ro n ic H C V
in fe c tio n it has b een a sso c ia te d w ith n o rm a lisa tio n o f serum A L T v a lu e s and im p rovem en t in h isto lo g ic a l injury, but a lso w ith H C V R N A recu rren ce o n d isco n tin u a tio n o f therapy [ D iB is c e g lie et al, 1 9 9 5 ].
IF N -a is k n o w n to m od u la te th e im m u n e re sp o n se b y a ctiv a tin g natural k ille r c e lls , p ro m o tin g c y to to x ic T ly m p h o cy te m aturation, in cr ea sin g c e ll su rface e x p r e ssio n o f M H C c la s s I a n tig e n s and b y d irect anti-viral m ec h a n ism s through d isru p tion o f viral rep lication [P eters, 1989; S a m u el, 1 9 9 1 ]. S in c e its d isc o v e r y , th e m a in sta y o f H C V treatm ent h as b een I F N -a . H o w e v e r , in itial stu d ies d em on strated p o o r treatm ent e f f ic a c y w ith su stain ed
response rates o f only up to 25% , but w ith 12 to 18 m onths treatm ent, this rate increased to 35% [Poynard et al, 1996], M ore recently, the com bination o f IF N -a and ribavirin for a period o f 24 or 48 w eeks w as associated w ith greater sustained response rates o f 35% and 43% respectively, com pared to 19% in the group treated w ith IF N -a alone for 48 weeks
[Poynard et al, 1998]. S im ilar results w ere achieved for a large Am erican based study [M cH utchinson et al, 1998], Factors associated with a poor response to treatm ent include genotype lb infection, advanced fibrosis on liver biopsy, m ale sex and older age.
M ore recently, pegylated IF N -a (P eg-IF N -a) has been introduced with even better response rates. P eg-IF N -a represents the addition o f a polyethyleneglycol m olecule to IF N -a
resulting in a biologically active m olecule w ith a longer half-life. Therefore, rather than adm inistering the drug three tim es a w eek as w ith IF N -a, P eg-IF N -a is adm inistered once weekly. An initial study reports sustained response rates o f 54% after 48 w eeks treatm ent o f P eg-IFN -a/ribavirin, com pared with 47 % in the IF N -a/ribavirin group [M anns et al, 2001 ]. S tratification indicated that a better treatm ent response w as associated with genotype 2/3 (82% ), HCV viral load < 2 m illion copies/m l (78% ) and no/m inim al fibrosis (57% ). H ow ever, as all o f the above studies have dem onstrated, IFN -a, Peg-IFN -a and ribavirin are associated with significant adverse effects that prohibit their use in a proportion o f patients who otherw ise w arrant treatm ent and necessitate w ithdraw al o f treatm ent in approxim ately 15%.
HCV, either with decom pensated liver disease or HCC, is one o f the m ajor indications for orthotopic liver transplantation (O L T)(D etre et al, 1996). In 1993 it surpassed alcoholic liver disease as the most com m on single indication for O LT. T he t'lve-year survival rate post transplantation for H C V is approxim ately 64% and graft survival rate is 57% . These outcom es are com parative to patients transplanted for alcoholic liver disease (65% and 59% respectively) but less than for cholestatic and autoim m une liver diseases (76% and 70% respectively). R e-infection o f the liver graft occurs alm ost universally but this is associated w ith insidious liver disease in the m ajority o f cases (B izollon et al, 1999). G raft HCV- related cirrhosis has been reported in up to 8% o f recipients after five years (G ane et al,
1996). This was thought to be due in part to genotype lb infection, w hich is unsupported by other studies (Zhou et al, 1996). An aggressive infection characterised histologically by fibrosing cholestasis and clinically by rapidly progressive graft dysfunction develops in approxim ately 10% o f liver recipients (S chluger et al, 1996). In this retrospective study o f 135 O LT recipients the m ean tim e to the onset o f cholestasis was 5.3 m onths and the tim e to retransplantation follow ing the onset o f cholestasis w as 4.1 months.