Relation of hepatitis C virus genotypes to risk factors and hepatic disease in Spanish patients

ORIGINAL ARTICLE Rela ion o hepa i is C i us geno ypes o isk ac o s and hepa ic disease in Spanish pa ien s l? Alonso Alonsol, A. O duh?, A. San Miguel', E. Dominguexl, M. A. B a o?, M. P Gu ie ex'?, J. M. Ei e?, L. Inglada3, J. 111. Gonxalex He nandez4 and A. Rod iguez ?i, es2 'Labo a o y, Regional Hospi al, Mon o e de Lemos, Lugo, 2Mic obiology Depa men and Clinical-Epidemiological Resea ch Uni , Uni e si y Hospi al, Facul y o Medicine, Valladolid, 31n e nal Medicine, Regional Hospi al, Medma del Campo, Valladolid, and 41n e nal Medicine, RJO Ho ega Hospi al, Valladolid, Spain Objec i e: To asce ain he p e alence o hepa i is C i us (HCV) geno ypes in Spain and hei dis ibu ion by isk ac o s. Me hods: The s udy co e ed 216 pa ien s wi h hepa i is C. O hese, 63 we e in a enous d ug use s (IVDU), 44 had ecei ed ans usions, and 30 we e hemodialyzed, and in 79 he isk ac o s we e unknown. An ibodies agains HCV we e de ec ed by second-gene a ion enzyme immunoassay (EIA) and con i med by immunoblo . HCV RNA p esence was in es iga ed by e e se ansc ip ion-polyme ase chain eac ion (RT-PCR), and a e e se hyb idiza ion es o he ampli ica ions was used o he geno yping. Resul s: The mos equen ly encoun e ed geno ypes we e Ib (48.1%), la (21.3%) and 3a (11.1%). HCV geno ypes la (42.8%) and 3a (20.6%) we e he mos p e alen geno ypes in IVDU pa ien s, while Ib was he mos equen in pa ien s wi h unknown isk ac o s (62.0%), ans used pa ien s (68.1%) and hemodialyzed pa ien s (50.0%). Mixed in ec ions we e de ec ed in nine cases (4.1%); h ee appea ed in IVDU pa ien s (4.7% o he o al IVDUs), wo in ans used pa ien s (4.5%) and ou (50%) in pa ien s wi h unknown isk ac o s. No s a is ically signi ican di e ences we e ound in a e age ages o he IVDU pa ien s wi h di e en geno ypes. Non-IVDU pa ien s ha ing geno ype 3a p esen ed he lowes a e age age o all. No signi ican s a is ical di e ences we e obse ed in alanine a nino ans e ase le els among pa ien g oups wi h di e en geno ypes (p0.05 in all cases). Sub ype Ib was p esen in six o he se en cases o ci hosis (85.7%) and in nine o he 18 cases o ac i e ch onic hepa i is (50.0%). Key wo ds: Hepa i is C, HCV geno ypes, epidemiology INTRO D U CTlO N Since he disco e y o he hepa i is C i us (HCV) in 1989 by in es iga o s a Chi on Co po a ion [l], a g ea deal o in o ma ion abou he gene ic di e si y o HCV has appea ed in wo ld li e a u e. The i s e idence o hs di e si y a ose &om he compa ison o a Japanese isola e (HCV J1) and an Ame ican one (HCV-1) [2], Co esponding au ho and ep in eques s: An onio O du ia, Depa amen o de Mic obiologia, Facul ad de Medicina, C/ Ramon y Cajal s/n, 47005 Valladolid, Spain Tel: +34 983 42 30 63 Accep ed 7 June 1997 +34 983 42 30 66 which e ealed di e ences in he NS3 and NS4 egions. La e on, di e ences we e demons a ed in he NS5 [3] and en elope [4] egions, sugges ing he exis ence o mul iple HCV ypes. The immedia e consequence o disco e ing he exis ence o HCV geno ypes was he in es iga ion o hei ela ion o a ied aspec s o he in ec ion. One o he aspec s mos s udied has been he geog aphic dis i- bu ion o he di e en geno ypes, which ha e been ound o be widely sp ead. Ths is especially ue o geno ype Ib, which seems o ha e he g ea es wo ld- wide p e alence [5]. Howe e , egional d e ences in geno ype dm ibu ion ha e been obse ed. In Japan, China and Taiwan, geno ypes lb, 2a and 2b cons i u e he as majo i y o a ian s ound [5,6], geno ype la 647 648 Clinical Mic obiology and In ec ion, Volume 3 Numbe 6, Decembe 1997 being e y in equen . In con as , in sou heas Asia geno ypes la and 3 a e equen ly ound [7]. Al hough he in o ma ion a ailable on geno ype ds ibu ion in Ah-ica is limi ed, i seems ha geno ype 4 p edomina es in wide zones o his con inen [6,8,9]. Howe e , Sou h Ah-ica cons i u es an excep ion, as geno ype 5 is he mos equen a ie y he e [6,10,11]. In Occiden al coun ies (Eu ope, he USA and Aus alia) and in Sou h Ame ica geno ypes 1, 2 and 3 a e he mos p e alen [5,6,8,12-141. Ano he aspec s udied has been he ela ion o p e alences o isk ac o s, wi h h e ences being e ealed by he s udies pe o med. Okamo o e al [7] ound h e en p e alences o geno ypes lb and 2a in blood dono s and Japanese pa ien s wi h non-A, non- B hepa i is. D iesel e al [15] desc ibed an ele a ed p e alence o ype 3a in IVDU pa ien s. The objec i e o ou s udy was o asce ain he p e alence o di e en HCV geno ypes in ou heal h a ea, as well as he possible in luence o isk ac o s on hei dis ibu ion and hei ela ion o hepa ic disease. PATIENTS AND METHODS In his s udy, 216 pa ien s diagnosed as ha ing hepa i is C we e included. All o hem p esen ed an ibodies agains HCV (an i-HCV) and HCV RNA de ec able in se a by e e se ansc ip ion-polyme ase chain eac ion (RT-PCR). Li e biopsy was pe o med in 37 pa ien s. The dis ibu ion o pa ien s by isk ac o s was as ollows: 63 (29.1%) we e in a enous d ug use s (IVDUs) (30.5k4.6 yea s), 44 (20.3%) had ecei ed ans usions (48.3k14.9 yea s) and 30 (13.8%) we e he nodialyzed (52.4k15.5 yea s), and in 79 cases (36.5%) no known isk ac o was ound (55.6k15.4 yea s). All pa ien s unde wen es ing o he p esence o HBs an igen and IgM agains hepa i is A i us (IMx, Abbo Labo a o ies, Chicago, IL, USA); he esul s we e nega i e in bo h cases o all subjec s. A second-gene a ion enzyme immunoassay (EIA) (Abbo Labo a o ies, Chicago, IL, USA) was used o sc een o an ibodies agains HCV. Immunoblo (Deciscan HCV, Sano i Diagnos ics Pas eu , Ma nes-la- Coque e, F ance) was used o con i m an ibodies agains HCV This es uses ecombinan p o eins de i ed om he co e (Cl) and NS3 egions, and syn he ic pep ides de i ed &om he co e (C2) and NS4 egions. To de ec HCV RNA, an RT-PCR (Amplico HCV, Roche Diagnos ic Sys ems, Inc., B anchbu g, NJ, USA) was used. A e e se hyb idiza- ion es o he ampli ica ion ob ained by RT-PCR (INNO-LiPA HCV, Innogene ics, Belgium) was used o HCV geno yping. Speci ic oligonucleo ide p obes de i ed &om he non-codmg egion 5‘ a e immobi- lized on ni ocellulose s ips by a poly-T glue. The ampli ica ions, p e iously ma ked wi h bio in, a e incuba ed wi h he s ips a 50°C o pe mi hei hyb idiza ion wi h he bound p obes. A e washing, hey a e incuba ed wi h s ep a idin conjuga ed wi h alkaline phospha ase a oom empe a u e. Colo de elopmen is pe o med by addi ion o ni oblue e azolium (NBT) and b omochlo oindol phospha e (BCIP). Each s ip has a o al o 15 bands. The i s is used as a con ol o he adh ion o conjuga e. The second con ains a uni e sal p obe which hyb idizes wi h he ampli ica ions o any geno ype. The ollowing 13 bands allow di e en ia ion o he ollowing HCV geno ypes and sub ypes acco ding o he scheme o Simmonds e al [16]: 1, la, lb, 2, 2a/2c, 2b, 3a, 3b, 4a and 5a. AU es s we e pe o med acco ding o he manu ac u e s’ ins uc ions. S a is ical analysis o he esul s was pe o med by he SAS p og am o pe sonal compu e s (SAS Ins i u e Inc., Ca y, NC, USA). Fishe ’s exac es ( wo- ail) was used o compa ison o p opo ions in he s anda d wo-way con ingency ables. Non-pa ame ic es s (ANOVA-based anks) we e used in compa ison o measu emen s. Values o p< 0.05 we e conside ed s a is ically signi ican . RESULTS The dominan geno ype was ype 1, ound in 164 o he pa ien s s udied (75.9%); he nex mos equen was 3a, p esen in 24 pa ien s (1 1 .l%) (Table 1). Geno- ypes 4a and 2 we e seldom ound, being p esen in only eigh (3.7%) and wo (0.9%) pa ien s espec i ely. In nine cases (4.1%), he geno ype was impossible o de e mine wi h he es used. Wi hin geno ype 1 he g ea es p e alence co esponded o sub ype lb, p esen in 104 pa ien s (48.1%), whde sub ype la was ound in 46 pa ien s (21.3%). In 14 cases (6.4%) classi ied as geno ype 1 i was impossible o de e mine he sub ype. Likewise, in one o he wo pa ien s in whom geno ype 2 was ound, he sub ype could no be de e mined, and he emaining case belonged o sub ype 2a/2c. A mixed in ec ion was ound in nine pa ien s (4.1%): in one case wi h geno ypes 1 and 2, ou cases wi h sub ypes la and lb, and ou cases wi h geno ypes la and 4a. Table 1 shows geno ype dis ibu ion acco ding o isk ac o s. Geno ype 1 was dominan in all g oups, wi h pe cen ages a ying om 60.3% in he IVDU pa ien s o 100% in he he nodialyzed pa ien s. Analysis o he dis ibu ion o sub ypes la and lb by isk ac o showed ha he sub ype p edominan in IVDU pa ien s was la (42.8%). Howe e , sub ype lb was he mos p e alen in pa ien s o unknown isk (62.0%) and in Alonso Alonso e al: Hepa i is C i us geno ypes and isk ac o s 649 Table 1 Geno ype dls ibu ion by isk ac o s IVDU T ans used Hemodialysis Unknown To al Geno ype No. (‘h) No. (‘YO) No. (%) No. (%) No (%) 1 1-NS 13 lb 2 2-NS 2a/2c 3a 4a NG Mixed la+lb la+4a 1+2 To al 38 ((60.3) 1 l(l.5) 27 (,42.8) 10 (15.8) 1 (1.5) 1 (1.5) 13 (20.6) 5 (7.9) 3 (4.7) 3 (4.7) 3 (4.7) 63 (100) 37 (84.0) 3 (6.8) 30 (68.1) 4 (9.0) 1 (2.2) 1 (2.2) I (2.2) 1 (2.2) 2 (4.5) 2 (4.5) 2 (4.5) 44 (100) 30 (100) 59 (74.6) 3 (10.0) 7 (8.8) 12 (40.0) 3 (3.8) 15 (50.0) 49 (62.0) 10 (12.6) 2 (2.5) 4 (5.0) 4 (5.0) 1 (1.2) 1 (1.2) 30 (100) 79 (100) 2 (2.5) 164 (75.9) 14 (6.4) 46 (21.3) 104 (48.1) 2 (0.9) 1 (0.4) l(0.4) 24 (11.1) 9 (4.1) 0 (4.1) 8 (3.7) 4 (1.8) 4 (1.8) 1 (0.4) 216 (100) l-NS, 2-NS=geno ypes 1 o 2 non-sub ypeable wi h he es used. NG=noii-geno ypeable wi h he es used. pa ien s wi h blood- ans usion an eceden s (68.1%). In he 30 he nodialyzed pa ien s he p e alence o sub ype la was 40.0% and ha o sub ype lb was 50.0%. On compa ing he p e alence o sub ypes la and lb in he di e en isk g oups s udied, we ound ha he p e a- lence o sub ype la was signi ican ly highe in IVDU pa ien s (42.8%) han in pa ien s wi h ans usion an e- ceden s (9.0%) (p<O.O01) o in hose wi h unknown isk ac o s (3.8%) (p<<O.OOl). In hese las wo g oups, sub ype lb p edomina ed. In he es o he com- pa isons, no s a is ically signi ican di e ences (p> 0.05) we e ound. Likewise, when he p e alence o geno- ype 3a in IVDU pa ien s (20.6%) was compa ed wi h ha in unknown isk ac o pa ien s (12.6%), no s a is- ically signi ican di e ences (p=0.253) we e ound. Howe e , compa ing ype 3a p e alence in IVDU pa ien s (20.6%) wi h ha in non-IVDU pa ien s (7.6%) did yield s a is ically signi ican di e ences (p = 0.01 6). On he o he hand, pa ien s wi h geno ype 3a p esen ed he lowes a e age age (32.7k8.1 yea s). In addi ion, geno ype 3a was associa ed wi h lowe a e age age han ha o pa ien s wi h geno ype 1 non- sub ypeable (54.7k14.9 yea s), geno ype lb (53.1 k15.8 yea s) and mixed geno ype la+lb (61.2k11.3 yea s) (p<0.05 in all cases) (Table 2). Sub ype la was de ec ed in a younge age g oup (37.95 15.5 yea s) han was sub ype lb (p<0.05). In addi ion, he ela ion be ween geno ypes and age in IVDU and non-IVDU pa ien s was analyzed. Non-IVDU pa ien s ha ing geno ype 3a showed lowe a e age age (36.4k9.4 yea s) han non-IVDU pa ien s in ec ed wi h o he geno ypes. Fu he mo e, ype 3a was associa ed wi h a lowe a e age age han in ec ion wi h geno ype lb (55.5k14.6 yea s) (p<0.05). Howe e , no s a is ically signi ican di e ences we e ound be ween he a e age ages o he IVDU pa ien s wi h he di e en geno ypes (Table 2). On he o he hand, we analyzed he ela ionship be ween geno ypes and ALT le els in pa ien s wi h Table 2 A e age age o pa ien s g ouped by geno ype IVTIU Non-IVDU To al Mean2 SII Mean SL> Mean k SII Geno nes (no.) (no.) (no.) 1 -NS la lb 2-NS 2a/2c 33 4a NG la+lb la+4a 1 +2 To l 28 (1) 30.624.6 (27) 30.424.1 (10) - 30 (1) 29 625.6 (13) 34.222.3 (5) 27.6 5.5 (3) 31.6ki.5 (3) ~ - 30.5k4.6 (63) 56.8k13.3 (13) 48.2k19.S (19) .5.5.52 14.6 (94) 32 (1) 36.4k9.4* (11) 53.6+11.1 (3) 49.5514.1 (6) 61.2k11.3 (4) 42 (1) 47 (1) 52.9215.5 (153) 54.7k14.9 (14) 37.9k15.5 (46) 53.1k15.8 (104) 32 (I) 30 (1) 32.7+8.1** (24) 41.5k11.8 (8) 42.2215.8 (9) 61.2211.3 (4) 34.226.8 (4) 47 (1) 46.4k 16.7 (216) l-NS, 2-NS=geno ypes 1 o 2 non-sub ypeable wi h he es used. NG=iion-geno ypeable wi h he es used. *p < 0.05 ype 3a e sus ype lb. **p < 0.05 ype 3a c sus I-NS, e sus lh, e sus la+lb. 650 Clinical Mic obiology and In ec ion, Volume 3 Numbe 6, Decembe 1997 Table 3 Pa ien s wi h pa hologic ALT le els and a e age ALT le els by geno ype DISCUSSION ALTb Geno aes” No. MeankSD 1-NS 12 166.9k 152.3 la 33 116.2k66.5 lb 85 125.1k85.5 2a/2c 1 48 3a 23 148.4k81.9 4a 5 203.4 208.0 NG 7 98.0k35.1 la+lb 4 155.0k77.2 la+4a 3 149.3k97.7 ’l-NS=geno ype 1 non-sub ypeahle wi h he es used. NG=non-geno ypeable wi h he es used. bA e age ALT le els in pa ien s wi h ele a ed ALT (J < 0.05 in all cases). Table 4 Dis ibu ion o hepa ic biopsy pa e ns by geno ype CH MC PCH ACH Ci hosis Ca cinoma GH Geno ype No No No No No. No No 1-NS la lh 2-NS 3a 4a la+lb la+4a To al ~~ 3 1 21 1 21 9 6 1 1 12 1 1 1 1 2 13618 7 1 1 CH = ch onic hepa i is o non-speci ied his ologic deg ee; MC=minimal changes; PCH =pe sis en ch onic hepa i is; ACH =ac i e ch onic hepa i is; GH =g anuloma ous hepa i is; LNS, 2-NS=non-sub ypeahle wi h he es used. pa hologic le els o ALT. No signi ican di e ences we e ound (p>0.05 in all cases) when he ALT le els we e compa ed in he same geno ype g oups (Table 3). Li e biopsy was pe o med in 37 pa ien s. In 18 (48.6%) o hese, a pa e n o ac i e ch onic hepa i is was ound, in se en (18.9%) hepa ic ci hosis, in six (16.2%) pe sis en ch onic hepa i is, and in ano he h ee only minimal changes (8.1%). Finally, one case each o hepa oca cinoma, o g anuloma ous ch onic hepa i is (p obably due o oxici y h ough isoniazid) and o non-classi ied ch onic hepa i is was ound. Table 4 p esen s he dis ibu ion o his ologic pa e ns acco ding o geno ype. Geno ype lb was ound in six o he se en cases o ci hosis (85.7%) and in nine o he 18 cases (50.0%) o ac i e ch onic hepa i is. Ou esul s show ha in he Spanish popula ion he highes p e alence co esponded o geno ype lb, wi h 48.1% o he cases, ollowed by geno ype la, wi h 22.3%, and geno ype 3a, wi h 11.1%. These da a show he g ea e p e alence o geno ype lb in Spain, jus as in o he Occiden al coun ies [5,6,17,18], apa kom he USA [5] and he no h o Eu ope [6,1Y] whe e geno ype la is he mos equen ly desc ibed. In con as o ou esul s, B a o e al [lY] ound geno ype la o be he mos p e alen , al hough his di e ence could be due o he small numbe o non-IVDU pa ien s in hei s udy (18 cases). On he o he hand, al hough in ou s udy geno ype 3a was he hi d mos equen ( ollowing ypes lb and la), i s p e alence was lowe han ha ound in o he Eu opean coun ies such as Sco land [12], Sweden [19] o F ance [18], whe e geno ype 3a p e alence is o e 20%. Geno ype 2 is p esen only a ely in ou heal h a ea. The p esence o geno ype 4a among Spanish people (eigh cases, 3.7%) is wo h no ing, as i is a ely desc ibed in coun ies o wes e n Eu ope [6, 8, 12, 181. The p esence o in ec ion by mo e han one geno- ype was inkequen in ou s udy (nine cases, 4.1%), in con as o he esul s o B a o e a1 [20], who ound 43.8% mul iple in ec ions. Mixed in ec ions a e a ely men ioned in he li e a u e, and he majo i y o au ho s epo mul iple in ec ion pe cen ages simila o ou s [17,18,21,22]. Howe e , he signi icance o mul iple in ec ions is deba able: on he one hand, a la ge numbe o mixed pa e ns a e due o c oss- eac ions, while on he o he hand, cu en geno yping echniques do no de ec mul iple in ec ions eliably and end o iden i j. p incipally he dominan geno ypes [23]. On he o he hand, an associa ion o some geno- ypes wi h speci ic isk g oups seems o exis . In Japan Okamo o e al [7] ound geno ype lb (I11 o Okamo o) o be mo e p e alen in pa ien s wi h non-A, non-B hepa i is. These au ho s also ound geno ype la o be mo e 6-equen in hemophiliacs, a ibu ing his o he US o igin o he coagula ion ac o s ecei ed. In a g oup o Ge man pa ien s, D iesel e al [15] ound geno ype lb o be he mos equen among all he pa ien s, wi h an ele a ed p e alence o geno ype 3a in he IVDU g oup. In ou s udy we ha e ound a clea dis ibu ion o geno ypes acco ding o isk ac o s: among pa ien s o unknown isk and hose wi h blood- ans usion an eceden s, geno ype lb was he mos p e alen (62.0% and 68.1%, espec i ely); among hemodialyzed pa ien s no s a is ical di e ences we e ound be ween geno ypes la and Ib (40.0% and 50.0% espec i ely), while among IVDU pa ien s geno ypes la (42.8% o all cases) and 3a (21.6%) we e he mos p e alen . Alonso Alonso e al: Hepa i is C i us geno ypes and isk ac o s 65 1 The low p e alence o geno ype 3a in IVDU pa ien s ound bo h in ou s udy and in ha o B a o e al [20] (11.5%) s a ids ou in con as o he p e a- lence obse ed in o he nea by Eu opean coun ies such as F ance (63%) [18] and I aly (64.3%) [21]. Simila di e ences a e ound i we compa e he p e a- lences o geno ype 3a among he non-IVDU pa ien s o hese coun ies wi h ou esul s. We eel ha he low p e alence o ype 3a compa ed wi h he le els exis ing in o he Eu opean coun ies may be due o he la e in oduc ion o his geno ype in o Spain. This could be suppo ed by he ac ha pa ien s ha ing geno ype 3a p esen ed he lowes a e age age (32.728.1 yea s), whe he he o ali y o pa ien s s udied is conside ed o he non-IVDU (36.429.4 yea s) and IVDU (30.6-+ 5.6) pa ien g oups a e analyzed sepa a ely. The disco e y o he gene ic di e si y o HCV ga e ise o he hypo hesis ha he g ea e o lesse se e i y o he clinical pic u e and i s e olu ion we e ela ed o speci ic geno ypes [24]. The s udies pe - o med o da e a e no : conclusi e. McOmish e al [13] ound a g ea e equency o abno mali ies o hepa ic unc ion in pa ien s in ec ed wi h ype 3 han in hose in ec ed wi h ypes 1 and 2, al hough hey doub he ue pa hogenic signi icance o his ac . Dusheiko e a1 [S] sugges he associa ion o geno ype 1 wi h mo e se ious his ologic lesions in he li e . In con as , o he au ho s [25] sugges ha a ela ion be ween geno ypes and se e i y o hepa ic disease is no e y likely. On he o he hand, al hough he equency wi h which dis inc deg ees o his ologic lesions a e ound a ies among he di e en s udies published [17,26-281, in conjunc ion pe sis en ch onic hepa i is (PCH) and ac i e ch onic hepa i is (ACH) a e he mos equen lesions, ollowed by ci hosis [29]. In ou s udy, ACH was he mos equen (18 o 37 cases), ollowed by ci hosis (se en cases), bu PCH was ound in only i e cases. Geno ype lb in ec ion has also been equen ly associa ed wi h he mos se e e his ologic lesions o he li e (especially ci hosis and hepa ocellula ca cinoma) [22,24,30,31]. We ha e ound simila esul s, as he six ci hosis pa ien s we e in ec ed by geno ype lb. Howe e , gi en ha geno ype lb is p edominan in ou heal h a ea, i is o be expec ed ha his geno ype would appea wi h g ea e equency in his ype o his ologic lesion. In any case, i is impo an o poin ou ha all he pa ien s wi h hepa ic ci hosis p esen ed geno ype lb. Acknowledgmen s We wish o hank D F ancisco Ma in Rod iguez o his assis ance in he elabo a ion and in e p e a ion o he s a is ical s udy, and C. J. Nolan o he help in ansla ing. Re e ences 1. Choo Q-L, Kuo G, Weine AJ, O e by LR, B adley DW, Hough on M. Isola ion o a cDNA clone de i ed om a blood-bo ne non-A, non-B i al hepa i is genome. Science 2. Kubo Y, Takeuchi K, Boonma S, e al. A cDNA Gagmen o hepa i is C i us isola ed om an implica ed dono o pos - ans usion non-A, non-B hepa i is in Japan. Nucleic Acids Res 1989; 17: 10367-72. 3. Takeuchi K, Boonma S, Kubo Y, e al. Hepa i is C i al cDNA clones isola ed om a heal hy ca ie dono impli- ca ed in pos ans usion non-A, non-B hepa i is. Gene 1990; 4. Takeuchi K, Kubo Y, Boonma S, e al. Nucleo ide sequence o co e and en elope genes o he hepa i is C i us genome de i ed di ec ly om human heal hy ca ie s. Nucleic Acids Res 1990; 18: 4626. 5. 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