Incidence and influence of GB virus C and hepatitis C virus infection in patients undergoing bone marrow transplantation

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1 Bone Mrrow Trnsplnttion, (1998) 21, Stockton Press All rights reserved /98 $ Incidence nd influence of GB virus C nd heptitis C virus infection in ptients undergoing one mrrow trnsplnttion H Akiym 1, N Nkmur 1, S Tnikw 1, H Skmki 1, Y Onozw 1, T Shiym 2, S Tnk 2, F Tsud 3, H Okmoto 4, Y Miykw 5 nd M Myumi 4 1 Hemtology Division nd 2 Liver Unit, Tokyo Metropolitn Komgome Hospitl, Tokyo; 3 Deprtment of Medicl Sciences, Toshi Generl Hospitl, Tokyo; 4 Immunology Division, Jichi Medicl School, Tochigi-Ken; nd 5 Miykw Memoril Reserch Foundtion, Tokyo, Jpn Summry: Mrkers of GB virus C (GBV-C) nd heptitis C virus (HCV) were sought in 80 ptients efore nd fter they underwent BMT in metropolitn hospitl in Tokyo etween 1990 nd RNA of GBV-C ws detected in 14 (18%) ptients efore BMT. Of the 55 ptients who hd een trnsfused, 14 (25%) possessed GBV-C RNA t frequency significntly higher thn in the 25 untrnsfused ptients who were ll negtive (P 0.01). HCV RNA ws detected in three of the 55 (5%) trnsfused ptients, ut in none of the 25 untrnsfused ptients. Ser t 3 months fter BMT were ville for 57 ptients. GBV-C RNA persisted in ll 10 ptients who were infected efore BMT, while it ws detected in five of the remining 47 (11%) ptients who were not. However, persistent nd/or ongoing GBV-C infection hd no pprecile influence on ptient moridity or mortlity. Two of the 57 ptients were positive for HCV RNA efore BMT nd this persisted fter BMT in oth. HCV RNA ecme positive in eight of the remining 55 (15%) ptients who were negtive efore BMT. Of the 14 ptients who received trnsfusions screened y the first-genertion test t BMT, seven (50%) ecme positive for HCV RNA, rte significntly higher thn the one of 41 (2%) ptients who received trnsfusions screened y the second-genertion test (P 0.001). These results indicte tht BMT ptients re t incresed risk of GBV-C infection trnsmitted y trnsfusions received efore nd t the time of BMT, nd tht the risk of HCV infection hs decresed fter the implementtion of the second-genertion nti-hcv test. Keywords: GBV-C; HCV; BMT Ptients undergoing BMT re t incresed risk of infection with lood-orne heptitis viruses such s heptitis B virus (HBV) nd heptitis C virus (HCV), which poses serious prolems. 1 These viruses re minly introduced either y the trnsfusions they received efore nd t BMT or y the Correspondence: Dr H Akiym, Hemtology Division, Tokyo Metropolitn Komgome Hospitl, , Honkomgome, Bunkyo-ku, Tokyo, 113, Jpn Received 12 Septemer 1997; ccepted 29 Decemer 1997 one mrrow donors. Since most ptients hve underlying diseses impiring their immune system nd receive myeloltive therpies efore BMT, infection with heptitis viruses tends to persist nd my induce severe heptitis. Furthermore, the infection my e modulted y the doptive immunity trnsferred y one mrrow donor who hs een infected or who hs overcome the infection. Recently, puttive heptitis virus designted GB virus C (GBV-C) or heptitis G virus (HGV) hs een discovered. 2,3 For the ske of convenience these re collectively referred to here s GBV-C. This is positive, singlestrnded RNA virus of pproximtely 9400 ses nd clssified in the Flviviride fmily. GBV-C previls worldwide, s estimted y the detection of its RNA in 1 5% of pprently helthy lood donors. 3 7 GBV-C is trnsmited y trnsfusion nd intrvenous drug use. 5,8 11 Coinfection with GBV-C nd HBV or HCV is frequent in ptients with chronic liver disese nd in intrvenous drug users. 3,8 GBV-C infection persists in ptients with compromised immunity, such s those on mintennce hemodilysis. 5 The ility of GBV-C to induce heptitis hs, however, s yet, not een estlished. Evidence of infection with GBV-C, HCV nd HBV ws sought in ptients with mlignnt hemtologicl diseses or plstic nemi efore nd fter they underwent BMT, nd the results were correlted with the numer of trnsfusions they received efore nd t the time of trnsplnttion. Ptients nd methods Study popultion From July 1990 to August 1996, totl of 155 ptients underwent BMT in the Hemtology Division of Tokyo Metropolitn Komgome Hospitl. Only the ptients who underwent llogeneic BMT nd whose pre-bmt ser re ville were included in this retrospective study. One hundred nd twenty-four ptients received llogeneic or syngeneic BMT nd in 80 ptients, pre-bmt ser were ville (Tle 1). They included 44 mles nd 36 femles with the men ( s.d.) ge of 32 ( 9) yers. During BMT, complete lood counts were performed three times week nd lood chemistries otined two to three times week. Ptients were evluted retrospectively for veno-occlusive disese (VOD) y the criteri of

2 1132 Tle 1 Chrcteristics Ptient chrcteristics H Akiym et l Age verge (rnge) 32 (17 53) yers Sex M/F 44/36 Dignosis CML 34 AML 13 ALL 15 SAA 8 MDS 8 Lymphom 2 Donor relted 67 unrelted 11 syngeneic 2 Preprtive regimen BU/CY 53 Ar-C/CY/TBI 19 CY/TLI 8 GVHD prophylxis CsA/MTX 76 Others 4 SAA = severe plstic nemi; MDS = myelodysplstic syndrome; TLI = totl lymphoid irrdition. McDonld et l, 12 GVHD, grft rejection, interstitil pneumoni, hemorrhgic cystitis nd vricell zoster virus infection. In 57, ser 3 months fter BMT were lso ville, while in 23 ptients, follow-up ser were not ville even though ll survived for more thn 3 months fter BMT. Bone mrrow trnsplnttion Preprtive therpy ws given minly ccording to the primry diseses nd source of one mrrow. Ptients with AML nd CML were treted with usulfn 16 mg/kg nd cyclophosphmide 120 mg/kg (BU/CY). Totl lymphoid irrdition (TLI) ws dded in ptients who received mrrow from unrelted donors. Ptients with severe plstic nemi were treted with cyclophosphmide 200 mg/kg nd TLI 700 cgy (CY/TLI) nd those with ALL were treted with Ar-C 8 g/m 2 nd cyclophosphmide 120 mg/kg, followed y totl ody irrdition (TBI), 1200 cgy (Ar-C/CY/TBI). Other diseses were treted with BU/CY or Ar-C/CY/TBI. All ptients except for those undergoing syngeneic BMT received cyclosporine nd short-term methotrexte s prophylxis for GVHD. Determintion of GBV-C RNA RNAs were extrcted from 100 l of serum with n extrction regent contining gunidinium isothiocynte nd phenol (ISOGEN-LS; Nippon Gene Co. Ltd, Tokyo, Jpn) nd dissolved in 5.3 l of distilled wter treted with diethylpyrocronte (Sigm Chemicl, St Louis, MO, USA). Smples were reverse trnscried nd the cdna ws mplified y PCR using primers tken from well-conserved res in the 5 untrnslted region of the genome, y the method descried previously. 9 For semi-quntittive nlysis of GBV-C RNA, seril smples of 10-fold dilution of extrcted RNAs were prepred, nd the smple of the highest dilution positive for GBV-C RNA ws determined. The result ws converted to represent the titer (10 N ) of the virus per ml of the test serum. Mrkers of HCV nd HBV infections Until Jnury 1992, lood trnsfused to ptients ws screened for ntiody to HCV (nti-hcv) y the first-genertion enzyme-linked immunosorent ssy (ELISA) using Ortho ELISA I (Ortho Dignostic Systems, Tokyo, Jpn) nd therefter y the second-genertion ELISA (Ortho ELISA II; Ortho Dignostic Systems). Ser from ptients were serilly diluted two-fold, nd nti-hcv ws ssyed y hemgglutintion using Aott HCV PHA 2nd Genertion (Dinot, Tokyo, Jpn). Ser inducing hemgglutintion t dilutions 2 5 were considered to e positive for nti-hcv. RNA of HCV ws determined in RNAs extrcted from 100 l of the test serum y commercil kit (Amplicor HCV detection kit; Jpn Roche, Tokyo, Jpn). Heptitis B surfce ntigen (HBsAg) nd the corresponding ntiody (nti-hbs) were determined y seril two-fold dilutions of the test serum using commercil kits (MyCell; Institute of Immunology Co. Ltd, Tokyo, Jpn), nd hemgglutintion t dilutions 2 2 ws considered rective. Sttisticl nlysis Frequencies etween groups were compred with the 2 test or Fisher s exct test using SttView (Acus Concepts, Berkeley, CA, USA). Group mens were compred using the Student s t-test. Results GBV-C, HCV nd HBV mrkers efore BMT Tle 2 gives prevlence rtes of virl mrkers in the 80 ptients efore BMT. GBV-C RNA ws detected in 14 (18%) ptients, HCV RNA in three (4%) nd HBsAg in none (P 0.001). No ptients were vccinted ginst HBV. All three ptients with HCV RNA were positive for Tle 2 Mrkers of GB virus C, heptitis C virus nd heptitis B virus infections in ptients efore BMT Virl mrkers Totl Trnsfusion P vlue n = 80 (%) (+) ( ) n = 55 (%) n = 25 (%) GBV-C RNA 14 (18) 14 (25) HCV RNA 3 (4) 3 (5) 0 Anti-HCV 5 (6) 5 (9) 0 HBsAg Anti-HBs 6 (8) 5 (9) 1 (4) Compred etween the ptients with previous trnsfusions nd those without.

3 nti-hcv; no ptients hd seronegtive HCV infection. Previously trnsfused ptients hd higher frequency of GBV-C RNA thn untrnsfused ptients (14 of 55 or 25% vs none of 25 or 0%, P 0.01). All three ptients with HCV RNA hd received trnsfusions; two of these were positive for GBV-C RNA. RNAs of GBV-C nd HCV in ptients with vrious underlying diseses re shown in Tle 3, with specil reference to the numer of trnsfused units (est estimted numer of exposures to lood donors) they hd received. All 13 ptients with AML hd received 50 or more units of lood, of whom 10 (77%) hd GBV-C RNA nd two were positive for HCV RNA. Both lso possessed GBV-C RNA. In contrst, of 21 ptients with other diseses who hd received 50 units, only two (10%) were positive for GBV-C RNA (P 0.001). However, ptients with diseses other thn AML hd received rther fewer lood units (men s.d.: vs units). Only two of the 21 (10%) ptients who hd received 50 units possessed GBV-C RNA, nd neither were positive for HCV RNA. Effects of GBV-C on BMT ptients VOD occurred in none of the 14 ptients with GBV-C RNA, nd in six of the 66 (9%) ptients without (P = 0.32). In the 14 ptients with GBV-C, the men of the mximum levels of totl iliruin during the first 3 months ws 0.9 mg/dl ( ) (Tle 4). Serum lnine minotrnsferse (ALT) levels were within three times the norml rnge up to 20 dys fter BMT in ll ut two ptients nd within twice the usul rnge etween dy 20 nd 3 months in ll ut two ptients. There were no differences in the incidence of grft-versus-host disese, grft rejection, interstitil pneumoni, hemorrhgic cystitis nd vricell zoster virus infection etween ptients with nd without GBV-C RNA. No differences were oserved in recovery of WBC to /l, reticulocytes 0.5% nd pltelets /l, or in the period during which pltelet trnsfusions were required (dt not shown). Tle 3 Prevlence rtes of GBV-C RNA nd HCV RNA in ptients strtified y disese nd numer of lood units efore BMT Disese n Age No. of Best estimted No. of exposures to (yers) ptients the lood donors trnsfused (%) n GBV-C HCV n GBV-C HCV RNA RNA RNA RNA CML ± 8 11 (32) ALL ± 9 15 (100) AML ± 8 13 (100) SAA 8 23 ± 3 8 (100) MDS 8 34 ± 9 7 (88) NHL 2 30 ± 11 1 (50) Totl ± 9 55 (69) (9%) (35%) (10%) MDS = myelodysplstic syndrome; NHL = non-hodgkin lymphom. H Akiym et l GBV-C RNA in ptients fter BMT In 57 of 80 ptients, ser otined t 3 months fter BMT were lso ville. GBV-C RNA persisted in ll 10 ptients who hd it efore BMT. Five of the remining 47 (11%) ptients who were negtive for GBV-C RNA efore BMT cquired it fter BMT (de novo infection) (Tle 5). In those five ptients, ALT levels efore BMT were within norml limits ( 40 IU/l), ut they incresed in three ptients fter BMT, one of whom ws coinfected with HCV nd hd the highest ALT level. Titers of GBV-C RNA rnged from 10 1 /ml to 10 4 /ml in these ptients ut there ws no correltion etween RNA titer nd level of ALT. Their mximum totl iliruin levels were less thn 1.4 mg/dl t 3 months nd no ptients developed VOD. All 57 ptients received trnsfusions during the course of BMT (totl: 931 units). There were no differences in the numers of lood units received etween the five ptients who contrcted de novo GBV-C infection nd the 42 ptients who did not (16 11 vs 14 11). HCV infection in ptients fter BMT Of the 57 ptients whose ser were ville t 3 months fter BMT, two possessed HCV RNA efore BMT which persisted. HCV RNA ecme positive in eight of the remining 55 (15%) ptients (Tle 6). After BMT, ALT levels incresed in seven to higher levels thn efore BMT. A modertely elevted level of ALT (60 IU/l) in the remining one ptient (Ptient No. 51) decresed to norml fter BMT. Anti-HCV ntiody ecme detectle in seven, while one other ptient ecme positive for nti- HCV ntiody without HCV RNA. Blood products were screened for HCV y the firstgenertion ELISA efore Ferury 1992 when the secondgenertion ELISA ws implemented. Of the 55 ptients without pre-trnsplnttion HCV RNA, 14 hd received trnsfusions t BMT screened y the first-genertion ELISA, nd eight (57%) of these ecme positive for HCV RNA fter BMT. By contrst, none of the 41 trnsfused ptients screened y the second-genertion ELISA developed HCV RNA t significntly lower frequency (P 0.001). Discussion GBV-C ws detected in 14 of the 80 (18%) BMT ptients whose ser were ville. In ddition, RNA of GBV-C ecme detectle in five of the 47 (11%) ptients within 3 months of BMT. Inclusive of the 10 ptients who were infected efore BMT, 15 of the 57 ptients (26%) were positive for GBV-C RNA fter BMT. The frequency of GBV-C infection in the BMT ptients ws much higher thn in the generl popultion of Jpn, which is reported s 1.2%. 13 Hence, ptients undergoing BMT re t incresed risk for GBV-C infection, s ws the cse for HBV nd HCV efore screening nd exclusion of contminted lood units. 1 These results confirm recent report from the UK documenting the detection of HGV RNA in 20 of 33 (61%) recipients of BMT 14 nd report of 42% from Spin

4 1134 Tle 4 Ptients with GBV-C RNA in serum efore BMT H Akiym et l Ptient No. Age nd sex Disese Trnsfused units Mximum ALT Mximum totl iliruin Before BMT At BMT Before dy 20 3 months Before 3 months HCV (IU/l) (mg/dl) RNA 48 28F AML F AML M ALL M AML F AML M SAA M AML F AML F CML M AML M MDS F AML F AML M AML Trnsfused units, est estimted numer of exposures to the lood donors. ALT, lnine minotrnsferse (norml: 40 IU/l). Tle 5 Ptients who developed GBV-C RNA in serum fter BMT Ptient Age nd Disese Trnsfused units Before BMT 3 months fter BMT Anti- No. sex HCV d Before At BMT Anti-HCV c ALT GBV-C HCV RNA Anti-HCV d ALT GBV- HCV BMT (IU/l) RNA (IU/l) C RNA RNA 53 27F CML I /ml F SAA II /ml M CML 0 16 II /ml M ALL II /ml F AML II /ml Trnsfused units; est estimted numer of exposures to the lood donors. ALT, lnine minotrnsferse (norml: 40 IU/l). c Anti-HCV, the method of nti-hcv mesurement of lood products used t BMT (Ortho ELISA I or II). d Anti-HCV, dt of nti-hcv of ptients ser mesured y Aott HCV PHA second genertion. Tle 6 Ptients who were infected with HCV fter BMT Ptient No. Age nd sex Disese Trnsfused units Before BMT 3 months fter BMT Before At BMT Anti-HCV c ALT HCV Anti-HCV d ALT HCV Anti-HCV d BMT (IU/l) RNA (IU/l) RNA 30 40F CML I M CML 4 9 I M SAA I M ALL 60 7 I F CML I F CML 0 34 I M CML 2 14 I M ALL 46 6 I Trnsfused units, est estimted numer of exposures to the lood donors. ALT, lnine minotrnsferse (norml: 40 IU/l). c Anti-HCV, the method of nti-hcv mesurement of lood products used t BMT (Ortho ELISA I or II). d Anti-HCV, dt of nti-hcv of ptients ser mesured y Aott HCV PHA second genertion.

5 None of the lood units or one mrrow donors were tested for GBV-C RNA. However, there is evidence to indicte trnsmission of GBV-C to ptients y trnsfusions they received efore nd during BMT. Before BMT, GBV-C RNA ws detected significntly more frequently in ptients who hd received trnsfusions thn in those who hd not (25 vs 0%, P 0.01). Furthermore, GBV-C RNA ws detected more frequently in ptients with AML who received more units of lood efore BMT thn in ptients with the other diseses (77 vs 10%, P 0.001). The incidence of VOD ws not rised in ptients with GBV-C, nd neither ws the incidence of severe liver dmge during the first 3 months fter BMT. Mximum levels of ALT were within three times the norml rnge nd the mximum levels of totl iliruin during the 3 months were less thn 2.1 mg/dl in ptients with GBV-C, suggesting n insignificnt ssocition etween GBV-C nd cute liver toxicity, which is lso consistent with previous reports. 14,15 At BMT, 657 lood units (plus 47 one mrrow dontions) were given to the 47 ptients negtive for GBV- C RNA. Assuming 1.2% of lood donors were infected with GBV-C, t lest eight contminted lood units were trnsfused. ALT levels fter BMT incresed in three of the five (60%) ptients who cquired GBV-C RNA. One of the three ptients with GBV-C RNA, however, ecme positive for HCV RNA which could hve een responsile for the elevted ALT. ALT levels were within three times the norml rnge t most in these ptients. In view of vriety of fctors potentilly inducing impired liver function in recipients of BMT, elevted ALT levels in the two ptients infected with GBV-C cnnot redily e scried only to GBV-C. 1 On the other hnd, HCV RNA is more relile mrker of HCV infection thn is nti-hcv in BMT ptients, since impired immune responses cn prevent seroconversion. HCV RNA ecme detectle in eight ptients fter BMT nd one of these ws negtive for nti-hcv. HCV RNA ws detected in the recipients of lood units screened y the first-genertion nti-hcv ELISA significntly more frequently thn in those screened y the second-genertion nti-hcv ELISA (57 vs 0%, P 0.001). Thus, HCV infection in recipients of BMT ppers to hve decresed s the sensitivity of the tests increses. These results indicte tht ptients with BMT re t incresed risk for GBV-C infection trnsmitted to them y trnsfusions they receive efore nd t the time of BMT, nd tht the risk of HCV infection hs decresed fter the implementtion of the second-genertion nti-hcv test. All in ll, there were no pprecile effects of persistent nd/or H Akiym et l ongoing GBV-C infection on the moridity or mortlity in the ptients who underwent BMT. References 1 Shuhrt MC, McDonld GB. Gstrointestinl nd heptic complictions. In: Formn SJ, Blume KG, Thoms ED (eds). Bone Mrrow Trnsplnttion. Blckwell Scientific: Msschusetts, 1994, pp Lery TP, Muerhoff AS, Simons JN et l. Sequence nd genomic orgniztion of GBV-C: novel memer of the Flviviride ssocited with humn non-a-e heptitis. J Med Virol 1996; 48: Linnen J, Wges J, Zhng-Keck ZY et l. Moleculr cloning nd disese ssocition of heptitis G virus: trnsfusiontrnsmissile gent. Science 1996; 271: Dwson GJ, Schluder GG, Pilot-Mtis TJ et l. Prevlence studies of GB virus-c infection using reverse trnscriptsepolymerse chin rection. J Med Virol 1996; 50: Msuko K, Mitsui T, Iwno K et l. Infection with heptitis GB virus C in ptients on mintennce hemodilysis. New Engl J Med 1996; 334: Brown KE, Wong S, Buu M et l. High prevlence of GB virus C/heptitis G virus in helthy persons in Ho Chi Minh City, Vietnm. J Infect Dis 1997; 175: Wng Y, Chen HS, Fn MH et l. Infection with GB virus C nd heptitis C virus in hemodilysis ptients nd lood donors in Beijing. J Med Virol 1997; 52: Aikw T, Sugi Y, Okmoto H. Heptitis G infection in drug users with chronic heptitis C (letter). New Engl J Med 1996; 334: Shimizu M, Osd K, Okmoto H. Trnsfusion-trnsmitted heptitis G virus following open hert surgery (letter). Trnsfusion 1996; 36: Wng JT, Tsi FC, Lee CZ et l. A prospective study of trnsfusion-trnsmitted GB virus C infection: similr frequency ut different clinicl presenttion compred with heptitis C virus. Blood 1996; 88: Alter HJ, Nktsuji Y, Melpolder J et l. The incidence of trnsfusion-ssocited heptitis G virus infection nd its reltion to liver disese. New Engl J Med 1997; 336: McDonld GB, Shrm P, Mtthews DE et l. Venocclusive disese of the liver fter one mrrow trnsplnttion: dignosis, incidence, nd predisposing fctors. Heptology 1984; 4: Yoshikw A, Fukud S, Itoh K et l. Infection with heptitis G virus nd its strin vrint, the GB gent (GBV-C), mong lood donors in Jpn. Trnsfusion 1997; 37: Skidmore SJ, Collinghm KE, Hrrison P et l. High prevlence of heptitis G virus in one mrrow trnsplnt recipients nd ptients treted for cute leukemi. Blood 1997; 89: Rodriguez-Inigo E, Toms JF, de Sori VGG et l. Heptitis C nd G virus infection nd liver dysfunction fter llogeneic one mrrow trnsplnttion: results from prospective study. Blood 1997; 90: