Bone Marrow Transplantation, (1998) 22, 499–501  1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt Detection of G /GB virus C after allogeneic bone marrow transplantation

P Ljungman1, R Halasz2,HHa¨gglund3,ASo¨nnerborg2 and O Ringde´n3,4

Departments of 1Hematology, 2Clinical Virology, 4Clinical Immunology and 3Transplantation Surgery, Huddinge University Hospital, Karolinska Institutet, Huddinge, Sweden

Summary: Patients and methods

Abnormal liver function before allogeneic BMT has Fifty consecutive allogeneic BMT recipients were studied. been associated with VOD. Hepatitis G virus/GB virus The patients were transplanted between January 1 and C (HGV) is a recently discovered virus suggested to be December 31 1995. Patient characteristics are shown in a cause of non-A, non-B, non-C, non-D and non-E hepa- Table 1. titis. The aim of this retrospective study was to analyze The bone marrow transplant procedure is well the risk for liver complications and time to engraftment described.8,9 Forty-five patients were conditioned with in patients infected with HGV. Fifty patients trans- cyclophosphamide 60 mg/kg for 2 days followed by single planted in 1995 were examined with RT-PCR for HGV fraction 10 Gy total body irradiation. Patients with unre- on samples collected before, and between 3 and 6 lated donors received ATG 3–5 mg/kg or OKT-3 (5 mg) for months after BMT. Seven patients had HGV detected 5 days in addition. Five patients received other conditioning before BMT. No patient became infected during or early regimens including busulphan and cyclophosphamide after the BMT. There were no differences in either pre- (n = 2), cyclophosphamide 50 mg for 4 days combined with or post-transplant liver function abnormalities, VOD, ATG 3–5 mg/kg for 4 consecutive days (n = 2), or cyclo- or time to neutrophil engraftment in patients who phosphamide 5 mg/kg for 4 days combined with thoraco- did or did not have HGV detected before BMT. We abdominal irradiation (n = 1). Prophylaxis against GVHD conclude that the importance of HGV was with a combination of cyclosporine and metho- for the development of post-transplant complications is trexate.8–10 limited. VOD was defined according to the criteria described by Keywords: hepatitis G virus; GB virus C; VOD; Jones et al.11 GVHD was defined according to Thomas et engraftment al.12 Engraftment was defined as the first of 3 consecutive days with an absolute neutrophil count (ANC) 0.5 × 109/l. All patients were studied before and repeatedly after BMT with measurements of s-AST, s-ALT, s-alkaline phospha- Liver function abnormalities are common after allogeneic tase, or s-bilirubin. These tests were performed at least BMT. Veno-occlusive disease (VOD) of the liver is a sev- three times a week until engraftment, at least twice weekly ere and frequently fatal complication. Several risk factors until day 100, and thereafter at all routine visits to the have been associated with development of VOD including clinic. Abnormal liver function tests were defined as more advanced disease, conditioning regimens containing busul- than double the upper normal value of these tests. Severe phan and abnormal liver function BMT.1,2 The roles of active and C virus before BMT in subsequent liver damage have been controversial with some Table 1 Patient characteristics studies indicating an increased risk of liver damage after BMT and other studies showing no increase in risk.3–6 HGV Characteristic HGV-positive HGV-negative (n = 7) (n = 43) is a recently discovered RNA virus that can be transmitted 7 by blood transfusions. Therefore, many BMT patients can Median age (years) (range) 40.1 (14.1–50.0) 26.0 (0.5–53.4) be assumed to be infected at the time of bone marrow trans- Donor type plantation. The aim of this study was to investigate the Unrelated 2 23 role of HGV in the development of liver damage in Mis-matched family 1 1 BMT recipients. Identical sibling 4 19 Diagnosis ALL 2 11 AML 1 5 CML 1 12 Aplastic anemia 0 6 Multiple myeloma 1 2 Correspondence: Dr P Ljungman, Dept of Hematology, Huddinge Univer- MDS 2 3 sity Hospital, S-14186 Huddinge, Sweden Other 0 4 Received 4 December 1997; accepted 9 April 1998 HGV infection in BMT patients P Ljungman et al 500 liver function test abnormalities were defined as more than Engraftment, VOD and GVHD 10 times the upper normal value of either of these four tests. There was no difference in the time to engraftment between patients with or without HGV before BMT (Figure 1; log- Virology rank test). Two (28%) patients with HGV developed VOD compared to six (14%) patients without HGV (P = NS; All patients were studied for HBsAg, and HCV RNA as Fisher’s exact test). None of seven HGV-positive patients part of a routine evaluation before, and for follow-up after developed acute GVHD of grade II compared to eight of BMT. Samples obtained for these routine investigations 43 patients without HGV infection (P = NS; Fisher’s were retrospectively analyzed for HGV RNA by reverse- exact test). transcriptase polymerase chain reaction (RT-PCR). Total RNA was extracted from 100 ␮l of serum by guanidinium extraction as described previously.13 cDNA synthesis was Discussion initiated by using reverse-transcriptase (Boehringer, Mannheim, Germany). HGV DNA was amplified by PCR HGV is a recently discovered virus that can be parenterally of the 5′ noncoding region by using outer primer pair sense transmitted.16 The prevalence of HGV in voluntary blood HGV-1X (nucleotides 205–222) and antisense HGV-2X donors in the USA is reported to be 1–2%. Thus, the likeli- (nucleotides 533–516) as described,14 nested with inner hood is high that multitransfused patients such as allogeneic primer pair, previously described.13,15 The nested PCR BMT recipients are infected with HGV. This is supported amplifies a product of approximately 245 bp. by our study since all patients who tested positive for HGV All stem cell donors were studied as a part of the routine before BMT had been transfused while none of the non- pre-transplant evaluation for HBsAg and either HCV RNA transfused patients had HGV RNA detected. The clinical or HCV antibodies by RIBA. Donor sera (n = 5) were ana- significance of HGV in different patient populations has lyzed for HGV RNA with RT PCR when available. been studied, and generally speaking this is low. Recently, Rodriguez-Inigo et al17 presented data on Spanish allo- geneic BMT recipients showing a very high incidence of HGV infection (42%) but no correlation with liver disease Results or engraftment kinetics. They also showed that 19% of patients contracted HGV at or early after BMT. Finally, HGV RNA detection before and after BMT almost half of their patients infected with HGV were also infected with HCV. Seven of 50 patients (14%) were HGV PCR positive before Our patient group differs from the patient group BMT. One patient had HBsAg detected while no patient described by Rodriguez-Inigo et al in that the frequency of was positive for HCV RNA before BMT. There were no HGV was only 14% and no patient contracted HGV during differences in the frequencies of abnormal liver function the transplant procedure, presumably reflecting the preva- tests in patients who were positive (2 of 7; 28%) or negative lence of HGV infection in the blood donor pool. We could (12 of 43; 28%) for HGV DNA before BMT. All patients only test a minority of the marrow donors but this is prob- who were HGV positive had received blood transfusions, ably of little importance since no patient contracted HGV while 15 of 43 patients without HGV had not received any infection after the transplant. Furthermore, none of our transfusion. No donor tested had either HGV or HCV DNA patients had HCV infection and therefore the possible con- before BMT. founding effects of HCV on both the HGV infected and Forty-eight patients were retested after BMT. Two uninfected patients can be disregarded. Despite these differ- patients died before engraftment and had no follow-up ences in the patient cohorts, our results are very similar to samples available for analysis. Six of the 48 patients stud- the previously published series, showing no difference in ied were HGV PCR-positive after BMT. All these patients were already HGV RNA-positive before BMT. No patient who was HGV PCR negative became PCR-positive after 1.0 HGV-neg HGV-pos the BMT. 0.8 /l 9

Liver function tests before and after BMT 0.6 Two of seven (28%) patients with HGV had abnormal liver function tests before BMT compared to 12 of 43 (28%) 0.4 = patients who did not have HGV detected (P NS; Fisher’s ANC >0.5 x 10 exact test). All seven patients (100%) with HGV had at 0.2

least one abnormal liver function test after BMT compared Proportion of patients with to 39 of 43 (91%) of patients without detectable HGV 0 (P = NS; Fisher’s exact test). Three of seven (43%) HGV- 0 5 10 15 20 25 30 35 positive patients had severe liver function test abnormalities Days after BMT compared to 11 of 43 (26%) patients without HGV Figure 1 Time to neutrophil engraftment in patients with and without (P = NS; Fisher’s exact test). HGV infection before BMT. HGV infection in BMT patients P Ljungman et al 501 the risk for either mild or severe liver disease after BMT. genotypes and liver disease in patients undergoing allogeneic Each of the two studies are small with a low number of bone marrow transplantation. Bone Marrow Transplant 1997; patients infected with HGV. However, the results of these 18: 237–240. studies taken together strongly argue against a significant 7 Wang T, Tsai F, Lee C-Z et al. A prospective study of trans- impact of HGV on liver function after allogeneic BMT. fusion-transmitted GB virus C infection: similar frequency but different clinical presentation compared with virus. This is in agreement with other patient categories suggest- Blood 1996; 88: 1881–1886. ing that severe liver disease caused by HGV is uncom- 8 Ringde´n O, Pihlstedt P, Markling L et al. Prevention of graft- 16,18 mon. versus-host disease with T-cell depletion or cyclosporin and There is an ongoing discussion regarding the role of methotrexate. A randomized trial in adult leukemic marrow HGV in the etiology of aplastic anemia occurring with or recipients. Bone Marrow Transplant 1991; 7: 221–226. without associated hepatitis.19–21 We detect no difference 9 Ringde´n O, Remberger M, Persson U et al. Similar incidence in the kinetics of marrow engraftment in HGV infected or of graft-versus-host disease using HLA-A, -B and -DR ident- uninfected patients. Thus, the results of our study together ical unrelated bone marrow donors as with HLA-identical sib- with the study of Rodriguez-Inigo et al argue against a lings. Bone Marrow Transplant 1995; 15: 619–625. direct effect of HGV on hematopoietic progenitors. How- 10 Aschan J, Ringde´n O, Andstro¨mEet al. Individualized prophylaxis against graft-versus-host disease in leukemic mar- ever, the possibility of an immune-mediated effect depress- row transplant recipients. Bone Marrow Transplant 1994; 14: ing hematopoiesis can not be ruled out. 79–87. 11 Jones R, Lee K, Beschorner W et al. Veno-occlusive disease of the liver following bone marrow transplantation. Transplan- Acknowledgements tation 1987; 44: 778–783. 12 Thomas E, Storb R, Clift R et al. Bone marrow transplan- This study was supported by the Swedish Cancer Fund and the tation. New Engl J Med 1975; 292: 895–902. Stockholm County Council’s research fund. 13 Chen M, So¨nnderborg A, Johansson B et al. Detection of hepatitis G virus (GB virus C) in saliva. J Clin Microbiol 1997; 35: 973–975. 14 Fischler B, Lara C, Chen M et al. Genetic evidence for References mother-to-infant of hepatitis G virus. J Infect Dis 1997; 176: 281–285. 1 McDonald G, Sharma P, Matthews DE et al. Veno-occlusive 15 Lara C, Halasz R, So¨nnerborg A et al. Detection of hepatitis disease of the liver after bone marrow transplantation: diag- G virus RNA in persons with and without known risk factors nosis, incidence, and predisposing factors. Hepatology 1984; for blood-borne viral infections in Sweden and Honduras. J 4: 116–122. Clin Microbiol 1998; 36: 255–257. 2 McDonald GB, Hinds MS, Fisher LD et al. Veno-occlusive 16 Alter H, Nakatsuji Y, Melpolder J et al. The incidence of disease of the liver and multiorgan failure after bone marrow transfusion-associated hepatitis G virus infection and its transplantation: a cohort study of 355 patients. Ann Intern Med relation to liver disease. 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