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Time on hemodialysis, transfusion requirement, and renal transplantation are risk factors for hepatitis G virus (HGV) infection in patients on maintenance hemodialysis, with a prevalence ranging from 3 to 57% in transversal studies (1-3, 5, 6, 8, 9, 12-15, 17).

This study analyzes the evolution of viremia (HGV RNA) and antibodies directed against the HGV envelope protein E2 (anti-E2) in a population of 58 hemodialyzed patients. They were prospectively followed up from 1995 until 1999 (mean follow-up time, 4.88 ± 0.42 years) and were screened every 12 months for the presence of both markers of HGV infection. Informed consent was obtained from all patients. In all the units, a strict environmental and equipment disinfection protocol was followed.

Serum samples were aliquoted and stored at 80°C until processing. They were thawed on ice only once before the reverse transcription-PCR (RT-PCR) amplification assay. RNA was extracted from 140 µl of serum by using a commercially available kit (Qiamp Viral RNA; Qiagen GmbH, Hilden, Germany). RT-PCR was performed according to the method described by Yoshiba et al. (18), using primers from the N3/helicase region. To avoid cross-contamination, PCR was performed under stringent conditions as recommended by Kwok and Higuchi (11). The amplified product was hybridized with a biotinylated, single-stranded DNA probe (PR3, 5' biotin GCCGGCCAGTTCTCHGCNMGGGGGGTNAATGCYATYGCCTATTA 3') and detected by a commercial assay (GEN-ETI-K DEIA; Sorin Diagnostics, Saluggia, Italy).

Serum anti-E2 antibodies were measured by an enzyme-linked immunosorbent assay (µPLATE Anti-HGenv; Boehringer GmbH, Mannheim, Germany) (7). Results were analyzed by optical density and were compared to the cutoff value with the help of kit-specific positive and negative controls, according to the manufacturer's instructions. Hepatitis C virus (HCV) antibodies were detected by a third-generation enzyme-linked immunosorbent assay (Abbott Diagnostics, Chicago, Ill.). HCV RNA was detected by RT-PCR (Amplicor HCV; Roche Diagnostics, Basel, Switzerland). Hepatitis B virus surface antigen (HBsAg) was analyzed by enzyme-linked immunosorbent assay (Abbott Diagnostics). All the samples of each patient were tested in the same run.

Data are presented as means ± standard deviations or, when indicated, as absolute number and percentage. The data from two independent groups were compared using the Mann-Whitney U test. For qualitative variables, chi square with Yates' correction or Fisher's exact test was used. A P value of <0.05 was considered significant.

According to the serial analysis of HGV infection markers (Table 1), patients were classified into four groups. Group 1 consisted of patients without evidence of infection (absence of HGV RNA and anti-E2 antibodies) throughout follow-up (n = 29). Group 2 consisted of five viremic patients. One of these presented with HGV RNA at the beginning of the study, with loss of viremia, although without development of anti-E2 antibodies, during follow-up. The other four, who showed no evidence of past infection at the beginning of the study, became HGV RNA positive during follow-up; all these patients remained HGV RNA positive at the end of the study. Group 3 consisted of patients with evidence of past infection (presence of anti-E2 antibodies but absence of HGV RNA) (n = 22). Thirteen of these patients lost their anti-E2 antibodies during follow-up; four of them presented with HGV viremia after the loss of anti-E2 antibodies. Of these four patients, three cleared their HGV viremia, without seroconversion at the end of the study, and one died. Group 4 consisted of two patients with no evidence of prior HGV viremia in whom anti-E2 antibodies were detected during follow-up.

Age (58.3 ± 13.5 versus 59.0 ± 14.4 years), male/female ratio (1.06:1 versus 0.92:1), and percentage of patients with HBsAg in the serum (5.7 versus 4.3) were similar in the group of patients with no evidence of infection (n = 35) and the group with evidence of past or active HGV infection (n = 23) at the beginning of the study. For patients without evidence of infection at the beginning, a significantly shorter time on hemodialysis (8.1 ± 4.5 versus 12.5 ± 6.8 years [P = 0.012]) and a nearly significantly lower percentage of individuals with HCV RNA in the serum (20.0 versus 47.8% [P = 0.05]) were found.

Group 3 patients (presence of anti-E2 antibodies at the beginning of the study) were subdivided into two subgroups according to the persistence or clearance of anti-E2 antibodies during follow-up. Patients who cleared their anti-E2 antibodies during follow-up presented a significantly lower baseline mean anti-E2 titer, indirectly determined by optical density (0.83 ± 0.24; range, 0.50 to 10), than those who retained these antibodies (1.57 ± 0.82; range, 0.80 to 3.0). The evolution of anti-E2 titers is shown in Fig. 1. Age (55.5 ± 16.2 versus 62.9 ± 11.8 years), male/female ratio (0.86:1 versus 0.80:1), time on hemodialysis (12.2 ± 6.2 versus 12.0 ± 7.0 years), percentage of HBsAg positivity (0 versus 11.1%), and percentage of HCV RNA positivity (38.4 versus 55.5%) were not significantly different for the subgroups of patients with persistence or clearance of anti-E2 antibodies.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Evolution of anti-E2 antibody titers, indirectly measured by optical density, in patients with evidence of past infection by HGV (presence of anti-E2 antibodies but absence of HGV RNA) (n = 22). Patients were subdivided into two subgroups according to the persistence (n = 9) (A) or clearance (n = 13) (B) of anti-E2 antibodies during follow-up.

Our study has demonstrated the existence of several different patterns of evolution for HGV infection markers in hemodialysis patients. Some of these patterns (persistent viremia, de novo infection during follow-up in those without anti-E2 antibodies, or persistent presence of anti-E2 antibodies) have been described previously (1, 3, 4, 6, 16).

Interestingly, a proportion of patients (13 of 58; 22.4%) with initial presence of anti-E2 antibodies lost them during follow-up. Antibody titers, indirectly measured by optical density, were significantly lower in these patients than in those who retained anti-E2 antibodies. Four of the patients who lost their anti-E2 antibodies developed HGV viremia. There are two possible explanations for this finding. First, a reservoir of HGV could have been present in these patients, and the disappearance of antibodies would then have allowed the replication of HGV and consequent viremia. Alternatively, they could have been reinfected in the absence of protective antibodies. Those HGV RNA-positive individuals who lost previous anti-E2 antibodies cleared the viremia. Although it has been thought that the clearance of viremia is linked with the appearance of anti-E2 antibodies (4), this was not the case in our study. The immunodeficiency associated with renal failure was probably implicated in this finding (10).

The existence of this group who lost anti-E2 antibodies during follow-up raises the possibility that HGV prevalence in transversal studies may be underestimated. More interestingly, the reappearance of HGV viremia in those who lost their HGV antibodies implies a temporally limited protection in patients previously infected with HGV.