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What Is the Role of GB Virus C Infection in Hepatitis C Virus/HIV Coinfection?
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Editorial
The Journal of Infectious Diseases Aug 15 2006;194:407-409
Mark D. Berzsenyi and Stuart K. Roberts
Department of Gastroenterology, Alfred Hospital, Victoria, Australia
See the article by Schwarze-Zander et al., below.
Much has been published over the past 10 years regarding the influence of GB virus C (GBV-C) on HIV infection. Several studies have reported that coinfection with HIV and GBV-C leads to a more favorable outcome in patients, with a delay in the development of AIDS, compared with the outcome in patients infected with HIV alone [1-6]. This has led some groups to look for the putative mechanism of this beneficial effect, and alterations in the cellular immune response have been implicated [7]. However, there is still considerable controversy regarding this interaction, because not all studies have shown a beneficial effect of GBV-C infection on the progression of HIV disease [8-11]. By contrast, there is no debate that GBV-C plays little role, if any, in hepatitis C virus (HCV) infection, with no effect on the progression of HCV-related liver disease or on the effectiveness of interferon (IFN)-based therapy [12-14]. Similarly, there is no dispute that the course of liver disease is accelerated in patients with HCV/HIV coinfection [15]. Indeed, HCV-related liver disease has been a significant cause of morbidity and mortality in HIV-infected patients during the era of highly active antiretroviral therapy (HAART). This has stimulated increased interest in HCV therapy in HCV/HIV-coinfected subjects, particularly given that HCV clearance rates of >40% are possible with combination pegylated (PEG)-IFN and ribavirin therapy.
What, then, do we know about the potential complex interactions among GBV-C, HCV, and HIV in triply infected subjects? For example, what effect does GBV-C have on HCV/HIV coinfection, and does clearance of GBV-C-either spontaneously or after the treatment of hepatitis C disease-affect the course of the progression of HIV disease? There is remarkably little published in this area, with the only prior report showing that patients infected with GBV-C, HCV, and HIV had an improved response to HAART [16]. In this issue of the Journal of Infectious Diseases, Schwarze-Zander et al. [17] look further into this important clinical scenario and suggest that the clearance of GBV-C during IFN-based therapy for HCV has no short-term effect on HIV control or immune status. Schwarze-Zander et al. evaluated 130 HCV/HIV-coinfected individuals as part of a larger study examining the efficacy of treatment with either PEG-IFN or non-PEG-IFN in combination with ribavirin for chronic hepatitis C infection. GBV-C RNA clearance occurred in the vast majority of HCV responders and was particularly associated with PEG-IFN plus ribavirin treatment and with patients with a higher baseline CD4+ cell count. This probably reflects the importance of the cellular immune response in viral clearance as well as the proven superior efficacy of PEG-IFN in the treatment of both HCV monoinfection and coinfection with HIV [18, 19]. Moreover, clearance of GBV-C did not seem to affect HIV load or CD4+ cell count, which suggests that, at least in the short term, immunological status was maintained. Other measures-such as the onset of AIDS and the long-term effect of GBV-C clearance on HIV markers-were not examined, which leaves unanswered the question as to whether patients who clear GBV-C are more vulnerable to the progression of HIV disease in the longer term.
Importantly, the study found no beneficial effect of GBV-C infection on baseline CD4+ cell count and HIV load, adding further fuel to the debate about whether GBV-C modulates the course of HIV infection. Why, then, is there a discordance between published studies examining this issue? Several confounding factors likely contribute, at least in part, to the discrepant results, including the introduction of HAART (which could mask the effect of GBV-C), differences between study populations, and possibly even the presence of HCV. Virological factors-including GBV-C genotype and viral load-might also potentially influence HIV-related clinical outcomes. In particular, differences in GBV-C genotype distribution among different populations could affect the progression of HIV disease. GBV-C has been shown to have a worldwide distribution, with variation in the proportion of different genotypes in various populations and geographical regions, which suggests a long evolutionary history that parallels prehistoric human migration [20]. It is therefore interesting to note that the authors found GBV-C genotype 2 infection to be associated with higher CD4+ cell counts, compared with those associated with genotype 1 infection. As to why genotype 2 is associated with a higher CD4+ cell count is unclear from the study, although it is interesting to note that others have reported variations in CD4+ cell counts among patients infected with genotype 2a [21]. Certainly, clinical isolates of GBV-C are known to differ in their ability to replicate in an in vitro model, and RNA sequence variability in key regulatory regions might therefore contribute to this phenomena [22]. Clearly, these data are food for thought and should be a catalyst for further large-scale prospective studies, from separate geographical regions, to determine whether particular genotypes are associated with reduced morbidity or mortality from HIV.
Another issue highlighted in the study is the role that antibodies to the E2 envelope glycoprotein play in defining past GBV-C infection. It has been previously accepted that antibodies to the GBV-C E2 protein serve as a useful marker for diagnosing clearance of GBV-C RNA and, as such, are a useful marker of past infection [23]. However, the study indicates that none of the individuals, after receiving treatment with IFN, who went on to temporarily clear GBV-C RNA or who had sustained RNA clearance, developed E2 antibodies during the follow-up period. This is not a new phenomenon; other researchers have shown that spontaneous resolution of GBV-C is not always followed by the appearance of E2 antibodies in an HIV/GBV-C-coinfected cohort and that E2 antibodies can exist in the presence of viral RNA. Perhaps the current definition of active and past GBV-C infection should be reconsidered for some patient populations [11]. To some extent, the use of HAART, changes in drug regimens, and drug holidays, in conjunction with the patient's immune function, may contribute to these findings. The initial work with clearance of GBV-C viremia and the development of E2 antibodies was performed in healthy blood donors who had functional immune systems [24]. The findings by Schwarze-Zander et al. [17] and others [11] raise serious questions about the usefulness of E2 antibodies as a marker of past GBV-C infection in populations infected with HIV or coinfected with HIV and HCV.
GBV-C has been previously described as a virus in search of a disease [25]. Exhaustive studies by many researchers have failed to reveal any associations of GBV-C with disease [7]. Perhaps now should be the time to consider whether GBV-C plays a role in the clinical management of HIV monoinfection or HCV/HIV coinfection. Furthermore, is it clinically important to know a patient's GBV-C status and GBV-C genotype, and does knowing that information make a difference in outcome? Could differential CD4+ cell counts among certain genotypes help explain some of the differing reports regarding the beneficial influence of GBV-C on HIV? At this point, there is still not enough information to answer these questions. However, the study by Schwarze-Zander et al. takes a small step in the right direction. Still, further studies in different populations and cohorts need to be performed to examine whether the association of GBV-C genotype 2 with CD4+ cell counts is maintained and whether these novel changes translate into improved clinical outcomes for patients. In addition, longer follow-up periods may be required to fully address whether the clearance of GBV-C as a result of HCV treatment adversely affects HIV-related outcomes.
Without question, the interactions among GBV-C, HCV, and HIV are complex. All the work performed to date in triply infected individuals has focused primarily on HIV-related outcomes; however, important questions still remain. Of great importance among these questions is the role that GBV-C infection plays in the progression of liver disease in the HCV/HIV-coinfected patient. For example, does clearance of GBV-C alter the course of HCV-related liver disease? Only once these questions are addressed will we be able to begin to fully appreciate the associations between GBV-C and HCV-HIV coinfection and to understand their potential clinical impact.
GB Virus C (GBV-C) Infection in Hepatitis C Virus (HCV)/HIV-Coinfected Patients Receiving HCV Treatment: Importance of the GBV-C Genotype
The Journal of Infectious Diseases Aug 15 2006;194:410-419
Carolynne Schwarze-Zander,1,4,a Jason T. Blackard,4,a Hui Zheng,7 Marylyn M. Addo,5 Wenyu Lin,4 Gregory K. Robbins,6 Kenneth E. Sherman,8 Dietmar Zdunek,2 Georg Hess,3 and Raymond T. Chung,4 for the AIDS Clinical Trial Group A5071 Study Team
1Department of Internal Medicine I, University of Bonn, Bonn, 2Roche Diagnostics, Penzberg, and 3Roche Diagnostics, Mannheim, Germany; 4Gastrointestinal Unit, 5Partners AIDS Research Center, and 6Infectious Diseases Unit, Massachusetts General Hospital, and 7Harvard Center for AIDS Research, Harvard Medical School, Boston, Massachusetts; 8Division of Digestive Diseases, University of Cincinnati College of Medicine, Cincinnati, Ohio
ABSTRACT
Background. Persistent GB virus C (GBV-C) coinfection leads to slower human immunodeficiency virus (HIV) progression. Despite the existence of multiple GBV-C genotypes, their relevance to the progression of HIV disease is unknown. We therefore investigated (1) the prevalence and genotype of GBV-C in hepatitis C virus (HCV)/HIV-coinfected patients and (2) the impact of HCV treatment on GBV-C RNA clearance.
Methods. We retrospectively studied 130 HCV/HIV-coinfected patients initiating HCV therapy. Anti-E2 enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction (PCR), and real-time PCR were used to detect and quantify GBV-C infection. GBV-C genotype was determined by sequencing the 5 untranslated region.
Results. GBV-C infection (past or current) was identified in 111 (85%) of the patients. Ongoing GBV-C replication was detected in 40 patients. Coinfection with GBV-C genotype 2 was associated with significantly higher CD4+ cell counts. After 24 weeks of HCV therapy, GBV-C RNA clearance was observed in 50% of patients, although this was not associated with changes in HIV load or with CD4+ cell counts. Sustained GBV-C RNA clearance was observed in 31% of patients with GBV-C RNA detected at baseline.
Conclusions. GBV-C coinfection was extremely common. GBV-C RNA clearance with HCV therapy was associated with neither short-term loss of HIV control nor impaired immune status. The association of GBV-C genotype 2 with higher CD4+ cell counts merits further study.
Background
GB virus C (GBV-C), which was first isolated in 1995, belongs to the Flaviviridae family and is the closest known relative of hepatitis C virus (HCV) [1, 2]. GBV-C was initially thought to induce hepatitis; however, subsequent investigations have failed to demonstrate GBV-C replication in hepatocytes or a causal association with hepatitis or other human diseases [3]. GBV-C viremia may persist for up to 16 years [4] and is diagnosed by the detection of viral RNA. In the majority of patients, viremia eventually resolves with the development of antibodies directed against the viral envelope protein E2 [5, 6] that appear to protect against GBV-C reinfection [7, 8].
Several groups have reported beneficial effects of GBV-C viremia on HIV disease, describing a slower progression of HIV disease to AIDS or death [9, 10]. Moreover, an association between GBV replication and lower plasma HIV loads and higher CD4+ cell counts has been demonstrated [11, 12], although these findings were not confirmed by other studies [13-15].
GBV-C, like HCV, has been shown to be sensitive to the antiviral actions of interferon (IFN) [16, 17]. IFNrelated clearance of GBV-C occurs in up to 60% of HCV- or HIV-infected patients [18]; however, very little is known about GBV-C RNA clearance in HIV/HCV/GBV-C triply infected patients treated with IFN or pegylated (PEG)-IFN plus ribavirin. With the increased survival conferred by the use of highly active antiretroviral therapy (HAART), complications of chronic HCV infection have emerged as an important cause of morbidity and mortality in HIV-infected individuals [19-21]. Indeed, several studies have shown an accelerated course of chronic HCV infection during HIV coinfection [22, 23]. Thus, the development of effective antiviral therapies for HCV in coinfected persons will be important for the long-term clinical management of HCV disease [24-26]. Nonetheless, the consequences of HCV therapy on the clearance of GBV-C have not been well characterized.
Phylogenetic analysis has revealed at least 5 major GBV-C genotypes; they exhibit geographical clustering in West Africa (genotype 1), Europe and the United States (genotypes 1 and 2), parts of Asia (genotype 3), Southeast Asia (genotype 4), and South Africa (genotype 5) [27-34]. However, the clinical significance of GBV-C genotypes during HIV coinfection is unknown.
Therefore, we sought to determine (1) the prevalence of GBV-C infection (past and current) and genotype distribution in a large cohort of HCV/HIV-coinfected patients and (2) the frequency of treatment-induced GBV-C RNA clearance in HIV/HCV/GBV-C triply infected patients and the potential effects of GBV-C RNA clearance on HIV disease. We hypothesized that distinct GBV-C genotypes may be cleared differentially by HCV therapy and that differences in GBV-C genotypes might influence important predictors of the progression of HIV disease.
DISCUSSION
In the present study, we analyzed the frequency, genotype distribution, and subsequent clearance of GBV-C infection among 130 HCV/HIV-coinfected patients receiving IFN or PEG-IFN plus ribavirin for the treatment of HCV infection. The overall prevalence of GBV-C infection (E2 antibody positivity and/or GBV-C viremia) was 85% in our cohort, which is similar to data from other populations with frequent exposure to blood products (including injection drug use) and from HIV-positive patients [8, 10, 38, 39]. In agreement with other studies, we found a higher prevalence of GBV-C viremia among younger patients, whereas E2 antibodies were detected more frequently in older patients [40]. This may be explained by a potential protective effect of E2 antibodies against GBV-C reinfection [4-6]. However, in our study, we found 11 patients who were simultaneously positive for GBV-C RNA and E2 antibody. Similar results have been found in other HCV/HIV-coinfected patients [41, 42], and they may be attributable to altered immune responses in the setting of HCV/HIV coinfection [43] or reinfection with GBV-C in the presence of E2 antibodies [7].
GBV-C RNA was cleared in 50% of HIV/HCV/GBV-C triply infected patients treated with IFN (or PEG-IFN) plus ribavirin. Consistent with the high degree of sequence homology between GBV-C and HCV, treatment-induced clearance of GBV-C RNA at week 24 was strongly correlated with an HCV virologic response (table 3). Moreover, factors predicting clearance during treatment were similar for the 2 viruses: GBV-C RNA clearance was associated with PEG-IFN plus ribavirin treatment and a higher baseline CD4+ cell count. Nonetheless, additional factors-such as host genetic determinants and adaptive immune responses-are likely to influence GBV-C RNA clearance.
At 24 weeks after treatment, sustained clearance of GBV-C was found in 31% of patients, whereas GBV-C RNA reappeared in another 19%. Sustained clearance of GBV-C was associated with baseline GBV-C load (figure 3) but not with CD4+ cell count, HCV virologic response, or HCV treatment arm. Although the factors that influence the sustained clearance of GBV-C are not known, studies in HCV/GBV-C-coinfected patients have shown a marginal effect or no effect of ribavirin on GBV-C sustained clearance [44, 45]. Moreover, the role of E2 antibody development in GBV-C RNA clearance remains unclear, because no patients with GBV-C relapse or a GBV-C sustained response developed E2 antibodies during the follow-up period. This may have been because of the efficient clearance of GBV-C by HCV therapy, but Van der Bij et al. [41] found, in a large HIV/GBV-C-coinfected cohort, that spontaneous resolution of GBV-C viremia was not necessarily followed by the appearance of E2 antibodies.
Several studies have described a beneficial effect of GBV-C viremia on the progression of HIV disease. GBV-C replication has been associated with lower HIV loads and higher CD4+ cell counts [9-11, 46]. Several mechanisms of GBV-C interference with the progression of HIV disease have been proposed, including postentry inhibition of replication, alteration of T helper cytokine profiles, and changes in chemokine coreceptor expression [47-49]. However, a beneficial impact of GBV-C on the progression of HIV disease has not been confirmed in all studies [13, 14, 50]; thus, other virologic and immunologic factors may partially explain these divergent results.
The existence of multiple GBV-C genotypes has led several authors to suggest that differences in GBV-C strains circulating within populations might affect the progression of HIV disease [50-52], although this has not been formally studied. We found no significant difference in baseline CD4+ cell count or HIV load according to GBV-C infection status. Nonetheless, important differences were observed based on the existing GBV-C genotype: GBV-C genotype 2 infection was associated with higher CD4+ cell counts, compared with genotype 1 infection. To our knowledge, until now there has been only a single report addressing the importance of genotype/subtype variations of GBV-C during HIV infection [51]. Those authors noted that CD4+ cell counts tended to be lower in patients infected with subtype 2a, compared with those infected with subtype 2b; however, other distinct genotypes were not circulating in the study population for further comparison. Therefore, it is possible that GBV-C genotype could at least partially account for the conflicting observations to date regarding the impact of GBV-C replication on the progression of HIV disease. In this respect, it is also interesting that GBV-C genotype 2 was marginally more sensitive to HCV therapy than was genotype 1 in our study (P = .09). Thus, given that an HCV virologic response and GBV-C RNA clearance may be linked and that GBV-C RNA persistence is associated with a decreased risk of death [41], it is tempting to speculate that GBV-C RNA clearance associated with HCV therapy might have an adverse effect on the progression of HIV disease.
In summary, we have shown a high overall prevalence of GBV-C in a large cohort of HCV/HIV-coinfected patients receiving IFN or PEG-IFN plus ribavirin to treat chronic HCV infection. Importantly, GBV-C RNA clearance did not appear to be associated with short-term loss of HIV RNA control. It is possible that the effects of GBV-C on HIV are outweighed by effective ART, given that the majority of our study patients were receiving HAART. In accordance with the ACTG A5071 study design, only those patients with virologic response at week 24 and virologic nonresponders with histologic improvement were treated for the full 48 weeks; thus, long-term follow-up of the majority of these HIV/HCV-coinfected patients was not possible. Furthermore, it is not known whether the selected cohort was fully representative of the HCV/HIV-coinfected population. Thus, additional prospective studies with several years of follow-up data will be necessary to confirm our findings and to determine whether treatment-induced clearance of GBV-C has deleterious effects on long-term HIV progression in HAART-naive and -experienced patients. We also noted a significant association between GBV-C genotype and CD4+ cell count that suggested a differential impact of GBV-C genotype on important immunologic parameters. Although other unmeasured factors may potentially influence these results, the findings have important implications for understanding the relationship and molecular interactions of GBV-C and HIV and the consequences of GBV-C infection for the management of HCV/HIV-coinfected patients undergoing IFN-based antiviral therapy.
RESULTS
GBV-C infection status. Of the 130 patients with HCV/HIV coinfection, 111 (85%) had evidence of past or present GBV-C infection. E2 antibody was detected in 71 (64%) of these 111 patients. GBV-C RNA was detected in 40 (36%) of the 111 patients, and E2 antibodies were also detected in 11 of the 40. Demographic and biochemical profiles of the study population according to GBV-C status are shown in table 1. More men than women were GBV-C RNA positive, and more women than men had no evidence of GBV-C infection (defined as the absence of GBV-C RNA and a lack of E2 antibodies) (P < .001). Furthermore, younger patients were more often GBV-C RNA positive, whereas older patients were more likely to have signs of past GBV-C infection (P = .004). There were no significant differences in baseline CD4+ cell count, HCV load, or HIV load according to GBV-C infection status.
GBV-C genotyping. The 5 UTR (256 nt) could be amplified from 39 (98%) of 40 GBV-C RNA-positive patients. Seven (18%) of the 40 patients were infected with GBV-C genotype 1, 31 (79%) with genotype 2, and 1 (3%) with genotype 3 (figure 1). The 5 UTR could not be amplified from 1 GBV-C-RNA positive patient.
Because previous studies did not explore the impact of different GBV-C genotypes on HIV disease, we were interested in further comparing demographic and clinical parameters between infections with GBV-C genotypes 1 and 2 (table 2). Patients with GBV-C genotype 1 infection were more likely to be nonwhite than were patients with genotype 2 infection (P = .031). At baseline, we noted significantly higher CD4+ cell counts in GBV-C RNA-positive patients infected with genotype 2 than in those infected with genotype 1 (534 vs. 308 cells/mL; P = .01). No significant differences in mean baseline HIV or HCV loads were found between GBV-C genotype 1- and genotype 2-infected patients. To further analyze CD4+ cell counts by GBV-C genotype, we adjusted for the potential confounding effects of race, HIV load, and ART use by including them in a multivariate linear-regression model; however, the difference in CD4+ cell counts between GBV-C genotype 1- and genotype 2-infected patients remained statistically significant (P = .015).
The mean baseline viral load for GBV-C genotype 1-infected patients was 7.21 log10 geq/mL, whereas the mean for genotype 2-infected patients was 7.25 log10 geq/mL (P = .33). In contrast to genotype 1-infected patients, 9 (29%) of 31 genotype 2-infected patients had GBV-C RNA loads below the limit of detection of the assay of 103 geq/mL. Interestingly, none of the patients (0/7) with GBV-C genotype 1 infection had an HCV virologic response at week 24, whereas 10 (32%) of 31 patients with GBV-C genotype 2 infection had an HCV virologic response. However, this was not statistically significant (P = .156) (table 2).
GBV-C response to IFN therapy. The response of GBV-C to IFN therapy was determined in the 40 GBV-C RNA-positive patients (figure 2). After 24 weeks of treatment with IFN or PEG-IFN plus ribavirin, 19 (50%) of 38 HIV/HCV/GBV-C-infected patients with available data had undetectable GBV-C loads (table 3): 11 (73%) of 15 patients who received PEG-IFN plus ribavirin had undetectable GBV-C RNA loads at week 24 of treatment, whereas only 8 (35%) of 23 patients receiving standard IFN plus ribavirin had undetectable GBV-C RNA loads at week 24 (P = .045). There was a strong association between GBV-C RNA clearance and HCV virologic response: of 10 patients who achieved HCV virologic response at week 24, 9 (90%) also had undetectable GBV-C RNA loads at week 24 (P = .007). In particular, the magnitude of reductions in HCV RNA load from baseline to week 24 was significantly higher in GBV-C responders than in GBV-C nonresponders (-2.09 vs. -0.37 HCV RNA log10 IU/mL, respectively; P = .0009). Interestingly, GBV-C RNA clearance was associated with higher baseline CD4+ cell counts (591 cells/uL for GBV-C responders vs. 419 cells/uL for GBV-C nonresponders; P = .03). In addition, there was a trend toward higher rates of GBV-C RNA clearance for genotype 2 than for genotype 1 infection (P = .09). After 24 weeks of HCV therapy, no correlation was found between GBV-C RNA clearance and baseline GBV-C, HCV, or HIV loads (table 3), nor were changes (from baseline to week 24 of HCV therapy) in HIV load or CD4+ cell count significantly different between GBV-C responders and nonresponders.
Of 19 patients who had cleared GBV-C after 24 weeks of HCV therapy, 6 (32%) had a recurrence of GBV-C RNA at a subsequent posttherapy follow-up visit ("GBV-C relapsers"). Two patients had similar GBV-C loads (±0.5 log10 copies/mL) before and after HCV treatment, whereas 1 patient had an increase in GBV-C viral load of >0.5 log10 copies/mL. In 3 patients, viral loads fell to below the limit of detection of the quantitative PCR (1 _ 103 geq/mL) but were nonetheless detectable by qualitative PCR.
Sustained clearance of GBV-C (defined as the following findings: GBV-C RNA positive at baseline, GBV-C RNA negative after 24 weeks of HCV treatment, and GBV-C RNA negative after 24 weeks of posttreatment follow-up) was observed in 11 (31%) of 36 HIV/HCV/GBV-C triply infected patients (figure 2). Interestingly, no patient developed E2 antibodies after clearance of GBV-C RNA. In contrast to GBV-C RNA clearance at week 24, sustained GBV-C RNA clearance was associated with a lower baseline GBV-C load (P = .0128 for the difference among the 3 groups) (figure 3).
At baseline, 90 patients were GBV-C RNA negative; however, E2 antibodies were detected in 71 (79%) of these patients. Sixty of these 90 patients were available for a reevaluation of GBV-C status at the 24-week follow-up visit (42-72 weeks after study entry) after the discontinuation of HCV treatment. No patient was newly infected or reinfected with GBV-C during this follow-up period, and no patient lost E2 antibody positivity (data not shown).
PATIENTS AND METHODS
Study design and patients. A total of 133 HCV/HIV coinfected patients was included in the study. All patients were enrolled as part of the prospective Adult AIDS Clinical Trials Group (ACTG) A5071 study evaluating the efficacy of IFNa-2a plus ribavirin versus PEG-IFN-a-2a plus ribavirin for chronic HCV infection in individuals coinfected with HIV [25]. By study design, an efficacy and safety assessment was performed for all patients at week 24 to determine whether they could continue to participate. HCV virologic responders (defined as those with an undetectable HCV RNA load at week 24) continued treatment until week 48. HCV virologic nonresponders at week 24 (57/67 patients in the IFN plus ribavirin arm and 37/66 patients in the PEG-IFN plus ribavirin arm) were asked to undergo liver biopsy, with continuation of treatment to week 48 for those with a >2-point drop in the total hepatic activity index from baseline (i.e., histologic responders) [35]. Thus, all patients were treated for at least 24 weeks, although only virologic responders (n = 39) and histologic responders (n = 25) completed 48 weeks of treatment. All patients were monitored for an additional 24 weeks of follow-up after the completion of therapy. HCV RNA loads were assessed using the Roche Cobas Amplicor 2.0 assay (Roche Diagnostics), which has a lower limit of detection of 60 IU/mL. HIV RNA loads were assessed using the Roche Cobas Amplicor 2.0 assay (Roche Diagnostics), which has a lower limit of detection of 50 copies/mL.
The presence of GBV-C RNA in serum was determined by reverse-transcription polymerase chain reaction (RT-PCR) before the initiation of HCV therapy (baseline). Three patients were excluded from further analysis because of a lack of available serum samples for GBV-C testing. Patients who were GBV-C RNA positive at baseline were further evaluated for the presence of GBV-C RNA at available follow-up visits, and patients who were GBV-C RNA negative at baseline were retested at the last available follow-up visit. All baseline and follow-up serum samples were also tested for the presence of E2 antibodies.
Detection and quantification of GBV-C RNA. Viral RNA was extracted from 140 uL of serum by use of the QIAmp Viral RNA Mini Kit (Qiagen), in accordance with the manufacturer's instructions. GBV-C RNA was detected by nested RT-PCR using primers corresponding to the 5 untranslated region (UTR) [11]. RNA was transcribed and amplified using the antisense primer 5-ATG CCA CCC GCC CTC ACC CGA A-3 (nt 494-473, according to GenBank accession number AY196904) and the sense primer 5-AAA GGT GGT GGA TGG GTG ATG-3 (nt 67-87) in a OneStep RT-PCR device (Qiagen). Amplification conditions were an initial cycle for 59 min at 50 C and 10 min at 94 C; 35 cycles for 30 s at 94 C, 1 min at 55 C, and 1 min at 72 C; and extension for 20 min at 72 C. The first-round PCR product was then subjected to nested PCR using internal antisense primer 5-CCC CAC TGG TCY TTG YCA ACT C-3 (nt 362-341) and sense primer, 5-AAT CCC GGT CAY AYT GGT AGC CAC T-3 (nt 107-131). After 35 cycles for 30 s at 94 C, 30 s at 55 C, and 1 min at 72 C, PCR products were analyzed by agarose gel electrophoresis for the presence of a 256-nt band.
The GBV-C load was quantified in all GBV-C RNA-positive samples by use of the LightCycler-RNA Master Hybridization System (Roche Diagnostics). Briefly, a 1-step RT-PCR was performed in glass capillaries. The following oligonucleotides were used in the PCR: 5-CGG CCA AAA GGT GGT GGA TG-3 (nt 61-80) and 5-CGA CGA GCC TGA CGT CGG G-3 (nt 246-228). For probing, a 5-LCRed640-marked oligonucleotide (5-CAA GGT GAC CGG GAT TTA CGA CCp-3) and a 3-fluorescein-marked oligonucleotide (5-CTC TTA AGA CCC ACC TAT AGT GGC T-3) were used. The lower limit of detection was 1000 genome equivalents (geq)/mL.
Detection of E2 antibody. As markers of GBV-C RNA clearance and prior exposure [8], serum E2 antibodies were detected using an immunoassay using recombinant E2 (uPlate Anti-Hgenv test; Roche Diagnostics), in accordance with the manufacturer's instructions. Plates were incubated with diluted (1 : 20) serum, and E2 antibodies were detected using anti-human IgG peroxidase conjugate and ABTS substrate. Serum samples were tested in duplicate. In accordance with the manufacturer's cutoff, an OD < 0.10 was considered to be negative, and an OD >0.10 was considered to be positive.
Phylogenetic analysis. The GBV-C genotype was determined by population-based amplification of the 5 UTR region, as described elsewhere [36]. PCR products were gel purified and sequenced by use of the internal PCR primers as sequencing primers. Sequences were aligned with database reference using Clustal X (version 1.64b) [37]. The reference sequences used to confirm GBV-C genotype included the following GenBank accession numbers: 1A, U59543 and U59540; 1B, U59555 and U59549; 2A, U59520 and U59521; 2B, U59529 and U59533; 3, U59538 and U59539; and 4, AB018667 and AB021287. The statistical robustness and reliability of the branching order within the phylogenetic tree was confirmed by bootstrap analysis using 100 replicates. Bootstrap values >70% were considered to be statistically significant.
Statistical analysis. We evaluated the associations between dichotomous variables, using Fisher's exact test. Comparisons of continuous outcomes between 2 groups with small group sizes were evaluated using the Wilcoxon rank sum test, adjusted for ties. Associations between a continuous variable and any categorical variable with >2 groups were evaluated using the Kruskal-Wallis test. Results of both the Wilcoxon rank sum test and the Kruskal-Wallis test were considered to be robust because they were free of normal distribution assumptions. All P values were 2-sided. P < .05 was considered to be statistically significant.
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