Maria Brezina

Biological dynamics of viral load in hemodialysis patients with hepatitis C virus

Abstract The biological dynamics of hepatitis C virus (HCV) viremia in uremic patients with chronic infection have not been fully characterized. We prospectively studied fluctuations of HCV-RNA in sera from 52 patients with end-stage renal disease who were undergoing maintenance hemodialysis (HD) and had chronic HCV infection. We measured HCV viremia monthly over the course of 13 months with the branched-chain DNA (bDNA) signal amplification assay and prospectively analyzed liver function, expressed by monthly serum aspartate (AST) and alanine aminotransferase (ALT) determinations. We observed three different patterns of HCV viremia: (1) patients persistently positive by bDNA assay (persistent viremia; 23 of 52 patients; 44%), (2) individuals with alternatively positive and negative results (intermittent viremia; 17 of 52 patients; 33%), and (3) patients persistently negative by bDNA assay (12 of 52 patients; 23%). The HCV viral load over the follow-up was greater among patients with persistent compared with intermittent viremia (persistent, 31.7 × 10 5 Eq/mL; range, 6.3 × 10 5 to 16.03 × 10 6 Eq/mL versus intermittent, 10.4 × 10 5 Eq/mL; range, 1.1 × 10 5 to 9.4 × 10 6 Eq/mL; P = 0.0001). In addition, patients with persistent viremia had over time greater AST and/or ALT activities than the intermittent group (AST: persistent, 26.5 IU/L; range, 9.6 to 73.7 IU/L versus intermittent, 21.3 IU/L; range, 8 to 56.8 IU/L; P = 0.001 and ALT: persistent, 14.7 IU/L; range, 3.7 to 57.9 IU/L versus intermittent, 10.9 IU/L; range, 2.3 to 52.1 IU/L; P = 0.001). In the group with persistent viremia, the mean difference between maximum and minimum values of HCV-RNA observed in each individual patient was 2.09 ± 0.7 natural logarithm (Log n ) and in intermittent viremic patients, 1.55 ± 1 Log n ( P = 0.045). The HCV load at study entry (19.4 × 10 5 Eq/mL) was rather low and did not change versus the end of follow-up in all patients ( P = not significant [NS]). In the entire group, the fluctuations in HCV-RNA levels over time between and within individuals were not significant ( P = NS). No difference in variability of HCV-RNA values over time between patients infected with different HCV genotypes was seen. In conclusion, three different patterns of HCV viremia in HD over time were assessed; one third of viremic patients had intermittent viremia, and those patients had less HCV-RNA, enzyme-linked immunosorbent assay, and aminotransferase activity than did patients with persistent HCV load. Larger fluctuations in HCV RNA levels occurred in patients with persistent than with intermittent HCV viremia. However, the viremic HCV load was low and relatively stable over a 13-month follow-up in our population. Studies with longer observation periods are warranted to understand fully the natural history of HCV in these immunosuppressed individuals.

Quantitative Assessment of HCV Load in Chronic Hemodialysis Patients: A Cross-Sectional Survey

Recent evidence has been accumulated showing that chronic hemodialysis (HD) patients have a very high prevalence of antibodies to hepatitis C virus (HCV). In contrast, there is little information addressing the virological characteristics of HCV infection in this population. Aim: To measure HCV viral load and to correlate this with demographic, biochemical, and clinical features of a large cohort of HCV-infected patients on chronic HD. Methods: 394 chronic HD patients were tested by branched-DNA signal amplification assay, anti-HCV enzyme-linked immunosorbent assay 2.0, and on the basis of the aspartate aminotransferase/alanine aminotransferase (AST/ALT) activity. Multivariate analysis by ordinal logistic regression model was performed: age, gender, race, time on HD, allocation of the patients among the HD units, etiology of end-stage renal disease, HBsAg status, anti-HCV positivity, HCV genotype, and AST/ALT levels were independent factors, and viremic levels of HCV in serum were assumed as dependent variables. Results: 88 (22.3%) patients showed serological and/or virological signs of HCV infection. 59 (15%) out of 394 had detectable HCV RNA in serum, the mean HCV load was 19.4 × 105 (95% CI, 6.06 × 107 to 6.2 × 104) Eq/ml. According to the criteria suggested by others [J Infect Dis 1994;169:1219–1225], there were 8 (13.5%) individuals with high-titer viremia (>1 × 107 Eq/ml) in the subset of viremic patients. A small subset (8/394 or 2%) of individuals was seronegative, but viremic; 29 (7%) out of 394 were seropositive without detectable HCV RNA in serum. Univariate analysis showed that the frequency of anti-HCV positivity was significantly higher in viremic patients as compared with individuals with no detectable HCV viremia: 51/59 (86%) vs. 29/335 (8.6%), p = 0.0001. Serum AST and ALT levels were significantly higher in viremic patients than in individuals with no detectable HCV RNA in serum: 23.8 (95% CI 60.8–9.3) vs. 17.1 (95% CI 50.4–5.8) U/l (p = 0.009) and 14.4 (95% CI 48.9–4.3) vs. 9.8 (95% CI, 37.3– 2.5) U/l (p = 0.008). Logistic regression analysis showed an association between HCV viremia and anti-HCV positivity (p = 0.00001) and ALT activity (p = 0.01). Conclusions: Hepatitis C virus infection is highly prevalent in the HD population; the viral load is relatively low, and it was associated with elevated hepatic enzyme levels and anti-HCV positivity. No other clinical characteristics were associated with HCV RNA levels. Seronegative but viremic patients were also found. Longitudinal studies with long follow-up periods are necessary to evaluate the course of HCV load over time in this population.

Detection of de novo Hepatitis C Virus Infection by Polymerase Chain Reaction in Hemodialysis Patients

Patients on chronic hemodialysis (HD) treatment have been identified by serological testing, including second- and third-generation enzyme-linked immunosorbent assay (ELISA), as a high-risk group for hepatitis C virus (HCV) infection. Previous studies have shown that de novo cases of HCV may occur in HD units in the absence of other parenteral exposures, which suggests the spread of HCV between patients. In addition, the reverse-transcription polymerase chain reaction (RT-PCR), which directly detects HCV virus, has identified HCV infection in chronic HD patients who are seronegative. The aim of this study was to determine the incidence of HCV infection detected by RT-PCR technology in a large cohort of chronic HD patients. One hundred and twenty chronic HD patients, HCV-negative by serological assays (second-generation ELISA) and molecular techniques (branched DNA and RT-PCR), were observed for a mean period of 9.5 months. They were tested monthly for serum alanine aminotransferase levels (ALT) and by second-generation ELISA. At the end of the follow-up period, they were again evaluated by branched DNA and RT-PCR testing. HCV RNA was detected in patients’ sera by RT followed by PCR using two separate primer sets from the 5′-untranslated region of the HCV genome. Southern blot was performed using a digoxigenin-labeled probe. Two patients who had HCV RNA detectable by RT-PCR at the end of the follow-up period remained branched-DNA-negative. Thus, the incidence of de novo acquisition of HCV infection in the current investigation was 2.1% per year. In 1 patient RT-PCR positivity and anti-HCV ELISA seroconversion occurred. The 2nd patient remained anti-HCV ELISA-negative, although viremic. In both patients, the onset of positivity by RT-PCR was associated with a rise of ALT levels into the ‘abnormal range’ in our laboratory. In these 2 patients, de novo acquisition of HCV infection was observed in the absence of obvious parenteral risk factors other than their presence in the HD environment. In conclusion, de novo acquisition of HCV infection may be undetected by ELISA and branched-DNA assays. The need to monitor chronic HD patients by serial ALT testing is emphasized. RT-PCR shoud be incorporated into diagnostic testing for HCV infection in chronic HD patients. RT-PCR technology can identify HCV in HD individuals with raised ALT activity.

Hepatitis B surface antigenemia in chronic hemodialysis patients: Effect of hepatitis B immunization

OBJECTIVE:Hemodialysis patients with hepatitis B surface antigen (HBsAg) are considered to have the hepatitis B virus (HBV) and are segregated to limit transmission. However, transient de novo HBsAg has been identified in hemodialysis patients shortly after vaccination. Our hypothesis is that immunization rather than actual HBV infection is the most common cause of de novo HBsAg among hemodialysis patients.METHODS:We performed a prospective study between January, 1998 and June, 2000 of de novo HBV infection in over 2400 hemodialysis patients who were screened monthly for HBsAg using a standard enzyme immunoassay. Positive results were confirmed with a neutralization assay. If the confirmatory test was positive, anti-hepatitis B core antibody IgM testing was performed.RESULTS:We identified nine patients with de novo HBsAg confirmed with the neutralization assay. In eight of the nine patients with de novo HBsAg (89%), HBsAg was temporally related to HBV immunization. In none of these eight patients was there a detectable anti-hepatitis B core antibody IgM, an elevated ALT level, or clinical history suggestive of recent hepatitis. The mean age (± SD) of the four men and four women was 56.4 ± 18.8 yr. HBsAg was detectable within 3 days of immunization in most patients.CONCLUSIONS:Our results suggest that HBV immunization is the most common cause of detectable HBsAg in hemodialysis patients. Hemodialysis patients should not be screened for HBV within a week of immunization, and caution should be exercised when interpreting HBsAg seropositivity within 4 wk of HBV immunization.

Epidemiology and Natural History of Hepatitis G Virus Infection in Chronic Hemodialysis Patients

Patients on chronic hemodialysis (HD) have recently been identified as having a high prevalence of hepatitis G virus (HGV) infection. The clinical significance of HGV in this population remains unclear, with no data available as to the acquisition and natural history of HGV infection in this group. Aims: To assess the prevalence and risk factors of HGV in a large cohort of chronic HD patients, and to evaluate the incidence and clinical consequences of HGV over time in this population. Methods: Paired sera from 292 patients undergoing chronic HD treatment in four units in the Los Angeles area were tested for HGV RNA before and after they had been on HD for a mean period of 9.7 ± 1.9 months. HGV was tested by a single-step RT-PCR using two couples of primers located in two different portions (5′UTR, NS5a) of the genome. The amplified products were detected by hybridization with 5′ biotin-labeled probes specific for each region. Results: At study entry there were 50 HGV RNA-positive patients, thus the HGV prevalence was 17% (50/292). The multivariate analysis by ordinal logistic regression model showed association (p = 0.0013) between HGV RNA and the location of patients among the HD units. No other significant associations were observed. Three (3/50 = 6%) HGV RNA-positive patients at study entry and 3 (3/41 = 7%) at the end of the follow-up showed a mild increase of alanine aminotransferase (ALT) activity in absence of other apparent causes of liver damage. 35 (70%) out of 50 HGV viremic patients had persistently detectable viremia during the study period; 15 (30%) had non-persistently detectable HGV RNA in the second serum specimen. There was no significant difference between the patients with persistently detectable HGV RNA and those who showed non-persistently detectable HGV viremia with regard to demographic, clinical or virological features. Six patients without detectable HGV viremia at the start of the study showed de novo HGV infection during the follow-up, thus the HGV incidence was 3.07% per year. These individuals did not simultaneously acquire HBV or HCV markers; de novo HGV infection was not associated with other demographic, clinical or virological features. One (16.7%) out of 6 individuals with HGV acquisition had persistently raised ALT levels and chronic HBsAg positivity. The prevalence of HGV was 14% (41/292) at the end of the observation period. Conclusions: The prevalence of HGV in our HD population was high; HGV positivity was strongly associated with the location of HD patients among the units; some HD individuals with current HGV infection showed biochemical signs of liver disease without other apparent causes. De novo acquisition of HGV occurred within HD units in the absence of evident parenteral risk factors for HGV other than their presence in the HD environment. A large portion of HGV viremic patients showed non-persistently detectable HGV viremia during the study. Acquisition of HGV was not associated with a rise in ALT activity unlike prior experience with de novo HCV in HD patients. Further investigations are warranted to explain the modes of HGV acquisition and the clinical significance of HGV in th HD population.

Comparison of the cost and effectiveness of two strategies for maintaining hepatitis B immunity in hemodialysis patients.

A decision-analysis model was developed to compare the strategies to maintain hepatitis B virus (HBV) immunity in hemodialysis patients who responded to the primary HBV vaccine. Our hypothesis is that the routine, annual administration of the vaccine booster to all hemodialysis patients (non-screening strategy) is more cost-effective than the current strategy of vaccination based on anti-HBs titers (screening strategy). Under baseline assumptions, the screening strategy was less costly and was associated with fewer HBV infections than the non-screening strategy. The results of our model did not support our hypothesis, and indicate that regularly screening patients for HBV immunity before revaccination is less costly and more effective than the empiric vaccination of hemodialysis patients.

Automated RIBATM HCV Strip Immunoblot Assay: A Novel Tool for the Diagnosis of Hepatitis C Virus Infection in Hemodialysis Patients

Hemodialysis (HD) patients remain a high-risk group for hepatitis C virus (HCV) infection. Serological assays (enzyme-linked immunosorbent assays, ELISAs) are the only tests currently approved by the Food and Drug Administration in the United States for the diagnosis of HCV. The RIBATM HCV Strip Immunoblot Assay (SIA) is an established method for supplemental testing of repeat reactive hepatitis C ELISA patients on HD. However, the current manual procedure is labor intensive, requiring subjective band scoring and result interpretation. Recently, the automated CHIRON® RIBATM HCV Processor System has been designed to perform RIBA supplemental testing. The CHIRON RIBA HCV Processor System consists of a bench-top instrument that provides objective evaluation of the RIBA immunoblot strips, by measuring the light differentially reflected from the developed bands and white background, creating a density of reflectance. The CHIRON RIBA HCV Processor System assesses the intensity of each of the reactive bands in relation to the intensity of the internal control bands on each RIBA HCV strip. Comparison between processor and manual protocols was performed using a large (n = 200) cohort of ELISA 3.0 HCV negative and positive patients on maintenance HD. The test characteristics of RIBA HCV 3.0 SIA were identical with manual and automated runs. The relative intensity values of antigenic bands by the CHIRON RIBA HCV 3.0 Processor System between anti-HCV positive and negative patients were significantly different; only 15 of 784 (1.9%) antigenic bands had borderline reactivities. The correlation of test results between manual and automated runs was very high (kappa value 0.989). Among positive results by RIBA HCV 3.0 SIA, there was a strong concordance between manual and automated runs with regard to the pattern of reactivity (kappa value 0.943). The discordant results between manual and automated protocols were attributable to increased variability of antigen scores close to the cutoff value for both tests. In conclusion, the CHIRON RIBA HCV 3.0 Processor System is capable of performing RIBA HCV 3.0 SIA in the HD population accurately with minimal operator involvement. The test characteristics of RIBA HCV 3.0 SIA were identical by manual and automated runs. There was a strong correlation between the results of the manual and automated runs; the few discordant results between the two procedures were mostly due to increased variability of antigen scores close to the cutoff value for both tests. The Centers for Disease Control and Prevention in the USA have recently included chronic HD patients among those persons for whom routine HCV testing is recommended; HCV-infected patients on HD often have a high rate of indeterminate results by manual RIBA technology which is operator dependent for band scoring and result interpretation. The CHIRON RIBA HCV 3.0 Processor System may be very useful for supplemental anti-HCV testing of ELISA repeat reactive specimens in clinical practice within dialysis units.

Original contributionHepatitis B surface antigenemia in chronic hemodialysis patients: effect of hepatitis B immunization

Hepatitis B surface antigenemia in chronic hemodialysis patients: effect of hepatitis B immunization

Quantitative assessment of HCV load in chronic dialysis patients: A cross sectional survey

UNLABELLED Recent evidence has been accumulated showing that chronic hemodialysis (HD) patients have a very high prevalence of antibodies to hepatitis C virus (HCV). In contrast, there is little information addressing the virological characteristics of HCV infection in this population. AIM To measure HCV viral load and to correlate this with demographic, biochemical, and clinical features of a large cohort of HCV-infected patients on chronic HD. METHODS 394 chronic HD patients were tested by branched-DNA signal amplification assay, anti-HCV enzyme-linked immunosorbent assay 2.0, and on the basis of the aspartate aminotransferase/alanine aminotransferase (AST/ALT) activity. Multivariate analysis by ordinal logistic regression model was performed: age, gender, race, time on HD, allocation of the patients among the HD units, etiology of end-stage renal disease, HBsAg status, anti-HCV positivity, HCV genotype, and AST/ALT levels were independent factors, and viremic levels of HCV in serum were assumed as dependent variables. RESULTS 88 (22.3%) patients showed serological and/or virological signs of HCV infection. 59 (15%) out of 394 had detectable HCV RNA in serum, the mean HCV load was 19.4 x 10(5) (95% CI, 6.06 x 10(7) to 6.2 x 10(4)) Eq/ml. According to the criteria suggested by others [J Infect Dis 1994;169:1219-1225], there were 8 (13.5%) individuals with high-titer viremia (>1 x 10(7) Eq/ml) in the subset of viremic patients. A small subset (8/394 or 2%) of individuals was seronegative, but viremic; 29 (7%) out of 394 were seropositive without detectable HCV RNA in serum. Univariate analysis showed that the frequency of anti-HCV positivity was significantly higher in viremic patients as compared with individuals with no detectable HCV viremia: 51/59 (86%) vs. 29/335 (8.6%), p = 0.0001. Serum AST and ALT levels were significantly higher in viremic patients than in individuals with no detectable HCV RNA in serum: 23.8 (95% CI 60.8-9.3) vs. 17.1 (95% CI 50.4-5.8) U/l (p = 0.009) and 14.4 (95% CI 48.9-4.3) vs. 9.8 (95% CI, 37.3- 2. 5) U/l (p = 0.008). Logistic regression analysis showed an association between HCV viremia and anti-HCV positivity (p = 0. 00001) and ALT activity (p = 0.01). CONCLUSIONS Hepatitis C virus infection is highly prevalent in the HD population; the viral load is relatively low, and it was associated with elevated hepatic enzyme levels and anti-HCV positivity. No other clinical characteristics were associated with HCV RNA levels. Seronegative but viremic patients were also found. Longitudinal studies with long follow-up periods are necessary to evaluate the course of HCV load over time in this population.

Biological dynamics of viral load in hemodialysis (HD) patients with hepatitis C virus

The biological dynamics of hepatitis C virus (HCV) viremia in uremic patients with chronic infection have not been fully characterized. We prospectively studied fluctuations of HCV-RNA in sera from 52 patients with end-stage renal disease who were undergoing maintenance hemodialysis (HD) and had chronic HCV infection. We measured HCV viremia monthly over the course of 13 months with the branched-chain DNA (bDNA) signal amplification assay and prospectively analyzed liver function, expressed by monthly serum aspartate (AST) and alanine aminotransferase (ALT) determinations. We observed three different patterns of HCV viremia: (1) patients persistently positive by bDNA assay (persistent viremia; 23 of 52 patients; 44%), (2) individuals with alternatively positive and negative results (intermittent viremia; 17 of 52 patients; 33%), and (3) patients persistently negative by bDNA assay (12 of 52 patients; 23%). The HCV viral load over the follow-up was greater among patients with persistent compared with intermittent viremia (persistent, 31.7 x 10(5) Eq/mL; range, 6.3 x 10(5) to 16.03 x 10(6) Eq/mL versus intermittent, 10.4 x 10(5) Eq/mL; range, 1.1 x 10(5) to 9.4 x 10(6) Eq/mL; P = 0.0001). In addition, patients with persistent viremia had over time greater AST and/or ALT activities than the intermittent group (AST: persistent, 26.5 IU/L; range, 9.6 to 73.7 IU/L versus intermittent, 21.3 IU/L; range, 8 to 56.8 IU/L; P = 0.001 and ALT: persistent, 14.7 IU/L; range, 3.7 to 57.9 IU/L versus intermittent, 10.9 IU/L; range, 2.3 to 52.1 IU/L; P = 0.001). In the group with persistent viremia, the mean difference between maximum and minimum values of HCV-RNA observed in each individual patient was 2.09 +/- 0.7 natural logarithm (Log(n)) and in intermittent viremic patients, 1.55 +/- 1 Log(n) (P = 0.045). The HCV load at study entry (19.4 x 10(5) Eq/mL) was rather low and did not change versus the end of follow-up in all patients (P = not significant [NS]). In the entire group, the fluctuations in HCV-RNA levels over time between and within individuals were not significant (P = NS). No difference in variability of HCV-RNA values over time between patients infected with different HCV genotypes was seen. In conclusion, three different patterns of HCV viremia in HD over time were assessed; one third of viremic patients had intermittent viremia, and those patients had less HCV-RNA, enzyme-linked immunosorbent assay, and aminotransferase activity than did patients with persistent HCV load. Larger fluctuations in HCV RNA levels occurred in patients with persistent than with intermittent HCV viremia. However, the viremic HCV load was low and relatively stable over a 13-month follow-up in our population. Studies with longer observation periods are warranted to understand fully the natural history of HCV in these immunosuppressed individuals.