Baseline hepatitis B surface antigen (HBsAg) as predictor of sustained HBsAg loss in chronic hepatitis B patients treated with pegylated interferon-α2a and adefovir.

BACKGROUND: In this study, we aimed to identify baseline predictors of response in chronic hepatitis B patients treated with a combination of pegylated interferon (PEG-IFN)-α2a and adefovir. METHODS: We treated 92 chronic hepatitis B patients (44 hepatitis B e antigen [HBeAg]-positive and 48 HBeAg-negative) with HBV DNA > 100,000 copies/ml (> 17,182 IU/ml) with PEG-IFN and adefovir for 48 weeks and followed them up for 2 years. Baseline markers for HBeAg loss, combined response (HBeAg negativity, HBV DNA levels ≤ 2,000 IU/ml and alanine aminotransferase [ALT] normalization) and hepatitis B surface antigen (HBsAg) loss were evaluated. RESULTS: Two years after the end of treatment, rates of HBeAg loss and HBsAg loss in HBeAg-positive patients were 18/44 (41%) and 5/44 (11%), respectively. In HBeAg-negative patients, rates of combined response and HBsAg loss were 12/48 (25%) and 8/48 (17%), respectively. HBeAg-negative patients with HBsAg loss had lower baseline HBsAg levels than those without HBsAg loss (mean HBsAg 2.35 versus 3.55 log10 IU/ml; P < 0.001). They also had lower HBV DNA levels and were more often (PEG-)IFN experienced. Baseline HBsAg was the only independent predictor of HBsAg loss (OR 0.02; P = 0.01). CONCLUSIONS: With combination therapy of PEG-IFN and adefovir for 48 weeks, a high rate of HBsAg loss was observed in both HBeAg-positive (11%) and HBeAg-negative (17%) patients 2 years after treatment ended. In HBeAg-negative patients, a low baseline HBsAg level was a strong predictor for HBsAg loss.

Detection of hepatitis B virus covalently closed circular DNA in paraffin-embedded and cryo-preserved liver biopsies of chronic hepatitis B patients.

BACKGROUND: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up. AIM: To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue. METHODS: Biopsies of 56 chronic hepatitis B patients were collected. Cryo-preserved and FFPE liver biopsies were available from 37 out of 56 patients. Paraffin was extracted with 1 ml xylene, followed by 100% alcohol and acetone. For the detection of cccDNA, selective primers were used. For quantification of hepatocytes a commercial Taqman beta-actin control kit was used. RESULTS: The cccDNA was detected in 80% of FFPE and in 100% of cryo-preserved liver specimens. Recovery of hepatocytes and cccDNA was approximately a 100-fold lower in FFPE liver tissue, but intrahepatic cccDNA levels were comparable. In FFPE and cryo-preserved liver tissue, intrahepatic cccDNA levels correlated strongly with HBV DNA, hepatitis B e antigen (HbeAg), and plasma cccDNA levels. HbeAg positive chronic hepatitis B patients had significantly higher intrahepatic cccDNA levels compared with HBeAg negative patients (P<0.05). In HBeAg positive patients, no difference in intrahepatic cccDNA levels were seen between patients with active (histological activity index score>3; HBV DNA>20 000 IU/ml) and inactive hepatitis (histological activity index score

Frequent HCV reinfection and superinfection in a cohort of injecting drug users in Amsterdam.

BACKGROUND/AIMS: This study investigates the occurrence of HCV reinfection and superinfection among HCV seroconverters participating in the Amsterdam Cohort Studies among drug users from 1985 through 2005. METHODS: HCV seroconverters (n=59) were tested for HCV RNA at five different time points: the last visit before seroconversion (t=-1), the first visit after seroconversion (t=1), six months after (t=2) and one year after (t=3) seroconversion, and the last visit prior to November 2005 (t=4). If HCV RNA was present, part of the NS5B region was amplified and sequenced. Additional phylogenetic analysis and cloning was performed to establish HCV reinfection and superinfection. RESULTS: Multiple HCV infections were detected in 23/59 (39%) seroconverters; 7 had HCV reinfections, 14 were superinfected, and 2 had reinfection followed by superinfection. At the moment of HCV reinfection, 7/9 seroconverters were HIV-negative: persistent HCV reinfection developed in both HIV-positive cases but also in 4/7 HIV-negative cases. In total, we identified 93 different HCV infections, varying from 1 to 4 infections per seroconverter. Multiple HCV infections were observed in 10/24 seroconverters with spontaneous HCV clearance (11 reinfections, 3 superinfections) and in 13/35 seroconverters without viral clearance (20 superinfections). CONCLUSIONS: HCV reinfection and superinfection are common among actively injecting drug users. This might further complicate the development of an effective HCV vaccine.

Cellular immune responses during high-dose interferon-alpha induction therapy for hepatitis C virus infection.

BACKGROUND: The effect that high-dose interferon (IFN)-alpha induction therapy for hepatitis C virus (HCV) infection has on cellular immune responses is currently unknown. METHODS: Thirty-one treatment-naive patients with chronic HCV infection received amantadine and ribavirin, combined with 6 weeks of high-dose IFN-alpha-2b induction therapy followed by weekly pegylated IFN-alpha-2b, for 24 or 48 weeks. Using IFN-gamma and interleukin (IL)-2 enzyme-linked immunospot (ELISpot) assays, we analyzed the pattern of cytokine secretion by structural and nonstructural HCV- and cytomegalovirus (CMV)-specific T cells before, during, and after therapy. RESULTS: HCV-specific T cell responses, which were predominantly IFN-gamma secreting and which correlated with alanine transaminase levels (r2 = 0.45; P = .001), were found before treatment in 10 of 15 patients with a sustained virological response (SVR) and in 11 of 16 in the non-SVR group. There was a striking loss of IFN-gamma and IL-2 HCV-specific T cells during therapy, predominantly in the SVR group. This response recovered after cessation of therapy, regardless of outcome. Suppression of CMV-specific T cell responses, in addition to total lymphocyte counts, was also observed. CONCLUSIONS: High-dose IFN-alpha induction therapy leads to a profound decline in IL-2- and IFN-gamma-secreting HCV- and CMV-specific T cells. These data indicate that restoration of T cell responses is unlikely to be causally linked to an early response or SVR to therapy.

Prediction of virologic response in difficult-to-treat chronic hepatitis C patients during high-dose interferon induction therapy.

OBJECTIVE: To determine (i) whether early viral kinetics or other markers during a modified treatment regimen are predictors of treatment outcome and (ii) whether fast responders can be treated for 24 weeks, without compromising the sustained virologic response (SVR) rate. MATERIAL AND METHODS: One hundred "difficult-to-treat" chronic hepatitis C patients (46 previous non-responders/relapsers (any genotype), 54 treatment-naive patients genotypes 1 and 4) were treated with triple antiviral induction therapy: amantadine hydrochloride and ribavirin, combined with 6 weeks interferon alfa-2b induction (weeks 1-2: 18 MU/day, weeks 3-4: 9 MU/day, weeks 5-6: 6 MU/day), thereafter combined with weekly peginterferon alfa-2b. Fast responders (>or=3 log(10) HCV RNA decline at week 4) were randomized to 24 or 48 weeks. Slow responders (<3 log(10) HCV RNA decline at week 4) were treated for 48 weeks. Treatment was stopped in patients with detectable HCV RNA at week 24. RESULTS: Thirty-six patients achieved SVR: 28 of 60 fast responders (47%) versus 8 of 32 slow responders (25%, p<0.05). Relapse rates among fast responders treated for 24 or 48 weeks were 27% and 20%, respectively (p=NS). SVR in fast responders was independent of baseline HCV RNA >or= or <600,000 IU/mL. All treatment-naive patients with HCV RNA <5 IU/mL at week 1 or 2 achieved SVR; all treatment-naive patients with HCV RNA >or=5 IU/mL at week 16 became non-SVR. In previous non-responders/relapsers, the predictive value for SVR was 83% if HCV RNA was <5 IU/mL at week 2; all previous non-responders/relapsers with HCV RNA >or=5 IU/mL at week 8 became non-SVR. CONCLUSIONS: With high-dose interferon induction, SVR and non-SVR can be predicted reliably within 16 weeks. Fast responders can be treated for 24 weeks, and SVR is independent of baseline viral load in fast responders.

Severe anemia in Malawian children.

BACKGROUND: Severe anemia is a major cause of sickness and death in African children, yet the causes of anemia in this population have been inadequately studied. METHODS: We conducted a case-control study of 381 preschool children with severe anemia (hemoglobin concentration, <5.0 g per deciliter) and 757 preschool children without severe anemia in urban and rural settings in Malawi. Causal factors previously associated with severe anemia were studied. The data were examined by multivariate analysis and structural equation modeling. RESULTS: Bacteremia (adjusted odds ratio, 5.3; 95% confidence interval [CI], 2.6 to 10.9), malaria (adjusted odds ratio, 2.3; 95% CI, 1.6 to 3.3), hookworm (adjusted odds ratio, 4.8; 95% CI, 2.0 to 11.8), human immunodeficiency virus infection (adjusted odds ratio, 2.0; 95% CI, 1.0 to 3.8), the G6PD(-202/-376) genetic disorder (adjusted odds ratio, 2.4; 95% CI, 1.3 to 4.4), vitamin A deficiency (adjusted odds ratio, 2.8; 95% CI, 1.3 to 5.8), and vitamin B12 deficiency (adjusted odds ratio, 2.2; 95% CI, 1.4 to 3.6) were associated with severe anemia. Folate deficiency, sickle cell disease, and laboratory signs of an abnormal inflammatory response were uncommon. Iron deficiency was not prevalent in case patients (adjusted odds ratio, 0.37; 95% CI, 0.22 to 0.60) and was negatively associated with bacteremia. Malaria was associated with severe anemia in the urban site (with seasonal transmission) but not in the rural site (where malaria was holoendemic). Seventy-six percent of hookworm infections were found in children under 2 years of age. CONCLUSIONS: There are multiple causes of severe anemia in Malawian preschool children, but folate and iron deficiencies are not prominent among them. Even in the presence of malaria parasites, additional or alternative causes of severe anemia should be considered.

Mannose-binding lectin deficiency facilitates abdominal Candida infections in patients with secondary peritonitis.

Mannose-binding lectin (MBL) deficiency due to variations in the MBL gene is associated with increased susceptibility to infections. In this study, the association between MBL deficiency and the occurrence of abdominal yeast infection (AYI) in peritonitis patients was examined. Eighty-eight patients with secondary peritonitis requiring emergency laparotomy were included. MBL genotype (wild type [WT] versus patients with variant genotypes), MBL plasma concentrations, and Candida risk factors were examined in patients with and those without AYI (positive abdominal yeast cultures during [re]laparotomy). A variant MBL genotype was found in 53% of patients with AYI and 38% of those without AYI (P = 0.18). A significantly higher proportion of variant patients had an AYI during early peritonitis (during first laparotomy) than WT patients (39% versus 16%, respectively; P = 0.012). Patients with AYI had lower MBL levels than did patients without AYI (0.16 microg/ml [0.0 to 0.65 microg/ml] versus 0.65 microg/ml (0.19 to 1.95 microg/ml); P = 0.007). Intensity of colonization (odds ratio [OR], 1.1; 95% confidence interval [CI], 1.0 to 1.1), MBL plasma concentrations of <0.5 microg/ml (OR, 4.5; 95% CI, 1.2 to 16.3), and numbers of relaparotomies (OR, 1.7; 95% CI, 1.0 to 2.8) were independently associated with AYI. In summary, deficient MBL plasma levels were independently associated with the development of AYI in patients with secondary peritonitis and seemed to facilitate early infection.

Monocyte-derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is characterized by an insufficient immune response, possibly owing to impaired function of antigen-presenting cells such as myeloid dendritic cells (DCs). Therapeutic vaccination with in vitro generated DCs may enhance the immune response. Subsets of DCs can originate from monocytes, but the presence of HCV in monocytes that develop into DCs in vitro may impair DC function. Therefore, we studied the presence of HCV RNA in monocytes and monocyte-derived DCs from chronic HCV patients. METHODS: Monocytes were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 6 days, and then with GM-CSF, IL-4, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2, IL-1beta and IL-6 for 2 days to generate mature DCs. HCV RNA was assessed by polymerase chain reaction. Surface molecules were assessed by flow cytometry. Cytokine production was assessed by cytokine bead array. RESULTS: HCV RNA was present in monocytes in 11 of 13 patients, but undetectable in mature DCs in 13 of 13 patients. The morphology of patient DCs was comparable with DCs from healthy controls, but the percentage of cells expressing surface molecules CD83 (P=0.001), CD86 (P=0.023) and human leucocyte antigen-DR (P=0.028) was lower in HCV patients. Compared with control DCs, patient DCs produced enhanced levels of IL-10 (P=0.0079) and IL-8 (P=0.0079), and lower levels of TNF-alpha (P=0.032), IL-6 (P=NS) and IL-1beta (P=0.0079). Patient and control DCs did not produce IL-12. CONCLUSIONS: Monocyte-derived DCs from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

Low-level HCV viraemia after initial response during antiviral therapy: transcription-mediated amplification predicts treatment failure.

BACKGROUND: In chronic hepatitis C patients with an initial virological response (IVR) during antiviral therapy (that is, HCV RNA becomes negative before week 16 of treatment) the significance of reappearing viraemia below the detection limit of PCR is not known. We studied this phenomenon in subsets of patients. METHODS: We assessed HCV RNA at weeks 16 and 20 of therapy by PCR and by more sensitive transcription-mediated amplification (TMA) in 23 patients with breakthrough or relapse and in 34 patients with sustained virological response (SVR). All patients participated in a high-dose-interferon induction study for difficult-to-treat patients. Therapy consisted of amantadine hydrochloride and ribavirin, combined with interferon-alpha2b induction during the first 6 weeks and thereafter combined with weekly pegylated interferon-alpha2b. RESULTS: Among the 57 IVR patients, we detected transient or persistent reappearance of low levels of HCV RNA in 10 of the 23 (43%) patients with eventual breakthrough or relapse; but in none of the 34 SVR patients. In 5 of 10 patients reappearing HCV RNA was only detectable by TMA. CONCLUSION: Reappearance of low levels of HCV RNA in patients with IVR predicts treatment failure.

Fourth human parechovirus serotype.

We identified a novel human parechovirus (HPeV) type (K251176-02) from a neonate with fever. Analysis of the complete genome showed K251176-02 to be a new HPeV genotype. Since K251176-02 could not be neutralized with antibodies against known HPeV serotypes 1-3, it should be classified as a fourth HPeV serotype.

Role of viruses in Kenyan children presenting with acute encephalopathy in a malaria-endemic area.

In malaria-endemic areas, it is difficult to differentiate between cerebral malaria (CM), bacterial meningitis, and viral encephalitis. We examined the cerebrospinal fluid of 49 children who fulfilled the World Health Organization's (WHO) definition of CM and in 47 encephalopathic children, without malaria, looking for viruses with polymerase chain reaction. In the children with CM, four (9%) had evidence of Herpes simplex virus 1 in the cerebrospinal fluid, whereas in the encephalopathy group without malaria, six (12%) were positive. A significant proportion of children who fulfil the WHO clinical definition of CM may have viral encephalitis.

Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays.

BACKGROUND: INNO-LiPA HBV Genotyping (LiPA HBV GT) and INNO-LiPA HBV PreCore (LiPA HBV PC) are commercially available assays for hepatitis B virus (HBV) characterization. These assays are labor-intensive and may be prone to exogenous DNA contamination due to their use of nested PCR amplification procedures and lack of contamination control measures. OBJECTIVE: Standardized, single-round INNO-LiPA PCR amplification protocols incorporating uracil N-glycosylase and automated sample processing by the MagNA Pure LC instrument were evaluated. STUDY DESIGN: HBV standards containing 10,000, 1000, 100, 10, and 0 IU/mL were analyzed to determine the analytical sensitivity and reproducibility of these modified procedures. One hundred clinical serum specimens with viral titers ranging from 390 to 16,900,000 IU/mL were tested by modified LiPA HBV GT, while 34 specimens with viral titers ranging from 378 to 11,600,000 IU/mL were tested by modified LiPA HBV PC. RESULTS: Modified LiPA HBV GT and LiPA HBV PC each yielded analytical sensitivities of 100% at an HBV DNA level of 1000 IU/mL and 90% at a level of 100 IU/mL. Among clinical specimens, success rates for both INNO-LiPA procedures were > or =94%. CONCLUSIONS: Both modified INNO-LiPA procedures were sensitive and reproducible, with improved efficiency and suitability for routine laboratory use.

No evidence of in vitro and in vivo porcine endogenous retrovirus infection after plasmapheresis through the AMC-bioartificial liver.

BACKGROUND: Currently a number of bioartificial livers (BAL) based on porcine liver cells have been developed as a treatment to bridge acute liver failure patients to orthotopic liver transplantation or liver regeneration. These xenotransplantation related treatments hold the risk of infection of treated patients by porcine endogenous retrovirus (PERV) released from the porcine cells, as in vitro infection experiments and transplantations in immunocompromised mice have shown that PERV is able to infect human cells. The Academic Medical Center (AMC)-BAL, unlike other BALs, is characterized by direct contact between porcine liver cells and human plasma, and might therefore be permissive for PERV transfer. METHODS: Prior to a clinical phase I trial, human plasma perfused through the AMC-BAL was investigated for PERV DNA and RNA. Moreover productive infectivity was analyzed by exposing the plasma to HEK-293 cells that were subsequently tested for PERV DNA, PERV RNA and reverse transcriptase activity. RESULTS: Although PERV DNA was detected in the perfused plasma, no productive infectivity was detected. Consequently fourteen patients were treated with the AMC-BAL and monitored for PERV transmission. Immediately after treatment the plasma of the patients was positive for PERV DNA, most probably due to porcine liver cell lysis. The PERV DNA was cleared within 2 weeks post-treatment and no PERV RNA was detected. No productive infectivity in human embryonic kidney (HEK)-293 cells exposed to plasma of treated patients was detectable. CONCLUSION: To conclude, no release of infective PERV particles from the AMC-BAL was observed. Therefore we consider the AMC-BAL as safe, however careful surveillance of patients will be continued.

Multicenter evaluation of the VERSANT hepatitis B virus DNA 3.0 assay.

The VERSANT hepatitis B virus (HBV) 3.0 Assay (branched DNA [bDNA]) (referred to herein as VERSANT 3.0) was evaluated at four external sites for analytical sensitivity, specificity, reproducibility, linearity of quantification, and limits of detection. In addition, each of the test evaluation sites provided HBV DNA-positive clinical samples that were previously analyzed by one of three commercially available HBV DNA quantitative tests: Digene Hybrid Capture II HBV DNA Test (Digene); VERSANT HBV DNA 1.0 Assay (bDNA) (VERSANT 1.0); and COBAS AMPLICOR HBV Monitor Test (COBAS AMPLICOR). These samples were reexamined using VERSANT 3.0. The results from these studies showed that VERSANT 3.0 has high specificity (99.3%), excellent reproducibility (between-run coefficient of variation [CV] = 1.6 to 9.4%; within-run CV = 6.5 to 20.7%), and a broad linear range of quantification (2.0 x 10(3) to 1.0 x 10(8) HBV DNA copies/ml) that facilitate the monitoring of HBV DNA levels at diagnosis and throughout the course of treatment. In general, correlation was very good between results obtained from clinical samples analyzed by VERSANT 3.0 and the comparative HBV DNA quantitative assays (VERSANT 1.0, R(2) = 0.900; Digene, R(2) = 0.985; COBAS AMPLICOR, R(2) = 0.771). The greatest differences in comparative quantitation occurred at HBV DNA levels approaching the limits of the dynamic ranges for the comparative assays. The performance characteristics of the new VERSANT 3.0 assay demonstrated that it provides a reliable and robust method for routinely monitoring serum HBV DNA levels in assessing disease activity and determining response to antiviral treatment.