Annemarie Berger

Serum microRNA-122 levels in different groups of patients with chronic hepatitis B virus infection

Summary.  miR‐122 is a liver‐specific microRNA, which also circulates in the blood. The levels of miR‐122 in serum and plasma correlate with hepatic necroinflammation in patients with hepatitis B virus (HBV) infection. Here, we investigated whether miR‐122 levels correlate with surrogate markers for viral replication and translation. Furthermore, we examined whether miR‐122 levels differ in the different groups of HBV‐infected patients and whether miR‐122 levels may be useful to identify patients with higher or lower risk for liver disease progression. Therefore, RNA was extracted from sera of therapy‐naïve patients with HBV infection (n = 89) and from healthy volunteers (n = 19). The concentration of miR‐122 was assessed by quantitative real‐time reverse‐transcription PCR. HBs antigen and HBV DNA levels were quantified as surrogate parameters for HBV replication and translation. Liver biopsies were examined according to the histological activity index and the degree of fibrosis was assessed. We found that the miR‐122 serum concentration correlated with the level of ALT, HBV DNA and HBs antigen (r = 0.259, P < 0.05; r = 0.225, P < 0.05; r = 0.508, P < 0.001, respectively). The miR‐122 serum levels discriminated the different patient groups infected with HBV from healthy subjects (P < 0.001), and inactive carrier patients with high (>3500 IU/mL) or low (<3500 IU/mL) levels of HBs antigen could be differentiated by the miR‐122 serum concentration (P < 0.05). As serum miR‐122 levels strongly correlated with HBs antigen, it might be an indicator for viral translation. Furthermore, serum miR‐122 levels discriminated HBV carrier patients with high or low risk for disease progression and may, thus, be an additional marker for risk stratification.

Multicenter Evaluation of a New Automated Fourth-Generation Human Immunodeficiency Virus Screening Assay with a Sensitive Antigen Detection Module and High Specificity

ABSTRACT Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibody that were available on the international market until now have antigen detection modules with relatively poor sensitivity and produce a higher rate of false-positive results than third-generation enzyme immunoassays (EIAs). The new Cobas Core HIV Combi EIA with an improved sensitivity for HIV p24 antigen was compared to alternative fourth- and third-generation assays, the p24 antigen test, and HIV type 1 (HIV-1) RNA reverse transcriptase PCR (RT-PCR). A total of 94 seroconversion panels (n = 709 sera), samples from the acute phase of infection after seroconversion (n = 32), anti-HIV-1-positive specimens (n = 730) from patients in different stages of the disease, 462 subtyped samples from different geographical locations, anti-HIV-2-positive sera (n = 302), dilutions of cell culture supernatants (n = 62) from cells infected with different HIV-1 subtypes, selected performance panels from Boston Biomedica Inc., 7,579 unselected samples from blood donors, 303 unselected daily routine samples, 997 specimens from hospitalized patients, and potentially interfering samples (n = 1,222) were tested with Cobas Core HIV Combi EIA. The new assay showed a sensitivity comparable to that of the Abbott HIV-1 AG Monoclonal A for early detection of HIV infection in seroconversion panels. The mean time delay of Cobas Core HIV Combi EIA (last negative sample plus 1 day) in comparison to that for HIV-1 RT-PCR for 87 panels tested with both methods was 2.75 days. The diagnostic window was reduced with Cobas Core HIV Combi EIA by between 3.6 and 5.7 days from that for third-generation assays. The specificities of Cobas Core HIV Combi EIA in blood donors were 99.84 and 99.85% (after repeated testing). Overall, 30 repeatedly reactive false-positive results out of 10,031 HIV-negative samples were obtained with Cobas Core HIV Combi EIA. Our results show that a fourth-generation assay with improved specificity such as Cobas Core HIV Combi EIA is suitable for blood donor screening because of its low number of false positives and because it detects HIV p24 antigen with a sensitivity comparable to that of single-antigen assays.

Slower Progression of HIV-1 Infection in Persons with GB Virus C Co-Infection Correlates with an Intact T-Helper 1 Cytokine Profile

Context Patients infected with HIV-1 progress to AIDS more slowly if they are co-infected with hepatitis G virus, also called GB virus C (GBV-C), than if they are not. The mechanism of this effect of GBV-C infection is not known. Contribution Among 80 asymptomatic HIV-1infected patients, T-helper 1 cytokine profiles changed unfavorably in those without GBV-C infection and remained stable in those with GBV-C infection. Implications Co-infection with GBV-C may slow progression of HIV-1 infection through a mechanism related to T-helper 1 cytokines. The Editors Hepatitis G virus, also called GB virus C (GBV-C), is a recently identified RNA virus belonging to the Flaviviridae family (1, 2). It is usually transmitted parenterally (2, 3). The prevalence of GBV-C viremia ranges from 20% to 24% among persons who use intravenous drugs (4, 5). Higher rates have been seen in patients with HIV-1 infection, regardless of intravenous drug use (4, 6). In recent surveys, HIV-1infected persons with GBV-C co-infection had better AIDS-free survival rates and higher CD4+ cell counts than HIV-1infected patients who were GBV-C negative (7-10). It has also been shown that GBV-C inhibits HIV-1 replication in vitro (11). Our objective was to evaluate AIDS-free survival rates, plasma HIV-1 viral load, and selected immunologic variables in 80 HIV-1seropositive patients with and without GBV-C co-infection. We sought to determine possible immunologic mechanisms involved in these co-infection scenarios. Methods The study was initiated between January and March 1989 at the Institute of Infectious Diseases, University of Catania, Catania, Italy. Institutional review boards at the Institute of Infectious Diseases, University of Catania, approved the follow-up protocol. Signed informed consent was obtained from each patient. Among 319 HIV-1seropositive patients, 240 used intravenous drugs, 60 were homosexual men, and 19 had received many blood transfusions. Eighty of these 319 patients (25%), all of whom used intravenous drugs, were asymptomatic and were enrolled in a prospective follow-up study to evaluate the progression of HIV-1related disease. All 80 patients underwent physical examination and routine blood biochemistry examinations every 6 months. Blood samples were obtained annually from each patient, and serum was stored at 80 C until use. The analyses for the current study were begun in January 1997. Quantitative plasma HIV-1 RNA levels and circulating levels of specific interleukins (ILs)IL-2, IL-4, IL-10, and IL-12were retrospectively determined in all serum samples. We determined GBV-C RNA levels in all serum specimens collected at the beginning of the study and at the end of follow-up. Analyses were repeated in January 2001. Anti-E2 antibodies were detected by using the Enzymun-Test Anti-HGenv (Boehringer Mannheim Corp., Indianapolis, Indiana). Plasma HIV-1 RNA copy numbers were determined by using the nucleic acid sequence-based amplification method (NASBA, Organon Teknika, Boxtel, the Netherlands). The sensitivity limit of the assay was 400 copies/mL. Interleukin-2 was analyzed by using a quantitative enzyme immunoassay (Predicta, Genzyme Diagnostics, Cambridge, Massachusetts) with a sensitivity limit of 4 pg/mL. Interleukin-4 was tested by using a quantitative enzyme immunoassay (InterTest, Genzyme Diagnostics) with a sensitivity limit of 0.045 ng/mL. Interleukin-10 was measured by using a competitive enzyme immunoassay (Cytokit Red 10, Genzyme Diagnostics), which had a range of detection between 0.195 and 200 ng/mL. Interleukin-12 levels were determined by using an enzyme immunoassay provided by R&D Systems (Oxon, United Kingdom) that had a lower sensitivity limit of 5 pg/mL. We measured GBV-C RNA level by using reverse transcriptase polymerase chain reaction, as described elsewhere (12). Statistical Analysis Plasma HIV-1 RNA levels were logarithmically transformed to normalize their distribution. Categorical variables were analyzed by using the Fisher exact test. The group means were compared by using the Student t-test. Variations of all interim values of plasma HIV-1 RNA level, CD4+ cell count, and IL levels were analyzed within each group by using two-way analysis of variance. We compared GBV-C RNApositive and GBV-C RNAnegative groups by using the MannWhitney U test to examine percentage variations from baseline values to values at the end of follow-up. Curves reflecting variations by time in immunologic and virologic variables were compared by using univariate repeated-measures analysis that followed an analysis-of-variance structure (13). Progression to AIDS was defined as the development of an opportunistic infection or malignant condition. We used the Kaplan-Meier method to evaluate the effect of GBV-C infection on AIDS-free survival by assuming that GBV-C and HIV-1 infection status was fixed at the beginning of follow-up. A P value less than 0.05 was considered statistically significant. Statistical analyses were performed by using SAS software, version 6.12 (SAS Institute, Inc., Cary, North Carolina). Role of the Funding Sources The funding sources had no direct control over the analysis of the study. Results The mean age of the study patients (SD) was 24.6 2.2 years. Fifty-two patients were men, and 28 were women. Mean duration of intravenous drug use (SD) was 24.2 1.8 months, and mean duration of known HIV-1 seropositivity (SD) was 9.2 1.6 months. The mean baseline CD4+ cell count (SD) was 471 55 109 cells/L. Fifty-eight patients declined to take any antiretroviral drug, and 6 were treated with zidovudine alone throughout the follow-up period. In 16 patients, didanosine was added to zidovudine, starting in 1993. In January 1997, follow-up was interrupted and all 80 patients began to receive different highly active antiretroviral therapy (HAART). At this time, 6 patients were asymptomatic, 24 had stage B disease, and 50 had stage C disease, according to stages defined by the U.S. Centers for Disease Control and Prevention. At the end of follow-up, the mean CD4+ cell count (SD) was 77 33 109 cells/L. Seventeen of the 80 serum specimens collected at the beginning of follow-up (21%) were positive for GBV-C RNA. All of these 17 patients maintained GBV-C viremia to the end of the follow-up period, and none of the 63 patients who were GBV-C RNA negative acquired the infection. Patients who were GBV-C negative and those who were GBV-C positive did not significantly differ in age, sex, duration of intravenous drug use and HIV-1 seropositivity, or rate of hepatitis C virus and hepatitis B virus infection (Table). Table. Epidemiologic and Virologic Variables Measured at Baseline and at the End of Follow-up Clinical Outcomes At the end of the 8-year follow-up, 3 of 17 GBV-Cpositive patients (18%) remained asymptomatic (stage A), 7 (41%) had stage B disease, and 7 (41%) had stage C disease. In the 7 patients with stage C disease, the following AIDS-defining diseases were observed: AIDS dementia complex (n = 7 [100%]), Pneumocystis carinii pneumonia (n = 4 [57%]), esophageal candidiasis (n = 3 [43%]), and Toxoplasma encephalitis infection (n = 1 [14%]). Three of 63 GBV-C RNAnegative patients (5%) had stage A disease, 17 (27%) had stage B disease, and 43 (68%) had stage C disease. Among those with stage C disease, the following diseases were observed: P. carinii pneumonia (n = 18 [42%]), esophageal candidiasis (n = 11 [26%]), Toxoplasma encephalitis infection (n = 5 [12%]), cryptococcal meningitis (n = 4 [9%]), intestinal cryptosporidiosis (n = 3 [7%]), Kaposi sarcoma (n = 2 [5%]), AIDS dementia complex (n = 2 [5%]), and disseminated cytomegalovirus disease (n = 1 [2%]). According to Kaplan-Meier curves showing progression to AIDS in HIV-1infected persons, cumulative AIDS-free survival rates at 24 and 48 months, respectively, were 0.5 (95% CI, 0.3 to 0.6) and 0.4 (CI, 0.3 to 0.5) in the GBV-Cnegative group and 0.9 (CI, 0.7 to 1.0) and 0.7 (CI, 0.5 to 0.9) in the GBV-Cpositive group (P = 0.02 for between-group comparisons). Mean AIDS-free survival time was 73 months (CI, 59 to 87 months) among GBV-Cpositive persons and 45 months (CI, 35 to 54 months) among GBV-Cnegative persons. Immunologic and Virologic Evaluations The Figure shows the cross-sectional averages of IL levels, CD4+ cell counts, and HIV RNA levels during the follow-up period. Annual plasma HIV-1 RNA levels significantly increased during follow-up in both the GBV-Cnegative and GBV-Cpositive groups, whereas CD4+ cell counts significantly decreased (P < 0.01 for all comparisons). In the GBV-Cnegative group, IL-4 and IL-10 levels increased significantly from baseline (P = 0.01 and P = 0.004, respectively) but IL-2 and IL-12 concentrations decreased significantly throughout the entire follow-up period (P = 0.005 and P = 0.01, respectively). In contrast, in the GBV-Cpositive group, none of the measured cytokine levels changed significantly during follow-up. The Table shows the percentage variation from baseline to the end of follow-up in plasma HIV-1 RNA levels, CD4+ cell counts, and cytokine concentrations between the two groups. Figure. Cross-sectional averages of interleukin ( IL ) levels, CD4+ cell counts, and HIV-1 RNA levels in GB virus C ( GBV-C )-positive and GBV-Cnegative patients during 8 years of follow-up. P P Response to HAART In January 1997, all 80 patients began taking HAART. Among the 17 GBV-Cpositive patients, 10 received zidovudine, lamivudine, and saquinavir and 7 received zidovudine, lamivudine, and indinavir. Among the 63 GBV-Cnegative patients, 20 were treated with zidovudine, lamivudine, and saquinavir; 20 were treated with zidovudine, lamivudine, and indinavir; 13 were treated with zidovudine, didanosine, and indinavir; and 10 were treated with zidovudine, lamivudine, and ritonavir. Fourteen patients, 2 in the GBV-Cpositive group and 12 in the GBV-Cnegative group, stopped taking HAART between 1997 and 2001 because of personal preference, lack of adh

Severe acute respiratory syndrome (SARS)—paradigm of an emerging viral infection

Abstract An acute and often severe respiratory illness emerged in southern China in late 2002 and rapidly spread to different areas of the Far East as well as several countries around the globe. When the outbreak of this apparently novel infectious disease termed severe acute respiratory syndrome (SARS) came to an end in July 2003, it had caused over 8000 probable cases worldwide and more than 700 deaths. Starting in March 2003, the World Health Organization (WHO) organised an unprecedented international effort by leading laboratories working together to find the causative agent. Little more than one week later, three research groups from this WHO-coordinated network simultaneously found evidence of a hitherto unknown coronavirus in SARS patients, using different approaches. After Koch’s postulates had been fulfilled, WHO officially declared on 16 April 2003 that this virus never before seen in humans is the cause of SARS. Ever since, progress around SARS-associated coronavirus (SARS-CoV) has been swift. Within weeks of the first isolate being obtained, its complete genome was sequenced. Diagnostic tests based on the detection of SARS-CoV RNA were developed and made available freely and widely; nevertheless the SARS case definition still remains based on clinical and epidemiological criteria. The agent’s environmental stability, methods suitable for inactivation and disinfection, and potential antiviral compounds have been studied, and development of vaccines and immunotherapeutics is ongoing. Despite its grave consequences in humanitarian, political and economic terms, SARS may serve as an example of how much can be achieved through a well-coordinated international approach, combining the latest technological advances of molecular virology with more “traditional” techniques carried out to an excellent standard.

Low vitamin D serum concentration is associated with high levels of hepatitis B virus replication in chronically infected patients

Vitamin D is an important immune modulator that plays an emerging role in inflammatory and metabolic liver diseases, including infection with hepatitis C virus (HCV). In contrast, the relationship between vitamin D metabolism and chronic hepatitis B (CHB) is less well characterized. Therefore, we quantified 25(OH)D3 serum levels in a cohort of 203 treatment‐naïve patients with chronic hepatitis B virus (HBV) infection and tested for their association with clinical parameters of CHB. Of 203 patients, 69 (34%), 95 (47%), and 39 (19%) had severe vitamin D deficiency (25(OH)D3 <10 ng/mL), vitamin D insufficiency (25(OH)D3 ≥10 and <20 ng/mL), or adequate vitamin D serum levels (25(OH)D3 ≥20 ng/mL), respectively. In both uni‐ and multivariate analyses, HBV DNA viral load (log10 IU/mL) was a strong predictor of low 25(OH)D3 serum levels (P = 0.0007 and P = 0.000048, respectively) and vice versa. Mean 25(OH)D3 serum concentrations in patients with HBV DNA <2,000 versus ≥2,000 IU/mL were 17 versus 11 ng/mL, respectively (P < 0.00001). In addition, hepatitis B early antigen (HBeAg)‐positive patients had lower 25(OH)D3 serum levels than HBeAg‐negative patients (P = 0.0013). Finally, 25(OH)D3 and HBV DNA serum levels showed inverse seasonal fluctuations. Conclusion: Low 25(OH)D3 serum levels are associated with high levels of HBV replication in patients with CHB. This represents a major difference from chronic hepatitis C, where numerous previous studies have shown a lack of correlation between HCV viral load and vitamin D serum levels. Inverse seasonal fluctuations of 25(OH)D3 and HBV DNA serum levels are suggestive of a functional relationship between both variables. (Hepatology 2013;58:1270–1276)

First transmission of human immunodeficiency virus Type 1 by a cellular blood product after mandatory nucleic acid screening in Germany

BACKGROUND: In February 2007, a 63‐year‐old man underwent surgery. Retrospective testing with nucleic acid testing (NAT) showed that the patient was human immunodeficiency virus Type 1 (HIV‐1) positive 10 days after transfusion. The transfusion‐transmitted infection had been identified by a donor‐related lookback started in April 2007 after anti‐HIV seroconversion.

High Frequency of HCV Infection in Individuals with Isolated Antibody to Hepatitis B Core Antigen

Although isolated antibody to hepatitis B core antigen (anti-HBc) is frequently nonspecific or may be the only serological marker of past self-limiting hepatitis B, where antibodies against the surface antigen have disappeared, isolated anti-HBc seropositivity is frequently associated with chronic hepatitis B in HIV- and HCV-infected individuals. Of 5,520 samples that tested positive for anti-HBc (IMx and AxSYM CORE, Abbott, Delkenheim, Germany) at the Institute of Virology, University Clinic Frankfurt during the time interval from January 1994 to February 1996, 643 (11.6%) were isolated anti-HBc-reactive in the IMx and AxSYM CORE assays (inhibition values >90%). There was a statistically significant association between isolated anti-HBc seropositivity and HCV and HIV/HCV coinfection (p < 0.05). A total of 190 samples were available for further testing. Six (3.2%) of 190 isolated anti-HBc-positive samples were considered false-positive since they were only positive in the AxSYM or IMx CORE assay and a linear decrease of the measured signal could not be observed in dilution series. Of 184 serum samples tested with nested PCR using primers of the S genome region, only 6 (3.3%) were HBV DNA-positive. Anti-HBc-IgM antibody could be detected in 3 (1.6 %) of the tested samples using the IMx CORE-M. With the more sensitive VIDAS HBc IgM specific IgM antibody was detected in 15 (8.5%) of 177 samples at concentrations ranging from 10 to >200 Paul Ehrlich Institute U/ml. HIV or HCV coinfection was present in 28.1% and 37.5% of isolated anti-HBc-positive individuals, respectively. We conclude from our observations that only a limited proportion of anti-HBc-isolated individuals are potentially infectious, however anti-HBc-IgM which is detectable in any form of liver disease associated with HBV infection was present in more than 8% of the individuals. Of isolated anti-HBc-positive sera 37% were positive for anti-HCV, suggesting that anti-HCV antibody testing should be performed in isolated anti-HBc-positive individuals.

Evaluation of Two New Automated Assays for Hepatitis B Virus Surface Antigen (HBsAg) Detection: IMMULITE HBsAg and IMMULITE 2000 HBsAg

ABSTRACT In recent years the diagnostic industry has developed new automated immunoassays for the qualitative detection of hepatitis B virus (HBV) surface antigen (HBsAg) in serum and plasma samples that are performed on analyzers that permit a high-speed throughput, random access, and primary tube sampling. The aim of the present study was the evaluation of two new automated HBsAg screening assays, IMMULITE HBsAg and IMMULITE 2000 HBsAg, from Diagnostic Products Corporation. The new HBsAg assays were compared to well-established tests (Auszyme Monoclonal [overnight incubation, version B], IMx HBsAg, AxSYM HBsAg, and Prism HBsAg [all from Abbott] and Elecsys HBsAg [Roche Diagnostics]). In the evaluation were included seroconversion panels, sera from the acute and chronic phases of infection, dilution series of various HBsAg standards, HBV subtypes and S gene mutants. To challenge the specificity of the new assays, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. IMMULITE HBsAg and IMMULITE 2000 HBsAg, although not as sensitive as the Elecsys HBsAg assay, were equivalent to the AxSYM HBsAg assay and showed a higher sensitivity than the Auszyme Monoclonal B and IMx HBsAg systems for detection of acute infection in seroconversion panels. The specificities (100%) of both IMMULITE assays on unselected blood donors and potentially interfering samples were comparable to those of the alternative assays after repeated testing. In conclusion, the new IMMULITE HBsAg and IMMULITE 2000 HBsAg assays show a good sensitivity for HBsAg detection compared to other well-established tests. The specificity on repeatedly tested samples was equivalent to that of the alternative assays. The rapid turnaround time, primary tube sampling, and on-board dilution make it an interesting assay system for clinical laboratory diagnosis.

Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-Bound Fusion Inhibitor Following HIV-1 Infection

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR

BACKGROUND Molecular detection has been shown to be superior to tissue culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. OBJECTIVES In this study, a qualitative molecular assay based on automated RNA extraction with the MagNA Pure LC and real-time PCR on the LightCycler (LC) instrument was evaluated and compared with an in-house molecular assay. STUDY DESIGN A total of 109 CSF specimens were investigated for the comparative study. The detection limit of the new molecular assay was determined with 10-fold dilutions of two enterovirus strains and with the Third European Union Concerted Action Enterovirus Proficiency Panel. RESULTS With the enterovirus strains, the detection limit of the LC assay was found to be 0.1 TCID(50) (50% tissue culture infective dose). When samples of the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, both molecular assays gave identical results to the expected results, which were based upon the results of three reference laboratories using a total of four different molecular methods before distribution of the panel. When clinical specimens were tested, there was a correlation between the LC assay and the in-house assay in 105 of 109 cerebrospinal fluids. CONCLUSIONS The new molecular assay allows rapid detection of enterovirus RNA in CSF. It was found to be labor saving and showed sufficient sensitivity.