R. Stefan Ross

Lack of evidence for an association between TTV infection and severe liver disease

BACKGROUND In 1997 a new human virus, TTV, was identified. The clinical significance of the TTV infection, however, remains unknown. OBJECTIVE Establishment of the prevalence of TTV DNA in different population groups in Germany and the assessment of the possible clinical significance of TTV infection. STUDY DESIGN Detection of the TTV DNA by PCR in blood donors, patients with end-stage liver disease, and multiple transfused patients with haemotological disorders. RESULTS TTV DNA was detected in 16 of 122 (13.1%) volunteer blood donors, in 34 of 77 (44.2%) patients with end-stage liver disease, and in 21 of 38 (55.3%) multiple transfused patients. There was no difference in the prevalence of the TTV DNA in end-stage liver disease patients with regard to sex, age, presence of HCV and HBV infection markers, and etiology of liver disease. Phylogenetic analysis of the amplified DNA fragments from 12 randomly selected TTV infected subjects demonstrated that in Germany at least two putative TTV genotypes and four subtypes are circulating. CONCLUSIONS (i) TTV is widely spread in German population; (ii) one of the possible ways of its transmission is blood transfusion; (iii) TTV infection most probably does not generally lead to the development of the end-stage liver disease.

Immunodominant B-cell domains of hepatitis C virus envelope proteins E1 and E2 identified during early and late time points of infection

BACKGROUND/AIMS We characterized immunoreactive B-cell domains of hepatitis C virus (HCV) envelope proteins E1 and E2 by a peptide ELISA using sera of patients who were infected by the same isolate of HCV (HCV-AD78). METHODS Fifty-four overlapping peptides which corresponded to the sequence of E1 and E2 of isolate HCV-AD78 were used to detect specific antibodies. Three groups of HCV-AD78 related sera were analyzed. Two groups were from sera obtained at early time points of infection (months 4-15) from patients who later resolved infection (group A), or who later developed chronic disease (group B). Group C sera were from later time points of chronic disease. As a control, sera of chronic HCV patients who did not have HCV-AD78 infection were also analyzed (group D). RESULTS In group A, 25 of the 54 peptides produced OD405 above the cut-off, whereas 17 peptides produced such values in group B. Only 10 and 3 peptides yielded such values in groups C and D, respectively. The overall prevalence of antibodies against peptides was high in the early phase of infection (means of 28.7+/-14.8% and 25.9+/-14.5% in groups A and B, respectively). At later time points of chronic infection (group C), the overall prevalence was lower (mean 18.6+/-15.4%). Group D sera produced the lowest overall prevalence (mean 13.2+/-14.1%). Three peptides, covering aa271-290, aa481-500 and aa551-570, were recognized significantly more frequently (p<0.05) by group A sera than group B sera. CONCLUSIONS We conclude that more linear epitopes of the HCV envelope are recognized with a high prevalence of antibodies, as was suggested previously. However, most B-cell domains of the HCV envelope induce a similarly high antibody response in patients who resolve infection or develop chronic disease.

Detection of infections with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus by analyses of dried blood spots - performance characteristics of the ARCHITECT system and two commercial assays for nucleic acid amplification

BackgroundNowadays, dried blood spots (DBS) are primarily used to obtain diagnostic access to risk collectives such as intravenous drug users, who are prone to infections with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Before DBS analyses can be used in this diagnostic context, however, a comprehensive evaluation of its performance characteristics must be conducted. To the best of our knowledge, the current study presents for the first time such essential data for the Abbott ARCHITECT system, which is currently the worldwide leading platform in this field of infection diagnostics.MethodsThe investigation comprised 1,762 paired serum/DBS samples and a total of 3,524 determinations with the Abbott ARCHITECT HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24-antigen/anti-HIV 1/2 assays as well as with the artus HBV LC PCR and VERSANT HCV RNA qualitative (TMA) tests.ResultsIn the context of DBS testing, a specificity of 100% was recorded for the seven serological and molecular biological assays. The analytical sensitivity of HBsAg, anti-HBc, anti-HBs, anti-HCV, HIV-1-p24-antigen/anti-HIV 1/2, HBV DNA, and HCV RNA detections in DBS eluates was 98.6%, 97.1%, 97.5%, 97.8%, 100%, 93%, and 100%, respectively.Discussion/conclusionsThe results obtained indicate that it is today possible to reliably detect HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24 antigen/anti-HIV 1/2 with state-of-the-art analytical systems such as the Abbott ARCHITECT in DBS eluates even when a comparatively high elution volume of 1,000 μl is used. They also provide evidence for the inherent analytical limits of DBS testing, which primarily concern the anti-HBc/anti-HBs system for individuals with HIV infections and nucleic acid tests with relatively low analytical sensitivity.

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

Comparative evaluation of the immunogenicity of combined hepatitis A and B vaccine by a prospective and retrospective trial

In the past, immunogenicity of hepatitis A and B vaccines needed to be questioned in persons of advanced age, especially in those of 40 years and older. We performed a comparative multicenter prospective and retrospective study with the combined hepatitis A and B vaccine Twinrix® to identify factors influencing the results of the vaccination in a population of all age groups. Out of 489 subjects enrolled, 241 were vaccinated in a prospective study (group 1) and 248 subjects in a retrospective study (group 2) in 17 German centers with median age of 40.1 (14-79) years. Following three applications of the combined hepatitis A/B vaccine we found 96.2% with protective antibodies against HAV and 88.7% were protected against HBV. With increasing age the subjects developed decreasing anti-HBs antibody levels whereas the seroprotection rate was significantly reduced by age (p

Performance characteristics of the VERSANT hepatitis C virus RNA 1.0 (kPCR) assay.

HCV RNA assays are of central importance for virological diagnostics and for clinical planning and monitoring of an antiviral combination treatment of chronic HCV infections. The objective of the pre-market evaluation of the VERSANT HCV RNA 1.0 Assay (kPCR) was to collect analytical performance data for this new method of HCV RNA quantification and to compare them with the high standards that exist in this context. The assay exhibited a specificity of 100%. The mean intra- and inter-assay imprecision was 14.1% and 14.6%, respectively. The detection limit was determined to be 16IU/ml (95% confidence interval: 11.9-30.6IU/ml) and consequently corresponded to the manufacturers claims (i.e. 15IU/ml). The test exhibited linearity for all HCV genotypes in a broad range from 15 to 10(8)IU HCV RNA/ml. Hence, the kPCR assay in general is well suitable for HCV RNA determinations in clinical practice. However, in a methodological comparison, a considerable under-quantification of the concentrations of HCV genotype 2 and 3 isolates was detected. Provided that the assays manufacturer will quickly remedy this shortcoming, the VERSANT HCV RNA 1.0 (kPCR) can be called a completely reliable technique for HCV RNA quantification in routine virological diagnostics.

Natural prevalence of resistance-associated variants in hepatitis C virus NS5A in genotype 3a-infected people who inject drugs in Germany

BACKGROUND People who inject drugs (PWID) are the most important risk group for incident Hepatitis C virus (HCV) infection. In PWID in Europe HCV genotype 3a is highly prevalent. Unfortunately, many of the recently developed directly acting antiviral drugs against HCV (DAAs) are suboptimal for treatment of this genotype. Detection of resistance-associated variants (RAV) in genotype 3a may help to optimize treatment decisions, however, robust protocols for amplification and sequencing of HCV NS5A as an important target for treatment of genotype 3a are currently lacking. OBJECTIVES The aim of this study was to establish a protocol for sequencing of HCV NS5A in genotype 3a and to determine the frequency of RAVs in treatment-naïve PWID living in Germany. STUDY DESIGN The full NS5A region was amplified and sequenced from 110 HCV genotype 3a infected PWID using an in-house PCR protocol. RESULTS With the established protocol the complete NS5A region was successfully amplified and sequenced from 110 out of 112 (98.2%) genotype 3a infected PWID. Phylogenetic analysis of sequences from PWID together with unrelated genotype 3a sequences from a public database showed a scattered distribution without geographic clustering. Viral polymorphisms A30K and Y93H known to confer resistance in a GT3a replication model were present in 8 subjects (7.2%). CONCLUSIONS A protocol for amplification of nearly all GT3a samples was successfully established. Substitutions conferring resistance to NS5A inhibitors were detected in a few treatment-naive PWID.

Transmission of Hepatitis C Virus

To the Editor: With regard to our article on the transmission of hepatitis C virus from a patient to an anesthesiology assistant and subsequently to five other patients (Dec. 21 issue),1 we wish to...

Concordance between self-reported and measured HIV and hepatitis C virus infection status among people who inject drugs in Germany

BackgroundPeople who inject drugs (PWID) are disproportionately affected by both HIV and hepatitis C infection (HCV). Awareness of infection status is essential to ensure linkage to appropriate healthcare for those infected, who need treatment and regular follow-up, as well as for uninfected individuals, who need access to targeted testing and counselling services. In this paper we compare self-reported HIV and HCV status with serological markers of infection among PWID recruited through respondent driven sampling.MethodsFrom 2011 through 2014, biological and behavioural data was collected from 2,077 PWID in Germany. Dried blood spots from capillary blood samples were collected and screened for HCV antibodies, HCV RNA and HIV-1/-2 antibodies. HIV reactive samples were confirmed by Western blot.ResultsLaboratory testing revealed that 5 % were infected with HIV and 81 % were aware of being infected. Chronic HCV infection was detected in 41 % of the participants, 2 % had an acute HCV infection, 22 % had a cleared infection, and 34 % were unexposed to HCV. The concordance between self-reported and measured HCV status was lower than for HIV, with 73 % of those with chronic HCV infection being aware of their infection.ConclusionsWe found a relatively high awareness of HIV and HCV infection status among PWID. Nevertheless, access to appropriate testing, counselling and care services targeted to the needs of PWID should be further improved, particularly concerning HCV.Trial registrationEthical approval was received from the ethics committee at the medical university of Charité, Berlin, Germany in May 2011 and with an amendment approved retrospectively on 19/11/2012 (No EA4/036/11). The German Federal Commissioner for Data Protection and Freedom of Information approved the study protocol retrospectively on 29/11/2012 (III-401/008#0035).

Epidemiology of hepatitis A, B and C among adults in Germany

Ten years after seroepidemiological data were obtained in the German National Health Interview and Examination Survey 1998 (GNHIES98), German Health Interview and Examination Survey (DEGS1) data contribute to a population-based, representative surveillance of hepatitis A and B immunity and of the serological markers for hepatitis C in Germany. The prevalence of antibodies against the hepatitis A virus is 48.6 %. In comparison to the situation 10 years ago, seroprevalence is significantly higher among 18- to 39-year-old adults and is significantly lower in those aged 50-79 years. The association between age and seroprevalence has changed, indicating a decrease in naturally acquired hepatitis A immunity. Individual and population immunity has to be achieved through vaccination. Prevalence of hepatitis B antibodies indicates that 5.1 % of adults have been exposed to the virus, significantly fewer than 10 years ago (7.9 %). Prevalence of hepatitis B surface antibodies indicates that 22.9 % of adults have been vaccinated against hepatitis B. Vaccination coverage has increased in all age groups and is highest in the younger age groups. These positive trends can be attributed to the general recommendation since 1995 to vaccinate against hepatitis B. For hepatitis C, the prevalence of antibodies in the general population is 0.3 %. Germany thus remains a low-HCV-endemic country. An English full-text version of this article is available at SpringerLink as supplemental.