Wolfgang Jilg

Serological pattern "anti-HBc alone": report on a workshop.

In areas with low hepatitis B virus (HBV) endemicity such as most parts of Europe and the United States “anti‐HBc alone” is found in 10–20% of all individuals with HBV markers, i.e., 1–4% of the population. In about 10% of these individuals HBV DNA is detected by PCR, the proportions varying greatly depending on the population studied, being highest in individuals coinfected with hepatitis C virus (HCV) (above 35%) and HIV (above 85%). A small proportion of individuals with “anti‐HBc alone” are in the window phase of an HBV infection or in a stage of late HBV immunity. For the large proportion of these individuals this is not the case and they are thought to have an unresolved HBV‐infection or a chronic infection in a late or “low grade” productive state. Currently, limited studies have been performed concerning the clinical aspects of individuals with “anti‐HBc alone” and suspected chronic HBV infection. The majority of these individuals seem to be healthy. Some chronic carriers with “anti‐HBc alone,” however, do present signs of chronic hepatitis. Individuals with “anti‐HBc alone” are potentially infectious. This is exemplified by a few case reports of HBV transmission to sexual contacts, perinatal transmission between mother and newborns and in blood recipients. Recommendations are given in relation to both the diagnostic and therapeutic procedures in the individuals with “anti‐HBc alone” and in the blood banking and transplantation services. J. Med. Virol. 62:450–455, 2000.

High genetic variability of the group-specific a-determinant of hepatitis B virus surface antigen (HBsAg) and the corresponding fragment of the viral polymerase in chronic virus carriers lacking detectable HBsAg in serum

Chronic carriers of hepatitis B virus (HBV) usually show hepatitis B surface antigen (HBsAg) in their sera, which is considered the best marker for acute and chronic HBV infection. In some individuals, however, this antigen cannot be detected by routine serological assays despite the presence of virus in liver and peripheral blood. One reason for this lack of HBsAg might be mutations in the part of the molecule recognized by specific antibodies. To test this hypothesis, the HBV S gene sequences were determined of isolates from 33 virus carriers who were negative for HBsAg but showed antibodies against the virus core (anti-HBc) as the only serological marker of hepatitis B. Isolates from 36 HBsAg-positive patients served as controls. In both groups, a considerable number of novel mutations were found. In isolates from individuals with anti-HBc reactivity only, the variability of the major hydrophilic loop of HBsAg, the main target for neutralizing and diagnostic antibodies, was raised significantly when compared with the residual protein (22. 6 vs 9.4 mutations per 1000 amino acids; P<0.001) and with the corresponding region in the controls (22.6 vs 7.5 exchanges per 1000 residues; P<0.001). A similar hypervariable spot was identified in the reverse transcriptase domain of the viral polymerase, encoded by the same nucleotide sequence in an overlapping reading frame. These findings suggest that at least some of the chronic low-level carriers of HBV, where surface antigen is not detected, could be infected by diagnostic escape mutants and/or by variants with impaired replication.

Test performance characteristics of Anti-HEV IgG assays strongly influence hepatitis E seroprevalence estimates.

Hepatitis E virus (HEV) seroprevalences of 0.3%-53% were reported from industrialized countries. Because these estimates may be influenced by detection assays, this study compares 3 frequently used tests for HEV detection: the MP Diagnostics HEV immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), the Axiom Diagnostics HEV IgG enzyme immunoassay (EIA), and the Mikrogen recomLine HEV IgG assay. Sera from 200 healthy healthcare workers and 30 individuals with acute HEV infection were analyzed. Among the healthy individuals, HEV IgG was found in 4.5% by the MP Diagnostics assay, in 29.5% by the Axiom Diagnostics assay, and in 18% by the Mikrogen assay. Among individuals with acute HEV infection, positive results were obtained for 83.3%, 100%, and 96.7%, respectively. Thus, the 3 assays show clear differences in diagnostic sensitivity.

Detection of hepatitis E virus (HEV) from porcine livers in Southeastern Germany and high sequence homology to human HEV isolates

BACKGROUND Hepatitis E virus (HEV) has been identified as an emerging cause of infectious hepatitis over the last years in developed countries. In contrast to travel associated hepatitis E, zoonotic sources of infection are suspected for autochthonous cases in Europe. OBJECTIVE Since pigs are known reservoirs of HEV, we tested porcine livers sold as food in Southeastern Germany for the presence of hepatitis E virus RNA. STUDY DESIGN We purchased 200 porcine liver samples in 81 butcher shops and grocery stores in Regensburg, Germany. Nucleic acid preparations were tested for the presence of HEV RNA by quantitative real-time PCR (RT-qPCR). HEV isolates from positive samples were characterized by partial sequencing of ORF1 and ORF2 regions in the HEV genome and by phylogenetic analysis. RESULTS Specimens from eight (4%) of 200 purchased pig livers had detectable HEV RNA amounts. Sequence determination and phylogenetic analysis allowed two novel isolates to be classified as HEV genotype 3, subgenotype 3a (swR437) and 3c (swR269), respectively. Both novel swine HEV isolates showed high sequence homology to isolates obtained from patients with acute HEV infection from the same geographic region. CONCLUSIONS These results support the suggested role of undercooked pig products in food as a source of zoonotic HEV infection for humans. It remains to be clarified if this mechanism of transmission is responsible for the surprisingly high anti-HEV IgG prevalence recently observed in some European countries and the USA.

Sensitive and accurate quantitation of hepatitis B virus DNA using a kinetic fluorescence detection system (TaqMan PCR)

The laboratory diagnosis of hepatitis B virus (HBV) infection is based mainly on serological assays. Yet the detection and quantitation of viral DNA is necessary when addressing directly the question of infectivity or when monitoring the viral load during therapy. Standard hybridization assays allow for exact quantitation, but their sensitivity is limited to 10(5)-10(6) viral genomes per ml of serum. The most sensitive tests for HBV DNA are nested PCR systems, which recognize virtually one molecule of the target DNA per reaction. However, these assays only provide very coarse quantitative statements. To take advantage of both methods, a new assay for HBV DNA is described based on the commercial TaqMan system. This assay is capable of quantifying HBV DNA from the theoretical lower limit up to 10(10) genome equivalents per ml of serum and, thus, covers the complete range of naturally occurring states of infections. The method was calibrated on the basis of serial plasmid dilutions and compared with a well-established nested PCR system. More than 100 HBV positive sera and serial dilutions of the Eurohep standard for both ad and ay subtypes were analyzed. The assay reliably detected all HBV positive samples. It shows minimal run-to-run deviations, allows for quantitation that covers eight orders of magnitude, and finally, completely avoids the risk of cross-contamination by PCR products. Thus, this technique combines the sensitivity of PCR amplification and the quantitation potential of hybridization tests and it is time efficient and safer.

Prevalence of hepatitis E virus-specific antibodies in humans with occupational exposure to pigs

Due to the increasing number of non-travel-associated hepatitis E virus (HEV) infections observed in several industrialised countries including Germany, there is a substantial interest in the characterisation of risk factors and transmission routes relevant to autochthonous HEV infections. Autochthonous cases are believed to be the result of a zoonotic HEV transmission from pigs, wild boars and deer. Recently, a high prevalence of HEV-specific antibodies in the German domestic pig population has been demonstrated. Thus, one may assume a higher prevalence of HEV-specific antibodies in humans with occupational exposure to pigs. In this study, sera obtained from 24 slaughterers, 14 meat inspectors, 46 pig farmers and 22 veterinarians were tested for the presence of HEV-specific antibodies using a line immunoassay. For comparison, sera obtained from 116 age- and gender-matched blood donors were also included. Twenty eight per cent (28.3%; 30/106) of the swine-exposed humans and 15.5% (18/116) of the blood donors without contact to pigs exhibited IgG-antibodies determined as reactive (i.e. borderline or positive) against HEV. Thus, an increased risk of HEV infection in humans occupationally exposed to pigs and particularly for slaughterers (41.7%; 10/24) was demonstrated.

Seroprevalence study in forestry workers from eastern Germany using novel genotype 3- and rat hepatitis E virus-specific immunoglobulin G ELISAs

Hepatitis E virus (HEV) is the causative agent of an acute self-limiting hepatitis in humans. In industrialized countries, autochthonous cases are linked to zoonotic transmission from domestic pigs, wild boar and red deer. The main route of human infection presumably is consumption of contaminated meat. Farmers, slaughterers and veterinarians are expected to be risk groups as they work close to potentially infected animals. In this study, we tested four Escherichia coli-expressed segments of the capsid protein (CP) of a German wild boar-derived HEV genotype 3 strain for their diagnostic value in an indirect immunoglobulin G (IgG) ELISA. In an initial validation experiment, a carboxy-terminal CP segment spanning amino acid (aa) residues 326–608 outperformed the other segments harbouring aa residues 112–608, 326–660 and 112–335. Based on this segment, an indirect ELISA for detection of anti-HEV IgG antibodies in human sera was established and validated using a commercial line immunoassay as reference assay. A total of 563 sera from forestry workers of all forestry offices of Brandenburg, eastern Germany and 301 sera of blood donors from eastern Germany were surveyed using these assays. The commercial test revealed seroprevalence rates of 11% for blood donors and 18% for forestry workers. These rates are in line with data obtained by the in-house test (12 and 21%). Hence, the in-house test performed strikingly similar to the commercial test (sensitivity 0.9318, specificity 0.9542). An initial screening of forestry worker and blood donor sera with a corresponding CP segment of the recently discovered Norway rat-associated HEV revealed several strong positive sera exclusively in the forestry worker panel. Future investigations have to prove the performance of this novel IgG ELISA in large-scale seroepidemiological studies. In addition, the observed elevated seroprevalence in a forestry worker group has to be confirmed by studies on groups of forestry workers from other regions. The epidemiological role of ratHEV in human disease should be assessed in a large-scale study of risk and non-risk groups.

Prevalence of markers of hepatitis B in the adult German population

The prevalence of hepatitis B virus markers was investigated in 5305 individuals considered to be representative for the adult German population. After adjustment of the data according to the age and sex distribution in the whole German population an anti‐HBc prevalence of 8.71% (95% confidence interval, 7.94–9.48%) and an HBsAg carrier rate of 0.62% (95% confidence interval, 0.40–0.84%) were calculated. Anti‐HBc prevalence increased with age from 4.12% in the youngest group to 15.66% in the 61–70‐year‐old. The percentage of HBsAg carriers showed a maximum of 1.12% in the 41–50‐year‐old individuals and decreased significantly in the older age groups. 1.40% (95% confidence interval, 1.08–1.72%) of individuals had anti‐HBc only. There was a trend to higher rates of this pattern in males than in females; a significantly higher percentage of persons with anti‐HBc only was found in anti‐HBc‐positive individuals below 31 years than in older individuals. Five participants with anti‐HBc only (7.7%, or about 0.1% of the whole population) showed HBV‐DNA despite the absence of HBsAg. 3.1% of anti‐HBc positive individuals where also positive for anti‐HCV, that was significantly higher than the percentage of anti‐HCV‐positives among persons without any HBV marker (0.46%). This study provides a comprehensive picture of the current hepatitis B situation in Germany, showing new data especially on the distribution of HBsAg in the general population and on the subgroup of individuals with anti‐HBc only. J. Med. Virol. 63:96–102, 2001.

Prevalence of antibodies against hepatitis C virus in the adult German population

OBJECTIVE The prevalence of anti-HCV in Germany has been determined for blood donors and certain risk groups, but the burden of disease in the general population remains unknown. The aim of this study was to determine the prevalence of anti-HCV in a study group representing the normal adult German population. DESIGN A total of 5312 individuals aged 18-70 years were randomly selected from small, middle-sized and big cities in five different German states. Sera were tested for anti-HCV by enzyme immunoassay and immuno dot assay, as well as for anti-HBc and, in the case of a positive result, for anti-HBs and HBsAg. Serological typing was performed in anti-HCV-positive persons. RESULTS Thirty-nine individuals were anti-HCV positive; indeterminate results (with antibodies against the viral core protein only) were obtained in 20. There was a tendency to higher prevalence rates with increasing age as well as to a higher prevalence in women. Serological typing revealed the presence of genotype 1 in the vast majority of participants (82%); only a minority showed genotype 3 (7.2%) or other genotypes (7.2%). Markers of HBV were seen in 43.6% of the anti-HCV positive individuals, with nearly one third (29.4%) of the double-infected showing anti-HBc as the only marker for HBV. CONCLUSIONS According to our data, an anti-HCV prevalence of 0.63% (95% confidence interval, CI, 0.42-0.84%) can be assumed in the general adult German population, with higher values in older people and women. Nearly half of the anti-HCV positive individuals also show markers of hepatitis B virus.

Long‐term surveillance of haematopoietic stem cell recipients with resolved hepatitis B: high risk of viral reactivation even in a recipient with a vaccinated donor

Summary.  Reactivation of resolved hepatitis B virus (HBV) infection is increasingly recognized in patients with severe immunosuppression. We monitored seven patients with pretransplant antibodies to hepatitis B surface antigen (anti‐HBs) and hepatitis B core antigen (anti‐HBc) for HBV reactivation after allogeneic haematopoietic stem cell transplantation (allo‐HSCT). Reverse seroconversion (from anti‐HBs to HBsAg) was observed in six recipients occurring 12, 14, 16, 22, 31 and 39 months after allo‐HSCT, respectively. The only patient without HBV reactivation had the highest pretransplant anti‐HBs titre and died after the shortest follow‐up period (25 months). A novel HBV surface mutant (D144G/G145E) was isolated from one recipient of stem cells from a donor vaccinated against HBV. Another surface mutant (P142L/G145R) was detected in a recipient from a non‐immune donor. Serum ALT elevation was measured in only two of the six patients with viral reactivation, followed by spontaneous clearance of HBsAg in one of them. Antiviral treatment reduced viral load in five patients, but the emergence of YMDD motif polymerase mutations resulted in lamivudine resistance in two patients. In conclusion, the risk of reactivation of a resolved HBV infection is close to 100% in allogeneic stem cell recipients and vaccination of the donor does not always warrant reliable protection.