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 manufacturer's 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 assay's 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.

[New approaches to the treatment of the flavivirus infections].

Since spontaneous mutagenesis and quasi-species rearrangements of the RNA-containing viruses, as well as an absence of both viral and cellular RNA reparation systems, causes resistance to originally effective antiviral drugs, combination therapy with nucleoside and non-nucleoside inhibitors of the viral enzymes in combination with immunomodulators is recommended. The use of specific immunoglobulins does not result in complete elimination of the flaviviruses but rather in possible antibody-dependent enhancement of the flavivirus infection by means of increased penetration of complexes of virions with specific antibodies into cells with receptors for Fc-fragments of immunoglobulins.

Analytical performance characteristics and clinical utility of a novel assay for total hepatitis C virus core antigen quantification.

The detection and quantification of hepatitis C virus (HCV) core antigen in serum or plasma by the use of different assay formats have previously been shown to represent useful markers of viral replication. In the present study, the intrinsic performance characteristics and the potential clinical utility of a novel assay for the quantification of total HCV core antigen were comprehensively assessed by using clinical serum samples and specimens contained in various evaluation panels. The Architect HCV Ag assay showed a specificity of 100%. The intra- and interassay coefficients of variation ranged from 3.6 to 8.0% and from 4.7 to 9.5%, respectively. Except for HCV genotype 2 isolates, the analytical sensitivity was always less than 10 fmol core antigen/liter, corresponding to approximately 500 to 3,000 IU of HCV RNA/ml. Linearity was guaranteed throughout the dynamic range (10 to 20,000 fmol/liter). When seroconversion panels were tested, the assay was not inferior to HCV RNA detection and reduced the preseroconversion period by 4 to 16 days. The results obtained by core antigen and HCV RNA quantification for 385 clinical specimens were correlated by regression analysis (r = 0.857), but the calculated conversion equation differed significantly from the line of identity. Monitoring of viral kinetics by use of either core antigen or RNA concentrations in 38 HCV-infected patients undergoing antiviral combination therapy resulted in very similarly shaped curves in all cases. Finally, the Architect HCV Ag assay was also shown to enable high-throughput screening of in vitro HCV RNA replication. With these results taken together, the Architect HCV Ag assay proved to be a specific, reproducible, highly sensitive, and clinically applicable test format which will find its future place in the context of virological HCV diagnostics.

[Human bocavirus].

Human bocavirus (HBoV) is a newly identified parvovirus associated with acute respiratory infections in young children in different parts of the world. It is not inconceivable that this virus is also capable of causing acute gastroenteritis and asymptomatically persisting in infected children. HBoV is the third widespread human respiratory virus after respiratory syncytial virus and rhinovirus. Polymerase chain reaction remains the most reliable of HBoV detection in clinical samples. Phylogenetic analysis shows the presence of at least 2 circulating variants (genotypes) of HBoV.

Lack of de novo hepatitis C virus infections and absence of nosocomial transmissions of GB virus C in a large cohort of German haemodialysis patients.

To determine the prevalence and incidence of hepatitis C virus (HCV) infections among haemodialysis patients, a large prospective multicentre trial was conducted in the German Federal State of North Rhine-Westphalia. Sera obtained from the recruited patients in two separate sampling rounds run 1 year apart were analysed for both anti-HCV antibodies and HCV RNA. HCV RNA positive samples were also genotyped by direct sequencing of an HCV core fragment. In the first and second rounds, 150 (5.2%) of 2909 and 114 (5.4%) of 2100 patients were anti-HCV positive, respectively, and 4% of individuals were viraemic. Evaluation of potential risk factors in a case-control study indicated that the factors 'foreign country of birth', 'blood transfusions given before 1991' and 'duration of treatment on haemodialysis' were associated with the risk of HCV infection. Among the 2100 patients of whom 'paired' serum samples from both rounds were available for testing, not a single 'de novo' HCV infection could be recorded. The fact that in a subset of about 20% of these patients no nosocomial GB virus C (GBV-C) transmission occurred during the observational period suggests that the lack of HCV seroconversions was not only attributable to the isolation of HCV-infected patients but also to the strict adherence to so-called universal hygienic precautions for infection control maintained in the participating dialysis centres.

Transmission of hepatitis C virus in an orthopedic hospital ward.

Healthcare-associated infections with hepatitis C virus (HCV) hitherto have been observed mainly in hemodialysis settings as well as in hematology and oncology wards. In this communication, molecular and epidemiologic investigations to elucidate an HCV outbreak in an orthopedic ward are reported. One hundred and thirty-five patients hospitalized in the ward and 104 staff members were tested. In addition to extensive epidemiologic reviews and hygienic inspections, direct sequencing of HCV PCR fragments and phylogenetic analysis of more than 300 partial HCV sequences obtained by end-point limiting-dilution real-time PCR assay were carried out. Six patients were infected with very closely related HCV variants. Patient-to-patient spread of the virus was inferred to have started from one patient with previous HCV infection to the other five patients during their hospital stay. Inspections did not reveal substantial breaches in basic infection control practices and did not identify a specific activity that might have led to nosocomial transmission. As a result of the investigations, the hospital corrected the documentation of all medical and nursing activities undertaken in the ward, abandoned the use of all multidose saline and other medication vials, and included explicitly recommendations for the safe preparation and administration of injectable drugs into internal infection control guidelines. Thereafter, no further nosocomial transmissions of HCV have been recorded in the orthopedic ward. The events observed suggest that nosocomial transmission of HCV is not limited to hemodialysis, hematology or oncology settings, and they also reinforce the mandatory adherence to basic infection control practices.

Outcome of an exercise to notify patients treated by a general surgeon infected with the hepatitis C virus.

BACKGROUND: Health-care workers infected with the hepatitis C virus (HCV) and performing exposure-prone procedures may expose their patients to the risk of nosocomial HCV infection. OBJECTIVE: To assess the number of provider-to-patient transmissions of HCV among former patients of an HCV-infected general surgeon. RESULTS: The notification exercise covered 1461 individuals, on whom the surgeon performed 1683 operations. Eighty-two percent of these patients were tested for markers of HCV infection, and all but six subjects turned out to be not infected with the virus. Two of the anti-HCV positive patients were already infected before their operations, one individual was not available for further molecular analyses, and three subjects harboured HCV isolates that belonged to a different subtype (i.e. 1b) than the variant detected in the surgeon's serum. CONCLUSION: In this retrospective survey, no provider-to-patient transmission of HCV was detected among 1192 former patients of an infected general surgeon. This finding, one more time, suggests that such nosocomial transmission events are probably very rare. Consequently, recommendations for the management and guidance of HCV-infected health-care workers should carefully balance the workers' rights against justified patients' interests.

Evidence for a complex mosaic genome pattern in a full-length hepatitis C virus sequence.

The genome of the hepatitis C virus (HCV) exhibits a high genetic variability. This remarkable heterogeneity is mainly attributed to the gradual accumulation of mutational changes, whereas the contribution of recombination events to the evolution of HCV remains controversial so far. While performing phylogenetic analyses including a large number of sequences deposited in the GenBank, we encountered a full-length HCV sequence (AY651061) that showed evidence for inter-subtype recombination and was, therefore, subjected to a detailed analysis of its molecular structure. The obtained results indicated that AY651061 does not represent a "simple" HCV 1c isolate, but a complex 1a/1c mosaic genome, showing five putative breakpoints in the core to NS3 regions. To our knowledge, this is the first report on a mosaic HCV full-length sequence with multiple breakpoints. The molecular structure of AY651061 is reminiscent of complex homologous recombinant variants occurring among other members of the flaviviridae family, e.g. GB virus C, dengue virus, and Japanese encephalitis virus. Our finding of a mosaic HCV sequence may have important implications for many fields of current HCV research which merit careful consideration.

Towards a better resolution of hepatitis C virus variants: CLIP sequencing of an HCV core fragment and automated assignment of genotypes and subtypes.

Commercially available assays for typing of hepatitis C virus (HCV) isolates satisfy the current clinical needs. They are, however, limited in their ability to identify the multitude of existing HCV subtypes correctly. Therefore, these kits should only be used cautiously in epidemiological studies and will also not meet future clinical demands which might arise, e.g., from the application of HCV subtype-specific antiviral compounds. In an attempt to overcome the drawbacks of commercial typing procedures based on the analysis of the 5' untranslated region (5' UTR), an approach was developed which relies on CLIP sequencing of an HCV core fragment with automated assignments of types and subtypes via an originally created "core-specific" sequence database. The performance characteristics of the new technique were evaluated in comparison to the Trugene 5' NC Genotyping Kit. The core-based sequencing method could regularly detect HCV isolates of genotypes 1-6 with an analytical sensitivity of 5000 IU/ml. The accuracy of typing results obtained by the Trugene test was 97% (genotypes) and 81% (subtypes). The core-linked approach classified all HCV strains correctly on the level of genotypes and led to an adequate subtype assignment in 96% of all cases. This analytical performance characteristics recorded for the newly devised typing technique was superior to those reported for all commercially available assays, including a most recently released new generation of the line probe assay. Consequently, CLIP sequencing of an HCV core fragment with subsequent automated assignment of types and subtypes can be confidently used in clinical laboratory practice to answer current and also future questions in the context of HCV typing.

Transcription-mediated amplification linked to line probe assay as a routine tool for HCV typing in clinical laboratories.

Typing of hepatitis C virus (HCV) isolates is currently a prerequisite for adequate tailoring of antiviral combination therapy. In many diagnostic laboratories, there seems to be a tendency toward convenient and time-saving procedures utilizing amplification products, which are already available from preceding qualitative or quantitative HCV ribonucleic acid (RNA) assays. In this context, we evaluated the performance characteristics of a combination of techniques, i.e., transcription-mediated amplification-line probe assay (TMA-LiPA), which links highly sensitive TMA of HCV RNA to the VERSANT HCV Genotype Assay (version 1). A total of 100 clinical samples were genotyped by TMA-LiPA. The obtained results were compared to those recorded by the original, nested reverse transcription (RT)-polymerase chain reaction (PCR)-based VERSANT assay, the core-related GEN-ETI-K DEIA, and phylogenetic analyses of partial sequences from the HCV core and NS5B regions. TMA-LiPA assigned the correct genotype to all 100 HCV isolates. For subtyping of genotype 1 and 2 isolates, TMA-LiPA only showed discriminatory powers of 82% and 53%, respectively. Thus, TMA-LiPA in our hands turned out as a convenient and time-saving routine procedure for HCV typing which currently provides sufficient information for clinical purposes. Like all 5'untranslated region (UTR)-based assays, the technique is limited, however, in its potentials to resolve the complexity of existing HCV subtypes.

[Human metapneumovirus as a common cause of respiratory tract disease].

Human metapneumovirus (HMPV), the newly identified paramyxovirus, causes respiratory infections in children, immunosuppressed patients, and the elderly in different countries of the world. The epidemiology and clinical manifestations of HMPV infection are similar to those in human respiratory syncytial virus infection. The diagnosis of HMPV infection is based on the polymerase chain reaction detection of viral RNA or the recording of rising serum antibody titers. There are at least two genotypes and several subtypes of HMPV in the human population. The use of cell cultures and laboratory animals have provided new evidence for the pathogenesis of HMPV infection, the specific features of antiviral immunity and enabled recombinant HMPV vaccine candidates to be designed.

Fatal pneumonia associated with human metapneumovirus (HMPV) in a patient with myeloid leukemia and adenocarcinoma in the lung.

We describe a clinical case of a 59 old caucasian male who was delivered to the hospital for severe pneumonia associated to human metapneumovirus. The patient suffered from a leukemia and an adenocarcinoma in the lung and died two weeks after submission due to fatal respiratory failure.

Two cases of severe obstructive pneumonia associated with an HKU1-like coronavirus.

BACKGROUND: During the last few years a number of previously undescribed viruses, including human metapneumovirus, coronaviruses SARS, NL63 and HKU1, and bocavirus, were identified in nasopharyngeal samples from patients with signs of respiratory infections. These viruses may cause mild to life-threatening infections. OBJECTIVES: Nasopharyngeal samples from hospitalized pediatric patients with respiratory disease were analysed for the presence of coronaviruses and other well known and newly identified respiratory viruses. RESULTS: Two clinical cases of a severe obstructive pneumonia, which were associated with the presence of RNA of a novel variant (subtype) of HKU1 coronavirus in the nasopharyngeal aspirates, were identified. DISCUSSION: The detection of a HKU1-like coronavirus in pediatric patients in the current study complement the most recent independent finding of similar or closely related coronaviruses in patients with respiratory diseases in France (Vabret et al. 2006) and Norway (Jonassen et al., see accompanying manuscript). These observations indicate a wide dissemination of HKU1-like coronaviruses in Europe.

Genotyping of hepatitis C virus isolates by a new line probe assay using sequence information from both the 5'untranslated and the core regions.

The correct assessment of hepatitis C virus (HCV) genotypes and subtypes by commercial assays is of utmost importance mainly for the therapeutic management of patients suffering from HCV infections. In this study, the performance characteristics of a newly designed genotyping assay were evaluated that does not rely exclusively on sequence information derived from the 5'untranslated region but also takes into account part of the HCV core. One hundred and ten clinical specimens were tested by this new assay prior to its commercialisation. The obtained typing results were compared to those recorded by the 5'UTR-based Versant HCV Genotyping Assay, version 1, the core-related Gen-Eti K DEIA, and phylogenetic analyses of partial HCV core and NS5B sequences. The HCV genotypes and subtypes identified by the newly devised kit were completely in line with the assignments achieved by DEIA and phylogenetic analyses. In particular, all 64 HCV strains belonging to subtypes 1a or 1b were recognised correctly, and HCV 6e and 6f isolates were adequately assigned to subtypes 6c-l. Thus, the second generation of the Versant genotyping assay could overcome the drawbacks of its exclusively 5'UTR-based predecessor and will turn out to be a reliable tool for HCV typing in clinical laboratories.

High prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates.

RNA of the newly identified human metapneumovirus (HMPV) was detected in nasopharyngeal aspirates of 11 of 63 (17.5%) young children with respiratory tract disease. Markers of infection caused by another member of the Pneumovirinae subfamily of the family Paramyxoviridae, respiratory syncytial virus (RSV), were identified in 15 of these patients (23.8%). Three patients were simultaneously infected with HMPV and RSV. Studies of the clinical characteristics of HMPV-infected children did not reveal any difference between HMPV-infected patients and a control population of RSV-infected patients with regard to disease severity, but the duration of symptoms was significantly shorter for HMPV-infected patients. Phylogenetic analysis of the amplified viral genome fragments confirmed the existence and simultaneous circulation within one epidemic season of HMPV isolates belonging to two genetic lineages.

Quantitation of hepatitis C virus RNA by third generation branched DNA-based signal amplification assay.

Quantitation of hepatitis C virus (HCV) RNA has become an important tool in different clinical settings and is used extensively for pretreatment evaluation of patients infected chronically with HCV. In this study, the performance characteristics of the third generation branched DNA-based signal amplification assay (bDNA 3.0) for HCV RNA quantitation were established. The new assay version showed an analytical specificity of 98%. Mean intra- and between-run imprecisions were 6.8 and 11.2%, respectively. The assay was linear over its entire dynamic range. Quantitation appeared to be unaffected by the genotypic variability of HCV. A comparison of bDNA 3.0 with the second generation bDNA assay calibrated against the international WHO HCV RNA standard, and the PCR-based Cobas Amplicor HCV Monitor 2.0 revealed a fairly good correlation among the assays. Twenty-nine and 11% of the paired quantitative results differed by more than log(10)0.5 (i.e. three-fold). All three assays after calibration against the WHO standard also yielded clinically comparable results with regard to the tailoring of interferon alpha/ribavirin treatment duration in patients infected by HCV genotypes 1, 4, and 5.

[Infection of mice with TT virus]. / Infitsirovanie myshei virusom TT.

The sera pool of the human blood containing TTV DNA were administered to mice intravenously and then every 10 days examinations were made of blood serum and different organs of inoculated mice by polymerase chain reaction for presence of the viral nucleotide sequences. The viral DNA was revealed in the blood serum, the liver, kidneys and other organs 20 days after infection and persisted in most of the animals during 80 days. The data obtained show the possibility of TTV-infection of mice.

Prevalence of TTV DNA and GBV-C RNA in patients with systemic sclerosis, rheumatoid arthritis, and osteoarthritis does not differ from that in healthy blood donors.

OBJECTIVE: To determine the prevalence of GB virus-C (GBV-C) RNA and TT virus (TTV) DNA in patients with systemic sclerosis (SSc), rheumatoid arthritis (RA), and osteoarthritis (OA) as well as to compare the autoantibody pattern in patients with SSc with and without evidence of viral infection. PATIENTS AND METHODS: The study included 168 patients (84 SSc, 41 RA, and 43 OA) diagnosed according to the American College of Rheumatology criteria and 122 volunteer blood donors. The presence of GBV-C RNA and TTV DNA in serum was assessed by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-nested PCR, respectively. Autoantibodies in patients with SSc were determined by enzyme linked immunosorbent assay (ELISA) and Hep-2 immunofluorescence. RESULTS: TTV-DNA was detected in 10/84 (12%) patients with SSc, 9/41 (22%) patients with RA, 3/43 (7%) patients with OA, and 16/122 (13%) blood donors. GBV-C RNA was present in 4/84 (5%) patients with SSc, 2/43 (5%) patients with OA, and 5/122 (4%) blood donors. No patient with RA was positive for GBV-C RNA. One patient with SSc and one patient with OA showed a double infection with GBV-C and TTV. 74/84 (88%) patients with SSc were positive for at least one autoantibody species tested: 18/84 (21%) showed anticentromeric autoantibodies, 55/84 (66%) a speckled (36/84 (43%) fine, 19/84 (23%) coarse), and 20/84 (24%) a homogeneous nuclear Hep-2 pattern, and 21/84 (25%) had antinucleolar autoantibodies. Anti-Scl-70 antibodies were found in 31/84 (37%) and anti-RNP antibodies in 5/84 (6%) patients with SSc. No differences in the autoantibody pattern in patients with SSc with or without viral infection could be detected. CONCLUSION: The prevalence of GBV-C RNA and TTV DNA in serum samples from patients with SSc, RA, and OA was low and comparable with that in blood donors. A continuing infection with TTV and or GBV-C was not associated with a significant change in the autoantibody pattern in patients with SSc. These data provide no evidence for an association between GBV-C and/or TTV infections and SSc and/or arthritis (RA and OA).

Differences in HCV antibody patterns in haemodialysis patients infected with the same virus isolate.

Eight cases of de novo hepatitis C virus (HCV) infection in a haemodialysis unit in Rio de Janeiro, Brazil, were retrospectively studied. HCV viraemia was demonstrated by RT nested PCR in seven of the seroconverters. Genotyping showed that six patients were infected with a genotype 1b strain and one with a genotype 1a strain. A phylogenetic analysis of nucleotide sequences of the HCV core region revealed that five of the six 1b isolates form a separate cluster when compared with other 38 HCV 1b core sequences randomly chosen from the GenBank. The revealed sequence similarities indicated the nosocomial spread of a single HCV strain within the unit. To investigate whether the patients infected with the same viral isolate display similar patterns of antibody response to individual proteins, serial serum samples were examined. A line immunoassay for qualitative and semi-quantitative determination of specific antibodies against recombinant and synthetic HCV antigens was employed. Despite infection with the same virus strain, the patients sera demonstrated different patterns of reactivity against individual structural and nonstructural HCV proteins immediately after seroconversion. For each patient, however, antibody responses remained mostly stable throughout the follow-up of 8 to 24 months.