Evaluation of an antigen-antibody "combination" enzyme linked immunosorbent assay for diagnosis of hepatitis C virus infections.

BACKGROUND: Development of "combination" assays detecting in parallel, within a single test, Hepatitis C Virus (HCV) antigens and antibodies, not only reduces the window period in HCV-infection but also costs. Reduction of costs is important for developing countries where money and personal resources are limited. METHODS: We compared the Monolisa® HCV Antigen-Antibody Ultra (Bio-Rad Laboratories Limited, Marnes La Coquette, France) with the AXSYM HCV version 3.0 (Abbot Diagnostics, Germany)-the latter assay detecting only antibodies to HCV. Seventy three HCV-PCR positive and negative samples were tested. RESULTS: Although the two assays showed comparable results, two samples from a bone marrow transplant (BMT) patient of viral loads 7.8 × 105 and 8.9 × 106 IU/mL could not be detected by the Monolisa® HCV Antigen-Antibody Ultra assay. Failure to detect the two samples with viral loads considered above threshold of detection for antigen proteins suggested a lack of sensitivity by this assay to discover viral capsid protein in patient samples. Genotyping of these samples revealed genotype 1b, a HCV-subtype which is widespread and should thus be easily detected. CONCLUSION: We conclude that although this assay depicts high sensitivity and specificity in detecting antibodies to HCV, it seems not to add further benefit in our study population to detect HCV infections by enhanced sensitivity due the potential contingency to trace viral capsid antigens.

Isolate KAV: a new genotype of the TT-virus family.

A novel DNA sequence belonging to a new genotype of TT virus (TTV) was detected by long-distance PCR in the serum of a chronically HCV-infected patient. The isolate was designated KAV according to the patient's initials. Extending the sequence to full length revealed a 3705-nt viral genome, which is about 100 nucleotides shorter than the other TT-viruses. KAV showed common features with the TTV family, such as the organization of open reading frames and conserved noncoding regions. The largest open reading frame of KAV (ORF 1) was about 40 aa shorter than that of other TT-viruses. Overall sequence homology with known TTV isolates was less than 66%. Phylogenetic analysis poses KAV in one major group with three recently published TTV sequences. So KAV can be considered as a new genotype of the TTV family (provisionally designated genotype 28).

The mRNAs of prekallikrein, factors XI and XII, and kininogen, components of the contact phase cascade are differentially expressed in multiple non-hepatic human tissues.

Recently RT-PCR studies had demonstrated the expression of plasma prekallikrein (PPK) mRNA in extrahepatic tissues. The questions arose whether that is illegitimate or regular expression, and whether the mRNAs of blood coagulation factors XI and XII, and high molecular weight kininogen, components of the contact activation cascade of blood coagulation are also expressed in non-hepatic tissues. These questions were addressed in the present study by employing quantitative RT-PCR. The relative mRNA levels of the respective proteins determined in 16 human tissues indicate legitimate extrahepatic transcription of at least three of the genes. Transcription of all genes was highest in the liver, but only PPK mRNA was detected in all 16 tissues, especially high levels in pancreas, kidney, testis, spleen and prostate. We conclude from these results that PPK is synthesized in significant amounts in non-hepatic tissues and that this locally synthesized PPK may have special local functions.

Prevalence of GB virus C/hepatitis G virus RNA and anti-E2 glycoprotein antibodies in homosexual men with HIV coinfection.

BACKGROUND: The objective of this cross-sectional, nonrandomized, prospective study was to generate data on the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) in a cohort of HIV-infected homosexuals from Munich. PATIENTS: A total of 71 HIV-infected homosexual men were analyzed for prevalence of GBV-C RNA and antibodies to the E2 envelope glycoprotein (E2Ab). 475 healthy volunteer blood donors in southern Bavaria served as a control group. RESULTS: The prevalence of GBV-C RNA was 27% (control group: 2.3%) and the prevalence of E2Ab was 35% (control group: 6%). The total prevalence for present and past infection was 62%. The differences between the HIV-infected patients and the control group were significant (p < 0.0001). GBV-C RNA and E2Ab were not detected simultaneously in any serum sample. The E2Ab positive patients were older than the GBV-C RNA positives (mean 46 years versus 39 years, p = 0.0350). The GBV-C RNA and E2Ab negative patients were older than the GBV-C RNA positives (mean 47 years versus 39 years, p = 0.0236). The E2Ab positive patients had suffered sexually transmitted diseases more frequently than the patients negative for markers of GBV-C infection (p = 0.0308). E2Ab positive patients also had higher mean levels of alanine aminotransferase compared to patients without evidence of GBV-C infection (p = 0.0164). 59.4% of all individuals were anti-HBc IgG positive. CONCLUSION: The data can be interpreted as indirect evidence for sexual transmission of GBV-C.

Sequence analysis of the HIV-1 protease coding region of 18 HIV-1-infected patients prior to HAART and possible implications on HAART.

BACKGROUND: The majority of HIV-infected patients are treated with highly active antiretroviral therapy (HAART) consisting of a combination of inhibitors of the protease (PIs) and the reverse transcriptase (RTIs). Analysis of mutations within these enzymes which are associated with development of resistance to the applied inhibitors is of major clinical importance. In particular, pre-existing mutations in previously untreated individuals may adversely influence the efficacy of HAART. OBJECTIVES: The sequences of the protease coding regions of 18 HIV-1-infected patients were analysed prior to HAART. STUDY DESIGN: DNA was extracted from whole blood samples of HIV-1-infected treatment-naive patients. The protease coding region was amplified by nested PCR and sequenced directly. The resulting amino acid substitutions were analysed for known mutations associated with known resistance to PIs. RESULTS: In all 18 analysed individuals we found 1-10 amino acid substitutions per patient in their HIV-1 protease coding region. These mutations occurred altogether at 27 positions of the 99 amino acids of the protease coding region. Seven of these mutated positions are associated with described resistance to PIs. Altogether, 15 of the 18 patients (83%) carried at least one such resistance-conferring alteration in their protease coding region. All patients are currently followed up during their present therapy to detect possible resistance formation to the applied PIs. CONCLUSIONS: A large variety of pre-existing mutations associated with resistance to PIs was observed prior to their treatment. As none of the patients ever received HAART before and infection with resistant viral strains is very unlikely, these amino acid substitutions evidently reflect natural polymorphism of the HIV-1 protease coding region.

A polymerase chain reaction-based protocol for the detection of transmission of pig endogenous retroviruses in pig to human xenotransplantation.

BACKGROUND: Xenotransplantation of pig organs and tissues to humans bears the risk of infection of immunosuppressed recipients by porcine endogenous retrovirus (PERV) released from the transplanted tissue. However, when diagnosing potential PERV transmission, it is essential to exclude microchimerism, i.e., persisting pig cells in analyzed bioptic material of xenotransplanted patients, which give rise to false positive PERV signals. Polymerase chain reaction (PCR) is so far the only suitable method to diagnose a cross-species transfer of PERV, but the exclusion of microchimerism might be a serious problem because most of the presently employed primer pairs detect PERV sequences with higher sensitivity than primers used for the detection of contaminating pig sequences. METHODS: We designed and evaluated a novel and improved primer set for detection of pig sequences as well as complementing positive control primers on the basis of mitochondrial cytochrome B, an approved marker for phylogenetic studies. We further established primer pairs derived from the long terminal repeat/leader region of PERV isolated from a Duroc German Landrace cross-bred pig and tested their sensitivity in comparison with known PERV- and pig-specific PCR markers. RESULTS: In standard PCR assays, the new cytochrome B-derived primers are at least 10 times more sensitive than the presently used PERV retroviral polymerase gene and mammalian beta-actin primers. When tested in a tissue culture infection model, PERV transmission to human 293 cells can be unambiguously demonstrated, even in the presence of up to 10% pig cells. One of the primer combinations derived from the PERV DuxDL3791 long terminal repeat/leader region amplifies with even lower sensitivity than primers detecting porcine beta-globin, thus permitting the exclusion of microchimerism also via chromosomal loci. CONCLUSIONS: The availability of the new PCR markers allows the proposal of a rigorous setup for the routine detection of PERV transmission after xenotransplantation.

Molecular comparison of Mycoplasma hominis strains isolated from colonized women and women with various urogenital infections.

Twenty Mycoplasma hominis strains isolated from colonized women and women with various urogenital infections were investigated for genetic and antigenic homogeneity by different methods. Restriction fragment length polymorphism analysis demonstrated heterogeneity for all strains, with one exception. Two strains sequentially isolated from one patient showed identical patterns. Otherwise, no clonal clustering could be detected within the strains isolated from either of the diagnostic groups. In contrast, SDS-PAGE analysis and the comparison of the immunoblot pattern revealed antigenic similarities of strains isolated from patients with bacterial vaginosis, chorioamnionitis, premature rupture of membranes and preterm delivery as well as endometritis but showed obvious differences in comparison to strains isolated from colonized women.

Methods for the differentiation of microorganisms.

Advances in analytical and diagnostic assays based on novel nucleic acid analyses techniques have revolutionized the application of molecular differentiation of microorganisms. Phenotypic typing schemes are now broadly supplemented by new genotyping methods which allow a more refined and detailed differentiation of closely related microorganisms, bacterial strains, isolates and pathogens on the DNA level. Bio-, sero- and phagetyping, antibiotic susceptibility tests, immunoblotting as well as multilocus enzyme- or polyacrylamide gel electrophoresis are now supported by the analysis of plasmid or chromosomal DNA restriction profiles, ribotyping, pulsed-field gel electrophoresis and polymerase- or ligase-chain reaction-based methods or direct sequencing technique to differentiate microorganisms. Some of these molecular techniques are also used in the field of virology to analyse and differentiate closely related sub- or genotypes. Few examples for the analysis and investigation of these usually small genomes will also be given.

The natural course of hepatitis C virus infection 18 years after an epidemic outbreak of non-A, non-B hepatitis in a plasmapheresis centre.

BACKGROUND: The natural history of hepatitis C virus (HCV) infection is variable and factors determining the course of the illness are unclear. AIMS: To determine the natural course of HCV infection in a well characterised group of patients 18 years after an epidemic outbreak of non-A, non-B hepatitis at a plasmapheresis centre. METHODS: Between 1994 and 1996, 20 of 30 affected individuals were studied. HCV infection was confirmed using second and third generation ELISA test kits. HCV RNA was detected by a polymerase chain reaction (PCR) method and HCV genotyping was performed by analysing amplicons from the conserved 5'-non-translated region generated by nested PCR. Thirty two liver biopsies were carried out in 14 patients. RESULTS: HCV antibodies were detected in all subjects. Eighteen patients had abnormal liver enzymes and 17 were HCV RNA positive, all of whom were infected with genotype 1a. Ninety per cent of this cohort showed evidence of chronic HCV infection with 50% having progressive liver disease and 20% cirrhosis 18 years after acute onset of non-A, non-B hepatitis. Considerable variation in disease outcome occurred between individuals and no correlation with clinical features of the acute illness was found. CONCLUSIONS: Variability in the consequences of HCV infection in cases infected with the same virus suggests that host factors are important in determining disease outcome. The factors which determine differences in the natural history of the disease still remain to be elucidated.

Transverse myelitis associated with Mycoplasma pneumoniae infection.

A 14-year-old boy developed acute transverse myelitis with severe abdominal pain, bladder dysfunction, weakness, and sensory loss of the lower extremities. Magnetic resonance imaging revealed a segmental expanded central edema affecting parts of the spinal cord, including the caudal medulla oblongata. Antibody response to Mycoplasma pneumoniae was negative in microparticle agglutination assays (1:40 in the acute serum and 1:160 in the convalescent serum) and complement fixation tests (1:20 and 1:10). However, analysis of acute-phase serum revealed a specific IgA and IgG response but no IgM response. Detection of M. pneumoniae in the cerebrospinal fluid by nested polymerase chain reaction and in nasopharyngeal aspirate by culture confirmed an M. pneumoniae infection. Treatment with doxycycline (100 mg daily) was started on the second day after admission to the hospital and continued for 14 days; the patient recovered completely and was discharged 20 days after onset of the disease, with no signs of neurological deficits.

Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae.

In this prospective study, the use of a culture-enhanced PCR assay for the detection of Mycoplasma pneumoniae, followed by hybridization with a specific probe (MP-HPCR) or without hybridization (MP-PCR), and the use of a nested PCR (MP-NPCR) were evaluated. Clinical samples (190 specimens) from 190 patients with respiratory complaints were incubated in culture broth overnight and then subjected to PCR. The results of the PCR were compared to those obtained by culture, the direct antigen test, and serologic testing by microparticle agglutination and by immunoblotting in unclear cases. The sensitivities were 19 CFU for MP-PCR, 1.9 CFU for MP-HPCR, and 0.019 CFU for MP-NPCR. PCR amplification of the beta-globin gene was possible in 98% of cases: after dilution of the beta-globin-negative samples, all samples were reactive. Correlation between negative MP-NPCR results and negative serology results was found in 89% of cases; a positive correlation was found with 10% of the patients. Samples from three immunocompromised patients were MP-NPCR positive but serologically negative. High respiratory colonization by M. pneumoniae (>10(5) CFU/ml) in patients with acute respiratory disease could be detected by culture, MP-PCR, and MP-NPCR. These results indicate that MP-PCR and MP-NPCR are reliable methods for the detection of M. pneumoniae in respiratory tract samples of patients with respiratory complaints.

Variability of the Hepatitis B Surface Protein in HBV-Infected Liver Transplant Recipients.

Variations in the major surface proteins (HBsAg) of hepatitis B virus (HBV) have been implicated in the high rate of reinfection in HBV-infected recipients of orthotopic liver transplantations (OLT). Sera from 6 OLT patients positive for HBsAg and from 3 recipients negative for it prior to transplantation were analyzed over several years, and 39 HBsAg sequences were compared. Despite anti-HBs immunoprophylaxis resulting in the disappearance of HBsAg, HBV DNA was detectable by a sensitive nested PCR in almost all sera. In 1 patient, a significant temporary shift in HBV subtypes was observed, indicating a mixed infection or the presence of multiple HBV populations in this patient; this was also true for other patients. Amino acid substitutions compared to wild-type HBV subtypes in 7 patients and variations within patients in 5 patients were detectable over time; the 'escape mutation' at amino acid position 145 was detected in 2 patients. Our data suggest that the high rate of reinfection in OLT recipients seems not to be associated with specific sequence variations in the major HBs gene, but shows a remarkable inter- and intraindividual variability. Obviously, no correlation between heterogeneity in this gene and clinical outcome was present. Copyright 1997 S. Karger AG, Basel

Polymerase chain reaction for diagnosis and identification of distinct variants of Crimean-Congo hemorrhagic fever virus in the United Arab Emirates.

Viral hemorrhagic fever has re-emerged in the United Arab Emirates (UAE) since November 1993. Genomic RNA of Crimean-Congo hemorrhagic virus (C-CHFV) was detected by a newly developed, nested reverse transcriptase polymerase chain reaction (RT-PCR) in the sera of four (25.0%) of 16 suspected cases of viral hemorrhagic fever. The RT-PCR was based on oligonucleotide primers deducted from the small RNA segment encoding the nucleoprotein of the virus. By comparison with a nucleotide sequence of a C-CHFV isolate from a Chinese sheep, a divergence of 10.0-11.8% was detected in the C-CHFV variants causing the UAE outbreak. In the four positive sera, three phylogenetically distinct C-CHFV variants were amplified and confirmed by direct sequencing of the PCR fragments. These C-CHFV sequences were obtained directly from sera of infected humans without prior propagation in cell culture. The RT-PCR allows rapid detection of genomic C-CHFV RNA in clinical specimens and study of the molecular epidemiology of this infection.

Three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B afzelii, and B. garinii) identified from cerebrospinal fluid isolates by pulsed-field gel electrophoresis and PCR.

A total of 36 European Borrelia burgdorferi sensu lato cerebrospinal fluid isolates (mainly from southern Germany) were analyzed by pulsed-field gel electrophoresis (PFGE) for large restriction fragment pattern (LRFP) and linear plasmid profiles. Analyzing this large panel of isolates, we detected all three species of B. burgdorferi sensu lato pathogenic for humans in cerebrospinal fluid from patients with Lyme neuroborreliosis by PFGE typing after MluI digestion: 21 B. garinii (58%), 10 B. afzelii (28%), and 4 B. burgdorferi sensu stricto (11%) strains as well as 1 isolate with bands characteristic of both B. afzelii and B. garinii. Species classification by PFGE typing was confirmed by 16S rRNA-specific PCR. Eighteen isolates (11 B. garinii, 6 B. afzelii, and 1 B. burgdorferi sensu stricto isolate) were further characterized by LRFP with four different restriction enzymes (ApaI, KspI, SmaI, and XhoI). All B. afzelii isolates showed identical patterns for each restriction enzyme group. Considerable heterogeneity was demonstrated within the B. garinii group. Subsequent analysis of plasmid profiles revealed only marginal differences for B. afzelii strains but different patterns for B. garinii isolates. In one B. afzelii strain we found a linear plasmid of about 110 kbp not described before. LRFP analysis by PFGE is a suitable tool for the molecular characterization of B. burgdorferi sensu lato strains and allows determination not only of the species but also of the subtypes within B. garinii.

Travel-related vector-borne virus infections in Germany.

Laboratory diagnosis of imported, vector-borne virus diseases during a 22-month-period in Munich, Germany, is summarized. IN 13/317 Germans returning from the Mediterranean with suspected sandfly fever, acute sandfly fever, serotype Toscana, was confirmed serologically: 84.6% of the infections were acquired in Italy. Of 249 German tourists with febrile disease returning from the tropics, acute infection with dengue virus was diagnosed serologically in 26 (10.4%): most infections were acquired in Thailand (57.7%). In a seroepidemiological study of 670 German aid workers who had spent two years in the tropics, 49 (7.3%) were positive for antibodies to dengue, 9 (1.3%) to chikungunya, and 1 (0.1%) to Sindbis virus. Of 17 Middle Eastern patients with suspected viral haemorrhagic fever, genomic Crimean-Congo haemorrhagic fever virus RNA was amplified in 4 (23.5%) by semi-nested reverse transcriptase polymerase chain reaction, and confirmed by molecular characterization of nucleic acid. With the increase in travel to and from endemic areas, imported vector-borne virus infections are increasingly important in Germany.

In vivo processing of Pr160gag-pol from human immunodeficiency virus type 1 (HIV) in acutely infected, cultured human T-lymphocytes.

The processing of the HIV-1 Pr160gag-pol precursor polyprotein was analyzed in freshly HIV-1-infected MT-4 cultured cells. Single intermediates of the processing cascade were characterized by immunoblotting using distinct antisera. A potent inhibitor of the HIV protease (PR), Ro 31-8959, was employed to block cleavage by the mature PR, thus allowing insights into initial stages of the gag-pol (auto)-catalytical processing. While most known gag-pol cleavages were blocked in the presence of the inhibitor, the cleavage site between the gag-NC and the pol-p6 domains was still cleaved even in presence of high amounts (1 microM) of inhibitor, leading to the accumulation of a novel 114-kDa polyprotein comprising p6-PR-RT-IN. In the absence of inhibitor no accumulation of p114 was observed. In inhibitor-treated, HIV-1-infected cells a p6-PR intermediate was also detected, indicating subsequent cleavage of the PR/RT scissile bond. These results demonstrate initial cleavage(s) of the gag-pol precursor hydrolyzed by a proteolytic activity different from the mature PR and indicate that p114 (p6-PR-RT-IN) and p6-PR intermediates could play an essential role in the PR activation process.

Nested RT-PCR for detection of sandfly fever virus, serotype Toscana, in clinical specimens, with confirmation by nucleotide sequence analysis.

A single tube, reverse transcriptase/polymerase chain reaction (RT-PCR) was developed and evaluated for detecting a 400-bp product of the small RNA of sandfly fever virus, serotype Toscana (TOS). For more sensitive detection of genomic TOS RNA, a nested PCR amplifying a 243-bp cDNA within the RT-PCR product was established. Nucleotide sequence analysis of first- and second-round PCR products using the dideoxy cycle sequencing technique confirmed a previously published sequence of the TOS reference strain (ISS. Phl.3). By nested PCR, genomic TOS RNA was amplified from two consecutive sera taken 3 and 7 weeks after the onset of illness in one patient, and from CSF of a second patient obtained at the onset of meningitis. Authenticity of amplified PCR products was confirmed by nucleotide sequence analysis, revealing a sequence identical to the TOS reference strain. RT-PCR and nested PCR are useful for laboratory diagnosis and studies of the molecular epidemiology of TOS infection.

Resistance of HIV type 1 to proteinase inhibitor Ro 31-8959.

During replication of human immunodeficiency virus type 1 (HIV-1), proteolytic cleavage of Gag and Gag-Pol precursor proteins into different functional protein subunits is catalyzed by the viral proteinase, and this enzyme is the target of the antiviral proteinase inhibitor, Ro 31-8959. We investigated in vitro which HIV mutants with reduced sensitivity to Ro 31-8959 emerged during proteinase inhibition treatment; from three different HIV-1 strains, comparable progeny virus resistant to proteinase inhibitor were found, whereas the same experimental protocol detected no resistant HIV-2 mutants. Molecular analysis of the mutations underlying resistance revealed a multistep mechanism in which an amino acid exchange was common to all resistant isolates, and in all experiments preceded further exchanges at position 90 (leucine to methionine) and/or at position 54 (isoleucine to valine). For wild-type strains the 90% inhibitory concentrations of Ro 31-8959 were close to 20 nM, whereas HIV-1 mutants with all 3 amino acid exchanges had more than 50-fold increased 90% inhibitory concentrations (above 1000 nM). The primary event (Gly-48 to valine) occurs at the hinge of the flaps of the proteinase, thus hampering entry of the inhibitor to the active center and suggesting steric hindrance. Detailed knowledge of this stereotypic process could open inhibitor design, thus preventing conceivable escape of resistant virus on proteinase inhibitor action.

Mapping of immunodominant CD4+ T lymphocyte epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection.

In acute and chronic viral disease the specific response of CD4+ T lymphocytes to certain viral proteins is an essential part of antiviral effector mechanisms. In hepatitis C virus infection, the contribution of the immune system and particularly of CD4+ T lymphocytes to the pathogenesis of disease is unknown. We serially determined the peripheral blood CD4+ T lymphocyte response to several recombinant hepatitis C virus proteins (core, NS3, NS4, NS5) and 17 overlapping synthetic peptides derived from the core sequence over up to 48 months in 43 patients with chronic hepatitis C; of these, 16 had been treated with interferon alfa (IFN). Twelve of 27 untreated patients, 4 of 4 sustained responders to IFN, 7 of 8 patients with a transient response, and 1 of 4 nonresponders showed a proliferative response to hepatitis C virus proteins. The hepatitis C virus core protein was the most immunogenic protein, and fine analysis with peptides indicated amino acids 23 to 42, 66 to 85, and 131 to 150 as immunodominant regions. In a subgroup of nine patients, proliferation assays were performed before or during IFN. In this subgroup, sustained responders but not those with a transient or no response to IFN showed a specific CD4+ immune reaction to hepatitis C viral antigens (P < .05). Infection with hepatitis C virus genotype 3a was significantly associated with a sustained response to IFN (P < .05). In general, a CD4+ T lymphocyte response was more common in patients with chronic hepatitis C who responded to interferon-alpha as compared with nonresponders.(ABSTRACT TRUNCATED AT 250 WORDS)