Evaluation of the new LCx HIV RNA quantitative assay: comparison with the Cobas Amplicor HIV Monitor assay.

In the present study we evaluated the performance of the new LCx HIV RNA quantitative kit (Abbott Laboratories, Delkenheim, Germany) for the quantitative detection of HIV-1 RNA in human plasma in comparison to the Cobas Amplicor HIV-1 Monitor assay (Roche Diagnostics, Branchburg, N.J.), including samples containing a variety of HIV-1 subtypes. LCx and Cobas were compared using archived EDTA plasma samples collected from HIV-infected patients. Considering the lower limit of the linear range of 50 copies/ml, the detection rate of the LCx was 139 out of 174 (79.9%) versus 131 out of 174 (75.3%) of the Cobas. Overall agreement was 95.4% (166/174) at a cut-off of 50 copies. LCx and Cobas results on clinical samples were found linearly associated (r2 = 0.900) and strongly correlated (r = 0.949). The mean viral load in the 174 frozen patient samples was 3.25 log10 copies/ml by LCx compared to 2.71 log10 copies/ml by Cobas. Considering only samples with a viral load > or =50 copies/ml, the average difference was -0.132 log copy/ml. Using a panel consisting of 9 plasma samples spiked with 9 different HIV-1 cultured isolates (A-H, and O) LCx detected the 9 subtypes with a high degree of precision, i.e., 9-33% coefficient of variation. As expected, the Cobas failed to detect the group O isolates. The results of the remaining samples showed a higher degree of variation (when testing four replicates of the subtype panel) than the LCx of 14.2-40.3%. Nevertheless, the results were comparable with the LCx data.

Lack of monomeric IgM anti-hepatitis C virus (HCV) core antibodies in patients with chronic HCV infection.

Antibodies of the IgM class against the hepatitis C virus core antigen are found in up to 70% of patients with either acute or chronic hepatitis C. The sedimentation rate of such IgM was analyzed by rate-zonal centrifugation of nine sera taken from seven patients, two acutely and five persistently infected with hepatitis C virus. All patients had circulating high-molecular weight (i.e. pentameric) IgM, indicating that the production of low molecular weight IgM, commonly observed in other persistent viral infections, does not apply to hepatitis C virus and cannot be used to distinguish acute from chronic hepatitis C virus infection.

Hepatitis E virus (HEV) infection in haemodialysis patients. The Multicentre Haemodialysis Cohort Study on Viral Hepatitis.

BACKGROUND: The aim of this study was to determine the prevalence of hepatitis E virus (HEV) infection among patients undergoing haemodialysis (HD) and to evaluate whether chronic haemodialysis is associated with an increased risk of HEV infection. METHODS: Serum samples from 420 HD patients and 316 healthy volunteers were tested for IgG and IgM antibodies to HEV (anti-HEV). Anti-HEV IgG positive sera were confirmed using synthetic peptides. RESULTS: Anti-HEV IgG was confirmed in 27/420 (6.4%) of the HD patients and in 7/316 (2. 2%) of the reference group (P=0.07). However, multiple logistic regression analysis showed that the prevalence of anti-HEV IgG was not significantly higher in HD patients compared with the reference group, after controlling for age and sex. No patient was found positive for anti-HEV IgM. The presence of anti-HEV was associated with sex in HD patients (P=0.04). No significant association was found between anti-HEV and underlying renal disease, anti-HCV, anti-HBc, blood transfusions, history of elevated transaminases, history of clinical hepatitis and renal transplantation. A marginal association, which was observed with the duration of haemodialysis in univariate analysis (P=0.07), was not confirmed in multivariate analysis. CONCLUSIONS: Chronic haemodialysis is not associated with an increased risk of exposure to HEV, and the high prevalence of anti-HEV IgG in HD patients reported in uncontrolled studies is possibly due to the confounding effect of age and sex.

Seroepidemiology of hepatitis E virus in the Egyptian Nile Delta.

The seroendemicity of hepatitis E virus (HEV) in an entire village population located in the Egyptain Nile Delta is described. Serum specimens were obtained from 68% of the total population of 1,850 villagers. The lack of serum specimen was greatest in the youngest age group (< 5). Commercially available enzyme immunoassays (EIA) for antibody to hepatitis A virus (anti-HAV), to hepatitis B virus core antigen (anti-HBc), to second-generation hepatitis C virus (anti-HCV) core and nonstructural antigen, and to hepatitis E virus (HEV) were used. Only repeated reactive sera were coded as positive. Stool specimens were examined for Schistosoma mansoni by the Kato method and standard methods for the examination of the liver and spleen by ultrasonography were used. Unadjusted for nonrespone, the seroprevalence of anti-HEV was 17.2% (SE +/- 1.1). Anti-HEV seroprevalence increased by age and was not associated statistically with any of the other viral markers including HCV. Anti-HAV seroprevalence was consistently > 95%, even in the youngest age group (< 5). The overall sero-endemicity of HEV was higher than reported elsewhere and appears not to have been introduced into the village population recently.

Hepatitis E virus infection in a cohort of patients with acute non-A, non-B hepatitis.

BACKGROUND/AIMS: The aim of this study was to determine the frequency of hepatitis E virus infection in a cohort of patients with acute non-A, non-B hepatitis in Greece. METHODS: Serial serum samples of 198 patients with acute non-A, non-B hepatitis and a single serum specimen from 316 healthy subjects were tested for IgG and IgM antibodies to hepatitis E virus (anti-HEV). RESULTS: Anti-HEV IgG was found in 15/198 (7.6%) of acute non-A, non-B hepatitis patients and 7/316 (2.2%) of healthy controls (p=0.007). Anti-HEV IgM was found in 2/198 (1.0%) acute non-A, non-B hepatitis patients and in none of the healthy subjects. Neither anti-HEV IgM (+) case reported any risk factor and neither had travelled in areas endemic for hepatitis E virus infection. HEV-RNA was detected by reverse transcription polymerase chain reaction in one patient. The prevalence of anti-HEV IgG was 7/45 (15.6%), 1/46 (2.2%), 5/30 (16.7%) and 2/77 (2.6%) in acute non-A, non-B hepatitis reporting transfusion, intravenous drug use, occupational/hospitalization, and unknown transmission, respectively (p=0.007). Anti-HEV IgG was found in 13/122 (10.7%) and 2/76 (2.6%) of acute non-A, non-B hepatitis patients positive and negative for anti-HCV, respectively (p=0.03). A similar association was found with anti-HBc (p=0.007). The prevalence of anti-HEV IgG was significantly higher in cases reporting transfusion [OR=7.3, 95% C.I. 1.4-37.7, p=0.017] and occupational/hospitalization [OR=6.8, 95% C.I. 1.2-38.2, p=0.029], as transmission category after controlling for age. CONCLUSIONS: These findings indicate that: (a) hepatitis E virus may be a cause - although not a frequent one - of sporadic or community-acquired acute non-A, non-B hepatitis in Greece; (b) hepatitis E virus may share transmission routes with hepatitis B and C viruses; and (c) the hypothesis that hepatitis E virus may be transmitted by parenteral routes deserves careful consideration.

Hepatitis E virus infection in acute hepatitis in Spain.

In order to study the prevalence of hepatitis E virus (HEV) infection in developed countries, IgG and IgM anti-HEV were determined in serum samples from 382 patients with acute viral hepatitis (244 hepatitis A, 48 hepatitis B and/or D, and 90 non-A, non-B, non-C hepatitis), 76 healthy subjects, 55 hemophiliacs and 50 patients on hemodialysis. IgG anti-HEV antibodies were detected and confirmed by a synthetic peptide-based EIA in 5 (5.6%) non-A, non-B, non-C acute hepatitis, in 3 (6.5%) B and D acute hepatitis, in 10 (4%) acute A hepatitis, in 3 (5.5%) of 54 healthy adults in none of the hemophiliacs and in 3 (6%) patients on hemodialysis. IgM anti-HEV antibodies were only detected in two cases of acute hepatitis B and/or D. Analysis of serial serum samples demonstrated IgG anti-HEV seroconversion in 3 of the 18 confirmed cases; one of them was also positive for IgM anti-HEV. All 3 acute anti-HEV-positive hepatitis cases occurred in adults, were community-acquired (two of them were intravenous drug addicts) and had a self-limited course. These results demonstrate that HEV is a minor cause of acute hepatitis in Spain. A similar low rate of IgG anti-HEV antibodies was detected in patients with different diseases, suggesting that HEV has a very low epidemiological impact. An apparent association of HEV infection with hepatitis B and D suggests a possible parenteral transmission of a mainly enteral pathogen.

Association between hepatitis C virus and hepatocellular carcinoma using assays based on structural and nonstructural hepatitis C virus peptides.

Stored sera from 181 Greek patients with hepatocellular carcinoma (HCC), 35 patients with metastatic liver cancer, and 416 hospital controls with diagnoses other than malignant neoplasm or liver disease were examined with first and second generation hepatitis C virus (HCV) enzyme immunoassays as well as with five HCV supplemental assays based on structural and nonstructural HCV peptides. Second generation HCV enzyme immunoassays were more sensitive than first generation assays. However, both assays had suboptimal specificity using the standard reactivity criterion (absorbance of sample to cutoff greater than or equal to 1.0). Specificity was improved by centrifugation and by using a sample's optical density to cutoff ratio greater than or equal to 3.0 or supplemental assays; in this instance the prevalence of antibodies to HCV was 13.3% (24 of 181), 0 (0 of 35), and 1.4% (6 of 416) in HCC, metastatic liver cancer, and hospital controls, respectively. A similar estimation of prevalence of antibody to HCV in HCC (12.5% or 4 of 32) was obtained when the recombinant immunoblot assay, second generation, was used to screen a random sample of HCC patients. The relative risk linking HCV to HCC was estimated as 10.4 (95% confidence interval, 4.2-26.0; P less than 0.0001). These data suggest that the prevalence of antibodies to HCV in HCC using stored sera has been previously overestimated even though the evidence of a causal association of HCV with HCC persists.

Abbott HCV EIA 2nd generation: a new screening assay.

An enzyme immunoassay for the detection of antibodies against structural and nonstructural domains of the hepatitis C virus (EIA-2) was compared to a first generation test (EIA-1) that can only detect antibodies against one nonstructural antigen (c100). EIA-2 revealed elevated detection rates as assessed in high risk and patient populations as well as lower repeat reactive rates in volunteer blood donors. Research test systems proved an increase in apparent specificity.

Antibody responses to hepatitis C virus by second-generation immunoassays in a cohort of patients with bleeding disorders.

The antibody responses and the prevalence patterns of antibodies to hepatitis C virus (anti-HCV) in a cohort of patients (n = 210) with bleeding disorders were studied using a first-generation and a second-generation enzyme immunoassays (EIA-1, EIA-2) as well as a second-generation recombinant immunoblot assay (RIBA-2). The anti-HCV prevalence as determined by EIA-1 and EIA-2 was 183/210 (87.1%) and 197/210 (93.8%), respectively (p = 0.0026). None of the 17 EIA-2(+)/EIA-1(-) samples was scored nonreactive by RIBA-2. At follow-up, samples of 123 patients were tested. Twenty-nine out of 111 patients reactive by EIA-1 seroreverted according to EIA-1 while the seroreversion rate with EIA-2 was 0 out of the 121 (p < 10(-8)). The anti-HCV prevalence by EIA-2 was 150/154 (97.4%) in anti-HIV-1-positive individuals and 47/56 (83.9%) in the anti-HIV-1-negative ones (p = 0.001). However, high assay signals (OD 492 nm > 2.0) were observed in 94/150 (62.7%) and 45/47 (95.7%) of the anti-HIV-1-positive and -negative patients, respectively (p = 10(-5)). The decreasing anti-HCV reactivity among anti-HIV-1-positive individuals was mainly due to diminishing c33c reactivity. Seroconversion to anti-HCV was observed in 3/7 (42.9%) cases with acute icteric non-A, non-B hepatitis by both EIA-1 and EIA-2, while the remaining 4 cases had detectable levels of anti-HCV 1-18 months before the acute episode.

Rapid and automated identification of Enterobacteriaceae with the abbott MS-2 system and API-20E versus conventional methods.

The ability of the MS-2 (Abbott Laboratories, Diagnostic Division, Dallas, Texas) to rapidly identify Enterobacteriaceae, was evaluated. The results of the MS-2 and of the Analytab API-20E test strip were compared with those obtained from conventional tubed media. The MS-2 and API-20E completely agreed with the conventional method for 92% and 95% of the organisms, respectively. Only 0.5% (MS-2) to 0% (API-20E) of the organisms tested led to complete disagreement. Approximately 6% (MS-2) and 3.5% (API-20E) of the organisms would have shown complete agreement if additional tests had been done. For 1.5% of the organisms the octal code could not be found in the API-20E profile index. It was concluded that the MS-2 system provides a rapid (5 to 6 h) and accurate method for the routine identification of Enterobacteriaceae.

RIT 2214, a new biosynthetic penicillin produced by a mutant of Cephalosporium acremonium.

A number of lysine-requiring auxotrophs of Cephalosporium acremonium were investigated for incorporation of side-chain precursors and for accumulation of beta-lactam compounds. One of the auxotrophs, Acremonium chrysogenum ATCC 20389, producing cephalosporin C and penicillin N only if grown in media supplemented with DL-alpha-amino-adipic acid (DL-alpha-AAA), was found to use L-S-carboxymethylcysteine (L-CMC) as a side-chain precursor for the synthesis of a new penicillin (RIT 2214). No corresponding cephalosporin was detected. The penicillin present in the culture filtrate, was concentrated by adsorption on activated carbon and successive column chromatography on Amberlite IRA-68 and Amberlite XAD-4. Final purification was achieved by cellulose column chromatography. RIT 2214 was identified as 6-(D)-[(2-amino-2-carboxy)-ethylthio]-acetamido]-penicillanic acid by spectral analysis, bioactivity spectrum, elucidation of side-chain structure and finally by semisynthesis. Its biological properties were also evaluated.