HBsAg-negative mono-infection with hepatitis B virus genotype G.

Infection with a genotype G strain of hepatitis B virus (HBV-G) often occurs as a co-infection with HBV genotype A. In mono-infection with HBV-G, the production of hepatitis B surface antigen (HBsAg), HBe antigen and anti-HBe seems diminished, hampering the serological diagnosis of HBV-G mono-infection. To corroborate this notion, we studied in detail a series of samples of a blood donor with transient HBV-G infection. In this donor, during the temporary presence of HBV DNA and the seroconversion to HBcore antibodies (anti-HBc), no HBsAg or hepatitis B e antigen was detected. During follow-up, no anti-HBe appeared. Multiple resistance mutations to lamivudine were present, demonstrating primary infection with a resistant HBV strain. Cloning and sequencing indicated that no other HBV genotype but genotype G was present. Like other HBV-G isolates, the DNA sequence of the HBsAg a-determinant showed no mutations that could explain the failure to detect HBsAg. Our findings demonstrate that HBV genotype G mono-infection occurs and that routine serology is unsuitable for its detection.

The Influence of Neocate in Paediatric Short Bowel Syndrome on PN Weaning.

Clinical management of short bowel syndrome remains a multistage process. Although PN is crucial, early introduction of enteral feeding is mandatory. We describe retrospectively 4 patients with an ultrashort bowel who could be weaned off PN on very short terms after introduction of an amino-acid-based formula (Neocate). Patient 1 had congenital short bowel with 50 cm small bowel and 30 cm colon. He had persistent diarrhoea on a semielementary formula. When Neocate was introduced he could be weaned from PN within 6 months. Patient 2 needed multiple surgical interventions because of NEC at D 27. He maintained 40 cm small bowel and an intact colon and remained PN dependent on semielemental formula. After introducing Neocate, PN could be weaned within 3 months. In the next 2 patients, Neocate was introduced as initial enteral feeding after bowel resection following antenatal midgut volvulus. Patient 3 had 20 cm small bowel and an intact colon. PN was weaned after 2 months. Patient 4 had 9 cm small bowel and an intact colon. PN was weaned after 13 months. In all patients Ileocaecal valve (ICV) was preserved. No consensus is reached on the type of formula to use for short bowel syndrome. Compared to recent data in the literature, the weaning period in these 4 patients was significantly shortened on an aminoacid based formula. The reason for this may lie in the antiallergic properties of this formula. We recommend the use of an amino-acid-based formula to induce earlier weaning of PN.

Parvovirus B19 genotypes 1 and 2 detection with real-time polymerase chain reaction assays.

BACKGROUND AND OBJECTIVES: Parvovirus B19 (B19V) DNA screening has been introduced to comply with European regulations for certain plasma products. Current commercial and some in-house B19V DNA assays fail to detect or under-quantify the recently identified genotypes 2 and 3. In this report, we describe 2-year experience with B19V DNA screening using the commercial assay from Roche (detecting only genotype 1) combined with an in-house assay (detecting genotypes 1, 2 and 3). This dual testing approach enables the identification of molecular variants of B19V. MATERIALS AND METHODS: Between 2005 and 2007, approximately 2.6 million plasma donations were screened for B19V DNA loads exceeding 10(6) IU/ml using the Roche and the in-house real-time polymerase chain reaction assay. RESULTS: A total of 232 plasma units were identified with B19V DNA loads above 10(6) IU/ml. Concordant results were observed for the majority of B19V positive samples; however, three of these showed discrepant results between the two assay systems. One was a B19V genotype 2 strain not detected by the Roche assay; another was a B19V genotype 1 strain with a mismatch in the 3'-end of the reverse primer and therefore under-quantified by the Roche assay; and the third one was also a B19V genotype 1 strain that gave an unusual amplification plot in the in-house assay due to a mismatch in the probe-binding site. CONCLUSIONS: New, high viral load, B19V genotypes 2 and 3 infections are rare in blood donors tested by Sanquin. One case was found while testing 2.6 million donations. The prevalence of B19V genotype 1 variants not detected by commercial or in-house assays might be in the same range or even higher than the prevalence of B19V genotype 2 viruses, which remain undetected.

No evidence of West Nile virus infection in Dutch blood donors.

BACKGROUND AND OBJECTIVES: West Nile virus (WNV) can be transmitted by transfusion through infected blood components. This study aimed to determine the prevalence of WNV infection among Dutch blood donors to assess whether WNV is a possible threat for the Dutch blood supply. MATERIALS AND METHODS: Plasma samples from 61 992 blood donations were pooled in 7,749 test pools of eight donations using a Tecan robot. These samples were collected between April and October 2004. The pools were tested for the presence of WNV RNA by using the Procleix WNV assay. RESULTS: No WNV RNA-positive pools were detected. Based on Poisson distribution statistics, extrapolation of our data to all the Dutch donations in 2004 revealed that between 0 and 55 cases of WNV infection could be expected. CONCLUSIONS: No evidence of the presence of WNV RNA in Dutch blood donor samples from 2004 was found. However, surveillance of this emerging infection is of importance to safeguard the blood supply in the future because the transmission cycle of WNV is complex and hard to predict.

Evaluation of COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen HBV DNA, HCV RNA and HIV-1 RNA amplification and detection.

BACKGROUND AND OBJECTIVES: This report describes the evaluation of the COBAS AmpliPrep instrument for fully automated generic nucleic acid extraction in conjunction with hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, and human immunodeficiency virus (HIV)-1 RNA COBAS AmpliScreen amplification and detection using serial dilutions of the WHO international standards (IS) and the PeliCheck reference panels. MATERIALS AND METHODS: Serial diluted samples of the WHO IS and the PeliCheck reference panels were tested 24 times to determine the HBV DNA, HCV RNA and HIV-1 RNA detection limits by Probit analysis. The existence and extent of cross-contamination were assessed by testing alternating high titre HBV DNA-positive and -negative samples. The specificity of the AmpliPrep-AmpliScreen test for HBV was determined by testing 232 minipools consisting of six donations, all negative for HCV/HIV-1 nucleic acid testing (NAT) and HBsAg. In addition, a HBV genotypes A-G panel was tested. RESULTS: The respective 95% detection limits (and 95% CI) on the WHO IS and on the PeliCheck reference panels were 6.7 (4.3-13) IU/ml and 123 (68-301) gEq/ml for HBV DNA, 23 (11-106) IU/ml and 126 (84-233) gEq/ml for HCV RNA, and 187 (108-422) IU/ml and 183 (108-434) gEq/ml for HIV-1 RNA. Based on the WHO IS and the PeliCheck reference panels, no significant differences in sensitivity for HBV and HCV were found between AmpliPrep and the licensed MultiPrep extraction method. The sensitivity of AmpliPrep-AmpliScreen for HIV-1 was probably twofold lower as compared to the MultiPrep-AmpliScreen method. No cross contamination was observed. All 232 minipools were HBV NAT-negative. The AmpliPrep-AmpliScreen test for HBV detected HBV genotypes A-G with equal sensitivity. CONCLUSIONS: The AmpliPrep instrument combined with the AmpliScreen assays for HBV, HCV and HIV-1 is robust and suitable for NAT donor screening. The sensitivity criteria for HIV-1 and HCV as defined by the Paul Ehrlich Institute and the Food and Drug Administration for minipool NAT screening are met by this system. SINGLE SENTENCE SUMMARY: Generic COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen detection for HBV, HCV and HIV-1.

Multicenter performance evaluation of a transcription-mediated amplification assay for screening of human immunodeficiency virus-1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA in blood donations.

BACKGROUND: The performance of the recently launched Procleix Ultrio (Chiron/Gen-Probe) human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) blood screening assay was evaluated in a European multicenter study. STUDY DESIGN AND METHODS: Serial dilutions of reference materials were tested to determine the detection limits. Robustness and specificity were assessed by testing alternating high-load HCV RNA-positive and -negative samples, and 2912 test pools of eight donations. The added value of minipool and single-donation HBV nucleic acid testing protocols was compared to the currently used Prism (Abbott GmbH & Co. KG) hepatitis B surface antigen (HBsAg) and Auszyme (Abbott GmbH & Co. KG) dynamic HBsAg tests in 15 HBV seroconversion panels. RESULTS: The 95 percent detection limits (and 95% confidence interval [CI]) on the WHO International Standards was 26 (16-58) IU per mL for HIV-1 RNA, 4.6 (3.7-6.5) IU per mL for HCV RNA, and 11 (7.3-22) IU per mL for HBV DNA. No cross-contamination was observed. Testing 2912 pools of eight donations revealed 16 initial reactive samples; 11 were confirmed. The specificity after initial testing and percentage of invalid results were 99.83 and 0.48 percent, respectively. The HBV window-period (WP) reductions relative to HBsAg seroconversion in Prism and Auszyme dynamic HBsAg were, respectively, 6 days (95% CI, 3-8) and 9 days (95% CI, 7-12) in 1:8 minipool (MP) testing. CONCLUSION: The performance characteristics of Procleix Ultrio assay and the Procleix HIV-1 and HCV assay are comparable. The sensitivity for HIV-1 and HCV met the directives of the Paul-Ehrlich Institute and the FDA. The assay can reduce the WP for HBV by 6 days to 2 weeks when used in small MP (<1:8) or single-donation screening protocols.

Multicenter evaluation of the NucliSens EasyQ HIV-1 v1.1 assay for the quantitative detection of HIV-1 RNA in plasma.

The Nuclisens EasyQ HIV-1 v1.1 assay (Biomerieux) is a real-time detection method combined with NASBA technology designed to measure plasma HIV-RNA. Its performance was assessed in 1008 clinical specimens collected from individuals infected with clade B (774) and non-B (234) HIV-1 variants at four European laboratories. The results were compared with those obtained using three other commercial viral load assays: Cobas Amplicor Monitor HIV-1 v1.5 (Roche), Versant HIV-1 RNA assay (Bayer) and Nuclisens HIV-1 QT (Biomerieux). Overall, the linearity, specificity and reproducibility of the EasyQ assay was comparable with that from the other tests. The correlation coefficient (R) between methodologies was 0.85 for Amplicor; 0.87 for Versant; and 0.91 for Nuclisens. The specificity of the assay was 99.4%. Of note, Versant missed 17% of specimens with non-B subtypes which could be detected by EasyQ, while Amplicor provided similar results than EasyQ. HIV-1 group O specimens were only detected by the EasyQ assay. In conclusion, the performance of the EasyQ assay seems to be similar to that of other HIV-1 viral load tests currently on the market, but it is more sensitive than Versant for HIV-1 non-B subtypes and shows a wider dynamic range than Amplicor. Moreover, as it incorporates the advantage of real-time detection procedures, it facilitates high throughput and short turnaround time.

Quantitative real-time detection of parvovirus B19 DNA in plasma.

BACKGROUND: As of 2004, the European Pharmacopoeia demands that plasma pools for production of anti-D immunoglobulin should not contain more than 104 IU per mL of parvovirus B19 (B19V) DNA. Hence, before pooling, highly viremic donations have to be identified, and after pooling the level of B19V DNA must be determined. The performance of a new real-time B19V DNA PCR test (Roche, Mannheim, Germany) was studied, using a DNA extractor (NucliSens, bioMerieux, Boxtel, the Netherlands) for isolation of nucleic acid, and using a DNA quantification test (LightCycler apparatus, Roche, Mannheim, Germany) for amplification and detection. STUDY DESIGN AND METHODS: Dilutions of the international B19V DNA standard and reference preparations were tested to determine the precision, linear range, and accuracy of the assay and to calculate the factor for conversion of B19V DNA copies to IUs. The internal control signals, invalid test results, and the effect of cryo-poor plasma were studied as a measure for robustness. Routine performance was assessed by testing 164 manufacturing pools (not screened for B19V) and 1048 test pools of 480 donations each. RESULTS: The copies-to-IU conversion factor was calculated to be 3.34 (95% CI, 3.07-3.63). The assay appears linear between 10(3) and 10(7) IU per mL. Between 10(3) and 10(5) IU per mL, the test can discriminate samples differing a factor two in B19V DNA content. Overall, 0.78 percent of the test results were invalid. Of 127 B19V DNA negative control plasma samples, 7 were contaminated with low levels of B19V DNA. Of 164 nonscreened manufacturing plasma pools, 92 contained B19V DNA (56%); 13 contained more than 10(4) IU per mL. Of 503,040 donations, 29 contained more than 5 x 10(6) IU per mL B19V DNA (1:17,346). CONCLUSION: The B19V DNA quantification test (LightCycler, Roche ) is suitable for quantitative, routine, in-process measurement of B19V DNA levels in plasma pools, using the DNA extractor (NucliSens, bioMerieux) for nucleic acid isolation.

Sensitivity of HCV RNA and HIV RNA blood screening assays.

BACKGROUND: The FDA requirement for sensitivity of viral NAT methods used in blood screening is a 95-percent detection limit of 100 copies per mL, whereas the NAT screening system should have a sensitivity of at least 5000 copies per mL per individual donation. According to the Common Technical Specifications of the European Directive 98/79/EC for in vitro diagnostics, viral standard dilutions (calibrated against the WHO standard) should be tested at least 24 times for a statistically valid assessment of the 95-percent detection limit. STUDY DESIGN AND METHODS: Viral standard dilution panels (PeliCheck, VQC-CLB) were prepared for HCV RNA genotypes 1 and 3 and for HIV RNA genotypes B and E. In a multicenter study, 23 laboratories tested the panels all together in 8 to 91 test runs per NAT method. RESULTS: The following 95-percent detection limits (and 95% CIs) were found on the HCV RNA genotype 1 reference panels (shown as geq/mL): Gen-Probe TMA, 85 (64-118); AmpliScreen, 126 (83-225); AmpliScreen with NucliSens Extractor, 21 (13-44); Amplicor with NucliSens Extractor, 69 (50-102), and Amplicor with Qiagen extraction technology, 144 (74-102). On HIV RNA genotype B dilution panels, the following 95-percent detection limits were found (shown as geq/mL): Gen-Probe TMA, 31 (20-52); AmpliScreen, 126 (67-311); AmpliScreen with NucliSens Extractor, 37 (23-69), and NucliSens QL assay, 123 (51-566). HIV RNA genotype E panels were detected with equal sensitivity as HIV RNA genotype B panels. In the Gen-Probe TMA assay, the 50-percent detection limits on HIV RNA type B and type E were 3.6 (2.6-5.0) and 3.9 (2.4-5.8) geq per mL, respectively. The HCV RNA genotype 1 and 3 standards were detected with equal sensitivity. CONCLUSION: The differences in sensitivity between NAT assays can be explained by the input of isolated viral nucleic acid in the amplification reactions. The FDA requirements for sensitivity of NAT blood screening assays can be met by the Gen-probe TMA, as well as by the AmpliScreen assays, particularly when combined with the NucliSens Extractor.

Sustained virological response in chronic hepatitis C patients after a 6- and a 36-month interferon-alpha2b treatment schedule: a multicenter, randomized, controlled study.

BACKGROUND: In patients with chronic hepatitis C (HCV) Interferon-alpha (IFN) treatment for 12-18 months is more effective than 6 months in inducing a sustained virological response. METHODS: In a multicenter, randomized, controlled trial, 88 patients with chronic HCV were enrolled (47 treated with IFN-alpha2b and 41 constituted an untreated control group). Treatment consisted of 5 million units (MU) IFN thrice a week (tiw) for 8 weeks and subsequently 2.5 MU IFN tiw for 16 weeks ('standard treatment'). After week 24 ('long-term treatment'), in virological non-responders treatment was continued using 5 MU IFN tiw for up to week 156, whereas in virological responders IFN was discontinued. In case of a virological relapse, treatment with 5 MU IFN tiw was restarted and continued up to week 156. RESULTS: Sustained virological response rate was 6/47 (13%) after standard treatment and increased to 19/47 (40%) after long-term treatment (McNemar paired test; P = 0.002). Of the 18 patients with a breakthrough or relapse during or after standard treatment, 14 (78%) became sustained virological responders upon long-term treatment. Of the 4 patients who did not have a sustained virological response after long-term treatment, 3 did not receive complete treatment due to side effects and/or non-compliance. In patients who failed to respond to standard treatment, no virological response was observed during long-term treatment. In the control group, no spontaneous clearance of HCV was observed. CONCLUSIONS: Long-term IFN (re)treatment enhanced the virological sustained response rate significantly and was particularly effective in patients with a breakthrough or relapse following standard treatment.

A survey of liver pathology in needle biopsies from HBsAg and anti-HBe positive individuals.

AIMS: To use laboratory data and liver biopsies, prospectively obtained from hepatitis B surface antigen (HBsAg) and anti hepatitis B e antigen (anti-HBe) positive patients, for the assessment of: (1) the relation between biopsy length/number of portal tracts and sampling error; (2) the relation between the severity of piecemeal necrosis and the new grading terminology (minimal, mild, moderate, and severe chronic hepatitis); and (3) liver pathology, which has not been studied in patients with this specific serological profile. METHODS: The study group (n = 174) included 104 patients with normal aminotransferase concentrations and no cases with clinically apparent cirrhosis. The specimen length and number of portal tracts were measured at light microscopy examination. Sampling error analysis was related to the discrepancies between aminotransferase concentrations versus histological grade. Detailed histological scorings were undertaken by the reference pathologist and compared with laboratory and hepatitis B virus (HBV) DNA precore sequence data. RESULTS: Sampling error seemed to be a constant feature, even for biopsies > or = 20 mm, but increased dramatically in biopsies < 5 mm long and/or containing less than four portal tracts. Between 25% and 30% of biopsies, graded as "mild" or "moderate" activity showed features of moderate and severe piecemeal necrosis, respectively. Ten per cent of the patients with normal aminotransferase values had stage III-IV hepatic fibrosis, and 20% had piecemeal necrosis. Only cytoplasmic, not nuclear, core antigen expression was a strong predictor of high hepatitis B viraemia. There was no association between precore stop codon mutations, grade/stage of liver disease, and hepatitis B core antigen (HBcAg) expression. CONCLUSIONS: The specimen available for light microscopical examination should be > 5 mm long and should contain more than four portal tracts. In addition, the new grading terminology might give the clinician an inappropriately mild impression of the severity of piecemeal necrosis. Furthermore, even in the presence of normal aminotransferase concentrations, considerable liver pathology can be found in 10-20% of HBsAg and anti-HBe positive individuals; such pathology is not associated with the occurrence of precore stop codon mutations.

Characterization of the quantitative HCV NASBA assay.

A quantitative nucleic acid sequence-based amplification assay (NASBA-QT) for detection of hepatitis C virus RNA (HCV-RNA) was evaluated and compared with the HCV branched-DNA (bDNA) assay (Chiron Corporation) and the HCV MONITOR assay (Roche Diagnostic Systems). For this evaluation five panels were designed: (1) serial dilutions of genotype 1b in-vitro HCV-RNA; (2) standards of in-vitro HCV-RNA genotypes 1a, 1b, 2, 3, 4, and 5; (3) a proficiency panel consisting of 12 HCV-RNA positive plasma samples of different genotypes and HCV-RNA concentrations and a genotype 1a and 1b 3-fold dilution series; (4) a panel of 67 HCV-RNA positive plasma samples obtained from patients with HCV infection and (5) an HCV-RNA positive control sample, diluted 50-fold in 25 different HCV-RNA negative plasma samples. The quantitative detection limit was found to be 10(3) copies per 100 microl and the qualitative detection limit 10(2.3) per 100 microl. The amplification efficiency was independent of the plasma matrix, but dependent on the HCV genotype. The HCV NASBA-QT assay was more than 10 times as sensitive as the bDNA assay while the quantitative results of both assays were highly concordant. The HCV NASBA-QT assay was comparable in sensitivity with the HCV MONITOR assay, but the HCV MONITOR assay yielded consistently lower values. It is concluded that the HCV NASBA-QT assay is a reliable assay for quantitative HCV-RNA detection in various settings.

European multi-centre validation study of NucliSens Extractor in combination with HCV Amplicor 2.0 assay for HCV-NAT screening of plasma pools.

The NucliSens Extractor in combination with the 2.0 version of the Roche Cobas HCV Amplicor test has been validated by five European blood screening laboratories in a multi-centre study. For testing the performance characteristics of this HCV-NAT method, the European Pharmacopoeia validation guidelines were followed. The CLB VQC reference reagents were used for testing robustness and sensitivity. After a technical improvement in the extraction stations, the NucliSens Extractor appeared to be contamination-free as was proved by testing negative controls alternating with samples containing a high HCV-RNA concentration. The Pelicheck HCV-RNA genotype 1 dilution panel was tested 74 times in the five laboratories and an overall 95% detection limit of 80 genome equivalents (geq)/ml was found. In one laboratory the Pelicheck panel was tested in 25 runs and here a 95% detection limit of 32 geq/ml was achieved. In this laboratory the Pelispy HCV-RNA run control samples of 140 geq/ml were consistently picked up in all extractor stations. In addition the laboratories have tested a WHO HCV-RNA genotype 1 standard dilution series 39 times and a Pelicheck HCV-RNA genotype 3 reference panel in 32 test runs. The limiting dilution analysis enabled us to compare the detection efficiency of the NucliSens-Amplicor method for the genoype 1 and genotype 3 isolates and to calibrate the reference reagents against each other. The combined Nuclisens-Amplicor method was found to detect the genotype 3 isolate in the Pelicheck HCV-RNA panels with 2-3 fold lower efficiency than the genotype 1 standard (assuming that the historical calibration of the genotype 3 against the genotype 1 standard is correct). In this study of a single method 1 IU of the WHO HCV-RNA standard was found to be equivalent to 5.1 geq of the VQC HCV-RNA standard (95% confidence intervals 3.1-9.1 geq). To avoid confusion with the use of the CLB VQC reagents we accept the NIBSC collaborative study in which calibration by a variety of methods showed that the Pelispy 380 geq/ml run control is equivalent to 100 IU/ml of the WHO standard. This multi-centre validation study demonstrates that the 95% detection limit of the NucliSens HCV Amplicor method lies far below the detection limits required by the international regulatory bodies.

Hepatitis G virus RNA and hepatitis G virus-E2 antibodies in Dutch hemophilia patients in relation to transfusion history.

The prevalence of hepatitis G virus (HGV)-RNA and HGV-E2 antibodies was studied in a cohort of Dutch hemophilia patients in relation to clotting products used, age, and coinfection with hepatitis C. Between 1991 and 1995, blood samples were taken from 294 patients with hemophilia A, B, or von Willebrand disease. From each patient one fresh frozen sample was tested for HGV cDNA polymerase chain reaction (PCR) and HCV cDNA PCR. Alanine aminotransferase (ALT) tests were performed on plasma samples of all patients. The presence of HGV-E2 antibodies was tested on plasma samples from a subset of 169 patients representing all age groups. Based on the origin and viral safety of the products used, three subgroups of patients were distinguished. Group A: patients who used viral noninactivated factors derived from small and large donor pools; group B: patients who used factors prepared with inadequate viral inactivation techniques derived from small and large donor pools; and group C: patients treated only with optimally viral inactivated large pool clotting factor or recombinant clotting factor concentrate. The prevalence of HGV-RNA was 18%. In group A patients the prevalence was 71%, in group B 50%, and in group C 6%. When related to age, the highest prevalence of HGV-RNA (35%) was seen in patients born between 1980 and 1989. The prevalence of HGV-E2 antibodies increased with age. Of HGV-RNA-negative patients born before 1950, 96% tested positive. HGV viremia did not affect ALT levels, neither in HCV-RNA positive nor in HCV-RNA negative patients. HGV infection is frequently seen in patients with hemophilia. In older age groups a lower rate of HGV-RNA positivity is seen coinciding with a higher rate of antienvelope antibodies.

Stability of hepatitis C virus RNA during specimen handling and storage prior to NASBA amplification.

The influence of different anticoagulants and pre-amplification storage conditions on the stability of hepatitis C virus (HCV)-RNA, as detected by the quantitative HCV NASBA assay (NASBA-QT), was studied. The HCV-RNA load remained stable for at least 15 months when serum or plasma samples (EDTA and heparin) were directly frozen at -70 degrees C in lysis buffer. At 4 degrees C, the HCV-RNA load in serum or plasma stored with lysis buffer did not decline for at least 14 days. At 30 degrees C, however, the load declined significantly after 7 days. When clotted, whole blood was stored at 4 degrees C, the HCV-RNA load was stable for 72 h. However, when EDTA-anticoagulated whole blood was stored at 4 degrees C, the HCV-RNA load declined significantly after 48 h. In paired plasma and serum samples at baseline the HCV-RNA levels were similar. Heparin did not influence the efficiency of the HCV NASBA-QT assay. Clotted blood as well as EDTA or heparin anticoagulated blood can be used for quantifying HCV-RNA using the NASBA-QT assay. Blood samples should be stored at 4 degrees C after collection and serum or plasma separated within 24 h. Preferably, after separation, samples should be frozen in lysis buffer at -70 degrees C until NASBA-QT analysis.

Validation of a new immunoblot assay (LiaTek HIV III) for confirmation of human immunodeficiency virus infection.

BACKGROUND: Used as a supplemental assay, new anti-human immunodeficiency virus (HIV) immunoblots, employing recombinant and synthetic antigens, appeared to resolve the majority of samples with false-reactive Western blot results. Would it be possible to completely replace the Western blot by an immunoblot for confirmation and exclusion of HIV infection? STUDY DESIGN AND METHODS: The sensitivity of the new LiaTek HIV III immunoblot assay (Organon Teknika, Turnhout, Belgium) was tested on 416 Western-blot positive samples (386 HIV-1, 22 HIV-2, 1 HIV-1/2, and 7 HIV-O) and on 45 HIV-1 seroconversion samples. The specificity was tested on 146 samples from noninfected donors with false-positive results on a HIV screening test. RESULTS: All Western-blot-positive samples tested positive in the immunoblot (sensitivity: 100%). The immunoblot could not discriminate between HIV-1 and HIV-2 infection in 22 of 416 (5%) samples. The LiaTek assay showed reactivity in 28 of 45 seroconversion samples, whereas the Western blot reacted in 30 of 45 seroconversion samples. With false-positive donor samples, the immunoblot was indeterminate in 10 of 146 samples (specificity: 93%), and the Western blot was indeterminate in 44 of 146 samples (specificity: 70%). CONCLUSION: Like the Western blot, the immunoblot runs the risk of missing samples that are reactive by enzyme immunoassay during the early stage of HIV infection. Nevertheless, considering its superior specificity on false-positive donor samples, it appears that the immunoblot offers a cost-effective alternative to the Western blot assay for confirmation and exclusion of HIV infection.

Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen.

BACKGROUND: Hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B e antigen (anti-HBe) commonly coexist, and laboratory tests are often requested to assess histological hepatitis activity. An optimum panel of tests has not been found and the usefulness of hepatitis B virus (HBV) DNA assays in this context has not been established. We assessed various blood tests to find which best predicted hepatitis activity. METHODS: Routine plasma biochemical liver tests and serum HBV DNA (hybridisation and PCR assays) were assessed prospectively in 123 patients positive for HBsAg and anti-HBe. We scored histological hepatitis activity (hepatitis activity index) and determined whether chronic active hepatitis (chronic hepatitis with portal and periportal lesions) was present. We analysed the relation between laboratory data and the hepatitis activity index or risk of chronic active hepatitis by multiple regression and multiple logistic regression, respectively. FINDINGS: The analyses provided models for predicting either the hepatitis activity index or the risk of chronic active hepatitis. Aspartate aminotransferase was the most important test in the two models. The contribution of HBV DNA and other assays, especially alanine-aminotransferase activity, were of no practical importance. INTERPRETATION: Because screening by aspartate-aminotransferase activity could not be improved by the addition of other assays or HBV DNA, patients positive for HBsAg and anti-HBe could be screened for chronic active hepatitis with a single assay and counselling of patients can be improved if proper reference values are used.

New hepatitis B virus mutant form in a blood donor that is undetectable in several hepatitis B surface antigen screening assays.

BACKGROUND: Envelope mutant forms of hepatitis B virus (HBV), impairing HBV antibody recognition, have been reported with mutations in single or multiple sites of the hepatitis B surface antigen (HBsAg) group-specific "a" determinant. Blood donors infected with such an HBsAg mutant form of HBV may escape detection by HBsAg screening assays and therefore may affect the safety of the blood supply. CASE REPORT: A repeat blood donor became HBsAg-reactive in an enzyme immunoassay. Confirmatory testing yielded negative results for HBsAg in a radioimmunoassay and in four enzyme immunoassays used in blood donor screening. The specificity of the HBsAg reactivity in the first enzyme immunoassay was confirmed by HBsAg neutralization with antibody to HBsAg. Additional HBV confirmatory test results were positive for antibody to hepatitis B core antigen and antibody to hepatitis B e antigen; negative for antibody to HBsAg and for hepatitis B e antigen; and positive for HBV DNA. DNA sequence analysis of the "a" determinant region of HBsAg revealed amino acid substitutions from Q (Gln) to R (Arg) at codon 129 and from M (Met) to T (Thr) at codon 133. CONCLUSION: This case illustrates the presence of HBsAg mutant forms of HBV in a West European blood donor population that were undetected by several HBsAg screening assays. Adaptation of HBsAg screening is indicated to overcome deficiencies in sensitivity in detecting HBsAg mutant forms of HBV. Screening for antibody to hepatitis B core antigen or HBV DNA may also detect blood donors infected with HBsAg mutant forms of HBV