[show abstract][hide abstract] ABSTRACT: BACKGROUND: The second triplex transcription-mediated amplification (TMA) assay version (Ultrio Plus, Novartis Diagnostics) uses an additional reagent enhancing the disruption of hepatitis B virus (HBV) particles and release of DNA for the target capture probe. This study compares the performance of this new assay version with the previous one (Ultrio). STUDY DESIGN AND METHODS: For analytical sensitivity assessment the World Health Organization HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) international standards and various genotype dilution panels were used. Individual donations (IDs) from 9980 first-time donors were screened simultaneously by serology and both TMA assay versions. RESULTS: The 50 and 95% limits of detection (LODs) for HBV using Ultrio Plus were 0.8 (0.6-1.0) and 4.6 (3.2-7.2) IU/mL, respectively, 2.4 (1.4-4.8)-fold more sensitive than Ultrio. The TMA assay versions had comparable LODs for HIV-1 and HCV. The improvement factors on analytical sensitivity panels of HBV Genotypes A to G ranged from 1.3 to 7.3 and 50% LODs (95% confidence interval) reduced from 12.5 (10-15) to 3.8 (3.2-4.4) copies/mL. One Ultrio Plus HBV Genotype D yield sample missed by the Ultrio assay in the donor screening study was detected with ninefold higher sensitivity. The specificities of ID nucleic acid test (ID-NAT) and serologic testing in a similar repeat test algorithm were 100 and 99.41%, respectively. CONCLUSION: More efficient target capture chemistry in the new TMA assay version significantly improved sensitivity and diminished variability in detecting HBV strains of various genotypes. We recommend a triplicate ID-NAT repeat test strategy to eliminate discriminatory tests on false-non-repeat-reactive (anti-HBc-nonreactive) donations.
[show abstract][hide abstract] ABSTRACT: Since 2004 Polish blood donors have been tested for parvovirus B19 (B19V) DNA. The screening testing has been performed in donors of plasma for fractionation and anti-D and anti-HBs production and donors of erythrocytes used for immunization. AIM is to present methods of the testing, quality control and results in period 2004-2010. MATERIAL AND METHODS: Testing was performed in individual donation testing (IDT) in Regional Blood Transfusion Center (RBTC) in Lublin or in pools of 24 in Institute of Haematology and Transfusion Medicine in Warsaw (IHTM). Quantitative testing with real-time PCR was preceded with nucleic acid isolation on silica based methods (Prepito Viral DNA/RNA, Chemagen and QIAamp DNA Mini Kit, QIAGEN). Amplification was performed initially with home made method and later with commercial assay (Artus Parvo B19 RG PCR Kit on Rotor Gene 6 000). In total 17 625 donations were tested: 8 539 in pools and 9 090 individually. Beside routine external quality control programmes in which both laboratories participated (Proficiency Study VQC,Amsterdam, Holand; EQA Programe, Glasgow, Scotland), panel containing negative samples, positive with very high DNA B 19V level and plasma infected with genotype 2 was prepared for RBTC in Lublin. RESULTS: B19V infection frequency was 1:980 donations, low viraemic donations were detected most frequently (1:1 037). It was identified only one donation with DNA load that could cause potential health risk for plasma product recipients (1:17 625). In one of the donors B 19V DNA was observed for 3 years and 3 months. In acute or persistent phase of infection no clinical or laboratory symptoms (morphology of peripheral blood, ALT) were observed. Due to risk of underestimation of viral load connected with viral genome polymorphism all donations with B19V positive result were not allowed to be clinically used.
[show abstract][hide abstract] ABSTRACT: Hepatitis B virus (HBV) genotypes have distinct geographical distributions and influence severity of clinical outcome and response to antiviral therapies. HBV polymorphism in HBV surface antigen (HBsAg) positive first time blood donors from Poland was examined. HBV serological markers and HBV DNA were tested in 170 samples. Whole genome (n = 53) or specific region sequences: pre-S/S and basic core promoter/precore (BCP/PC) region (91 and 154 samples, respectively) were phylogenetically analyzed. The median age of infected donors was 21 years. Anti-HBs, anti-HBe and hepatitis B e antigen were detected in 5%, 92.4% and 10.5% of tested donors, respectively. The HBV DNA load ranged between unquantifiable and 3.1 x 10(10) IU/mL (median: 4.10 x 10(3) IU/mL). Genotypes A2 (81.2%) and D (18.8%) co-circulated. Phylogenetic analyses revealed differences between the genotypes. Viral load and level of HBsAg tended to be lower in genotype D. The median HBsAg/HBV DNA ratio expressed in IU/mL was one for both genotypes, but very low or very high ratios appeared more frequent in genotype D infections. Higher amino acid variability in the surface proteins (median: 4%vs 1.5%; P = 0.01) and in the major hydrophilic region was observed in genotype D (P = 0.01). BCP/PC region analysis revealed the double mutation 1762T/1764A in 49/125 (39.2%) genotype A2 and 6/29 (20.7%) genotype D strains (P = 0.08). Mutations in PC and BCP regions correlated neither with HBsAg nor HBV DNA levels. HBV genotype A2 is dominant in HBsAg positive donors in Poland. Minority genotype D strains are significantly more substituted than genotype A2 strains potentially affecting the course of infection.
Journal of Viral Hepatitis 09/2009; 17(6):444-52. · 3.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nucleic acid testing (NAT) for hepatitis B virus (HBV) has been performed in Poland since 2005 on samples seronegative for hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (anti-HCV), and anti-human immunodeficiency virus (anti-HIV). Tools included 24-donation pool testing (PT) using Cobas Amplicor or in individual donations (ID) by Procleix Ultrio. Seven of 761,666 (1:108,800) and 21/250,191 (1:11,900) HBV DNA-positive donations were identified and confirmed by alternative methods. HBV DNA load ranged between 11.6 and 4.6 x 10(4) IU/mL in 11 samples and could not be quantified in 17 samples. HBV genotypes A (56%) and D (4%) were found. The analysis of combined results from index, follow-up, and look-back samples identified four groups: (1) Two cases tested HBsAg positive with alternative, more sensitive, assays; (2) Four cases were in the pre-seroconversion window period; (3) Eight cases had a fluctuating pattern of HBV DNA and anti-HBs detection (recovered infection); and (4) twelve cases carried anti-HBc without anti-HBs, which might correspond to either chronic or recovered "occult" HBV infection. One donor with no HBV markers in the follow-up was excluded, and another was in the window period preceding anti-HBs. HBV NAT identified more confirmed positive donors than HCV or HIV NAT, and 1:250,000 could not be detected by anti-HBc screening. Serological and molecular studies on follow-up and look-back samples are important to classify donors. In conclusion, further studies are needed to determine whether the considerably higher yield of HBV DNA detection obtained with individual donation screening improves blood safety compared with anti-HBc screening.
[show abstract][hide abstract] ABSTRACT: To improve the blood transfusion safety, according to the international recommendations anti-HCV negative blood donors are screened for HCV RNA. For decreasing the costs, the pools of 48 donor samples are tested. Until now 2,500,000 Polish donors were tested and HCV RNA was detected in 40. Anti-HCV was detected in plasma of all donors available for follow up for more than three months. No clinical symptoms were observed but in 20 donors slightly elevated ALT level was found. In the follow up, after detection of anti-HCV in 9/11 donors elevated ALT (167-2043 U/l) was observed. In two donors, out of 4 observed for more than 12 months HCV RNA was not detected in the follow up whereas anti-HCV were still present. In 7 donors probable source of infection was identified. The early detection of HCV infection is a serious challenge not only to transfusion medicine but also to general practitioners and hepatologists due to the necessity of further monitoring and treatment of infected individuals.
Polski merkuriusz lekarski: organ Polskiego Towarzystwa Lekarskiego 11/2004; 17(100):321-5.
[show abstract][hide abstract] ABSTRACT: Summary Background: The aim of the study was to investigate the sensitivity of Procleix HIV1/HCV Assay for HIV RNA and HCV RNA detection, and to analyse the frequency of viral nucleic acid detection in blood donors with various EIA test results. Material and methods: The 95% sensitivity of the Procleix test was 6.2 IU HCV RNA /ml and 44.5 IU HIV RNA /ml. HCV RNA was detected in 71/392 (18.1%) anti-HCV positive and HIV RNA in 21/557 (3.8%) EIA HIV-reactive donors. The frequency of HCV RNA detection correlated with signal/cut-off ratios (S/C ratio) of EIA. HCV RNA was found in 68/105 (64.8%) of the donors if it was >4, in 1/85 (1.2%) if it was between 2.00 and 3.99, and in 2 out of 202 donors (1%) if it was between 1.00 and 1.99. HIV RNA was detected in all 21 blood donors positive in EIA and Western Blot. Results and conclusions: The study demonstrated that Procleix HIV1/HCV Assay is very sensitive and can be used in the reference laboratory to confirm active infection in donors with positive results. We observed a low frequency of active infection in Polish blood donors with repeated reactive results in HCV and HIV EIA. The high S/C ratio value (> 4) of EIA is a good predictor of HCV RNA detection, but HCV RNA can also be detected in single donors with low ratio values of the EIA test.