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Paturel A, Casuscelli di Tocco F, Bousquet D, Plissonnier ML, Grand X, Tak H, Berby F, Scholtès C, Testoni B, Zoulim F, Levrero M. A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B. JHEP Rep 2024; 6:101124. [PMID: 39328324 PMCID: PMC11424956 DOI: 10.1016/j.jhepr.2024.101124] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Revised: 05/16/2024] [Accepted: 05/23/2024] [Indexed: 09/28/2024] Open
Abstract
Background & Aims Circulating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of covalently closed circular DNA (cccDNA) and may help to evaluate HBV treatment activity. Different research assays have been proposed and, although two PCR-based research use only investigational assays have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays. Methods HBV RNA-producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 unit length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP domain (Y63F) of the polymerase, and the ε-loop of the pregenomic (pg)RNA (mutation A1G). Results The clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virion-like particles and only a minority in small extracellular vescicles. Nanopore sequencing of 5'RACE products shows that the majority of the Huh7-3D29-secreted HBV RNAs start at the 5' end of pgRNA and pgRNA-derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from one flask. Conclusion We generated a clonal cell line that produces high quantities of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assay calibration. Impact and implications Several investigational assays and two research use only assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of covalently closed circular DNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNA quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients' sera (e.g. pgRNA, HBx transcripts). This new RNA standard can be used to calibrate all PCR-based assays for circulating HBV RNA quantification to evaluate, in a non-invasive manner, the size of the transcriptionally active cccDNA pool and the activity of novel strategies aimed at curing HBV infection.
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Affiliation(s)
- Alexia Paturel
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
- University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France
| | - Francesca Casuscelli di Tocco
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
- University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France
| | - Delphine Bousquet
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
- University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France
| | - Marie-Laure Plissonnier
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
| | - Xavier Grand
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
- University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France
| | - Hyosun Tak
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
- University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France
| | - Françoise Berby
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Department of Hepatology, Hospices Civils de Lyon, France
| | - Caroline Scholtès
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
- University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France
- Laboratoire de Virologie, Institut des Agents Infectieux, Hospices Civils de Lyon, Lyon, France
| | - Barbara Testoni
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
| | - Fabien Zoulim
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
- University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France
- Department of Hepatology, Hospices Civils de Lyon, France
| | - Massimo Levrero
- IHU Lyon, Lyon Hepatology Institute, Lyon, France
- Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France
- University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France
- Department of Hepatology, Hospices Civils de Lyon, France
- Department of Internal Medicine, SCIAC and the IIT Center for Life Nanoscience, Sapienza University, Rome, Italy
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Jung Y, Song J, Park HG. Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification. Sci Rep 2021; 11:8399. [PMID: 33863981 PMCID: PMC8052315 DOI: 10.1038/s41598-021-87948-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2020] [Accepted: 03/24/2021] [Indexed: 02/02/2023] Open
Abstract
Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5' phosphorothioate (PS)-modified overhang, a target recognition site, and a 3' self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3' end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3' end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.
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Affiliation(s)
- Yujin Jung
- Department of Chemical and Biomolecular Engineering (BK 21+ Program), KAIST, Daehak-ro 291, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Jayeon Song
- Department of Chemical and Biomolecular Engineering (BK 21+ Program), KAIST, Daehak-ro 291, Yuseong-gu, Daejeon, 34141, Republic of Korea
| | - Hyun Gyu Park
- Department of Chemical and Biomolecular Engineering (BK 21+ Program), KAIST, Daehak-ro 291, Yuseong-gu, Daejeon, 34141, Republic of Korea.
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Salama II, Sami SM, Elserougy SM, Emam HM, Salama SI, Elhariri HM, Hemeda SA, Hassanain AI, Abdel Mohsen AM, Fouad WA, El Etreby LA, Said ZN. Humoral Immune Memory to Hepatitis B Vaccine after Primary Vaccination of Children and Adolescents in Assiut, Egypt. Oman Med J 2020; 35:e175. [PMID: 33083033 PMCID: PMC7538637 DOI: 10.5001/omj.2020.117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2020] [Accepted: 04/14/2020] [Indexed: 02/05/2023] Open
Abstract
Objectives We sought to assess the prevalence of hepatitis B virus (HBV) seroprotection among vaccinated children in the Assiut governorate, Egypt, and assess a booster dose immune memory response among non-seroprotected children. Methods Using a multistage cluster sample, 566 children were recruited from three clusters: one urban and two rural. Children were aged from nine months to 16 years old. All participants received the full three doses of the compulsory HBV vaccine during infancy. Serum hepatitis B surface antigen (HBsAg), total anti-hepatitis B core (anti-HBc) antibodies, and quantitative detection of anti-HBs were measured using enzyme-linked immunosorbent assay. Repeatedly positive samples for HBsAg/anti-HBc were submitted for quantitative HBV DNA detection using real-time polymerase chain reaction. Non-seroprotective participants (anti-HBs < 10 IU/L) were given a booster dose of HBV vaccine. Two weeks later, a blood sample was taken from each child to assess an anamnestic response. Results The seroprotection rate was 53.2%, and only two children had HBV breakthrough infection (0.4%) with positive serum anti-HBc and HBV DNA. Age was the only significant predictor for non-seroprotection with an adjusted odds ratio (OR) of 3.2, 9.4, and 9.9 among children aged 5-10, 11-15, and > 15 years, respectively, compared to younger children (p < 0.001). About 85% of non-seroprotected children developed an anamnestic response after receiving the booster dose, and 84.3% of responders had a good response (3 100 IU/L). Undetectable pre-booster titer was found to be the only risk factor for non-response to booster with OR = 3.2 (p < 0.010). About 95.7% of children who were not responding to booster dose developed immune response after receiving the three doses of HBV vaccine. Conclusions Older age of children was the only significant predictor for HBV non-seroprotection. High anamnestic response rate signifies the presence of immune memory with long-term protection despite the waning of anti-HBs over time. However, some children with pre-booster undetectable anti-HBs titers may be unable to develop anamnestic response, and a second vaccination series might be necessary for HBV protection for these children.
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Affiliation(s)
- Iman I Salama
- Community Medicine Research Department, National Research Center, Cairo, Egypt
| | - Samia M Sami
- Child Health Department, National Research Center, Cairo, Egypt
| | - Safaa M Elserougy
- Department of Environmental and Occupational Medicine, National Research Center, Cairo, Egypt
| | - Hanaa M Emam
- Dermatology and Venereology, National Research Center, Cairo, Egypt
| | - Somaia I Salama
- Community Medicine Research Department, National Research Center, Cairo, Egypt
| | - Hazem M Elhariri
- Community Medicine Research Department, National Research Center, Cairo, Egypt
| | - Samia A Hemeda
- Community Medicine Research Department, National Research Center, Cairo, Egypt
| | | | - Aida M Abdel Mohsen
- Community Medicine Research Department, National Research Center, Cairo, Egypt
| | - Walaa A Fouad
- Community Medicine Research Department, National Research Center, Cairo, Egypt
| | - Lobna A El Etreby
- Community Medicine Research Department, National Research Center, Cairo, Egypt
| | - Zeinab N Said
- Microbiology and Immunology Department, Faculty of Medicine (for Girls), Al-Azhar University, Cairo, Egypt
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Standardization of Nucleic Acid Tests: the Approach of the World Health Organization. J Clin Microbiol 2019; 57:JCM.01056-18. [PMID: 30257900 DOI: 10.1128/jcm.01056-18] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The first World Health Organization (WHO) international standards (ISs) for nucleic acid amplification techniques were established two decades ago, with the initial focus on blood screening for three major viral targets, i.e., hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1. These reference materials have subsequently found utility in the diagnosis and monitoring of a wide range of infectious diseases in clinical microbiology laboratories worldwide. WHO collaborating centers develop ISs and coordinate international studies for their evaluation. The WHO Expert Committee on Biological Standardization is responsible for the endorsement of new standardization projects and the establishment of new and replacement ISs. Potencies of ISs are defined in international units (IU); the reporting in IU for assays calibrated with an IS (or secondary standards traceable to the IS) facilitates comparability of results for different assays and determination of assay parameters such as analytical sensitivities.
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Salama II, Sami SM, Said ZN, Salama SI, Rabah TM, Abdel-Latif GA, Elmosalami DM, Saleh RM, Abdel Mohsin AM, Metwally AM, Hassanin AI, Emam HM, Hemida SA, Elserougy SM, Shaaban FA, Fouad WA, Mohsen A, El-Sayed MH. Early and long term anamnestic response to HBV booster dose among fully vaccinated Egyptian children during infancy. Vaccine 2018. [PMID: 29530634 DOI: 10.1016/j.vaccine.2018.02.103] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
OBJECTIVE To evaluate early and long term anamnestic response to a booster dose of HBV vaccine among non-seroprotected children. SUBJECTS AND METHOD A national community based project was carried out on 3600 children aged 9 months to 16 years, fully vaccinated during infancy. They were recruited from 6 governorates representing Egypt. It revealed that 1535 children (42.8%) had non sero-protective anti-HBs (<10 IU/L) and were HBsAg or anti-HBc negative. A challenging dose of 10 μg of mono-valent Euvax HBV vaccine was given to 1121/1535 children. Quantitative assessment of anti-HBs was performed to detect early (2-4 weeks) and long term (one year) anamnestic responses. RESULTS Early anamnestic response developed among 967/1070 children (90.3%).Children having detectable anti-HBs (1-9 IU/L) significantly developed early anamnestic response (90%) compared to 85% with undetectable anti-HBs (<1 IU/L), P < 0.001. Multiple logistic analysis revealed that undetectable anti-HBs, living in rural residence and children aged 15-16 years were the most significant predicting risk factors for the absence of early anamnestic response (<10 IU/L), with AOR 2.7, 2.7 & 4.7 respectively. After one year, long term anamnestic response was absent among 15% of children who previously showed early response. Poor early anamnestic response and undetectable pre-booster anti-HBs were the significant predicting risk factors for absent long term anamnestic response, with AOR 18.7 & 2.7 respectively. CONCLUSION Immunological memory for HBV vaccine outlasts the presence of anti- HBs and HBV vaccination program provides effective long term protection even in children showing waning or undetectable concentrations of anti-HBs. This signifies no need for a booster dose especially to healthy children.
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Affiliation(s)
- Iman I Salama
- Community Medicine Research Department, National Research Centre, Egypt.
| | - Samia M Sami
- Child Health Department, National Research Centre, Egypt
| | - Zeinab N Said
- Microbiology and Immunology Department, Faculty of Medicine (for Girls), Al-Azhar University, Cairo, Egypt
| | - Somaia I Salama
- Community Medicine Research Department, National Research Centre, Egypt
| | - Thanaa M Rabah
- Community Medicine Research Department, National Research Centre, Egypt
| | | | | | - Rehan M Saleh
- Community Medicine Research Department, National Research Centre, Egypt
| | | | - Ammal M Metwally
- Community Medicine Research Department, National Research Centre, Egypt
| | | | - Hanaa M Emam
- Dermatology and Venereology Department, National Research Centre, Egypt
| | - Samia A Hemida
- Community Medicine Research Department, National Research Centre, Egypt
| | - Safaa M Elserougy
- Environmental and Occupational Medicine Department, National Research Centre, Egypt
| | | | - Walaa A Fouad
- Community Medicine Research Department, National Research Centre, Egypt
| | - Amira Mohsen
- Community Medicine Research Department, National Research Centre, Egypt
| | - Manal H El-Sayed
- Pediatrics Department, Faculty of Medicine, Ain-Shams University, Cairo, Egypt
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Complete Genome Sequence of the WHO International Standard for Hepatitis B Virus DNA. GENOME ANNOUNCEMENTS 2017; 5:5/7/e01576-16. [PMID: 28209818 PMCID: PMC5313610 DOI: 10.1128/genomea.01576-16] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
The World Health Organization (WHO) international standard (IS) for hepatitis B virus (HBV) DNA for use in nucleic acid amplification assays was characterized by determining the complete genome sequence, which was assigned genotype A. This information will aid the design, development, and evaluation of HBV DNA amplification assays.
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A collaborative study to establish the 3rd WHO International Standard for hepatitis B virus for nucleic acid amplification techniques. Biologicals 2017; 46:57-63. [PMID: 28082100 DOI: 10.1016/j.biologicals.2016.12.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2016] [Revised: 12/01/2016] [Accepted: 12/12/2016] [Indexed: 11/21/2022] Open
Abstract
Nucleic acid amplification techniques (NAT) are routinely used for clinical diagnostics and monitoring hepatitis B virus (HBV) infections, and are implemented on a voluntary basis for blood screening. A collaborative study was performed to evaluate a replacement WHO International Standard for HBV for the standardization of NAT. Two lyophilised HBV candidates were evaluated by 16 laboratories worldwide, alongside the existing HBV International Standard. The overall mean potency estimates for the candidate samples 1 and 2, relative to sample 3 (2nd HBV International Standard), from quantitative assays, were 5.93 and 5.98 log10 International Units (IU)/mL respectively. The variability in individual laboratory mean estimates for samples 1-3 for quantitative assays was ∼0.3 log10 IU/mL. The inter-laboratory variability for qualitative assays was higher. Accelerated thermal degradation studies indicate that both lyophilised candidates are stable and suitable for long-term use. Overall, the results suggested that both candidates were suitable as replacement International Standards. Sample 1 (NIBSC code 10/264) was established as the 3rd WHO International Standard for HBV for NAT with an assigned potency of 850,000 IU/mL (∼5.93 log10 IU/mL), when reconstituted in 0.5 mL of nuclease-free water. It is intended for the calibration (in IU) of secondary reference materials used in HBV NAT.
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Jia J, Ma Y, Zhao X, Huangfu C, Zhong Y, Fang C, Fan R, Lv M, Zhang J. Existence of various human parvovirus B19 genotypes in Chinese plasma pools: genotype 1, genotype 3, putative intergenotypic recombinant variants and new genotypes. Virol J 2016; 13:155. [PMID: 27639978 PMCID: PMC5027099 DOI: 10.1186/s12985-016-0611-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2016] [Accepted: 09/09/2016] [Indexed: 12/31/2022] Open
Abstract
Background Human parvovirus B19 (B19V) is a frequent contaminant of blood and plasma-derived medicinal products. Three distinct genotypes of B19V have been identified. The distribution of the three B19V genotypes has been investigated in various regions or countries. However, in China, data on the existence of different B19V genotypes are limited. Methods One hundred and eighteen B19V-DNA positive source plasma pool samples collected from three Chinese blood products manufacturers were analyzed. The subgenomic NS1/VP1u region junction of B19V was amplified by nested PCR. These amplified products were then cloned and subsequently sequenced. For genotyping, their phylogenetic inferences were constructed based on the NS1/VP1-unique region. Then putative recombination events were analyzed and identified. Results Phylogenetic analysis of 118 B19V sequences attributed 61.86 % to genotype 1a, 10.17 % to genotype 1b, and 17.80 % to genotype 3b. All the genotype 3b sequences obtained in this study grouped as a specific, closely related cluster with B19V strain D91.1. Four 1a/3b recombinants and 5 new atypical B19V variants with no recombination events were identified. Conclusions There were at least 3 subtypes (1a, 1b and 3b) of B19V circulating in China. Furthermore, putative B19V 1a/3b recombinants and unclassified strains were identified as well. Such recombinant and unclassified strains may contribute to the genetic diversity of B19V and consequently complicate the B19V infection diagnosis and NAT screening. Further studies will be required to elucidate the biological significance of the recombinant and unclassified strains. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0611-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Junting Jia
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China
| | - Yuyuan Ma
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China.
| | - Xiong Zhao
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China
| | - Chaoji Huangfu
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China
| | - Yadi Zhong
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China
| | - Chi Fang
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China
| | - Rui Fan
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China
| | - Maomin Lv
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China
| | - Jingang Zhang
- Laboratory for Viral Safety of National Centre of Biomedical Analysis, Beijing Institute of Transfusion Medicine, No. 27 Taiping road, Haidian District, Beijing, 100850, China.
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Datta S, Budhauliya R, Chatterjee S, Veer V, Chakravarty R. Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR). Mol Diagn Ther 2016; 20:297-305. [PMID: 26993322 DOI: 10.1007/s40291-016-0195-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. OBJECTIVES This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. METHODS Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. RESULTS Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. CONCLUSIONS This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.
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Affiliation(s)
- Sibnarayan Datta
- Molecular Virology Laboratory, Defence Research Laboratory (DRDO), Tezpur, Assam, 784001, India.
| | - Raghvendra Budhauliya
- Molecular Virology Laboratory, Defence Research Laboratory (DRDO), Tezpur, Assam, 784001, India
| | - Soumya Chatterjee
- Molecular Virology Laboratory, Defence Research Laboratory (DRDO), Tezpur, Assam, 784001, India
| | - Vijay Veer
- Molecular Virology Laboratory, Defence Research Laboratory (DRDO), Tezpur, Assam, 784001, India
| | - Runu Chakravarty
- Hepatitis Research Laboratory, ICMR Virus Unit, Kolkata (ICMR), Kolkata, 700010, West Bengal, India
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Molecular Detection and Characterization of Hepatitis B Virus. Mol Microbiol 2016. [DOI: 10.1128/9781555819071.ch32] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Salama II, Sami SM, Said ZNA, El-Sayed MH, El Etreby LA, Rabah TM, Elmosalami DM, Abdel Hamid AT, Salama SI, Abdel Mohsen AM, Emam HM, Elserougy SM, Hassanain AI, Abd Alhalim NF, Shaaban FA, Hemeda SA, Ibrahim NA, Metwally AM. Effectiveness of hepatitis B virus vaccination program in Egypt: Multicenter national project. World J Hepatol 2015; 7:2418-2426. [PMID: 26464758 PMCID: PMC4598613 DOI: 10.4254/wjh.v7.i22.2418] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2015] [Revised: 07/10/2015] [Accepted: 09/16/2015] [Indexed: 02/06/2023] Open
Abstract
AIM To assess the effectiveness of hepatitis B virus (HBV) vaccination program among fully vaccinated children.
METHODS A national community based cross-sectional study was carried out in 6 governorates representing Egypt. A total of 3600 children aged from 9 mo to 16 years who were fully vaccinated with HBV vaccine during infancy were recruited. Face to face interviews were carried out and sera were evaluated for hepatitis B surface antigen (HBsAg), anti-HBV core antibodies (total) and quantitative detection of hepatitis B surface antibody using enzyme linked immunoassays techniques. Samples positive to HBsAg/anti-HBV core antibodies were subjected to quantitative HBV-DNA detection by real time polymerase chain reaction with 3.8 IU/L detection limit.
RESULTS Sero-protection was detected among 2059 children (57.2%) with geometric mean titers 75.4 ± 3.6 IU/L compared to 3.1 ± 2.1 IU/L among non-seroprotected children. Multivariate logistic analysis revealed that older age and female gender were the significant predicting variables for having non sero-protective level, with adjusted odds ratio 3.3, 9.1 and 14.2 among children aged 5 to < 10, 10 to < 15 and ≥ 15 years respectively compared to those < 5 years and 1.1 among girls compared to boys with P < 0.01. HBsAg was positive in 0.11% and breakthrough infection was 0.36% and 0.39% depending on positivity of anti-HBc and DNA detection respectively. The prevalence of HBV infection was significantly higher among children aged ≥ 7 years (0.59%) compared to 0.07% among younger children with odds ratio equal to 8.4 (95%CI: 1.1-64.2) and P < 0.01.The prevalence was higher among girls (0.48%) than boys (0.29%) with P > 0.05.
CONCLUSION The Egyptian compulsory HBV vaccination program provides adequate protection. Occult HBV infection exists among apparently healthy vaccinated children. Adherence to infection control measures is mandatory.
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Affiliation(s)
- Iman I Salama
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Samia M Sami
- Child Health Department, National Research Center, Cairo 12311, Egypt
| | - Zeinab Nabil Ahmed Said
- Micro-biology and Immunology Department, Faculty of Medicine (for girls), Al-Azhar University, Cairo 11754, Egypt
| | - Manal H El-Sayed
- Pediatrics Department, Faculty of Medicine, Ain-Shams University, Cairo 11566, Egypt
| | - Lobna A El Etreby
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Thanaa M Rabah
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Dalia M Elmosalami
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Amany T Abdel Hamid
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Somaia I Salama
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Aida M Abdel Mohsen
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Hanaa M Emam
- Dermatology and Venereology Department, National Research Center, Cairo 12311, Egypt
| | - Safaa M Elserougy
- Environmental and Occupational Medicine Department, National Research Center, Cairo 12311, Egypt
| | - Amal I Hassanain
- Child Health Department, National Research Center, Cairo 12311, Egypt
| | - Naglaa F Abd Alhalim
- Micro-biology and Immunology Department, Faculty of Medicine (for girls), Al-Azhar University, Cairo 11754, Egypt
| | - Fatma A Shaaban
- Child Health Department, National Research Center, Cairo 12311, Egypt
| | - Samia A Hemeda
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Nihad A Ibrahim
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
| | - Ammal M Metwally
- Community Medicine Research Department, National Research Center, Cairo 12311, Egypt
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12
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Seiz PL, Slanina H, Ziebuhr J, Gerlich WH, Glebe D, Schüttler CG. Studies of nosocomial outbreaks of hepatitis B in nursing homes in Germany suggest a major role of hepatitis B e antigen expression in disease severity and progression. Int J Med Microbiol 2015; 305:663-72. [PMID: 26338147 DOI: 10.1016/j.ijmm.2015.08.016] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) causes acute or chronic hepatitis B. Local outbreaks of HBV infections in skilled nursing facilities is a matter of growing concern in developed countries. Here, we investigated two outbreaks of hepatitis B that recently occurred in nursing homes in Germany. The outbreak at location A was associated with acute fulminant hepatitis with fatal outcome in several cases, while individuals infected at location B developed asymptomatic or mild hepatitis B. Sequence analysis of viruses involved in these outbreaks revealed different, but unique HBV strains for each location. Each of the strains produced high viremia of more than 10(9) virions/mL serum. We found that the mild course of hepatitis B at location B was caused by a circulating wild-type HBV genotype A2 strain, which is commonly found in Central Europe. Complete genome sequences of isolates obtained from infected patients revealed nearly 100% sequence identity at the nucleotide level as well as expression of HBV e protein (HBeAg), a known T cell tolerogen in the incubation or chronic phases of HBV infection. By contrast, the outbreak at location A was associated with an HBV genotype D2 variant that lacked HBeAg expression, suggesting that immunopathology and selection of specific HBV variants played a major role in the severe (or even fulminant) acute hepatitis observed at location A. Importantly, all patients were diagnosed with type 2 diabetes mellitus, a known risk factor for healthcare-associated transmission of HBV. The study leads us to suggest that, besides strict adherence to hygiene standards, additional efforts are required to reduce the risk of HBV transmission and fulminant disease progression in healthcare settings and nursing homes. In this context, a general screening for HBsAg and active hepatitis B vaccination should be considered for people living in nursing homes, especially for those with diagnosed diabetes or other predisposing factors for HBV transmission.
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Affiliation(s)
- Pia L Seiz
- Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, German Center for Infection Research, Biomedical Research Center Seltersberg, Schubertstr. 81, 35392 Giessen, Germany
| | - Heiko Slanina
- Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, German Center for Infection Research, Biomedical Research Center Seltersberg, Schubertstr. 81, 35392 Giessen, Germany
| | - John Ziebuhr
- Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, German Center for Infection Research, Biomedical Research Center Seltersberg, Schubertstr. 81, 35392 Giessen, Germany
| | - Wolfram H Gerlich
- Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, German Center for Infection Research, Biomedical Research Center Seltersberg, Schubertstr. 81, 35392 Giessen, Germany
| | - Dieter Glebe
- Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, German Center for Infection Research, Biomedical Research Center Seltersberg, Schubertstr. 81, 35392 Giessen, Germany.
| | - Christian G Schüttler
- Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, German Center for Infection Research, Biomedical Research Center Seltersberg, Schubertstr. 81, 35392 Giessen, Germany
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13
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Baylis SA, Chudy M, Nübling CM. Standardization of NAT for Blood-Borne Pathogens. Transfus Med Hemother 2015; 42:211-8. [PMID: 26557812 DOI: 10.1159/000435872] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2015] [Accepted: 05/31/2015] [Indexed: 12/15/2022] Open
Abstract
Assays based on nucleic acid amplification technology (NAT) are increasingly used for screening of blood and for diagnosis or monitoring of patients. Both regulatory requirements for blood screening and international recommendations for the treatment of patients are based on common reference materials available globally for the standardization of NAT assays. World Health Organization International Standards (WHO ISs) and International Reference Panels (WHO IRPs) are primary reference materials. The characterization and manufacture of WHO reference materials as well as their evaluation is performed on behalf of the WHO by collaborating centers; their establishment is decided upon by the WHO Expert Committee on Biological Standardization (ECBS). The potency of the first WHO IS is defined by the 'international unit' (IU) which should be maintained upon replacement of the IS. The IU, unlike copy number or genome equivalent, is defined by the IS with a physical existence, is available worldwide, and allows traceability and comparability of results. The anticipated use of WHO ISs is the calibration of secondary standards or the validation of essential assay features, e.g. limit of detection.
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Affiliation(s)
- Sally A Baylis
- Department of Virology, Paul-Ehrlich-Institut, Langen, Germany
| | - Michael Chudy
- Department of Virology, Paul-Ehrlich-Institut, Langen, Germany
| | - C Micha Nübling
- Department of Virology, Paul-Ehrlich-Institut, Langen, Germany ; World Health Organization, Essential Medicines and Health Products Department, Geneva, Switzerland
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14
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Gerlich WH. Reduction of Infectivity in Chronic Hepatitis B Virus Carriers among Healthcare Providers and Pregnant Women by Antiviral Therapy. Intervirology 2014; 57:202-11. [DOI: 10.1159/000360949] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
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15
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Occult HBV infection: a faceless enemy in liver cancer development. Viruses 2014; 6:1590-611. [PMID: 24717680 PMCID: PMC4014712 DOI: 10.3390/v6041590] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2014] [Revised: 03/13/2014] [Accepted: 03/20/2014] [Indexed: 12/12/2022] Open
Abstract
The hepatitis B virus (HBV) represents a worldwide public health problem; the virus is present in one third of the global population. However, this rate may in fact be higher due to occult hepatitis B virus infection (OBI). This condition is characterized by the presence of the viral genome in the liver of individuals sero-negative for the virus surface antigen (HBsAg). The causes of the absence of HBsAg in serum are unknown, however, mutations have been identified that produce variants not recognized by current immunoassays. Epigenetic and immunological host mechanisms also appear to be involved in HBsAg suppression. Current evidence suggests that OBI maintains its carcinogenic potential, favoring the progression of fibrosis and cirrhosis of the liver. In common with open HBV infection, OBI can contribute to the establishment of hepatocellular carcinoma. Epidemiological data regarding the global prevalence of OBI vary due to the use of detection methods of different sensitivity and specificity. In Latin America, which is considered an area of low prevalence for HBV, diagnostic screening methods using gene amplification tests for confirmation of OBI are not conducted. This prevents determination of the actual prevalence of OBI, highlighting the need for the implementation of cutting edge technology in epidemiological surveillance systems.
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Said ZN, Sayed MHE, Salama II, Aboel-Magd EK, Mahmoud MH, Setouhy ME, Mouftah F, Azzab MB, Goubran H, Bassili A, Esmat GE. Occult hepatitis B virus infection among Egyptian blood donors. World J Hepatol 2013; 5:64-73. [PMID: 23646231 PMCID: PMC3642725 DOI: 10.4254/wjh.v5.i2.64] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/01/2012] [Revised: 11/28/2012] [Accepted: 12/23/2012] [Indexed: 02/06/2023] Open
Abstract
AIM To identify blood donors with occult hepatitis B virus (HBV) infection (OBI) to promote safe blood donation. METHODS Descriptive cross sectional study was conducted on 3167 blood donors negative for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV Ab) and human immunodeficiency virus Ab. They were subjected to the detection of alanine aminotransferase (ALT) and aspartate transaminase (AST) and screening for anti-HBV core antibodies (total) by two different techniques; [Monoliza antibodies to hepatitis B core (Anti-HBc) Plus-Bio-Rad] and (ARC-HBc total-ABBOT). Positive samples were subjected to quantitative detection of antibodies to hepatitis B surface (anti-HBs) (ETI-AB-AUK-3, Dia Sorin-Italy). Serum anti-HBs titers > 10 IU/L was considered positive. Quantitative HBV DNA by real time polymerase chain reaction (PCR) (QIAGEN-Germany) with 3.8 IU/mL detection limit was estimated for blood units with negative serum anti-HBs and also for 32 whose anti-HBs serum titers were > 1000 IU/L. Also, 265 recipients were included, 34 of whom were followed up for 3-6 mo. Recipients were investigated for ALT and AST, HBV serological markers: HBsAg (ETI-MAK-4, Dia Sorin-Italy), anti-HBc, quantitative detection of anti-HBs and HBV-DNA. RESULTS 525/3167 (16.6%) of blood units were positive for total anti-HBc, 64% of those were anti-HBs positive. Confirmation by ARCHITECT anti-HBc assay were carried out for 498/525 anti-HBc positive samples, where 451 (90.6%) confirmed positive. Reactivity for anti-HBc was considered confirmed only if two positive results were obtained for each sample, giving an overall prevalence of 451/3167 (14.2%) for total anti-HBc. HBV DNA was quantified by real time PCR in 52/303 (17.2%) of anti-HBc positive blood donors (viral load range: 5 to 3.5 x 10(5) IU/mL) with a median of 200 IU/mL (mean: 1.8 x 10(4) ± 5.1 x 10(4) IU/mL). Anti-HBc was the only marker in 68.6% of donors. Univariate and multivariate logistic analysis for identifying risk factors associated with anti-HBc and HBV-DNA positivity among blood donors showed that age above thirty and marriage were the most significant risk factors for prediction of anti-HBc positivity with AOR 1.8 (1.4-2.4) and 1.4 (1.0-1.9) respectively. Other risk factors as gender, history of blood transfusion, diabetes mellitus, frequent injections, tattooing, previous surgery, hospitalization, Bilharziasis or positive family history of HBV or HCV infections were not found to be associated with positive anti-HBc antibodies. Among anti-HBc positive blood donors, age below thirty was the most significant risk factor for prediction of HBV-DNA positivity with AOR 3.8 (1.8-7.9). According to HBV-DNA concentration, positive samples were divided in two groups; group one with HBV-DNA ≥ 200 IU/mL (n = 27) and group two with HBV-DNA < 200 IU/mL (n = 26). No significant difference was detected between both groups as regards mean age, gender, liver enzymes or HBV markers. Serological profiles of all followed up blood recipients showed that, all were negative for the studied HBV markers. Also, HBV DNA was not detected among studied recipients, none developed post-transfusion hepatitis (PTH) and the clinical outcome was good. CONCLUSION OBI is prevalent among blood donors. Nucleic acid amplification/HBV anti core screening should be considered for high risk recipients to eliminate risk of unsafe blood donation.
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Affiliation(s)
- Zeinab N Said
- Zeinab N Said, Enas K Aboel-Magd, Microbiology and Immunology Department, Faculty of Medicine (for Girls), Al-Azhar University, 11511 Cairo, Egypt
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Chudy M, Hanschmann KM, Kress J, Nick S, Campos R, Wend U, Gerlich W, Nübling CM. First WHO International Reference Panel containing hepatitis B virus genotypes A-G for assays of the viral DNA. J Clin Virol 2012; 55:303-9. [PMID: 22981623 DOI: 10.1016/j.jcv.2012.08.013] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2012] [Revised: 08/09/2012] [Accepted: 08/16/2012] [Indexed: 12/18/2022]
Abstract
BACKGROUND WHO International Standards (IS) are provided for the calibration and validation of diagnostic and screening assays, e.g. for hepatitis B virus (HBV). HBV forms numerous subgenotypes and the current IS for HBV DNA reflects subgenotype A2. OBJECTIVE A reference panel with the most prevalent subgenotypes should facilitate evaluation of genotype-specific detection efficiencies. STUDY DESIGN 215 HBV positive plasma samples collected worldwide were characterized for HBV markers and sequenced. Fifteen subgenotype A1, A2, B2, B4, C2, D1, D3, E, F2 and G samples were selected for the panel. The lyophilized samples were tested in parallel with the IS in an international collaborative study with 16 laboratories using 13 different nucleic acid amplification techniques (NATs). RESULTS Eight of 13 NAT had a HBV DNA detection efficiency which was independent of the genotype and consistent with the IS, while with five assays, certain deviations were noted, particularly with genotype F which was under quantitated or even missed by three assays. The panel was accepted by the WHO as the "1st WHO International Reference Panel for HBV Genotypes for HBV NAT-Based Assays". CONCLUSIONS The evaluation of HBV DNA assays should include many different genotypes. The WHO Reference Panel is universally available for manufacturers of HBV DNA assays, diagnostic laboratories and control authorities to facilitate standardized validation of HBV genotype specific detection efficiency of both diagnostic (quantitative and qualitative) and screening NAT assays.
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Affiliation(s)
- M Chudy
- Paul-Ehrlich-Institut, Section of Molecular Virology, Langen, Germany
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19
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Said ZNA. An overview of occult hepatitis B virus infection. World J Gastroenterol 2011; 17:1927-38. [PMID: 21528070 PMCID: PMC3082745 DOI: 10.3748/wjg.v17.i15.1927] [Citation(s) in RCA: 109] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/08/2010] [Revised: 01/18/2011] [Accepted: 01/25/2011] [Indexed: 02/06/2023] Open
Abstract
Occult hepatitis B virus (HBV) infection (OBI), alternatively defined as occult hepatitis B (OHB), is a challenging clinical entity. It is recognized by two main characteristics: absence of HBsAg, and low viral replication. The previous two decades have witnessed a remarkable progress in our understanding of OBI and its clinical implications. Appropriate diagnostic techniques must be adopted. Sensitive HBV DNA amplification assay is the gold standard assay for detection of OBI. Viral as well as host factors are implicated in the pathogenesis of OBI. However, published data reporting the infectivity of OBI by transfusion are limited. Several aspects including OBI transmission, infectivity and its relation to the development of chronic liver diseases and hepatocellular carcinoma have to be resolved. The aim of the present review is to highlight recent data on OBI with a focus on its virological diagnosis and clinical outcome.
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Amini-Bavil-Olyaee S, Pourkarim MR, Schaefer S, Mahboudi F, Van Ranst M, Adeli A, Trautwein C, Tacke F. Single-step real-time PCR to quantify hepatitis B virus and distinguish genotype D from non-D genotypes. J Viral Hepat 2011; 18:300-4. [PMID: 20367802 DOI: 10.1111/j.1365-2893.2010.01308.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Hepatitis B virus (HBV) viral load and its genotype play important roles in clinical outcome, management of disease and response to antiviral therapy. In many parts of the world such as Europe or the Middle East, distinguishing HBV genotype D from non-D is most relevant for treatment decisions, because genotype D-infected patients respond poorly to interferon-based therapeutic regimens. Here, we developed an in-house real-time PCR to concordantly assess HBV genotype (D vs non-D) based on melt curve analysis and quantify the viral load. Genotype distinction was established with control plasmids of all HBV genotypes and validated with 57 clinical samples from patients infected with six different HBV genotypes. Our in-house real-time PCR assay could discriminate HBV genotype D from non-D using single-step melt curve analysis with a 2 °C difference in the melt curve temperature in all samples tested. Viral load quantification was calibrated with the WHO HBV international standard, demonstrating an excellent correlation with a commercial kit (r = 0.852; P < 0.0001) in a linear range from 3.2 × 10(2) to 3.2 × 10(10) IU/mL. In conclusion, we developed a rapid, simple and cost-effective method to simultaneously quantify and distinguish HBV genotypes D from non-D with a single-step PCR run and melt curve analysis. This assay should be a useful diagnostic alternative to aid clinical decisions about initiation and choice of antiviral therapy, especially in geographical regions with a high prevalence of HBV genotype D.
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Sakata H, Matsubayashi K, Takeda H, Kishimoto S, Ihara H, Sato S, Kato T, Tadokoro K, Ikeda H. GENOTYPIC SPECIFICITY OF CHEMILUMINESCENT ENZYME IMMUNOASSAY SCREENING FOR HUMAN PARVOVIRUS B19 ANTIGEN IN BLOOD DONORS. ACTA ACUST UNITED AC 2011. [DOI: 10.3925/jjtc.57.146] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
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Lim LG, Aung MO, Seet BL, Tan C, Dan YY, Lee YM, Sutedja DS, Fernandes M, Lee GH, Koay E, Lim SG. Alanine aminotransferase is an inadequate surrogate marker for detecting lamivudine resistance. World J Gastroenterol 2010; 16:4691-6. [PMID: 20872970 PMCID: PMC2951520 DOI: 10.3748/wjg.v16.i37.4691] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/13/2010] [Revised: 04/24/2010] [Accepted: 05/01/2010] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the accuracy of serum alanine aminotransferase (ALT) in diagnosing lamivudine resistance and factors that contributed to abnormal serum ALT. METHODS This was a retrospective study of chronic hepatitis B patients on lamivudine therapy who were followed for 3-mo with liver function tests and hepatitis B virus (HBV) DNA measurement. Lamivudine resistance was defined as HBV DNA ≥ 1 log from nadir on at least 2 occasions, confirmed by genotyping. Serum ALT levels in patients with lamivudine resistance were compared to serum ALT levels in those without lamivudine resistance. RESULTS There were 111 patients with and 117 without lamivudine resistance. The area under the receiver operating characteristic of serum ALT to diagnose lamivudine resistance was 0.645 ± 0.037. Serum ALT > 42.5 U/L gave the best diagnostic accuracy with sensitivity = 61%, specificity = 60%, positive predictive value = 60%, negative predictive value = 61%, positive likelihood ratio = 1.53 and negative likelihood ratio = 0.65 for predicting lamivudine resistance, missing 39% of resistant patients. Using other serum ALT cutoffs, diagnostic accuracy was lower. By multivariate analysis, baseline abnormal serum ALT was associated with abnormal ALT during resistance (OR = 5.98, P = 0.003), and males were associated with serum ALT flares during resistance (OR = 8.9, P = 0.016). CONCLUSION Serum ALT is inadequate for diagnosing lamivudine resistance and has implications where viral resistance testing is suboptimal and for reimbursement of rescue therapy.
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Baylis SA, Chudy M, Blümel J, Pisani G, Candotti D, José M, Heath AB. Collaborative study to establish a replacement World Health Organization International Standard for parvovirus B19 DNA nucleic acid amplification technology (NAT)-based assays. Vox Sang 2009; 98:441-6. [PMID: 20003130 DOI: 10.1111/j.1423-0410.2009.01288.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
BACKGROUND AND OBJECTIVES The aim of the study was to replace the 1(st) World Health Organization International Standard for parvovirus B19 DNA for nucleic acid amplification technique (NAT)-based assays (code 99/800). Two lyophilized preparations (coded 99/800 and 99/802) had been evaluated in the original collaborative study. The present study re-evaluates these two preparations in terms of potency, stability and encapsidation of virus DNA. MATERIALS AND METHODS The 1(st) International Standard (99/800) and 99/802 were re-coded as Samples 1 and 2, respectively. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after storage at 20 degrees C for 7 years. Nuclease treatment was used to investigate the encapsidation of virus DNA. RESULTS Data were returned from a total of six different quantitative NAT-based assays. The results of the present study confirm those of the original, with no significant differences found in estimated international units (IU)/ml for the 1(st) International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (99/802). Accelerated thermal degradation studies demonstrate that both samples are very stable, with no loss of potency after storage at 20 degrees C for 7 years. Both lyophilized preparations contained the majority of B19V DNA encapsidated in virions. CONCLUSIONS On the basis of the data presented in this collaborative study, Sample 2 (code number 99/802) was established as the 2(nd) International Standard for parvovirus B19 DNA for NAT-based assays with a potency of 10(6) IU/ml (500 000 IU/vial).
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Affiliation(s)
- S A Baylis
- National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.
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