1
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Hepatitis B among University Population: Prevalence, Associated Risk Factors, Knowledge Assessment, and Treatment Management. Viruses 2022; 14:v14091936. [PMID: 36146743 PMCID: PMC9501279 DOI: 10.3390/v14091936] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 08/25/2022] [Accepted: 08/29/2022] [Indexed: 11/17/2022] Open
Abstract
Background: Very few studies have been reported on hepatitis B in the State of Azad Jammu and Kashmir, Pakistan, and none of them are specific to the prevalence and causes of hepatitis B spread among educational institutes. This study aimed to estimate the prevalence of hepatitis B infection and its associated risk factors among the University of AJ and K population. Methods: An observational, cross-sectional, and analytical study was conducted with 7015 students and employees. Hepatitis B was detected by rapid immunochromatographic tests (ICTs), enzyme-linked immunosorbent assay (ELISA), and real-time quantitative PCR. A questionnaire and interview method was used to assess the disease knowledge and associated risk factors with hepatitis B through Chi-square, Fisher’s exact test, and paired t-test. Results: Of the participants, 150 (2.13%) were found positive for the hepatitis B surface antigen (57.3% male and 42.7% female). Only 0.3% participants were found fully vaccinated against the hepatitis B virus. Among ethnic groups, the Syed tribe was found more prevalent for hepatitis B infection (40.6%), while use of contaminated mourning blades (95% CI: p = 0.0001) was found as an overlooked risk factor. Hepatitis preventive awareness sessions were found to be very significant (p = 0.0001). Conclusions: The study showed that an overlooked risk factor is playing a key role in the spread of HBV in a tribe living worldwide, which must be addressed globally to eradicate hepatitis B. In Pakistan, a country-wide annual HBV vaccination program should be launched to control hepatitis B.
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Ghorbani M, Shokri R, Kia V, Yari F, Sharifi Z, Paryan M. New design and optimization of an in-house quantitative TaqMan Real-Time PCR-based assay for the detection and monitoring of occult hepatitis B virus (genotype A-J) infection. Indian J Med Microbiol 2022; 40:560-566. [DOI: 10.1016/j.ijmmb.2022.07.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2022] [Revised: 06/18/2022] [Accepted: 07/06/2022] [Indexed: 11/26/2022]
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3
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Saboor Soomro R, Shah IA, Saboor A, Bhutto AUB, Memon S. Sensitivity and Specificity of Hepatitis B Virus Screening via Rapid Immunoassay Chromatographic Test. Cureus 2021; 13:e12909. [PMID: 33654594 PMCID: PMC7906272 DOI: 10.7759/cureus.12909] [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] [Indexed: 11/16/2022] Open
Abstract
Introduction: In low-income and high hepatitis B and C virus burden countries like Pakistan, it is important to develop cheap yet efficient strategies in diagnosing as well as treating hepatitis. The aim of this study is to assess the sensitivity and specificity of serum hepatitis B surface antigen (HbsAg) via Rapid Immunoassay Chromatographic Test (RICT) for the screening of hepatitis B, compared to the gold standard laboratory-based method. Methods: The study was conducted in the Hepatology Clinic of Civil Hospital, Sukkur. All records of the clinic from June 1, 2018, to December 31, 2018, were accessed for identification of the records in which hepatitis B screening via RICT and then confirmatory polymerase chair reaction (PCR) by gene amplification with forward and reverse primers was done. Results: There were 151 samples in this study. There were 32 (94.1%) true-positive and three (5.8%) false-negative samples. There were two (2.5%) false-positive and 114 (97.4%) true-negative samples. The sensitivity of HbsAg detection via RICT for the screening of 1-1B V was 91.43%, specificity was 98.28% and the accuracy was 96.69%, compared to PCR. Conclusion: The RICT method has high sensitivity and specificity. In low-income and high-hepatitis B virus-burden countries like Pakistan, it serves as a very efficient screening tool that is easy to use, cheaper in cost, and gives rapid and accurate results.
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Affiliation(s)
| | - Iftikhar Ali Shah
- Internal Medicine, Ghulam Muhammad Mahar Medical College Hospital, Sukkur, PAK
| | - Abdul Saboor
- Urology, Ghulam Muhammad Mahar Medical College Hospital, Sukkur, PAK
| | | | - Sidra Memon
- Internal Medicine, Jinnah Sindh Medical University, Karachi, PAK
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4
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Teh CP, Chook JB, Ngeow YF, Tong TYK, Tee KK, Bong JJ, Mohamed R. Primer and probe conservation issue in the quantification of hepatitis B virus DNA. Rev Med Virol 2020. [DOI: 10.1002/rmv.2182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Affiliation(s)
- Chye Phing Teh
- Department of Biological Sciences School of Science and Technology Sunway University Petaling Jaya Selangor Malaysia
- Department of Medical Sciences School of Healthcare and Medical Sciences Sunway University Petaling Jaya Selangor Malaysia
| | - Jack Bee Chook
- Department of Medical Sciences School of Healthcare and Medical Sciences Sunway University Petaling Jaya Selangor Malaysia
| | - Yun Fong Ngeow
- Department of Pre‐Clinical Sciences Faculty of Medicine and Health Sciences Universiti Tunku Abdul Rahman Kajang Malaysia
| | - Tommy Yuh Koon Tong
- Department of Biological Sciences School of Science and Technology Sunway University Petaling Jaya Selangor Malaysia
| | - Kok Keng Tee
- Department of Medical Microbiology Faculty of Medicine University of Malaya Kuala Lumpur Malaysia
| | - Jan Jin Bong
- Sunway Medical Centre Petaling Jaya Selangor Malaysia
| | - Rosmawati Mohamed
- Department of Medicine Faculty of Medicine University of Malaya Kuala Lumpur Malaysia
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5
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Shariati M, Sadeghi M. Ultrasensitive DNA biosensor for hepatitis B virus detection based on tin-doped WO3/In2O3 heterojunction nanowire photoelectrode under laser amplification. Anal Bioanal Chem 2020; 412:5367-5377. [DOI: 10.1007/s00216-020-02752-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2020] [Revised: 05/10/2020] [Accepted: 06/02/2020] [Indexed: 12/29/2022]
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6
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Portilho MM, Mendonça ACDF, Bezerra CS, do Espirito-Santo MP, de Paula VS, Nabuco LC, Villela-Nogueira CA, Lewis-Ximenez LL, Lampe E, Villar LM. Usefulness of in-house real time PCR for HBV DNA quantification in serum and oral fluid samples. J Virol Methods 2018; 256:100-106. [PMID: 29514044 DOI: 10.1016/j.jviromet.2018.03.001] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2017] [Revised: 02/26/2018] [Accepted: 03/03/2018] [Indexed: 02/07/2023]
Abstract
For quantification of hepatitis B virus DNA (HBV DNA), commercial assays are used with serum or plasma samples, but oral fluid samples could be an alternative for HBV diagnosis due to ease of collection. This study aims to develop in-house real time PCR using synthetic curve for HBV DNA quantification for serum and oral fluid samples. Samples were collected from 103 individuals (55 HBsAg reactive and HBV DNA reactive by commercial assay and 48 without HBV markers) and submitted to two in-house real time PCR assays for HBV pre-S/S region with different standard curves: qPCR plasmidial and qPCR synthetic. A total of 27 serum samples were HBV DNA positive by qPCR plasmidial and 40 with qPCR synthetic (72% and 85% of concordance, respectively). Quantitative PCR synthetic presented efficiency of 99% and sensitivity of 2log10 copies/mL. Among oral fluid samples, five and ten were detected using qPCR plasmidial and synthetic, respectively. This study demonstrated that qPCR synthetic using serum samples could be used as alternative for HBV DNA quantification due to its sensitivity. In addition, it was possible to quantify HBV DNA in oral fluid samples suggesting the potential of this specimen for molecular diagnosis of HBV.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Elisabeth Lampe
- Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil
| | - Livia Melo Villar
- Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
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7
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Papatheodoridis G, Triantos C, Hadziyannis E, Zisimopoulos K, Georgiou A, Voulgaris T, Vlachogiannakos I, Nikolopoulou V, Manolakopoulos S. Serum HBsAg kinetics and usefulness of interferon-inducible protein 10 serum in HBeAg-negative chronic hepatitis B patients treated with tenofovir disoproxil fumarate. J Viral Hepat 2015; 22:1079-87. [PMID: 26146764 DOI: 10.1111/jvh.12434] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2015] [Accepted: 06/04/2015] [Indexed: 01/27/2023]
Abstract
The kinetics of serum HBsAg and interferon-inducible protein 10 (IP10) levels in patients with chronic hepatitis B infection treated with tenofovir are unclear. We evaluated the changes of HBsAg levels and the predictability of IP10 for HBsAg decline in 160 HBeAg-negative patients receiving tenofovir for ≥12 months. Serum samples taken before and at 6, 12, 24, 36 and 48 months after tenofovir were tested for HBsAg levels. In 104 patients, serum samples before tenofovir were tested for IP10 levels. Compared to before tenofovir, HBsAg levels decreased by a median of 0.08, 0.11, 0.24, 0.33 and 0.38 log10 IU/mL at 6, 12, 24, 36 and 48 months, respectively (P < 0.001). HBsAg kinetics did not differ between nucleos(t)ide analogue(s) naive and experienced patients. The 12-, 24-, 36- and 48-month cumulative rates of ≥0.5 log10 HBsAg decline were 8%, 16%, 24% and 41% and of HBsAg ≤100 IU/mL were 9%, 12%, 14% and 18%, respectively. The only factor associated with HBsAg ≤100 IU/mL was lower HBsAg levels before tenofovir (P < 0.001), while HBsAg decline ≥0.5 log10 was associated with higher IP10 levels (P = 0.002) and particularly with IP10 > 350 pg/mL (P < 0.001). In conclusion, tenofovir decreases serum HBsAg levels in both nucleos(t)ide analogue(s) naive and experienced patients with HBeAg-negative chronic hepatitis B infection. After 4 years of therapy, HBsAg ≤100 IU/mL can be achieved in approximately 20% of patients, particularly in those with low baseline HBsAg levels. HBsAg decline is slow (≥0.5 log10 in 40% of patients after 4 years) and is associated only with higher baseline serum IP10 levels.
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Affiliation(s)
- G Papatheodoridis
- Department of Gastroenterology, Athens University Medical School, Laiko Hospital of Athens, Athens, Greece
| | - C Triantos
- Department of Gastroenterology, University Hospital of Patras, Patras, Greece
| | - E Hadziyannis
- 2nd Department of Internal Medicine, Athens University Medical School, Hippokratio Hospital of Athens, Athens, Greece
| | - K Zisimopoulos
- Department of Gastroenterology, University Hospital of Patras, Patras, Greece
| | - A Georgiou
- Department of Gastroenterology, Athens University Medical School, Laiko Hospital of Athens, Athens, Greece
| | - T Voulgaris
- Department of Gastroenterology, Athens University Medical School, Laiko Hospital of Athens, Athens, Greece
| | - I Vlachogiannakos
- Department of Gastroenterology, Athens University Medical School, Laiko Hospital of Athens, Athens, Greece
| | - V Nikolopoulou
- Department of Gastroenterology, University Hospital of Patras, Patras, Greece
| | - S Manolakopoulos
- 2nd Department of Internal Medicine, Athens University Medical School, Hippokratio Hospital of Athens, Athens, Greece
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8
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Nikolopoulos GK, Paraskevis D, Psichogiou M, Hatzakis A. HBV-DNA levels predict overall mortality in HIV/HBV coinfected individuals. J Med Virol 2015; 88:466-73. [PMID: 26288334 DOI: 10.1002/jmv.24357] [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] [Accepted: 08/14/2015] [Indexed: 12/15/2022]
Abstract
The coinfection of Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) has been associated with increased death rates. However, the relevant research has mostly relied on serologic HBV testing [HBV surface antigen (HBsAg)]. The aim of this work was to explore the relationship of HBV viraemia with overall mortality among HIV/HBV coinfected individuals. The analysis included 1,609 HIV seropositives of a previously described cohort (1984-2003) with limited exposure to tenofovir (12%) and a median follow-up of approximately 5 years. Those with persistent expression of HBsAg were further tested for HBV-DNA. The data were analyzed using Poisson regression models. Totally, 101 participants were chronic carriers of HBsAg (6.28%). Of these, 81 were tested for HBV-DNA. The median HBV-DNA levels were 3.81 log (base-10) International Units (IU)/ml. A third (31%) of those tested for HBV-DNA had received tenofovir. Before developing acquired immune deficiency syndrome (AIDS), the adjusted incidence rate ratio (IRR) for all-cause mortality of coinfected patients with HBV viraemia above the median value versus the HIV monoinfected group was 3.44 [95% confidence interval (CI): 1.05-11.27]. Multivariable regressions in the coinfected group only (n = 81) showed that one log-10 increase in HBV-DNA levels was associated with an elevated risk for death (IRR: 1.24, 95%CI: 1.03-1.49). HBV-DNA levels predict overall mortality in the setting of HIV/HBV coinfection, especially during the period before developing AIDS, and could thus help prioritize needs and determine the frequency of medical monitoring.
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Affiliation(s)
- Georgios K Nikolopoulos
- Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece.,Hellenic Center for Disease Control and Prevention, Amarousio, Greece
| | - Dimitrios Paraskevis
- Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece
| | - Mina Psichogiou
- First Department of Propaedeutic Medicine, Laiko General Hospital, Medical School, University of Athens, Athens, Greece
| | - Angelos Hatzakis
- Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece
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9
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Giamblanco N, Conoci S, Russo D, Marletta G. Single-step label-free hepatitis B virus detection by a piezoelectric biosensor. RSC Adv 2015. [DOI: 10.1039/c5ra03467a] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Probe densityvs.genome recognition selectivity.
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Affiliation(s)
- Nicoletta Giamblanco
- Laboratory for Molecular Surfaces and Nanotechnology (LAMSUN)
- Department of Chemical Sciences
- University of Catania and CSGI
- 95125 Catania
- Italy
| | | | | | - Giovanni Marletta
- Laboratory for Molecular Surfaces and Nanotechnology (LAMSUN)
- Department of Chemical Sciences
- University of Catania and CSGI
- 95125 Catania
- Italy
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10
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Datta S, Chatterjee S, Veer V. Recent advances in molecular diagnostics of hepatitis B virus. World J Gastroenterol 2014; 20:14615-14625. [PMID: 25356025 PMCID: PMC4209528 DOI: 10.3748/wjg.v20.i40.14615] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/27/2013] [Revised: 02/13/2014] [Accepted: 06/05/2014] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) is one of the important global health problems today. Infection with HBV can lead to a variety of clinical manifestations including severe hepatic complications like liver cirrhosis and hepatocellular carcinoma. Presently, routine HBV screening and diagnosis is primarily based on the immuno-detection of HBV surface antigen (HBsAg). However, identification of HBV DNA positive cases, who do not have detectable HBsAg has greatly encouraged the use of nucleic acid amplification based assays, that are highly sensitive, specific and are to some extent tolerant to sequence variation. In the last few years, the field of HBV molecular diagnostics has evolved rapidly with advancements in the molecular biology tools, such as polymerase chain reaction (PCR) and real-time PCR. Recently, apart of PCR based amplification methods, a number of isothermal amplification assays, such as loop mediated isothermal amplification, transcription mediated amplification, ligase chain reaction, and rolling circle amplification have been utilized for HBV diagnosis. These assays also offer options for real time detection and integration into biosensing devices. In this manuscript, we review the molecular technologies that are presently available for HBV diagnostics, with special emphasis on isothermal amplification based technologies. We have also included the recent trends in the development of biosensors and use of next generation sequencing technologies for HBV.
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11
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Yao CY, Fu WL. Biosensors for hepatitis B virus detection. World J Gastroenterol 2014; 20:12485-12492. [PMID: 25253948 PMCID: PMC4168081 DOI: 10.3748/wjg.v20.i35.12485] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/04/2014] [Revised: 03/01/2014] [Accepted: 04/16/2014] [Indexed: 02/06/2023] Open
Abstract
A biosensor is an analytical device used for the detection of analytes, which combines a biological component with a physicochemical detector. Recently, an increasing number of biosensors have been used in clinical research, for example, the blood glucose biosensor. This review focuses on the current state of biosensor research with respect to efficient, specific and rapid detection of hepatitis B virus (HBV). The biosensors developed based on different techniques, including optical methods (e.g., surface plasmon resonance), acoustic wave technologies (e.g., quartz crystal microbalance), electrochemistry (amperometry, voltammetry and impedance) and novel nanotechnology, are also discussed.
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12
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Papatheodoridis GV, Manolakopoulos S, Margariti A, Papageorgiou MV, Kranidioti H, Katoglou A, Kontos G, Adamidi S, Kafiri G, Deutsch M, Pectasides D. The usefulness of transient elastography in the assessment of patients with HBeAg-negative chronic hepatitis B virus infection. J Viral Hepat 2014; 21:517-24. [PMID: 24750382 DOI: 10.1111/jvh.12176] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2013] [Accepted: 08/15/2013] [Indexed: 12/09/2022]
Abstract
Histological severity is often mandatory for the management of HBeAg-negative chronic HBV patients. We evaluated the performance of transient elastography (TE) in this setting. We included 357 untreated HBeAg-negative patients with ≥ 1 reliable liver stiffness measurement (LSM-kPa) by TE: 182 inactive carriers with HBV-DNA < 2000 (n = 139) or 2000-19 999 IU/mL (n = 43) and 175 patients with chronic hepatitis B (CHB). In carriers, HBV-DNA > 2000 and/or LSM > 6.5 were considered as biopsy indications. LSMs did not differ between carriers with low and high viremia, but were lower in carriers than in patients with CHB (5.8 ± 1.7 vs 9.0 ± 5.6, P < 0.001) offering moderate differentiation between these two groups (AUROC: 0.705). LSMs did not change significantly in carriers after 16 (12-24) months. In carriers with a liver biopsy, Ishak's staging scores were similar between cased with low and high viremia but higher in cases with LSM > 6.5 than ≤ 6.5 kPa. Moderate fibrosis (stages: 2-3) was detected in 0/10 carriers with only HBV-DNA > 2000 IU/mL, 2/10 (20%) carriers with only LSM > 6.5 and 5/10 (50%) carriers with both HBV-DNA > 2000 and LSM > 6.5 (P = 0.009). In patients with CHB, LSMs correlated significantly with grading and staging scores and offered excellent accuracy for ≥ moderate, ≥ severe fibrosis or cirrhosis (AUROC ≥ 0.919-0.950). TE can be helpful for the noninvasive assessment of HBeAg-negative chronic HBV patients. In conclusion, LSMs offer excellent accuracy for fibrosis severity in HBeAg-negative patients with CHB and can identify carriers with high risk of moderate fibrosis, which may be present in up to 35% of carriers with LSM > 6.5 kPa and 50% of carriers with LSM > 6.5 kPa and HBV-DNA > 2000 IU/mL.
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Affiliation(s)
- G V Papatheodoridis
- 2nd Department of Internal Medicine, Athens University Medical School, 'Hippokration' General Hospital of Athens, Athens, Greece
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dos Santos ADO, Souza LFB, Borzacov LM, Villalobos-Salcedo JM, Vieira DS. Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western Amazon region of Brazil. Virol J 2014; 11:16. [PMID: 24472141 PMCID: PMC3906887 DOI: 10.1186/1743-422x-11-16] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Accepted: 01/14/2014] [Indexed: 12/16/2022] Open
Abstract
Background Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to develop an in-house real-time PCR-based method, which was both ultra-sensitive and efficient offering an alternative method for nucleic acid testing (NAT). Methods A precore fragment with 109 bp was cloned and serial diluted to standard curve construction. The calibration of the HBV - DNA values was performed against OptiQuant® HBV-DNA Quantification Panel, Acrometrix Europe B.V.). Results From our in-house plasmid we prepared serial dilutions ranging from 2 × 103 – 2 × 109 copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r2 = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 μl. We found a strong correlation between the two methods (r2 = 0.9965 and p < 0.0001). The regression line give us the following equation: Log 10 (IU/mL) = 0.9038Log 10 (copies/mL) − 1.0643, suggesting that 1 IU/mL = 15 copies/mL. Conclusions Therefore, we can affirm that the qHBVRO PCR can detect HBV DNA in individuals with hepatitis B at any stage of the disease showing high capacity for NAT screening in hepatitis b donors. This results of sensitivity could provide an advance for automation in blood banks and increasing safety of patients who receive blood transfusions.
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14
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Kessler HH. Comparison of currently available assays for detection of hepatitis B virus DNA in the routine diagnostic laboratory. Expert Rev Mol Diagn 2014; 5:531-6. [PMID: 16013971 DOI: 10.1586/14737159.5.4.531] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Infection with the hepatitis B virus (HBV) continues to present diagnostic and therapeutic challenges worldwide. Today, many routine diagnostic laboratories have implemented assays based on molecular techniques for the detection of HBV DNA. However, the standard algorithm for specific diagnosis of HBV infection still relies on serologic testing. Molecular assays are employed for pretreatment evaluation, clinical staging and monitoring of antiviral therapy. Furthermore, molecular methods are essential for identification of mutations in the HBV genome. Although a continuous improvement of assay performance has been observed during recent years, lack of comparability of different molecular assays remains a problem to be resolved in the future. The limited range of linearity when employing conventional PCR will be overcome by using real-time assays.
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Affiliation(s)
- Harald H Kessler
- Molecular Diagnostics Laboratory and National Reference Laboratory for Hepatitis A, B, C, Institute of Hygiene, Medical University Graz, Universitaetsplatz 4, A-8010 Graz, Austria.
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15
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Ntziora F, Paraskevis D, Haida C, Manesis E, Papatheodoridis G, Manolakopoulos S, Elefsiniotis I, Karamitros T, Vassilakis A, Hatzakis A. Ultrasensitive amplification refractory mutation system real-time PCR (ARMS RT-PCR) assay for detection of minority hepatitis B virus-resistant strains in the era of personalized medicine. J Clin Microbiol 2013; 51:2893-2900. [PMID: 23804383 PMCID: PMC3754615 DOI: 10.1128/jcm.00936-13] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Accepted: 06/19/2013] [Indexed: 02/07/2023] Open
Abstract
Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymerase region. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resistance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a molecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay, was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type (WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A, and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ratio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinations of mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitivity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral populations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecies were also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be a useful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evaluation with prospective studies before they can be implemented in daily practice.
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Affiliation(s)
- Fotinie Ntziora
- Department of Hygiene, Epidemiology, and Medical Statistics, University of Athens Medical School, Athens, Greece
| | - Dimitrios Paraskevis
- Department of Hygiene, Epidemiology, and Medical Statistics, University of Athens Medical School, Athens, Greece
| | - Catherine Haida
- Department of Hygiene, Epidemiology, and Medical Statistics, University of Athens Medical School, Athens, Greece
| | - Emanuel Manesis
- Division of Internal Medicine, University of Athens Medical School, Athens, Greece
| | | | | | | | - Timokratis Karamitros
- Department of Hygiene, Epidemiology, and Medical Statistics, University of Athens Medical School, Athens, Greece
| | - Alexis Vassilakis
- Department of Hygiene, Epidemiology, and Medical Statistics, University of Athens Medical School, Athens, Greece
| | - Angelos Hatzakis
- Department of Hygiene, Epidemiology, and Medical Statistics, University of Athens Medical School, Athens, Greece
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16
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T-Cell Response to Hepatitis B Core Antigen: Identification of Prior Exposure to and Confirmatory Testing for Screening for Anti-HBc. J Biomark 2013; 2013:812170. [PMID: 26317023 PMCID: PMC4437383 DOI: 10.1155/2013/812170] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2013] [Revised: 09/08/2013] [Accepted: 09/08/2013] [Indexed: 01/05/2023] Open
Abstract
Background. During routine donor screening in the blood bank, it is not uncommon to find isolated reactivity for anti-HBc in the absence of detectable HBV DNA in a first donation but absence of reactivity to anti-HBc in subsequent donations, suggesting a false-positive result for anti-HBc. Study Design and Methods. The blood donor population was screened between January 2010 and October 2011. We selected 2,126 donations positive only for anti-HBc from a total of 125,068 donations. During the process, OBI donors were identified, and their HBcAg-specific T-cell response was analyzed and compared to donors with chronic (HBsAg positive) and recovered (anti-HBc only) infection. We analyzed correlations between signal levels (Co/s) in the competitive assay for anti-HBc and HBV DNA detection. Results. In the 21-month study period, 21 blood donors with anti-HBc alone were identified as OBI (1 in each 5955 donors). The relevant finding was the observation that anti-HBc only subjects with Co/s ≥ 0.1 did not have either HBcAg-specific T-cells or detectable HBV DNA and OBI subjects presented with Co/s ≤ 0.1 and HBcAg T-cell response. In the subset of 21 OBI subjects, 9 donors remained positive for HBcAg T-cell response after four collections. In all 9 samples, we observed HBV DNA fluctuation. Conclusion. Our data suggest that HBcAg-specific T-cell response could be used to confirm anti-HBc serological status, distinguishing previous exposure to Hepatitis B virus from anti-HBc false-positive results.
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Ali M, Idrees M, Ali L, Hussain A, Ur Rehman I, Saleem S, Afzal S, Butt S. Hepatitis B virus in Pakistan: a systematic review of prevalence, risk factors, awareness status and genotypes. Virol J 2011; 8:102. [PMID: 21375760 PMCID: PMC3058090 DOI: 10.1186/1743-422x-8-102] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2011] [Accepted: 03/06/2011] [Indexed: 12/13/2022] Open
Abstract
In Pakistan, there are estimated 7-9 million carriers of hepatitis B virus (HBV) with a carrier rate of 3-5%. This article reviews the available literature about the prevalence, risk factors, awareness status and genotypes of the HBV in Pakistan by using key words; HBV prevalence, risk factors, awareness status and genotypes in Pakistani population in PubMed, PakMediNet, Directory of Open Access Journals (DOAJ) and Google Scholar. One hundred and six different studies published from 1998 to 2010 were included in this study. Weighted mean and standard deviation were determined for each population group. The percentage of hepatitis B virus infection in general population was 4.3318% ± 1.644%, healthy blood donors (3.93% ± 1.58%), military recruits (4.276% ± 1.646%), healthcare persons (3.25% ± 1.202%), pregnant women (5.872% ± 4.984), prisoners (5.75% ± 0.212%), surgical patients (7.397% ± 2.012%), patients with cirrhosis (28.87% ± 11.90%), patients with HCC (22% ± 2.645%), patients with hepatitis (15.896% ± 14.824%), patients with liver diseases (27.54% ± 6.385%), multiple transfused patients (6.223% ± 2.121%), opthalmic patients (3.89% ± 1.004%) and users of injectable drugs (14.95% ± 10.536%). Genotype D (63.71%) is the most prevalent genotype in Pakistani population. Mass vaccination and awareness programs should be initiated on urgent basis especially in populations with HBV infection rates of more than 5%.
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Affiliation(s)
- Muhammad Ali
- Division of Molecular Virology, National Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal Bank Road, Thokar Niaz Baig, Lahore, Pakistan
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Giannousis IP, Manolakopoulos SG, Hadziyannis E, Georgiou A, Papatheodoridis GV. Changes of serum levels of keratin-18 fragments in hepatitis B e antigen-negative chronic hepatitis B patients under oral antiviral therapy. Antivir Ther 2011; 16:505-14. [DOI: 10.3851/imp1775] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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Koumbi L, Bertoletti A, Anastasiadou V, Machaira M, Goh W, Papadopoulos NG, Kafetzis DA, Papaevangelou V. Hepatitis B-specific T helper cell responses in uninfected infants born to HBsAg+/HBeAg- mothers. Cell Mol Immunol 2010; 7:454-458. [PMID: 20657604 PMCID: PMC4002954 DOI: 10.1038/cmi.2010.34] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2010] [Revised: 05/20/2010] [Accepted: 05/26/2010] [Indexed: 11/09/2022] Open
Abstract
Vertically transmitted hepatitis B virus (HBV) usually causes chronic infection. While combined active-passive immunoprophylaxis in neonates of hepatitis B surface antigen-positive (HBsAg(+)) mothers at birth prevents vertical transmission, it is not yet clear whether neonates encounter the virus or its products in the absence of hepatitis B e antigen (HBeAg). This study was undertaken to investigate HBV antigen-specific T-cell responses in vaccinated neonates of HBsAg(+)/HBeAg(-) mothers. Blood was collected from 46 HBsAg(+) mothers and their neonates (subjects) as well as 24 age-matched controls. All neonates of HBsAg(+) mothers received appropriate immunoprophylaxis, and HBsAg and hepatitis B surface antibody (anti-HBs) antibody titers were determined after completion of the vaccination course. Peripheral blood mononuclear cells (PBMCs) from infants at birth, 1 and 6 months of age were stimulated with recombinant HBsAg, hepatitis B core antigen (HBcAg) and mitogen, and interferon (IFN)-γ concentrations were determined by ELISA. HBsAg-induced production of IL-2, IL-5, IL-6 and IL-10 was assessed using a cytometric bead array kit on cells from 6-month-old neonates post-vaccination. All neonates were HBsAg(-) and responded to vaccination. Increased IFN-γ production following HBcAg stimulation was seen in 30.4% of neonates born to HBsAg(+)/HBeAg(-) mothers. Subjects demonstrated significantly higher IL-2 production post-HBsAg stimulation, whereas IL-5, IL-6 and IL-10 cytokine responses were not significantly different. Almost one-third of uninfected neonates developed viral antigen-induced IFN-γ production, suggesting that they had been exposed to virions or viral derivatives. This encounter, however, did not impair their T-cell responses to vaccination.
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Affiliation(s)
- Lemonica Koumbi
- Second Department of Pediatrics, Allergy Research Center, University of Athens, Athens, Greece.
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Koumbi LJ, Papadopoulos NG, Anastassiadou V, Machaira M, Kafetzis DA, Papaevangelou V. Dendritic cells in uninfected infants born to hepatitis B virus-positive mothers. CLINICAL AND VACCINE IMMUNOLOGY : CVI 2010; 17:1079-1085. [PMID: 20463102 PMCID: PMC2897267 DOI: 10.1128/cvi.00074-10] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/24/2010] [Revised: 04/20/2010] [Accepted: 05/04/2010] [Indexed: 12/27/2022]
Abstract
Plasmacytoid dendritic cells (pDCs) play a central role in antiviral immunity, detecting viruses via Toll-like receptors (TLR) and producing in response vast amounts of type I interferons (IFNs). Hepatitis B virus (HBV) causes chronic infection after vertical transmission. This study investigated whether an HBV-infected maternal environment might influence DC numbers and pDC function in uninfected infants. Blood was collected from inactive HBsAg carrier and control mothers and their infants at birth and 1 and 6 months of age. HBV DNA was measured in maternal and neonatal perinatal sera using real-time PCR. The circulating frequencies of myeloid DCs (mDCs) and pDCs were determined in the babies by flow cytometry. Peripheral blood mononuclear cells (PBMCs) and cord blood pDCs were stimulated with resiquimod, and alpha interferon (IFN-alpha) production and the pDC phenotype were assessed. The effect of the common-cold virus, rhinovirus (RV), on resiquimod stimulation was also determined. HBV DNA was detected in 62.3% of the mothers and 41% of their infants. DC numbers and pDC functions were similar between subjects and controls and were not correlated with maternal or neonatal viremia. RV infection did not induce pDC maturation until the age of 6 months, and it reduced TLR7-dependent resiquimod-induced IFN-alpha production similarly in both groups. Although the DC system is immature at birth, DCs of uninfected neonates of HBV-positive mothers are competent to initiate and maintain T-cell responses. RV is a weak inducer of IFN-alpha production until the age of 6 months and inhibits IFN-alpha responses triggered by the TLR7 pathway.
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Paraskevis D, Beloukas A, Haida C, Katsoulidou A, Moschidis Z, Hatzitheodorou H, Varaklioti A, Sypsa V, Hatzakis A. Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR) for the quantification of HBV-DNA. Virol J 2010; 7:57. [PMID: 20226057 PMCID: PMC2848214 DOI: 10.1186/1743-422x-7-57] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2010] [Accepted: 03/12/2010] [Indexed: 12/17/2022] Open
Abstract
Background Improved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR). Results Previously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant® HBV-DNA Quantification Panel, Accrometrix Europe B.V.). The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67%) to the Procleix Ultrio discriminatory HBV test (dHBV) (70%) in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979). Conclusions We report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.
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Affiliation(s)
- Dimitrios Paraskevis
- Department of Hygiene Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece.
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Meng S, Zhan S, Li J. Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays. Virol J 2009; 6:226. [PMID: 20025781 PMCID: PMC2803455 DOI: 10.1186/1743-422x-6-226] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2009] [Accepted: 12/22/2009] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV) DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H), we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure. RESULTS The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits. CONCLUSIONS The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays.
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Affiliation(s)
- Shuang Meng
- National Center for Clinical Laboratories, Beijing Hospital, Beijing, PR
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A novel real-time PCR assay for determination of viral loads in person infected with hepatitis B virus. J Virol Methods 2009; 165:9-14. [PMID: 20026193 DOI: 10.1016/j.jviromet.2009.12.009] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2009] [Revised: 12/08/2009] [Accepted: 12/10/2009] [Indexed: 11/21/2022]
Abstract
A novel LUX (Light Upon eXtension) primer-based real-time PCR assay was developed and evaluated in this study, which was designed to provide a cost-effective, specific and highly sensitive method for viral load determination of hepatitis B virus (HBV). The assay employed an effective and rapid nucleic acid extraction system based on magnetic beads. To evaluate its efficacy, this new viral DNA preparation method was compared with QIAamp Blood Mini Kit and the results showed a good correlation (r=0.971; P<0.001). The performance of the LUX real-time assay was validated by testing serial dilutions of HBV plasmid DNA (5 to 5 x 10(8)copies/reaction) and a good linear relationship was obtained between the Ct values and the log(10) concentration of the HBV DNA. The assay possessed high sensitivity and the detection limit of this system was as few as 25 copies/ml of serum. A total of 91 positive serum samples were detected to evaluate further the assay and the high specificity was confirmed by melting curve analysis. This assay provides an ideal tool for monitoring the treatment efficacy and studying the relationship between HBV viral load and the stage of disease.
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Bergallo M, Costa C, Terlizzi ME, Sidoti F, Margio S, Astegiano S, Ponti R, Cavallo R. Development of a LUX real-time PCR for the detection and quantification of human herpesvirus 7. Can J Microbiol 2009; 55:319-25. [PMID: 19370075 DOI: 10.1139/w08-134] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Human herpesvirus 7 is a highly seroprevalent beta-herpesvirus that, following primary infection, remains latent in CD4+ T cells and determines a persistent rather than a latent infection in various tissues and organs, including the lung and skin. This paper describes the development of an in-house light upon extension real-time PCR assay for quantification of human herpesvirus 7 DNA in clinical samples. The efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated by evaluating the human herpesvirus 7 load in bronchoalveolar lavages and skin specimens, chosen as 2 persistency sites, from healthy and pathological individuals. The real-time PCR assay developed in this study could be a useful tool to detect and quantify human herpesvirus 7 DNA in different clinical specimens to elucidate its epidemiological and pathogenic roles.
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Affiliation(s)
- Massimiliano Bergallo
- Virology Unit, Department of Public Health and Microbiology, University of Turin, Via Santena 9, Turin 10126, Italy
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Katsoulidou A, Paraskevis D, Magiorkinis E, Moschidis Z, Haida C, Hatzitheodorou E, Varaklioti A, Karafoulidou A, Hatzitaki M, Kavallierou L, Mouzaki A, Andrioti E, Veneti C, Kaperoni A, Zervou E, Politis C, Hatzakis A. Molecular characterization of occult hepatitis B cases in Greek blood donors. J Med Virol 2009; 81:815-25. [PMID: 19319945 DOI: 10.1002/jmv.21499] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The use of sensitive nucleic acid testing for hepatitis B virus in blood donors revealed a number of HBV DNA(+) cases among HBsAg(-) donors, a status known as occult HBV infection. The purpose of this study was the serological and molecular characterization of occult HBV infection in Greek blood donors. A prospective study was undertaken in order to identify occult HBV infection cases in blood donors. As part of the routine screening of blood donations in Greece, blood units were screened individually by a multiplex HIV-1/HCV/HBV nucleic acid assay. Initially reactive samples were retested with discriminatory assays. HBV DNA(+)/HBsAg(-) samples were tested further for HBV serological markers and HBV DNA was quantified by real-time PCR. Molecular characterization was performed by sequencing the envelope and polymerase genes of HBV. Preliminary screening revealed 21 occult cases with the following patterns: anti-HBc only: 7 donors, anti-HBc/anti-HBs: 7 donors, anti-HBc/anti-HBe: 5 donors, anti-HBc/anti-HBs/anti-HBe: 2 donors. In all cases, the HBV DNA load was <351 IU/ml. Sequencing was successful in 10 donors (classified within genotype D) revealing several amino acid substitutions related to diagnostic escape and antiviral resistance. HBsAg diagnostic failure and low viral replication in occult HBV infection carriers could possibly be attributed to multiple changes in envelope and polymerase regions, respectively.
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Affiliation(s)
- Antigoni Katsoulidou
- Department of Hygiene and Epidemiology, Athens University Medical School, Athens, Greece
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Development of a RT real-time PCR for the detection and quantification of human rhinoviruses. Mol Biotechnol 2009; 42:350-7. [PMID: 19291427 PMCID: PMC7091102 DOI: 10.1007/s12033-009-9164-x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2009] [Accepted: 03/04/2009] [Indexed: 10/25/2022]
Abstract
Human Rhinoviruses (HRV) are the most common viral agents, being responsible for upper as well as lower respiratory tract infections. Evidence demonstrating that HRV disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of HRV infections and the increasing number of immunocompromised patients underline the need for including HRV in virological diagnostics of acute lower respiratory tract illness. This article describes the development and optimization of a reverse transcription (RT) real-time PCR assay for quantification of HRV RNA in clinical samples. Efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated on bronchoalveolar lavage (BAL) specimens obtained from immunocompetent and immunocompromised patients.
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Chen MH, Hsiao LT, Chiou TJ, Liu JH, Gau JP, Teng HW, Wang WS, Chao TC, Yen CC, Chen PM. High prevalence of occult hepatitis B virus infection in patients with B cell non-Hodgkin's lymphoma. Ann Hematol 2008; 87:475-80. [PMID: 18327583 DOI: 10.1007/s00277-008-0469-9] [Citation(s) in RCA: 87] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2007] [Accepted: 02/14/2008] [Indexed: 01/05/2023]
Abstract
Several reports recently found that patients with B cell non-Hodgkin's lymphoma (NHL) had a higher carrier rate of hepatitis B surface antigen (HBsAg). The current study aimed to examine the hepatitis B virus (HBV) infection status of NHL patients in Taiwan, an HBV-endemic area. Serum HBV and serum hepatitis C virus were measured in 471 NHL patients and 1,013 non-lymphoma cancer patients enrolled between February 2000 and March 2007. Furthermore, nested polymerase chain reaction of HBV-DNA was used to examine the sera from selected patients in these two populations and healthy volunteers for the presence of occult HBV infection. The infection rates (as indicated by the rates of HBsAg and occult HBV) were compared between different groups. There was a higher incidence of HBV infection in B cell NHL patients (23.5%), especially patients with diffuse large B lymphoma, than solid tumor patients (15.6%, P = 0.001). Among HbsAg-negative patients, those with B cell NHL had a higher prevalence of occult HBV infection (6%) than those with non-lymphoma solid tumors and healthy volunteers, 0% and 0.9%, respectively (P = 0.005). B cell NHL patients, even HBsAg-negative B cell NHL patients, but not T cell NHL patients, have a higher incidence of HBV infection than patients with solid tumors. Our findings support the etiologic role of HBV infection in B cell NHL.
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Affiliation(s)
- Ming-Huang Chen
- Division of Hematology & Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
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Li X, Huang Y, Song C, Zhao M, Li Y. Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection. Anal Bioanal Chem 2007; 388:979-85. [PMID: 17468857 DOI: 10.1007/s00216-007-1281-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2007] [Revised: 03/21/2007] [Accepted: 03/26/2007] [Indexed: 11/29/2022]
Abstract
A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer-dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method.
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Affiliation(s)
- Xiaomin Li
- The Key Laboratory of Bioorganic Chemistry & Molecular Engineering and Institute of Analytical Chemistry, College of Chemistry & Molecular Engineering, Peking University, Beijing, China
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Olioso D, Boaretti M, Ligozzi M, Lo Cascio G, Fontana R. Detection and quantification of hepatitis B virus DNA by SYBR green real-time polymerase chain reaction. Eur J Clin Microbiol Infect Dis 2007; 26:43-50. [PMID: 17216291 DOI: 10.1007/s10096-006-0223-y] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
A single-round real-time polymerase chain reaction (PCR) assay based on SYBR green dye technology for the detection and quantification of hepatitis B virus (HBV) DNA in serum was evaluated and compared with a qualitative nested PCR and the Cobas Amplicor HBV Monitor assay (Roche Molecular Diagnostics, Milan, Italy). The performance of the real-time PCR assay was evaluated in a routine clinical laboratory setting with a total of 212 clinical specimens. The sensitivity of the real-time PCR corresponded to 31 IU/ml (70 copies/ml), and comparison with the qualitative nested PCR showed significant concordance for 94% of samples. The linear curve over 7 log units, spanning 10(3)-10(9) IU/ml (2.28 x 10(3) to 2.28 x 10(9) copies/ml), was observed in the quantitative determination. The interexperimental variability coefficient of the assay ranged from 0.22 to 0.39 and the intraexperimental variability coefficient from 0.24 to 0.41. By excluding values outside of the dynamic ranges of both tests, the HBV Monitor and the real-time PCR gave an agreement within +/-1 log unit for 90% of samples, while those for the remaining 10% were found to be above 1 log unit but less than 1.5 log units. When the results inside and outside the dynamic range of the HBV Monitor were examined, 90% of the results were in agreement. In conclusion, the real-time PCR based on SYBR green technology proved suitable for routine diagnostic purposes, showing good sensitivity, high specificity, high reproducibility, and good linearity over a broad dynamic range of quantification.
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Affiliation(s)
- D Olioso
- Dipartimento di Patologia, Sezione di Microbiologia, Università di Verona, Verona, Italy
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Katsoulidou A, Moschidis Z, Sypsa V, Chini M, Papatheodoridis GV, Tassopoulos NC, Mimidis K, Karafoulidou A, Hatzakis A. Analytical and clinical sensitivity of the Procleix Ultrio HIV-1/HCV/HBV assay in samples with a low viral load. Vox Sang 2007; 92:8-14. [PMID: 17181585 DOI: 10.1111/j.1423-0410.2006.00857.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND AND OBJECTIVES The Procleix Ultrio human immunodeficiency virus type 1 (HIV-1)/hepatitis C virus (HCV)/hepatitis B virus (HBV) (Ultrio) assay simultaneously detects HIV-1 RNA, HCV RNA and HBV DNA in individual blood donations. The main objective of the study was to assess the analytical and clinical sensitivity of the multiplex and discriminatory probe assays in samples with a low viral load. MATERIAL AND METHODS The VQC HIV RNA genotype B, HCV RNA genotype 1 and HBV DNA genotype A standard dilutions were tested in 26 repeats. The probability of detection by Ultrio was compared with previously obtained data of the Procleix Duplex HIV-1/HCV assay on the same reference panels. A selection of 121 anti-HIV-1, 138 anti-HCV and 190 HBsAg positive samples from patients receiving antiviral therapy were tested. The majority of patient samples had a viral load below the detection limit of the diagnostic nucleic acid test assays, which made them suitable to evaluate the performance of the multiplex and discriminatory assays on yield cases with a similar low viral load. RESULTS The 95% and 50% detection end-points of the Ultrio assay along with the corresponding 95% confidence intervals are 53.7 (32.9-117.2) and 8.6 (6.2-12.1) geq/ml for HIV-1 RNA, 30.3 (19.0-62.4) and 5.2 (3.7-7.2) geq/ml for HCV RNA and 393.7 (147.9-6978) and 54.5 (22.4-143.8) geq/ml for HBV DNA. The analytical sensitivity of Ultrio expressed as a potency factor relative to previously obtained Duplex results on the same HIV-1 RNA and HCV-RNA standard dilutions was 1.09 (0.20-6.10) and 1.11 (0.21-5.89), respectively. The assay detected all 22 HIV-1 infected patients with viral load > 50 copies/ml, and 41 of 99 patients (41%) with viral load < 50 copies/ml, of which 23 (56%) were detected by the discriminatory assay. All 47 patients with HCV RNA load > 521 IU/ml and 10/91 polymerase chain reaction-negative patients with viral load < 50 IU/ml tested positive in Ultrio assay of which five were missed in the discriminatory test. The assay detected 53/55 HBV infected patients (96%) with viral load > 250 copies/ml and 108/135 patients (80%) with viral load < 250 copies/ml of which 17 (16%) were missed by the discriminatory test. CONCLUSIONS The new Procleix Ultrio assay is as sensitive as the Procleix Duplex assay for HIV-1 and HCV detection meeting the requirements of universal guidelines. The ability of the assay to detect HBV DNA in low viral load samples could be useful for screening blood. Inevitable negative results of discriminatory probe assays caused by stochastic sample variation will reduce the chance of recognizing low viraemic blood donors detected by individual donation nucleic acid test.
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Affiliation(s)
- A Katsoulidou
- Department of Hygiene and Epidemiology, Athens University Medical School, Athens, Greece
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32
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Lu YQ, Han JX, Qi P, Xu W, Zu YH, Zhu B. Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA. World J Gastroenterol 2006; 12:7365-70. [PMID: 17143958 PMCID: PMC4087500 DOI: 10.3748/wjg.v12.i45.7365] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA.
METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified.
RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 104/mL, 6.3 × 102/mL and 1.6 × 103/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 109/mL, 2.08 × 106/mL and 4.40 × 107/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 105/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate.
CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.
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Affiliation(s)
- Yan-Qin Lu
- Shandong Medicinal Biotechnology Center, Shandong Academy of Medical Sciences, Key Laboratory of Ministry of Health for Biotech-Drugs, Jinan 250062, China
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Hwang SH, Cha CH, Kim YL, Kwon OJ, Oh HB. Performance Evaluation of Real-Q HBV Quantification Kit for HBV DNA by Real-Time PCR. Ann Lab Med 2006; 26:442-8. [DOI: 10.3343/kjlm.2006.26.6.442] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Affiliation(s)
- Sang-Hyun Hwang
- Department of Laboratory Medicine, Pusan National University Hospital, Busan, Korea
| | - Choong-Hwan Cha
- Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Korea
| | - Yoo-Li Kim
- BioSewoom Institute of Bioscience & Biotechnology, Seoul, Korea
| | - Oh-Joong Kwon
- BioSewoom Institute of Bioscience & Biotechnology, Seoul, Korea
| | - Heung-Bum Oh
- Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Korea
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Mazet-Wagner AA, Baclet MC, Loustaud-Ratti V, Denis F, Alain S. Real-time PCR quantitation of hepatitis B virus total DNA and covalently closed circular DNA in peripheral blood mononuclear cells from hepatitis B virus-infected patients. J Virol Methods 2006; 138:70-79. [PMID: 16962180 DOI: 10.1016/j.jviromet.2006.07.019] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2006] [Revised: 07/12/2006] [Accepted: 07/19/2006] [Indexed: 12/29/2022]
Abstract
It remains unclear whether hepatitis B virus (HBV) replicates in extrahepatic tissues, and particularly in peripheral blood mononuclear cells (PBMCs), which may serve as a reservoir for the maintenance of infection. A real-time PCR assay for the quantitation of total and covalently closed circular (ccc) HBV DNA in serum and in PBMCs was developed. This assay was highly sensitive (detection limit: 27 IU/mL), linear over a wide range (9 log10), and was displayed high inter- and intra-assay reproducibility for the quantitation of total DNA. Genotypes A to E were detected and the results were consistent with those obtained with the COBAS Amplicor HBV Monitor Test. The specificity of the methodology was increased by prior treatment with an enzyme that digests relaxed circular DNA, and the elimination of background signals from virus adsorbed to the surface of PBMCs. HBV DNA was detected in the serum and PBMCs of 12 HBsAg-positive patients, with less than 1% in the cccDNA form. In conclusion, the quantitation of total and ccc HBV DNA in PBMCs is potentially useful as a non-invasive marker, and may help to increase our knowledge of the natural history of hepatitis B.
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Affiliation(s)
- A A Mazet-Wagner
- Laboratoire de Bactériologie-Virologie EA3175, Faculté de Médecine, Université de Limoges, 2 rue du Dr Marcland, 87000 Limoges, France
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Hsia CC, Purcell RH, Farshid M, Lachenbruch PA, Yu MYW. Quantification of hepatitis B virus genomes and infectivity in human serum samples. Transfusion 2006; 46:1829-35. [PMID: 17002641 DOI: 10.1111/j.1537-2995.2006.00974.x] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
BACKGROUND Hepatitis B virus (HBV) infections are still a major health issue, with approximately 350 million people chronically infected with HBV worldwide. Information about the minimum copy number of HBV genomes required for infection would be useful as a reference for drug and vaccine development; for monitoring HBV patients during treatment; for screening of blood, organ, and tissue donors; and for regulating nucleic acid amplification assays for HBV. STUDY DESIGN AND METHODS Serum samples from chronic carriers (hepatitis B surface antigen-positive and antibody to HBV core antigen-positive) of the three most common subtypes of HBV were studied; their infectivity titers had been evaluated previously in chimpanzees. The genotypes of the HBV samples were determined by DNA sequences and type-specific amino acids of the S gene of HBV. Copy numbers of HBV DNA were quantified by real-time TaqMan polymerase chain reaction (PCR) and by nested PCR applied to limiting dilutions. The copy number determined for each inoculum was compared with previously defined chimpanzee infectivity titers. RESULTS The genotypes of the HBV adw, ayw, and adr inocula were A, D, and C, respectively. The concentration of HBV DNA was determined to be 5.4 x 10(9), 2.5 x 10(9), and 3.1 x 10(8) genome equivalents (geq) per mL for serum samples containing the adw, ayw, and adr, respectively. The chimpanzee infectivity titers per milliliter of these initial HBV-containing serum samples were previously determined to be 10(7.5) for adw, 10(7.5) for ayw (MS-2 strain), and 10(8) for adr. CONCLUSION The minimal copy number of HBV DNA in chronic carriers of HBV that can infect the chimpanzee model was estimated to be from 3 to 169 geq based upon the three well-characterized inocula.
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Affiliation(s)
- Chu Chieh Hsia
- Division of Emerging and Transfusion Transmitted Diseases, CBER, FDA, Bethesda, Maryland, USA.
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36
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Abstract
Management of hepatitis B virus (HBV) infected patients involves serological diagnosis, quantitation of HBV-DNA and measurement of HBV drug resistance. Different serological markers such as HBsAg, anti-HBs, anti-HBc (total and IgM), HBeAg and anti-HBe are assessed by immunoassays in order to define the infection status. The emergence of surface mutants however is a continuous challenge to design more effective immunoassays. Commercially available quantitative HBV-DNA assays with increased sensitivity and wider linear range give a more accurate estimate of viral replication and contribute decisively in the initiation and the monitoring of the response to HBV therapy. Genotypic drug resistance assays are important diagnostic tools, since the administration of nucleoside/nucleotide analogues to HBV infected patients leads to the development of drug resistance patterns very much dependent on the treatment regimen. Special issues have to be taken into consideration regarding HBV/HIV-1 co-infected patients, since concominant HIV and HBV replication results in higher rates of HBV replication. Current efforts are focused on the standardization of HBV-DNA assays (qualitative and quantitative), of HBV drug resistance assays as well as in the development of new assays and markers that will help in the prognosis and management of HBV infection (quantitative detection of pre-core mutants and HBV ccc-DNA assays).
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Elia G, Decaro N, Martella V, Campolo M, Desario C, Lorusso E, Cirone F, Buonavoglia C. Detection of equine herpesvirus type 1 by real time PCR. J Virol Methods 2006; 133:70-5. [PMID: 16309751 DOI: 10.1016/j.jviromet.2005.10.024] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2005] [Revised: 10/17/2005] [Accepted: 10/20/2005] [Indexed: 11/16/2022]
Abstract
A real-time PCR assay was developed for detection and quantitation of equid herpesvirus type 1 (EHV-1). The sensitivity of the assay was compared with an established nested-PCR (n-PCR). The real-time PCR detected 1 copy of target DNA, with a sensitivity 1 log higher than gel-based n-PCR. The assay was able to detect specifically EHV-1 DNA in equine tissue samples and there was no cross-amplification of other horse herpesviruses. Real-time PCR was applied to determine EHV-1 load in tissue samples from equine aborted fetuses. The high sensitivity and reproducibility of the EHV-1-specific fluorogenic PCR assay, combined with the wide dynamic range and the high throughput, make this method suitable for diagnostic and research applications.
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Affiliation(s)
- Gabriella Elia
- Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, 70010 Valenzano, Bari, Italy.
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38
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Hsiao LT, Chiou TJ, Liu JH, Chu CJ, Lin YC, Chao TC, Wang WS, Yen CC, Yang MH, Tzeng CH, Chen PM. Extended lamivudine therapy against hepatitis B virus infection in hematopoietic stem cell transplant recipients. Biol Blood Marrow Transplant 2006; 12:84-94. [PMID: 16399572 DOI: 10.1016/j.bbmt.2005.09.001] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2005] [Accepted: 09/02/2005] [Indexed: 12/17/2022]
Abstract
Lamivudine has demonstrated efficacy in the treatment and prevention of hepatitis B virus (HBV) reactivation after hematopoietic stem cell transplantation (HSCT). However, most of these studies involved short durations of prophylaxis, so there is significant concern regarding lamivudine resistance in these patients. Between March 1984 and November 2002, 71 HBV surface antigen-positive HSCT recipients, including a subgroup of 16 who received pretransplantation lamivudine therapy, which was continued into the posttransplantation period to prevent reactivation hepatitis, were enrolled onto our study. The efficacy of lamivudine therapy was first evaluated for the subgroup of 16 patients in terms of treatment response, lamivudine resistance, and viral recurrence after discontinuation by using virologic assays. Efficacy was then evaluated for all patients in terms of the hazards of lamivudine therapy for reactivation hepatitis after transplantation. During a median lamivudine therapy period of 73 weeks (range, 19-153 weeks), the initial response showed a median reduction of 2.54 log10 in serum HBV DNA (-0.28 to 6.72 range). Lamivudine-resistant mutations were detected in 10 (63%) of 16 patients during therapy, and 1 (12%) of 16 patients finally developed a viral breakthrough. At a median follow-up of 30 months after discontinuation, 3 (27%) of 11 cases had recurrence of HBV infection. Despite the emergence of the mutations, no deaths were due to HBV reactivation or severe cases of hepatitis. In the Cox proportion regression model regarding reactivation hepatitis after transplantation of all enrolled patients, lamivudine therapy was found to be the only favorable factor for the event, with a hazard ratio of 0.122 (95% confidence interval, 0.016-0.908; P = .040). In conclusion, extended lamivudine therapy is safe and effective for the prevention of HBV reactivation in an HSCT setting and significantly decreases reactivation hepatitis after transplantation.
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Affiliation(s)
- Liang-Tsai Hsiao
- Division of Medical Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
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Lole KS, Arankalle VA. Quantitation of hepatitis B virus DNA by real-time PCR using internal amplification control and dual TaqMan MGB probes. J Virol Methods 2006; 135:83-90. [PMID: 16551481 DOI: 10.1016/j.jviromet.2006.02.004] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2005] [Revised: 02/07/2006] [Accepted: 02/09/2006] [Indexed: 12/29/2022]
Abstract
Hepatitis B virus (HBV) DNA quantitation is used extensively for monitoring of antiviral treatment of HBV infection. A real-time PCR assay was developed using a TaqMan minor groove binder probe and primers corresponding to HBV pre-core region for HBV DNA quantitation. A 228 bp fragment from this genomic region of HBV was cloned and serial dilutions of plasmid DNA were used as an external DNA standard. Comparison of the real-time PCR quantitation results from 35 clinical serum samples with those obtained by COBAS Amplicor HBV DNA monitor kit (Roche Diagnostics) revealed a significant correlation (r = 0.92) for all the samples. The assay showed wide dynamic linear range between 2.5 x 10(2) and 2.5 x 10(10) copies/ml serum. Sera from 25 healthy individuals tested negative indicating the high specificity of the assay. The median coefficients of variation for both intra- and inter-experimental variability were 4.9% and 10.6%, respectively, which indicated remarkable reproducibility. An internal amplification control (IC) was developed to detect the presence of PCR inhibitors in the samples to avoid false negative results. The IC had the same primer binding sites but different internal sequence and it competed with the virus-derived target. The optimum concentration of IC was found to be 100 copies/reaction. The assay was validated by testing serial dilutions of the WHO international HBV DNA standard. Since conserved regions were considered during primer and probe design, the assay should be applicable to all HBV genotypes. The real-time assay will be useful for monitoring HBV-infected patients in routine diagnostic laboratories and in clinical practice enabling analysis of a wide dynamic range of HBV DNA in a single, undiluted sample.
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Affiliation(s)
- Kavita S Lole
- Hepatitis Division, National Institute of Virology, Pune, India
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40
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Abstract
The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.
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Affiliation(s)
| | | | - T. Lion
- Corresponding author. Tel.: +43 1 40470 489; fax: +43 1 40470 437.
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41
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Sum SSM, Wong DKH, Yuen JCH, Lai CL, Yuen MF. Comparison of the COBAS TaqMan HBV test with the COBAS Amplicor monitor test for measurement of hepatitis B virus DNA in serum. J Med Virol 2006; 77:486-90. [PMID: 16254975 DOI: 10.1002/jmv.20481] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Quantitation of low hepatitis B virus (HBV) DNA levels in patients with chronic hepatitis B is important for monitoring natural history of disease and treatment efficacy. This study aimed to compare the quantitation range and analytical sensitivity of the newly developed COBAS TaqMan HBV test (TaqMan test) with the COBAS Amplicor HBV Monitor Test (Amplicor test), using the Eurohep HBV reference plasma and serum samples from patients. Serial dilutions (2.7x10(1)-2.7x10(8) copies/ml) of the Eurohep HBV reference plasma and 50 serum samples from chronic hepatitis B patients were tested by both assays. The TaqMan test could detect seven (2.7x10(2)-2.7x10(8) copies/ml) of eight dilutions of the reference plasma, while the Amplicor test could only detect three of them (2.7x10(3)-2.7x10(5) copies/ml). The HBV DNA values measured by the TaqMan test correlated very well with the theoretical Eurohep standard values (r=0.998, P<0.001). There were good correlations between the HBV DNA levels measured by the two assays on both the Eurohep reference plasma (r=0.993, P<0.001) and serum samples from patients (r=0.904, P<0.001). Compared to the Amplicor test, the TaqMan test had a higher sensitivity (50 vs. 300 copies/ml), shorter assay time (6 vs. 10 hr), and wider dynamic range (8 vs. 3 logs), and was more cost-effective in a clinical setting. These data indicate that the TaqMan test is an excellent tool for HBV DNA quantitation.
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Affiliation(s)
- Simon Siu-Man Sum
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong
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42
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Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JDC, Wengenack NL, Rosenblatt JE, Cockerill FR, Smith TF. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006; 19:165-256. [PMID: 16418529 PMCID: PMC1360278 DOI: 10.1128/cmr.19.1.165-256.2006] [Citation(s) in RCA: 833] [Impact Index Per Article: 43.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.
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Affiliation(s)
- M J Espy
- Mayo Clinic, 200 First St. SW, Hilton 470, Rochester, MN 55905, USA.
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43
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Lindh M, Hannoun C. Dynamic range and reproducibility of hepatitis B virus (HBV) DNA detection and quantification by Cobas Taqman HBV, a real-time semiautomated assay. J Clin Microbiol 2005; 43:4251-4. [PMID: 16081992 PMCID: PMC1233936 DOI: 10.1128/jcm.43.8.4251-4254.2005] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The Cobas Taqman assay for hepatitis B virus (HBV) DNA showed linear detection over 7 logs for genotypes A to D. The coefficient of variation was 1.2% at > or =1,000 IU/ml and 22.0% at 10 IU/ml. In 97 clinical samples, the log HBV DNA/ml differed by 0.11 between Cobas Amplicor and Cobas Taqman (r2 = 0.97).
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Affiliation(s)
- Magnus Lindh
- Department of Clinical Virology, Göteborg University, Guldhedsgatan 10B, 413 46 Göteborg, Sweden.
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44
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Sypsa VA, Mimidis K, Tassopoulos NC, Chrysagis D, Vassiliadis T, Moulakakis A, Raptopoulou M, Haida C, Hatzakis A. A viral kinetic study using pegylated interferon alfa-2b and/or lamivudine in patients with chronic hepatitis B/HBeAg negative. Hepatology 2005; 42:77-85. [PMID: 15962284 DOI: 10.1002/hep.20738] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
We studied viral dynamic parameters in 44 chronic hepatitis B/hepatitis B e antigen (HBeAg)(-) patients treated with pegylated interferon alfa-2b (PEG-IFN) 100 or 200 microg weekly or lamivudine 100 mg daily or the combination of PEG-IFN 100 or 200 microg with lamivudine. Patients receiving PEG-IFN monotherapy exhibited viral load oscillations between weekly injections, which were resolved by the addition of lamivudine. The median pharmacological delay was estimated at 4.1, 5.8, and 1.8 hours in PEG-IFN monotherapy, PEG-IFN 100/200 microg + lamivudine, and lamivudine monotherapy, respectively (P = .44). The median half-life of free virus was 12.7 hours (range, 2.4-69.2 hours). The mean antiviral effectiveness of PEG-IFN 100/200 microg monotherapy was lower than that of lamivudine (82.6% vs. 96.4%; P = .005). The mean effectiveness of PEG-IFN 100 microg + lamivudine and PEG-IFN 200 microg + lamivudine was 92.8% and 94.4%, respectively. The half-life of infected cells ranged from 2.7 to 75 days. The median half-life of infected cells in patients receiving the combination regimens of PEG-IFN and lamivudine was similar to that of lamivudine patients (5.0 days vs. 6.0 days, P = .77). In conclusion, the addition of pegylated interferon alfa-2b in lamivudine treatment was found to neither enhance the potency of blocking HBV production nor the decay rates of infected cells. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
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45
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Konnick EQ, Erali M, Ashwood ER, Hillyard DR. Evaluation of the COBAS amplicor HBV monitor assay and comparison with the ultrasensitive HBV hybrid capture 2 assay for quantification of hepatitis B virus DNA. J Clin Microbiol 2005; 43:596-603. [PMID: 15695651 PMCID: PMC548123 DOI: 10.1128/jcm.43.2.596-603.2005] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Performance characteristics of the COBAS Amplicor HBV Monitor test (Roche Diagnostics), which measures hepatitis B virus (HBV) DNA quantitatively, were evaluated and compared with the Ultrasensitive HBV Hybrid Capture 2 (HC2; Digene Corporation) assay. Linearity and within-run precision were assessed for both methods by using eight HBV DNA-positive samples serially diluted to obtain a range of <100 to 500,000 HBV DNA copies/ml and run in triplicate. Agreement between the methods was studied with 100 clinical samples. HC2 assay performance near the limit of detection was investigated through repeat testing of 149 samples with HC2 and testing of 37 samples with HC2 results of <4,700 HBV DNA copies/ml by Amplicor assay and a qualitative PCR assay. The linearity experiment for Amplicor had regression of observed values compared to expected values (y = 1.073x - 0.247; R(2) = 0.993, n = 32; for HC2, y = 0.855x + 0.759, R(2) = 0.729, n = 18). Within-run standard deviation of log HBV DNA copies/ml ranged from 0.003 to 0.348 (Amplicor) and 0.027 to 0.253 (HC2). Agreement assessed by Deming regression was poor [Amplicor = 1.197(HC2) - 0.961; R(2) = 0.799, standard error of the estimate (SEE) = 0.710, n = 94]. Near the lower limit of detection, 32 of 149 repeat HC2 results were <4,700 HBV DNA copies/ml. Of the 37 samples with HC2 results of <4,700 HBV DNA copies/ml, HBV DNA was not detected in 15 samples, while HBV DNA was detected by at least one PCR method in 12 samples. Amplicor is linear from 200 to 200,000 HBV DNA copies/ml with undiluted samples, and this range can be expanded through dilution. Inconsistent HC2 results near the limit of detection justify use of a grey zone.
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Affiliation(s)
- Eric Q Konnick
- ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA.
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46
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José M, Gajardo R, Jorquera JI. Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples under long-term storage. Biologicals 2005; 33:9-16. [PMID: 15713552 DOI: 10.1016/j.biologicals.2004.10.003] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2004] [Revised: 10/13/2004] [Accepted: 10/24/2004] [Indexed: 11/28/2022] Open
Abstract
The implementation of nucleic acid amplification technology (NAT) for detection of HCV, HIV-1 and HBV has undoubtedly contributed to the viral safety of blood, reducing the window period. One important matter related to the stability of RNA/DNA is the effect of the storage conditions on samples. In a previous work, we studied the stability of HCV RNA in plasma samples after storage at different temperatures. This work is an update on the follow-up of a sample containing 100 IU/ml HCV RNA for 5 years at -20 degrees C, showing no decrease in the initial titre. The nucleic acid stability of other viruses, such as HIV-1 and HBV, has also been studied. At -20 degrees C, samples containing HIV-1 were followed up for approximately 3 years and the results obtained show no decay in HIV-1 RNA detectability. Regardless of the HIV-1 RNA concentration, samples stored at 5 degrees C maintain their titre for at least 14 days. At 25 degrees C, the HIV-1 RNA half-life was determined at nearly 7 days. The HBV DNA, at 5 degrees C and 25 degrees C, is stable for at least 28 days, regardless of the initial titre.
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Affiliation(s)
- Marta José
- Research and Development Area, Instituto Grifols, S.A., Poligon Llevant, C/Can Guasch, 2, 08150-Parets del Vallès, Barcelona, Spain.
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Shyamala V, Arcangel P, Cottrell J, Coit D, Medina-Selby A, McCoin C, Madriaga D, Chien D, Phelps B. Assessment of the target-capture PCR hepatitis B virus (HBV) DNA quantitative assay and comparison with commercial HBV DNA quantitative assays. J Clin Microbiol 2005; 42:5199-204. [PMID: 15528715 PMCID: PMC525161 DOI: 10.1128/jcm.42.11.5199-5204.2004] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Recent clinical studies suggest that hepatitis B virus (HBV) load and genotype may be independent predictors of responses to antiviral therapies. However, it is difficult for clinicians to accurately determine viral loads in patient samples because results--both the values and the units of measure--can vary greatly among different tests. Accordingly, the World Health Organization (WHO) has produced the first international standard for HBV DNA for nucleic acid amplification technology (NAT) assays. In the present study, we describe the performance of the target-capture PCR HBV DNA quantitative assay for the quantitation of HBV DNA in clinical samples and reference panels. The range of quantitation was between 50 and 10(10) IU/ml. The sensitivity and accuracy of the target-capture PCR assay were demonstrated by using the HBV panel from Quality Control for Medical Diagnostics (QCMD) and the WHO HBV DNA standard. The target-capture PCR assay quantitated the six genotype A members of the QCMD panel and dilutions of the WHO HBV DNA standard within an accuracy of 74 to 142%. Compared to current serological methods, the assay offers window period reductions of 19 days prior to HBV surface antigen and 26 days prior to HBV e antigen detection. The target-capture PCR assay was also compared with four commercially available NAT assays, and the various units of measure were standardized with respect to the international units of the WHO HBV DNA standard. The target-capture PCR assay is a sensitive, accurate, high-throughput, rapid, and reproducible assay for the determination of HBV loads.
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48
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Sum SSM, Wong DKH, Yuen MF, Yuan HJ, Yu J, Lai CL, Ho D, Zhang L. Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA. J Clin Microbiol 2004; 42:3438-40. [PMID: 15297480 PMCID: PMC497581 DOI: 10.1128/jcm.42.8.3438-3440.2004] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (ad and ay subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10(1) to 10(7) copies per reaction (1.0 x 10(2) to 1.0 x 10(9) copies ml(-1)). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (r = 0.901; P < 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.
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Affiliation(s)
- Simon Siu-Man Sum
- Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong
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Dewhurst-Maridor G, Simonet V, Bornand JE, Nicod LP, Pache JC. Development of a quantitative TaqMan RT-PCR for respiratory syncytial virus. J Virol Methods 2004; 120:41-9. [PMID: 15234808 DOI: 10.1016/j.jviromet.2004.03.017] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2002] [Revised: 03/23/2004] [Accepted: 03/23/2004] [Indexed: 11/19/2022]
Abstract
Respiratory syncytial virus (RSV) is a ubiquitous RNA virus of the family Paramyxoviridae that may interfere with graft tolerance and with other interstitial lung diseases. The low viral titre observed in the immunodeficient transplanted patients requires a highly sensitive detection method. Although different tests already exist for the detection of RSV, reverse transcription-polymerase chain reaction (RT-PCR) has been shown to have the best sensitivity. In this study, a SYBR Green assay was established for the detection of RSV A and RSV B in a common screening test, and two quantitative TaqMan RT-PCRs were developed to quantify both RSV subgroups separately. Standard dilutions obtained from RSV cell infections were included in each test, and the assay was normalised using a housekeeping gene. RSV was found in 16% of the transplanted patients tested. The quantitative TaqMan assay is fast, reproducible, specific and very sensitive, and could facilitate considerably the detection of RSV virus. This would in-turn facilitate studies on the role of RSV in graft rejection.
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Affiliation(s)
- G Dewhurst-Maridor
- Division of Clinical Pathology, CMU, Michel-Servet 1, Ch-1211 Geneva 4, Switzerland
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Aliyu SH, Aliyu MH, Salihu HM, Parmar S, Jalal H, Curran MD. Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system. J Clin Virol 2004; 30:191-5. [PMID: 15125876 DOI: 10.1016/j.jcv.2003.11.005] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Revised: 10/20/2003] [Accepted: 11/11/2003] [Indexed: 11/25/2022]
Abstract
BACKGROUND The diagnosis of hepatitis B virus (HBV) has until recently been based on traditional serologic methods targeting viral antigens and antibodies to viral proteins. The development of molecular methods allowing for the quantitation of HBV DNA is proving clinically valuable for monitoring therapy and detecting early treatment failures. OBJECTIVES Here we report a new real-time (LightCycler) quantitative PCR for the detection of HBV DNA based on sequence specific hybridisation probes (designed in-house), targeting the HBV surface antigen. STUDY DESIGN The assay was evaluated using a 10-fold dilution series of standard HBV DNA [Eurohep standard reference 1, genotype A, HBsAg subtype adw with a unitage of 10(6) WHO. i.u./ml] and 89 clinical serum samples. The performance was measured against a quantified standard HBV DNA working reagent (NIBSC code 98/780) and the sensitivity compared with our conventional thermal-block PCR. RESULTS AND CONCLUSION Real-time PCR detected HBV DNA in 45% (40/89) and thermal-block PCR in 16% (14/75) of clinical samples. Results for 26 samples were below the detection limit of the thermal-block PCR but could be quantified by real-time (LightCycler) PCR. The LightCycler assay was at least 5 logs more sensitive than thermal-block PCR and could detect HBV in a linear range between 5 and 10(7) i.u. per reaction. The broad generic nature of the PCR primers coupled with the enhanced sensitivity and specificity of the fluorescent hybridisation probes makes this assay potentially valuable for both routine diagnostic and epidemiological work.
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Affiliation(s)
- Sani Hussein Aliyu
- Health Protection Agency, Clinical Microbiology and Public Health Laboratory Addenbrookes Hospital, Box 236, Hills Road, Cambridge CB2 2QW, UK.
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