Detection of Hepatitis B Virus Covalently Closed Circular DNA in the Plasma of Iranian HBeAg-Negative Patients With Chronic Hepatitis B

Tajik, Zahra; Keyvani, Hossein; Bokharaei‐Salim, Farah; Zolfaghari, Mohammad Reza; Fakhim, Shahin Abdollahi; Keshvari, Maryam; Alavian, Seyed Moayed

doi:10.5812/hepatmon.30790

Cited by 6 publications

(8 citation statements)

References 23 publications

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“…There is no doubt that cccDNA exists in HBV-infected hepatocytes, but it is controversial whether extra-hepatic tissues contain cccDNA and whether cccDNA is released into the serum. Some reports have shown that cccDNA is present in patients’ sera and that it is a marker of off-treatment virological relapse [ 11 , 26 , 32 , 34 , 114 ], whereas others have refuted the existence of cccDNA in serum and have used DNA extracted from CHB patients’ sera as a negative control in the quantification of intracellular cccDNA [ 39 , 42 , 63 ]. Do some non-liver cells, such as PBMCs, support HBV infection and formation of cccDNA?…”

Section: Conclusion and Discussionmentioning

confidence: 99%

“…The supernatant is further extracted with phenol chloroform and precipitated with ethanol [ 20 , 32 , 33 , 42 , 45 , 65 , 66 ]. The QIAamp ® DNA blood & tissue Mini Kit (QIAGEN, Hilden, Germany) and NucleoSpin ® Tissue kit (Macherey-Nagel, Düren, Germany) have also been used to extract total DNA in many reports [ 26 , 30 , 31 , 35 , 38 , 39 , 40 , 41 , 44 , 67 , 68 , 69 ]; however, the HBV DNA species and ratios recovered from the QIAGEN and NucleoSpin ® Tissue kit have never been validated by Southern blot and compared to the conventional total DNA method. Because cccDNA accounts for a small proportion of total DNA, this method is not optimal for specific cccDNA detection.…”

Section: Preparation Of Cccdna Samplesmentioning

confidence: 99%

“…To further increase the sensitiveness and accuracy, Singh et al applied two hybridization fluorescence resonance energy transfer (FRET) probes in LightCycler TM (Roche Diagnostic, Mannheim, Germany) real-time PCR [ 41 ] which were able to maintain a cccDNA:rcDNA specificity ratio greater than 1:10,000. Due to the inherent speed, simplicity and sensitivity of qPCR, subsequent efforts to optimize general real-time PCR conditions [ 11 , 12 , 13 , 26 , 32 , 33 , 34 , 38 , 54 , 63 , 69 , 85 , 86 , 87 , 88 , 89 ] (including primers, probe design and sample handling) to effectively distinguish cccDNA from other HBV DNA forms have been made without sacrificing the high amplification efficiency and sensitivity of the method.…”

Section: Cccdna Detection and Quantificationmentioning

confidence: 99%

“…The widely accepted method for cccDNA detection is Southern blotting, which is insensitive, complex, time-consuming and not suitable for high-throughput drug screening. To resolve this problem and facilitate research on cccDNA, many new methodologies, including polymerase chain reaction (PCR)-based methods, Invader assays, in situ hybridization, and surrogates, have recently been applied to detect and quantify cccDNA [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. In this review, we summarize and compare currently available approaches for cccDNA detection.…”

Section: Introductionmentioning

confidence: 99%

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Detection of HBV Covalently Closed Circular DNA

Zhao

Yuan

et al. 2017

Viruses

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Chronic hepatitis B virus (HBV) infection affects approximately 240 million people worldwide and remains a serious public health concern because its complete cure is impossible with current treatments. Covalently closed circular DNA (cccDNA) in the nucleus of infected cells cannot be eliminated by present therapeutics and may result in persistence and relapse. Drug development targeting cccDNA formation and maintenance is hindered by the lack of efficient cccDNA models and reliable cccDNA detection methods. Southern blotting is regarded as the gold standard for quantitative cccDNA detection, but it is complicated and not suitable for high-throughput drug screening, so more sensitive and simple methods, including polymerase chain reaction (PCR)-based methods, Invader assays, in situ hybridization and surrogates, have been developed for cccDNA detection. However, most methods are not reliable enough, and there are no unified standards for these approaches. This review will summarize available methods for cccDNA detection. It is hoped that more robust methods for cccDNA monitoring will be developed and that standard operation procedures for routine cccDNA detection in scientific research and clinical monitoring will be established.

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Section: Conclusion and Discussionmentioning

confidence: 99%

Section: Preparation Of Cccdna Samplesmentioning

confidence: 99%

Section: Cccdna Detection and Quantificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection of HBV Covalently Closed Circular DNA

Zhao

Yuan

et al. 2017

Viruses

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“…The HBV DNA load was determined using a real-time PCR assay, as described previously (20). Detection of HBV cccDNA in liver tissue specimens was conducted by a probe-based real-time PCR method, using a Rotor-Gene device (QIAGEN, Germany), as previously explained (21).…”

Section: Detection Of Viral Load and Cccdnamentioning

confidence: 99%

Molecular Investigation of Occult Hepatitis B Virus Infection Among Patients with Liver Cancers

Javanmard,

Marjani,

Karbalaie Niya

et al. 2023

Int J Cancer Manag

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Background: Hepatitis B virus (HBV) is still the leading cause of hepatocellular carcinoma (HCC). HBV could persist in the low replicate state that is defines as occult hepatitis B virus infection (OBI). Recently, OBI has been defined important in the development and/or exacerbation of HCC and other liver diseases. Objectives: This study tried to determine the frequency of OBI among liver tumor samples. Methods: This cross-sectional study was performed among patients with HCC and intrahepatic cholangiocarcinoma (iCCA) in hospitals affiliated with Iran University of Medical Sciences. Liver tumor samples (Fresh frozen and formalin fixed paraffin embedded) were processed for isolation of DNA and then subjected to molecular assays, including PCR examinations of HBV genes (S, X, and C), determination of viral loads, detection of HBV-cccDNA, phylogenetic analysis, and assessment of mutations in HBsAg and RT regions. Results: In total, there were 93 participants, including HCC (n = 60), iCCA (n = 33); among which, 15% were detected positive for OBI. The OBI among HCC and iCCA were 18.3% and 9.1%, respectively. The mean intrahepatic viral load was 1.3 × 105 ± 1.4 × 102 copies/µl, and 5 had detectable cccDNA. The OBI subjects belonged to the HBV genotype D and subgenotype D1. In the HBsAg protein, T45N (33.3%) and P105A (25%) were the most prevalent mutations. N53K (33.3%), Y54H (25%), L80I (33.3%), and A113G (25%) were missense mutations that were observed in the RT domain. Conclusions: HBV-DNA was detected among liver tumor samples with negative HBsAg status. The results of viral load and cccDNA tests are important in identification and prognosis of liver diseases. It’s needed more precise molecular and cohort studies to clarify the functions of OBI in liver diseases.

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Detection of HBV genome in the plasma and peripheral blood mononuclear cells of Iranian HBsAg negative patients with HIV infection: occult HBV infection

et al. 2018

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The presence of hepatitis B virus (HBV) DNA in the absence of traceable hepatitis B surface antigen (HBsAg) in the plasma specimen of patients is defined as occult HBV infection (OBI). This study aimed to detect HBV-DNA in the plasma and peripheral blood mononuclear cells (PBMCs) of Iranian HBsAg negative patients with human immunodeficiency virus (HIV) infection. This cross-sectional study was conducted on 172 patients with HIV infection from September 2015 to August 2017. The patients were tested for serological parameters (HBsAg, HBcAb, HBeAg and HBeAb) against HBV infection. Moreover, they were tested for HBV viral load (using COBAS TaqMan 48 Kit, Roche, USA) in plasma and the presence of the HBV genome in PBMC specimens using real-time PCR. The mean age of the patients was 35.4 ± 13.4 years. Of the 172 studied patients, 109 (63.4%) were male. In this study, 151 (87.8%) patients were negative for HBsAg, 111 (64.5%) patients were negative for all HBV infection serological markers, 9 (5.2%) patients were only positive for HBsAg and 29 (16.9%) patients were only positive for HBcAb. Moreover, five (3.3%) patients with HBsAg negative had OBI (in the plasma sample of four patients and PBMC specimens of all five patients, HBV-DNA was detected). The present study revealed that 3.3% of the patients with HIV infection had occult HBV infection. Presumably, designing prospective studies to identify this infection in patients with HIV infection is informative and valuable.

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Detection of Hepatitis B Virus Covalently Closed Circular DNA in the Plasma of Iranian HBeAg-Negative Patients With Chronic Hepatitis B

Cited by 6 publications

References 23 publications

Detection of HBV Covalently Closed Circular DNA

Detection of HBV Covalently Closed Circular DNA

Molecular Investigation of Occult Hepatitis B Virus Infection Among Patients with Liver Cancers

Detection of HBV genome in the plasma and peripheral blood mononuclear cells of Iranian HBsAg negative patients with HIV infection: occult HBV infection

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