Quantitative Detection Of Hepatitis Research Articles

Accuracy of Saliva as a Diagnostic Medium for Hepatitis B Virus Infection: A Quantitative ELISA Analysis

Context: Diagnosis of hepatitis B virus infection (HBV) has been traditionally serum-based with its attendant risks and invasive procedure. Massive research interests are being directed to the use of saliva for screening, diagnosis, and monitoring of several infectious diseases, including HBV. Various reports suggest that saliva contains biomarkers that are comparable with that of sera. It can also be obtained with minimal risk to the clinician and patient as well. Aim: To determine the diagnostic accuracy of saliva in quantitative detection of hepatitis B surface antigen (HBsAg) using enzyme-linked immunosorbent assay (ELISA). Settings and Design: This cross-sectional analytical study was performed on HBV seropositive patients at the Gastroenterology clinic of the University of Benin Teaching Hospital, Benin City, Edo State. Materials and Methods: Under standardized conditions, equal amounts of blood and saliva samples of 43 HBsAg seropositive patients were analyzed using ELISA to quantitatively detect the concentration of HBsAg. Statistical Analysis Used: The analysis relied on Spearman’s correlation coefficient, linear regression analysis, and Bland–Altman plots to describe the correlational, predictive, and agreements between measurements of HBsAg in sera and saliva. The statistical significance was set at P < 0.05, while a 95% confidence level was used to construct intervals. Results: All participants had detectable levels of HBsAg in both saliva and serum with mean titers of 1.70 ± 0.35 ng/ml and 2.80 ± 0.77 ng/ml, respectively. Correlational and linear regression showed poor fit and predictive relationships of the HBsAg levels. Bland–Altman analysis showed good agreement and no significant bias in their diagnostic agreements. Conclusion: Saliva can be reliably used in screening and diagnosis of HBV infection. There was agreement in their levels independently and within their averages. It may be premature to rely on saliva for quantitative assay of HBsAg in treatment monitoring.

Clinical Value of Two and a Half Hepatitis B Combined with Quantitative Detection of Hepatitis B Virus DNA in Diagnosis of Chronic Hepatitis B

Clinical Value of Two and a Half Hepatitis B Combined with Quantitative Detection of Hepatitis B Virus DNA in Diagnosis of Chronic Hepatitis B

Effect of alcohol on the progress of hepatitis B cirrhosis.

Hepatitis B virus (HBV) and alcohol are primary causes of cirrhosis. Alcohol can result in replication of HBV, an increase in oxidative stress, a compromised immune response to the virus and an increase in liver inflammation, all of which can result in progression of cirrhosis. The aim was to explore the interaction of alcohol with HBV and to show the effect of different levels of alcohol intake on liver fibrosis and cirrhosis. We selected 90 patients with hepatitis B cirrhosis and divided them into three groups: non-drinking, moderate drinking and excessive drinking. Indicators of fibrosis (type III procollagen, type IV collagen, laminin, hyaluronic acid), HBV-DNA load, transaminases, quantitative detection of hepatitis B surface antigen (HBsAg), Child-Pugh scoring system rating and the number of complications were tested at three time points: 0, 3 and 6 months after quitting drinking and after medical treatment. We found that all indicators of fibrosis, HBV-DNA load, alanine (ALT) and aspartate (AST) transaminases in the excessive drinking group were highest among the three groups at any time. There were almost no differences between the moderate drinking and non-drinking groups at 0, 3 and 6 months after quitting drinking and treatment. We also found no difference among the three groups in quantitative detection of HBsAg at any time. It was observed that there are more patients with excessive drinking were in Child-Pugh C class and had more complications compared with the other two groups. Patients with chronic HBV infection and an excessive drinking habit activate HBV-DNA which increases liver inflammation, thus accelerating the progress of liver cirrhosis. Moderate drinking had no significant effect on the progress of liver cirrhosis. Hepatitis B cirrhosis patients with excessive drinking had more complications and were more likely to be in Child-Pugh C class compared with the other groups.

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay.

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R 2 = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.

Quantitative detection of hepatitis C virus (HCV) RNA with novel artus® HCV QS-RGQ Kit version 2

Quantitative detection of hepatitis C virus (HCV) RNA with novel artus® HCV QS-RGQ Kit version 2

Quantitative detection of hepatitis B virus (HBV) DNA with the novel artus® HBV QS-RGQ Kit version 2

In most research laboratories the use of EDTA tubes for the isolation of plasma DNA from tumor patients is standard. Unfortunately these tubes do not allow for an extended storage of samples before processing and prevent EDTA tubes from being shipped at ambient temperature. The aim of our study was to compare the quantity and quality of plasma DNA isolated from EDTA and PAXgene® Blood ccfDNA Tubes in different downstream applications.We enrolled 29 patients in our study. Blood samples were drawn into EDTA and PAXgene® Blood ccfDNA Tubes and were processed on day 0 and day 7 after storage at ambient temperature. The plasma DNA from 10 patients was isolated manually. For the DNA isolation from the plasma of 19 additional patients we used the automated QIAsymphony system. The total amount DNA from all samples was measured with a quantitative real-time PCR assay. In addition the amount of methylated mSHOX2 plasma DNA was determined.While the 7 day storage lead to an increased amount of total DNA in almost all EDTA tubes, this effect was only seen in very few PAXgene® Blood ccfDNA Tubes. The stabilization solution which prevents the lysis of blood cells had no effect on the method for quantification of methylated sequences in these samples.The quantity and quality of plasma DNA from both types of blood draw tubes are comparable. DNA from PAXgene® Blood ccfDNA was successfully used for PCR-based quantification of total amount of cell-free DNA and for methylation analysis as well.

Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads

Hepatitis B virus infection is one of the major causes of hepatitis, liver cirrhosis and liver cancer. In this study, we used highly luminescent quantum dot-beads (QBs) as signal amplification probes in the sandwich immunochromatographic assay (ICA) for ultrasensitive and quantitative detection of hepatitis B virus surface antigen (HBsAg) in human serum. Various parameters that influenced the sensitivity and stability of the QB-based ICA (QB-ICA) sensor were investigated. Two linear independent regression equations for detection of serum HBsAg were expressed with Y=0.3361X−0.0059 (R2=0.9983) for low HBsAg concentrations between 75pgmL−1 and 4.8ngmL−1, and Y=0.8404 X−2.9364 (R2=0.9939) for high HBsAg concentrations in the range from 4.8ngmL−1 to 75ngmL−1. The detection limit of the proposed ICA sensor achieved was 75pgmL−1, which is much higher than that of the routinely-used gold nanoparticle based ICA. The intra- and inter-assays recovery rates for spiked serum samples at HBsAg concentrations of 75pgmL−1, 3.75ngmL−1 and 18.75ngmL−1 ranged from 90.14% to 97.6%, and coefficients of variation were all below 7%, indicating that the QB-ICA sensor has an acceptable accuracy for HBsAg detection. Additionally, the quantitative method developed showed no false positive results in an analysis of 49 real HBsAg-negative serum samples, and exhibited excellent agreement (R2=0.9209) with a commercial chemiluminescence immunoassay kit in identifying 47 HBsAg-positive serum samples. In summary, due to its high fluorescence intensity, the sandwich QB-ICA sensor is a very promising point-of-care test for rapid, simple and ultrasensitive detection of HBsAg, as well as other disease-related protein biomarkers.

Rapid and quantitative detection of hepatitis B virus.

Despite availability of a universal vaccine, hepatitis B virus (HBV) infection has a huge impact on public health worldwide. Accurate and timely diagnosis of HBV infection is needed. Rapid developments have been made in the diagnostic and monitoring methods for HBV infection, including serological and molecular assays. In clinical practice, qualitative hepatitis B surface antigen (HBsAg) testing has long served as a diagnostic marker for individuals infected with HBV. More recently, HBsAg level has been used to predict treatment outcome when determined early during treatment or at baseline. However, identification of HBV DNA positive cases that do not have detectable HBsAg has encouraged the application of molecular tests. Hence, combination of quantitative detection of HBV DNA and HBsAg can be used to discriminate patients during the course of HBV infection and to monitor therapy. This article reviews the most commonly used quantitative methods for HBsAg and HBV DNA.

Effectiveness of hepatitis B virus vaccination program in Egypt: Multicenter national project.

To assess the effectiveness of hepatitis B virus (HBV) vaccination program among fully vaccinated children. A national community based cross-sectional study was carried out in 6 governorates representing Egypt. A total of 3600 children aged from 9 mo to 16 years who were fully vaccinated with HBV vaccine during infancy were recruited. Face to face interviews were carried out and sera were evaluated for hepatitis B surface antigen (HBsAg), anti-HBV core antibodies (total) and quantitative detection of hepatitis B surface antibody using enzyme linked immunoassays techniques. Samples positive to HBsAg/anti-HBV core antibodies were subjected to quantitative HBV-DNA detection by real time polymerase chain reaction with 3.8 IU/L detection limit. Sero-protection was detected among 2059 children (57.2%) with geometric mean titers 75.4 ± 3.6 IU/L compared to 3.1 ± 2.1 IU/L among non-seroprotected children. Multivariate logistic analysis revealed that older age and female gender were the significant predicting variables for having non sero-protective level, with adjusted odds ratio 3.3, 9.1 and 14.2 among children aged 5 to < 10, 10 to < 15 and ≥ 15 years respectively compared to those < 5 years and 1.1 among girls compared to boys with P < 0.01. HBsAg was positive in 0.11% and breakthrough infection was 0.36% and 0.39% depending on positivity of anti-HBc and DNA detection respectively. The prevalence of HBV infection was significantly higher among children aged ≥ 7 years (0.59%) compared to 0.07% among younger children with odds ratio equal to 8.4 (95%CI: 1.1-64.2) and P < 0.01.The prevalence was higher among girls (0.48%) than boys (0.29%) with P > 0.05. The Egyptian compulsory HBV vaccination program provides adequate protection. Occult HBV infection exists among apparently healthy vaccinated children. Adherence to infection control measures is mandatory.

Detection of hepatitis B virus with a non-labeled molecular beacon and a nucleic acid dye SYBR Green I

A high sensitive and selective method for sequence-specific DNA detection is developed using a non-labeled molecular beacon (MB) and a nucleic acid dye SYBR Green I. It is demonstrated by an oligonucleotide sequence of wild-type hepatitis B virus as a model system. Under the optimum conditions, the fluorescence intensity of SYBR Green I exhibit good linear dependence on quantity of target DNA in the range of 4 × 10^-11-400 × 10^-11 mol/L. The fitted regression equation is DI = 1.9556<italic>C</italic> + 31.4659 with a correlation coefficient of 0.9956 (<italic>R</italic>²), and the detection limit is 2 × 10^-11 mol/L (3<italic>s</italic>). The proposed method has a good precision, simple operation, fast detection speed, low detection limit, high accuracy and high sensitivity. The quantitative detection of hepatitis B virus is realized by this proposed method based on asymmetric PCR technology.

Quantitative Detection of Hepatitis A, Rotavirus and Genogroup I Norovirus by RT‐qPCR in Fresh Produce from Packinghouse Facilities

AbstractFood‐borne outbreaks caused by virus have been associated with the consumption of fresh produce. However, it is difficult to track the source of contamination due to the lack of sensitive methods of detection. In this study, we evaluated the presence of norovirus (NoV), rotavirus (RV) and hepatitis A virus (HAV) in fresh produce from the northwestern part of Mexico by reverse transcription quantitative PCR (RT‐qPCR). Fresh produce was sampled from field and packinghouse facilities during different stages of the packing process. Surfaces of equipment, as well as water used for disinfection were also sampled. A total of 46 samples were analyzed for the quantification of virus: HAV was found in 28.2% of the samples, NoV in 32.6% and RV in 13.0%. Eleven samples were positive with at least two different viruses. These results show a high prevalence of these viruses in fresh produce and highlight the need to establish further virological surveys for this kind of products.Practical ApplicationsViruses are considered important causal agents of food‐borne diseases. Many outbreaks implicate the consumption of fresh produce, mainly because fresh vegetables are usually consumed raw or with minimum processing. In spite of this, in many undeveloped countries, analyses for microbial quality imply testing for bacterial but not for viruses' contamination. For that reason, it is necessary to generate information on the risk of virus transmission in fresh produce. In that way, producers can be warned of the risk of virus contamination, and they could implement good manufacturing practices to improve sanitary conditions in fresh produce processing plants for consumer protection to diminish food‐borne outbreaks. We evaluated the prevalence of enteric viruses in fresh produce, water and surfaces of equipment by reverse transcriptase quantitative PCR, a sensitive method not commonly used for sanitary monitoring of food products.

To establish a magnetic beads based nucleic acid extraction method and apply for quantitative detection of hepatitis B virus DNA

Objective To facilitate the establishment of fully automatic clinical diagnostic nucleic acid assay, a study was carried out for developing a fast and convenient method of extracting highly purified viral nucleic acid from human blood with magnetic beads. Method Five KHB (Shanghai Kehua Biological Engineering Company Limited) internal strong positive HBV serum samples were used in the process, for the method development, screening and selecting the magnetic beads based on magnetism, suspension power and extraction effects of HBV DNA, the optimization of the selected key components of extraction reagents were performed, which including the quantity of magnetic beads（1-1.75 μl/ml）, the buffer pH of lysis (4-9), washing conditions with or without alcohol, elution for proliferation with or without magnetic beads, and etc.The evaluation for the extraction method was carried out including linearity, precision, sensitivity, specificity, anti-interference and resistance to contamination.The magnetic beads method was compared with domestic mature boiling method in a quantitative test with 100 HBV serum samples, the magnetic beads method was also compared with the interntional “Gold standard” Roche COBAS Tagman test method （Roche method） in a quantitative test with 50 HBV serum samples and the results were statistically compared for their linear regression. Results Optimization experiment result showed superparamagnetic beads A was the best among the candidates (C,B,D) in terms of their physical properties and extraction effects of DNA.The optimum condition of the assay were as follow:magnetic beads best used at 1.25 μl/ml, buffer pH 7-8 of lysis, ethanol was eliminated from washing buffer, proliferation can be carried out with magnetic beads.The parallel comparison experiments with five KHB internal strong positive HBV serum sample and gradient diluted serum sample between beads method and boiling method were carried out.The results showed that the linear range of beads method was 4×101-4×108 IU/ml and that of boiling method was 4×102-4×108 IU/ml.The sensitivity for beads method was 30 IU/ml and that of boiling method was 100 IU/ml.The precision CV of beads method was 6.0% and boiling method was 11.9%.The false positive rate was 0 for beads method vs 3.1% for boiling method.The anti-interference ability experiment showed that when sample contained sodium heparin concentration exceed 62.5 U/ml, there was significant drop of HBV DNA detection with boiling method and no HBV DNA was detected when sodium heparin concentration in the sample reached 1000 U/ml.However, sample sodium heparin concentration had no effect on HBV DNA detection with beads method.The comparison study between beads method and boiling method was carried out by use of 100 clinical HBV positive serum samples.The result showed that the sensitivity for beads method was 86% vs boiling method 74%.The correlation between the two method was R2=0.948，P＜0.05.The comparison study between beads method and Roche method was carried use of 50 clinical HBV DNA positive serum samples and the result showed that sensitivity for beads method was 95%(49/50) vs Roche method was 100%（50/50）.The correlation was R2=0.963，P＜0.05. Conclusions The established method for automatic nucleic acid extraction with magnetic bead displayed characteristic of high yield nucleic acid extraction, broader testing range, accurate quantification and convenient in operation.This can be one choice for nucleic acid extraction and applicable for clinical automatic diagnostic system.(Chin J Lab Med,2012,35:843-850) Key words: Hepatitis B virus; DNA; viral; Magnetics

Improvement for sensitivity of biosensor with total internal reflection imaging ellipsometry (TIRIE)

The biosensor based on the total internal reflection imaging ellipsometry (TIRIE) is realized as an automatic analysis method for protein interaction processes in real-time, with high throughput and label-free. An evanescent wave is used as the optical probe to monitor bio-molecular interactions on a chip surface with a high sensitivity due to its phase sensitive property. In this paper, the technique is optimized with a polarization setting, a spectroscopic light source and a low noise CCD detector to improve the performance of the biosensor in sensitivity and detection limit, as evidenced by a quantitative detection of Hepatitis B virus surface antigen (HbsAg) with concentrations of 8, 16, 32, 64, 125 and 250ng/ml. The sensitivity is increased by one order of magnitude and the detection limit has been extended more than 50 times for HbsAg detection.

Electrochemical detection of hepatitis C virus based on site-specific DNA cleavage of BamHI endonuclease

We have developed a new electrochemical approach for qualitative and quantitative detection of hepatitis C virus (HCV) based on the site-specific DNA cleavage of BamHI endonuclease.

Quantitative detection of hepatitis B virus covalently closed circular DNA in sera of chronic hepatitis B patients with a newly established assay

To quantitatively detect hepatitis B virus covalently closed circular DNA (HBV cccDNA) in sera of chronic hepatitis B patients with a newly established assay. Primers and probe were designed in highly conservative region of HBV DNA. DNA was extracted from 175 sera samples of chronic hepatitis B patients, and was treated with plasmid-Safe-ATP-dependent Dnase(PSAD) to eliminate the relaxed circular DNA (rcDNA). The products were amplified by real-time PCR with primers spanning. The detection rate of serum HBV cccDNA was found to correlate directly with serum HBV DNA loading. HBeAg positive chronic hepatitis B patients had higher serum HBV cccDNA levels than HBeAg negative chronic hepatitis B patients. The method is good because of the high specificity. It can be used for detection of HBV cccDNA. DNA;

Quantitative Detection of Hepatitis A Virus and Enteroviruses Near the United States-Mexico Border and Correlation with Levels of Fecal Indicator Bacteria

For decades, untreated sewage flowing northward from Tijuana, Mexico, via the Tijuana River has adversely affected the water quality of the recreational beaches of San Diego, California. We used quantitative reverse transcription-PCR to measure the levels of hepatitis A virus (HAV) and enteroviruses in coastal waters near the United States-Mexico border and compared these levels to those of the conventional fecal indicators, Escherichia coli and enterococci. Over a 2-year period from 2003 to 2005, a total of 20 samples were assayed at two sites during both wet and dry weather: the surfzone at the mouth of the Tijuana River and the surfzone near the pier at Imperial Beach (IB), California (about 2 km north of the mouth of the Tijuana River). HAV and enterovirus were detected in 79 and 93% of the wet-weather samples, respectively. HAV concentrations in these samples ranged from 105 to 30,771 viral particles/liter, and enterovirus levels ranged from 7 to 4,417 viral particles/liter. The concentrations of HAV and enterovirus were below the limit of detection for all dry weather samples collected at IB. Regression analyses showed a significant correlation between the densities of both fecal bacterial indicators and the levels of HAV (R2>0.61, P<0.0001) and enterovirus (R2>0.70, P<0.0001), a finding that supports the use of conventional bacterial indicators to predict the levels of these viruses in recreational marine waters.

Quantitative detection of hepatitis E virus RNA and dynamics of viral replication in experimental infection

Hepatitis E virus (HEV) RNA has been detected in the stool and serum of patients with HEV infection and experimentally infected nonhuman primates. However, dynamics of HEV levels in the stool and serum during clinical and subclinical infections have not been determined. A real-time reverse transcription polymerase chain reaction assay, using SYBR Green I in a LightCycler, was developed and optimized to allow quantification of HEV RNA in the stool and serum of both genotype 1 and 2 isolates. The specificity of the assay was confirmed by testing known HEV-RNA-positive and -negative stool and serum specimens and the sensitivity was evaluated using a synthetic HEV RNA standard. Profiles of viraemia and faecal shedding in two chimpanzees inoculated with an isolate of HEV genotype 1 showed the appearance of virus in the stools on day 4 postinoculation (5.65-6.85 log copies/mg) and in the serum on day 7 postinoculation (6.0-6.93 log copies/mL). Peak HEV RNA levels in the stool and serum coincided with peak alanine aminotransferase (ALT) levels observed on day 22 postinoculation in the two chimpanzees. At the time of detection of IgG anti-HEV in serum, viral RNA was no longer detectable in the stool or serum and ALT values had returned to normal levels in both chimpanzees, suggesting the efficacy of the immune response in terminating viral replication. Quantitative evaluation of HEV RNA in humans may allow determining the role of virus levels in the pathogenesis and transmission of HEV.

Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold.

Quantitative Detection of Hepatitis C Virus (HCV) RNA in Saliva and Gingival Crevicular Fluid of HCV-Infected Patients

The search for hepatitis C virus (HCV) in body fluids other than blood is important when assessing possible nonparenteral routes of viral transmission. However, the role of oral fluids in HCV transmission remains controversial. Here we quantitatively determined HCV RNA in saliva and gingival crevicular fluid (GCF) of anti-HCV-positive patients. Most patients (14 of 18; 78%) whose saliva specimens were negative had HCV RNA in their GCF. Most patients (20 of 26; 77%) had higher HCV RNA levels in their GCF than in their saliva. Although there was not a statistically significant correlation between the serum viral load and HCV level in saliva or GCF, patients with low serum HCV loads were less likely to have detectable HCV in their saliva. These findings have important implications for medical personnel and suggest that epidemiological studies designed to understand the significance of the oral route of transmission of HCV are warranted.

Quantitative detection of hepatitis B surface antigen by chemiluminescent microparticle immunoassay during lamivudine treatment of chronic hepatitis B virus carriers

The usefulness of fully automated chemiluminescent microparticle immunoassay (Architect HBsAg QT) for monitoring serum levels of hepatitis B virus (HBV) during antiviral therapy remains unclear. Using this assay, hepatitis B surface antigen (HBsAg) was measured in 20 patients with chronic hepatitis B before and during lamivudine treatment. At the start of therapy, 12 patients had detectable hepatitis B e antigen (HBeAg) and 8 did not. The median serum HBV DNA level and HBsAg concentration (25th-75th centile) were 7.2 (6.1-7.8) log genome equivalents/ml and 3,932 (1,585-12,330) IU/ml, respectively. The HBsAg concentration was significantly higher in HBeAg positive than in HBeAg negative patients (P=0.031). There was a significant correlation between the HBsAg concentration and HBV DNA level (r=0.490, P=0.027). The HBsAg concentration negatively correlated with patient age (r=-0.395, P=0.085). After the start of lamivudine therapy, HBV DNA levels fell rapidly in all patients. Serum HBsAg concentrations also fell in most patients, but to a lesser extent. When drug-resistant variants emerged, serum HBsAg usually increased before biochemical breakthrough. Although HBV DNA was elevated persistently after the emergence of drug-resistant variants, the increase in HBsAg was transient. In some patients, the increase in HBsAg preceded the increase in HBV DNA. Monitoring of serum HBsAg concentrations with the use of Architect HBsAg QT, in addition to measurement of HBV DNA levels, is helpful for evaluating the response to lamivudine treatment and for the early detection of drug-resistant strains.