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Hepatitis B virus(HBV)

Hepatitis B virus(HBV) infection is a worldwide health problem plagued, there are around 350 million patients with chronic hepatitis B infection, the annual number of deaths caused by chronic hepatitis B up to 1.2 million, is listed as 9 major cause of death worldwide one of the diseases . China is a big country of hepatitis B, about 125 million people carry the hepatitis B virus, chronic hepatitis B patients in which there are about 30 million (accounting for about 1/12), hepatitis B incidence rate showed an increasing trend in the statutory report infectious diseases in our country over the years The incidence of hepatitis B and has among the highest incidence of chronic hepatitis B virus infection in clinical diagnosis and treatment become very difficult social and economic problems.

HBsAg is the surface antigen of the hepatitis B virus (HBV). It is the earliest sign of an active hepatitis B infection. This antigen may be present before symptoms of an HBV infection are present.

HBcAg is present on the surface of core particles. HBcAg and core particles are not present in the blood in a free form, but are found only as internal components of virus particles. It has strong antigenicity, and can stimulate the body to produce HBcAb. HBcAb lgG exists for a long time in the blood, and it is for the non protective antibodies. HBcAb lgM shows that HBV is the replication state.

HBeAg is the extracellular form of HBcAg, hence why the presence of both are markers of viral replication, and antibodies to these antigens are markers of a decline in replication. Multiple protein products can be produced from the same DNA sequence. When "ORF Core" and "Pre C" are translated together, the result is "HBeAg".It stimulates the production of HBeAb, which has a protective effect on HBV infection. Usually thinking that the appearance of HbeAb is a sign of good prognosis.

The two and half pair test and quantitative detection of HBV DNA

The main serum markers of hepatitis B are Hepatitis B Virus surface antigen (HBsAg), HBV surface antibody (HBsAb), HBV e antigen (HBeAg), HBV e antibody (HBeAb), and HBV c antibody (HBcAb). (They are commonly referred to as the two and a half pairs and are usually used to identify HBV infections.)  The first two pair of markers are obvious but because HBV c antigen is quickly degenerated in the serum, it cannot be tested in the serum.  If any one of these markers test positive, it means that the person has had a HBV infection. If HBsAg, HBeAg,and HBcAb test positive, it means that the HBV is replicating actively and the patient is infectious.

More recently molecular diagnostic methods have been used to quantify the levels of HBV DNA in serum as a marker of viral replicative activity. The detection and quantification of HBV DNA is reported to have prognostic value for the outcomes of acute and chronic HBV infections. Quantification of HBV DNA may be a more useful measure than HBeAg as genetic variants of HBV may continue to replicate at high level without secreting HBeAg. Quantification of HBV DNA can be useful to assess the efficacy of antiviral therapy as a more direct method of detecting viral replication than HBV serologic markers.

Diagnosis of acute or chronic hepatitis B virus (HBV) infection is based on the presence of HBV serologic markers such as hepatitis B surface antigen (HBsAg) and hepatitis B core IgM antibody (anti-HBc IgM), or the presence of HBV DNA detected by molecular assays. Although the diagnosis of acute and chronic HBV infection is usually made by serologic methods, detection and quantification of HBV DNA in serum

The presence of HBV DNA in serum is a reliable marker of active HBV replication. HBV DNA levels are detectable by 30 days following infection, generally reach a peak at the time of acute hepatitis, and gradually decrease and disappear when the infection resolves spontaneously. In cases of acute viral hepatitis with equivocal HBsAg test results, testing for HBV DNA in serum may be a useful adjunct in the diagnosis of acute HBV infection, since HBV DNA can be detected approximately 21 days before HBsAg typically appears in the serum.

Chronic HBV infection fail to clear the virus and remain HBsAg-positive. Such cases may be further classified as chronic active (replicative) HBV (high HBV levels, hepatitis Be antigen [HBeAg]-positive) or chronic inactive (nonreplicative) HBV (low or undetectable HBV DNA levels, HBeAg-negative). HBV DNA levels in serum are useful in determining the status of chronic HBV infection, by differentiating between active and inactive disease states. Patients with chronic active HBV are at greater risk for more serious liver disease and are more infectious than patients with inactive HBV infection. Reactivation of inactive chronic HBV infection (HBeAg-negative state) may occur with or without reappearance of HBeAg in serum. In patients with HBeAg-negative disease, detection of HBV DNA is the only reliable marker of active HBV replication.

Hepatitis B virus reproduces by making copies of its viral DNA nucleosides and nucleotides. The nucleoside analogues fool the hepatitis B virus into thinking they are normal building blocks for DNA. Essentially, the virus is unable to reproduce. Nucleoside analogues do not prevent all viral reproduction, but they can substantially lower the amount of virus in the body. Over time, the hepatitis B virus can become resistant to nucleoside/nucleotide analogues. Once this happens, the viral load rises again. Thus quantitative detection of HBV-DNA appears relatively complex and dynamic the results.

The significance of fluorescent quantitative HBV-DNA detection of PCR 

(1) The clinical threshold with < 103 copies/ml at HBV-DNA quantitative detection can be used as index of no replication or low level status. The patient with chronic hepatitis B has HBV-DNA > 105 copies/ml in serum. If alanine aminotransferase (ALT) levels is abnormal, should be considered for treatment.

(2) Quantitative PCR of HBV-DNA directly amplifies HBV DNA.It is earliest most sensitive and most reliable indicators for viral replication, then make early diagnosis of HBV infection. HBV-DNA content was positively related with infection of HBV. The content of HBV-DNA in patient serum is higher, and its infectivity is strong.

(3) The patients commonly are treated with interferon and lamivudine. If in the Clinic HBV-DNA decrease to below 105 copies/ml after medication, it is drug complete response . If quantitatively declined by > 2 orders of magnitude(102), it is partly drug response. If it fails to meet the standards, it belongs to low or no respond.

Interferon treatment in the research:

①The reaction of interferon with low level of replication of HBV is highly greater than high level of replication . If the content of serumin HBV-DNA is low, HBsAg almost become negative. If the content of serumin HBV-DNA is high, there is poor effect.

②During the treatment, if the level of replication rapidly decreased, it could be cured. On the contrary, if the level of HBV-DNA decreased slowly, or not obvious changes, most of all are ineffective treatment. 

③After treatment, using quantitative PCR for monitoring. If HBV-DNA has been negatvie for more than one year, it counld not been recurrence. If HBV-DNA remains low after termination of treatment, it has great possibility to be recurrence. Lamivudine of nucleoside analogues has significantly reduced viral load. In the short-term, its effect is significant. But in the long-term, it can lead to mutations of HBV gene, and the patient is easy to produce tolerance, and easy to recur after drug withdrawal.

(4) The level of HBV-DNA in serum determines whether or not HBV reinfects after liver transplantation. Because the HBV mainly infects the liver. Suggestion that taking anti-HBV medicine before transplantation to decrease concentration of HBV-DNA at the greatest degree. It Helps prevent HBV infection after liver transplantation, enhancing the effect of liver transplantation.

(5) Quantitative detection of PCR Hepatitis B before pregnancy contributes to choose available time for pregnancy. when viral load is low, it is good opportunity for pregnancy. During the woman with hepatitis B is pregnant, regular PCR examination helps to make a part of the patients timely and correctly diagnose, reducing the risk of intrauterine infection. Propagation between mother and infant occurs in the fetal and perinatal period. Especially for double positive mothers of HBsAg and HBeAg. If replication level of HBV-DNA is relatively high, the occurrence of fetal transmission and perinatal infection is higher. Because HBV can be transmitted directly to the baby through breast milk, breast-feeding women (except the blood test) should be detected the milk HBV-DNA, in order to determine whether it is available to feed breast milk. 

Hepatitis C virus (HCV)

Hepatitis C is an infectious disease affecting primarily the liver, caused by the hepatitis C virus (HCV). The infection is often asymptomatic, but chronic infection can lead to scarring of the liver and ultimately to cirrhosis, which is generally apparent after many years. In some cases, those with cirrhosis will go on to develop liver failure, liver cancer, or life-threatening esophageal and gastric varices.

Hepatitis C is a worldwide problem. The hepatitis C virus (HCV) is a major cause of both acute and chronic hepatitis. The World Health Organization (WHO) estimates about 3% of the world’s population has been infected with HCV and that there are more than 170 million chronic carriers who are at risk of developing liver cirrhosis and/or liver cancer.

Patients with hepatitis C should not donate blood or organs. One exception is in patients with HCV who require liver transplantation.

Hepatitis C is caused by a spherical, enveloped, single-stranded RNA virus belonging to the family Flaviviridae, genus Flavivirus. The HCV genome consists of a single, open reading frame and 2 untranslated, highly conserved regions, 5'-UTR and 3'-UTR, at both ends of the genome. The genome has approximately 9500 base pairs and encodes a single polyprotein of 3011 amino acids that are processed into 10 structural and regulatory proteins.

The natural targets of HCV are hepatocytes and, possibly, B lymphocytes. Viral clearance is associated with the development and persistence of strong virus-specific responses by cytotoxic T lymphocytes and helper T cells.

In most infected people, viremia persists and is accompanied by variable degrees of hepatic inflammation and fibrosis. Findings from studies suggest that at least 50% of hepatocytes may be infected with HCV in patients with chronic hepatitis C. RNA-dependent RNA polymerase, an enzyme critical in HCV replication, lacks proofreading capabilities and generates a large number of mutant viruses known as quasispecies. These represent minor molecular variations with only 1-2% nucleotide heterogeneity. HCV quasispecies pose a major challenge to immune-mediated control of HCV and may explain the variable clinical course and the difficulties in vaccine development.

The significance of fluorescent quantitative HCV-RNA detection of PCR 

(1) The clinical threshold with < 80 copies/ml at HCV-RNA quantitative detection can be used as index of no replication or low level status. Patients with HCV-RNA > 103 copies/ml in the serum should receive antiviral treatment. The efficacy of antiviral therapy in patients with HCV-RNA < 107 copies/ml often is better than the patient with higher HCV-RNA content.

(2) Quantitative detection of PCR HCV-RNA can greatly enhance the rate of detection. After 1 ~ 3W for exposure to HCV, it could be detected by HCV-RNA in peripheral blood. The immunological index of HCV has only one anti-HCV antibody. The period of HCV antibody detection is about three months. When clinical symptoms in patients with HCV infection, only 50% to 70% of patients are anti-HCV positive, and after 3 months about 90% patients are anti-HCV positive. A few patients infected with HCV do not produce anti-HCV. So the patients infectedc with HCV who do not produce antibody can be detected by HCV-RNA (+).

(3) Chronic hepatitis C is anti-HCV positive at long term. it only means infecting with HCV and the corresponding immune response , and does not means still presence or copy of HCV. Only the quantitative detection HCV-RNA identifies the activites of virus and the level of replication. Some patients are anti -HCV positive, but show normal levels of ALT, HCV-RNA (-). By the way, it does not exclude the HCV gene mutation, or false negative results caused by the inhibitory effect of PCR.

(4) HCV-RNA viral load positively related with the anti -HCV concentration and ALT level. the concentration of anti -HCV and abnormal rate of ALT increased with the increase of HCV-RNA load. HCV-RNA often turns to negative before the ALT returns to normal, but ALT returns to normal and HCV RNA continues to be positive. 

(5) The level of HCV-RNA viral load is no absolute correlation with disease severity and disease progression,  but it can be used as observed indexes of antiviral efficacy. Hepatitis C treatment has certain effect. The rate of HCV-RNA turned to negative can be as high as from 50% to 80%, but after drug withdrawal, about half of HCV-RNA turn to positive. The time of recurrence is 6 ~ 12 months after the treatment. If ALT is persistently normal after treatment, and HCV-RNA in serum negative, the patient may be cured. Whether or not Anti-HCV turns to negative, which can not be used as indexes of antiviral efficacy. 

(6) The replication of HCV mainly is in the liver. recurrence after liver transplantation is related with the level HCV HCR-RNA and the extent of immune inhibition after transplantation .

(7) HCV mainly propagates through blood transfusion and blood products. Instrument and injector without strict sterilization also are the important way to spread HCV through broken skin and mucosa. Medical disputes grow in this era. It is necessary for the patients to carry out quantitative detection of HCV-RNA before and after blood transfusion.