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MedTech Outlook | Monday, May 01, 2023
Molecular diagnostics significantly advance the detection, treatment, and response monitoring of Hepatitis B and Hepatitis C infection. This technology accurately identifies minute traces of viral DNA, enabling earlier and more precise diagnosis, which improves intervention timelines.
One of the major health issues facing the world today is the hepatitis B virus (HBV). Numerous clinical symptoms of HBV infection can include serious hepatic consequences such as liver cirrhosis and hepatocellular cancer. Currently, HBV surface antigen (HBsAg) immunodetection is the main method used for routine HBV screening and diagnosis.
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The adoption of nucleic acid amplification-based techniques, which are very sensitive, specific, and susceptibly tolerant of sequence variation, has been considerably encouraged by the detection of HBV DNA-positive cases without detectable HBsAg. Recent developments in molecular biology methods like real-time PCR and polymerase chain reaction (PCR) have hastened the field of HBV molecular diagnostics. Several isothermal amplification assays, such as loop-mediated isothermal amplification, transcription-mediated amplification, ligase chain reaction, and rolling circle amplification, have recently been used for HBV diagnosis in addition to PCR-based amplification techniques. Real-time detection options and connection with biosensing devices are also provided by these assays.
Clinical testing is crucial because specific diagnostic techniques are needed to distinguish HBV infection from other hepatitis-causing viruses. Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), human antibodies against these antigens (anti-hepatitis B surface antigen, hepatitis B core antibody, etc.), and, in some cases, the presence of viral nucleic acids (HBV DNA) are used to diagnose HBV. These antigens can be found primarily in the blood, followed by the liver and other extrahepatic sites. Acute/chronic, current/past HBV infections can be separated based on the presence or absence of a combination of antigens/antibodies.
Since the virus's discovery, the immunological detection of HBsAg has played a significant role in the laboratory diagnosis of HBV infection. The problem of HBsAg non-detection due to diagnostic-escape changes in the epitopes or low antigen levels, however, has greatly underlined the significance of applying reliable molecular biology tools for the effective detection of HBV. Nevertheless, the field of molecular diagnostics for HBV has also been substantially expanded by the development of molecular-based assays.
The HBV DNA can be amplified using molecular techniques such as polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), rolling circle amplification (RCA), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), or nucleic acid sequence-based amplification (NASBA).
Since many individuals spontaneously eliminate hepatitis infection, antibody testing for the detection of HBV and HCV does not determine if the patient is currently infected. In this situation, molecular diagnostics are crucial for identifying viral DNA. The open system kits measure and detect DNA specific to HBV and HCV to track treatment effectiveness. Currently, HBV testing kits that can validate genotypes A, B, C, D, and H are required, while HCV testing kits can validate genotypes 1-6.
With exclusive primers and probes that target highly conserved gene areas that are unaffected by a virus mutation, both viral load qPCR kits must have 100 per cent clinical sensitivity and specificity. The HCV kit needs to perform consistently with EDTA plasma samples.
Although viral DNA can be discovered in peripheral blood mononuclear cells, human hepatocytes are the primary site of HBV replication. Envelope binding to an unidentified receptor mediates entry. Nucleocapsids are translocated into the nucleus after entry and virion uncoating, where cellular DNA repair enzymes finish virion DNA synthesis. The resulting covalently closed circular DNA (cccDNA), which is mediated by host polymerase, serves as the template for the transcription of viral mRNA. Core protein, encapsidated full-length genomic RNA, and viral polymerase are organised into replication-competent nucleocapsids in the cytoplasm.
Viral polymerase uses reverse transcription of genomic RNA to create genomic DNA. Encapsidated, relaxed, open circular DNA can be released from the host cell by a process requiring cytosolic packaging (along with polymerase) by envelope glycoproteins, budding into the endoplasmic reticulum, and releasing after Golgi transit. It can also be transported to the nucleus to become an additional mRNA template.
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