Hepatitis C is an infection of the liver caused by the Flaviviridae HCV virus, which consists of one or more RNA molecules. As a rule, a series of diagnostic measures are taken to establish hepatitis C in a patient. PCR is an analysis that accurately confirms the diagnosis. Often the conclusion is provided by doctors when the patient already has the first signs of the disease.
What is hepatitis C
Liver damage by a viral infection transmitted through blood or sexual contact is called hepatitis C in medicine. The causative agent is an RNA-containing virus of non-cellular infectious agents of the Flaviviridae family. The infection can stay in the body for a long time without showing itself. Therefore, only if a microorganism is detected using a blood test using PCR RNA RNA, hepatitis C (as a diagnosis) is considered justified.
Flavivirus is not produced in artificially grown isolated cells. In the process of reproduction, the infectious agent creates immunologically distinct modifications. These factors prevent the body from givingappropriate protective response, and experts are having difficulty developing an effective vaccine.
The virus is transmitted parenterally. For infection, it must enter in sufficient quantities directly into the bloodstream.
What tests are done for hepatitis C
Viral pathologies of the liver are diagnosed using laboratory tests. Clinical studies of hepatitis C are carried out by studying foreign and dangerous antigens or protein compounds (antibodies) in the patient's biomaterial, mainly in the blood.
- Enzymatic immunoassay (ELISA). The method is based on the detection of lgM antibodies in the blood. Immunoglobulin is considered the largest and is a pentamer. It manifests itself in the primary protective response of lymphocytes to an unknown foreign substance.
- Radioimmunoassay (RIA) - quantitative determination of lgM immunoglobulin using a labeled radioactive isotope of iodine.
- PCR of hepatitis C - determination of viral RNA in the biomaterial (blood). This analysis reliably confirms the diagnosis.
IgG testing is not reliable. Its presence in the blood serum can indicate not only the presence of a flavivirus, but also another past infection that has the same pathogen.
What determines PCR analysis
Polymerase chain reaction (PCR) is based on the identification of DNA regions specific for certain pathogens in samples of material for research (epithelium, blood). The analysis makes it possible to identify pathogenic smallest organisms (viruses) by detecting their RNA or DNA in biological material.
Laboratory procedure for the detection of antibodies Hepatitis C PCR is carried out in 3 stages:
- Select. The studied biomaterial is purified from impurities and DNA is obtained in the mobile phase of the chromatographic column.
- Amplification. The analysis is carried out in the device-thermostat cycler. It heats and cools the test tubes with a certain cyclicity. For one study, up to 35 cycles are performed. The result is a sufficient number of DNA fragments to detect, identify and evaluate the pathogen.
- Electrophoresis. The resulting fragments are placed in a gel with different saturation of agarose and electrophoresis is performed. The resulting electropherogram is analyzed on a computer.
Not only the standard PCR study is used, but also the Real Time PCR analysis. This is real time diagnostics. The method allows for qualitative and quantitative analysis, which makes it possible to identify the formation and dynamics of the disease, as well as prescribe therapy aimed at eliminating the cause of the origin of the pathology. Real-time PCR is also used to determine a pure culture of infectious agents (genotyping).
How is the polymerase chain reaction test done
For diagnosis, any human biological fluid can be used. Biomaterial for testing for hepatitis C is usually blood.
Sampling for diagnostics is carried out in the proceduraloffice. To take blood, use disposable test tubes with a substance that inhibits blood clotting. Activities focused on preventing the spread of infectious agents help to avoid the entry of another strain of microorganisms from outside.
Blood for PCR of hepatitis C is taken in the morning on an empty stomach. The sample is sent immediately to the laboratory. It is allowed to store the biomaterial at a temperature of +4 to +8 degrees Celsius. Containers are labeled and provided with directions. Test results are available within 48 hours, sometimes sooner.
Types of PCR
The range of application of the polymerase chain reaction is quite wide. Infectionists determine using PCR hepatitis B, diseases transmitted by blood-sucking insects, AIDS, tuberculosis. In oncology, using this method, tumor cells are detected at an early stage.
There are about fourteen types of analysis. The use of one or another type depends on the scope and final results of the analyzes. Some types of PCR are indispensable where the result is needed within 20 minutes.
To diagnose hepatitis C, 3 types of polymerase chain reaction are used:
- Qualitative assessment can be either positive, indicating the presence of an infection in the body, or negative, indicating the absence of a flavivirus.
- Real-time PCR (quantitative analysis) - determines the quantitative RNA of the pathogen in IU/ml.
- Genotyping is an analysis that reveals the type (genotype) of the virus.
To accurately identify and determine the disease, followed byall three types of studies are used to prescribe optimal therapy.
Qualitative polymerase chain reaction analysis
An analysis is mandatory for everyone who has an ELISA that has detected antibodies to HCV. The qualitative PCR for hepatitis C is the standard susceptibility test. The method is aimed only at detection, no counting or isolation of other substances is performed.
To detect antibodies, special test systems are used with a sensitivity threshold of at least 50 IU/ml. If antibodies are detected, other clarifying tests are prescribed. If the result is negative, no further testing will be done.
In some cases, a false negative result may be the result of incompetent laboratory staff or poor quality reagents. For insurance, it is better to retake the analysis elsewhere.
Quantitative PCR
The method is used to directly study the number of flavivirus in one reaction cycle. For accurate measurement, DNA fragments used for hybridization or fluorescently labeled primers are used. There is an economical detection option using the SYBL Green dye. The dye is wedged into a small groove in the DNA and, when irradiated with a laser, turns blue.
Concentration is determined by the apparatus-amplifier in digital equivalent. Values may vary slightly between labs and should therefore be compared with reference values.
Quantitative PCRhepatitis C helps to choose the optimal dosage of drugs and determine the duration of therapy. The frequency of the study depends on the stage of the disease, the type of genotype and the prescribed course of treatment.
Genotype determination
The hepatitis C virus has a variable genetic structure. Numerous modifications make it impossible to create a vaccine, and also complicate therapy. A total of 11 genotypes and 100 subtypes have been identified and recorded. In the countries of the former USSR, in persons infected with hepatitis C, PCR mainly detects genotypes 1b and 3.
In the presence of any of the genotypes, cirrhosis or liver cancer can frolic. That is why it is so important to detect the virus in time.
For some patients, certain drugs designed to fight HCV are toxic. Genotyping allows you to determine the type of protein and prescribe an effective drug.
In the results of typing tests there is a number with a lowercase Latin letter indicating the genotype of the virus. If HCV is detected but not typed, this means that the person has a genotype that is not typical for a certain geographical area.
Analysis results
The doctor deals with deciphering the results. Only data from several studies (together with anamnesis and examination) can give an overall real picture of the medical history.
- In a he althy person, a test for a qualitative reaction in the biomaterial does not detect anything. If the value "detected" is indicated, the infection is confirmed, and the patientneeds further diagnosis followed by treatment.
- Determination of the number of infectious agents makes it possible to assess the viral load on the body. In normal quantitative PCR hepatitis C, the pathogen is not detected. An indicator of up to 810^5 is regarded as a low load and, with proper therapy, guarantees a favorable outcome. Higher values require in-depth examination and determination of long-term therapy, for the positive outcome of which no one can vouch.
- Positive genotyping results indicate which genotype is recognized. A negative result indicates either the absence of a flavivirus or the presence of a genotype that is not typical for this region.
What does a positive PCR test indicate
Any serious disease requires a comprehensive diagnosis. A positive hepatitis C PCR confirms the diagnosis, but predictions can only be made after additional laboratory and instrumental studies.
Detection of the virus does not give a complete picture of the pathology. It is necessary to identify its type and nature, to determine how it affects the liver and other organs. Early detection of HCV often has a favorable therapeutic outcome.
Negative PCR with positive ELISA
When observing the symptoms characteristic of liver damage, there is a high probability that the infection has already entered the body. Therefore, you should contact a hepatologist or an infectious disease specialist. During the interviews, the specialist will collect an anamnesis and prescribe the necessary examination.
Ifthe results of studies on hepatitis C PCR is negative, and the enzyme immunoassay is positive, this indicates the presence of antibodies to the flavivirus in the blood. This usually happens when the infection enters the body in a small amount, so the immune system coped with it on its own. But such people are still considered infected and are required to be examined every 6 months. If, with such test results, a person is denied a free medical examination, it is better to take a PCR for a fee to maintain he alth, and if the results are positive, contact a specialist for diagnosis and treatment.
Advantages and disadvantages of the method
When establishing the diagnosis of hepatitis C, PCR has several advantages:
- Early detection of pathogen.
- Accurate definition of the virus.
- Efficiency of diagnostics.
- Minimum error rate.
- High sensitivity.
Disadvantages of the method:
- High cost of analysis. The test requires expensive equipment, reagents and highly qualified medical staff. All together adds up to a considerable amount.
- Strict requirements for the transportation of biomaterial.
Treatment of inflammation of the liver
PCR analysis for hepatitis C virus is considered fundamental to diagnosis, but not definitive. For the therapy to be effective, a number of additional laboratory and instrumental studies are required. After passing alldiagnostic measures the doctor prescribes treatment:
- Diet 5 is prescribed.
- Alcohol is completely excluded.
- Joint reception of Interferon and Ribavirin for 25 days.
- Courses of hepaprotectors "Essentiale", "Karsil", "Phosphogliv".
- In special cases, discrete plasmapheresis is used.
Medicines prescribed by a hepatologist are used regularly, without changing the dosage. The duration of taking the drugs is determined by the doctor after passing the control tests. After completing the course of therapy, you must visit the doctor every six months.
PCR-diagnostics allows to identify the causative agent of hepatitis C at an early stage. This contributes to the effectiveness of therapy and the prevention of the transition of the disease into such dangerous forms as cirrhosis and hepatocellular carcinoma.
