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I. ДИАГНОСТИКА И ЛЕЧЕНИЕ
MARKERS OF SEROLOGICAL DIAGNOSIS OF VIRAL HEPATITIS B
УДК 616.36-00207:57.083.33
Abdilova G.B., Nurakhova A.D., Maymakova A.M., Abdigalieva G.K., Baichalova A.D.
National Scientific Surgery Center under the name of A.N.Syzganov, Almaty, Kazakh Medical University Continuing Education, Almaty
ABOUT THE AUTHORS
Abdilova Gulnur Bekmurzaevna - Head of the CDL, e-mail address: gulnur_abdilova@mail. ru, telephone 87019911346
Nurakhova Alma Dandybaevna - PhD, doctor-laboratory CDL, e-mail address: nad7788@mai.ru, telephone 87776850298
Abstract
In this paper the analysis of enzyme immunoassay research of viral hepatitis markers in the data extracted from the _
blood serum of patients treated and examined in National Scientific Surgery Center under the name of A.N.Syzganov in
2015. The article is devoted to the diagnosis of viral hepatitis B. To date, viral hepatitis (VH) is the most urgent problem Keywords
of medical science and public health throughout the world, which is due to their widespread prevalence, high incidence nked ¡mmunosorbent assay, viral
of the population and polyetiology. hepatitis, viral hepatitis B.
«В» вирусты гепатит ауруыньщ серологияльщ диагнозыньщ маркерлер1
Абдилова Г.Б., Нурахова А.Д., Маймакова А.М., Абдигалиева Г.К., Байчалова А.Д.
«A.H.Cbi3faH0B атындаш ¥FXO» AK,, Алматы к,.,
Уздшз 6rniM беру бойынша Казак медицина университет!, Алматы к.
Ацдатпа
«А.Н.Сызеанов атындаеы YFXO» АК-та 2015 жылы емделген жэне денсаулыеын тексерткен пациенттерд!^ канынан бел!нген сарысуынан алынеан «В» вирусты гепатилнщ маркерлер1н иммунды-ферментт! зерттеу деректерн¡н талдауы осы жумыста усынылеан.
Бул макала «В» вирусты гепатилн диагностикалау мэселесне арналеан. Казрп танда (ВГ) вирусты гепа-титтер аурулары дyниежyзi бойы кеннен таралуына, туреындардын аурушандыеынын денгей¡ жоеары болуына жэне олардын полиэтиологиялыеына (аталмыш аурудын этиологиясына эртYрл¡ себептер пайда болуы жeн¡ндег¡ кезкарас) орын алуына байланысты, 6yкл элемде, медицина еылымында жэне денсаулык сактау idнде де бул ауру ен езект болып табылады.
АВТОРЛАР ТУРАЛЫ
Абдилова Гульиур Бекмурзаевна - КДЗ мецгерушю,
злектронды адресi: gulnur_abdilova@mail. ru, телефон 8701991134
Нурахова Алма Дандыбаевна - M.f.K., КДЗ лаборант-даргерi,
злектронды адрес1: nad7788@mai.ru, телефон 87776850298
Туйш сездер
иммунды- ферментнтт анализ, вирусты гепатиттер, «В» вирусты гепатит
Маркеры серологического диагноза вирусного гепатита В
Абдилова Г.Б., Нурахова А.Д., Маймакова А.М., Абдигалиева Г.К., Байчалова А.Д.
AO «ННЦХ им. А.Н.Сызганова», г. Алматы,
Казахский медицинский университет непрерывного образования, г. Алматы
ОБ АВТОРАХ
Абдилова Гульнур Бекмурзаевна - Заведующая КДЛ,
злектронный адрес: gulnur_abdilova@mail. ru, телефон 8701991134
Нурахова Алма Дандыбаевна - к.м.н., Врач-лаборант КДЛ, злектронный адрес: nad7788@mai.ru, телефон 87776850298
Аннотация
В работе выполнен анализ данных иммуноферментных исследований маркеров вирусного гепатита В, выделенных из сывороток крови пациентов, лечившихся и обследовавшихся в ННЦХ им. А.Н.Сызганова в 2015 г. Статья посвящена вопросу диагностики вирусного гепатита В. На сегодняшний день вирусные гепатиты (ВГ) представляют собой наиболее актуальную проблему медицинской науки и здравоохранения во всем мире, что обусловлено их широкой распространенностью, высокой заболеваемостью населения и полиэтиологичностью.
Ключевые слова
ультрафильтрация, модифи-цирленген ультрафильтрация, жасанды канайналым
At the present stage of the study of the problem of viral hepatitis B, the Australian antigen, which is the surface antigen of the hepatitis B virus (HBsAg), is the main indicator of hepatitis B virus. As a result of this discovery, the American scientist B. Blamberg received the Nobel Prize (1977). Determination of the causative agent of hepatitis B (Dane particle) (HBV) and the execution of its study contributed to the discovery of additional information. At first, it was believed that HBV is a pathogenic agent only for human or anthropoid apes. But subsequent studies have led to the discovery that similar viruses are found in different animals: North American marmots, earth squirrels, Peking ducks and other birds.
The data obtained allowed all these viruses, including the human hepatitis B virus, to be included in the newly discovered family of Hepadnaviridae. HBsAg in its chemical structure is represented by proteins, glycoproteins, lipoproteins and lipids of cellular origin, which account for up to 30% of the total composition. HBV contains a double-stranded DNA ring molecule, which has approximately 3200 nucleotides in length, with variations from 3020 to 3320. The viral genome consists of four genes: the S gene (programming HBsAg), it has three Pre-S1, Pre -S2 and S; C-gene (programming HBcAg), including Pre-C and C zones; P-gene, which encodes the enzyme to DNA polymerase acting as a reverse transcriptase; X-gene, which carries information on the synthesis of X-protein. Significant information content of HBV DNA is determined by the fact that the open reading frames partially overlap each other. HBV DNA has the ability to integrate into the genome of various cells and stay there for many years. The nucleus of the hepatitis B virus, in addition to the DNA of the virus, also contains an enzyme DNA polymerase that can act as a reverse transcriptase. This enzyme is necessary for the assembly of a single-stranded portion of the short chain of HBV DNA, with the synthesis of RNA-replicative pregenoma with simultaneous transcription and translation, that is, for the formation of virus-specific proteins. In addition to the proteins needed to create HBV particles, the virus DNA programs data on non-structural proteins, such as HBeAg, HBsAg, which are also in demand for the life of the virus. Swedish scientists L.Magnius and J. Espmark in 1972 announced the discovery of a new serological hepatitis B system, which was established as an e-antigen (HBeAg) and antibodies to it (anti-HBe). The resulted antigen was detected by serum samples of individuals who are sick with acute and chronic HBsAg-positive hepatitis B and «carriers» HBsAg. It was determined that in sera with a positive response detected when HBV DNA polymerase and HBV DNA are detected, HBeAg is
often determined rather than anti-HBe. HBeAg is important in the pathogenesis of different stages of chronic hepatitis B [1,8].
It was found that the H-protein HBV plays a role in carcinogenesis and promotes the development of primary liver cancer that is associated with the hepatitis B virus. It also has an effect on the reproduction of HBV and is able to initiate the replication of other viruses such as human immunodeficiency virus and HTLV-1. This circumstance indicates a negative value of the hepatitis B virus in co-and superinfection of patients with HIV infection, leading to the development of a negative clinical course and the prognosis of the disease. Investigation of DNA of different HBV isolates made it possible to detect the presence of 8 genotypes, which are identified by Latin letters from A to N. Moreover, a number of genotypes of the virus can be detected simultaneously in one territory, but a certain genotype may predominate [7].
There is an opinion that HBV is one of the changing DNA-containing virus. The complex cycle of replication, which includes the stage of reverse transcription, is favorable for its considerable muta-tional ability, and changes can occur in all genes of the virus. To date, more than 150 mutant strains of HBV are known. Most of these strains do not cause changes in the properties of the virus, its antigens or the course of the infectious process. Mutant strains of HBV associated with these changes have been identified, in addition, strains that determine resistance to various drugs (eg, lamivudine). The significant prevalence of hepatitis B and its pronounced transmission among high-risk groups (drug addicts, homosexuals, etc.) contribute to the formation of the possibility of a double infection with the formation of recombinant forms between different HBV genotypes. Recombinant forms of the virus have been identified that have simultaneously the DNA sequences of the following genotypes: B / C, A / D, A / B / C, A / E, A / G, C / D, C / F, C / G, C / unknown Genotype [2,5].
The main feature of the virus, which determines the large spread of HBV, is its high infectivity. The emergence of hepatitis B can range from 10 to 100 particles of the virus. Individual samples of sera containing HBV are pathogenic even in dilutions of 10-7 to 10-8. The concentration of virus particles in serum with the presence of HBsAg varies from 10100 particles per ml to values that can not be determined using immunoassay. The hepatitis B virus can be isolated from all the sex secrets of a person infected with this pathogen. It is widely believed that the hepatitis B virus is 100 times more infectious than HIV. The significant resistance of HBV to various environmental influences also ensures the wide spread of hepatitis B [6].
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ВЕСТНИК ХИРУРГИИ КАЗАХСТАНА № 1-2017
The aim of the work enzyme immunoassay research was to analyze the markers of viral hepatitis B, made in 2015 in diagnostic laboratory of National Scientific Surgery Center under the name of A.N.Syzganov.
Material and Methods
This paper analyzes the research immuno markers of viral hepatitis B, isolated from the blood serum of patients treated and examined in National Scientific Surgery Center under the name of A.N.Syzganov in 2015. A total of the following number of parameters: HBsAg - 2114; anti-Hbs antibody - 117; anti-HBc total antibodies - 103; anti-HBc IgM antibody - 103; HBeAg - 103; anti-HBe antibodies - 103 tests were carried out on the instrument «Cobas», which is an automatic analyzer which calculates the level of discrimination on the basis of measurements and Cal1 Cal2, outstanding results in the form of discriminatory level index (DLI), cutoff index - COI - sample the signal to discriminatory terms. Thus DLI<1,0 samples evaluated as negative and samples with DLI>1,0 considered positive.
Results and discussion
As a result, the received review results of the study the serum of patients for 2114 assays of HBsAg positive results were obtained in cases 130 and 1984 made negative cases; in the study on the anti-Hbs antibody analysis of 117 positive samples were 66, and the negative - 51; the results of determination on the anti-HBc total antibodies of 103 tests were positive 58 and negative - 45; when screening for anti-HBc IgM antibody of 103 tests positive there was only one, and negative were 102; the study sample to HBeAg positive of 103 tests were 2 result, and negative - 101; finally, the determination of anti-HBe antibodies of 103 tests positive were evaluated 27 samples and a negative - 76.
It is believed that the development of hepatitis B can be if small amounts of blood come from the patient. That is, virtually unseen visual quantities of infectious blood can cause infection. Moreover, together with the large infectivity of HBV, a significant transmission of the virus can be determined by the expressed resistance of the pathogen in environmental conditions and in various disinfection regimens [6].
The following concentration of HBV in liquid body fluids is observed:
1) High:
- blood;
- serum;
- wound discharge;
2) moderate:
- vaginal discharge;
- saliva;
- sperm;
3) low / undetectable
- urine;
- chair;
- breast milk;
- sweat;
- tears.
The main ways of spreading hepatitis B are parenteral, sexual, perinatal. Simultaneously with these routes, HBV can be transmitted in artificial way - when injecting infected blood, performing operations, visiting a dentist, various parenteral manipulations performed by poorly processed reusable objects, so-called iatrogenic infection. Therefore, people at high risk of HBV infection are recipients of donated blood and its drugs, in particular patients with hemophilia, hematological diseases; Patients with chronic hemodialysis centers; Persons undergoing multiple medical-diagnostic and instrumental procedures with damage to the skin and mucous membranes; As well as medical personnel who have professional contact with the blood of patients. A greater likelihood of infection by artificial means is noted in drug addicts and in persons subjected to tattooing and ritual procedures [3,6].
In the laboratory diagnosis of viral hepatitis B, an important role is played by enzyme immunoas-say (ELISA), since this method contributes to the establishment of a certain list of indicators that are of great diagnostic value. For example, the surface antigen of HBsAg is an early indicator of viral hepatitis B, it appears in the blood in the incubation period. In acute infection, HBsAg can be detected up to 5-6 months. If HBsAg will be detected in the blood for more than 6 months, this may indicate a chronic disease. The initial positive result on HBsAg should be verified by the confirmatory test system, which is based on the neutralization reaction. If a negative test result is obtained for HBsAg, this is usually assessed as the absence of infection in the patient. However, the diagnosis of hepatitis B is unambiguously denied, since the subject may be at the seronegative stage of the incubation period of the disease or as a result of modification the virus discards the surface shell (L-form). The next infectivity antigen (HBeAg) begins to circulate at an early stage of infection with the hepatitis B virus. If this marker is determined more than a month, it indicates possible pathological changes in the liver that are caused by the propagation of the pathogen. This indicator determines the period of active reproduction of the virus, is released by affected he-patocytes, and also indicates that the serum of the patient has an infectious ability. Chronic hepatitis B HBeAg (+) (positive) is the most dangerous. With
Table 1
Serological evidence of infection with hepatitis B virus in stages
The stage of infection Surface antigen HBsAg Antigen «e» HBeAg IgM- anti nuclear antibody anti-HBc IgM Are common antinuclear antibody anti-HBc anti-HBe anti-HBs
Acute (early) + + + + - -
Acute (permitted) + - + + + or - -
Chronic (high infectivity) + + - + - -
Chronic (low infectivity) + - - + + or - -
Healing (immunity) - - - + + or - + or -
Successful vaccination - - - - - +
HBeAg (+), active treatment with antiviral drugs is used. Removing HBeAg 1.5-2 months after the onset of infection shows a favorable outcome of the disease. But in nature there is a dephist variant of the virus, which does not form HBeAg. This variant of hepatitis B is chronic and is usually observed in the form of long asymptomatic periods, which are followed by exacerbation phases. Common antibodies to the nuclear antigen (anti-HBc) circulate long, indicate a hepatitis B infection or may be a sign of a transferred illness. Patients with a chronic infection usually have a high titer of anti-HBc antibodies. This marker can be determined for life, often in high titles. In blood services of foreign countries, this indicator is included in the list of compulsory studies of all donor blood for the presence of common anti-HBc antibodies without identifying classes of immunoglobulins G and M. Antibodies to the surface antigen (anti-HBs) indicate immunity to the causative agent of hepatitis B or immune Response to vaccination against this infection. Then at high titre anti-HBs-antibodies anti-HBc-antibodies are not formed. In acute hepatitis B, antibodies to the HBs antigen are detected in 80% of patients 1-3 months after the appearance of the HBs antigen. When determining the effectiveness of vaccination, quantitative determination of anti-HBs antibodies is mandatory. If within 4-12 weeks after the last dose of the vaccine, the level of anti-HBs is detected above 10 mlU / mL, then this is confirmed by an adequate response. Antibodies IgM to the nuclear antigen (anti-HBc IgM) are the first antibodies that appear during infection with the hepatitis B virus (1.5-2 months after the onset of the acute period of the infection). The detection of these antibodies in the blood is indicative of acute hepatitis B. Anti-HBc IgM is circulating for a period of several weeks to several months, after their titre is reduced by the development of reconvalescence. Detection
of anti-HBc IgM is particularly informative when anti-HBs antibodies have not yet emerged. Sometimes low titres of anti-HBc IgM can be detected in the presence of chronic viral hepatitis B. The next marker is antibodies to e-antigen (anti-HBe). Good prognostic value is caused by the disappearance of HBeAg and the emergence of anti-HBe antibodies. The process of seroconversion by e-antigen is very significant in determining the effectiveness of antiviral therapy. Thus, the diagnosis of hepatitis B is established when HBsAg and IgM antibodies to the nuclear antigen (anti-HBc IgM) are detected. If the positive result of the HBsAg assay is preserved for 3 months, it is a sign of a chronic infection. To determine the probability of infection from a particular patient, HBe antigen is detected. With a positive result, the presence of chronic hepatitis is possible, the causative agent is easily transmitted, since it is in the phase of constant reproduction. When infecting with mutant strains, HBe-antigen in the serum is absent (Table 1) [6].
At the present stage, it is considered that the value of HBsAg as the only and basic screening indicator of chronic HBV infection requires revision; The presence of anti-HBs is not an absolute sign of freeing the body from the virus; patients with latent HBV infection may be sources of the pathogen in posttransfusion hepatitis and liver damage in recipients of donor organs. The detection of HBsAg in the blood transfusion service and in transplantology as the only indicator of the presence of HBV does not guarantee the complete exclusion of hepatitis B cases in recipients. It is necessary to use diagnostic systems to detect anti-HBc and DNA HBV testing, including new highly sensitive variants of its detection; Latent HBV infection may contribute to worsening of the course of chronic diffuse liver diseases that have arisen for other reasons, primarily alcohol and hepatitis C, and is associated
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BECTHÈK XMPyPfMM KA3AXCTAHA № 1-2017
with a poorer response to antiviral therapy in these individuals; Prolonged immunosuppressive treatment can promote the activation of a latent infection with the development of severe liver damage up to fulminant hepatitis; In this regard, a detailed virologic examination is required before the beginning of such treatment, and if latent HBV infection is detected, constant control of the level of viremia (the quantitative determination of HBV DNA in the serum) and biochemical hepatic analyzes during and after therapy are required; The oncogenic potential of latent HBV infection is not excluded; In her presence, patients need a long, perhaps lifelong, observation in case of detection of hepatocellular carcinoma (dynamic ultrasound and determination of the level of alpha-fetoprotein); In patients with cryptogenic hepatitis having a latent HBV infection and signs of active liver damage (according to
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