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IMMUNOLOGY. METHODS OF TREATMENT OF THE SYNDROME OF ACQUIRED IMMUNE DEFICIT (AIDS)

INVENTION
Patent of the Russian Federation RU2251701

METHOD OF DIAGNOSTICS OF STAGES OF HIV INFECTION

METHOD OF DIAGNOSTICS OF STAGES OF HIV INFECTION

The name of the inventor: Sizyakina Lyudmila Petrovna (RU); Andreeva Irina Ivanovna (RU)
The name of the patent holder: Sizyakina Lyudmila Petrovna (RU); Andreeva Irina Ivanovna (RU)
Address for correspondence: 344022, Rostov-on-Don, per. Nakhichevan, 29, Rostov State Medical University, Patent Department
Date of commencement of the patent: 2004.06.07

The invention relates to the field of clinical immunology and can be used to determine the stages of HIV infection. The essence of the invention consists in testing the blood serum by the enzyme immunoassay method, determining the optical density of the initial sample of the serum and the absorbance of the initial serum sample incubated for 10 minutes with 100 μl of a 3.5 M solution of sodium isothiocyanate NaSCN and with a decrease in OP 40% or less diagnose the stage A - generalized lymphadenopathy, with a decrease in OP in the range of 40-60% diagnose stage B - pre-AIDS, and with a decrease in OP by 60% or more diagnose stage C - AIDS.

DESCRIPTION OF THE INVENTION

The invention relates to the field of medicine, mainly clinical immunology, namely to the immunodiagnosis of infection caused by the human immunodeficiency virus (HIV), and can be used in determining the stages of HIV infection.

HIV infection continues to be one of the most urgent problems of modern medicine. Since 1990, a pandemic has been registered, which means the spread of the disease across the planet, with over 200,000 HIV-infected people registered in Russia in the past 3 years. Spreading on the "new" territory, the disease does not go away from the previously captured. In addition, the biology of HIV infection is such that there are no natural mechanisms of confrontation, and only social factors are able to counteract further progression of the pandemic (PMKhaitov, GAIgnatyeva, IGSidorovich Immunology - M .: Medicine, 2000. - C . 291-295).

To really effective means to combat the spread of HIV should be attributed timely and quality diagnostics, the significance of which can be traced from two perspectives. First, revealing the fact of infection allows us to use effective methods of preventing further spread of the infection. Secondly, early diagnosis of HIV infection contributes to the timely appointment of sufficiently effective therapeutic measures that prolong the initial stages of HIV infection and thus significantly affect its progression. In this connection, the problem of choosing reliable criteria allowing quickly, reliably, timely and with minimal material costs to determine the stages of the HIV-associated infection process (LP Sizyakina, VN Chernyshev - HIV infection in children. Rostov-on-Don, 2001. - P.81).

Currently, the WHO has adopted a classification of the stages of the HIV-mediated infection process based on clinical manifestations, according to which the initial stage or stage of A-generalized lymphadenopathy (GLP) is determined, which is characterized not only by the absence of pathognomonic features, but also by the clinically stable condition of the patient. Stage B (pre-AIDS) is characterized by the growth of a variety of clinically relevant symptoms - bacillary angiomatosis, oropharyngeal candidiasis, cervical carcinoma, general infection syndromes such as fever or diarrhea for more than a month, leukoplakia language, idiopathic thrombocytopenic purpura, peripheral neuropathy. For stage C - AIDS (terminal), the characteristic clinical signs are pulmonary tuberculosis, relapsing pneumonia, tumors of diverse localization (CDC MMWR, 1992, v.41, NRR-17, p.22). Determination of the stages of the infectious process is necessary to justify the option of timely adequate therapy, which allows to extend the average life expectancy of HIV-infected people and to prevent the spread of infection. The analysis of the patent and scientific-medical literature revealed a number of ways to diagnose the stages of HIV infection.

In particular, the test used to determine the stages of HIV infection and serves as the basis for prescribing antiviral therapy is the evaluation of the viral load of peripheral blood by means of polymerase chain reaction (PCR) (AV Kravchenko, LV Serebrovskaya, and E. E. Golohvostova. Timaside, in combination with Chividum and Invirase in the complementary therapy of patients with HIV infection .-- Epidemiology and infectious diseases .-- 1998. - №5 - p.51-52). The essence of the method is the quantitative determination of copies of the RNA virus in the plasma. However, it should be noted that there is no clear correlation between the progression of the infectious process and the viral load of the plasma. In particular, stage A of HIV infection corresponds to a high level of the virus in the plasma. The next stage - B (pre-AIDS) is characterized by a significant reduction in the number of copies of the virus RNA, despite the clinical progression of the infection. In the terminal stage C (AIDS), unlimited progression of the HIV RNA level in the blood is recorded again (VV Shkarin, SN Sorinson, HIV / AIDS infection, Twenty years after the onset of the pandemic, N. Novgorod: In NGMA, 1999. - P.43). Thus, this test is not sufficiently informative to characterize the level of developing immunodeficiency. The disadvantages of the method include its high technological complexity and cost, namely: the need for strict adherence to special conditions - sterility and temperature conditions for blood collection, transportation and testing. In addition to certified diagnostic kits, not only very expensive instruments are needed, but also a complex of laboratory rooms, including sterile boxes. The direct method for determining the genome of the virus is laborious and time-consuming, takes at least 5 hours.

The method of laboratory diagnosis of HIV infection (patent No. 21223187 published in BI No. 34) provides for ion-exchange chromatography of serum with subsequent ELISA determination of the level of "hidden" antibodies. This method, which is important in predicting the course of HIV infection, is not focused on determining its stages.

The method for determining antibodies to the human immunodeficiency virus (patent No. 2032907 published in BI No. 10) can be used to establish the fact of HIV infection, but it is not related to the definition of the stage of infection.

The method of solid-phase enzyme immunoassay for the detection of antibodies to human immunodeficiency virus (fp. 4653296 / 30-13, BI No. 45) is aimed at increasing the sensitivity of the method for determining antibodies to HIV, but it can not be used to diagnose the stage of the infectious process.

Known is a method for evaluating stages of HIV infection (Clinical Immunology and Allergology, edited by G.Lolor, Jr. - M., Practice, 2000. - P. 585-587), which consists in determining the level of serum 2-microglobulin. The increase in the concentration of this protein, determined with the help of diagnostic sets of enzyme immunoassay, corresponds to the progression of HIV infection. Meanwhile, these changes are nonspecific, since such dynamics 2-microglobulin is possible not only with HIV infection, but also with tumor processes, liver cirrhosis, and lymphoproliferative diseases. Thus, this method is not sufficiently informative to evaluate the stages of HIV infection because of its non-specificity (A.Roit, J. Bristoff Immunology .- M .: Mir, 2000. - p.411).

The closest technical solution we accepted for the prototype is the method of diagnosing the stages of HIV infection (VVPokrovsky, et al., "Clinical diagnosis and treatment of HIV infection." - M .: GOU VUNMTS RF, 2001. - P.11 ), Which consists in determining the absolute number of peripheral blood CD4 + lymphocytes. This method consists of a series of successive steps: isolating the population of lymphocytes by layering 2 milliliters of blood into a pre-prepared ficoll solution and verographia with a density gradient of 1.077 g / cm 3 , centrifuging the resulting mixture on an OPN-3 centrifuge at a speed of 1,000 rpm for 35 minutes, removal of the formed "lymphocyte ring" into a separate tube, followed by a three-fold "wash" from the ficoll-verrographic mixture by centrifugation in the culture medium 199 at 1,500 rpm for 5 minutes. Further, monoclonal antibodies to CD4 surface antigen labeled with fluorochrome are added to the washed lymphocyte suspension, the resulting slurry is incubated for 40 minutes at 4 ° C, and then again washed on a centrifuge OPN-3 in medium 199 for 5 minutes at 1.5 thousand revolutions in a minute. After this, a determination is made of the relative amount of CD4 positive lymphocytes on the flow cytofluorimeter. It is this device that enables the most adequate estimation of the level of CD4 + cells. On the same day, the formula of the patient's blood is determined, after which the absolute amount of CD4 + lipocytes is calculated according to the formula: the number of leukocytes × 10 9 l ×% lymphocytes ×% CD4 + lymphocytes: 10,000. In this case, if the absolute number of CD4 + T cells is more than 0.5 × 10 9 / L, stage A is diagnosed; With an absolute amount of CD4 + lipocytes in the range 0.2-0.499 × 10 9 / l - stage B; With the absolute number of CD4 + lymphocytes less than 0.2 cells × 10 9 / l diagnose stage C - (CDC MMWR, 1992, v.41, NRR-17, p.22). The disadvantage of the method is the complexity of its implementation. In particular, the results of this test can be objective only with the use of highly specific certified reagents (monoclonal antibodies) and an extremely expensive device - a flow cytofluorimeter that requires support personnel - engineers, programmers. These diagnostic devices are located only in specialized centers of regional scale, which significantly complicates the possibility of assessing the stages of HIV infection. First of all, because the analysis is based on the characteristic of receptors of viable blood cells, therefore the study is limited to a time frame - the interval between blood sampling and its testing should not exceed 3-4 hours. The method is complicated by additional tests - the determination of the blood formula and the total number of leukocytes in order to calculate the main criterion - the absolute number of CD4 + peripheral blood lymphocytes. For the prototype is characterized by insufficient information because of the lability of indicators, not directly related to the fact of HIV infection. Changes in the number of CD4 + lymphocytes are highly non-specific and can be observed in other diseases. In this regard, the diagnostic significance of the test can be important only in the context of other criteria for HIV infection.

An object of the present invention is to simplify the method of diagnosing HIV infection stages.

This goal is achieved by examining the patient's blood serum, which is divided into 2 portions. Based on the enzyme immunoassay, the optical density of the initial serum sample is determined, reflecting the level of the specific anti-HIV antibodies in the patient and the second portion of OP, incubated for 10 min with 100 μl of a 3.5 M solution of sodium isothiocyanate and characterizing the level of anti-HIV- Immunoglobulins that did not destroy the association with the antigens of the ELISA plate when incubated with NaSCN. Based on the results of the% reduction in the initial serum OD values, the stage of HIV infection is determined: when the OD decreases by 40% or less, the stage A - generalized lymphadenopathy is diagnosed, with the decrease in optical density in the open interval 40-60% diagnose stage B - pre-AIDS , And with a decrease in OD by 60% or more, the stage C - AIDS is diagnosed.

The proposed method is carried out in the following way: the patient is taking venous blood in the amount of 2-3 milliliters, after which it is centrifuged at 3,000 rpm for 10 minutes in the OPN-3 centrifuge, the serum is separated, divided into 2 portions. A sample of serum is added to the well of the diagnostic ELISA test system with sorbed HIV antigens (in accordance with the order of the Ministry of Health and the Ministry of Health of the Russian Federation No. 170 dated August 16, 1994). Then, the solution is washed to remove serum components that are not bound to HIV antigens. Further, antibodies to human immunoglobulins, conjugated with an enzyme changing the color of the subsequently added chemical substrate reagent, are added to the well. In this case, if the serum contains anti-HIV antibodies, the contents of the well change color. Using the special device - the enzyme immunoassay, the optical density of the contents of the well is determined, which reflects the level of specific antibodies to HIV in the test sample.

At the same time, 100 μl of a 3.5 M solution of sodium isothiocyanate (NaSCN) was added to the second well of the diagnostic plate, in addition to the test serum, and the resulting solution was incubated for 10 minutes. Further, the sequence of steps described above is followed: the solution is washed to remove components of serum that have not been bound to HIV antigens, antibodies to human immunoglobulins conjugated with the enzyme changing the color of the subsequently added chemical substrate agent are added to the well, if there is an anti- -HIV-antibody content of the hole changes color. Using the enzyme immunoassay analyzer, the optical density of the contents of the well is determined, reflecting the level of specific antibodies to HIV after treatment with a 3.5 M solution of sodium isothiocyanate (NaSCN).

Further, the% change in the OD versus the initial serum sample is calculated, and when the OD values ​​are reduced by 40% or less, the stage A - generalized lymphadenopathy is diagnosed, with a decrease in OP in the open range of 40-60% diagnoses stage B - pre-AIDS, 60% or more diagnose stage C - AIDS.

The feasibility of the method is confirmed by the following clinical examples.

Example 1. Patient C., medical history No. 122/198. During the epidemiological investigation was identified as a contact with infected HIV. At clinical examination of signs of pathology it is not revealed. There were no complaints, the condition was satisfactory. Patient, according to the proposed method, a blood serum study was performed, as a result of which the following results were obtained: ODexex = 1500, OD inc = 1200. In connection with a decrease in OD by 20%, stage A of HIV infection is diagnosed.

Example 2. Patient D., medical history 68/103. At the planned dispensary examination, generalized lymphadenopathy, hepatospliomegaly, was revealed. In accordance with the claimed method, the patient was examined for blood serum. The value of OP ex = 1627, OP inc. = 897, a decrease in the value of OP = 45%, in connection with which the patient was diagnosed: HIV infection, stage B - pre-AIDS, adequate therapy was prescribed.

Example 3. Patient E., medical history 1203/109. Was in the infectious hospital with clinical manifestations of pneumocystis pneumonia and nephropathy. The patient underwent a serum blood test in accordance with the claimed method. OPex = 1020, OD inc = 340, decrease in optical density = 67%. The patient was diagnosed with AIDS according to the claimed method, adequate therapy was prescribed.

Example 4. Patient C., medical history 101/04. During the year he was on dispensary registration in the regional center for the prevention and fight against AIDS, he did not complain. In accordance with the claimed method, a serum blood test was performed. The value of OP ex = 1920, OP inc = 1152, reduction of OD = 40%, and therefore diagnosed: HIV infection, stage A - generalized lymphadenopathy.

Example 5. Patient S., a medical history 88/98. Identified as a contact with HIV-infected intravenous drug users. When a clinical examination, pneumonia was detected, the fact of a decrease in body weight of 16 kg during a half-year was established. In accordance with the claimed method, the patient was examined for blood serum. The value of OD exx = 1950, OD of INC = 780, decrease of OD value = 60%. The patient was diagnosed with HIV infection, stage C - AIDS, and adequate therapy was prescribed.

In accordance with the proposed method for assessing the stages of HIV infection, we tested 86 specimens of sera from infected patients on the basis of the NUPK "Clinical Immunology" of RostMU. Of these, 44 were diagnosed: HIV infection in stage A, as a decrease in OD values ​​was 40 or less, 32 patients were diagnosed with HIV infection in stage B-pre-AIDS (OD declined by more than 40 but less than , Than on 60%), and at 10 person the stage With (AIDS) was diagnosed, thus% of decrease OP was 60 and more.

We analyzed 42 case histories of people on inpatient treatment in a specialized HIV-1 department in the city of Rostov-on-Don, among them 20 people were diagnosed with HIV infection in stage A (GLAP), 16 - "HIV infection in stage B (pre-AIDS)", and in 6 - "AIDS". These patients we conducted studies according to the claimed method. The results obtained showed that in 3 of 20 people the diagnosis of "HIV infection in stage A" was erroneously diagnosed in these patients for stage B-pre-AIDS (OD decreased by more than 40 but less than 60 %), Which was subsequently confirmed by clinical signs, 4 patients from 16 with stage B (pre-AIDS) diagnosis were diagnosed with AIDS on the basis of the claimed method of diagnosing stages of HIV infection, since% reduction in OD = 60 or more. This allowed to timely adjust the therapy. In 100% of cases of the terminal stage of HIV infection C (AIDS), the diagnoses coincided (in all patients, the decrease in OP was equal to or exceeded 60%). At the same time, in order to diagnose stages of HIV infection in accordance with the claimed method, it was not necessary to use additional labor, diagnostic kits and expensive equipment.

Thus, the proposed method is simpler than the prototype.

CLAIM

A method for diagnosing stages of HIV infection by blood testing, characterized in that the serum is tested by enzyme immunoassay, the optical density is determined from the initial sample of the serum and the absorbance of the initial serum sample incubated for 10 min with 100 μl of a 3.5 M solution of isothiocyanate Sodium - NaSCN and with a decrease in OD by 40% or less, diagnose stage A - generalized lymphadenopathy, with a decrease in OD in the range 40-60% diagnose stage B - pre-AIDS, and with a decrease in OD by 60% or more diagnose stage C - AIDS .

print version
Date of publication 06.01.2007gg