RU2403869C1 - Method of detection of specific liver disease in leprosy patients - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to leprology, and can be used particularly for the detection of specific liver disease in the leprosy patients. Blood serum of the leprosy patients is analysed to determine the level of US-M.leprae and DIS-BSA leprosy mycobacteria antigen antibodies, and also the concentration of alpha-1-antitrypsin. If observing the level of antibodies to two antigens increased as compared to a norm and the concentration of alpha-1-antitrypsin increased higher than 346.7 mg/dl, liver disease is detected in the leprosy patients.

EFFECT: method provides higher accuracy.

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Description

The invention relates to medicine, namely leprosy, and can, in particular, be used to determine the specific liver damage in patients with leprosy.

From the practice of medicine, a method for determining liver damage in patients with leprosy is known, which consists in determining the activity of gamma-glutamyl transferase in blood serum (Tsemba V.P., Naumov V.Z., Yushchenko A.A., Umnova Z.G. ., Bezrukavnikova OA, Urlyapova NG Diagnostic informativeness of determining the activity of gamma-glutamyltransferase in the blood serum of patients with leprosy with chronic persistent hepatitis // Scientific-practical conference "Actual issues of treatment of sexually transmitted infections and chronic FIR dermatoses ": Abstracts of scientific rabot.- Ekaterinburg, 2002. - p.218). The disadvantages of this method is its use only in combination with other diagnostic methods, due to its lack of accuracy; an increase in enzyme activity occurs in other diseases (pancreatitis, coronary heart disease) and conditions (elevation, prolonged physical activity, pregnancy, circulatory failure) and is not always caused by chronic liver damage; false-positive test results can be associated with the administration of medications to patients (anticonvulsants, antitumor, isoniazid) and contribute to the overdiagnosis of pathological conditions from the liver, which reduces the accuracy of the method. All these shortcomings do not allow to obtain a specific technical result - improving the accuracy of the method.

There is also a method for determining liver damage in patients with leprosy, which consists in determining the amount of peroxide-modified heparin-containing apo-B-containing lipoproteins in blood serum. (Patent No. 2188431 of 08/27/2002). The disadvantages of this method are the inability to determine specific liver damage, the complexity and duration of implementation. In addition, to obtain the results, complex mathematical calculations are necessary. These shortcomings do not provide the opportunity to obtain a specific technical result - improving the accuracy of the method.

The closest way to the proposed is a method for determining leprosy mycobacteria in patients with disease regression, which consists in determining antibodies to leprosy mycobacteria using an enzyme-linked immunosorbent assay (Patent No. 2137137 of 09/10/1999).

The similarity of this method with the proposed one lies in the fact that they both belong to the field of medicine, namely leprology, and consist in determining antibodies to mycobacteria of leprosy.

The known method has the following disadvantages:

- insufficient accuracy of the method due to the fact that in a known manner it is impossible to determine the localization of a specific process;

- the known method includes the additional determination of cross-reacting antibodies to antigens of mycobacterium leprosy, which may occur in other diseases;

- the duration of the method due to the necessity of setting the reaction of counter immunoelectrophoresis;

- the ability to clarify the localization of a specific process only according to autopsy.

These shortcomings do not make it possible to obtain a specific technical result - improving the accuracy of the method.

Leprosy is a chronic infectious granulomatous disease caused by Mycobacterium leprae (M. leprae), characterized by a long incubation period, prolonged course, a variety of clinical manifestations, a tendency to periodic exacerbations, affecting mainly the skin, mucous membranes of the upper respiratory tract, peripheral nervous system, and support motor apparatus and internal organs. Of the latter, the liver is most often affected, in which chronic leprosy hepatitis usually develops, confirmed by clinical and laboratory-instrumental examination methods. Impaired liver function in patients with lepromatous type of leprosy is expressed in the deviation of protein, pigment, enzyme, carbohydrate metabolism, as well as in a violation of the antitoxic function of the liver. The leprosy process causes quite pronounced specific and nonspecific changes in the parenchyma and stroma of the organ. In the etiology of liver damage during leprosy, it is impossible to exclude the toxic component due to the long-term use of drugs for both specific anti-leprosy therapy and the treatment of concomitant pathology. Thus, the problem of liver damage during leprosy remains one of the most urgent in modern leprology. Its most important aspect is the timely detection of liver damage, especially since some patients may have a latent or slightly symptomatic course of the disease. At the same time, specific liver damage with leprosy is observed already in the early stages of the disease, and the onset of regression of the leprosy process does not indicate liver intactness. In addition, in the presence of functional insufficiency of the liver, the likelihood of adverse effects from anti-leprosy chemotherapy drugs that patients receive for a long time increases. In this regard, it seems relevant to search for new ways to determine liver damage.

In clinical practice, indicators of acute phase proteins are widely used, which is explained by the important role of the latter in the cascade of reactions of nonspecific defense of the body during pathological processes.

Along with immunoglobulins that can specifically bind foreign antigens, plasma contains another group of protective proteins - proteinase inhibitors. Widespread in tissues and biological fluids of proteolytic enzymes with high physiological activity, requires fine regulation of their action. In some pathological conditions caused by the inflammatory reaction, excessive activation of proteinases occurs, which can contribute to the destruction of tissue proteins. An important way to control proteolysis is the presence in the body of specific proteins - inhibitors of proteolytic enzymes, to the extent necessary, limiting their biological activity.

From 70 to 80% of the antiprotease activity of blood is accounted for by alpha-1-antitrypsin. Acting as the main inhibitor of neutrophilic elastase and an alternative inhibitor of the entire class of serine proteinases, alpha-1-antitrypsin naturally increases in inflammatory processes. Proteiasis inhibitors can be considered as indicators of the severity of the inflammatory reaction, the massiveness of tissue damage, the effectiveness of the therapy and the prognosis of the disease, and their determination is of great diagnostic value in inflammatory and destructive processes that occur in a number of diseases and conditions, including leprosy .

The chronic nature of the disease, generalization of the process, slow regression under the influence of chemotherapy can lead to the appearance of persistent forms of leprosy mycobacteria in various organs and tissues - liver, spleen, lymph nodes, nerves. Antibodies are markers of the persistence of leprosy mycobacteria in organs and tissues, leading to the development of specific complications of the leprosy process. Antibody titers reflect the degree of antigenic load, and their determination in blood serum allows us to evaluate the effectiveness of specific chemotherapy and may be one of the criteria for the cure of patients. At the same time, a serological examination of patients with leprosy in the regression stage allows the identification of individuals with persistent mycobacteria in organs and tissues and thus forms risk groups for relapse of the disease and the development of specific complications, including from the liver.

In the serological analysis of leprosy, the semisynthetic analogue of the specific carbohydrate epitope of phenolic glycolipid-1 from leprosy mycobacteria and the native preparation from leprosy mycobacteria, which is a complex of species-specific antigens, are used as antigens in serological analysis. The detection in the blood serum of patients with clinical regression of antibodies to these antigens is an indicator of the presence of mycobacteria leprosy in the body.

The present invention solves the main task of improving the accuracy of the method. The invention is expressed by a combination of essential features sufficient for the technical result provided by the invention.

The proposed method consists in the fact that when determining antibodies to mycobacteria of leprosy, alpha-1-antitrypsin in the blood serum is additionally determined and, with its value above 346.7 mg / dl, specific liver damage in leprosy patients is judged.

The proposed method is as follows.

We examined 55 patients with leprosy who are on inpatient and outpatient treatment at the Federal State Institution Research Institute for the Study of Leprosy of Roszdrav. All patients were divided into groups. The first group consisted of 30 patients who showed clinical, laboratory and instrumental (according to the results of an ultrasound study) signs of liver damage of a mixed etiology (specific, toxic, viral). The second group included 25 patients who did not have signs of liver damage at the time of the examination.

The content of alpha-1-antitrypsin in the blood serum is determined by immunoturbodimetric method using a commercial set of the company "Sentinel", Italy. Measurements are made on a Humalyzer 2000 programmable photometer (Germany). Statistical processing of the results is carried out using Student's criterion.

Antibodies to mycobacteria leprosy is determined as follows. We take blood from a finger using a scarifier in an amount of 0.1 ml in a dry tube. The blood tube is placed for 1 h in a thermostat at + 37 ° C, and then in the refrigerator (+ 4 ° C) for 1 h to retract the clot. The settled serum is aspirated into a dry tube and examined using an enzyme-linked immunosorbent assay (ELISA). For long-term storage, serum is frozen at -20 ° C. Two antigenic preparations from M. leprae are used: native - a water-soluble antigen from M.leprae, destroyed by ultrasound, and semi-synthetic (DIS-BSA) based on the carbohydrate epitope of phenolic glycolipid (FGL-1) from M.leprae.

Water-soluble antigens were obtained from M. leprae isolated from infected rat tissue with experimental leprosy using the Draper method (Draper P. Problems related to purification of M. leprae from armadillos tissues and standertiration of M. Leprae preparations - Jn .: Report on the Enlarged SC Meeting, Geneva, 1979. WHO Document, Annex 1, prat 1/79, p. 4-4).

M.leprae is isolated by differential centrifugation of tissue homogenates in a sucrose density gradient, and then separated from tissue particles in a two-phase polymer system consisting of 7% T-500 dextran (Pharmacia) and 5% polyethylene glycol with a molecular weight of 6000 daltons (BDH ) Purified M. leprae is subjected to ultrasonic destruction on the apparatus M E-100 (England) in physiological saline for 7 min at 18 kHz (kilohertz). The effect of destruction is controlled microscopically. The suspension of the destroyed M. Leprae is centrifuged (10,000 r 40 min d = 23 cm), the supernatant is used as a native antigen (SPL-M. Leprae). Semi-synthetic antigen (DIS-BSA) obtained from the WHO Bank.

ELISA is carried out on polystyrene microplates for single-use immunological reactions (Production Association Medpolymer TU 64-2-375-86).

Two tablets are sensitized at the same time: 1st — SPD-M.leprae, 2nd — semisynthetic antigen (DIS-BSA).

The first plate is sensitized with a UZD-M. Leprae in a carbonate-bicarbonate buffer pH 9.25 ± 0.25 (12.18 g Na ₂ CO ₃ , 3.47 g NaHCO ₃ and 0.2 NaN _{3 are} dissolved in 1 l of distilled water) at the rate of 5 μg of antigen per 1 ml of buffer in a volume of 0.1 ml per 1 well (18 hours at + 4 ° C). Then the tablet is washed 3 times in buffered saline and 3 times in distilled water with the addition of 0.05% tween - 20, after which 0.1 ml of a 1% solution of bovine serum albumin in 0.02 M phosphate saline buffer (pH 7.3-7.4) and incubated for 1 hour at 37 ° C. Then the wells are washed again using the above method and 0.1 ml of the studied sera of patients with leprosy diluted to 1: 400 with phosphate-buffered saline (pH 7.4) containing 1% calf serum are poured into the wells. The positive control (serum of patients with active leprosy) and the negative control (donor serum) in the same dilution (1: 400) are also placed on the tablet. Each serum sample is poured into 2 wells (duplicate). The plate is incubated at + 37 ° C for 1 hour, after which the washing process is repeated. Then, 0.1 ml of conjugate (rabbit antibodies to common human immunoglobulins labeled with peroxidase) was added to each well at a dilution of 1: 1000 and incubated for 1 hour at + 37 ° C. After washing, add the substrate mixture - 5-aminosalicylic acid with 0.05% hydrogen peroxide (to 100 ml of distilled water heated to 70 ° C add 80 mg of 5-aminosalicylic acid, the solution is cooled to room temperature and the pH is adjusted to 5.9-6, 0; to 18 ml of the resulting solution add 2 ml of a 0.05% hydrogen peroxide solution). The tablet is kept at room temperature for 60 minutes.

If serum antibodies to SPD-M.leprae are present in the serum, the substrate mixture stains dark brown (positive reaction), and the control wells remain colorless or slightly yellow (negative reaction). The reaction results are evaluated on a photometer (Minireder 590, USA) at a wavelength of 450 nm by the color intensity of the test sample and are expressed in units of optical density (OD). Samples are considered positive, whose OD indicators are 2 or more times higher than the indicators in the negative OD control. The OD of the negative control does not exceed 0.20.

At the same time, a second tablet is sensitized. DIS-BSA antigen is diluted in carbonate-bicarbonate buffer (pH 9.25 ± 0.25) at the rate of 2 μl of antigen per 1 ml of buffer, poured into 0.1 ml per well (1.18 g of Na ₂ CO ₃ ; 3, 74 g of NaHCO ₃ and 0.2 NaN _{3 are} dissolved in 1 liter of distilled water) and left at + 37 ° C for 18 hours. Then the tablet is washed 3 times in a buffered saline solution with 0.05% tween - 20 (34 g of NaCl in 4 l of distilled water, adjusting the pH to 7.4 2 m Na ₂ HPO ₄ ). After washing, 0.1 ml of a 1% solution of bovine serum albumin in 0.02 M phosphate-buffered saline (pH 7.3-7.4) is poured into the wells of the plate and incubated for 1 hour at a temperature of + 37 ° C. Then, the washing is performed again using the above method and the test sera (0.1 ml) are poured into the wells, leprosy patients diluted to 1: 200 with phosphate-buffered saline (0.584 g NaCl, 12.8 g Na ₂ HPO ₄ ; 2.62 g NaH ₂ PO ₄ containing 1% calf serum. Serums from patients with active leprosy (positive control) and donor serum (negative control) are diluted in the same way. The plate is incubated for 1 hour at a temperature of + 37 ° C, and then washed again. Then, 0.1 ml of conjugate (rabbit antibodies to human immunoglobulins Assa labeled with peroxidase) at a dilution of 1: 1000 and incubated for 1 hour at a temperature of + 37 ° C. After washing, add a substrate of 5-aminosalicylic acid with 0.05% hydrogen peroxide (add 100 ml of distilled water heated to 70 ° C) 80 mg of 5-aminosalicylic acid, the solution is cooled to room temperature and the pH is adjusted to 5.9-6.0; 2 ml of a 0.05% hydrogen peroxide solution are added to 18 ml of the resulting solution). The tablet is kept at room temperature for 40-60 minutes.

In the presence of antibodies to DIS-BSA in the blood serum, the substrate mixture stains dark brown (positive reaction), and the control wells remain colorless or slightly yellow (negative reaction). The reaction results are evaluated instrumentally with a photometer (Minireader 590, USA) at a wavelength of 450 nm by the color intensity of the test sample and are expressed in units of optical density (OD). Consider positive samples whose OD values are 2 or more times higher than the OD values in the negative control, the negative control OD does not exceed 0.15.

The results of the study showed that the concentration of alpha-1-antitrypsin in patients with leprosy with liver damage was higher compared to patients with leprosy who did not have this pathology, and amounted to 389.77 ± 43.1 mg / dl and 298.40 ± 15.68 mg / dl, respectively (p <0.05). Thus, the average alpha-1-antitrypsin (M) content in leprosy patients with liver damage (first group) was 389.77 mg / dl. Taking into account the standard error of the mean (t), the lower limit of the indicator under study in the first group is 346.7 mg / dl. It was adopted as a criterion, when the values of which in patients with leprosy were exceeded, liver damage was determined.

Serological examination of patients belonging to the first and second groups revealed an increase in the level of antibodies to mycobacterium leprosy antigens (USD-M.leprae and DIS-BSA) in patients with signs of liver damage (normal for USD-M.leprae <0.20 ; for DIS-BSA <0.15). The level of antibodies to both antigens in the second group was within acceptable values.

Thus, the positive results of a serological examination, together with an increased level of alpha-1-antitrypsin, indicate the persistence of leprosy mycobacteria in the liver, which is confirmed by clinical, laboratory (biochemical blood analysis) and instrumental (ultrasound) research methods.

The proposed method is achieved, in comparison with the prototype, improving the accuracy of the method.

The proposed method was successfully tested in the clinic of the Federal State Institution “Research Institute for the Study of Leprosy of Roszdrav” for 55 patients during 2007-2009.

Below are the results of testing.

Example 1

Patient M., born in 1932 Case history No. 1963.

DS: Leprosy, borderline-lepromatous type (BL), regression stage.

Pain and dyspeptic syndromes were clinically observed, according to laboratory and instrumental research methods (biochemical blood analysis, ultrasound of the abdominal organs): increased levels of aspartate aminotransferase, increased liver size, increased echogenicity and diffuse heterogeneous organ structure.

Determination of specific liver damage was carried out by the proposed method.

Serological examination of March 31, 2008

Blood (0.1 ml) was taken from a finger in a test tube, placed in a thermostat (1 h, + 37 ° С), then in the refrigerator (+ 4 ° С, 1 h). Serum was taken and titrated on phosphate-buffered saline (pH 7.4) containing 1% calf serum to obtain two dilutions: 1: 200 and 1: 400. In two wells (duplicate) of a polystyrene plate previously sensitized with an ultrasound scan of M.leprae (5 μg / ml), 0.1 ml of patient M.'s serum was added at a dilution of 1: 400; 0.1 ml (1: 400) of the serum pool of donors (negative control) was poured into two wells; 0.1 ml (1: 400) of the serum pool of patients with active leprosy was poured into two wells (positive control). The tablet was placed in a thermostat (+ 37 ° C, 1 h), then washed with phosphate-buffered saline (pH 7.4) containing 0.05% tween-20 (FSB-T), after which 0 was added to the wells, 1 ml conjugate (rabbit antibodies to common human immunoglobulins labeled with peroxidase) at a dilution of 1: 1000 and incubated for 1 h at 37 ° C. Then the plates were washed with FSB-T, 0.1 ml of substrate mixture (5-aminosalicylic acid with 0.05% hydrogen peroxide, pH 5.9-6.0) was added to the wells, kept at room temperature for 1 h and the reaction results were photographed . The OD in the negative control wells was 0.05. The OD in the wells with positive control was 0.98. The OD in the wells with serum of patient M. was 0.68.

Serum of patient M. in a dilution of 1: 200 in a volume of 0.1 ml was poured into two wells (duplicate) of a tablet previously sensitized with DIS-BSA. Two ml were poured into OD ml of the serum pool of donors (negative control), two wells were poured into 0.1 ml of the serum pool from patients with active leprosy (positive control) in the same dilution (1: 200) as the patient’s M serum ., and incubated at 37 ° C for 1 h. Then the wells were washed with PBS-T (pH 7.4) and 0.1 ml of conjugate (rabbit antibodies to human immunoglobulins of class M labeled with peroxidase) was added at a dilution of 1: 1000. The plate was incubated for 1 hour at 37 ° C, and then washed as described above. 0.1 ml of substrate mixture (5-aminosalicylic acid with 0.05% hydrogen peroxide, pH 5.9-6.0) was added to the wells. The tablet was held for 35-40 minutes at room temperature and the results were evaluated photometrically on a microphotometer at a wavelength of 450 nm. The OD of the negative control was 0.03. The OD of the positive control was 0.96. The serum OD of patient M. was 0.29.

The content of alpha-1-antitrypsin in the blood serum was determined by immunoturbodimetric method using a commercial kit company "Sentinel", Italy. Measurements were made on a Humalyzer 2000 programmable photometer (Germany). Statistical processing of the obtained results was carried out using Student's criterion. The concentration of alpha-1-antitrypsin in the blood serum of March 31, 2008 was 458.1 mg / dl.

Thus, the patient’s seropositivity in combination with an increased level of alpha-1-antitrypsin indicates the persistence of leprosy mycobacteria in the liver, which is confirmed by clinical, laboratory (blood chemistry) and instrumental (ultrasound) research methods.

Conclusion: the proposed method allows to reliably determine the specific liver damage in this patient.

Example 2

Patient A., born in 1945 Case history No. 3070.

DS: Leprosy, lepromatous (LLs) type, regression stage.

Unstable pain and dyspeptic syndromes were clinically observed, according to laboratory and instrumental research methods (biochemical blood analysis, ultrasound of the abdominal organs): an increase in the level of aspartate aminotransferase, an increase in the level of total protein with a low content of albumin, an increase in the size of the liver, increased echogenicity and a diffuse heterogeneous structure body.

Determination of specific liver damage was carried out according to the method described in example 1. Serological examination from 2.06.2008: antibodies to ultrasound-M.leprae - 0.87 (norm <0.20); antibodies to DIS-BSA - 0.19 (normal <0.15). The concentration of alpha-1-antitrypsin in blood serum from 2.06.2008 was 431.4 mg / dl.

Thus, the patient’s seropositivity in combination with an increased level of alpha-1-antitrypsin indicates the persistence of leprosy mycobacteria in the liver, which is confirmed by clinical, laboratory (biochemical blood analysis) and instrumental (ultrasound) research methods.

Conclusion: the proposed method allows to reliably determine the specific liver damage in this patient.

Example 3

Patient N., born in 1932 Medical history No. 3645.

DS: Leprosy, lepromatous (LLs) type, regression stage.

Clinically, there is no evidence of liver damage. According to ultrasound: the liver is enlarged, echogenicity is increased, the structure is diffuse-heterogeneous.

Determination of specific liver damage was carried out according to the method described in example 1. Serological examination from 02/04/2008: antibodies to SPL-M.leprae - 0.11 (norm <0.20); antibodies to DIS-BSA - 0.16 (norm <0.15). The concentration of alpha-1-antitrypsin in the blood serum of February 4, 2008 was 314.0 mg / dl.

Conclusion: a slight increase in the level of antibodies to only one of the two antigens (DIS-BSA) in combination with a concentration of alpha-1-antitrypsin below the established criterion does not allow to reliably determine the specific liver damage in this patient.

Example 4

Patient K., born in 1934 Case history No. 3829.

DS: Leprosy, lepromatous type (LLs), stage of regression.

Clinical and laboratory-instrumental signs of damage are absent. The determination of specific liver damage was carried out according to the method described in example 1. Serological examination from 05.26.2008: antibodies to ultrasound-M.leprae - 0.04 (norm <0.20); antibodies to DIS-BSA - 0.03 (norm <0.15). The concentration of alpha-1-antitrypsin in the blood serum of May 26, 2008 was 132.0 mg / dl.

Conclusion: the results indicate the absence of specific liver damage.

The proposed method is achieved, in comparison with the prototype, improving the accuracy of determining specific liver damage in patients with leprosy. The proposed method allows to reliably determine not only the localization of the pathological process, but also establish its etiology in vivo. In addition, the proposed method is possible early, preclinical, detection of signs of specific liver damage in patients with leprosy both in the active stage and in the regression stage. The proposed method does not require a counter immunoelectrophoresis reaction and complex mathematical calculations, which significantly reduces the time of its implementation, there is also no need for additional determination of cross-reacting antibodies to leprosy mycobacteria antigens, which can also occur in other diseases. The method can be reproduced in a clinical laboratory, is minimally invasive and not burdensome for the patient.

Thus, the authors proposed no one previously proposed method for determining specific liver damage in patients with leprosy.

The proposed method can be recommended for use in anti-leprosy and dermatovenereological institutions of the country's health system.

Claims (1)

A method for determining liver damage in patients with leprosy, including a study of blood serum, characterized in that they determine the level of antibodies in the blood serum of antibodies to leprosy antigens of the leprosy of UZD-M.leprae and DIS-BSA and the content of alpha-1-antitrypsin, and with increasing levels of two antigens relative to the norm and the content of alpha-1-antitrypsin above 346.7 mg / dl determine liver damage in patients with leprosy.