RU2124730C1 - Method of leprosy diagnosis - Google Patents

Abstract

FIELD: medicine, leprology. SUBSTANCE: blood is taken off in patient and clarified serum is analyzed by immunoenzymatic method. To detect antibodies to antigens of M. lepras in blood serum samples from patients with leprosy by immunoenzymatic method cultured mycobacteria - Mycobacterium fulu are used as a test-antigen. EFFECT: simplified method, improved precision of leprosy diagnosis. 2 ex, 3 tbl

Description

 The invention relates to medicine, namely to leprology, and can be used, in particular, for the diagnosis of leprosy.

 From the practice of medicine, a method is known for detecting antibodies to M. leprae in the blood serum of patients with leprosy using the enzyme-linked immunosorbent assay, where water-soluble proteins from human leprosy are used as test antigen (Alonso SM, Mangiaterra ML, Szarfman A. La reaccion de immunoperoxidasias indirecta aplicata a indirect la lepra humana // Medicina (Argent) .- 1978.-Vol. 38.- N 63.- P.541-544.). The method allows for serological diagnosis of leprosy, as well as studies to predict the disease. The disadvantage of this method is the invasiveness of the method of obtaining antigenic material: the need to take biopsy samples from skin lesions of patients with leprosy of people. The method cannot be introduced into clinical practice, because even with common processes, the amount of M.leprae in the skin leproomas of patients with leproomatous leprosy is not enough to obtain a diagnosticum.

 There is also a method of detecting antibodies to M.leprae antigens in the blood serum of patients with leprosy on the basis of enzyme-linked immunosorbent assay, where preparations from M. leprae obtained from tissues of experimentally infected nine-laden armadillos are used as test antigens (Stanford JL, Rook GAW, Samuel N. , Madlener F., Khamenei AA, Nemati T., Modabber F., Rees RJW // Preliminary immunological studies in search of correlates of protective immunity carried out on some Iranian leprosy patients and their families.- Lepr. Rev.- 1980.- Vol.51.- N 4.- P.303-314.). The method allows to detect specific antibodies to M. leprae in blood serum in patients with leprosy, as well as in individuals who are in close family contact with patients with leprosy. The disadvantages of the method include the need to reproduce on armadillos an experimental leprosy infection that develops over 2-5 legs. Armadillos have a limited habitat (South America), do not breed in captivity and require special conditions of detention. Isolation of M. leprae from the tissues of the armadillo involves the use of a multi-stage cleaning method, which in turn increases the cost and complicates the process of obtaining an antigenic diagnosticum.

 There is a method for diagnosing leprosy by an enzyme immunoassay, in which antibodies to M.leprae are determined in blood serum using Dis-BSA, which is a semisynthetic analogue of the specific carbohydrate epitope of M. leprae - phenolic glycolipid-1 (FGL-1) (Cho SN, Fujiwara T ., Hunter SW, Rea T.N., Gelber RH, Brennan PJ Use of an artifical antigen containing the 3,6-di-O-methyl-β-O-glycopyranosyl epitope for the serodiagnosis of leprosy // J. Infect. Dis. - 1984.- Vol. 140.- P.311-322). The method allows to detect in the blood serum of patients with leprosy antibodies to the species-specific antigen M.leprae - FGL-1. Antibody levels correlate with the clinical manifestations of the disease. The disadvantage of this method is the limited capabilities of the test system associated with the detection of antibodies to only one epitope of M. leprae. Antibodies to this antigen quickly disappear under the influence of a specific treatment and are not determined 1-1.5 years after the start of chemotherapy, while in most patients with leprosy M. leprae are detected in the skin and antibodies in the blood serum to other antigen are detected components of M.leprae.

 The closest method to the proposed one is a method for detecting antibodies to M.leprae in human serum and armadillo during leprosy, where drugs from M.leprae passaged in mice and rats were used as a specific test antigen (Dyachina M.N., Suchenko L .T., Yushchenko A.A., Ermolin G.A. Diagnostic test systems based on the enzyme-linked immunosorbent assay for leprosy // Journal of microbiol., Epidemiol., Immunobiol. 1985, N 1.- P. 64-68 ) Using the known method, it is possible to detect antibodies to M. leprae in the blood serum of patients with leprosy of people and armadillos with experimental leprosy infection, and antibody levels correlate with the degree of activity of the leprosy process. The method also allows you to predict the timing of the development of experimental leprosy infection in armadillos.

The disadvantages of the method in the duration and multi-stage implementation:
- modeling of the infectious process in experimental animals in order to accumulate bacterial mass takes an average of 1.5-2 years;
- obtaining purified M.leprae is associated with a multi-stage process of isolating the pathogen from animal tissue with experimental leprosy infection.

 The known method is adopted by the authors as a prototype.

 The aim of the invention is to simplify the method of diagnosis of leprosy.

 The goal of the invention is achieved by the fact that cultured mycobacteria - Mycobacterium lufu are used as an antigen in the enzyme immunoassay.

 A distinctive feature of the implementation of the method is the use of enzyme immunoassay (ELISA) as an antigenic diagnosticum of drugs from M.lufu, previously not used for these purposes. It is known that M. lufu is suitable as a test strain for the initial screening of anti-leprosy drugs in vitro.

 The analysis of patent and scientific literature showed that in the known methods for the diagnosis of leprosy M.lufu were not used.

 The lack of reproducible methods for cultivating M.leprae on culture media is a serious obstacle to the creation of diagnostic test systems. Laboratory animals (mice, rats, armadillos) in the body of which M.leprae breed are still the only source of bacterial mass used to create diagnostic drugs. The preparation of these preparations is further complicated by the fact that M. leprae obtained as a result of passage in laboratory animals must be isolated from infected tissues of the host organism. Cleaning the pathogen of leprosy from the tissues of experimental animals is a complex, time-consuming, multi-stage process, requiring the participation of qualified personnel, special equipment and reagents, which in turn also complicates the task of obtaining a diagnosticum.

 M. lufu, isolated from soil in Zaire (Portaels, 1980), are saprophytic acid-resistant mycobacteria that can grow on nutrient media. The authors propose M.lufu as a source of bacterial mass for the production of antigenic diagnosticum instead of currently used uncultivated in vitro M.leprae. The proposed method allows to reduce the time for obtaining a test antigen by 1.5-2 years, i.e. limit the time to 1-2 weeks.

 The use of cultivated M.lufu to obtain test antigen is a significant difference in the approach to the diagnosis of leprosy, and it is this difference that allowed to obtain a positive effect in the form of a simplification of the method. The proposed method eliminates the use of M.leprae, unable to grow on artificial nutrient media, as a material for obtaining antigenic diagnosticum.

The proposed method was carried out as follows: blood from a finger in an amount of 0.1 ml was collected in a test tube and placed for 1 hour in a thermostat (+37 ^o C), and then in the refrigerator (+4 ^o C) for 1 hour to retract the clot. Settled serum was examined using an enzyme-linked immunosorbent assay. For long-term storage, serum was frozen at -20 ^o C.

Used two water-soluble antigenic drugs:
- antigen from M.leprae, destroyed by ultrasound;
- antigen from M.lufu, destroyed by ultrasound.

The bacterial mass of M. leprae was accumulated by passage on mice and rats for 2 years. M. leprae was isolated from infected rat tissue with experimental leprosy according to the Draper method (Draper P. Problems related to purification of M. leprae from armadillos tissues and standartization of M. leprae preparations // Report on the Enlarged SC Mutina.- Geneva, 1979.- WHO Document. - Annex I, prot. 1 / 79.- P.4-4,), which includes differential centrifugation of tissue homogenates in a sucrose density gradient. Separation from tissue particles was carried out in a two-phase polymer system consisting of 7% T-500 dextran (Pharmacia) and 5% polyethylene glycol with
molecular weight 6 kDa (company BDH). Purified M.leprae was subjected to ultrasonic disruption on an MSE-100 apparatus (England) in physiological saline for 7 min at 18 kHz. The effect of destruction was controlled microscopically. The suspension of disrupted M.leprae was centrifuged (10,000 rpm), the supernatant was used as a native water-soluble antigen (USD-M.leprae).

M. lufu was cultured on Levenshtein-Jensen medium for 7 days. Antigens were obtained from cold-synchronized (at 4 ^° C. for 72 hours) M.lufu culture. Cell cultures were subjected to ultrasound disruption on an MSE-100 apparatus (England) in physiological saline for 7 min at 18 kHz. The effect of destruction was controlled microscopically. The suspension of destroyed mycobacteria was centrifuged (10,000 rpm), the supernatant was used as a native antigen (SPL-M.lufu).

 ELISA was carried out on polystyrene tablets for single-use immunological reactions (production association Lenmedpolymer TU 64-2-278-79). Two tablets were sensitized simultaneously: the first with antigens from M.leprae (UZD-M.leprae), the second with antigens from M.lufu (UZD-M.lufu).

The first plate was sensitized with ultrasound-M.leprae in carbonate-bicarbonate buffer pH 9.0-9.5 (1.18 g of Na ₂ CO ₃ 3.47 g of NaHCO ₃ and 0.2 g of NaN _{3 were} dissolved in 1 l of distilled water) at the rate of 27 μg of antigen per 1 ml of buffer in a volume of 0.1 ml per 1 well (18 hours at + 4 ^o C). Then, the plate was washed 3 times in buffered saline (34 g of NaCI in 4 L of water, bringing 2M Na ₂ PO ₄ pH to 7.4) with the addition of 0.05% Tween-20, after which 0.01 were added to the wells ml of a 1% solution of bovine serum albumin in 0.02 M phosphate-buffered saline (pH 7.4) and kept for 1 h at +37 ^o C. Then, the wells were washed again with the above method and the wells were filled with the studied sera of patients with leprosy diluted (1 : 400) phosphate-buffered saline (pH 7.3 - 7.4) containing 1% cattle serum in a volume of 0.1 ml. The positive control (serum of patients with active manifestations of the leprosy process) and the negative control (donor serum) in the same dilution (1: 400) were also placed on the tablet. Each serum sample was poured into 2 wells (duplicate). The plate was incubated at +37 ^° C. for 1 hour, after which the washing process was repeated. Then, 0.1 ml of conjugate (rabbit antibodies to common human immunoglobulins labeled with peroxidase) was added in each well at a dilution of 1: 1000 and incubated for 1 h at +37 ^° C. After washing, a substrate mixture of 5-aminosalicylic acid with 0, 05% hydrogen peroxide (80 mg of 5-aminosalicylic acid was added to 100 ml of distilled water heated to 70 ^° C, the solution was cooled to room temperature and pH 5.5 - 6.0 was adjusted. 2 ml of 0.05 was added to 18 ml of the resulting solution % hydrogen peroxide solution). The tablet was kept at room temperature for 60 minutes. If serum antibodies to USD-M.leprae were present in the serum, the substrate mixture was stained dark brown (positive reaction), and control wells remained colorless or slightly yellowish (negative reaction). The reaction results were evaluated instrumentally with a photometer (Minireader MR 590, USA), at a wavelength of 450 nm, by the color intensity of the test sample and expressed in units of optical density (OD). Samples were considered positive, the OD indices of which were 2 or more times higher than the indices in the negative control. The OD of the negative control did not exceed 0.2.

At the same time, a second tablet was sensitized. Antigens from M. lufu (UZD-M.lufu) were diluted in carbonate-bicarbonate buffer pH 9.0-9.5 at the rate of 33 μg of antigen per 1 ml of buffer in a volume of 0.1 ml per 1 well (1.18 Na ₂ CO ₃ , 3.74 NaHCO ₃ and 0.2 NaN _{3 are} dissolved in 1 l of distilled water) and left for 18 hours at +37 ^o C). Then the tablet was washed 3 times in buffered saline (34 g of NaCl in 4 l of water, bringing 2M Na ₂ PO ₄ pH to 7.4) with the addition of 0.05% tween-20. 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) was poured into the wells of the plate and kept for 1 h at +37 ^° C. Then, washing was performed again. by the above method, the studied sera of patients with leprosy (in a volume of 0.1 ml) diluted 1: 400 with phosphate-buffered saline pH 7.4 (0.584 g NaCI, 12.8 g Na ₂ HPO ₄ , 2.62 g NaHPO were poured into the wells ₄ ) containing 1% cattle serum. In the same way, sera from patients with active leprosy (positive control) and donor serum (negative control) were diluted. The plate was incubated for 1 h at +37 ^o C, and then washed again. 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 kept for 1 h at +37 ^o C. After washing, a substrate of 5-aminosalicylic acid was added with 0.05 % hydrogen peroxide. The tablet was kept at room temperature for 40-60 minutes. If serum antibodies to SPL-M.lufu were present in the serum, the substrate mixture was stained dark brown (positive reaction), and control wells remained colorless or slightly yellowish (negative reaction). The reaction results were evaluated using a photometer (Minireader MR 590, USA), at a wavelength of 450 nm, by the color intensity of the test sample and expressed in units of OD. Samples were considered positive, the OD indices of which were 2 or more times higher than the indices in the negative control. OD of the negative control did not exceed 0.3.

 The essence of the invention is illustrated in table. 1, which presents the results of an enzyme-linked immunosorbent assay of blood serum of patients with leprosy and control groups using various antigens.

 Thus, it follows from the data in Table 1 that the antibody level indices in all the studied groups obtained in the test system using antigens from M. leprae are completely comparable with those obtained in ELISA, where the test antigen is preparations from M. lufu.

 The results of ELISA of the sera of patients with leprosy using antigens from M. leprae and from M.lufu are presented in table 2.

 Thus, from the data table. Figure 2 shows that the results of ELISA using the ultrasound-M.leprae antigen completely correlate (ρ = 0.999) with the results of the ELISA, where the M.Duf ultrasound was used as a test antigen.

 The authors evaluated the sensitivity, specificity and effectiveness of the enzyme immunoassay. The diagnostic sensitivity of ELISA was taken as a percentage of the frequency of truly positive ELISA results in patients with active manifestations of the leprosy process.

 The diagnostic specificity of ELISA was taken as a percentage of the frequency of truly negative ELISA results in individuals without leprosy.

For diagnostic efficacy, the percentage expression of the ratio of the true ELISA results to the total number of results was taken. The calculation of sensitivity, specificity and effectiveness was carried out taking into account the recommendations of Griner PF, Mayewsky RG, Mushlin AY Selection and interpretation of diagnostic tests and procedures // Ann. Inter. Med.- 1981.- V.94, N 4.- P. 555-600 using the formula:
sensitivity = IP / B • 100%;
specificity = IO / NB • 100%;
efficiency = PI + IO / PI + LP + LO + IO • 100%,
where 4 PI - truly positive results; B - the total number of patients with active manifestation of the leprosy process; AI - true negative results; NB - the total number of patients with leprosy; LP - false positive results; LO - false negative results.

Using the above formulas, a comparative assessment of diagnostic test systems based on IFD with antigens from M.leprae and M.lufu was carried out (Table H)
Thus, from the data table. 3 it can be seen that at 100% sensitivity, the test system, in which the ultrasound - M.lufu was used as an antigen, has comparable specificity and effectiveness with the test system, where the ultrasound - M.leprae was used as antigen.

 The proposed method was successfully tested at the research institute for the study of leprosy of the Ministry of Health of the Russian Federation in 1994-1995.

 The following are the results of testing the method.

 Example 1. Patient A (and / b N 3844) was first admitted to the research institute for the study of leprosy 05/29/93 g. On admission: leprosy process is common. Eyebrows and eyelashes are sparse, there is a deep diffuse infiltration of the skin of the face, auricles and earlobes, a large number of dermal and hypodermal leprosy. Elbow, peroneal nerves are thickened, painful on palpation. Stem type anesthesia up to the upper third of the shoulders, thighs. Bacterioscopic index (BIN) = 1.83+. Histological examination of skin biopsy: actively forming granuloma of the lepromatous structure (leprooma) against the background of leprosy erythema nodosum. Diagnosis: lepromatous type of leprosy.

Serological examination from 09/05/94, revealed the presence of antibodies to M. leprae:
- OP indicators with ultrasound-M.leprae - 0.85 (norm <0.20);
- OP indicators with ultrasound-M.lufii - 1.13 (norm <0.30).

 Thus, patient A at the time of the examination was seropositive. Indicators of optical density of the level of antibodies to SPL-M.leprae and SPL-M.lufu were comparable to each other and more than 3 times higher than the background level. Clinical, histological and bacterioscopic studies confirmed the results of serological analysis.

 Example 2. Patient L. (and / b N 2461) was first admitted to the research institute for the study of leprosy June 21, 2005. On admission, diffuse facial infiltration, leprosy on the extremities were noted, peroneal nerves were thickened, painful on palpation. In scraping from the nasal mucosa, in the skin scarification M.leprae was found. Histological examination of a skin biopsy sample: infiltrate of a lepromatous structure with the presence of M.leprae. Diagnosis: lepromatous type of leprosy. He received specific treatment for 7 years and was discharged in 1962 for outpatient treatment.

 In 1991, the patient entered the clinic of the research institute for the study of leprosy with exacerbation of the leprosy process. Examination of the skin revealed focal pink infiltration with clear edges in the abdomen, in the buttocks with a transition to the lumbar region. The ulnar and peroneal nerves are thickened. Anesthesia to the upper third of the shoulders and upper third of the hips. M.leprae was found in the skin scarific and tupf preparation. Histological examination of skin biopsy: an actively forming infiltrate of lepromatous structure. Diagnosis: lepromatous type of leprosy. Relapse.

A serological examination dated March 30, 1993 revealed the presence of antibodies to M. leprae:
- OP indicators with ultrasound-M.leprae - 0.72 (norm <0.20);
- OP indicators with ultrasound-M.lufu - 0.87 (norm <0.30).

 Thus, patient L at the time of the examination was seropositive. Indicators of optical density of the level of antibodies to SPL-M.leprae and SPL-M.lufu were comparable to each other and 3 times higher than the background level. Clinical, histological and bacterioscopic studies confirmed the results of serological analysis.

 Example 3. Patient G. (and / b N 1765) was admitted to the research institute for the study of leprosy 9.03.48, with a diagnosis of lepromatous type of leprosy. He received specific treatment for 15 years. Currently, the leprosy process is in a regressive state. Histological examination of skin biopsy specimen: lepromatous structure infiltrate in a state of distinct regression, not containing leprosy mycobacteria. BIN = 0. The course of the disease is relapse-free. The patient is under medical supervision.

Serological examination from 10/31/94 did not reveal the presence of antibodies to M.leprae:
- OP indicators with ultrasound-M.leprae - 0.04 (norm <0.20);
- OP indicators with ultrasound-M.lufu - 0.12 (norm <0.30).

 Thus, patient G, at the time of the examination, was seronegative. Indicators of optical density of the level of antibodies to SPL-M.leprae and SPL-M.lufu were comparable to each other and did not exceed the background level. Clinical, histological and bacterioscopic studies confirmed the results of serological analysis.

The proposed method is achieved by simplifying the diagnosis of leprosy in comparison with the prototype, which consists of:
to reduce the time for obtaining an antigenic diagnosticum by 1.5-2 years, since there is no need to reproduce experimental leprosy infection in laboratory animals in order to accumulate bacterial mass;
to reduce costs (qualified personnel, reagents, equipment) associated with the cleaning of mycobacteria from the tissues of experimental animals.

 Thus, the authors proposed a new, no one previously proposed, method for diagnosing leprosy.

 The proposed method can be recommended for use in scientific and clinical laboratories of anti-leprosy institutions of the health system.

Claims (1)

 A method for the diagnosis of leprosy, consisting in the detection of antibodies to M. Leprae antigens in the blood serum of patients with leprosy, characterized in that cultured mycobacteria Mycobacterium lufu are used as a testantigen in an enzyme immunoassay.

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