RU2310393C1 - Method for predicting exacerbation of leprous neuropathies in patients at leprosy - Google Patents

Abstract

FIELD: medicine, leprology.

SUBSTANCE: the present innovation deals with predicting exacerbation of leprous neuropathies in patients at leprosy and neuropathies of nonleprous etiology. In patients at leprosy one should detect antibodies to antigen of peripheral nerves in blood serum. According to the availability of antibodies to ceramide in blood serum and at their increased level from 0.26 U optic density and higher one should predict the exacerbation of leprous neuropathies in patients at leprosy.

EFFECT: higher accuracy of prediction.

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Description

The invention relates to medicine, namely to leprology, and can be, in particular, used to predict exacerbations of leprosy neuropathies and neuropathies of non-leprosy etiology.

From the practice of medicine, it is known about the prediction of exacerbations of leprosy neuropathies, based on subjective data - patient complaints of sharp pain in the nerve trunk; objective data - edema of surrounding tissues, sharp pain of peripheral nerves during palpation, thickening of nerve trunks, increase in sensitive, motor, trophic disorders [Alamdarov I.N., Verbina N.K., Belopasov V.V. Clinic and treatment of leprosy neuritis. Method. recommendations. - Astrakhan, 1974. - 10 p.]. A disadvantage of the known forecasting method is the lack of accuracy of the method associated with the subjective assessment of signs of exacerbation of leprosy neuropathies, as well as the late diagnosis of exacerbation of leprosy neuropathies with pronounced clinical manifestations, which makes it impossible to obtain a specific technical result - improving the accuracy of the method.

There is also a method for predicting exacerbations of leprosy neuropathies in patients with leprosy, based on an electromyographic study of peripheral nerves and muscles. With exacerbation of leprosy neuropathies, type II electromyograms are more often observed both with active reduction and with synergy. During the rest period, the appearance of potentials of fasciculations with a higher voltage was observed on the electromyogram [Belopasov V.V. On the significance of changes in the nervous and vascular systems in the pathogenesis of some leprosy dystrophies: Dis. ... cand. honey. sciences. - Astrakhan, 1973. - 215 p.]. The disadvantage of this method for predicting leprosy neuropathies is the complexity of the electromyographic method and the inaccessibility of equipment (high cost) for practical medical institutions. In addition, these changes on the electromyogram are recorded in the presence of pronounced clinical signs of exacerbations of leprosy neuropathies, which indicates the lack of accuracy of the method and does not allow to obtain a specific technical result - improving the accuracy of the method.

The closest way to the proposed one is the method of RU 2171689 C1, 08/10/2001, which consists in determining the level of optical density of antibodies. The similarity of this method with the proposed one lies in the fact that both of them belong to the field of medicine, namely to leprology, and are based on the determination of antibodies to the peripheral nerve antigen in the blood serum of patients with leprosy.

The disadvantage of this method is:

- insufficient accuracy of the method due to the lack of data on the relationship of antibody levels to ceramide with the course of leprosy neuropathies, the degree of damage to peripheral nerves and the activity of leprosy neuropathies;

- the inability to predict exacerbations of leprosy neuropathies.

Thus, the above disadvantages do not allow to obtain a specific technical result - improving the accuracy of the method.

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 solution of the problem by the proposed method consists in the fact that exacerbation of leprosy neuropathies is predicted by the presence of antibodies to ceramide in the blood serum of patients with leprosy and by increasing their level 5-10 months before the onset of clinical signs of exacerbations of leprosy neuropathies from 0.26 units of optical density and higher in patients with leprosy.

Improving the accuracy of the proposed method makes it possible to weaken the activity of the process or even to prevent it by reducing the disabling consequences for the patient with the complex treatment of leprosy neuropathies prescribed earlier than the detected clinical signs of exacerbations of leprosy neuropathies when detecting elevated levels of antibodies to ceramide in the blood serum of patients with leprosy.

Leprosy is still the cause of severe damage to the peripheral nervous system, leading to the development of disabling disorders. Over a quarter of all registered leprosy patients in the world are disabled, half of the newly identified leprosy patients already have clinical manifestations of neuropathy [WHO, 1997, 2000]. Given the prevalence and socio-economic damage caused by the disease, WHO has included leprosy in its tropical disease social program.

Morphological changes in the nerve during leprosy lead to disruption of tissue metabolism, causing the release of peripheral nervous tissue antigens (neuromarkers) into the bloodstream, followed by antibody response to them. Exacerbations of leprosy neuropathies occur in the process of anti-leprosy treatment, as a rule, increasing the disability of the patient. Therefore, it is important to establish a relationship between the activity of the process in the peripheral nerves and the detection in the blood serum of patients with leprosy antibodies to neuromarkers, to clarify their diagnostic and prognostic role in exacerbation of leprosy neuropathies. Comprehensive treatment of exacerbations of leprosy neuropathies, prescribed earlier than the detected clinical signs of exacerbation, can weaken the activity of the process or even prevent it, reducing the disabling consequences for the patient.

The proposed method was carried out as follows.

Blood was taken from a patient with leprosy from a vein (1-2 ml) into a test tube, which was placed in a thermostat (+ 37 °) for 1 hour, then in a refrigerator (+ 4 °) at night. In the morning, the tube was centrifuged (3 thousand revolutions × 15 min), the serum was aspirated into Eppendorf tubes and placed in a freezer (-20 °) until use.

Antibodies to ceramide in serum were determined by enzyme immunoassay (ELISA). The reaction was carried out in several stages. Ceramides (Sigma) were diluted in absolute alcohol and at a concentration of 4 μg / ml were adsorbed into the wells of a polyvinyl chloride plate (Dynatech). 50 μl of antigen was added to each well and incubated overnight at room temperature (+ 20 °). The next day, the tablet was washed three times with buffered saline (pH 7.3) without tween (PBS) using an automatic louse (PP-2428, Dubna, Moscow). Then, 100 μl of a blocking solution (0.5% bovine serum albumin solution in phosphate-buffered saline pH 7.4 - FSB) was added to each well of the plate, incubated for 60 min at + 37 ° in a thermostat, followed by washing with a PBS plate ( 3 times). Then, 50 μl of the studied blood serum samples were introduced into the wells at a dilution of 1: 200, the plate was incubated for 60 min at + 37 ° C, followed by washing it with PBS (3 times). 50 μl of antibodies against human class M immunoglobulins labeled with peroxidase (Vector-Best, Novosibirsk) were added to the washed wells in a working dilution of 1: 500. The plates were incubated for 60 min at + 37 ° C followed by washing with PBS (5 times). To manifest the reaction, 50 μl of substrate-buffer mixture containing 0.12% hydrogen peroxide and 0.04% orthophenylenediamine in citrate-phosphate buffer (pH 5.0) was added to the wells and left at + 20 ° for 20 min. The reaction was stopped by adding 50 μl of sulfuric acid at a concentration of 0.5 mol / L to each well.

The reactions were taken into account by measuring the absorbance in the wells on an enzyme-linked immunosorbent photometer (EFOS 9305, Sapphire, Moscow), at a wavelength of 490 nm. A pool of blood serum from 30 donors was used as a negative control, and a blood serum of a patient with leprosy neuropathy with a high level of antibodies to ceramide was used as a positive control. Antibody levels were evaluated in units of optical density (OD). A positive result was taken that was 2 or more times higher than the OD in wells with a donor pool.

The proposed method was successfully tested at the Federal State Institution “Research Institute for the Study of Leprosy of Roszdrav” for 4 years for 133 patients with leprosy, of which 57 patients were with exacerbations of leprosy neuropathies.

The essence of the invention is illustrated in the table and examples.

Table 1 The frequency of detection of antibodies to ceramide abs ./% Fluctuations in ceramide antibody levels (optical density units) Patients with exacerbations of leprosy neuropathies n = 57 44 / 77.2% 0.26-1.6 Patients without exacerbations of leprosy neuropathies n = 6З 25 / 39.7% 0.01-0.18 Donors n = 30 2 / 6.6% 0.01-0.12

Table 1 shows that in patients with exacerbations of leprosy neuropathies, antibodies to ceramide are detected 1.9 times more often than in patients without exacerbation of leprosy neuropathies. Patients with exacerbations of leprosy neuropathies have elevated levels of antibodies to ceramide, while in patients without exacerbation of leprosy neuropathies, levels of antibodies to ceramide correspond to donor values.

Example 1

Patient B., born in 1937 (medical history 2498). Diagnosis: undifferentiated type of leprosy in the regression stage. Chronic specific polyneuropathy with sensitive, amyotrophic, motor disorders. It was monitored for 4 years, from 1999 to 2002.

Neurological status: ulnar and peroneal nerves are thickened on both sides, painless on palpation. Atrophy of the I interosseous space of the hands, hypothenar, interosseous muscles of the hands, more on the right, tibial muscle on the left. A marked decrease in sensitivity to the shoulder joints and gluteal and inguinal folds with enlightenment on the palmar surfaces of the hands and plantar surfaces of the feet. There are no mutations, trophic ulcers.

In June 2001, the patient began to complain of periodic pain in the legs of a shooting character. Objectively: clinical signs of exacerbation of specific neuropathy of the peroneal nerve on both sides: the peroneal nerves are thickened, painful on palpation of the external ankle on both sides. Specific treatment was prescribed: corticosteroids (prednisone per os), desensitizing, diuretics, painkillers, B vitamins, vitamin E, phonophoresis with hydrocortisone on the elbow and peroneal nerves. Complex treatment led to the disappearance of signs of exacerbation. During the observation, the patient was repeatedly sampled (the sampling time is indicated in the table and graph) and blood serum was tested for the presence of antibodies to ceramide using an enzyme-linked immunosorbent assay. The reaction was carried out in several stages. Ceramides (Sigma) were diluted in absolute alcohol and at a concentration of 4 μg / ml were adsorbed into the wells of a polyvinyl chloride plate (Dynatech). 50 μl of antigen was added to each well and incubated overnight at room temperature (+ 20 °). The next day, the tablet was washed three times with buffered saline (pH 7.3) without tween (PBS) using an automatic louse (PP-2428, Dubna, Moscow). Then, 100 μl of a blocking solution (0.5% bovine serum albumin solution in phosphate-buffered saline pH 7.4 - FSB) was added to each well of the plate, incubated for 60 min at + 37 ° in a thermostat, followed by washing with a PBS plate ( 3 times). Then, 50 μl of the studied blood serum samples were introduced into the wells at a dilution of 1: 200, the plate was incubated for 60 min at + 37 ° C, followed by washing it with PBS (3 times). 50 μl of antibodies against human class M immunoglobulins labeled with peroxidase (Vector-Best, Novosibirsk) were added to the washed wells in a working dilution of 1: 500. The plates were incubated for 60 min at + 37 ° C followed by washing with PBS (5 times). To manifest the reaction, 50 μl of substrate-buffer mixture containing 0.12% hydrogen peroxide and 0.04% orthophenylenediamine in citrate-phosphate buffer (pH 5.0) was added to the wells and left at + 20 ° for 20 min. The reaction was stopped by adding 50 μl of sulfuric acid at a concentration of 0.5 mol / L to each well.

The reaction was taken into account by measuring the absorption in the wells on an enzyme-linked immunosorbent photometer (EFOS 9305, Sapphire, Moscow), at a wavelength of 490 nm. A pool of blood serum from 30 donors was used as a negative control, and a blood serum of a patient with leprosy neuropathy with a high level of antibodies to ceramide was used as a positive control. Antibody levels were evaluated in units of optical density (OD). A positive result was taken that was 2 or more times higher than the OD in wells with a donor pool.

The dynamics of serological indicators are presented in table 2 and figure 1.

table 2 Date (month, year) of blood sampling Ceramide antibody levels (absorbance values) Clinical manifestations of exacerbation of leprosy neuropathy IV-1999 0.15 Are absent XI-2000 0.10 Are absent I-2001 0.26 Are absent III-2001 0.30 Are absent VI-2001 0.50 Are available IX-2001 0.55 Are available XII-2001 0.35 Are available III-2002 0.32 Are available VI-2002 0.20 Are absent IX-2002 0.10 Are absent XII-2002 0,03 Are absent

As can be seen from table 2 and figure 1, the level of antibodies to ceramide in patient B. began to increase (0.26 - I / 2001; 0.30 - III / 2001) 5 months before the clinical signs of exacerbation of leprosy neuropathy (0.50 - VI / 2001). Antibody levels were increased (0.55 - IX / 2001; 0.35 - XII / 2001; 0.32 - III / 2002) for 8 months, and then after treatment decreased to normal values ( 0.20 - VI / 2002; 0.10 - IX / 2002; 0.03 - XII / 2002).

Thus, by the presence of antibodies to ceramide in the patient's blood serum and their increase 5 months before the onset of clinical signs of exacerbation of leprosy neuropathy from a level of 0.26 units of optical density and above, an exacerbation of leprosy neuropathy was predicted.

Example 2

Patient E., born in 1931 (medical history 3850). Diagnosis: lepromatous type of leprosy, active stage. Chronic specific polyneuropathy with sensitive disorders, in the acute stage. It was monitored for 4 years from 1998 to 2002.

Patient E. was admitted with complaints of pain in the upper limbs. During a neurological examination, signs of exacerbation of leprosy neuropathy were revealed: ulnar nerves were thickened on both sides, painful on palpation, hypesthesia on infiltrates. Specific treatment was prescribed: corticosteroids, desensitizing, diuretics, painkillers, B vitamins, vitamin E, phonophoresis with hydrocortisone. Complex treatment led to the disappearance of signs of exacerbation. Clinical signs of exacerbation of leprosy neuropathies gradually disappeared by IV / 1999. In June 2000, complaints of aches, shooting pains in the upper and lower extremities, weakness in the hands and feet reappeared. Examination revealed clinical signs of exacerbation of leprosy neuropathy: ulnar and fibular nerves are thickened, painful on palpation. By IX / 2001, signs of exacerbation of leprosy neuropathies disappeared under the influence of appropriate treatment.

During the observation, the patient was repeatedly sampled blood (sampling time is shown in the table and figure 2) and blood serum was tested for the presence of antibodies to ceramide using the enzyme immunoassay method described in example 1.

The dynamics of serological indicators are presented in table 3 and figure 2.

Table 3 Date (month, year) of blood sampling Ceramide antibody levels (absorbance values) Clinical manifestations of exacerbation of leprosy neuropathy IV / 1998 0.68 Are available VIII / 1998 0.65 Are available Xi / 1998 1.05 Are available I / 1999 0.35 Are available IV / 1999 0.15 Are absent IX / 1999 0.08 Are absent Xi / 1999 0.53 Are absent III / 2000 0.70 Are absent VI / 2000 0.85 Are available X / 2000 1.15 Are available XII / 2000 1,54 Are available III / 2001 0.90 Are available VII / 2001 0.50 Are available XI / 2001 0.21 Are absent I / 2002 0.18 Are absent III / 2002 0.09 Are absent

From the data presented in table 3 and figure 2, it is seen that patient E. twice during the observation period suffered exacerbations of leprosy neuropathies. High levels of antibodies to ceramide in 0.68 - IV / 1998 and 0.65 - VIII / 1998, 1.05 - XI / 1998, 0.35 - I / 1999 corresponded to clinical signs of exacerbation leprous neuropathy. Remission was observed from IV / 1999 to IX / 1999. Then, an increase in antibody levels to ceramide of 0.53 - XI / 1999 and 0.70 - III / 2000 was again noted, and clinical signs appeared only in VI / 2000 with an optical density index of 0.85, that is, 8 months before the clinically diagnosed exacerbation of leprosy neuropathy. After the treatment, the levels of antibodies to ceramide decreased to indicators of the donor background (0.09).

Thus, by the presence of antibodies to ceramide in the patient's blood serum and their increase 8 months before the onset of clinical signs of exacerbation of leprosy neuropathy from a level of 0.53 optical density units or higher, an exacerbation of leprosy neuropathy was predicted.

Example 3

Patient M., born in 1936 (medical history 3118). Diagnosis: undifferentiated type of leprosy in the regression stage. Chronic specific polyneuropathy with severe motor, sensory, amyotrophic disorders, residual stage. The patient was followed up for 4 years from 1999 to 2002.

Neurological status: ulnar and ear nerves on both sides are thickened, painless on palpation. Steppage from two sides. Atrophy of the muscles of the hands. Mutations at the level of the main phalanges of the II-V fingers of the hands on the right and at the level of the terminal phalanges on the left. Flexion contracture of the fingers of the left hand and I finger of the right hand. Anesthesia to the shoulder joints and gluteal and inguinal folds. Trophic ulcers of the plantar surface of the feet on both sides.

Since IX / 2000, the patient began to complain of pain of a shooting nature in the upper and lower extremities. Examination revealed clinical signs of exacerbation of leprosy neuropathy: peripheral nerve trunks are thickened, painful on palpation.

During the observation, the patient was repeatedly sampled blood (sampling time is shown in the table and figure 3) and blood serum was tested for the presence of antibodies to ceramide using the enzyme-linked immunosorbent assay described in example 1.

The dynamics of serological indicators are presented in table 4 and figure 3.

Table 4 Date (month, year) of blood sampling Ceramide antibody levels (absorbance values) Clinical manifestations of exacerbation of leprosy neuropathy I-1999 0.05 Are absent V-1999 0.04 Are absent VII-1999 0.10 Are absent XI-1999 0.72 Are absent IV-2000 0.60 Are absent IX-2000 0.70 Are available XII-2000 0.63 Are available III-2001 0.40 Are available VI-2001 0.50 Are available XII-2001 0.20 Are absent VI-2002 0.05 Are absent

As can be seen from table 4 and figure 3, the level of antibodies to ceramide in patient M. began to increase 0.72 - XI / 1999, 10 months before the clinical signs of exacerbation of leprosy neuropathy (0.70 - IX / 2000) . Antibody levels were increased 0.63 - XII / 2000; 0.40 - III / 2001; 0.50 - VI / 2001 for 9 months, and then after treatment decreased to normal levels 0.20 - XII / 2001; 0.05 - VI / 2002

Thus, by the presence of antibodies to ceramide in the blood serum of the studied patient and their increase 10 months before the onset of clinical signs of exacerbation of leprosy neuropathy from a level of 0.72 optical density units or higher, an exacerbation of leprosy neuropathy in this patient with leprosy was predicted.

Example 4

Patient V., born in 1943 (medical history 2263). Diagnosis: lepromatous type of leprosy in the regression stage. Chronic specific polyneuropathy with sensitive, motor, trophic disorders. It was monitored for 3 years from 1999 to 2002.

Neurological status: ulnar and peroneal nerves are thickened, painless on palpation. Anesthesia to the elbow and knee joints. Atrophy of the interosseous muscles of the hands and feet, tenar, hypotenar on both sides. Slight flexion contracture of the fingers of the hands on both sides. Trophic ulcers of the plantar surface of the feet on both sides.

During the observation, clinical signs of exacerbation of leprosy neuropathy were recorded. In May 2001, the patient began to complain of periodic moderate pain in the upper extremities of a breaking nature. Objectively: the ulnar nerves are thickened on both sides, painful on palpation in the ulnar fossa. Specific treatment was prescribed: corticosteroids, desensitizing, diuretics, painkillers, B vitamins, vitamin E, phonophoresis with hydrocortisone. Complex treatment led to the disappearance of signs of exacerbation. Clinical signs of exacerbation of leprosy neuropathies gradually disappeared in March 2002.

During the observation, the patient was repeatedly sampled blood (sampling time is shown in the table and figure 4) and blood serum was tested for the presence of antibodies to ceramide using the enzyme-linked immunosorbent assay described in example 1.

The dynamics of serological indicators are presented in table 5 and figure 4.

Table 5 Date (month, year) of blood sampling Ceramide antibody levels (absorbance values) Clinical manifestations of exacerbation of leprosy neuropathy X / 1999 0.09 are absent I / 2000 0.05 are absent IV / 2000 0.12 are absent VI / 2000 0.10 are absent I / 2001 0.15 are absent V / 2001 0.04 are available IX / 2001 0,03 are available XII / 2001 0.05 are available III / 2002 0,07 are absent IX / 2002 0,03 are absent

As can be seen from table 5 and figure 4, the patient in May 2001 recorded clinical signs of exacerbation of leprosy neuropathy, and the level of antibodies to ceramide in the patient did not increase during the entire observation period (antibody level ranged from 0.03 to 0.15 ), that is, these indicators corresponded to those of the donor pool.

Thus, antibodies to ceramide remained low for 4 and 11 months before the clinical signs of exacerbation of leprosy neuropathy, which was observed only in 9.2% of the examined patients.

The proposed method achieves a positive result, which consists in increasing the accuracy of the method, weakening the activity of the process or even preventing it by reducing the disabling consequences for the patient with the complex treatment of leprosy neuropathies, prescribed earlier than the clinical signs of exacerbations of leprosy neuropathies.

The proposed method establishes a relationship between the level of antibodies to ceramide and the activity of leprosy neuropathies. The proposed method objectively assesses in comparison with known methods the state of exacerbation of leprosy neuropathies in 90.8% of patients in the absence of clinical signs of exacerbation of leprosy neuropathies - by the level of antibodies to ceramide.

The proposed method is economically viable, not difficult to set up, the equipment (tablets, reader) is of domestic production and is available for clinical laboratories.

The proposed method can be recommended in the practice of anti-leprosy institutions and neurological clinics in the diagnosis and treatment of neuropathies of non-lethal etiology.

Claims (1)

A method for predicting exacerbations of leprosy neuropathies in patients with leprosy, which consists in determining antibodies to the peripheral nerve antigen in the blood serum of patients with leprosy, characterized in that exacerbations are predicted by the presence of antibodies to ceramide in the blood serum and an increase in their level from 0.26 units of optical density and higher leprosy neuropathies in patients with leprosy.

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