RU2140644C1 - Method of prognostication of development of chronic course of mite-borne systemic borreliosis in children - Google Patents

Abstract

FIELD: medicine, particularly, methods of prognostication of development of chronic forms of mite-borne systemic borreliosis in children; applicable in clinical practice. SUBSTANCE: method consists in that determined simultaneously in patient's blood serum in common system of reactions is concentration of antibodies to borrelias, level of specific immune complexes and non-specific immune complexes containing no complement. If, at the end of acute period, antibodies amount to less than 0.20 unit, with non complement-containing specific immune complexes, no concentration of immune complexes containing no complement more than 40 units, development of chronic form of mite-borne systemic borreliosis is prognosticated. Application of claimed method of prognostication resulted in improvement of disease termination and reduced transition in chronic forms from 20-18% in 1995-96 down to 11% in 1997. EFFECT: improved terminations of disease and prevented transition from acute course of disease to chronic one. 1 cl

Description

 The invention relates to medicine, more specifically to infectology, and is intended to predict the development of a chronic course of tick-borne systemic borreliosis (KSB) in patients in the acute period of infection.

 KSB or Lyme borreliosis is a ubiquitous infectious disease associated with the activity of ixodid ticks, the main guardians and carriers of infection, the endemic foci of which are located throughout Russia, including the Leningrad Region. KSB is characterized by the staged development of the disease, which is characterized by the presence of an acute period of the disease, proceeding in the form of an erythema or general infection form (stage 1 infection), or with the development of neuroborreliosis and cardiopathy (stage 2). However, with insufficient antibiotic therapy, the disease can go into a chronic 3rd stage, which is manifested by damage to the joints (arthritis), skin (focal scleroderma, atrophic acrodermatitis), heart (pancarditis), nervous system (encephalomyelitis, encephalopathy with psychopathological disorders, polyradiculoneuropath).

 The acute period of the disease proceeds mainly favorably and leads to recovery. However, the chronic stage of the disease becomes the greatest threat to health when infected with borrelia, since it is characterized by dissemination of the infection, difficulty in healing, and the possibility of subsequent disability. In the development of chronic forms of infection, both the persistence of the pathogen is important, which is proved by the isolation of Borrelia 5 or more years after the onset of the disease from the skin, cerebrospinal fluid, synovial fluid of patients, as well as circulating immune complexes, as well as autoimmune mechanisms.

 There are various methods for detecting immune complexes (IR), both specific and non-specific, in patients with many infectious forms. (Herrmann K., Lohrisch I.et al. Detection of antibodies after immune complex splitting in serum of patients with bullous pemfphigoid and systemic lupus erythematosus // Br. J. Dermatol., 1987, V.99, p. 635-40; Dobson SRM, Taber LH, Baughn RECharacterization of the components in circulating immune complexes from infants with congenital syphilis // J.Infect.Dis.l988, V.158, p. 940-47). Such complexes are also tested in KSB along with the formation of antibodies (AT) (Hardin JA, Steere AC, Malawista SE // Immune complexes and the evolution of Lyme Arthritis // N. Engl.J. Med.l997, V.301, p 1358-63; Coyle PK, Schutzer SE et al. Cerebrospinal fluid immune complexes in patients exposed to Borrelia burgdorferi: detection of borrelia-specific and non-specific coplexes // Ann. Neurol., 1990, V.28 (6), p .739-44). Most often, the isolation of such complexes is carried out using ethylene glycol, followed by their cleavage in zones of pH> 10.0 and then testing specific AT by ELISA, immunoblotting or any other test.

 At the same time, the disadvantages of all these methods for detecting IR are the following: first, the detection of complexes is not accompanied by a characteristic of the degree of avidity of specific ATs in such complexes, and secondly, although highly sensitive reactions are used in the laboratory determination of IR and free ATs, they they cannot finally characterize the state of the infectious process at the time of the examination.

 Closest to the proposed invention is a method for determining IR in CFS in seronegative cases for the detection of free AT (Schutzer SE, Koyle PK, Belman AL et al. Sequestrastion of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease // Lancet, 1990, V .335, p. 312-15).

 The method is based on the precipitation of IR using 7.0% polyethylene glycol (m.v. 6000) at pH 8.4 overnight, centrifugation at 8320g for 15 minutes, followed by resuspension in borate buffer at pH 10.2, to dissociate IR . Further, the presence of specific ATs was determined in an ELISA.

 The disadvantages of this method can be attributed the same as for analogues, the lack of avidity characteristics when testing this IR, which can characterize to some extent the severity of the acute period of CSF. Another disadvantage of the method described in the prototype is the lack of revealing the role of complement (KO) in the formation of specific IR, since the presence of KO proteins in the complex should reduce the degree of their dissociation. And finally, when performing the prototype method, there is no way to predict the timing of the process.

 These disadvantages are eliminated in the proposed method for predicting KSB in children by determining specific AT and IR in order to timely correct therapy, improve the outcome of the disease, prevent the transition of the acute course of the disease to chronic.

 The goals are achieved by the fact that in the blood serum of patients simultaneously determine in a single reaction system the concentration of AT to Borrelia, the level of specific IR containing proteins of KO, as well as non-specific IR that do not contain KO, and the presence of AT at the end of the acute period is less than 0.20 relative units (OE), the absence of complement-specific specific IR and the concentration of IR that do not contain KO, more than 0.40 OE judge the possible development of chronic forms of KSB.

 The authors first proposed the use of a comprehensive assessment of the parameters determined in the blood of patients at the end of the acute period, specific AT, as well as two types of IR: specific, containing complementary proteins and non-specific, not carrying complementary proteins and sorbing them when predicting the development of chronic forms of borreliosis additional making. This total score, reproduced by the claimed method, makes it possible to determine the patient’s state of specific immunity (AT), as well as the ability of his immune system to eliminate the pathogen from the body. In the case of patients with a high content of complexes that do not contain CO, it is natural that the degree of excretion of borrelia from the body is sharply delayed, since in the absence of IR complement its sorption on phagocytes (macrophages, skin histiocytes) slows down. This creates the possibility of IR entering into other tissues and organs and the development of a pathological process there, associated with both IR and the pathogen eliminated from this complex. On the other hand, in the presence of patients with IC with sorbed KO, its excretion occurs sooner, which leads to recovery. The development of the chronic course of KSB is especially common in patients who, at the end of the acute period, lack not only specific complement-containing IRs in the blood, but also free ATs.

 The method is as follows.

Combine (50 μl) of the test serum of the patient at the end of the acute period of CSF and 50 μl of Borrelia burgdorferi antigen at a dilution of 1: 100, add 100 μl of KO (2 hemolytic units) and the mixture is placed in a thermostat for 2 hours at +37 ^o C. Next to the hemolytic system is added to the mixtures in the form of a 0.8% suspension of mutton erythrocytes and 10 doses of anti-mutant hemolytic serum. The controls in the method are: control of the antigen without adding the test sample, control of the test sample with a medial-veronal buffer instead of the antigen, and control of complement and hemolytic system without the test sample and antigen.

After the appearance of hemolysis in the control of the test sample and the control of the hemolytic system, all mixtures (experimental and control) are centrifuged at 3000 g to precipitate non-hemolyzed red blood cells. Next, the degree of hemolysis is determined by the level of oxidase enzymes released into the supernatant from hemolized red blood cells. For this, after centrifugation in 50 μl of supernatants, a 0.08% solution of orthophenylenediamine in 3.0% hydrogen peroxide is added, the reaction is stopped after 20 minutes with 1N H ₂ SO ₄ and the color reaction extinction value is determined at a wavelength of 495 nm using an enzyme immunoassay analyzer of any company.

The results of this determination represent three variants of the detected components:
- an immune complex that does not contain complement is determined by the formula (K _co -K _ip ): K _co , where K _co - complement control; K _un - control of the test sample (IR value is indicated at values of 0.10-1.5 OE);
- specific antibodies are determined by the formula (K _ag - A _op ): K _ag , where K _ag - antigen control; A _op - test sample (test serum + antigen) (in this definition, specific ATs are indicated with a negative value in the range - 0.10-1.0 OE);
- the immune complex containing complement proteins is determined by the previous formula in the case of obtaining a positive mathematical value in the range from +0.10 to +1.0.

 An unfavorable prognosis in the possible development of chronic forms of the disease is established by the proposed method according to the following parameters. In the presence of specific antibodies at the end of the acute period within 0.10-0.20 OE or their complete absence in 60%, patients subsequently develop repeated exacerbations in the form of the appearance of migrating recurrent erythema, or the progression of the disease with damage to other organs and systems in the form polyradiculoneuropathies, plexopathies, arthritis, etc. The main laboratory indicator of the prognosis in terms of chronicity of the disease was the level of complement-containing IR. These complexes are determined by the increase in complementary activity upon addition of the Borrelia burgdorferi antigen and KO to the test serum. With this arrangement of testing, separate complementary proteins emerge from IR with KO and enhance the activity of the added KO. Such complement-containing complexes are completely absent in patients with an unfavorable prognosis, but they are noted in 63.5% with full recovery. At the same time, ICs that do not contain KO and substantially sorb it within> 0.40 OE are found in patients who are diagnosed with chronic forms of KSB in 80.0% during clinical examination.

 The sum of three main parameters: the absence of complement-containing IR, the presence of complexes that do not contain CO, and the low level or absence of specific ATs make it possible to predict the development of chronic KSB in children in 90.0% of cases (see table).

 The table also shows that IR without CO are detected in 80.0% of patients with subsequent chronicization of the process, while complexes with the presence of CO are not detected in any patient. Complexes without KO in the acute process are almost also specific, which is clearly shown in the prototype. At the same time, such a difference in complexes, in contrast to the prototype, characterizes the infectious process that develops in patients at the stage of early convalescence. Patients in whom the disease acquires a chronic course at the end of the acute period in the blood contain mainly complexes that do not contain KO. These complexes are weakly visible and, as you know, dissociate with the release of the pathogen, followed by its dissemination in the tissues. In contrast, with complete recovery, such complexes are tested with a minimum frequency (12.5%), and in almost two thirds of cases, specific complexes with the presence of complementary proteins in their structure are detected. It becomes obvious that with complete recovery in patients, specific complexes blocked by complement circulate, which leads to their more rapid elimination from the body due to sorption by macrophages and tissue phagocytes.

 At the same time, these two IRs characterize some aspects of the nonspecific resistance of the organism. With a complete recovery in patients, enough complementary proteins to associate with IR, while a deficiency of CO leads to a chronic process, in particular by reducing the affinity of the resulting IR.

 On the other hand, the level of detectable AT in the subsequent development of chronic forms of infection is slightly lower than in patients with recovery. Minimum levels of AT in the first case are found in 80.0% of patients, while in the second - in 50.0%.

 When comparing all three parameters - AT, IR with KO and IR without KO, it is obvious that the level of the latter is more important for the forecast, since the difference in frequency in favor of timing is 67.5% for this parameter, for IR with KO it is somewhat less - 62.5%, and for AT it does not exceed 30.0%.

 However, for a reliable forecast, it is necessary to evaluate all three identified components in total. So, at the end of the acute period of CSF, 90.0% of patients with subsequent chronicity satisfy the necessary requirements of the method in all respects, while there were no such patients with full recovery. Among these patients, 79.1% were identified by one parameter, and 8.3% by two parameters.

 The method is easy to carry out, it is a single system of binding reactions of CO with a different level of accounting for enzyme testing and can be performed in any virological and serological laboratories. The economic efficiency of the method is unconditional, since it does not require special expensive enzyme immunoassays. All the ingredients for the reaction (Borrelia burgdorferi antigen, complement, lamb erythrocytes, hemolytic serum and orthophenylenediamine) do not have a high cost.

 The described forecasting method can be illustrated with specific examples: in the 1st case with the development of the chronic course of the disease, in the 2nd in the case of recovery.

 Example 1. Patient Petya V., 8 years old. East b N1792, diagnosis: KSB - 1st stage, erythema form. East b N2920, diagnosis: KSB - stage 3, widespread polyradiculoneuropathy with autonomic and sensitive disorders, chronic course.

From the anamnesis it is known that it was bitten by a tick on 05/07/95 in the right behind-the-ear area in the village of Kapralovo, Pushkin district, Leningrad region. 05/18/95, (after 11 days) the child rose to a temperature of 39 ^o C, appeared weakness, decreased appetite, erythema at the site of suction of the tick. He was hospitalized at the Research Institute of Children's Infections on the 2nd day of illness.

 Upon admission, the patient showed annular erythema of the right parotid region with a diameter of 15 cm with swelling of soft tissues, accompanied by soreness and itching, an increase in posterior cervical, occipital lymph nodes, and symptoms of intoxication. In the blood test dated 05/20/95 - specific AT, no CIC with KO + was found, the level of IR without KO was 0.59 OE. The diagnosis was made on the basis of epidemiological data (information about a tick bite), as well as a typical clinical picture of the disease (annular erythema, regional lymphadenitis). Prescribed antibiotic therapy - erythromycin 0.25 g 4 times a day for 10 days. Against the background of the therapy, the fever persisted for 3 days, erythema disappeared on the 6th day of treatment. An ECG examination dated 05/20/95 (3rd day of illness) revealed mild impairment of repolarization, from 05/31/95, without deviations from the norm.

 By the time of discharge from the hospital on June 1, 1995, the patient had moderate regional lymphadenitis, the remaining clinical symptoms of the disease regressed. In the blood test dated June 31, 1995 (day 14 of illness), specific AT and LIC with KO + are absent, the level of IR without KO is 0.70 OE.

 From 09.95 g (4 months after the onset of the disease), the boy developed pains in the popliteal and ulnar fossa, aggravated by physical exertion, which bothered for a month, then remission began. From 04.96 g. (11 months after the onset of the disease) the pains resumed, intensified, there were sensations of freezing, coldness of the legs, which led to the need for re-hospitalization at the NIIDI Neuroinfection Clinic. On admission, pronounced autonomic disorders (hyperhidrosis and hypothermia of the palms and feet), disorders of surface sensitivity (pain and temperature) of a polyneuritic type were revealed. Hyperreflexia, symptoms of tension of nerve trunks. When electroneuromyography from 04/15/96, there was a decrease in the speed of the impulse and the amplitude of the potential of mainly sensitive nerves, which indicates the axonal nature of the lesion. Thermal imaging revealed hypothermia of the feet and hands. In the blood test dated April 14, 1996, specific ATs are absent, the level of SIC with KO + was 0.34 OE, the level of IR without KO was 0.88 OE. Based on the clinical picture, functional and laboratory research methods, the child was diagnosed with KSB - stage 3, widespread polyradiculoneuropathy with autonomic and sensitive disorders, and chronic course. The patient received a second course of antibiotic therapy - zinacef at the rate of 80 thousand per kg of body weight for 21 days, vascular preparations, vitamins.

 Example 2. Patient Vladimir N., 12 years old. East b N3457, diagnosis: KSB - 1 stage, erythema form.

 From the anamnesis it is known that it was bitten by a tick in the interscapular region on July 12, 1996 in the village of Lembolovo in the Vsevolozhsk district of the Leningrad region. The tick was detected and removed by parents only on the 10th day of suction. On July 23, 1996, erythema appeared at the site of a tick bite against a background of normal temperature. He was hospitalized at the Research Institute of Children's Infections on the 2nd day of illness. Upon admission, the patient found in the interscapular region, with spread to the right scapula, annular erythema with a diameter of 12 cm with a pale center and edematous hyperemic edges, an increase in mainly right axillary lymph nodes to 2 cm. On an ECG examination from 07.25.96 (3- the first day of illness) and from 06/06/96 (on the 15th day of illness) deviations from the age norm were not detected. In the blood analysis dated July 24, 1996, specific AT and IR without KO were not detected, LIC with KO + amounted to 0.33 OE. The patient received zinnat 500 mg 2 times a day for 14 days. Erythema disappeared on the 3rd day of therapy, lymph nodes decreased. In the blood test dated August 6, 1996 (on the 16th day of illness), there were no LICs with KO + and IR without KO, the level of specific AT was 0.34 OE. During a two-year follow-up observation of the patient with repeated laboratory examinations, data indicating the transition of the disease to the chronic stage were not obtained.

 Using the proposed laboratory criteria and the results of a long-term follow-up of 150 patients who had an acute stage of the disease from 3 months to 15 years for two to three years, it was possible to identify a reliable correlative dependence of these data and diagnose chronic forms of the disease in 27 patients.

 The proposed method for predicting the development of chronic KSB allows even in the acute period of infection to identify the possibility of an unfavorable course of borreliosis in children and timely correction of therapy.

 The accumulated experience in the treatment of KSB in children has shown the high therapeutic efficacy of antibiotics such as penicillin, doxycycline, cefuroxime and other cephalosporin preparations, the use of which leads to the body's sanation from borrelia. Using this forecasting method for 3 years allowed to reduce the development of chronic forms of borreliosis, which in 1995 amounted to 20.0%, in 1996 - 18.0%, while in 1997 - 11.0% , which indicates the effectiveness of the method both socially and economically.

Claims (1)

 A method for predicting the chronic course of tick-borne systemic borreliosis in children by determining specific antibodies and immune complexes, characterized in that the concentration of antibodies to borrelia, the level of specific immune complexes and nonspecific immune complexes containing no complement are determined simultaneously in a unified reaction system, and by the presence of antibodies at the end of the acute period of less than 0.20 relative units, the absence of complement-containing specific immune complexes and The centers of immune complexes that do not contain complement, more than 0.40 relative units judge the development of chronic forms of tick-borne systemic borreliosis.

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