RU2251695C2 - Method for predicting the flow of erysipelas infection in children - Google Patents

Abstract

FIELD: medicine, immunological diagnostics.

SUBSTANCE: in patients' coprofiltrates simultaneously in one diagnostic kit on should detect the presence of erysipelas viral antigen and erysipelas viral antibodies and at antigen content being above 1.3 relative units and antibodies being below 0.30 relative units it is possible to predict the flow of infection during 6-8 d, and at antigen content being above 1.3 relative units and antibodies being above 0.60-0.80 relative units one should predict the flow of infection during 2-3 d. The method enables to predict the development of complications.

EFFECT: higher accuracy of prediction.

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Description

This invention relates to medicine, more specifically to infectology, and is intended to predict rotavirus infection in children in the early days of the disease.

Acute intestinal infections (acute intestinal infections) remain one of the urgent problems of pediatrics. This is due to their widespread worldwide, continuing high mortality, especially in children under the age of 5 years. In Russia, children with acute intestinal infections steadily occupy the 2nd-3rd place among all infectious diseases (Uchaykin V.F., 1999, Novokshenov A.I. et al., 2000; Bern S., Glass R.I., 1994). Gastroenteritis of viral etiology account for more than 50% of all acute intestinal infections in children. Rotaviruses detected in 58.3% of all viral gastroenteritis are of leading etiological significance.

Rotavirus infection - an acute infectious disease of humans and animals caused by rotaviruses, belongs to the group of intestinal infections, characterized by damage to the gastrointestinal tract by the type of gastroenteritis. The disease usually has an acute onset with a triad of symptoms: fever, vomiting, and diarrhea. In some cases, a gradual development of symptoms is described with an increase in the severity of the process and the development of dehydration. In the clinical picture of rotavirus gastroenteritis during the period of developed clinical manifestations, the leading ones are the syndromes of gastroenteritis and intoxication. Gastroenteritis syndrome is characterized by the development of diarrhea, decreased appetite, the appearance of rumbling and abdominal pain, nausea and vomiting. Vomiting usually precedes or appears simultaneously with the development of diarrhea. Repeated vomiting is repeated, repeated, but for all it is short-lived, 1-3 days. The duration of a febrile reaction averages 2-4 days, in isolated cases it lasts up to 8 days (Mashilov V.P., 1997; Vasiliev et al., 2000). The pain is localized mainly in the upper abdomen or is diffuse, it can be of varying intensity. Almost always, it is accompanied by a loud rumbling in the stomach.

Diarrhea is characterized by abundant watery foamy stools of yellow color without pathological impurities. In some cases, there is a greenish staining and an admixture of transparent mucus mixed with feces (Uchaikin V.F., 1999; Gracheva N.M., Avakov A.A., Blokhina I.T., 1998). The frequency of defecation is from 2-5 times a day to 20 or more times. The duration of diarrhea is 3-6 days, in some cases up to 8 days. Loss of fluid and electrolytes due to vomiting and diarrhea can be significant and lead to the development of dehydration (exsicosis). With dehydration of the I-II degree, thirst, dry mouth, weakness, pallor, dehydration of the III degree are characterized, along with these symptoms, also hoarseness, muscle spasms of the extremities, acrocyanosis, decreased skin turgor, decreased diuresis. The described cases of rotavirus disease with a fatal outcome relate to this particular type of complications. Exsikosis syndrome most often develops on 3-4 days from the onset of gastroenteritis, thus, a timely diagnosed and correctly predicted disease allows you to take adequate measures to prevent the development of complications.

Currently, diagnostic methods for rotavirus infection and prognosis are carried out according to the clinical features of the course of the disease and virological laboratory diagnostic methods. The prognosis of the course of rotavirus infection is carried out according to the severity of individual clinical manifestations characteristic of rotavirus gastroenteritis. A predictably longer and more severe course of the disease in children is expected in the case of the simultaneous development of all symptoms, while the manifestation of only one symptom allows one to predict a milder and faster course of the disease.

However, the diagnosis of rotavirus gastroenteritis, and therefore the prognosis only according to the clinical picture, presents certain difficulties due to the absence of symptoms strictly pathognomonic for this pathology, and therefore the diagnosis of rotavirus gastroenteritis needs laboratory confirmation (Gracheva N.M., Avakov A .A., Blokhina I.T., 1998).

There are a number of ways to diagnose and predict rotavirus infection using virological methods. The high sensitivity of these methods makes it possible to use it for the diagnosis and prediction of rotavirus gastroenteritis in the early stages of the disease. These methods include enzyme-linked immunosorbent assay (ELISA), the reaction of neutralization of hemagglutination (RNGA) and the reaction of inhibition of hemagglutination (RTGA).

The disadvantage of all these methods is the need to use at least two diagnosticums to detect rotavirus and anti-rotavirus antibodies, which complicates the diagnosis and prognosis due to the high cost of diagnosticums and the duration of the methods.

Closest to the proposed invention is a diagnostic method with an attempt to predict the course of rotavirus infection by testing antibodies to rotaviruses in ELISA (Vasiliev B.Ya., Vasilieva RI, Lobzin Yu.V. Acute intestinal diseases. Rotaviruses and rotavirus infection). The method is highly specific, sensitive. It consists in detecting antibodies in the blood of patients on tablets with immobilized rotavirus antigen. It is performed standardly, like all enzyme immunoassay tests: the patient's serum is introduced into the wells of such panels, 2 hours after washing, human IgG bkb IgM, orthophenylenediamine or other peroxidation developers are introduced into these plates.

The disadvantages of this method are the ability to test antibodies only in the blood, since only antibodies of these classes of immunoglobulins appear in the reaction. Local antibodies such as IgA will not be detected in such a reaction. Another drawback is that with a full examination you need 3 types of different ELISA - diagnosticums - to detect antigen, antibodies of the IgM and IgG class.

Thus, using this method, it is difficult to predict in the early stages of the disease and prescribe therapy in a timely manner, and therefore prevent the development of complications.

In order to eliminate these drawbacks, the authors propose a fundamentally new method for predicting the course of rotavirus infection using enzyme-linked immunosorbent assay for the presence of rotavirus antigen and antiviral antibodies in coprofiltrates of patients, which differs in that one diagnosticum is used in the enzyme-linked immunosorbent assay for antigen testing, moreover, along with testing antigen carry out its sorption in coprofiltrates of patients, and in such samples with the removed antigen determined yayut local rotavirus antibodies. To do this, samples with desorbed antigen are combined in an amount of 50 μl with an equal volume of a standard rotavirus antigen containing 0.400-0.500 optical activity units when tested in ELISA, the samples are kept in an incubator at 37 °, introduced into a standard test system for testing rotavirus antigen and determine the level of not neutralized antigen. The method is characterized in that the level of local antibodies in relative units is calculated in coprofiltrates and a long course of infection is predicted by the presence of antigen> 1.3 relative units in the absence of antibodies, and in the presence of antigen in the same concentration and antibody level of the order of 0.600-0.800 relative units - the course of the infection within 2-3 days.

The method is as follows.

From the feces of patients prepare 10-20% suspension in saline and maintain it overnight at + 4 ° C. The prepared samples are clarified by centrifugation at 2000 rpm and stored at -20 ° C before setting the method

When setting up the method, the obtained samples are added to the wells of an immuno-enzyme test system for the detection of human rotavirus antigens (manufactured by NPP "Aquapast") in an amount of 100 μl, incubated for 2 hours at 37 ° C in a humid chamber. The collected liquid after sorption with no viral antigen is used to determine antibodies.

In the sorbed material, the concentration of rotavirus antigen is determined by standard injection of the conjugate into the wells of the conjugate and carrying out the enzyme reaction using tetramethylbenzidine, the results are measured by measuring the optical density at a wavelength of 450 nm using a multichannel spectrophotometer. The presence of rotavirus antigen is confirmed by the ratio of the experimental sample to the negative control> 1.3 (relative units).

In the liquid after sorption of the test material, the total anti-rotavirus antibodies are determined. For this purpose, the supernatant after the first stage of sorption is combined with a standard rotavirus test antigen pre-treated containing 0.400-0.500 rel. Such a test virus can be a standard preparation of an industrial rotavirus antigen or a substrate prepared from the feces of patients with rotavirus infection. A mixture of the supernatant and the standard test virus of 50 μl is placed in a thermostat at 37 ° C for 2 hours, then the material is introduced into the wells of the test system for determination of rotavirus antigen and the level of test virus not neutralized by antibodies is detected according to the standard scheme for determining antigen in the specified diagnostic test system. The control in this case was the wells with the standard test virus introduced into them without samples from patients.

The level of detected antibodies is calculated by the formula A = (Vop.-Vk) / Vk, where:

And - the level of detectable antibodies in relative units (from 0.10-0.80 relative units),

Woop. - rotavirus antigen in the experimental sample after inactivation by antibodies,

Bk - rotavirus antigen in the control sample.

The next step of the proposed method is to forecast the course of rotavirus infection in patients with rotavirus antigen in the feces (more than 1.3 rel.units) and the absence of anti-rotavirus antibodies (less than 0.30 rel. Units), in contrast to patients with the same level antigen and the presence of antibodies at a concentration of 0.600-0.800 relative units

The technical result of this invention is to improve the accuracy of the forecast.

The authors first proposed to use a comprehensive assessment of antigen and antibody indicators to predict the course of rotavirus infection in children.

Preliminary sorption of coprofiltrates to remove antigen allows for more complete identification of local antibodies belonging to different classes of immunoglobulins - IgA, IgM and IgG, not included in the structure of specific immune complexes. These antibodies are detected using a test virus - in the form of a coprofiltrate containing rotavirus antigen in the range of 0.400-0.500 rel. In special studies, we found that this is the optimal concentration for the detection of antiviral antibodies. A lower dose of the virus, within 0.150-0.300 rel.units, creates a high percentage of false-positive reactions (up to 20%), a large dose (> 0.600 rel. Units) leads to the detection of minimum antibody concentrations.

Thus, this method allows the use of a single enzyme-linked immunosorbent assay (to detect rotavirus antigen) to test both antigen and antibodies. Antibodies are determined in total of all classes, which makes it possible to test them regardless of their structure and origin. At the same time, this creates the conditions for the use of only one type of enzyme-linked immunosorbent diagnostic.

In the initial examinations with testing of the test virus and its working dose, the time spent doubles with respect to time to detect one antigen and is 5-6 hours. Subsequently, with repeated use of the test virus, the time spent is practically extended by 30 minutes with respect to the time of the initially tested antigen (2.5-3 hours).

Our method adds in the diagnosis of rotavirus infection 43.9% confirmation of the detection of specific Ag or At

The main clinical symptoms that determine the severity of the course of the disease in these patients were more pronounced. Intoxication syndrome was observed in 75% of cases and lasted an average of 3 days. In the early days of the disease, febrile fever was observed in 66.7% of patients; the duration of the entire febrile period averaged 3.8 days. Vomiting lasted up to 3 days in 66.7% of patients, exicosis syndrome of the 1st or 2nd degree developed in 75% of the cases we observed. In this group of patients, the duration of diarrhea characteristic of rotavirus infection reached 6 days in 16.7% of patients, and averaged 4.3 days. The duration of the entire period of rotavirus gastroenteritis in this group was from 6 to 8 days.

In the presence of both Ar and At in coprofiltrates in high concentrations from 0.60 to 0.80, the prognosis of the course of infection was more favorable. So, intoxication syndrome was observed in 61.1% of patients and lasted no more than 2 days. The febrile period was shorter than in the first group of patients, and averaged 2.9 days, with febrile digits reaching body temperature of only 44.4% in the first days of the disease. The duration of vomiting did not exceed 2 days and occurred in 61.1% of cases. Exicosis developed in 38.9% of cases, mainly I degree. The duration of diarrhea did not exceed 3 days. Thus, the duration of rotavirus infection in this group of patients was no more than 3 days.

Thus, our proposed method is easy to perform, it is a unified system of ELISA reactions, can be performed in any virological laboratories and allows to predict the course of rotavirus infection in the early stages of the disease only by virological indicators (antigen and antibody levels in coprofiltrates). The result of such forecasting is the timely appointment of rational therapy, and therefore the prevention of the development of complications. The economic efficiency of the method is unconditional, since it does not require the inclusion of several tests in the diagnostic system.

The method is as follows: using coprofiltrate supernatant after antigen sorption in a classical ELISA, these supernatants are combined with a pool of feces containing rotavirus antigen in the amount of 0.400-0.500 rel.ed., incubated for 2 hours and introduced into a panel with immobilized anti-rotavirus antibodies infection. To reduce the level of test antigen more than 0.30 rel. establish the presence of antibodies in the patient's coprofiltrate. The prognosis is carried out by assessing the amount of both antigen and antibodies: by the presence of antigen in the coprofiltrate (> 1.3) in the absence of antibodies (0.30 rel. Units), a longer course of infection from 6 to 8 days can be predicted. In the presence of high concentrations of antigen (> 1.3 rel. Units) and antibodies (0.69-0.80 rel. Units) - for no more than 3 days.

The described forecasting method can be illustrated with specific examples:

Example 1

Patient Kolya M., 1 year 4 months, East. b. No. 608, diagnosis: Acute rotavirus gastroenteritis, moderate.

From the anamnesis it is known that he fell ill acutely, at first there was a watery foamy stool, 5 times during the day, after a few hours the body temperature rose to 38 ° C. The next morning, a single vomiting and watery stool without pathological impurities were noted. At the same time, the child was lethargic, drowsy, lacked appetite, refused to take water. He was hospitalized at the Research Institute of Children's Infections on the 2nd day of illness.

On admission, intoxication syndrome was expressed, body temperature 37.2 ° C, watery diarrhea continued up to 4 times during the day, with palpation of the abdomen moderate soreness and rumbling along the intestine. The child was admitted in a state of exicosis of the first degree. The diagnosis was made on the basis of laboratory virological data (ELISA, TEM), as well as a typical clinical picture of gastroenteritis. In the analysis of feces, rotavirus Ag was found in the amount of 1.47 conventional units, and specific antibodies were found to be 0.19. Applying our method, we predicted an unfavorable course of rotavirus gastroenteritis in this case and tried to prevent possible complications. Assigning adequate therapy from the first hours of admission, it was possible to avoid the growth of exsicosis and aggravation of the course of infection. However, since the child was already in a state of exicosis, it was not possible to prevent the duration of gastroenteritis. Clinical recovery occurred only on the 8th day.

Example 2

Patient Sasha A., 3 g. 9 months, East. b. No. 1606, diagnosis: Acute rotavirus gastroenteritis, mild.

From the anamnesis it is known: he fell ill acutely, simultaneously with repeated vomiting, watery stool without pathological impurities appeared, more than 10 times during the day. The next day, body temperature rose to 37.5 ° C, vomiting continued, diarrhea stopped on the 2nd day of illness. The child was hospitalized at the clinic of the Research Institute for Children's Infections on the second day of the disease, in the evening.

On admission: intoxication syndrome is quite pronounced (weakness, adynamia, lack of appetite), repeated vomiting, manifestations of exicosis of the first degree. In the ELISA analysis of feces, Ar - 1.35, At - 0.72. The diagnosis was also made on the basis of virological confirmation. Our prognosis in this case was favorable, despite the development of exicosis at the prehospital stage, since specific antibodies in high concentrations were determined in coprofiltrates. The child was prescribed standard therapy. Syndromes of intoxication, exsicosis, fever and vomiting stopped on the 2nd - 3rd day of treatment, diarrhea did not recur. The child was discharged home with clinical recovery.

We examined 120 patients who underwent acute rotavirus gastroenteritis, which allowed us to identify a reliable correlative dependence of these data, to diagnose the infection in the early stages of the disease and to predict the severity of the course in 55 patients. This method allowed to reduce the development of severe forms of the disease, prevent the increase in the severity of clinical symptoms at the hospital stage, prevent the development of a complicated course due to an early assessment of the possible severity and duration of the course of rotavirus infection and as a result of the timely implementation of the necessary therapy.

Claims (1)

A method for predicting the duration of the course of rotavirus infection in children, characterized in that the patient’s coprofiltrates in one diagnostic simultaneously determine the presence of rotavirus antigen and antibodies to rotavirus by sorbing antigen in coprofiltrates and connecting the supernatant with the feces pool containing rotavirus antigen in an amount of 0.400-0.500 rel . units, kept in a thermostat, followed by introduction into a panel with immobilized antibodies, the presence of antibodies is established to reduce the level of test antigen and when the antigen content is more than 1.3 rel. units and antibodies less than 0.30 rel. units they predict the course of infection in 6-8 days, and when the antigen content is more than 1.3 rel. units and antibodies greater than 0.60-0.80 rel. units Predict the course of infection for 2-3 days.