RU2450274C1 - Early diagnostic technique for ebola hemorrhagic fever in individuals presumably infected with such virus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: blood serum is sampled from individuals presumably infected with such virus for the first time in 3-4 hours and for the second time - in 15-18 hours after presumed infection. Each serum sample is examined for a level of hemolytic complement activity (HCA) by any known technique. If observing the HCA in 1.5-2.0 times and more for a specified period of time, the fact of body infection is stated, and the absence or minor change of the HCA shows the absence of body infection. The HCA level can be evaluated by unified technique showing intact sheep erythrocyte lysis with commercial hemolytic anti-sheep rabbit serum.

EFFECT: use of the technique enables earlier diagnosing of Ebola hemorrhagic fever that will allow prescribing a complete complex of therapeutic actions immediately.

2 cl, 1 tbl, 4 dwg

Description

The invention relates to methods for early diagnosis of Ebola hemorrhagic fever and can be used in medicine and virology.

The detection of antigens (viral particles) of the Ebola virus in the blood is known, which occurs at the end of the incubation period or during the height of the disease, which corresponds to 4-6 days from the moment of infection. The following methods are used for this:

- virus isolation in cell culture and staging of bioassays in sensitive laboratory animals

1. Peters et al., 1996: Peters C. J., Sanchez Anthony, Rollin Pierre E. Filoviridae: Marburg and Ebola Viruses. 1161-1176. In: Fields Bernard N., Knipe David M., Howley Peter M. Field's Virology Third Edition Volume 1 1996. Lippincott-Raven Publishers, Philadelphia PA.

2. Saijo et al., 2001: Saijo Masayuki, Niikura Masahiro, Morikawa Shigeru, Kurane Immunofluorescence Method for Detection of Ebola Virus Immunoglobulin G, Using HeLa Cells Which Express Recombinant Nucleoprotein. Journal of Clinical Microbiology. 2001; 39 (2): 776-778. [PubMed: 11158150].

3. Mekki and Van Der Groen, 1981: Mekki A.A., Van Der Groen G. A comparison of indirect immunofluorescence and electron microscopy for the diagnosis of some haemorrhagic viruses in cell cultures. Journal of Virological Methods. 1981; 3 (2): 61-69. [PubMed: 7024293].

- detection of virus RNA in samples from an infected person by PCR and real-time PCR methods.

1. Leroy et al., 2000: Leroy E.M., Baize S., Lu C.Y., McCormick JB, Georges AJ, Georges-Courbot MC, Lansoud-Soukate J, Fisher-Hoch SP. Diagnosis of Ebola haemorrhagic fever by RT-PCR in an epidemic setting. J Med Virol. 2000; 60 (4): 463-467.

2. Drosten et al., 2002: Drosten C., Gottig S., Schilling S., Asper M, Panning M, Schmitz H, Gunther S. Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean- Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin. Microbiol. 2002; 40 (7): 2323-2330. [PubMed: 12089242].

3. Drosten et al., 2003: Drosten C, Kummerer BM, Schmitz H, Gunther S. Molecular diagnostics of viral hemorrhagic fevers. Antiviral Res. 2003; 57 (1-2): 61-87. [PubMed: 12615304].

- detection of viral particles by electron microscopy and indirect electron microscopy.

The detection of antibodies in the blood serum of ill people is known, which is carried out no earlier than 3 days from the moment of infection (early antibodies of the IgM class) and antibodies of the IgG class appearing on days 10-14 by enzyme-linked immunosorbent assay:

1. Ksiazek et al., 1999: Ksiazek TG, Rollin PE, Williams AJ, Bressler DS, Martin ML, Swanepoel R, Burt FJ, Leman PA, Khan AS, Rowe AK, Mukunu R, Sanchez A, Peters C. J. Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigen, and IgG and IgM antibody findings among EHF patients in Kikwit, Democratic Republic of the Congo, 1995. J. Infect Di. 1999; 179 (Suppl 1): S177-S187. [PubMed: 9988182].

2. Merzlikin et al., 1995: Merzlikin N.V., Chepurnov A.A., Istomina N.N., Ofitserov VI, Vorob'eva MS. Development and application of an immunoenzyme test system for diagnosing Ebola fever. Vopr. Virusol. 1995; 40 (1): 31-35. [PubMed: 7740786].

3. Leroy et al, 2000B: Leroy E.M., Baize S., Volchkov V.E., Fisher-Hoch SP, Georges-Courbot MC, Lansoud-Soukate J, Capron M, Debre P, McCormick JB, Georges AJ. Human asymptomatic Ebola infection and strong inflammatory response. Lancet. 2000; 355 (9222): 2210-2215. [PubMed: 10881895].

The disadvantages of the known methods are that the diagnosis of Ebola hemorrhagic fever disease is still possible only if viral particles are detected in the blood 4-6 days after infection, which coincides with the end of the incubation period and the development of the clinical picture.

A known method for determining in vitro complement activity according to the classical activation pathway for various diseases (lupus erythematosus, acute pneumonia, bronchial asthma, rheumatoid arthritis), which can occur with the use of the complement system (RF patent No. 2124209, IPC G01N 33/559, publ. 27.12 .1998).

However, this method is used to diagnose diseases whose symptoms have already manifested, and is not intended for early rapid diagnosis of especially dangerous infections.

The closest analogue (prototype) is a method for detecting the specificity of activated lymphocytes in the body (RF patent No. 2343486, IPC G01N 33/53, published January 10, 2010), which includes the following stages: a) dilution of antigen or antigens capable of activating lymphocytes in the body, an environment containing, in addition to the standard ingredients used for cell cultures, neutralizing antibodies against cytokines that can induce cell proliferation, and / or cytokines that can induce apoptosis of a mononuclear cell s cells or inhibit cell activation or cell proliferation;

b) the preparation of a suspension of mononuclear cells containing activated lymphocytes intended for testing in the medium;

C) incubation of the mixture of antigen and suspension containing activated lymphocytes obtained in stages a) and b) in the wells of a tablet for cell cultures; and

d) determining the presence of antigen-specific activated lymphocytes by comparing the differences in the detected signals coming from the test wells and control wells of the tablet. The method is intended for the diagnosis of hemorrhagic fevers, including the Ebola virus. Scheme for determining the cellular activity of lymphocytes: the reaction uses a mixture of sensitized lymphocytes, human mononuclear cells and commercial neutralizing antibodies against various cytokines. The incubation time is 20 hours in a CO ₂ incubator at a temperature of 37 ° C. After incubation, the intravital dye MTT is introduced into the cell suspension. The results of cell staining are taken into account by a minder or in a light microscope.

However, the method described in the patent is high-tech, requires the use of complex and expensive equipment. The method of preparing the components for the reaction includes the following steps:

- obtaining cell lines containing human HLA and mH antigens by genetic engineering, takes several weeks;

- the creation of a line of b-cells, cloning takes 3-5 weeks;

- the preparation of specific antigens of each item for sensitization of human lymphocytes takes 1 month;

- monoclonal neutralizing antibodies (Fab fragments of IgM) are expensive commercial preparations (1 mg of each drug costs from 2000 rubles).

The main disadvantage of using this method for the early diagnosis of viral infections (including hemorrhagic fevers of HFRS, Lassa, Kyasanur forest disease, Argentine GL, Bolivian GL, Congo-Crimean GL, etc.) - a functional change in lymphocytes occurs at the end of the incubation period, t. e. 3-4-5 days after infection, which makes this method of little relevance for the early diagnosis of infectious diseases.

The technical result of the claimed invention is an earlier diagnosis (in the first hours after a likely infection) of Ebola hemorrhagic fever.

The specified technical result is achieved by the fact that the method for early diagnosis of Ebola hemorrhagic fever in individuals suspected to be infected with the virus involves obtaining the first blood serum samples from the body 3-4 hours after its probable infection and the second receiving 15-18 hours after it suspected infection. In each serum sample, the level of complement hemolytic activity (HAC) is determined by any known method and, in the case of an increase in HAC indices of 1.5-2.0 or more times in samples obtained 15-18 hours after the alleged infection compared to background values (serum samples obtained after 3-4 hours from the time of the alleged infection), ascertain the fact of infection of the body, and in the absence or insignificant change in HAC, ascertain the absence of infection of the body. The level of HAC is determined by a unified method for determining HAC by hemolysis of intact mutton erythrocytes with the addition of commercial hemolytic anti-mutton rabbit serum.

Our proposed method for early diagnosis of Ebola hemorrhagic fever disease is technically simple, does not require expensive components. The total time of the study, including the preparatory period, does not exceed 3 hours.

Figure 1 shows the graphs of changes in HAC during infection of guinea pigs with active drugs of the lethal and non-lethal strains of the Ebola virus (LVE and non -letVE), inactivated with the preparation of VE. In figure 2. a graph of changes in the hemolytic activity of complement in the blood of guinea pigs in the first 30 hours after infection with LHE is given. The solid line shows the average complement activity, the dots show the results of measurements of complement activity in individual animals. The scatter of titration results for each serum sample did not exceed 10 units. Dotted lines indicate the region of normal values of complement activity, based on our experimental data. Figure 3 shows a graph of changes in the blood glucose in the blood of guinea pigs during infection of guinea pigs with active preparations of herpes viruses, influenza and non-lethal strain of VE. Figure 4 shows a graph of changes in HAC in the blood of guinea pigs during infection of guinea pigs with a lethal strain of CE and HAC in the blood of an infected person (surfactant).

A method for the early diagnosis of Ebola hemorrhagic fever disease is as follows.

For a person who is presumably infected with the Ebola virus, for example, who had an accident while working with the indicated object in a research laboratory, they take the first blood serum samples from the body no later than 3-4 hours after its probable infection (to obtain background HAC values) and after 15-18 hours after his alleged infection. In each serum sample, the level of complement hemolytic activity (HAC) is determined by any known method. The determination of HAC can be carried out by two methods:

- determination of the functional activity of the complement system by hemolysis of ram intact red blood cells with hemolytic anti-mutton serum (commercial preparation, the cost of a box with 5 serum ampoules does not exceed 200 rubles, working dilution of commercial serum 1: 300-1: 1200, which allows you to use 1 ampoule for 50-100 definitions);

- determination of the concentration of C ₃ complement components in blood serum, is tested in ELISA using a test system to determine the C ₃ factor _of any manufacturer (usually 1 set is designed for 50-100 determinations).

The components of the reaction can be stored in a refrigerator at a temperature of + 8 ° C for a long period (from 1 month for a mixture of intact red blood cells and up to 1 year for a commercial test system). The preparation time of the tested serum samples does not exceed 1 hour, the reaction time is no more than 1 hour. The total time of the study, including the preparatory period, does not exceed 3 hours.

The level of HAC is determined, for example, by a unified method for determining HAC by the hemolysis of intact mutton erythrocytes of commercial hemolytic anti-mutton rabbit serum.

Determination of hemolytic complement activity in blood serum was carried out on model animals - guinea pigs. Assessment of the hemolytic activity of complement was carried out by a micromethod according to [Sokolov MI, Sinitsky AA, Remezov PI Virological and serological studies for viral infections. // Leningrad, "Medicine". - 1972. - 216 s] in its own modification. HAC titration was carried out in polystyrene 96-well round-bottom plates for immunological reactions. Dilutions of serum samples were prepared in the ratio 1/10, 1/12, 1/15, 1/17, 1/20, 1/25 and then to 1/120 in steps of 5. 50 μl of physiological saline, 25 μl were added to each well. dilutions of complement-containing serum, 50 μl of hemolytic system was added. The hemolytic system was prepared by combining equal parts of a 2% suspension of sheep erythrocytes and commercial anti-sheep hemolytic serum. Incubation of the reaction mixture, consisting of samples of serum, saline and hemolytic system, was carried out in an thermostat at a temperature of 37 ° C for 45-60 minutes. Analysis was carried out visually. The last dilution of serum, in which 100% erythrocyte hemolysis was still observed, was taken as 1 hemolytic unit (ED) [Sokolov MI, Sinitsky AA, Remezov PI Virological and serological studies for viral infections. // Leningrad, "Medicine". - 1972. - 216 p.].

In the case of an increase in HAC by 1.5-2.0 or more times in paired sera obtained after 3-4 hours and after 15-18 hours from the time of the alleged infection, the fact of infection of the body is ascertained, and in the absence or insignificant change in HAC, the absence of infection of the body.

The most important advantage of the proposed method is that a preliminary diagnosis of Ebola hemorrhagic fever can be made after 18-24 hours from the date of the alleged infection (in cases of accidents during laboratory, research, diagnostic work or when medical personnel carry out medical manipulations with patients with Ebola hemorrhagic fever) . All other generally accepted methods allow to make a preliminary diagnosis of Ebola hemorrhagic fever no earlier than 4 days after infection, i.e. at the end of the incubation period against the background of already developing clinical manifestations.

The results of experimental studies to assess the possibility of early diagnosis of Ebola hemorrhagic fever of the claimed method.

1. The dynamics of the hemolytic activity of complement in the blood of guinea pigs with lethal and non-lethal infections of Ebola, herpes simplex virus type 2 and influenza A was investigated (Figure 1).

2. A correlation was established between the dynamics of the increase in complement activity in the blood of animals with the lethality of the ongoing infection. In animals demonstrating a lethal infection, the hemolytic activity of complement rapidly increased after infection with the Ebola virus, and already 15 hours after infection, the activity indicators of complement in 100% of guinea pigs exceeded the background values by more than 2 times (Fig. 2). By the end of 1 day, their decline began, which reached the initial level by the end of the incubation period (3-4 days after infection) and then decreased to zero 2-3 days before death.

3. Infection of guinea pigs with non-EVE, as well as other viral infections (influenza, herpes), leads to activation of complement only at the end of the incubation period (3-4 days after infection), followed by a “plateau-like” increased level of complement for the period of the acute course of the disease and the normalization of HAC indicators during the recovery period (figure 3).

4. A relationship has been established between the values of HAC in the blood of infected guinea pigs and the timing of death of these same animals. The more HAC indicators exceed normal values (39.2 ± 5.3 PIECES), the sooner the death of the animal occurs. The revealed feature is characteristic of Ebola hemorrhagic fever, but was not found in laboratory animals with other lethal infections (herpes, rabbit hemorrhagic disease) (Table 1).

5. The study of blood serum of a person with Ebola, taken 18 hours after infection, on the 2nd day and then 1 time per day throughout the period of the disease, showed an increase in HAC 18 hours after infection by 2.25 times, maintaining elevated values throughout the incubation period. After a rise in temperature (day 7), a sharp decrease in HAC was observed: every day, HAC indices decreased by half, reached zero values on the 11th day from the moment of infection and remained so until death (14th day from the moment of infection) (Fig. .four). The results of the study of HAC in the blood serum of a person who became ill and died of Ebola fever, completely coincided with the experimental data of laboratory studies when modeling Ebola fever in guinea pigs (figure 4).

The revealed phenomenon of a sharp increase in HAC (15-18 hours after infection) during the lethal course of the disease can have prognostic value and can be used for early diagnosis of Ebola. This is especially important, since biochemical, hematological changes in the peripheral blood, indicating infection, including and liver damage (massive cytolysis) in guinea pigs infected with VE and in humans appears either at the end of the incubation period (4-5 days) or at the height of the disease (Akinfeeva L.A., Aksenova O.I., Vasilevich I.V., Ginko Z.I., Zarkov K.A., Zubavichene N.M., Katkova L.R., Kuzovlev O.P., Kuzubov V.I., Lokteva L.I., Ryabchikova E . I. and others. The case of Ebola virus hemorrhagic fever. J. "Infectious Diseases", 2005, v.3, No. 1, pp. 85-88).

Table 1 Correlation between serum guinea pigs HAC indices 18 h after infection with EV and the terms of their death *. Fallen animals / total
animals HOOK (UNIT) ** Deadlines (day) 4/20 85.0 ± 3.5 7-8 4/20 77.5 ± 5.5 9 5/20 66, 2 ± 5.9 10-13 4/20 55.0 ± 3.5 16-18 3/20 39.2 ± 5.3 no doom Note * - the correlation coefficient was 0.94. ** - standard deviation, determined by the Fischer-student formula.

The inventive method is relevant in the event of a probable intra-laboratory infection. Thus, the proposed method for early confirmation of the fact of human infection with the Ebola virus (during the first day) will immediately enable the full range of medical and preventive measures to be deployed.

Claims (2)

1. A method for the early diagnosis of Ebola hemorrhagic fever in individuals suspected to be infected with the virus, including obtaining blood serum samples of the body 3-4 hours and 15-18 hours after its alleged infection and determining the level of complement hemolytic activity in each serum sample (HAC ) by any known method and, in the case of an increase in HAC by 1.5-2.0 or more times during the indicated time, the fact of infection of the organism is ascertained, and in the absence or insignificant change of HAC to they note the absence of infection of the body. 2. The method according to claim 1, characterized in that the level of HAC is determined by a unified method for determining HAC by the hemolysis of intact mutton erythrocytes of commercial hemolytic anti-mutton rabbit serum.

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