RU2310852C1 - EXPRESS-DIAGNOSIS METHOD FOR DIAGNOSING HEMOPHILIC INFECTION OF b TYPE IN CHILDREN - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves using hemagglutination reaction for detecting type b hemophilic bacillus antigen in blood serum. Both liquor and blood serum are used as material under study. 1.2% monodisperse polystyrene latex suspension is used as immunoglobulin carrier particles of d=0.8 mcm (type b hemophilic bacillus antibodies). Agglutination reaction is carried out on object plate having 50 mcl of sensibilized IgG against type b hemophilic bacillus antigens and 20 mcl of liquor or 20 mcl of blood serum. Results are recorded 2-3 min later after having mixed the reagents. Type b hemophilic bacillus antigens availability in the material under study is proved by availability or lack of visually detectable latex agglutinate particles.

EFFECT: high expressive power and diagnosis accuracy.

4 tbl

Description

The invention relates to new medical biotechnology and can be used in clinical practice as a method of early diagnosis of hemophilic infection "b" in children.

Hemophilic infection "b" is an extremely urgent problem in the world, as it is characterized by a wide spread, severe course and high mortality among young children. Actual data accumulated in recent years indicate that the infection caused by H. influenzae type “b” (Hib infection) is widespread in Russia and is an urgent public health problem.

The causative agent of hemophilic infection "b" is Haemophilus influenzae type "b", which belongs to gram-negative microorganisms, the virulence markers of which, as is known, are the strains belonging to the capsular type "b". A hemophilic infection takes second place after pneumococcal, complicating the course of acute respiratory viral infections. Currently, only 10 of its existing species (more than 20) are considered as causative agents of bronchitis, pneumonia and otitis. In recent years, there has been an increase in the incidence of meningitis, meningoencephalitis, and generalized forms of diseases caused by hemophilic bacillus type b. The latter are often complicated by subdural effusion, heart attack of the brain and other organs, improper secretion of antidiuretic hormone, prolonged course, which determines the adverse outcomes of the disease (mortality up to 19% according to the USA) and residual consequences in the form of intellectual insufficiency.

The widespread prevalence of hemophilic infection, the diversity of clinical forms, the severity of the course with adverse outcomes, as well as high rates of morbidity and mortality, especially among children, require improved diagnostic methods for these diseases.

Isolation and identification of the pathogen is currently difficult due to the emergence of strains resistant to penicillin and other antibiotics, the lack of diagnostic drugs and the lack of culture media of domestic production.

The currently used algorithm for bacteriological diagnosis of Hib infection, regulated by order of the Ministry of Health No. 535 of April 22, 1985, provides for confirmation of the diagnosis by isolating the pathogen culture. However, the method is very cumbersome, its implementation requires up to 3 or more days, while the effectiveness of the method remains low.

A known microscopic method for making an indicative diagnosis by studying smears from material (blood, CSF) obtained from patients with suspected hemophilic infection (Kolukanov I.E., Chaika N.A. Hemophilus infection. St. Petersburg, 1998, 60 pp.). However, the evaluation of the microscopic picture of the preparations seems especially difficult with respect to hemophilic bacteria and, in particular, H. influenzae type “b,” since the detection of small encapsulated gram-negative rods randomly located relative to each other in the smear can only suggest the presence of a hemophilic bacillus.

Known methods for the rapid diagnosis of Hib infection, the essence of which is to identify antigens of hemophilic bacillus type “b” in biological fluids (CSF, blood and urine) using immunodiffusion reactions (RID), counter immunoelectrophoresis (WIEF) and radioimmunoassay (RIA) (Kolukanov I.E., Chaika N.A. // Hemophilus infection. / St. Petersburg, 1998, 60 p.). However, when these reactions are formulated, there are a large number of cross-reactions with polysaccharides of other capsular microorganisms, for example pneumococci, meningococci, and others, which ultimately does not ensure the accuracy of the diagnosis. Despite the high sensitivity and specificity of the methods, their use is limited by technical complexity and the possibility of irradiation of personnel when using RID.

Optimal results in the diagnosis of hemophilic infection of type "b" were obtained using enzyme-linked immunosorbent assay - ELISA. The principle of the method is that the surface of a solid-phase carrier is sensitized by mouse monoclonal antibodies against H. influenzae “b”. When introducing into the wells of the test material (CSF, blood serum of the patient) in the presence of the Hib antigen in it, the antigen + antibody complex is formed, which is associated with the surface of the carrier. After addition of an antispecies serum labeled with an enzyme, labeled antibodies interact with the antigen + antibody complex to form a new complex. To identify it, add a solution of the substrate, which under the influence of the enzyme changes color, which indicates a positive result (Kolukanov I.E., Chaika N.A. // Hemophilus infection. / St. Petersburg, 1998, 60 pp.).

However, despite the high sensitivity of the described ELISA, it was not promising in terms of the limited possibility of its use for widespread use. The difficulty in obtaining mouse Hib monoclonal antibodies, and in connection with this, the lack of standard test systems for ELISA, to date, significantly reduces the accuracy of diagnostic results.

Closest to the proposed method for the rapid diagnosis of hemophilic infection "b" is a method for detecting Hib antigens in materials obtained from patients using the latex agglutination reaction - RAL (Bogdanovich T.M et al. // Isolation, identification and determination of sensitivity to H. influenzae antibiotics /. "Clinical Microbiology and Antimicrobial Chemotherapy, 2000, No. 2, Volume 2, S.93-109). The principle of the reaction is agglutination on a glass of latex particles, on the surface of which antibodies against H. influenzae “b” are adsorbed when mixed with the patient's blood serum or CSF in order to detect antigen in them. The reaction is technically simple, sensitive and specific. To detect H. influenzae "b" antigens abroad and in our country, commercial latex agglutination kits have been tested, including the "Slidex meningite - Kit 5" bioMerieux (France) diagnostic latex kit, which is expensive and, moreover, with very limited shelf life (no more than 3 months) .In addition, the reaction has a limited area of use of the test material, since it allows (according to the instructions) to detect antigen only in the cerebrospinal fluid. Meanwhile, quite often (up to 30% of cases) with invasive infection caused by hemophi with bacillus bacillus, septic forms of the disease develop, in which the pathogen circulates in the blood.Thus, despite the high sensitivity of RAL in the early diagnosis of hemophilic “b” meningitis, it is absolutely not applicable in the diagnosis of sepsis of hemophilic etiology.

Based on this, the development of a fundamentally new method for early diagnosis of Hib infection is a very urgent task. The authors solved this problem by declaring a method for detecting antigens of hemophilic bacillus type “b” in the blood serum and CSF of patients with hemophilic infection using the latex agglutination reaction. In addition to cerebrospinal fluid, blood serum can be used as test material, as well, and 1.2% suspension of monodispersed polystyrene latex as immunoglobulin carrier particles (antibodies against Haemophilus influenzae type “b”). RAL is carried out on a glass slide, the results are recorded 2-3 minutes after mixing the reagents and the presence or absence of visually observed agglutinates of latex particles judges the presence of haemophilus influenzae type "b" antigens in the test material.

The technical result is to increase the accuracy, rapidity, which ensures the appointment and conduct of timely therapy.

This result is achieved by the fact that revealing the hemophilic bacillus antigen in the latex agglutination reaction, the authors propose to isolate immunoglobulin G from hyperimmune rabbit serum immunized with Act-Hib conjugated vaccine, which sensitizes 1.2% of the suspension of monodispersed polystyrene latex particles (d = 0.8 mmk), then 50 μl of sensitized latex and 20 μl of cerebrospinal fluid or 20 μl of blood serum are applied to a glass slide, and they are mixed by the presence or absence of visually observed agglutinates after 2-3 minutes with a positive The results of the reaction with a blood serum sample are diagnosed with a generalized form of hemophilic infection “b” with a cerebrospinal fluid sample - acute purulent meningitis of hemophilic etiology “b”.

For the first time, to increase accuracy, the authors use 1.2% suspension of monodispersed polystyrene latex with the characteristics most suitable for sorption of immunoglobulins G against antigens of the hemophilic bacillus b. So, by working out the parameters of sorption of latex particles by antibodies to antigens of the hemophilic bacillus b, the authors were able to choose the optimal size and concentration of latex particles and use monodispersed polystyrene latexes with a particle diameter of 0.8 mmk with a concentration of 1.2% particle suspension for effective sensitization of latex particles.

The authors successfully used the rabbit model to obtain high-quality hyperimmune serum with a high content of fractions of antibodies of the immunoglobulin G class against the antigens of the hemophilic bacillus b due to the testing of the high-quality commercial conjugate vaccine Act-Hib, Pasteur Merrier, France). Thus, the authors were able for the first time to obtain the maximum number of antibodies against "Hib" for sensitization of latex particles, which ensures the most accurate diagnosis. It is not clear that the authors obtained the results, as it turns out, with the fact that a specific class G immunoglobulin fraction is obtained from the hyperimmune serum of rabbits immunized with a vaccine (Act-Hib) containing purified polysaccharide H. influenzae “b”, the main typical pathogen antigen. Rabbits are immunized with a vaccine based on the Hib conjugated capsular polysaccharide - polyribosyl ribitol phosphate (PRF) with tetanus toxoid used as a protein carrier, with the help of which the authors were able to obtain hyperimmune serum with a high IgG content against antigens of the hemophilic bacillus b.

As experimental studies showed, as a result of which a fact was revealed, namely, the carrier protein (tetanus toxoid), which is part of the vaccine, does not cause the formation of specific anti-toxic tetanus antibodies. Its function is to participate in the mechanism of presentation of PRF antigen to T-lymphocytes and, thus, in the induction of a T-dependent immune response. Anti-PRF antibodies produced during the humoral immune response as a result of vaccination, according to the researchers, belong to the IgG class (Parke JC, Schneerson R., Reimer M., Robbinson JB et al. / Clinical and immunologic responses to H. influenzae type b tetanus toxoid conjugate vaccine in infants ingected at 3.5.7 and 18 mounths of age.// J. pediatr., 1991; 118: 184-190).

Thus, based on the foregoing, the authors for the first time, using mechanical sorption of polystyrene latex particles with a specific IgG class immunoglobulin (at a concentration of 10 μg / ml), obtained a highly specific preparation for the detection of type b hemophilic bacillus antigens. For the first time, the authors experimentally selected sensitization conditions for obtaining a latex diagnosticum: 0.5 ml diluted with phosphate buffer 1:10 immunoglobulins of class G against antigens of hemophilic bacillus "b" at a concentration of 1.0 mg / ml were added to 2 ml of 1.5% latex (the final concentration of latex is 1.2%, the concentration of class G immunoglobulin is 0.4 mg / ml). For effective sorption of antibodies to latex particles, the following incubation mode was selected: on a shaking apparatus at a temperature of 18-20 ° C, for two hours, then at a temperature of 37 ° C for 18 hours.

The authors propose using a commercial standard preparation, Act-Hib vaccine, containing a certain amount of PRF (polyribosyl ribitol phosphate), the main specific component of the capsular material of the hemophilic bacillus, which specifically reacts with a specific class G immunoglobulin adsorbed on lathes, as a positive control. particles.

The proposed method is as follows. A patient who is admitted to a hospital with a suspected invasive hemophilic infection (purulent meningitis or sepsis of a hemophilic nature) is sampled from a blood serum or cerebrospinal fluid (CSF) sample, which is immediately sent to a bacteriological laboratory for diagnosis: simultaneously with bacteriological culture for isolation causative agent of Hib infection and formulation of the express method - latex agglutination reaction. The method of the express method, that is, the latex agglutination reaction, is as follows. RAL is carried out at room temperature (18-20 ° C). When examining one CSF or blood serum sample, 50 μl of diagnostic latex — latex sensitized with a specific class G anti-Hib immunoglobulin — is applied to a glass slide, to which 20 μl of the test material is added and mixed. After 1-3 minutes, the RAL result is taken into account. The time for setting up and recording RAL is 20-30 minutes from the moment samples are delivered to the laboratory. A sample containing Hib antigens (vaccine Act-Hib) serves as a positive control, and a sample of 1.2% suspension of unloaded latex is a negative control.

RAL results are taken into account visually using a 4-cross system:

4+ - the formation of large flakes of agglutinate on the enlightened background of a drop;

3+ - the formation of large agglutinate flakes on the background of a drop without enlightenment;

2+ - the formation of a distinct fine-grained precipitate throughout the drop;

1+ - weak grit throughout the drop.

- uniformly homogeneous milky white suspension, lack of agglutination (negative RAL).

The result is evaluated as positive in the case of 4+, 3+ and 2+ in the first and third drops, the result is evaluated as negative if the first drop retains a uniform milky-white color, identical to the drop with a control negative sample (second drop).

The inventive method can be confirmed by the following examples.

Example 1

Surname, name P. Darya

Paul F

Date of birth / age 07/09/02

No. of the disease history No. 6918

Date received 08/18/05 Diagnosis: generalized hemophilic infection

Liquor Blood Sowing Neg. H. influenzae "b" - culture LAR - according to the author's method Neg. H. influenzae "b" - antigens LAR - “Kit-5” Neg. Neg. Cytosis 2880/3 Formula n 95% Sugar 3.5 mmol / l Protein 0.33 g / l Gram stain Gram (-) sticks

As can be seen from the data (example 1), RAL was positive only with a sample of blood serum and was negative with a sample of cerebrospinal fluid. Thus, an early etiological diagnosis of a generalized infection (sepsis) of hemophilic etiology “b” was established based on the detection of hemophilic bacillus antigens “b” using RAL only in samples of the patient's blood serum. While RAL using the Kit-5 commercial latex diagnostic kit turned out to be negative both with a cerebrospinal fluid sample and a blood serum sample, which indicates the great effectiveness of the method of express diagnostics developed by the authors of other clinically significant forms of hemophilic infection, for example, generalized forms ( sepsis). The result is confirmed by the "gold" standard - the allocation of the culture of the pathogen N. influenzae "b" from the patient’s blood.

Example 2

Surname, name K. Ilya

Paul M

Date of birth / age 10/18/98

No. of medical history 3076

Date of receipt 02.04.05, Diagnosis: acute bacterial meningitis of a hemophilic nature

Liquor Blood Sowing H.influenzae "b" - culture Neg. LAR - according to the author's method H. influenzae “b” - antigens H. influenzae “b” - antigens LAR - “Kit-5” H. influenzae "b" - antigens Neg. Cytosis 50944/3 Formula n 98% Sugar 2.9 mmol / L Protein 0.66 g / l Gram stain Gram (-) sticks

As can be seen from the above data (example 2), an early etiological diagnosis was established on the basis of a positive reaction in the author's RAL, that is, on the basis of the detection of hemophilic bacillus antigens “b” in both the blood and cerebrospinal fluid of a patient with acute bacterial meningitis, the reaction was negative with a sample blood using a commercial kit-5 latex diagnosticum. The result is confirmed by the "gold" standard - the allocation of the culture of the pathogen N. influenzae "b" from the patient's cerebrospinal fluid.

Example 3

Surname, name K. Nastya

Paul F

Date of birth / age 04/23/00

No. of medical history No. 7994

Date of receipt 09/27/05

Diagnosis: generalized form of meningococcal infection

Liquor Blood Sowing N. meningitidis N. meningitidis LAR - according to the author's method Neg. Neg. LAR - “Kit-5” Neg. Neg. Cytosis 12544/3 Formula n 98% Sugar 3.0 mmol / L Protein 0.49 g / l Gram stain Gram (-) Diplococci

As can be seen from the above data (example 3), the latex agglutination reaction using the inventive method with blood serum and cerebrospinal fluid samples in a patient diagnosed with a generalized form of meningococcal infection established by isolation of a pure culture of N. meningitides turned out to be negative, which confirms the accuracy of early etiological diagnosis of Hib infection.

When developing the method, we used samples of cerebrospinal fluid (48 samples) and blood serum (38 samples) from sick children who were in the clinic of acute neuroinfections of the Research Institute for Children's Infections (St. Petersburg) in the period 2003-2005, with the diagnoses of “acute bacterial meningitis, meningoencephalitis and generalized infection of hemophilic etiology. " The results of RAL clinical trials testified to the high specificity of the author's RAL, since the% coincidence of positive results when comparing the standard (culture) method and RAL was 100%. In addition, the tests indicated a high sensitivity of the RAL. So, in eight negative samples of cerebrospinal fluid, obtained from patients with acute bacterial meningitis, and nine negative samples of blood serum from patients with sepsis, not confirmed by the cultural method (that is, without isolating the culture of the pathogen), hemophilic bacillus antigens were detected using only RAL " b ”, which made it possible to confirm additionally the etiological diagnosis of acute bacterial meningitis of hemophilic etiology in 16.7% of cases, generalized hemophilic infection in 17, 15% of cases (Table 1).

Table 1
The results of a comparative evaluation of methods for isolating the culture of hemophilic bacillus b, detection of antigens of hemophilic bacillus b in cerebrospinal fluid and blood using the author's RAL and commercial kit-5 latex diagnostic kit Clinical forms of hemophilic infection "b" Total cases confirmed by the isolation of the culture of hemophilic bacillus type "b" and in the RAL (the author and using the Kit-5 kit) All cases confirmed by the identification of antigens of hemophilic bacillus type type "b" only in the author's RAL The total number of cases confirmed by the identification of antigens of hemophilic bacillus type "b" only in the RAL using the kit "Kit-5" "Acute bacterial meningitis, hemophilic etiology meningoencephalitis" n = 48 cases, samples 40, 83.3% 8, 16.7% 8, 16.7% “Generalized hemophilic infection” (“sepsis”) n = 35 cases, samples 29, 82.85% 9, 17.15% - (Hib antigens not detected)

Thus, the assessment of specificity, sensitivity and accuracy, as well as the results of a comparative assessment of the results of RAL using the commercial Kit-5 kit, allow us to consider that the method developed by the authors can be recommended for widespread use in practical public health for making an early etiological diagnosis of Hib - infections. The method is characterized by high specificity, accuracy and rapidity and is suitable for establishing an early etiological diagnosis with other clinically significant forms of hemophilic infection "b".

Claims (1)

A method for the rapid diagnosis of hemophilic infection "b" in children by detecting antigens of the hemophilic bacillus "b" in the latex agglutination reaction, characterized in that immunoglobulin G is isolated from hyperimmune rabbit serum immunized with Act-Hib conjugated vaccine, which sensitizes 1,2 % suspension of monodispersed polystyrene latex particles (d = 0.8 mmk), then 50 μl of sensitized latex and 20 μl of cerebrospinal fluid or 20 μl of blood serum are applied to a glass slide, mixed by the presence or absence of the observed visual but agglutinates after 2-3 minutes with a positive reaction with a blood serum sample are diagnosed with a generalized form of hemophilic infection "b", with a sample of cerebrospinal fluid - acute purulent meningitis of hemophilic etiology "b".