RU2247383C2 - Method for serological diagnosis of chlamydium conjunctivitis - Google Patents

Abstract

FIELD: medicine, ophthalmology, immunology.

SUBSTANCE: invention relates to a method for immunological diagnosis of chlamydium conjunctivitis. Method involves the simultaneous determination of antibodies of classes IgA and IgG in serum blood against chlamydium antigen by immunoenzyme assay in conjunctivitis coarse 10-120 days. Conjunctivitis of chlamydium etiology is diagnosed in the concentration of IgA from 0.600 to 1.800, and in the concentration of IgG from 0.800 to 2.700 U of optical density. Invention provides enhancing precision and reducing time for carrying out the diagnosis.

EFFECT: improved method for diagnosis.

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Description

The invention relates to medicine, namely to ophthalmology, and can be used to diagnose chlamydial conjunctivitis.

A known method for serological diagnosis of ophthalmic chlamydia by enzyme-linked immunosorbent assay, based on the detection of serum antibodies to chlamydial antigens of classes Ig M, Ig A and Ig G. Determination of their concentrations in the blood allows to establish the chlamydial etiology of the disease, its course (acute, chronic, chronic with relapses ) In the interval between 5 and 20 days after the onset of clinical symptoms of the disease, antibodies of these three classes consistently arise against the chlamydia antigen. Ig M is a marker of the acute stage, it is determined within 5 days after the onset of the disease. Within 10 days, Ig M changes to Ig A; for a short time they are present simultaneously. At the same time, or with a slight delay of 2-3 weeks, Ig G can be determined. The progression of the disease, its transition to a chronic form, is characterized by the appearance of antibodies of class Ig A and Ig G.

A standard approach in the serological diagnosis of chlamydial infection of any localization is a two-time examination of the patient with an interval of 2-3 weeks and, according to a 4-fold or higher definition of antibodies of class Ig A and G in the blood serum against the chlamydia antigen, a chlamydial etiology of the disease is established. However, repeated studies after 2-3 weeks significantly extend the diagnostic time, and the determination of the concentration of specific antibodies by titration makes them expensive.

The closest in technical essence is the method of serological diagnosis of urogenital chlamydia by enzyme-linked immunosorbent assay (ELISA), which consists in a single quantitative quantitative determination of all three classes (Ig A, G and M) of antibodies against chlamydia antigen. Moreover, in the case of primary chlamydial infection, this method provides two options: the first is Ig G in the titer <100, Ig A <50 or ≥50, and Ig M≥50; the second is Ig G≥100, Ig A≥50 and Ig Mi50. For the diagnosis of chronic (protracted) chlamydial infection, its relapse or reinfection, Ig G400, Ig A≥200 and Ig M <50 are determined. This method has significant drawbacks. So, specific antibodies of the Ig M class exist for a short time - from the 5th to the 10th day of the disease, which does not allow them to be determined in patients visiting a doctor, as a rule, at a later date. The method does not allow to determine the optimal timing of the disease, in which it is necessary to conduct it. It has a high cost due to the quantitative determination of the level of antibodies by titration and provides for the diagnosis of only chronic forms and relapse of this infection. The effectiveness of this method is also low (M.A. Bashmakova et al., 2000. 68 p.).

The technical result of the invention is to increase the frequency of diagnosis of chlamydial conjunctivitis, reducing the number of false-positive reactions and reducing the duration of the study.

The technical result is achieved by the method of serological diagnosis of chlamydial conjunctivitis, which consists in the determination of two classes of antibodies against chlamydial antigen in the blood serum of patients using enzyme-linked immunosorbent assay, characterized in that antibodies of classes Ig A and Ig G against chlamydial antigen in the blood serum are determined simultaneously by enzyme-linked immunosorbent assay the duration of conjunctivitis is 10-120 days and within the threshold concentration of Ig A from 0.600 to 1.800 and Ig G from 0.800 to 2.700 in units of optical density dissolved chlamydial conjunctivitis etiology.

The method is as follows. In a patient with conjunctivitis, fasting blood is taken in an amount of 5.0 ml from a vein into a sterile tube.

Then, the necessary reagents provided for this method are added by automatic pipette. IFA is carried out on an Uniplan automatic photometer (Russia).

Diagnostic principle: antibodies of classes Ig A and Ig G against the Chlamidia trachomatis antigen present in the patient’s serum bind to chlamydia antigens adsorbed on the surface of the polystyrene plate wells, forming an “antigen-antibody” complex labeled with the enzyme. And the last process in ELISA is the transformation of the enzyme label into the corresponding signal, measured using a spectrophotometer. Visually positive samples are stained yellow. ELISA results are recorded using a spectrophotometer, measuring the optical density at a wavelength of 450 nm. And when determining antibodies against the chlamydia antigen at a concentration of Ig A from 0.600 to 1.800 and Ig G from 0.800 to 2.700 in units of optical density in the patient's blood serum obtained from 10 to 120 days of the disease, conjunctivitis of chlamydial etiology is diagnosed.

Using the developed method, a study was carried out of the blood serum of 43 patients with chlamydial conjunctivitis (the main group) in terms of the disease from 10 to 120 days. The control group consisted of 18 healthy individuals. The results are presented in the table.

Table
Serological diagnosis of chlamydial conjunctivitis using ELISA Study groups Number of observations Chlamydia antibody detection rate Ig A (0.6-1.8 in units of OD) Ig G (0.8-2.7 in units of OD) Ig A + Ig G abs. hours % abs. hours % abs. hours % Main 43 27 62.8 34 79.0 32 74,4 Control 19 3 15.8 6 31.6 1 5.2

In 27 (62.8%) of the 43 examined patients with chlamydial conjunctivitis, antibodies of class Ig A were detected in blood serum, and in 34 (79.0%) - of class Ig G to chlamydia. At the same time, these antibodies were detected in 32 patients within the threshold values of their concentrations noted above, which laboratory confirmed the clinical diagnosis of chlamydial eye infection in 74.4% of cases, increasing the frequency of diagnosis. In addition, the proposed method reduced the number of expected false-positive results to 5.2%, since in healthy individuals individually Ig A and Ig G against chlamydia antigen are determined respectively in 15.8 and 31.6% of cases and together in 5.2 % The study lasted for 2-2.5 hours, which is significantly shorter compared to 2-3 weeks required for the study of paired sera.

As can be seen from the studies, it is not possible to diagnose chlamydial conjunctivitis separately for one of the two antibodies to the chlamydial antigen due to their occurrence in healthy individuals (Ig A in 15.8%, Ig G in 31.6% of cases), and in case of simultaneous detection of antibodies to the chlamydia antigen of both classes (Ig A and Ig G) is much less likely false-positive results (in 5.2% of cases). It should also be noted that among patients with conjunctivitis of unclear etiology who applied to the clinic of the Ufa Research Institute of Eye Diseases, the developed method diagnoses chlamydial conjunctivitis in 42.8%, which justifies the implementation of appropriate therapeutic and preventive measures.

Example 1. In one of the hospitalized patients with conjunctivitis in the department for the study of infectious diseases of the eyes of the Ufa Eye Research Institute of Eye Diseases on the 10th day of the disease, when carrying out an enzyme-linked immunosorbent assay of a patient's serum, the Ig A antibody values were 0.899, and the Ig G class value was 0.920 units of optical density. In accordance with the developed method, chlamydial conjunctivitis was diagnosed. The correct result of the study was also confirmed by the detection of chlamydia DNA in a conjunctival scraping by PCR. The diagnostic study lasted only 2 hours, which is much shorter than other known serological diagnostic methods.

Example 2. In the office of infectious eye diseases of the Ufa Research Institute of Eye Diseases, one of the patients with conjunctivitis on the 90th day of illness took blood from a vein. When conducting an enzyme immunoassay, the values of the antibodies against the Chl antigen. trachomatis of class Ig A amounted to 1,544, and class Ig G - 1,683 in units of optical density and chlamydial conjunctivitis was detected in the patient.

Example 3. In the eye cabinet of the municipal clinic of Ufa, one of the patients with conjunctivitis of unclear etiology took blood on the 7th day of illness. Chlamydial conjunctivitis was not diagnosed, because in accordance with the developed method, when conducting an enzyme-linked immunosorbent assay, the values of antibodies to the chlamydia IgA class antigen were 0.350, and that of class Ig G 0.420, which did not correspond to the threshold values of their levels according to the proposed method.

Thus, the proposed method makes it possible for a short time to laboratory diagnose chlamydial conjunctivitis in 74.4% of cases, provided that two classes of antibodies to the chlamydial antigen are simultaneously determined within the threshold values of their concentration (Ig A from 0,600 to 1,800 and Ig G from 0,800 to 2,700 in units of optical density) during the disease from 10 to 120 days. Due to the simplicity of formulation and accessibility, this method is recommended for widespread implementation in medical institutions.

Claims (1)

A method for serological diagnosis of chlamydial conjunctivitis, which consists in determining antibodies against the chlamydial antigen in the blood serum of a patient by enzyme-linked immunosorbent assay, characterized in that antibodies of classes Ig A and Ig G against the chlamydial antigen in the blood serum are determined simultaneously by enzyme-linked immunosorbent analysis in conjunctivitis periods of 10-120 days and within the threshold values of the concentration of Ig A from 0.600 to 1.800 and Ig G from 0.800 to 2.700 in units of optical density, conjunctivitis of chlamydial etiology is diagnosed.