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12МЕДИЦИНСКИЕ НАУКИ
MODERN LABORATORY DIAGNOSTICS OF M. PNEUMONIAE Kudratova Z.E.1, Muhamadieva L.A.2, Rustemova Z.R.3, Kuvandikov G.B.4
1Kudratova Zebo Erkinovna - Assistant, DEPARTMENT OF CLINICAL LABORATORY DIAGNOSTICS;
2Muhamadieva Lola Atamuradovna - Doctor of Medical Sciences, Head of the Department, DEPARTMENT 3 OF PEDIATRICS AND MEDICAL GENETICS;
3Rustemova Zarema Ridvanovna - Assistant; 4Kuvandikov Golib Berdirasulovich - Assistant, DEPARTMENT OF CLINICAL LABORATORY DIAGNOSTICS, SAMARKAND STATE MEDICAL INSTITUTE, SAMARKAND, REPUBLIC OF UZBEKISTAN
Abstract: according to literature and scientific publications, M. pneumoniae infection in children is one of the separate topics for discussion. Despite the progress achieved in the study of mycoplasmosis of the respiratory system by identifying the characteristics of the pathogen and the features of its inflammatory process, there are a number of problems associated with the diagnosis of mycoplasma infection (MI) in children. Keywords: research methods, atypical infections, hemagglutination reaction, mycoplasma infection.
First of all, it should be noted that the clinical manifestations of myocardial infarction are nonspecific, they have become habitual for clinicians. When confirming the diagnosis, giving priority to laboratory methods of diagnosing MI, we often encounter ambiguous results that raise new questions. The multitude of research methods used to diagnose atypical infections and having different levels of sensitivity and specificity, as well as the lack of standardized diagnostic approaches, make it difficult to compare the results obtained from scientific research and do not always meet the practical needs of clinicians. To diagnose MI, the bacteriological method is not used in routine practice due to the need for special media for cultivation and the duration (2-3 weeks) of growth pathogen. Methods for detecting antibodies (AT) to M. pneumoniae, such as growth inhibition test (RIR), metabolic inhibition test (RIM), mycoplasmicide test and complement fixation test (CFT), also do not find practical application due to the mandatory use of living cultures of mycoplasmas [1]. The most informative methods of laboratory diagnostics of MI are recognized as the reaction of immunofluorescence (RIF) and molecular genetic methods (PCR diagnostics), as well as the determination of specific antibodies in the study of paired sera using enzyme immunoassay (ELISA) or indirect hemagglutination reaction (RNGA) [1].
Direct detection of the pathogen, in addition to the bacteriological method, can be carried out by detecting the M. pneumoniae antigen using RIF, for which test systems based on monoclonal antibodies have been created. This method has high specificity and sensitivity for detecting mycoplasmas in any clinical material, incl. in nasopharyngeal washes [1-3]. However, RIF may not be available to a wide range of diagnostic laboratories. In addition, this study is time-limited.
It is advisable to detect the mycoplasma antigen in the first 10 days of the disease, since at a later date it can be included in the circulating immune complexes [1]. The direct methods of rapid diagnosis of infections include the molecular genetic method (PCR diagnostics), aimed at identifying the DNA of the pathogen. This method has high specificity and sensitivity. And allows the detection of M. Pneumoniae from the first days of the disease. A negative result of PCR diagnostics in most cases excludes infection, provided that the rules for obtaining biological material are observed and that the test controls the
absence of inhibition of the reaction by components of biological samples. However, violation of the rules for organizing the laboratory and conducting research can lead to a false-positive result when the laboratory premises are contaminated (contaminated) with DNA amplification fragments.
The development and improvement of methods of molecular genetic diagnostics and the use of PCR with hybridization-fluorescent detection of the pathogen DNA in real time, which provides maximum sensitivity, specificity and contamination safety, makes it possible to diagnose with a certain reliability acute MI. In this case, the PCR study allows detecting the DNA of the pathogen within the first 3 weeks (1-21 days) from the onset of the disease [4] and even after the start of antibacterial therapy. Nevertheless, at the present stage, the widespread practical application of PCR diagnostics is limited by the high level of costs associated with the need for special equipment of laboratories, the purchase of expensive equipment and reagents. For this reason, in modern foreign guidelines for the management of community-acquired pneumonia (CAP) in children, the PCR method for detecting atypical infections is not included in the number of mandatory ones, in contrast to the methods of serological diagnostics aimed at detecting specific antibodies in paired blood sera [5, 6]. It should also be borne in mind that the PCR study does not allow differentiating an acute infection from the persistence of the mycoplasma pathogen, the prerequisites for which are laid down by the ability of mycoplasmas to attach and parasitize on the cell membrane of human cells with the help of tip-organelles, becoming inaccessible to AT, complement and other protective factors during localization of host cell membranes in invaginates, as well as the ability of M. Pneumoniae to suppress phagocytic activity of cells owner. For the serological diagnosis of atypical infections, most practical laboratories use ELISA, which is convenient due to the availability of domestic test systems and the possibility of a quick response, or RNGA [7, 8].
A sign of acute infection is the presence of antibodies of the IgM class, which can be detected starting from the 5-7th day after the first symptoms of the disease appear. By the 6th week of the disease, IgM antibodies are usually not detected.
Antibodies of the IgA class appear from the 2nd week of illness, but disappear faster than antibodies of other classes. IgG-antibodies are recorded at 2-3 weeks from the onset of the disease at the maximum level of antibodies of the IgM class and can persist at a low level for a long time. Since after the transferred MI, stable immunity is not formed, cases of reinfection are possible.
However, during mycoplasma reinfection, which, like an acute infection, requires antibacterial therapy, IgM antibodies are not produced. False negative results of the serological method of research can be reduced by examining the blood serum obtained after the 7th day from the onset of the disease. At the same time, the possibility of a false positive result is not excluded for two reasons. There is a possibility of nonspecific positive results due to cross-over values with normal human antibodies [1, 6], and also the likelihood of asymptomatic M. pneumoniae infection. In a study by R. NirPaz et al. registered the presence of IgM-AT in 20% of apparently healthy children of primary and secondary school age without signs of respiratory infections within 30 days [9]. There are reports of the detection of antibodies specific to M. pneumoniae during serological examinations of apparently healthy adults [2]. The use of paired sera, the first of which is taken in the earliest stages of the disease, and the second - after 10-14 days, helps to solve the listed problems. An increase in AT titers by 4 or more times in paired sera or seroconversion are considered diagnostically significant for MI [1, 3]. The sensitivity and specificity of ELISA when using paired sera reaches 60-80 and 90100%, respectively [4, 5, 10]. However, the serological method of research using paired sera, increasing the reliability of MI diagnosis, is of greater importance for its retrospective diagnosis in CAP. Setting ourselves the task of detecting antibodies
specific to M. pneumoniae, starting from the 6th day of illness, we could not fulfill this condition, since we found ourselves in a situation typical for outpatient practice [7,8].
Diagnosis of M. pneumoniae and C. pneumoniae infections rarely begins from the first days of the disease due to late referral of patients due to the peculiarities of the course of atypical infections. The reason for the study, as a rule, is a lingering cough or lingering obstructive bronchitis, which does not have a convincing connection with allergies, or the lack of effect of the prescribed anti-bacterial therapy for pneumonia [9].
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