Method for detection of most significant pathogens of non-gonococcus urogenital infections (chlamydia trachomatis, mycoplasma hominis, ureaplasma urealyticum, cytomegalovirus, herpes simplex i/ii) in children by using method of polymerase chain reaction (pcr)

FIELD: medicine, infectious diseases.

SUBSTANCE: invention relates to laboratory diagnosis of non-gonococcus urogenital infections in children. Method involves assay in children of first years of life the following pathogens of urogenital infections: Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Cytomegalovirus, Herpes simplex I/II by PCR method. Method involves simultaneous assay in mixture of 4 substrates: urine, saliva, blood and lacrimal secretion taken from a single patient. Advantage of invention involves enhancing precision in detection of pathogens.

EFFECT: improved detecting method.

2 ex

 

The invention relates to medicine, in particular for laboratory diagnosis of infectious diseases, specifically nongonococcal infections.

Nongonococcal infections (NOUGAT) group clinically diverse diseases of bacterial and viral etiology, affecting mainly the organs of the urinary system. A person's susceptibility to NOUGAT high - close to 100%. Diseases are characterized by the tendency to chronicity and persistence. Strong immunity is not produced. Currently, the weakening of the immune status of the organism associated with environmental pollution, stress pressure, NOUGAT becoming more relevant and socially significant. Clinical manifestations are diverse, it is difficult to trace the connection of certain types of pathogens with specific forms of pathology. Women pathogens NOUGAT cause inflammatory processes, such as urethritis, cystitis, vaginitis, colpitis, endometritis, salpingitis, oophoritis[3, 4, 7].

The essential influence of NOUGAT on the reproductive function. During pregnancy pathogens NOUGAT activated and can cause spontaneous abortions and premature births. Some of the possible consequences of genital inflammation are disorders of ovulation, fallopian tubes that m which can lead to infertility [5, 9].

One of the major problems associated with NOUGAT, are of intrauterine infection. Female patients and carriers of pathogens NOUGAT - in 40-60% of cases transmit infection to newborns. Infection of the fetus may occur in utero or during passage through the birth canal and depends on the location and severity of the inflammatory process. The sooner there is an infection of the fetus, the more significant may be the pathological changes. In utero infection by pathogens NOUGAT affects the respiratory system, eyes, kidneys, liver, Central nervous system, which can even become a cause of fetal death [6, 12].

In children with intrauterine infection history can develop diseases of the respiratory system infectious-allergic nature: obstructive bronchitis, asthma, juvenile arthritis, liver and spleen, polylithionite, accompanied by prolonged fever [8].

The analysis of literature data and the results of their research allowed to determine the range of the most common and important pathogens of NOUGAT - Clamydia trachomatis. Garbage lasma hominis, Ureaplasma urealyticum, Cytomegalovirus, Herpes symplex I/II.

It is advisable to examine this range of infectious agents newborns and children in the first year of life with prematurity, malnutrition, anemia, defeat TSN is, by hepatosplenomegaly, jaundice of unknown origin, as well as children with suspected intrauterine infection and children born to mothers infected with pathogens NOUGAT.

In these risk groups noted the following incidence of certain types of pathogens NOUGAT: Cytomegalovirus - 53,6%; Ureaplasma urealyticum, and 22.6%; Mycoplasma hominis - 19,0%; Clamydia trachomatis - 3,5%; Herpes symplex I/II types- 2,8% [6, 12].

Clamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Cytomegalovirus, Herpes symplex I/II types are contaminants and parasites mucous membranes. They can be present in almost any biological liquids of an organism.

Traditional objects for the detection of pathogens NOUGAT children are urine, saliva, blood, tear detachable. The frequency of detection of infectious agents in a variety of different substrates. Often agents NOUGAT logged in urine and saliva, often in the lacrimal discharge and significantly less in the blood. Typically, infected is one of the substrates, in 10-15% of cases the causative agent detected in two substrates (urine - saliva; saliva, lacrimal detachable), in a few cases infected with three objects.

The most promising method for the detection of pathogens NOUGAT is a method of polymerase chain reaction (PCR). Currently, it is widely used in practical diagnosis of infectious diseases. The advantages of PCR PE the ed classical methods are especially evident in the diagnosis of infections caused by natural or trudnosorbiruemye in laboratory conditions by microorganisms. To this group of infectious agents include agents NOUGAT.

PCR due to the multiple increase (amplification) unique DNA fragment of the desired pathogen allows to achieve high sensitivity and identifies 10-100 cells or virions in a sample.

The use of hybridization of nucleic acids as a starting stage for PCR makes it possible to achieve high specificity (100%) and to avoid false-positive results. Unlike microbiological methods, PCR does not require obtain pure cultures of pathogens. Explore directly assay from patients: biopsies, mucosal scrapings, blood, urine, saliva, feces, and other PCR automated using programmable thermostatic devices (thermocycler, amplifiers). This reduces analysis time, reduces its complexity. Using PCR method allows to get the result within one working day (6-7 hours) [11].

In the analyzed scientific literature devoted to the examination of children on pathogens NOUGAT PCR, similar studies conducted in either one of the substrates, or analyze multiple substrates separately.

Presented as options prior Uro is nya equipment the following works: - publication [2].

In this work were selected strokes with eyes, smear processed (identified nucleic acid and PCR revealed Chlamydia trachomatis. As a target for detection used a fragment of the 16S RNA gene. In the group of patients with clinical signs of trachoma chlamydia detected in 74.2%of patients without clinical symptoms of 3.4%. The authors concluded that this method is good for the diagnosis of ocular chlamydia because of the excellent sensitivity and specificity.

Publication [1]

In this study we investigated blood cells, urine, swabs from the throat (separately) for the presence of cytomegalovirus. We detected viral DNA by PCR and mRNA late viral antigen RT-PCR. In patients with clinical symptoms of CMV infection (current cytomegalovirus infections) viral DNA was detected significantly more frequently (100%)than in the group immunodeficient children without symptoms of current cytomegalovirus infections (25%). Messenger RNA of CMV were detected in all patients with severe, current cytomegalovirus infections not detected in children who do not have symptoms of the infection.

Both of these approaches have their drawbacks. In the study, only one object is a high probability of false-negative results. The infectious agent is not detected because it is not present in this substrate, but in this case it is different. The study of various objects separately on a full range of vozbuditelyu vysokozatratno and increases the duration of the study. Thus, for studies of urine, saliva, blood, tear detachable 5 infectious agents requires 4 prosopographic and 20 PCR assays.

The aim of the invention is to increase efficiency, streamlining and reducing the cost of complex (5 most important pathogens of NOUGAT) PCR screening of newborns and children during the first years of life.

In our proposed version of the study identification of pathogens NOUGAT is carried out in a mixture of substrates (urine, saliva, blood, tear detachable) from the same patient.

When preparing the mixture for the PCR detection of pathogens NOUGAT take 4 of the substrate from one person, while the dilution effect, in comparison with polerowanie negligible. A comparative study of a sample of 50 children who have analyzed the mixture and the substrate separately, was not received false-positive results when processing biological mixture.

The method is as follows.

Example 1.

From the patient under examination taken in separate disposable plastic tubes, urine, saliva, lacrimal discharge, blood; preparing a mixture of substrates containing 50 μl of each of them.

To 200 μl of the mixture add 600 ál lyse solution, 15 μl of the internal control sample, 30 μl of the sorbent. The duration of the lysis at 65° - 8 minutes.

For vyd the population of nucleic acids from a mixture of substrates can be used the classical phenol-chloroform method [10]. The most technologically advanced option for sample preparation are sorption technologies for extraction of DNA and RNA.

We spent processing the mixture of substrates set of reagents "DNA-Sorb B" development and production and the Central research Institute of epidemiology public health Ministry. When using this kit reagents after treatment is not observed inhibition of PCR.

The next stage of washing is carried out in accordance with the instruction to "DNA-Sorb B".

For PCR detection Clamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Cytomegalovirus, Herpes symplex I/II use test systems series "AmpliSens" development and production of the Central research Institute of epidemiology health Ministry according to the instructions or similar systems of other companies.

Detection of the amplification product is performed by the method of agarose gel electrophoresis [10]. Visualization electrophoregram carried out using the system for image processing "Biotest".

Example 2.

Because the probability of detecting pathogens NOUGAT in the blood are small (less than 1%), when carrying out practical research can be limited to the analysis of a mixture of urine, saliva, lacrimal discharge. These substrates are mixed in 50 μl.

To handle this variant mixture, set "DNA-Sorb And" production znii health Ministry. It has a lower cost, the procedure of using this set easier and less will continue the flax. If no mixture of blood given sufficient sample preparation, PCR inhibition was not observed.

To 150 μl of the mixture add 450 ál lyse buffer, 11 ál of internal control and 22 μl of the sorbent, the time of lysis at 65° - 8 minutes Subsequent stage of washing is carried out in accordance with the instruction to "DNA-Sorb.

For PCR detection Clamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Cytomegalovirus, Herpes symplex I/II use test systems series "AmpliSens" development and production of the Central research Institute of epidemiology health Ministry according to the instructions or similar systems of other companies.

Detection of the amplification product is performed by the method of agarose gel electrophoresis [10]. Visualization electrophoregram carried out using the system for image processing "Biotest".

A positive effect.

Simultaneous PCR detection of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Cytomegalovirus, Herpes symplex I/II allows to detect most of the atypical pathogens, occurring in infants and children during the first years of life.

According to our results, the frequency of detection of bacteria of the genera Mycoplasma, Chlamydia, viruses of the group of herpes in children amounted to (collectively): blood - 1-5%; detachable tear - 8-10%; urine - 20-25%; in the saliva - 30-37%; mixture of substrates is 48-50%. The use for research of the mixture, and not one of the substrates increases e is the efficiency of detection of pathogens NOUGAT 25-30%.

The proposed testing algorithm can significantly reduce costs, this is due to the fact that at the first stage investigated the mixture, and not substrates separately. The presence of the researcher all 4 substrates optionally allows you to specify what the material presence of microorganisms identified in the first stage. The saving effect is due to the fact that in the second stage, the individual substrates are investigated not on 5 infectious agents, and those that are identified in the mixture of substrates in a mixture in 68% of cases detected monoinfection, the Association of the 2 pathogens found in 27% of cases and only 5% of the Association of 3 or more pathogens). Thus, when the savings value of the examination is not reduced.

When PCR study of mixtures of substrates from 554 children (newborns, children in the first year of life, children from 1 year to 7 years) at the same time on 5 types of pathogens NOUGAT was identified 279 (50.5%) and infected. At 190 (34,3%) of them revealed 1 infectious agent, 74 (13,4%) - Association of 2 infectious agents, 11 (2,0%) - 3, 1 (0,2%) - 4.

To study mixtures of substrates was conducted 554 sample preparation and 2770 PCR studies. Clarify which of substrates with was not conducted. During this phase of the work additionally, it would take more 1116 prosopographic and 1500 PCR studies. For about the westline expanded research 554 children using the technology mixes must 1670 prosopographic and 4270 PCR studies. For similar work in the study of 4 substrates separately would 2216 of prosopographic 11080 and PCR studies.

Therefore, when working with a mixture of substrates from 554 patients (detailed analysis) be conducted at 546 prosopographic and 6810 PCR reactions less than in the study of the same number of patients according to the traditional scheme.

Our experience in conducting practical tests have shown that for a diagnosis and deciding on the appropriate therapy doctor, as a rule, sufficient study of a mixture of substrates. In some cases, severe clinical picture, additionally investigate the blood. In this embodiment, detection of pathogens NOUGAT effect of saving even more.

Literature

1. Nishihara H, Ito M, Matsumoto N, Nakano T, lhara T, Kamiya H, Sakurai M. Detection of human cytomegalovirus DNA in are immunocompromised children by polymerase chain reaction.// Clin Diagn Virol. - 1995 - v.3 - No. 1 - s-81.

2. Zou J, Liu S, Xiao W, Yang X. Evaluation of polymerase chain reaction for detection of Chlamydia trachomatis in eye swabs/.// Yan Ke Xue Bao - 2000, - v.16 - 124-126.

3. Granite NR. The chlamydia. M: Honey. book. - 2000. - 192 S.

4. Granite NR. Herpes virus infection. M: Honey. book. - 2001. - 80 S.

5. Dmitriev G.A. Modern methods of diagnosis of the most common sexually transmitted infections. Consilium medicum. - Dermatology, venereology. - 2002. No. 4. - s-259.

6. Evsukova I.I., Patrus the VA E.N., Savicheva A.M. and other Topical issues of the clinic, diagnosis and treatment of chlamydial infection in newborn infants.//Obstetrics and gynecology. - 1995. No. 1. - s-36.

7. Kozlov V.I., Puchner A.F., Viral, chlamydial, mycoplasmal diseases of the genitals. M. - 1995. - 261 S.

8. Konopleva T.V., Lukashkin E.F., V. Mazepa. and other Use of polymerase chain reaction in the diagnosis of mycoplasmal infections in young children.// In the collection of scientific. "The health of the population of Nizhny Novgorod region". Nizhny Novgorod. - 2002, - p.39-43.

9. The Moors I.I. Violation of reproductive function in patients with urogenital clamidiosis and a ureaplasmosis.//Journal of dermatology. - 1992. No. 11, p.72-75.

10. Maniatis T., Fritsch E., Sambrook J. Molecular cloning. - M.: Mir, 1984. - 480 S.

11. Medical and laboratory technology. The Handbook. Vol.2. Ed. Karpischenko A.I. - St. Petersburg. - Intermedica. - 1999. - 654 S.

12. Simakova MG Intrauterine infection.// Sbornik nauch. Tr. - M., - 1994., - s-57.

The method of examination of children on the most significant pathogens of nongonococcal infections using PCR method, characterized in that the study on identification of pathogens (Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Cytomegalovirus, Herpes symplex I/II) subjecting the mixture of the four biological substrates - urine, saliva, blood, tear detachable from one p is they.



 

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