RU2292551C1 - Early stage diagnosis method for determining prenatal infection in newborns - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves applying polymerase chain reaction diagnostic methods for detecting perinatal infection pathogenic agents in afterbirth. Preparation for detecting DNA- and RNA-containing pathogenic agents, is obtained as follows. 1.0x1.0 cm large placenta samples are taken from fetal membrane rupture places and in intervillous lacuna in the afterbirth center. The samples are placed into reservoir filled with sterile physiologic saline to be kept in storage not longer than 4 h at+4°C temperature. Then the samples are grinded and alkaline extraction is carried out with 200 mcl adding 200 mcl of solution containing 1.5 M NaCl, 0.3 M NaOH, 0.1% Sacrosyl solution and 0.5% sodium dodecylsulfate. The sample is left for 16-18 h at temperature of +4°C. Then the suspension is shaken, centrifuged at 5000 rpm during 5 min. The so produced suspension is transferred into test-tube having volume of 1.5 ml and extraction with chloroform and phenol and denaturation with ethanol is carried out.

EFFECT: high accuracy of measurements.

Description

The invention relates to medicine, more specifically to laboratory diagnosis of intrauterine infections in newborns.

Currently, there is a method for predicting the risk of intrauterine infection in a newborn, including the determination of inflammatory and dystrophic changes by histological methods in all parts of the placenta: in different parts of the placenta, villi and intervillous space, in the membranes of the placenta, in the placenta and its vessels, in the center of the placenta, on the periphery, on the maternal and fetal surfaces (RU No. 2168946). By the combination of revealed inflammatory-dystrophic changes in various structural parts of the placenta, the degree of risk of intrauterine infection in newborns is predicted (V.A. Tsinserling, V.F. Melnikova, 2002),

The known method has several disadvantages:

- investigated a large number of samples from different parts of the placenta;

- non-pathognomonic, nonspecific inflammatory-dystrophic changes are revealed, which can be caused not only by various pathogens, but also by other causes, for example, birth traumatic injury, metabolic disorders, etc .;

- assessment of inflammatory-dystrophic changes is subjective;

- histological examination of the placenta takes 2 from 10 to days.

The closest analogue prototype is a method for identifying pathogens of infections in autopsy material by setting up a polymerase chain reaction (PCR), in which pieces of tissue are weighed, 100 mg is ground in a mortar in liquid nitrogen to a powder. Then, to isolate RNA, the powder is transferred to a homogenizer and instructions for the isolation of RNA are followed. To isolate DNA, an equal volume (0.1 ml) of sterile physiological saline is added to the obtained powder, mix thoroughly and the required volume of material is selected according to the instructions for DNA extraction (Interlabservice Instructions for the collection, transportation and pretreatment of biopsy and autopsy material, Instructions for sets of reagents for the selection of nucleic acids, Moscow, 2004).

The disadvantages of the prototype are:

- the need for different types of sample preparation to isolate DNA-containing and RNA-containing viruses;

- increased safety measures when working with liquid nitrogen;

- taking a certain amount of autopsy material, namely 100 mg;

- increased costs for additional equipment associated with working with liquid nitrogen.

The objective of the invention is to offer an objective and quick way to predict the risk of intrauterine infection in newborns.

The technical result is the identification of specific causative agents of intrauterine infections by PCR in placenta samples taken in the membrane at the site of rupture and in the intervillous space in the center of the placenta.

The technical result is achieved as follows. For research, take 2 pieces of the placenta measuring 1.0 × 1.0 cm in two places. The first is at the site of rupture of the membranes, since this is the most likely place for the presence and reproduction of the pathogen with an ascending path of infectious agents entering the placenta and the fetus from the lower parts of the genital tract. The second sample is taken in the intervillous space in the center of the placenta, where the most active blood flow and the most likely presence of the pathogen that got into the placenta and fetus by the hematogenous route. Then both pieces are placed in a container with sterile saline that covers their surface. Samples are delivered to the laboratory within 2-4 hours at + 4 ° C and immediately begin processing. Storage of samples is not allowed, as this increases the loss of RNA due to degradation. Both pieces of the placenta are transferred from the container to the mortar, 1.0 ml of culture medium, for example, Needle MEM medium, is added so that, if necessary, part of the sample can be examined on tissue culture or bacteriological media. Then the pieces are crushed so that cell lysis, protein denaturation, neutralization and removal of impurities in the subsequent stages of sample preparation occur more successfully. Inadequate lysis leads to the fact that cell detritus masks specific DNA, reduces the likelihood of its contact with DNA polymerase and increases the possibility of false-negative results.

Then 200 μl of the crushed suspension is transferred into an Eppendorf type tube and 200 μl of an alkaline mixture of the following composition is added: 1.5 M NaCl, 0.3 M NaOH, 0.1% sarcosyl solution, 0.5% sodium dodecyl sulfate. Leave the sample for 16-18 hours at + 4 ° C. After this, the suspension is shaken in a microcentrifuge and centrifuged for 5 minutes at 5000 rpm. 200 μl of the supernatant is transferred into 1.5 ml Eppendorf tubes.

Then produce phenol-chloroform extraction and denaturation with ethanol. The result is a pure nucleic acid preparation suitable for PCR or from-PCR. Grinding, alkaline lysis and phenolic extraction can get rid of impurities in autopsy material rich in products of necrosis and autolysis of tissues, decay of nuclei and cells, impaired metabolism of proteins, lipids, enzymes and other PCR inhibitors. Thus, a high-purity preparation is obtained that is suitable for the effective identification of pathogens.

The resulting preparation is used to identify pathogens of perinatal infections, both RNA- and DNA-containing (e.g. Enteroviruses, Rubella virus, Herpes simplex virus I, II types, Cytomegalovirus, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticumt, Listeria mon ) by subsequent staging of amplification and detection of PCR products by horizontal electrophoresis. Next, the result is photographed and stored in the archive.

The advantages of the proposed method:

- reduction in the number of samples in the study: only 2 samples are taken at the sites of the most probable virus multiplication during the ascending and hematogenous pathways of the pathogen penetration into the placenta and fetus;

- high purity of the nucleic acid preparation, suitable for PCR, is achieved by the removal and neutralization of impurities, which are rich in autopsy material, by grinding, alkaline treatment, phenol-chloroform extraction and denaturation with ethanol;

- reduction of research time to 2 days;

- one technically simple method of sample preparation for PCR is used to identify a complex of RNA- and DNA-containing causative agents of perinatal infections, while reducing RNA loss is achieved by a short time interval between sampling and the beginning of sample preparation - no more than 4 hours;

- the ability to identify specific pathogens contributes to the adequate treatment of the newborn and appropriate rehabilitation therapy for the mother.

Example 1

A study of the placenta in a woman M., 30 years old, whose pregnancy proceeded with the threat of miscarriage at 14 weeks against a background of chronic pyelonephritis. The baby was born at 36 weeks, with a body weight of 2800 g. Two pieces of the placenta, 1.0 × 1.0 cm in size, were taken for examination. One was at the rupture of the membranes, the second was in the intervillous space in the center of the placenta. Samples were delivered to the laboratory on the same day and processed 2 hours after sampling. Both pieces were ground in a mortar, pre-treated with chrompeak and sterilized, with the addition of MEM needle culture medium. An equal volume of the alkaline mixture was added to 200 μl of the resulting suspension and left for 16 hours at + 4 ° C. After this, shaking and centrifugation were performed for 5 minutes at 5000 rpm. 200 μl of the supernatant was transferred to a 1.5 ml Eppendorf type tube, then phenol-chloroform extraction was carried out, the obtained nucleic acid preparation was used for amplification. Identification of RNA-containing viruses (Enteroviruses, Rubella virus) was performed using Isogen kits. Detection of DNA-containing pathogens (Herpes simplex virus I, II types, Cytomegalovirus, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Listeria monocytogenes) - sets of the company "Litech". In the test sample, Cytomegalovirus was detected. To confirm the results of PCR, the samples were studied by the fast culture method on a tissue culture of PEC. Identified and identified Cytomegalovirus. During histological examination of the placenta, inflammatory and dystrophic changes were recorded: fibrinoid deposits on the surface of the villi, pseudo infarcts, calcifications, dissociated maturation of the villi, dystrophic changes in the stroma of the villi, in the vessel walls. According to the results of PCR diagnostics and histological examination, it was concluded that there is a high risk of intrauterine cytomegalovirus infection. The newborn was expressed petechiae, jaundice, impaired liver function. Recommendations are given on transferring a child from the maternity hospital to the neonatal pathology unit with treatment, including the use of anticytomegalovirus immunoglobulin, viferon, and other appropriate treatment.

Example 2

Samples of the placenta in a woman K., 24 years old, were studied. The first pregnancy was with the threat of miscarriage at 24 weeks and gestational pyelonephritis. The baby was born at 39-40 weeks old with a body weight of 3140 g. The placenta was studied by the NDP method as described in Example 1. Ureaplasma urealyticum was found in the test sample. An histological examination of the placenta revealed inflammatory-dystrophic changes: a fibrinoid, pronounced dystrophic changes in the stroma of the villi, in the walls of blood vessels, and a fibroblastic reaction. Based on these data, a conclusion is drawn about a high risk of intrauterine ureaplasma infection in a child. In the neonatal period, the following were identified: intrauterine growth retardation by the hypotrophic type, a long adaptation period, perinatal encephalopathy, neuromuscular dystonia syndrome and vegetovisceral disorders. Recommended treatment with antibiotics of the macrolide group, for example, rovamycin.

Claims (1)

A method for early diagnosis of intrauterine infections in newborns, consisting of a PNR diagnosis of causative agents of perinatal infections in the placenta, characterized in that placenta samples 1.0 × 1.0 cm in size are taken at the site of rupture of the membranes and in the intervillous space in the center of the placenta, placed in a container with sterile physiological saline, stored for no more than 4 hours at 4 ° C, after which the samples are crushed, 200 μl are taken and alkaline extraction is carried out, adding 200 μl of a solution containing 1.5 M NaCl, 0.3 M NaOH to the suspension , 0.1% sarcosyl solution, 0.5% dodecyl sodium sulfate, leave the sample for 16-18 hours at 4 ° C, then the suspension is shaken, centrifuged at 5000 rpm for 5 minutes, the resulting suspension is transferred to a 1.5 ml tube and chloroform-phenol extraction and denaturation with ethanol are carried out.