RU2310851C1 - Method for diagnosing intrauterine viral infection in babies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves carrying out morphological study of placenta, determining IgM and IgG antibodies in mCBR reaction, analyzing retroplacentary blood, baby and mother blood, additionally determining viral antigens, specific immune complexes with antigens and specific low-avidity antibodies of IgG₃ and IgG_1-2.

EFFECT: high accuracy and specificity in examining mother and baby; enhanced effectiveness in selecting specific antiviral and immunomodulating therapy.

Description

The invention relates to medicine, namely to the diagnosis of intrauterine viral infections in young children. Early detection of perinatal infection allows for dispensary monitoring of children and timely therapy.

Among the risk factors for the pathology of pregnancy and the birth of children with severe psychosomatic disorders, one of the first places belongs to viral infections, especially those prone to a latent-chronic course, such as herpesvirus infections. There is an increase in the frequency of IUI caused by herpes viruses (herpes simplex virus type 1 and 2 (HSV 1/2), Ebstein-Bar virus, human cytomegalovirus (CMV), influenza viruses, rubella, B19 parvoviruses.

Identification of intrauterine infection of children with various viruses is very important. About 500 viruses are known that can infect the human body and cause acute and persistent forms of the infectious process. The immune rearrangement that occurs in the woman’s body during pregnancy creates the conditions for the activation of latent infection, which increases the likelihood of intrauterine infection of the fetus. Viral infections are especially dangerous during pregnancy, as they can cause a serious infectious process, leading to disability or mortality in newborns. Indicators of a high risk of IUI are acute viral infection or an exacerbation of chronic latent viral infection during pregnancy.

The frequency of intrauterine infection with various viruses, their specific tropism to immunocompetent cells, the ability to induce immunosuppression and persist for a long time in the body of children, manifested in the form of a variety of pathologies, is the basis for the development of methods for determining the risk of intrauterine infection of a child based on a virological study of retro-placental blood for clinical examination of children with intrauterine viral infections and timely prescription of antiviral and immuno-landmark ovannoy therapy.

Despite the fact that the effect of many viral infections on the mother-placenta-baby system is well described, perinatal infection with certain viruses, such as the Ebstein-Barr virus (EBV), remains poorly understood. The development of methods for more accurate, specific and timely diagnosis of intrauterine infection is very relevant at the present time.

Known methods for the diagnosis of intrauterine viral infections using morphological methods. So, in the work of Zinserling V.A. and Melnikova V.F. "Perinatal infections. Issues of pathogenesis, morphological diagnosis and clinical and morphological comparisons." Practical Guide. St. Petersburg: “Elby St. Petersburg” - 2002 shows the data of pathomorphological investigation of the afterbirth, carried out using morphometric, macroscopic and microscopic methods. The authors described the morphological features of various viral placentitis, developed criteria for assessing the risk of intrauterine infection in children based on examination of the placenta. Despite the fact that the morphological examination of the placenta shows structural changes in the placenta, however, this method has a number of significant drawbacks: a morphological study provides only a qualitative assessment of the changes in the placenta, which does not provide a specific analysis of virus damage, does not always determine the implementation of the infection in the child due to the function of the placenta, in addition, this method depends on certain conditions, such as the experience and skill of the morphologist, the quality of the equipment used to conduct the study, Akim, all this does not ensure the accuracy and specificity of the diagnosis.

Closest to our proposed method is a method for the diagnosis of intrauterine infections in pregnant women and children, given in the work of Markov I.S. "Diagnosis and treatment of herpetic infections and toxoplasmosis": Sat. articles. - K .: ArtEk, 2002. The diagnosis of intrauterine infections was carried out by determining specific antibodies for HSV type 1 and 2, CMV, EBV and toxoplasmosis antibodies, IgM and IgG classes in pregnant women, as well as in newborns in cord blood. The presence of viral DNA in blood plasma was studied by PCR. For ELISA used commercial diagnostic kits. A blood test for antibodies was performed once, PCR monitoring of the replicative activity of pathogens in pregnant women was carried out. According to the testimony, an ultrasound scan of the fetus and placenta was performed. When identifying the replicative activity of pathogens based on the detection of specific IgM and viral DNA, antiviral therapy was prescribed.

This method has the following disadvantages. The use of PCR diagnostics, serological detection of IgM and IgG antibodies using ELISA does not allow for a comprehensive, informative and reliable assessment of the results. To detect the DNA of viruses (HSV, CMV), the detection of a full-fledged viral antigen is not provided, in addition, a special laboratory is required. For PCR analysis, it is necessary to know special guidelines for the collection, transportation, storage, sample preparation of biological material. PCR diagnostics are mandatory on the day the material is taken. An ELISA test showing the presence of IgG antibodies to viruses in the blood is not a clear sign of the activity of the infectious process, since antibodies, for example, to EBV, are detected up to 85-90% in healthy donors, on the other hand, inhibition of the immune response, antibody genesis is violated, and in newborns, the determination of serological markers is ineffective due to an unformed immune system, in addition, there may be no IgM antibodies in the initial stage of the disease, since they are formed deferred. When diagnosed with ELISA in 30% of cases false-positive and doubtful results are detected. In this case, it is recommended to re-analyze the samples in parallel with the serum of the patient’s data taken after 7-14 days, which requires the use of additional labor-intensive methods, which are not always informative, and do not ensure the expressness of diagnosis and the accuracy of diagnosis. False positive results can be shown in serums containing non-specific proteins present in serum, for example, class M rheumatoid factor. The above disadvantages reduce the accuracy of diagnosis of intrauterine viral infections by this method. In addition, ultrasound scanning of the fetus and placenta is a non-specific method that is not informative enough to diagnose intrauterine infection.

In order to eliminate these shortcomings, the authors propose a method for diagnosing intrauterine viral infections in children by virologically determining a set of indicators: virus antigens, specific immune complexes loaded with antigens, early low-avidity antibodies in retro-placental blood, blood of a newborn and mother's blood.

The technical result of the present invention is to improve the accuracy and specificity of the diagnosis of an active infectious process in a child. This result is achieved by the fact that by morpho-virological examination of the placenta, determination of IgM and IgG in the blood of mother and child, the authors in the mRSC reaction examine retro-placental blood, the blood of the child and mother, additionally determine viral antigens, specific immune complexes with antigens, specific low-form IgG antibodies ₃ , IgG _1-2 , and with values in the retro-placental blood of the viral antigen> 0.10 OE; level of specific immune complexes> 0.06 OE; antibody titers> 1:20 identify the specific causative agent of viral placentitis, and in the presence of a virus antigen in the blood serum of the child> 0.10 OE; LIC> 0.06 OE; IgM in titer>1:20; one of - IgG ₃ , IgG _1-2 , IgG ₄ in titers in a 2-4-fold increase in comparison with mother titers of the same IgG subclasses; upon detection of one of IgG ₃ , IgG _1-2 , IgG ₄ in the titer> 1:20, and in the mother of the other of IgG ₃ , IgG _1-2 , IgG _{4, the} child is diagnosed with intrauterine viral infection.

The authors first proposed a comprehensive virological study of retro-placental blood, the blood of a newborn and mother, since examination of only one of the above objects does not give a complete picture of fetal infection due to the protective role of the placenta and the characteristics of newborn antigenogenesis (insufficient production of IgM and transplacental origin of IgG from the mother). The placenta, according to the authors, in addition to the mechanical barrier to the penetration of viruses, is of interest as an organ that provides immunological protection of the fetus against a pathogenic agent during physiological immunosuppression of the mother during pregnancy, exacerbated by a viral infection. We have shown that in 27% of cases there is a mismatch between the pathomorphological conclusion of the examination of the placenta and the virological analysis of retroplacental blood, therefore it is necessary to examine the blood of the child and mother, since according to our data, the implementation of confirmed viral infection in the placenta was detected in 76% of children and 24% of newborns were healthy, whereas if you rely only on the data from a survey of the placenta, an erroneous diagnosis of the intrauterine infection would be made by these really healthy children. The data obtained indicate that the IUI of the placenta does not mean the inevitable development of infection in the child. But it is also known that viral damage to the placenta can lead to prenatal pathology, the development of infectious diseases in children with a typical clinic, the development of somatic diseases that are diagnosed immediately at birth or develop after some time (delayed pathology), the birth of an apparently healthy baby, which indicates the need for examination of retroplacental blood.

As the authors showed, examination of the mother is advisable because the immune status of children in the neonatal period and in the first years of life is largely associated with the features of the course of pregnancy in their mothers. The concentration of IgM in serum in newborns is usually (by the prototype method) 8-10 times lower than the concentration of IgG.

Diagnosis of viral infections in newborns is difficult due to the frequent absence of clinically pronounced symptoms. The detection of specific antibodies of the IgM class in young children clearly indicates infection (however, in newborns and young children this class of specific immunoglobulins is rarely detected). The authors proved that the identification of various subclasses of low-form IgG antibodies (IgG ₃ , IgG _1-2 ) is of great help in the diagnosis of intrauterine infections.

The authors first proposed to use the mRSC method to identify a number of virological criteria: viral antigen, specific immune complexes with antigenic load and specific antibodies of the IgM and IgG classes of various subclasses (IgG ₃ , IgG _1-2 , IgG ₄ ), indicating the presence of an active infection process on moment of examination. An additional definition of low-avidity antibodies (IgM, IgG ₃ , IgG _1-2 ), which are produced in the early stages of the infection process and indicate an exacerbation of the disease at the moment, contributes to the timely and more accurate diagnosis of intrauterine infections in children.

The authors developed diagnostically significant quantitative criteria for the level of viral antigen in the blood serum, the concentration of SIC and the titers of specific antibodies of various Ig subclasses.

The method is as follows: using modified CSC (mRSC) in the extract of a retroplacental clot and blood serum of the mother and child, the determination of virus antigens and specific immune complexes (SICs) are carried out taking into account the prevalence of virus antigens in the structure, determination of AT of various Ig subclasses (IgM, IgG ₃ , IgG _1-2 , IgG ₄ ). The result of mRSCs was evaluated in a quantitative test using the principle of orthophenyl diamine (RPD) cleavage by oxidase enzymes derived from destroyed ram red blood cells, the test ingredient of RAC, and the enzyme content was evaluated on an AIF-C-01 C immunoassay analyzer at a wavelength of 492 nm.

Determination of AH viruses. In the serum of 50 μg of blood of patients add 50 μg of diagnostic serum containing AT to viruses (in titer 1/1000), and two doses of complement (50 μg). After one hour of contact, a hemolytic mixture is introduced into the system (0.85% suspension of mutton erythrocytes and 10 doses of antiserum to them). Assessment of experience is carried out after 30 minutes The level of AH viruses calculated by the formula:

AG VEB = (K _control -K _experience ) / K _control

where K _control is a quantitative indicator of enzymes in the control of the studied serum;

K _experience - a quantitative indicator of enzymes in the test serum

For the minimum AH content, a value of> 0.10 OE was taken.

Determination of specific immune complexes (SIC) with prevalence of hypertension. The setting of this experiment is similar to the experiment for the determination of AH, but for the presence of SIC with AH in serum, the result is considered that gives a negative value in the calculation according to the specified formula. This indicator reflects the relationship between AH and AT in the complex, when the addition of highly avid AT and exogenous complement led to the destabilization of the complex and the elimination of complementary proteins from it, enhancing erythrocyte hemolysis. The level of SIC with AH> 0.06 OE was considered to be diagnostically significant.

Differentiation of AT into subclasses M and G is carried out using a 2% suspension of cysteine amino acid that destroys class M antibodies, the determination of IgG _1-2 , IgG ₃ and IgG ₄ subclasses is carried out by calculation, taking into account the degree of complement sorption on immune complexes of patients AT, changes in the concentration of AT after removing them with a 5% suspension of Staph. Cowan 1, as well as the destruction of their 2% suspension of cysteine.

The described method was first tested on a small material, the good results obtained prompted the authors to draw up the application material for the invention and to further widely use the diagnostic method. A virological examination of 96 retroplacental blood clots from placentas with morphological signs of viral placentitis was performed, virologically intrauterine viral infections such as HSV 1/2, CMVI, and EBV infection were confirmed in 70 (72.9%) cases. During a virological examination of mother-child pairs, the diagnosis of viral intrauterine infection was confirmed in 76.0% of the subjects. The method can be illustrated with specific examples:

Example 1. Patient W., 1 month. Outpatient card number 263.

Diagnosis: intrauterine EBV infection, exacerbation.

In conclusion, a pathomorphological study of the placenta was diagnosed with DNA-viral placentitis with chronic subcompensated placental insufficiency.

Examination of a retroplacental clot using mRSC revealed the presence of specific antibodies to EBV of the IgG _1-2 subclass in the titer of 1:40, HSV HS (0.3 OE) of specific antibodies to the HSV of the IgG ₃ subclass in the titer of 1:56, which indicated a combined lesion the placenta by different herpes viruses and made it possible to establish a diagnosis - combined HSV and EBV placentitis.

In the blood serum of the patient’s mother, LICs with VEB AH (0.1 OE), specific antibodies to EBV of the IgG _1-2 subclass in the titer 1:28, specific antibodies to HSV of the IgG ₄ subclass in the titer 1:80 were found. In the blood serum of the child, specific antibodies to EBV of the IgG ₃ subclass in the titer of 1:32 were detected, which indicated that the child was infected only with EBV (the presence of diagnostic titers for EBV of the IgG ₃ subclass against IgG _1-2 in the mother showed that these antibodies specifically to the child and the course of his active infectious process), and the infection of the boy with HSV detected in the mother and in the afterbirth did not take place, since placental tissue apparently delayed the pathogen.

Based on the data obtained, the child was diagnosed with intrauterine EBV infection, exacerbation.

Example 2. Patient Z., 1 month. Outpatient card number 307.

Diagnosis: Intrauterine CMV infection, chronic form.

In conclusion, a pathomorphological study of the placenta was diagnosed with DNA-viral placentitis with chronic placental insufficiency and acute decompensation.

Examination of a retroplacental clot using mRSC revealed the presence of specific antibodies to CMV subclass IgG ₄ in a titer of 1:28, this made it possible to diagnose CMV placentitis.

In the blood serum of the mother of the patient, specific antibodies to CMV of IgG ₄ subclass in titer 1:20 were detected. Specific antibodies to CMV of IgG ₄ subclass in titer 1:60 were detected in the blood serum of the child, which indicated the chronic course of CMVI in the child, since more than 2-fold IgG ₄ seroconversion to the mother was detected.

Based on the data obtained, the child was diagnosed with intrauterine CMV infection, chronic form.

Example 3. Patient M., 1 month. Outpatient card number 315.

Diagnosis: Combined intrauterine viral infection. HSV infection, exacerbation. CMV infection, chronic form.

In the conclusion of the pathomorphological study of the placenta, the diagnosis was made: purulent chorioamnionitis, subchorial intervillitis, endovasculitis of the umbilical cord vessels. DNA-viral placentitis with chronic placental insufficiency and acute decompensation.

Examination of a retro-placental clot using mRSC revealed the presence of specific antibodies to HSV subclass IgG _1-2 in a titer of 1:32 and specific antibodies to CMV subclass IgG ₄ in a titer of 1:60, this allowed the diagnosis of combined HSV and CMV placentitis.

In the blood serum of the patient’s mother, specific antibodies to HSV of the IgG _1-2 subclass in the titer of 1:64 and specific antibodies to CMV of the IgG ₄ subclass in the titer of 1:28 were found. In the blood serum of the child revealed HSV HS (0.4 OE), specific antibodies to HSV subclass IgG ₃ in a titer of 1:60, indicating an active HSV infection in the child at the time of examination, since the elimination stage of the virus, which is judged by subclass specific antibodies, it was different in mother and child, if the mother had already passed the chronic phase, then the child showed exacerbation markers (HS HS and IgG ₃ ). Also, specific antibodies to CMV of IgG ₄ subclass in titer 1:80 were detected in the child, which indicated a chronic course in the girl of CMVI, since more than 2-fold IgG ₄ seroconversion in the child to maternal antibodies of the same subclass was detected.

Based on the data obtained, the child was diagnosed with a combined intrauterine viral infection. HSV infection, exacerbation. CMV infection, chronic form.

Example 4. Patient T., 1 month. Outpatient card number 329.

Diagnosis: healthy.

In conclusion, a pathomorphological study of the placenta was diagnosed with DNA-viral placentitis with chronic subcompensated placental insufficiency.

Examination of a retroplacental clot using mRSC revealed the presence of specific antibodies to EBV of the IgG ₄ subclass in the titer of 1:40, specific antibodies to CMV of the IgG ₄ subclass in the titer of 1:56, this indicated a combined lesion of the placenta by different herpes viruses and made it possible to establish a diagnosis of combined EBV and CMV placentitis.

In the blood serum of the patient’s mother, specific antibodies to EBV of the IgG ₄ subclass in the titer of 1:30, specific antibodies to HSV of the IgG ₄ subclass in the titer of 1:42 were found. Specific antibodies to EBV of IgG ₄ subclass in the titer of 1:20 were revealed in the blood serum of the child, which indicated the maternal origin of these antibodies and the absence of EBV infection in the child himself; no markers of HSV infection were detected in the boy. The results obtained show the protective function of the placental barrier in this case.

Based on the data obtained, the child was not diagnosed with intrauterine viral infection.

The method proposed by the authors has high accuracy, specificity and ease of execution, does not require a special laboratory and sophisticated equipment. Allows you to examine the child and mother for a specific pathogen, first identified in retro-placental blood, which makes the study more targeted, and also increases the cost-effectiveness of diagnosis.

The present method for the diagnosis of intrauterine viral infections in children allows for the follow-up of children with confirmed infection, as well as for antiviral and immunomodulating therapy and can be used in children's clinics.

Diagnosis of PGVI in retroplacental blood method indicators diagnostic values reversing IDGC viral antibodies > 0.10 OE SIC > 0.06 OE IDGC IgM titer> 1:20 IgG ₃ titer> 1:20 IgG _1-2 titer> 1:20 IgG ₄ titer> 1:20 Diagnosis of PGVI in children child indicators diagnostic values viral antibodies > 0.10 OE SIC > 0.06 OE IgM titer> 120 Diagnosis of PGVI in children, taking into account the vertical seroconversion of specific antibodies from the mother indicators diagnostic values mother child IgG ₃ titer> 1:20 2-4 times more maternal tigers of the same antibody subclasses IgG _1-2 titer> 1:20 IgG ₄ titer> 1:20 Diagnosis of PGVI in a child, depending on the subclass of antibodies in the mother child mother IgG ₃ IgG _{1-2 or} IgG ₄ lgG _1-2 IgG _{3 or} IgG ₄ IgG ₄ IgG _{3 or} IgG _1-2

Claims (1)

A method for diagnosing intrauterine viral infections in children by morphological examination of the placenta, determination of IgM and IgG antibodies in the blood of the mother and child, characterized in that retroplacental blood, the blood of the child and mother are examined in the mRSC reaction, viral antigens, specific immune complexes with antigens are additionally determined, specific low-avidity antibodies IgG ₃ , IgG _1-2 and with values in the retro-placental blood of the viral antigen> 0.10 OE; level of specific immune complexes> 0.06 OE; antibody titers> 1:20 identify a specific causative agent of viral placentitis, and in the presence of a child antigen of the virus> 0.10 OE; LAC> 0.06 OE; IgM antibodies in titer>1:20; one of the antibody subclasses - IgG ₃ , IgG _1-2 , IgG ₄ in titers in 2-4-fold increase in comparison with mother titers of the same IgG subclasses; upon detection of one of the antibody subclasses - IgG ₃ , IgG _1-2 , IgG ₄ in the titer> 1:20, and in the mother of the other - IgG ₃ , IgG _1-2 , IgG _{4, the} child is diagnosed with intrauterine viral infection.