RU2641609C1 - Method for determination of indications for antiherpetic therapy in case of hhv-6 infection in children with acute respiratory diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for determination of indications for antiherpetic therapy in case of HHV-6 infection in children with acute respiratory diseases is proposed. The PCR method with hybridization-fluorescent detection in "real time" mode is used to determine the number of HHV-6 DNA copies. For more than 100 copies/10⁵ cells in the blood, indications for antiherpetic therapy are determined.

EFFECT: possibility of infectious process correction, excluding the unreasonable use of antiherpetic drugs and preventing recurrence.

1 dwg, 1 tbl, 3 ex

Description

The invention relates to pediatrics and infectious diseases and is intended to determine indications for antiherpetic therapy in children with acute respiratory diseases and human herpes simplex virus type 6 infection.

Herpes virus infection is one of the most important medical and social problems due to its wide spread and extremely large range of clinical manifestations and complications. Herpesvirus infection in humans is caused by herpes simplex viruses I, II, III, IV (Epstein-Barr virus), V (cytomegalovirus), types VI, VII and VIII. According to a global review of herpesvirus studies, herpes infection / incidence is increasing from year to year. The variety of clinical manifestations, the characteristics of pathogens, the possibility of their spread by almost all known routes of transmission allowed the WHO Regional Office for Europe to attribute herpesvirus infections to the group of diseases that determine the future of infectious diseases in the current century. It is important to note that according to modern epidemiological studies, not only an increase in the number of patients with herpes infection, but also more severe forms of the disease with frequent exacerbations [Human Herpesviruses HHV-6A, HHV-6B, and HHV-7, is recorded. Diagnosis and Clinical Management / Edited by L. Flamand [et al.]. - Amsterdam, Elsevier, 2014.341.

After the initial infection and the development of manifest forms of the disease, the herpes virus is in the latent state for life in the human body under the control of the immune system in 85-90% of cases. However, in 10-15% of people with weakened immunity, reactivation of the infection with clinical manifestations of the disease occurs.

Like other representatives of the subfamily Betaherpesvirinae, HHV-6 penetrates the cell and replicates, it also has the ability to persistence and latency in the body. Persistence is the ability of herpes viruses to continuously multiply in infected cells, and latency is the lifelong preservation of viruses in implicit form in the ganglia.

Currently, it is believed that infection occurs mainly in early childhood. After the initial infection of the child, several options for the development of the disease are possible: from the classic Roseola infantum to an undifferentiated disease, accompanied by a fever, irritability, otitis media, seizures, rash.

However, often an active HHV-6 infection is asymptomatic, but the virus can cause serious diseases, especially in people with weakened immune systems.

Recent studies have shown that HHV6, like other herpesvirus infections, can cause not only acute, but also chronic forms of the disease in children. This fact plays an important role in the formation of a group of frequently ill children, leading to the development of a secondary immune imbalance with predominant damage to the B-cell link. The course of active herpesvirus infections in young and older children aggravates the clinical manifestations of acute respiratory infections (prolonged febrile fever, febrile seizures, damage to the lower respiratory organs). Diagnosis of herpesvirus infection includes an interpretation of the characteristic clinical picture, as well as virological and immunological studies. A common diagnostic method is the determination of IgM and IgG titers for antigens of viruses, as well as their avidity. However, to determine the stage of infection, it is fundamental to study various media of the patient's body (saliva, urine, blood) in order to isolate the virus. The diagnosis is established based on the detection of an increased virus content by determining the viral genome, a variety of which is the polymerase chain reaction (PCR). The advantages of the method are high specificity (100%), sensitivity (90-97%), speed of research (several hours).

There are a number of serological methods for determining markers of HHV-6 infection: immunofluorescence method, enzyme-linked immunosorbent assay (ELISA), immunoblot, immunoprecipitation. The most commonly used ELISA, although the method has a number of disadvantages. The determination of the titer of specific IgM is used to diagnose acute infection or reactivation. However, not all children with primary infection have IgM antibody production. and approximately 5% of healthy adults have IgM antibodies to HHV-6 (Human Herpesviruses HHV-6A, HHV-6B, and HHV-7. Diagnosis and Clinical Management / Edited by L. Flamand [et al.]. - Amsterdam, Elsevier. 2014 .-- 341).

Serological diagnosis is complicated by the cross-reaction with antibodies of other DNA viruses, for example, human herpes virus type 7 (HHV-7), due to the similarity of genomes by 42.5%. In addition, heterotypic reactivation of IgM synthesis, for example, occurs in CMV and EBV infections.

Virus isolation in cell cultures is a way to prove the presence of infectious viral particles in a sample. Detection of antigens is difficult due to the decrease in the number of circulating antibodies, the presence of cross-reactivity, distribution of antigenic determinants, nonspecific staining and low sensitivity.

Antiviral drugs are effective against active HHV-6 infection, but indications for use (as well as dosing regimens) in children are still unclear.

The closest analogue of the proposed method is a method of the same purpose, including the implementation of antiherpetic therapy depending on the severity of the clinical condition, that is, only in patients with complicated forms - pathology of the nervous system and erythema multiforme, and when detecting virus DNA in blood plasma with a qualitative method (Myukke N.A. Herpetic infection of the 6th type in children (Abstract of dissertation, candidate of medical sciences, 2006). However, as was shown in later works (Vashura L.V., Savenkova M.S., Zavadenko N.N., Koltunov I.E., Karazhas N.V., Rybalkina T.N., Kalugina M.Yu. ., Bullikh A.V. Convulsive syndrome in children: the role of herpesvirus infections // Infection of Children 2014. - No. 2, pp. 48-52. Vashura L.V. Convulsive attacks in children with herpesvirus infections: differential diagnosis and outcomes. Author. Diss., 2016), after suffering uncomplicated forms without specific therapy, infection with febrile seizures is possible. In addition, with a qualitative study and a positive analysis result, the severity of the disease is not always determined. Incorrect diagnosis of the stage of the disease in children often leads to the appointment of antiviral therapy in patients with latent forms. Antiviral drugs have a number of side effects and do not affect the latency of the virus. In this regard, the exact establishment of indications for the appointment of antiherpetic therapy is extremely relevant in pediatric practice. In the case of HHV-6 primary infection, mainly during the first year of life, the presence of maternal IgG can affect the diagnostic results. Thus, in the presence of symptoms similar to the primary HHV-6 infection, the use of a quantitative method to determine the indications for antiherpetic therapy is required to establish a diagnosis. This method is a highly sensitive PCR diagnostic method with which clinically healthy individuals can also determine the minimum amount of virus DNA in the blood, which does not require the appointment of therapy.

The task of the invention is to develop a laboratory diagnostic method for determining indications for antiherpetic therapy in children with HHV-6 infection with acute respiratory diseases.

The technical result of the invention is the ability to correct the infectious process by the timely implementation of antiherpetic therapy, as well as the exclusion of unreasonable use of antiherpetic drugs and the prevention of recurrence.

The technical result is achieved by determining using the PCR method with hybridization-fluorescence detection of the results of the analysis in real time in the blood.

The method for detecting and quantifying DNA virus using polymerase chain reaction (PCR) in real time is based on multiple copying of a specific DNA fragment using the DNA polymerase enzyme, which is a marker for this type of infectious disease agent. PCR is a direct method for detecting virus DNA and has a high degree of sensitivity. Real-time PCR has a number of advantages, such as speed of execution (2 hours for combined amplification and reading), a wide range of signal linearity (less than 10 to 10 ⁸ genomic copies), all fluorescent signals are received during amplification without opening the reaction tubes, the stability of the fluorescent probe, the presence of 96 wells, which allows for the production of large series of tests (Human Herpesviruses HHV-6A, HHV-6B, and HHV-7. Diagnosis and Clinical Management / Edited by L. Flamand [et al.]. - Amsterdam , Elsevier, 2014 .-- 341).

We examined 236 patients older than 1 year to 17 years: of them 136 children with acute respiratory infections admitted to the children's infectious diseases department, and 100 clinically healthy children during the medical examination (control group).

The levels of viral load in the blood were analyzed by real-time PCR (Table 1. Results of a PCR study of blood).

136 blood samples of patients of the main group were studied (in children older than 1 year to 17 years with acute respiratory infections), 5-9 copies / 10 ⁵ cells were found in 25%, from 10 to 99 copies / 10 ⁵ cells in 43% 19% - 100-999 copies / 10 ⁵ cells, 11% - 1000-9999 copies / 10 ⁵ cells, 2% of children more than 10,000 copies / 10 ⁵ cells. In all 100 patients of the control group, the load was no more than 99 copies / 10 ⁵ cells (Fig. 1. Viral load in the blood (PCR)).

The detection of HHV-6 DNA in the patient’s blood in an amount of more than 100 copies / 10 ⁵ cells correlated with the severity of the clinical condition. In children who had a high level of HHV-6 DNA in the blood - more than 100 copies / 10 ⁵ cells, the clinical picture was dominated by the classical forms characteristic of HHV-6 monoinfection, such as sudden exanthema and febrile seizures. All this confirms the presence in the patient of an active form of infection with HHV-6 and should determine the further management tactics of the patient.

In children who had a low level of HHV-6 DNA in the blood - less than 100 copies / 10 ⁵ cells, the clinical picture was dominated by nonspecific symptoms of acute respiratory disease, combined with mixed infections of HHV-6 and respiratory viruses.

Antiherpetic therapy was performed for children with ARI (16 patients) who had high levels of HHV-6 DNA more than 100 copies / 10 ⁵ cells. The comparison group consisted of children with ARI who had high levels of HHV-6 DNA more than 100 copies / 10 ⁵ cells who did not have antiherpetic therapy. Control examination of patients was performed twice: on the 5th day of therapy, and also 1 month after the start of antiherpetic treatment. The study included PCR blood. On the 5th day of therapy, 61% of children on the background of antiherpetic treatment did not have HHV-6 DNA in their blood. In the group of children without antiherpetic therapy, HHV-6 DNA was found in blood in 69% of patients, i.e. more often than in groups of children who received antiherpetic treatment, which was continued. Children were examined again after 1 month from the start of treatment. 82% of children on the background of antiherpetic treatment after 1 month from the start of therapy showed no signs of active HHV-6 infection. In the group of children without antiherpetic therapy, the proportion of patients with active forms of infection was 56%, which is higher than in the groups of children receiving antiviral treatment.

Thus, the minimum number of copies (less than 100 copies / 10 ⁵ cells) or their absence in the blood is significantly more often determined in clinically healthy children (p <0.05, Fisher’s exact test, Chi-square test). A virus level in the blood of more than 100 copies / 10 ⁵ cells characterizes the course of the disease that requires antiherpetic therapy (Table 1).

The method is as follows. Blood is taken from a child with an acute respiratory disease, and the number of copies of HHV-6 DNA is determined using real-time PCR with hybridization-fluorescence detection of the analysis results. If there are more than 100 copies / 10 ⁵ cells in the blood, indications for antiherpetic therapy are determined.

Examples

Clinical example 1

Darima M., 1 year 4 months, was admitted to the infectious diseases ward with complaints of fever to febrile numbers. The child is sick for 2 days - a rise in temperature to 38.8, rhinitis. On the first day of the disease was examined by an ambulance doctor, Analgin was introduced, with a slight effect. On the second day of the disease, the temperature rises to 39, sluggish, after taking Ibuprofen in an age dose for 2 hours, the temperature remains up to 39.5. The child was taken to hospital. Amid anxiety, tonic-clonic convulsions first occurred without loss of consciousness. Docked on their own. On examination, the condition of the child of moderate severity is capricious. The skin is pale pink, clean. Visible mucous membranes are clean, moist, the pharynx is moderately hyperemic, there are no raids. The posterior wall of the granular, tonsils enlarged 1 tbsp. Nasal breathing is slightly difficult, mucous discharge. Lymph nodes of the cervical groups more than 3 in the group, enlarged to 10-12 mm, axillary - up to 10 mm, all of them are soft-elastic to the touch, mobile, painless, the skin above them is not changed. In the lungs, puerile breathing is carried out evenly, no wheezing. There is no meningeal symptomatology.

Clinical blood test at admission to the hospital: Hb - 125 g / l, Eritre. - 4.54 × 10 ¹² / l, Leikots. - 3.98 × 10 ⁹ / l, thrombotic. - 277 × 10 ⁹ / l, n / a - 6, s / I - 17, mon. - 12, lymph. - 65, ESR - 19 mm / hour. A biochemical blood test without features, CRP is normal.

Virological examination data.

Blood PCR: detected HHV-6 DNA 16122 copies of DNA / 10 ⁵ cells, which indicates the active form of HHV-6 infection in a child, requiring the use of antiherpetic therapy.

On the 4th day of the disease, the temperature dropped to normal values and a spotty-papular rash appeared on the abdomen and extensor surfaces of the upper extremities, which confirmed the laboratory diagnosis of HHV-6 infection by the development of the classic form of infection in children - childhood roseola (sudden exanthema).

Clinical blood test on day 7 of the disease: Hb - 127 g / l, Eritre. - 4.7 × 10 ¹² / l, Leikots. - 6.3 × 10 ⁹ / l, Trombots. - 311 × 10 ⁹ / l, n / a - 2, s / a - 19, mon. - 10, lymph. - 68, ESR - 16 mm / hour.

Thus, the child has a primary active infection of HHV type 6. Febrile convulsive attack. Sudden exanthema.

In connection with a timely diagnosis, antiherpetic therapy was immediately started (Acyclovir 200 mg × 4 r / d in combination with recombinant interferon-α2β with taurine at a dose of 125000 ME 2 times a day) and concomitant therapy.

2 months after the illness, the child was consulted on an outpatient basis. SARS once, the temperature of 37.8 febrile seizure was not. There are no complaints at the time of the inspection. There was granular pharyngitis, tonsil hypertrophy of the 1st degree, microlymphadenopathy (lymph nodes of the cervical groups more than 3 in the group, up to 10 mm, axillary up to 10 mm). Clinical blood test: Hb - 130 g / l, Eritre. - 5.01 × 10 ¹² / l, Leukots. - 11.0 × 10 ⁹ / l, Thrombocyte. - 350 × 10 ⁹ / l, n / a - 1, s / I - 30, mon. - 7, lymph. - 61, ESR - 8 mm / hour.

A PCR blood test showed a negative result. The treatment is over. Additional therapy was not carried out.

When examined after 4 months, the child is clinically healthy, when examined - without features. Serological examination revealed Ig G to HHV-6 in a diagnostic titer of 1: 540. There was no convincing data for the activity of HHV-6. Repeated examination after 1 year is recommended.

Thus, this clinical example clearly demonstrates the typical development of acute primary infection with HHV-6, the onset of which proceeded under the guise of ARVI. Early diagnosis of infection by determining the level of HHV-6 DNA in the blood using PCR with hybridization-fluorescence detection of the results of the analysis in real time of more than 100 copies / 10 ⁵ cells allowed to prescribe antiherpetic therapy before the appearance of classical clinical manifestations of HHV-6 infection in in the form of a rash. This contributed to the rapid transition of the infection into a latent form and prevented the recurrence of febrile convulsive attacks, as a manifestation of exacerbation of HHV-6 infection, in a child against the background of subsequent acute respiratory viral infections.

Clinical example 2

Vanya P., 3 years old. Examined as part of a clinical examination in a kindergarten. Mom does not complain at the time of the examination. Objectively healthy.

Before the examination, the last acute respiratory disease was 6 months. back.

From 1 g to 7 months Adenoiditis. ARVI suffered 3 times.

Clinical blood test: Hb - 131 g / l, Eritre. - 4.84 × 10 ¹² / l, Leikots. - 8.83 × 10 ⁹ / l, Trombots. - 276 × 10 ⁹ / l, n / a - 1, s / a - 23, mon. - 7, lymph. - 68, eosinophids - 1, ESR - 3 mm / hour.

HHV-6 DNA was isolated in blood 17 copies / 10 ⁵ cells.

Serological studies did not detect specific IgM and IgG for HHV-6.

During dynamic observation of the child within 1 month of catarrhal phenomena in the child was not observed.

Upon re-examination after 30 days, the HHV-6 DNA in the smear and blood was not determined. Serological studies did not detect specific IgM and IgG for HHV-6.

This clinical example demonstrates the detection of the minimum amount (less than 100 copies / 10 ⁵ cells) of HHV-6 DNA in the blood of a clinically healthy child, which can be detected by a highly sensitive PCR method with hybridization-fluorescence detection of the results of the analysis in real time. Such a finding without clinical manifestations of HHV-6 infection at the time of examination and during 5 months of observation may indicate a latent infection that does not require the appointment of therapy.

Clinical example 3

Rodion A., 3 years old, was admitted to the infectious diseases ward with complaints of fever to febrile numbers, an attack of tonic-clonic seizures. Within 2 days, he had ARVI, rhinitis, did not receive treatment, attended kindergarten. Against the background of increasing T to 38.8, tonic-clonic seizures with loss of consciousness, lasting 2-3 minutes, stopped on their own. The child was hospitalized.

A child under 2 years of acute respiratory viral infection with temperatures up to 39 ° C, there were no episodes of seizures. At the age of 2 years he entered the infectious diseases department with a diagnosis of acute respiratory viral infections with a febrile seizure attack. On the 2nd day of hospitalization, against the background of normalization of temperature, a spotty-papular rash appeared on the skin. In this regard, an infection with HHV-6 was suspected and a blood sample was taken for analysis for the isolation of virus DNA in the blood. HVG-6 DNA was detected in the blood in an amount of 120,000 copies / 10 ⁵ cells, which was an indication for antiherpetic therapy. However, at the insistence of the mother, the child was discharged. The boy did not receive the prescribed anti-herpes therapy.

Later, there was a repeated episode of febrile seizure in the background of fever with SARS. Upon admission to the hospital, a moderate condition.

The skin is pale pink, clean from rash. Visible mucous membranes are pink, clean, moist, the pharynx is hyperemic, the posterior wall of the pharynx is granular, there are no deposits. Subcutaneous fat is moderately developed, evenly distributed. Edema, no pastiness. Lymph nodes of the cervical groups are single, up to 1.0 cm, soft-elastic, b / w. Nasal breathing is free. There is no shortness of breath. Percussion - a clear pulmonary sound, symmetrical across all pulmonary fields. In the lungs, puerile breathing is carried out uniformly in all departments, no wheezing. Clinical blood test: Hb - 117 g / l, Eritre. - 4.2 × 10 ¹² / l, Leikots. - 16.9 × 10 ⁹ / l, Trombots. - 199 × 10 ⁹ / l, n / a - 4, s / I - 28, mon. - 8, lymph. - 60, ESR - 12 mm / hour.

Serological blood tests revealed immunoglobulins G to HHV 6 in a titer of 1: 350 (ELISA, Vector-best test system). Human herpes virus DNA was detected in the blood in the amount of 2300 copies / 10 ⁵ cells by PCR.

Based on the history (frequent acute respiratory infections, febrile seizures), clinical presentation (seizures with loss of consciousness), laboratory tests (DNA and HHV-6 antigens in the blood and oropharyngeal swab, antibodies to the virus), Chronic persistent HHV infection 6 was diagnosed type in the reactivation stage. There are indications for antiherpetic therapy.

During the stay of the child in the hospital, against the background of the therapy, the state of the child with positive dynamics does not have a fever, convulsions did not recur, and she feels well.

Monitoring of a clinical blood test, PCR blood test with hybridization-fluorescence detection of real-time analysis results for quantitative determination of HHV-6 DNA, ELISA to HHV6, after the end of the course of therapy revealed the absence of HHV-6 DNA in the blood.

This clinical example demonstrates the formation of chronic persistent infection with HHV-6 (manifested by repeated febrile seizures with ARVI) as a result of the lack of timely antiherpetic therapy at the time of diagnosis.

Thus, the proposed method allows timely implementation of antiherpetic therapy to prevent the formation of chronic forms of infection, as well as to exclude unreasonable use of antiherpetic drugs and prevent recurrence.

Claims (1)

A method for determining indications for antiherpetic therapy for HHV-6 infection in children with acute respiratory diseases, characterized in that in the blood using the PCR method with hybridization-fluorescence detection of the results of the analysis in "real time" determine the number of copies of HHV-6 DNA and with more than 100 copies / 10 ⁵ cells in the blood, indications for antiherpetic therapy are determined.

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