Predicting the severity of seasonal influenza in children

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Introduction. Influenza is an acute respiratory viral infection transmitted by airborne droplets and caused by influenza viruses [1]. Characteristic symptoms include fever and symptoms of general intoxication, which prevail over moderate catarrhal syndrome in the upper respiratory tract (mainly the nasopharynx, larynx and trachea) [2, 3]. Cytokines and parameters of the non-specific lipid peroxidation-antioxidant protection system are of great importance in the pathogenesis of influenza [4, 5, 6]. In assessing the severity of influenza in children, the analysis of the severity of intoxication, the level of inflammatory changes in the respiratory tract, the severity of hemorrhagic syndrome, and the presence of complications are currently used [7, 8, 9]. In the analysis of domestic and foreign literature, a method for predicting the course of pandemic influenza in children was described (Miromanova N.A., Zhamsueva D.R., 2013), where, using multivariate regression, indicators were identified with the determination of metalloprotease-9 (MMP-9), aspartate aminotransferase (AST), interleukin IL-1β, von Willebrand factor and tumor necrosis factor (TNF-α) in the blood serum. These factors largely determine the occurrence of endothelial dysfunction and hemorrhagic complications of pandemic influenza in children [10]. Therefore, the search for the most informative markers for predicting the severity of seasonal influenza in children is of interest for study.

Objective: to determine the cytokine profile, indicators of the lipid peroxidation-antioxidant protection system in children with seasonal influenza and to identify the most informative indicators for predicting the severity.

Material and methods. The study design is observational, multicenter, prospective cohort. Inclusion criteria: Children aged 1 month to 11 years; established diagnosis of "Influenza", the virus is identified, confirmed by polymerase chain reaction (PCR) in a smear from the oropharynx (qualitative); no competing infectious diseases; consent of the child's parents/legal representatives to participate in the study. The control group consisted of 112 practically healthy children, comparable by gender and age. Inclusion criteria for the control group: children aged 1 month to 11 years; children with health groups I, II (determined on the basis of the conclusion of the local pediatrician); children without signs of acute respiratory viral infection or 14 days after recovery; consent of the child's parents/legal representatives to participate in the study.

Conditions. The clinical part of the work was carried out on the basis of the Irkutsk Regional Infectious Diseases Clinical Hospital (chief physician, PhD Khabudaev V.A.), the State Regional Healthcare Institution State Children's Children's Clinical Hospital No. 1 (chief physician, head of the Department of Pediatric Surgery at Irkutsk State Medical University, professor Novozhilov V.A.), the laboratory part of the work was carried out on the basis of the laboratory of pathological physiology of the Scientific Center for Family Health and Human Reproduction Problems (director - MD, Corresponding Member of the Russian Academy of Sciences Rychkova L.V., chief physician, MD Bugun O.V.). Duration of the study. Patients were recruited during the epidemic rise in seasonal influenza and acute respiratory viral infections in 2018-2019, patients for the control group were randomly recruited from September to February 2024 during a visit to a local pediatrician during a routine examination, and the information obtained was processed and analyzed in 2024-2025. Description of medical intervention. A standard clinical, anamnestic, laboratory and instrumental examination, as well as an additional one (enzyme-linked immunosorbent assay and assessment of lipid peroxidation parameters, antioxidant protection (POL-AOZ)) were carried out at the Irkutsk Regional Infectious Diseases Clinical Hospital. Blood was collected from children on an empty stomach from the cubital vein, in vacuum tubes (Vacutainer) on the first day of the child's hospitalization in the infectious diseases hospital in a volume of 5 ml with subsequent delivery to the Clinic's laboratory no more than 60 minutes after blood collection. In case of delayed delivery, the blood was stored at a temperature from plus 4 to plus 60C (in the refrigerator) and then delivered to the laboratory in special transport containers. Blood serum was obtained by centrifuging the tubes at 3000 rpm for 10 minutes. The resulting serum was stored in disposable "Epindоrf" type tubes at minus 400C, defrosting, if necessary, was performed no more than once. In the Regional State Autonomous Healthcare Institution State Children's Children's Clinic No. 1, patients were recruited for the control group (blood sampling from the cubital vein and further transportation to the Clinic's laboratory. Primary outcome of the study. All patients who underwent examination and treatment in the hospital were discharged with improvement or recovery, no adverse outcomes were identified. Additional outcomes of the study. None identified.

Group analysis. The distribution of children by age groups was based on the periodization of childhood (Gundobin N.P., 1906): infancy 1 month - 1 year, early childhood from 1 year to 2 years 11 months 29 days; preschool age from 3 to 6 years 11 months 29 days; primary school age (girls - from 7 to 10 years, boys - from 7 to 11 years). To build the model, the children were divided into subgroups by severity: 82 children (78.8%) with a moderate form of the disease, 22 (21.2%) children with a severe form of influenza.

Methods for recording outcomes. Clinical and anamnestic research methods (collection of complaints, medical history, life history, vaccination status), physical examination (palpation, percussion, auscultation of organs and systems), standard general clinical methods (clinical blood test with formula, biochemical blood test, general urine test, molecular genetic method: detection of RNA/DNA of respiratory viruses in smears of the nasal and oropharynx by polymerase chain reaction (PCR), enzyme immunoassay of blood: detection of IgM, IgG to chlamydia, mycoplasma, whooping cough, HIV, enzyme immunoassay of feces: detection of IgM to rotavirus, norovirus, bacterial analysis of feces for acute bacterial intestinal infections; ECG, pulse oximetry; X-ray, ultrasound of organs as indicated). Spontaneous levels of cytokines (IL-1β, IL-4, IL-6, IL-8, TNF-α, INF-α, INF-γ) and highly sensitive C-reactive protein in blood plasma were determined by solid-phase enzyme-linked immunosorbent assay (ELISA) using diagnostic test systems manufactured by Vector-Best (Novosibirsk) on a Multiscan EX analyzer (Thermo Electron, Germany). The activity of the non-specific lipid peroxidation system was analyzed based on the content of unsaturated double bonds (UDB), primary – diene conjugates (DC) and secondary – ketodienes and conjugated trienes (KD – CT), end products – Schiff bases (SB) in blood plasma. The state of the antioxidant defense system (AOS) was assessed by the total antioxidant activity (AOA), superoxide dismutase (SOD) activity in the blood serum, the content of reduced and oxidized glutathione (GSH and GSSG) in erythrocytes (hemolysate), the level of α-tocopherol and retinol in the blood serum was determined by the fluorimetric method, measurements were made on an SF-2000 spectrophotometer (Russia), a BTS-350 spectrofluorophotometer (Spain) and fluorate 02 ABFF-T (Russia). The content of the final products of lipid peroxidation reacting with thiobarbituric acid (TBA-AP) was determined fluorimetrically using the Agat reagent kit, Russia. Ethical review. The study protocol was approved by the local ethics committee of the Federal State Budgetary Scientific Institution "Scientific Center for Family Health and Human Reproduction" (protocol No. 3.1.1 dated September 28, 2022). Statistical analysis of the data was performed in Statistica 10.0 (StatSoft, USA). The sample size was not pre-calculated. The distribution of the studied features was determined using the Kolmogorov-Smirnov test for sample sizes (n> 50) and the Shapiro-Wilk test for small samples (n<50). To describe quantitative features that do not correspond to the normal distribution law, the median (Me), upper and lower quartiles (Q1; Q3) were used. The reliability of differences in quantitative features with a distribution different from normal - using the Mann-Whitney test (U-test). The prognostic model was built using ROC analysis and logistic regression.

Results. Main results. Etiological structure of influenza: influenza A(H1N1) accounted for 13.5% (n = 14), A(H3N2) – 31.7% (n = 33), A(H1N1)sw2009 – 43.3% (n = 45), influenza B – 9.6% (n = 10), unsubtyped influenza A – 1.9% (n = 2). The average age of patients was 3.0 (1.75-5.0) years. Distribution of children by gender: 40 boys (38.5%), 64 girls (61.5%). Patients were admitted to hospital on the 2.0 (2.0; 4.0) day of illness. Prevalence of clinical symptoms: runny nose occurred in 76.9% of cases (n = 80), cough was observed in 96.2% of cases (n = 100), fever - in 97.1% of cases (n = 101), intoxication - in 70.2% of cases (n = 73). Additional results. In children with seasonal influenza, the concentrations of the studied cytokines in the blood serum significantly exceeded the upper limit of the reference range: the level of IL-1β in children with influenza was 940 times higher, IL-4 was 260 times higher, IL-8 was 63.44 times higher, TNF-α was 4.34 times higher, INF-γ was 3.97 times higher, IL-6 was 43.77 times higher, INF-α was 2090 times higher, and the level of high-sensitivity C-reactive protein (CRP) was 31.76 times higher, p<0.05. The highest synthesis of CRP was noted in young children; the highest level of TNF-α, IFN-γ, IL-6 was in infants, the level of IL-4 was higher in preschool children; the synthesis of IL-8 and IFN-α was highest in primary school children, but without statistically significant differences. The ratio of serum IL-4 to IFN-γ was 14.5 (0.5–32.0) in children with pneumonia and 1.37 (0.46–6.17) in children without pneumonia, p = 0.219.The area under the ROC curve reflecting the level of intoxication and IL-4 was 0.729, 95% CI (0.611; 0.846), p < 0.001, the sensitivity and specificity of the method were 61.2 and 78.6%, respectively. When analyzing the lipid peroxidation-antioxidant protection system in children with seasonal influenza, age differences were revealed: infants had a statistically significantly higher level of Dv.Sv., DK, KD-ST, SOD, in contrast to children of other age groups. Young children had lower levels of TBA-AP, children of primary school age had a high synthesis of SH, compared with children of other ages. In children of all age groups with influenza, the level of retinol is statistically significantly lower by 1.41 times, α-tocopherol by 1.46 times, AOA by 1.02 times, GSH and GSSG by 1.16 times, p<0.05. The further aim of the study was to examine clinical and laboratory data as risk factors for the development of severe influenza and to develop a prognostic model for moderate and severe influenza in children. For this purpose, all patients were divided into 2 groups according to the clinical criterion of "severity of the disease": 82 children with moderate disease, 22 children with severe influenza. Using the logistic regression method, the most informative indicators reflecting the prognosis of the severity of influenza were selected from 94 indicators, namely, the maximum body temperature during hospitalization, the presence of pneumonia, the level of highly sensitive CRP and KD-ST. The area under the ROC curve corresponding to the probability of pneumonia was 0.658, 95% CI (0.572; 0.737), p = 0.011, the sensitivity and specificity of the method were 96.19 and 35.48%, respectively. The area under the ROC curve reflecting the maximum value of body temperature during hospitalization was 0.808, 95% CI (0.731; 0.870), p < 0.0001, the sensitivity and specificity of the method were 99.05 and 74.19%, respectively. An increase in the level of CRP, odds ratio (OR) = 1.219, 95% CI (1.018; 1.461), an increase in the level of KD-ST OR = 0.084, 95% CI (0.008; 0.801). In order to predict the possible development of severe influenza, we calculated the SD (severe degree) coefficient using the formula: SD = 1/ 1 + e320.194 + 11.738 * X1 + 8.141 * X2 - 0.198 * X3 + 2.475 * X4, where 320.194 is the regression coefficient b0, 11.738; 8.141; 0.198; 2.475 are the non-standardized coefficients b, e is the base of the natural logarithm (2.72), X1 is the presence of pneumonia, X2 is the maximum body temperature during illness, X3 is the CRP level, X4 is the KD-ST level. The value of the SD coefficient = 0.001 corresponds to a high risk of developing a severe form of influenza in a child. The share contribution of the indicators is 36.8% for pneumonia, 34.2% for fever, 13.1% for CRP, and 13.0% for CD-ST. Discussion. The obtained results on the assessment of the severity of seasonal influenza in children based on a combination of clinical, metabolic, and immunological indicators require further research in this area on larger samples to confirm the identified patterns and trends, as well as to evaluate the effectiveness of the proposed prognostic model. Limitations of the study. Our study included children with seasonal influenza aged from 1 month to 11 years inclusive, which limits the use of the prognostic model in older children. Conclusion. The course of influenza at the height of the disease is characterized by an increased level of pro- and anti-inflammatory cytokines and highly sensitive CRP with a deficiency of antioxidant defense factors. The assessment of the maximum body temperature during the illness, the fact of pneumonia, as well as the level of highly sensitive C-reactive protein and KD-ST are specific markers for predicting moderate and severe seasonal influenza in children. The proposed algorithm allows predicting the severity at an early stage, which allows recommending its use in clinical practice.

Funding source. This study was not supported by any external sources of funding.

Patients’ consent. Written consent was obtained from all the study participants before the study screening in according to the study protocol approved by the local ethic committee.

Competing interests. The authors declare that they have no competing interests.

Contribution of the authors: E.D. Kazantseva - literature review, analysis of case histories, writing of the text, and statistical analysis of data; A.G. Petrova - writing and editing of the article, M.A. Darskaya - writing and editing of the article, I.M. Mikhalevich - literature review and statistical analysis of data, N.V. Semyonova - writing and editing of the article, L.F. Sholokhov - writing and editing of the article, L.V. Rychkova - writing and editing of the article, and L.I. Kolesnikova - writing and editing of the article. All authors confirm that they meet the international ICMJE criteria for authorship (all authors have made a significant contribution to the development of the concept, research, and article, and have read and approved the final version before publication).

About the authors

EKATERINA KAZANTSEVA

Scientific Centre for Family Health and Human Reproduction Problems

Author for correspondence.
Email: kat.smile7@yandex.ru
ORCID iD: 0000-0003-0692-2295

Pediatrician, postgraduate student, junior researcher at the Laboratory of Infectology and Immunoprophylaxis in Pediatrics,
Scientific Centre for Family Health and Human Reproduction Problems

Russian Federation

Alla G. Petrova

Scientific Centre for Family Health and Human Reproduction Problems

Email: rudial75@gmail.com
ORCID iD: 0000-0002-7965-8061
SPIN-code: 3962-5093

Doctor of Medical Sciences, Professor

Russian Federation, Irkutsk

Marina A. Darenskaya

Scientific Centre for Family Health and Human Reproduction Problems

Email: marina_darenskaya@inbox.ru
ORCID iD: 0000-0003-3255-2013
SPIN-code: 3327-4213
Scopus Author ID: 55453341400
ResearcherId: O-4490-2015

Dr. Sci. (Biol.), leader research associate

Russian Federation, Irkutsk

Natalya V. Semenova

Scientific Centre for Family Health and Human Reproduction Problems

Email: natkor_84@mail.ru
ORCID iD: 0000-0002-6512-1335
SPIN-code: 6606-0160

Dr. Sci. (Biol.), leader research associate

Russian Federation, Irkutsk

Lyubov I. Kolesnikova

Research Centre for Family Health and Human Reproduction

Email: iphr@sbamsr.irk.ru
ORCID iD: 0000-0003-3354-2992
SPIN-code: 1584-0281

MD, PhD, DSci (Medicine), Professor, Academician of the Russian Academy of Sciences, Scientific Director

Russian Federation, Irkutsk

Lyubov V. Rychkova

Scientific Centre for Family Health and Human Reproduction Problems

Email: clinica_zam@inbox.ru
ORCID iD: 0000-0002-0117-2563
SPIN-code: 1369-6575

MD, PhD, Dr. Sci. (Medicine), Professor, Corresponding Member of the RAS, Director

Russian Federation, 16 Timiryazeva str., Irkutsk, 664003

Isai M. Mikhalevich

Scientific Centre for Family Health and Human Reproduction Problems

Email: mim977@list.ru
ORCID iD: 0000-0003-4854-3277

Candidate of Medical Sciences, Associate Professor, Head of the Department of Pedagogical and Information Technologies at the Russian Medical Academy of Continuous Professional Education of the Ministry of Health of the Russian Federation, 664049, Irkutsk, Yubileiny, 100; employee of the Scientific Center for Family Health and Human Reproduction

Russian Federation, 16 Timiryazeva Street, Irkutsk, 664003, Russian Federation

Leonid F. Sholokhov

Scientific Centre for Family Health and Human Reproduction Problems

Email: lfshol@mail.ru

д.м.н., профессор, ведущий научный сотрудник лаборатории физиологии и патологии эндокринной системы ФГБНУ «Научный центр проблем здоровья семьи и репродукции человека»

Russian Federation, 16 Timiryazeva Street, Irkutsk, 664003, Russian Federation

References

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