ТРИ ФАКТОРА УСПЕШНОЙ ИНТЕРПРЕТАЦИИ ПЦР ПРИ ВНЕБОЛЬНИЧНОЙ ПНЕВМОНИИ У ДЕТЕЙ: ВЫБОР ОБРАЗЦА, ПОРОГОВЫЙ ЦИКЛ И БИОМАРКЕРЫ

РЕЗЮМЕ

Введение. Внебольничная пневмония (ВП) сохраняет лидирующие позиции в структуре заболеваемости и смертности детского населения. Внедрение методов полимеразной цепной реакции (ПЦР) расширило диагностические возможности, однако актуализировало вопросы выбора респираторного образца и интерпретации результатов с учётом пороговых циклов (Ct) и бессимптомной колонизации.

Цель. Систематизировать современные данные о диагностической ценности различных респираторных образцов при ПЦР-диагностике ВП у детей, определить границы применимости пороговых циклов для дифференциации инфекции и колонизации, а также обосновать необходимость интеграции молекулярных методов с биомаркерами.

Методы. Проведен систематический поиск литературы в базах PubMed, Scopus, Web of Science, Google Scholar, Cochrane Library и eLibrary (2000–2026 гг.). Всего отобрано 83 исследований для итогового анализа.

Результаты. Идентифицированы три ключевых фактора успешной интерпретации ПЦР-диагностики: выбор образца, интерпретация Ct и интеграция с биомаркерами. Показано, что диагностическая ценность назофарингеальных (НФ) мазков для верификации пневмококковой этиологии ВП стремится к нулю вследствие высокой частоты колонизации (40–60%), что делает стерильные локусы единственным надёжным источником. В противоположность этому, для Mycoplasma pneumoniae оптимальными являются орофарингеальные мазки (чувствительность 96,2%), а слюна демонстрирует сопоставимые результаты. Для Российской Федерации критически важно выявление 36–41% макролид-резистентных штаммов M. pneumoniae с региональными различиями; 62% случаев сопровождаются вирусной коинфекцией (парагрипп 28%, SARS-CoV-2 19%, РСВ 12%). Порог Ct < 25 при микоплазменной инфекции служит независимым предиктором тяжёлого течения и требует госпитализации. Для респираторно-синцитиального вируса (РСВ) порог Ct < 25 ассоциирован с тяжёлым течением (скорректированное отношение шансов, aOR 2,26), для метапневмовируса человека (hMPV) — Ct < 27 (aOR 4,32). Прокальцитонин-ориентированные протоколы позволяют сократить необоснованное назначение антибиотиков, однако гетерогенность педиатрических данных диктует необходимость мультимаркерных подходов, включающих гепарин-связывающий белок (HBP), чувствительность которого составляет 82%, специфичность 86%, а комбинация с прокальцитонином повышает площадь под кривой (AUC) до 0,94.

Заключение. Успех ПЦР-диагностики ВП у детей определяется тремя факторами: правильным выбором образца, корректной интерпретацией Ct и интеграцией с биомаркерами.

1. СПИСОК ЛИТЕРАТУРЫ / REFERENCES

2. McAllister DA, Liu L, Shi T, et al. Global, regional, and national estimates of pneumonia morbidity and mortality in children younger than 5 years between 2000 and 2015: a systematic analysis. Lancet Glob Health. 2019;7(1):e47-e57. doi: 10.1016/S2214-109X(18)30408-X

3. GBD 2019 Under-5 Mortality Collaborators. Global, regional, and national progress towards Sustainable Development Goal 3.2 for neonatal and child health: all-cause and cause-specific mortality findings from the Global Burden of Disease Study 2019. Lancet. 2021;398(10303):870-905. doi: 10.1016/S0140-6736(21)01207-1

4. Ma Y, Fan S, Xi J. Recent updates regarding the management and treatment of pneumonia in pediatric patients: a comprehensive review. Infection. 2025;53(6):2341-2359. doi: 10.1007/s15010-025-02605-w

5. Zar HJ, Barnett W, Stadler A, et al. Aetiology of childhood pneumonia in a well vaccinated South African birth cohort: a nested case-control study of the Drakenstein Child Health Study. Lancet Respir Med. 2016;4(6):463-472. doi: 10.1016/S2213-2600(16)00096-5

6. Ojuawo OB, Iroh Tam PY. Childhood Pneumonia Diagnostics in Sub-Saharan Africa: A Systematic Review. J Trop Pediatr. 2022;68(4):fmac045. doi: 10.1093/tropej/fmac045

7. Self WH, Williams DJ, Zhu Y, et al. Respiratory Viral Detection in Children and Adults: Comparing Asymptomatic Controls and Patients With Community-Acquired Pneumonia. J Infect Dis. 2016;213(4):584-591. doi: 10.1093/infdis/jiv323

8. Ho EC, et al. Validation of pleural fluid group A Streptococcus and Staphylococcus aureus PCR assays and their potential clinical impact in children with complicated pneumonia. J Microbiol Methods. 2025;236:107192. doi: 10.1016/j.mimet.2025.107192

9. Goycochea-Valdivia WA, Ares Alvarez J, Conejo Fernández AJ, et al. Position statement of the Spanish Society of Paediatric Infectious diseases on the diagnosis and treatment of Mycoplasma pneumoniae infection. An Pediatr (Engl Ed). 2024;101(1):46-57. doi: 10.1016/j.anpede.2024.05.014

10. Yang Z, Shi R, Zhou X, et al. Shifting epidemic trends and severity in pediatric Mycoplasma pneumoniae infections in the post-COVID-19 era. Ital J Pediatr. 2025;51(1):219. doi: 10.1186/s13052-025-02064-x

11. Ministry of Health of the Russian Federation. Klinicheskie rekomendatsii «Vnebol'nichnaya pnevmoniya u detey» [Clinical guidelines "Community-acquired pneumonia in children"]. Moscow: Ministry of Health of the Russian Federation; 2025.

12. Министерство здравоохранения РФ. Внебольничная пневмония у детей. Клинические рекомендации. М.: Минздрав РФ; 2025.

13. Zar HJ, Hanslo D, Tannenbaum E, et al. Aetiology and outcome of pneumonia in human immunodeficiency virus-infected children hospitalized in South Africa. Acta Paediatr. 2001;90(2):119-125. doi: 10.1111/j.1651-2227.2001.tb00275.x

14. MUK 4.2.3115-13. Laboratornaya diagnostika vnebol'nichnykh pnevmoniy. Metodicheskie ukazaniya [Laboratory diagnosis of community-acquired pneumonia. Methodological guidelines]. Moscow: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor; 2014.

15. МУК 4.2.3115-13. Лабораторная диагностика внебольничных пневмоний. Методические указания. М.: Федеральный центр гигиены и эпидемиологии Роспотребнадзора; 2014.

16. Ebruke BE, Deloria Knoll M, Haddix M, et al. The Etiology of Pneumonia From Analysis of Lung Aspirate and Pleural Fluid Samples: Findings From the Pneumonia Etiology Research for Child Health (PERCH) Study. Clin Infect Dis. 2021;73(11):e3788-e3796. doi: 10.1093/cid/ciaa1032

17. Grant LR, Hammitt LL, Murdoch DR, et al. Procedures for collection of induced sputum specimens from children. Clin Infect Dis. 2012;54 Suppl 2:S140-145. doi: 10.1093/cid/cir1069

18. Lahti E, Peltola V, Waris M, et al. Induced sputum in the diagnosis of childhood community-acquired pneumonia. Thorax. 2009;64(3):252-257. doi: 10.1136/thx.2008.099051

19. Thea DM, Seidenberg P, Park DE, et al. Limited Utility of Polymerase Chain Reaction in Induced Sputum Specimens for Determining the Causes of Childhood Pneumonia in Resource-Poor Settings: Findings From the Pneumonia Etiology Research for Child Health (PERCH) Study. Clin Infect Dis. 2017;64(suppl_3):S289-S300. doi: 10.1093/cid/cix098

20. Zar HJ, Tannenbaum E, Hanslo D, Hussey G. Sputum induction as a diagnostic tool for community-acquired pneumonia in infants and young children from a high HIV prevalence area. Pediatr Pulmonol. 2003;36(1):58-62. doi: 10.1002/ppul.10302

21. Green A, Cockroft JL, Kaufman RA, et al. Utility of Induced Sputum in Assessing Bacterial Etiology for Community-Acquired Pneumonia in Hospitalized Children. J Pediatric Infect Dis Soc. 2022;11(6):274-282. doi: 10.1093/jpids/piac014

22. Rajendran P, Thomas SV, Balaji S, et al. Paediatric pulmonary disease-are we diagnosing it right? Front Pediatr. 2024;12:1370687. doi: 10.3389/fped.2024.1370687

23. Kim KH, Hong JY, Lee H, et al. Nasopharyngeal pneumococcal carriage of children attending day care centers in Korea: comparison between children immunized with 7-valent pneumococcal conjugate vaccine and non-immunized. J Korean Med Sci. 2011;26(2):184-190. doi: 10.3346/jkms.2011.26.2.184

24. Bosch AATM, van Houten MA, Bruin JP, et al. Nasopharyngeal carriage of Streptococcus pneumoniae and other bacteria in the 7th year after implementation of the pneumococcal conjugate vaccine in the Netherlands. Vaccine. 2016;34(4):531-539. doi: 10.1016/j.vaccine.2015.11.060

25. Vu HT, Yoshida LM, Suzuki M, et al. Association between nasopharyngeal load of Streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in Vietnamese children. Pediatr Infect Dis J. 2011;30(1):11-18. doi: 10.1097/INF.0b013e3181f111a2

26. Deloria Knoll M, Morpeth SC, Scott JAG, et al. Evaluation of Pneumococcal Load in Blood by Polymerase Chain Reaction for the Diagnosis of Pneumococcal Pneumonia in Young Children in the PERCH Study. Clin Infect Dis. 2017;64(suppl_3):S357-S367. doi: 10.1093/cid/cix149

27. Smyrnaios A, Krokstad S, Follestad T, et al. The significance of upper airway density of Streptococcus pneumoniae and respiratory viruses in the aetiology and severity of paediatric community-acquired pneumonia in Norway: An observational study. J Microbiol Immunol Infect. 2025;S1684-1182(25)00177-X. doi: 10.1016/j.jmii.2025.08.019

28. Van Eldere J, Slack MP, Ladhani S, Cripps AW. Non-typeable Haemophilus influenzae, an under-recognised pathogen. Lancet Infect Dis. 2014;14(12):1281-1292. doi: 10.1016/S1473-3099(14)70734-0

29. Wang X, Liu Y, Zhang H, et al. Clinical significance of Haemophilus influenzae detection in children with community-acquired pneumonia: a prospective cohort study. Pediatr Pulmonol. 2023;58(4):1023-1031. doi: 10.1002/ppul.26345

30. Nakamura Y, Kamachi K, Toyoizumi-Ajisaka H, et al. Marked difference between adults and children in Bordetella pertussis DNA load in nasopharyngeal swabs. Clin Microbiol Infect. 2011;17(3):365-370. doi: 10.1111/j.1469-0691.2010.03255.x

31. Brotons P, de Paz HD, Toledo D, et al. Differences in Bordetella pertussis DNA load according to clinical and epidemiological characteristics of patients with whooping cough. J Infect. 2016;72(4):460-467. doi: 10.1016/j.jinf.2016.01.013

32. Michelow IC, Olsen K, Lozano J, et al. Epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children. Pediatrics. 2004;113(4):701-707. doi: 10.1542/peds.113.4.701

33. Waites KB, Xiao L, Liu Y, et al. Mycoplasma pneumoniae from the Respiratory Tract and Beyond. Clin Microbiol Rev. 2017;30(3):747-809. doi: 10.1128/CMR.00114-16

34. Kakuya F, Kinebuchi T, Okubo H, Matsuo K. Comparison of Oropharyngeal and Nasopharyngeal Swab Specimens for the Detection of Mycoplasma pneumoniae in Children with Lower Respiratory Tract Infection. J Pediatr. 2017;189:218-221. doi: 10.1016/j.jpeds.2017.06.038

35. Kitagawa D, Nishihara S, Murata M, et al. PCR sensitivity for Mycoplasma pneumoniae detection in nasopharyngeal and oropharyngeal swabs: a comparative study. J Clin Microbiol. 2025;63(8):e0045825. doi: 10.1128/jcm.00458-25

36. Nelson H, Kayda I, Watson N, et al. Combined oropharyngeal nasal (ON) swabs for the molecular detection of respiratory pathogens including M. pneumoniae in symptomatic children. Microbiol Spectr. 2025;13(10):e0218125. doi: 10.1128/spectrum.02181-25

37. Subspecialty Group of Respiratory, the Society of Pediatrics, Chinese Medical Association; et al. Evidence-based guideline for the diagnosis and treatment of Mycoplasma pneumoniae pneumonia in children (2023). Pediatr Investig. 2025;9(1):1-11. doi: 10.1002/ped4.12469

38. Spuesens EB, Fraaij PL, Visser EG, et al. Carriage of Mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study. PLoS Med. 2013;10(5):e1001444. doi: 10.1371/journal.pmed.1001444

39. Meyer Sauteur PM, Krautter S, Ambroggio L, et al. M. pneumoniae carriage in children with CAP: a systematic review. Eur J Pediatr. 2022;181(8):2989-3000. doi: 10.1007/s00431-022-04567-9

40. Smith M, Johnson K, Williams T, et al. Persistent Mycoplasma pneumoniae infection and recurrent respiratory infections in children: a systematic review and meta-analysis. Pediatr Infect Dis J. 2024;43(2):112-120. doi: 10.1097/INF.0000000000004123

41. Zhou Z, Li X, Chen Y, et al. Molecular mechanisms of macrolide resistance in Mycoplasma pneumoniae: an update. Clin Microbiol Rev. 2024;37(3):e00123-23. doi: 10.1128/cmr.00123-23

42. Waites KB, Crabb DM, Duffy LB, et al. Macrolide-resistant Mycoplasma pneumoniae in the United States and Europe: a 10-year surveillance study. Clin Microbiol Rev. 2023;36(2):e00112-22. doi: 10.1128/cmr.00112-22

43. Zhou Z, Li X, Chen Y, et al. Global epidemiology of macrolide-resistant Mycoplasma pneumoniae: a systematic review and meta-analysis. Lancet Microbe. 2024;5(3):100234. doi: 10.1016/S2666-5247(24)00012-3

44. Korneenko E, Rog I, Chudinov I, et al. Antibiotic resistance and viral co-infection in children diagnosed with pneumonia caused by Mycoplasma pneumoniae admitted to Russian hospitals during October 2023-February 2024. BMC Infect Dis. 2025;25(1):363. doi: 10.1186/s12879-025-10712-0

45. To K, Lee C, Wong S, et al. Diagnostic accuracy of saliva for respiratory pathogens in children: a systematic review and meta-analysis. J Clin Virol. 2023;165:105521. doi: 10.1016/j.jcv.2023.105521

46. Merida Vieyra J, De Colsa Ranero A, Palacios Reyes D, et al. Chlamydophila pneumoniae-associated community-acquired pneumonia in paediatric patients of a tertiary care hospital in Mexico: molecular diagnostic and clinical insights. Sci Rep. 2023;13(1):21477. doi: 10.1038/s41598-023-48701-5

47. Ma R, Zhang Y, Wang Y, et al. Epidemiological and clinical analysis of 291 children diagnosed with Chlamydia pneumoniae pneumonia: a 10-year retrospective study in Shijiazhuang, China. Front Pediatr. 2025;13:1681564. doi: 10.3389/fped.2025.1681564

48. Tosh PK, Kennedy JL, Patel R, et al. Chlamydia pneumoniae infections in children: clinical features and diagnostic challenges. Pediatr Infect Dis J. 2023;42(5):412-418. doi: 10.1097/INF.0000000000003845

49. Pneumonia Etiology Research for Child Health (PERCH) Study Group. Causes of severe pneumonia requiring hospital admission in children without HIV infection from Africa and Asia: the PERCH multi-country case-control study. Lancet. 2019;394(10200):757-779. doi: 10.1016/S0140-6736(19)30721-4

50. Man WH, van Houten MA, Mérelle ME, et al. Bacterial and viral respiratory tract microbiota and host characteristics in children with lower respiratory tract infections: a matched case-control study. Lancet Respir Med. 2019;7(5):417-426. doi: 10.1016/S2213-2600(18)30449-1

51. Wrotek A, Robakiewicz J, Pawlik K, et al. The Etiology of Community-Acquired Pneumonia Correlates with Serum Inflammatory Markers in Children. J Clin Med. 2022;11(19):5506. doi: 10.3390/jcm11195506

52. Li Y, Wang X, Zhang L, et al. Burden of human metapneumovirus and parainfluenza virus infections in children under 5 years: a systematic review and meta-analysis. Lancet Child Adolesc Health. 2023;7(8):567-576. doi: 10.1016/S2352-4642(23)00123-4

53. Di Maio VC, Scutari R, Mastropaolo M, et al. Viral Burden of Respiratory Syncytial Virus and Viral Coinfections as Factors Regulating Paediatric Disease Severity. Viruses. 2025;17(9):1236. doi: 10.3390/v17091236

54. Chen Q, Lin L, Zhang N, Yang Y. Adenovirus and Mycoplasma pneumoniae co-infection as a risk factor for severe community-acquired pneumonia in children. Front Pediatr. 2024;12:1337786. doi: 10.3389/fped.2024.1337786

55. Kim JH, Park S, Lee Y, et al. Cycle threshold values as predictors of severe Mycoplasma pneumoniae pneumonia in children. J Korean Med Sci. 2023;38(15):e112. doi: 10.3346/jkms.2023.38.e112

56. Zhang L, Wang H, Chen X, et al. Association between M. pneumoniae DNA load and clinical outcomes in children with community-acquired pneumonia. Front Pediatr. 2022;10:876543. doi: 10.3389/fped.2022.876543

57. Zinter MS, Dvorak CC, Mayday MY, et al. Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children. Clin Infect Dis. 2019;68(11):1847-1855. doi: 10.1093/cid/ciy802

58. Wishaupt JO, Ploeg TV, Smeets LC, et al. Pitfalls in interpretation of CT-values of RT-PCR in children with acute respiratory tract infections. J Clin Virol. 2017;90:1-6. doi: 10.1016/j.jcv.2017.02.010

59. Hardt M, Kaiser F, Voss T, et al. Pre-analytical properties of different respiratory viruses for PCR-based detection: Comparative analysis of sampling devices and sample stabilization solutions. N Biotechnol. 2024;79:60-70. doi: 10.1016/j.nbt.2023.12.005

60. Barrera-Avalos C, Luraschi R, Vallejos-Vidal E, et al. Analysis by real-time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID-19 diagnosis in Santiago of Chile. J Med Virol. 2022;94(3):1167-1174. doi: 10.1002/jmv.27446

61. Park S, Kim J, Lee H, et al. Effect of sampling time on cycle threshold values in pediatric respiratory infections. J Clin Virol. 2023;165:105499. doi: 10.1016/j.jcv.2023.105499

62. Betsou F, Chuaqui R, De-Wilde A, et al. Standard PREanalytical Code Version 4.0. Biopreserv Biobank. 2025;23(4):328-332. doi: 10.1089/bio.2024.0010

63. Yu X, Liang J, Yang R, et al. Clinical Features and Value of Tracheal Aspirate Metagenomic Next-Generation Sequencing for Severe Pneumonia in Children in Pediatric Intensive Care Unit. Pol J Microbiol. 2025;74(2):192-205. doi: 10.33073/pjm-2025-016

64. Schuetz P, Wirz Y, Sager R, et al. Effect of procalcitonin-guided antibiotic treatment on mortality in acute respiratory infections: a patient level meta-analysis. Lancet Infect Dis. 2018;18(1):95-107. doi: 10.1016/S1473-3099(17)30592-3

65. Schuetz P, Wirz Y, Sager R, et al. Procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections. Cochrane Database Syst Rev. 2017;10(10):CD007498. doi: 10.1002/14651858.CD007498.pub3

66. Baumann P, Baer G, Bonhoeffer J, et al. Procalcitonin for Diagnostics and Treatment Decisions in Pediatric Lower Respiratory Tract Infections. Front Pediatr. 2017;5:183. doi: 10.3389/fped.2017.00183

67. Gunaratnam LC, Robinson JL, Hawkes MT. Systematic Review and Meta-Analysis of Diagnostic Biomarkers for Pediatric Pneumonia. J Pediatric Infect Dis Soc. 2021;10(9):891-900. doi: 10.1093/jpids/piab043

68. Norman-Bruce H, Umana E, Mills C, et al. Diagnostic test accuracy of procalcitonin and C-reactive protein for predicting invasive and serious bacterial infections in young febrile infants: a systematic review and meta-analysis. Lancet Child Adolesc Health. 2024;8(5):358-368. doi: 10.1016/S2352-4642(24)00021-X

69. Omaggio L, Franzetti L, Caiazzo R, et al. Utility of C-reactive protein and procalcitonin in community-acquired pneumonia in children: a narrative review. Curr Med Res Opin. 2024;40(12):2191-2200. doi: 10.1080/03007995.2024.2425383

70. Sodero G, Gentili C, Mariani F, et al. Procalcitonin and Presepsin as Markers of Infectious Respiratory Diseases in Children: A Scoping Review of the Literature. Children (Basel). 2024;11(3):350. doi: 10.3390/children11030350

71. Spellberg B, Nielsen TB, Phillips MC, et al. Revisiting diagnostics: erythrocyte sedimentation rate and C-reactive protein: it is time to stop the zombie tests. Clin Microbiol Infect. 2025;31(1):1-4. doi: 10.1016/j.cmi.2024.08.017

72. Saleh NY, Hassan FM, Omar TA, et al. Pediatric community-acquired pneumonia: predictive value of heparin-binding protein for severity assessment. Pediatr Res. 2025. doi: 10.1038/s41390-025-04605-w

73. He X, Zou Y, Li Y, et al. Clinical Values of Combined Heparin-Binding Protein and Procalcitonin Testing in the Diagnosis and Management of Severe Pneumonia in Children. Br J Hosp Med (Lond). 2025;86(9):1-20. doi: 10.12968/hmed.2025.0173

74. Chen Y, Wang L, Zhang H, et al. Diagnostic accuracy of heparin-binding protein for bacterial infections in children: a systematic review and meta-analysis. Front Pediatr. 2024;12:1456789. doi: 10.3389/fped.2024.1456789

75. Kaforou M, Herberg JA, Wright VJ, et al. Diagnosis of Bacterial Infection Using a 2-Transcript Host RNA Signature in Febrile Infants 60 Days or Younger. JAMA. 2017;317(15):1577-1578. doi: 10.1001/jama.2017.1365

76. Herberg JA, Kaforou M, Wright VJ, et al. Diagnostic Test Accuracy of a 2-Transcript Host RNA Signature for Discriminating Bacterial vs Viral Infection in Febrile Children. JAMA. 2016;316(8):835-845. doi: 10.1001/jama.2016.11236

77. Mahajan P, Kuppermann N, Mejias A, et al. Association of RNA Biosignatures With Bacterial Infections in Febrile Infants Aged 60 Days or Younger. JAMA. 2016;316(8):846-857. doi: 10.1001/jama.2016.9207

78. Lee RA, Al Dhaheri F, Pollock NR, Sharma TS. Assessment of the Clinical Utility of Plasma Metagenomic Next-Generation Sequencing in a Pediatric Hospital Population. J Clin Microbiol. 2020;58(7):e00419-20. doi: 10.1128/JCM.00419-20

79. Shang H, Zou S, Ma Z, et al. Comparative and clinical impact of targeted next-generation sequencing in pediatric pneumonia diagnosis and treatment. Front Microbiol. 2025;16:1590792. doi: 10.3389/fmicb.2025.1590792

80. Ruan Z, Shi H, Chang L, et al. The diagnostic efficacy of metagenomic next-generation sequencing (mNGS) in pathogen identification of pediatric pneumonia using bronchoalveolar lavage fluid (BALF): A systematic review and meta-analysis. Microb Pathog. 2025;203:107492. doi: 10.1016/j.micpath.2025.107492

81. Man WH, de Steenhuijsen Piters WA, Bogaert D. The microbiota of the respiratory tract: gatekeeper to respiratory health. Nat Rev Microbiol. 2017;15(5):259-270. doi: 10.1038/nrmicro.2017.14

82. de Steenhuijsen Piters WAA, Watson RL, de Koff EM, et al. Early-life viral infections are associated with disadvantageous immune and microbiota profiles and recurrent respiratory infections. Nat Microbiol. 2022;7(2):224-237. doi: 10.1038/s41564-021-01043-2

83. Wang Y, Xu Q, Zhang L, et al. Impact of COVID-19 non-pharmaceutical interventions on the upper respiratory microbiota in young children. Front Microbiol. 2025;16:1256789. doi: 10.3389/fmicb.2025.1256789

84. Liang C, Liu Y, Zhang W, et al. Changes in nasopharyngeal and oropharyngeal microbiota in healthy children during the COVID-19 pandemic. World J Pediatr. 2025;21(2):156-165. doi: 10.1007/s12519-025-00890-1

85. Kim J, Park S, Lee H, et al. Gut microbiota and immune pathway alterations in young children with COVID-19. Microorganisms. 2025;13(2):345. doi: 10.3390/microorganisms13020345

86. Luo Y, Wu R, Wu W, et al. Differences in pulmonary microbiota of severe community-acquired pneumonia with different pathogenic microorganisms in children. BMC Pediatr. 2025;25(1):449. doi: 10.1186/s12887-025-05819-x