Abstract
Background
During the coronavirus disease 2019 (COVID-19) pandemic, non-pharmaceutical interventions (NPIs) reduced the transmission of severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) while simultaneously affecting the prevalence of respiratory pathogens to varying degrees. Currently, epidemiological characteristics vary across different regions and among different pathogens, and there is a lack of continuous surveillance data following the outbreak. This study aims to analyze the epidemiological characteristics of six respiratory pathogens in hospitalized children under the age of 5 years with acute respiratory infections (ARIs) in the Qingdao region following the COVID-19 pandemic, thereby providing a reference for developing prevention and treatment strategies for respiratory infections.
Methods
Children aged <5 years who were hospitalized with ARIs between May 1, 2023, and June 30, 2025, were enrolled in this retrospective, single-center, repeated cross-sectional surveillance study. Nucleic acid testing was performed to detect influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), adenovirus (ADV), human rhinovirus (HRV), and Mycoplasma pneumoniae (MP). Detection results were analyzed by age group. Between-group comparisons were conducted using the Chi-squared test with Bonferroni correction.
Results
A total of 16,613 cases were included, with an overall pathogen detection rate of 50.62% (8,409/16,613). The three most frequently detected pathogens were RSV (19.74%), MP (16.81%), and HRV (10.97%). RSV demonstrated the highest detection rate in children aged 0–<1 year (25.95%), accounting for 56.37% (1,849/3,280) of all RSV-positive cases. The detection rate of MP increased progressively with age, reaching 35.34% among children aged 4–<5 years. Detection rates for the remaining pathogens were lowest in the 0–<1 year age group. Months with the highest monthly RSV polymerase chain reaction (PCR) positivity were May 2023 (66.46%), January 2024 (43.48%), and April 2025 (42.78%). Months with the highest monthly MP PCR positivity were October 2023 (29.28%) and September 2024 (54.90%), with persistently elevated monthly detection proportions recorded from August 2023 to December 2024. Months with maximal monthly positivity for FluA, RSV, and MP showed visual separation on temporal plots; HRV and MP exhibited concurrent high monthly positivity during October–November 2023.
Conclusions
RSV and MP were the most frequently detected pathogens among the six tested targets in hospitalized children aged <5 years with ARIs in Qingdao, China. Infants aged <1 year exhibited the highest RSV PCR positivity rate, while MP PCR positivity rose progressively with age. Months with maximal RSV and MP detection proportions showed visually separated temporal maxima. FluA and FluB maintained low epidemic intensity throughout the study period, while HRV circulated continuously year-round.
Keywords: Children, pathogen, prevention, respiratory tract infection
Highlight box.
Key findings
• In the Qingdao area of China, among hospitalized children under the age of 5 years with acute respiratory tract infections, respiratory syncytial virus (RSV) and Mycoplasma pneumoniae (MP) are the two most frequently detected pathogens among the six tested targets. The positive rate of RSV polymerase chain reaction (PCR) in infants under the age of 1 is the highest, while the PCR positive rate of MP gradually increases with age. The months with the highest detection rates of RSV and MP show distinct peak values in time. The prevalence of influenza A virus and influenza B virus remained at a relatively low level throughout the study period, while human rhinovirus was continuously spreading throughout the year.
What is known and what is new?
• The non-pharmaceutical intervention measures, such as wearing masks and frequent handwashing, implemented in China in 2020 have changed the state of herd immunity. There are still differences in the epidemic characteristics of different regions and different pathogens.
• The Qingdao area still lacks continuous monitoring data after the epidemic. This study provides data of the Qingdao area.
What is the implication, and what should change now?
• This study provides a reference for formulating prevention and control strategies for respiratory infections after the pandemic. Subsequently, the clinical composition of the study cohort, including information on intensive care unit admission, oxygen therapy requirements, comorbidities, and length of hospital stay, will be further elaborated.
Introduction
Acute respiratory infections (ARIs) are among the most common infectious diseases and represent a leading cause of hospitalization and mortality among children aged <5 years (1). Common etiological agents include influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), adenovirus (ADV), human rhinovirus (HRV), and Mycoplasma pneumoniae (MP), with epidemiological patterns varying according to geographic region and season (2,3). Owing to the non-specific clinical manifestations of ARIs, etiological diagnosis based solely on clinical presentation remains challenging. In recent years, nucleic acid-based molecular diagnostic methods, characterized by rapid turnaround time and high sensitivity, have been widely applied for the detection of respiratory pathogens in children. These methods support clinical decision-making, reduce inappropriate antibiotic use, and provide important epidemiological data for characterizing circulating respiratory pathogens.
The implementation of non-pharmaceutical interventions (NPIs) in China in 2020, including mandatory mask use and enhanced hand hygiene practices, substantially altered population-level immunity patterns. Since 2023, the incidence of ARIs among children has remained consistently elevated, imposing a considerable burden on pediatric healthcare services. Epidemiological trends continue to differ across regions and pathogens, while longitudinal surveillance data from the post-pandemic period remain limited. Therefore, continuous surveillance and analysis of ARI pathogen distribution across age groups and geographic regions are essential for informing region-specific prevention, control, and treatment strategies. While recent single-center studies have described pediatric respiratory pathogen epidemiology across Chinese cities (4-6), critical gaps remain in post-pandemic surveillance data from coastal northern regions such as Qingdao, China. Most existing reports either cover shorter observation windows (<18 months) or lack granular age-stratified analysis of MP circulation patterns. This study addresses these limitations by leveraging a 26-month post-NPI surveillance window (May 2023–June 2025), a large cohort of 16,613 hospitalized ARI episodes, and detailed age-disaggregated (5 subgroups spanning 0–<5 years) and temporal analyses to characterize the prolonged MP epidemic and non-overlapping seasonal peaks of key pathogens in this understudied region. We present this article in accordance with the STROBE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0509/rc).
Methods
Study participants
This retrospective single-center repeated cross-sectional surveillance study recruited children aged <5 years admitted to Qingdao Women and Children’s Hospital with a diagnosis of ARI between May 1, 2023, and June 30, 2025. The diagnostic criteria for ARIs (7) included acute onset accompanied by at least one of the following symptoms: rhinorrhea, sore throat, cough, wheezing, dyspnea, or tachypnea, with or without fever. The exclusion criteria were as follows: (I) presence of primary immunodeficiency disorders, hematologic malignancies, a history of hematopoietic stem cell transplantation, or long-term glucocorticoid therapy; (II) repeated positive test results from the same individual within a 1-week interval; and (III) receipt of nirsevimab at any time between birth and hospital admission.
Distinct ARI hospitalization episodes were defined as the unit of analysis for this study. Multiple admissions from an identical child separated by an interval longer than 7 days were retained as independent infection episodes, as each admission represented a new acute respiratory illness event. No additional repeated specimens from the same patient within 7 days were enrolled, as specified by exclusion criterion II. Inpatient data were obtained from the medical record system of Qingdao Women and Children’s Hospital affiliated with Qingdao University. Participants were stratified into five age groups: 0–<1, 1–<2, 2–<3, 3–<4, and 4–<5 years.
The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Review Committee of Qingdao Women and Children’s Hospital (No. QFELL-YJ-2026-49; approval date: 2026-02-06). Informed consent was waived in this retrospective study.
Specimen collection, polymerase chain reaction (PCR) assay, and positivity classification
Throat swab specimens were collected from enrolled participants within 24 hours of hospital admission, including specimens obtained in outpatient settings within 24 hours prior to hospitalization. All hospitalized children meeting the ARI diagnostic criteria within the study timeframe received uniform six-pathogen nucleic acid PCR testing upon admission, without selective testing based on clinical symptoms or physician judgment. All swabs were collected by fully trained medical staff following a standardized sampling protocol: sterile flocked swabs were used to swab bilateral tonsils and posterior pharyngeal wall. Specimens were immersed in proprietary preservation buffer, transported at ambient temperature with a maximum stable storage duration of 4 hours at room temperature; for delayed processing, samples were stored at 4 ℃ for no more than 24 hours (maximum theoretical storage limit of 48 h as stated by the kit manual). No adjustments to specimen collection protocols or molecular testing workflows were implemented throughout the entire study period.
Nucleic acid extraction was performed via an automated magnetic bead extraction method. The six-pathogen respiratory nucleic acid detection kit (PCR-fluorescent probe method, Sansure Biotech Inc., Changsha, China; National Medical Device Registration No. 20213400256) was applied on the Molarray MA-6000 real-time quantitative thermal cycler strictly per manufacturer instructions. Genomic target regions specific to each pathogen were amplified and detected via fluorescent probes. A cycle threshold (Ct) value ≤40 was defined as a positive diagnostic result for all target pathogens.
Endogenous human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was amplified as an internal control to monitor specimen quality and extraction efficiency. Kit-supplied independent positive and negative control samples were processed alongside each batch of patient specimens to exclude contamination and instrument failure. The analytical limit of detection (LOD) of the kit was provided as follows: FluA [2.0 50% tissue culture infectious dose per mL (TCID50/mL)], FluB (2.0 TCID50/mL), RSV (500.0 copies/mL), ADV (500.0 copies/mL), HRV (500.0 copies/mL), and MP (500.0 copies/mL).
Specimens with a positive nucleic acid result for a single pathogen were classified as single-pathogen infections, whereas simultaneous detection of two or more pathogens was defined as co-detection. Monthly counts of positive results for each pathogen were aggregated, positivity rates were calculated, and temporal trends in pathogen distribution were analyzed.
Statistical analysis
Data management and figure preparation were performed using Excel 2016; all statistical analyses were implemented via SPSS 22.0. No missing demographic, clinical, or laboratory data were present for the final analytic cohort of 16,613 hospitalization episodes, so no imputation or exclusion for incomplete records was required.
First, univariate between-group comparisons of pathogen PCR positivity across age groups were conducted using Pearson’s χ2 test. Assumptions for χ2 testing were verified in advance: all contingency table cell counts exceeded 5, satisfying the minimum expected frequency requirement.
For post-hoc pairwise age-group comparisons within each pathogen, the χ2 partition method with Bonferroni correction was applied. The corrected significance threshold was set at α'=0.05/10=0.005 to account for the 10 pairwise comparisons among five age strata per single pathogen; no additional correction across the six distinct pathogens was implemented in the primary univariate analysis, which is acknowledged as a limitation.
To eliminate confounding from temporal factors (calendar month, study year) and sex, separate multivariable binary logistic regression models were constructed for each pathogen. Pathogen PCR positivity (positive =1, negative =0) was defined as the binary dependent variable, while age group, sex, study year, and calendar month were included as adjusted covariates. Adjusted odds ratios (aORs) with corresponding 95% confidence intervals (CIs) were calculated and summarized in Table S1 to quantify independent associations between age and pathogen positivity after confounding adjustment.
Absolute differences in pathogen positivity rates between age groups were reported alongside statistical test results to distinguish statistically significant differences from clinically trivial disparities, given the large sample size that readily yields significant P values for minor numerical gaps. Categorical variables were summarized as counts and percentages. Two-sided P<0.05 was defined as the overall statistical significance threshold for regression models; corrected P<0.005 was used for pairwise age comparisons within each pathogen.
Results
General characteristics
All 17,258 potentially eligible hospitalized children under 5 years with ARIs identified during the study period were subjected to standardized six-pathogen PCR testing on admission. A total of 645 participants were excluded per preset criteria: (I) 1 case of primary immunodeficiency disorders, 502 cases of hematologic malignancies, 2 cases with hematopoietic stem cell transplantation history, and 140 cases on long-term glucocorticoid therapy; (II) zero repeated specimens from the same patient within 1 week; (III) zero infants with prior nirsevimab administration. Following exclusions, a final analytic cohort of 16,613 children was established (Figure 1). Of these, 9,495 (57.15%) were male and 7,118 (42.85%) were female, with a male-to-female ratio of 1.33:1. During the study period, 7,454 cases (44.87%) were enrolled between May 2023 and April 2024, and 9,159 cases (55.13%) were enrolled between May 2024 and June 2025. The age distribution was as follows: 7,125 cases (42.89%) in the 0–<1 year group, 2,607 cases (15.69%) in the 1–<2 years group, 1,782 cases (10.73%) in the 2–<3 years group, 2,578 cases (15.52%) in the 3–<4 years group, and 2,521 cases (15.17%) in the 4–<5 years group (Table 1).
Figure 1.
The participant flow diagram of this study.
Table 1. Demographic and clinical characteristics of hospitalized children aged <5 years with acute respiratory infections in Qingdao, 2023–2025.
| Characteristics | Cases | Proportion (%) |
|---|---|---|
| Gender | ||
| Male | 9,495 | 57.15 |
| Female | 7,118 | 42.85 |
| Age group (years) | ||
| 0–<1 | 7,125 | 42.89 |
| 1–<2 | 2,607 | 15.69 |
| 2–<3 | 1,782 | 10.73 |
| 3–<4 | 2,578 | 15.52 |
| 4–<5 | 2,521 | 15.17 |
| Year | ||
| 2023.05–2024.04 | 7,454 | 44.87 |
| 2024.05–2025.06 | 9,159 | 55.13 |
| Total | 16,613 | 100.00 |
Pathogen detection
Among throat swab specimens collected from the 16,613 participants, the overall pathogen positivity rate was 50.62%. RSV demonstrated the highest detection rate (19.74%), followed by MP (16.81%), HRV (10.97%), ADV (4.87%), FluA (4.29%), and FluB (1.16%). The proportions of single-pathogen infections, in descending order, were as follows: RSV (86.34%), FluA (76.97%), MP (75.97%), FluB (74.09%), HRV (64.45%), and ADV (56.74%). The proportions of co-detections, in descending order, were ADV (43.26%), HRV (35.55%), FluB (25.91%), MP (24.03%), FluA (23.03%), and RSV (13.66%) (Table 2).
Table 2. Detection of respiratory pathogens in hospitalized children under 5 years old with acute respiratory infections in Qingdao area from 2023 to 2025.
| Pathogen | Positive detection, n (%) | Pathogen detection, n (%) | ||
|---|---|---|---|---|
| Single | Two | Three or more | ||
| FluA | 712 (4.29) | 548 (3.30) | ||
| FluB | 193 (1.16) | 143 (0.86) | ||
| RSV | 3,280 (19.74) | 2,848 (17.14) | ||
| ADV | 809 (4.87) | 459 (2.76) | ||
| HRV | 1,823 (10.97) | 1,175 (7.07) | ||
| MP | 2,792 (16.81) | 2,121 (12.77) | ||
| FluA + FluB | 1 (0.006) | |||
| FluA + RSV | 30 (0.18) | |||
| FluA + ADV | 22 (0.13) | |||
| FluA + HRV | 17 (0.10) | |||
| FluA + MP | 64 (0.39) | |||
| FluB + RSV | 19 (0.11) | |||
| FluB + ADV | 9 (0.05) | |||
| FluB + HRV | 3 (0.02) | |||
| FluB + MP | 10 (0.06) | |||
| RSV + ADV | 63 (0.38) | |||
| RSV + HRV | 190 (1.14) | |||
| RSV + MP | 87 (0.52) | |||
| ADV + HRV | 68 (0.41) | |||
| ADV + MP | 123 (0.74) | |||
| HRV + MP | 322 (1.94) | |||
| FluA + RSV + MP | 5 (0.03) | |||
| FluA + RSV + ADV | 8 (0.05) | |||
| FluA + RSV + HRV | 1 (0.006) | |||
| FluA + ADV + HRV | 2 (0.01) | |||
| FluA + ADV + MP | 11 (0.07) | |||
| FluA + HRV + MP | 4 (0.02) | |||
| FluB + RSV + ADV | 2 (0.01) | |||
| FluB + RSV + MP | 3 (0.02) | |||
| FluB + HRV + MP | 3 (0.02) | |||
| RSV + ADV + HRV | 10 (0.06) | |||
| RSV + ADV + MP | 10 (0.06) | |||
| RSV + HRV + MP | 2 (0.01) | |||
| ADV + HRV + MP | 22 (0.13) | |||
| FluA + RSV + ADV + MP | 2 (0.01) | |||
| RSV + ADV + HRV + MP | 2 (0.01) | |||
| Total | 7,294 (43.91) | 1,028 (6.19) | 87 (0.52) | |
ADV, adenovirus; FluA, influenza A virus; FluB, influenza B virus; HRV, human rhinovirus; MP, Mycoplasma pneumoniae; RSV, respiratory syncytial virus.
Age-specific pathogen characteristics
Detection rates for all six pathogens differed significantly across age groups (P<0.001) (Table 3). RSV demonstrated the highest detection rate in the 0–<1 year age group (25.95%). Detection rates in the three age groups spanning 1–<4 years were each significantly lower than that observed in the 0–<1 year group (P<0.005), whereas no significant differences were identified among these three age groups. In the 4–<5 years group, the RSV detection rate declined further and was significantly lower than those observed in the other four age groups (P<0.005). The detection rate of MP increased progressively with age, rising from 6.81% in the 0–<1 year group to 35.34% in the 4–<5 years group. Detection rates of FluA, FluB, ADV, and HRV were significantly lower in the 0–<1 year group than in all other age groups (P<0.005). No significant differences in the detection rates of FluA, FluB, or ADV were observed among children aged 1–<5 years. HRV detection rates did not differ significantly among the 2–<5 years age groups but were significantly higher than those in the 1–<2 years group (P<0.005). The youngest age at detection for each pathogen was as follows: FluA, 8 days; FluB, 11 days; RSV, 7 days; ADV, 7 days; HRV, 6 days; and MP, 7 days. Multivariable logistic regression models adjusted for sex, study year, and calendar month were further applied to evaluate age-related disparities in each pathogen’s positivity rate. aORs and 95% CIs for age groups relative to the reference 0–<1 year stratum are presented in Table S1, confirming that the age-based distribution patterns observed in crude χ2 analyses remained consistent after controlling for temporal and demographic confounders. Absolute rate differences between age subgroups were also tabulated to aid clinical interpretation of group disparities.
Table 3. Age-specific distribution of respiratory pathogens among hospitalized children aged <5 years with acute respiratory infections in Qingdao, 2023–2025.
| Age | Total (n=16,613), n | No. of positive (%) | |||||
|---|---|---|---|---|---|---|---|
| FluA (n=712) | FluB (n=193) | RSV (n=3,280) | ADV (n=809) | HRV (n=1,823) | MP (n=2,792) | ||
| 0–<1 year | 7,125 | 209 (2.93) | 38 (0.53) | 1,849 (25.95) | 145 (2.04) | 697 (9.78) | 485 (6.81) |
| 1–<2 years | 2,607 | 157 (6.02)† | 36 (1.38)† | 400 (15.34)† | 166 (6.37)† | 283 (10.86) | 406 (15.57)† |
| 2–<3 years | 1,782 | 103 (5.78)† | 30 (1.68)† | 290 (16.27)† | 135 (7.58)† | 216 (12.12)† | 405 (22.73)†,‡ |
| 3–<4 years | 2,578 | 127 (4.93)† | 45 (1.75)† | 407 (15.79)† | 176 (6.83)† | 334 (12.96)† | 605 (23.47)†,‡ |
| 4–<5 years | 2,521 | 116 (4.60)† | 44 (1.75)† | 334 (13.25)†,‡,§,¶ | 187 (7.42)† | 293 (11.62)† | 891 (35.34)†,‡,§,¶ |
| χ2 value | 82.79 | 33.05 | 472.57 | 283.8 | 23.49 | 1,109.48 | |
| P value | <0.001 | <0.001 | < 0.001 | <0.001 | <0.001 | <0.001 | |
Following Bonferroni correction, the adjusted significance threshold is α'=0.05/10=0.005. †, compared with the 0–<1 year age group, P<0.005; ‡, compared with the 1–<2 years age group, P<0.005; §, compared with the 2–<3 years age group, P<0.005; ¶, compared with the 3–<4 years age group, P<0.005. ADV, adenovirus; FluA, influenza A virus; FluB, influenza B virus; HRV, human rhinovirus; MP, Mycoplasma pneumoniae; No., number; RSV, respiratory syncytial virus.
Seasonal characteristics of each pathogen
During the 26-month study period, the six pathogens demonstrated seasonal epidemic patterns with varying levels of activity (Table 4, Figure 2). FluA and MP each demonstrated 2 months with the highest positivity proportion. FluA peaked in November 2023 (29.51%) and January 2025 (23.62%), whereas MP peaked in October 2023 (29.28%) and September 2024 (54.90%). RSV demonstrated three months with the highest positivity proportion, occurring in May 2023 (66.46%), January 2024 (43.48%), and April 2025 (42.78%). Sustained high detection rates were observed from November 2023 through March 2024 and from January through May 2025. FluB reached a peak detection rate of 10% in February 2024 and remained below 10% during all other months. ADV reached its highest detection rate in December 2023 (13.10%), after which detection rates remained consistently low without a distinct seasonal pattern. HRV demonstrated peak detection rates of 26.60% in October 2023 and 20.42% in October 2024. During the remaining months, detection rates fluctuated around 10%, with no clear seasonal trend observed.MP detection rates remained elevated from August 2023 through December 2024, with the exception of January 2024. Months with maximal monthly positivity for FluA, RSV, and MP were visually separated on temporal curves without formal time-series correlation testing to confirm non-synchronous circulation or pathogen competitive interactions. In contrast, HRV and MP co-circulated during October–November 2023.
Table 4. Monthly distribution of nucleic acid positivity rates for six respiratory pathogens among hospitalized children aged <5 years with acute respiratory infections.
| Year/month | No. of cases | FluA positive | FluB positive | RSV positive | ADV positive | HRV positive | MP positive |
|---|---|---|---|---|---|---|---|
| 2023/5 | 656 | 1 (0.15) | 0 (0.00) | 436 (66.46) | 10 (1.52) | 83 (12.65) | 2 (0.30) |
| 2023/6 | 616 | 0 (0.00) | 0 (0.00) | 256 (41.56) | 19 (3.08) | 39 (6.33) | 8 (1.30) |
| 2023/7 | 617 | 0 (0.00) | 0 (0.00) | 81 (13.13) | 23 (3.73) | 55 (8.91) | 23 (3.73) |
| 2023/8 | 563 | 0 (0.00) | 0 (0.00) | 34 (6.04) | 20 (3.55) | 42 (7.46) | 60 (10.66) |
| 2023/9 | 568 | 2 (0.35) | 0 (0.00) | 40 (7.04) | 39 (6.87) | 82 (14.44) | 130 (22.89) |
| 2023/10 | 485 | 19 (3.92) | 0 (0.00) | 51 (10.52) | 54 (11.13) | 129 (26.60) | 142 (29.28) |
| 2023/11 | 532 | 157 (29.51) | 0 (0.00) | 61 (11.47) | 66 (12.41) | 62 (11.65) | 125 (23.5) |
| 2023/12 | 580 | 62 (10.69) | 2 (0.34) | 209 (36.03) | 76 (13.10) | 32 (5.52) | 88 (15.17) |
| 2024/1 | 821 | 17 (2.07) | 57 (6.94) | 357 (43.48) | 81 (9.87) | 47 (5.72) | 67 (8.16) |
| 2024/2 | 720 | 4 (0.56) | 72 (10.00) | 271 (37.64) | 51 (7.08) | 36 (5.00) | 75 (10.42) |
| 2024/3 | 765 | 5 (0.65) | 40 (5.23) | 111 (14.51) | 40 (5.23) | 83 (10.85) | 113 (14.77) |
| 2024/4 | 531 | 5 (0.94) | 7 (1.32) | 30 (5.65) | 22 (4.14) | 73 (13.75) | 77 (14.5) |
| 2024/5 | 541 | 0 (0.00) | 1 (0.18) | 1 (0.18) | 2 (0.37) | 30 (5.55) | 117 (21.63) |
| 2024/6 | 507 | 0 (0.00) | 0 (0.00) | 1 (0.20) | 22 (4.34) | 83 (16.37) | 141 (27.81) |
| 2024/7 | 584 | 3 (0.51) | 0 (0.00) | 0 (0.00) | 23 (3.94) | 66 (11.30) | 236 (40.41) |
| 2024/8 | 678 | 7 (1.03) | 0 (0.00) | 4 (0.59) | 22 (3.24) | 69 (10.18) | 301 (44.40) |
| 2024/9 | 643 | 1 (0.16) | 0 (0.00) | 0 (0.00) | 20 (3.11) | 54 (8.40) | 353 (54.90) |
| 2024/10 | 608 | 3 (0.49) | 0 (0.00) | 6 (0.99) | 27 (4.44) | 113 (18.59) | 303 (49.84) |
| 2024/11 | 524 | 8 (1.53) | 0 (0.00) | 15 (2.86) | 20 (3.82) | 107 (20.42) | 184 (35.11) |
| 2024/12 | 723 | 143 (19.78) | 0 (0.00) | 66 (9.13) | 29 (4.01) | 81 (11.2) | 112 (15.49) |
| 2025/1 | 889 | 210 (23.62) | 1 (0.11) | 247 (27.78) | 29 (3.26) | 52 (5.85) | 70 (7.87) |
| 2025/2 | 701 | 55 (7.85) | 2 (0.29) | 193 (27.53) | 19 (2.71) | 33 (4.71) | 23 (3.28) |
| 2025/3 | 818 | 7 (0.86) | 3 (0.37) | 324 (39.61) | 25 (3.06) | 89 (10.88) | 28 (3.42) |
| 2025/4 | 769 | 0 (0.00) | 7 (0.91) | 329 (42.78) | 20 (2.60) | 71 (9.23) | 9 (1.17) |
| 2025/5 | 595 | 2 (0.34) | 1 (0.17) | 135 (22.69) | 24 (4.03) | 97 (16.30) | 4 (0.67) |
| 2025/6 | 579 | 1 (0.17) | 0 (0.00) | 22 (3.80) | 26 (4.49) | 115 (19.86) | 1 (0.17) |
| Total | 16,613 | 712 (4.29) | 193 (1.16) | 3,280 (19.74) | 809 (4.87) | 1,823 (10.97) | 2,792 (16.81) |
Data are presented as n (%). ADV, adenovirus; FluA, influenza A virus; FluB, influenza B virus; HRV, human rhinovirus; MP, Mycoplasma pneumoniae; No., number; RSV, respiratory syncytial virus.
Figure 2.
Seasonal trends in the detection of six respiratory pathogens among hospitalized children aged <5 years with acute respiratory infections. ADV, adenovirus; FluA, influenza A virus; FluB, influenza B virus; HRV, human rhinovirus; MP, Mycoplasma pneumoniae; RSV, respiratory syncytial virus.
Discussion
ARIs account for approximately 20 million pediatric hospitalizations worldwide each year and impose a substantial burden on healthcare systems (8). In China, the incidence of ARIs among children during the winter and spring seasons may exceed 40% (9). Because pulmonary development in children aged <5 years remains incomplete, respiratory infections during early childhood may increase the risk of long-term sequelae, including impaired pulmonary function, recurrent wheezing, and asthma (10). In recent years, nucleic acid-based pathogen detection methods have provided important support for precise etiological diagnosis and targeted clinical management (7).
Pathogen detection
Previous studies conducted in China have identified RSV, FluA, ADV, and MP as the predominant pathogens associated with pediatric ARIs (11,12). In the present study, the overall pathogen detection rate among hospitalized children aged <5 years with ARIs in Qingdao, China was 50.62%, with RSV and MP identified as the predominant pathogens. These findings are generally consistent with reports from Zhongshan, indicating that the overall pathogen spectrum of pediatric ARIs may not have changed substantially following the relaxation of epidemic prevention and control measures (4). Single-pathogen PCR positivity was most commonly observed for RSV, FluA, and MP. ADV and HRV were more often identified in specimens with co-detection of multiple pathogens. Notably, the six-pathogen detection kit used in this study provides semi-quantitative Ct values for positive results, though Ct data were not included in the primary analysis. For co-detections, we acknowledge that distinguishing causative pathogens from incidental detections requires integration of viral load metrics: pathogens with lower Ct values (higher viral loads) are more likely to drive clinical manifestations, while higher Ct values may indicate residual shedding or asymptomatic carriage. Post-hoc exploratory analysis of Ct values in co-infected cases revealed that RSV had significantly lower mean Ct values than co-detected pathogens in 82.3% of RSV-related co-detections, supporting its dominant role in symptomatic disease. Future studies should incorporate routine viral load reporting to improve etiological attribution in mixed infections.
Age distribution characteristics
RSV was the predominant pathogen identified in this study, with 56.37% of RSV-positive cases occurring in infants aged <1 year, consistent with previous domestic and international reports (13,14). Given the elevated mortality risk associated with RSV infection in this age group, infants aged <1 year should remain a primary target population for prevention and control strategies (15,16). MP is associated with widespread population susceptibility, and infection risk increases with cumulative exposure over time. MP pneumonia accounts for approximately 10–40% of community-acquired pneumonia cases in children (17). In the present study, MP detection rates increased progressively with age, consistent with findings reported from Hebei (5). ADV infections in this cohort were observed predominantly among children aged >1 year. However, infants with ADV infection are at increased risk of severe disease, indicating the need for heightened clinical vigilance in this population (18-20).
Seasonal epidemiological characteristics
To better characterize seasonal epidemic patterns, cases were further categorized into four meteorological seasons in Qingdao: spring (March–May), summer (June–August), autumn (September–November), and winter (December–February). Monthly positivity rates were aggregated to generate seasonal heatmaps for each pathogen. In the present study, RSV and MP demonstrated non-overlapping months with the highest positivity proportion. In China, RSV activity is most commonly observed during winter and spring. In northern regions, the RSV epidemic season typically extends from October to March of the following year, with a median duration of approximately 4.6 months [interquartile range (IQR), 4.1–5.4 months] across most provinces (21). The RSV epidemic pattern identified in Qingdao, China was generally consistent with that reported in northern China. MP infections occur throughout the year in China but are more prevalent during autumn and winter in northern regions (22). Community outbreaks are reported periodically at intervals of approximately 3 to 7 years and may persist for months to years (23).
FluA and MP each demonstrated two epidemic peaks in this cohort. FluA activity peaked in November 2023 (29.51%) and January 2025 (23.62%), with peak timing shifting earlier relative to RSV compared to typical northern hemisphere patterns, where FluA usually follows RSV activity in winter months (21). This decoupling of FluA and RSV peaks may reflect residual effects of post-pandemic immunity gaps or altered viral interference dynamics in the study period.
MP detection rates remained elevated from August 2023 through December 2024, representing a 17-month prolonged epidemic period in Qingdao. Notably, 24.03% of MP-positive cases were classified as co-detections, raising questions about whether low-load MP detections represent active infection or colonization. While MP bacterial load (reflected by Ct values) is critical for guiding macrolide treatment decisions, our current assay does not distinguish viable organisms from nucleic acid remnants. Clinicians in Qingdao Women and Children’s Hospital empirically consider MP as causative when Ct values are <30 in symptomatic children, though prospective studies correlating MP load with treatment response are needed to refine management algorithms.
HRV demonstrated continuous year-round circulation, with monthly positivity rates consistently above 5% and prominent peaks reaching 26.60% in October 2023 and 20.42% in October 2024. Unlike RSV or MP, HRV activity showed no clear seasonal nadir, consistent with its extensive serotype diversity enabling persistent transmission across climatic conditions (6). Our exclusion criterion removed children who received nirsevimab prior to admission, as the monoclonal antibody is routinely administered to high-risk infants (e.g., preterm birth, chronic lung disease) in Qingdao between September and November, immediately preceding the local RSV epidemic season. This targeted pre-season administration aligns with current expert consensus recommendations (24) and may partially explain the lower RSV detection rate in the 4–<5 years group, where fewer high-risk children remain eligible for prophylaxis. However, nirsevimab coverage in our study population was estimated at only 12.7% among eligible infants, limiting its impact on overall RSV epidemiology in this cohort.
Prevention strategies
Prevention and control strategies for ARIs should be tailored according to age-specific risk profiles. General preventive measures, including mask use and hand hygiene, remain broadly applicable; however, prolonged mask use is not recommended for children aged <3 years (24). Based on the findings of the present study, infants aged <1 year should be prioritized for RSV prevention. Administration of long-acting monoclonal antibodies before the RSV epidemic season may be considered for high-risk populations (24,25). Children aged 1–<5 years require targeted MP prevention, as MP PCR positivity rises substantially with age. Given the increasing prevalence of macrolide resistance, the development of effective vaccines against MP is urgently needed, although MP vaccines for human use remain under development (26,27). Prevention of FluA, FluB, ADV, and HRV should remain a priority across all pediatric age groups. Annual influenza vaccination represents a cost-effective strategy for influenza prevention, and vaccination before the influenza season is recommended for children aged >6 months (28). The absence of an effective HRV vaccine, largely due to extensive serotype diversity, highlights the importance of continuous surveillance and comprehensive prevention strategies.
The establishment of a long-term sentinel surveillance system for pediatric ARIs is recommended to characterize local pathogen distributions and monitor temporal epidemiological trends, thereby supporting the development of evidence-based prevention and control strategies. From a public health perspective, sustained implementation of NPIs at both community and household levels, combined with accelerated vaccine development and expanded immunization coverage, may contribute to reducing the burden of ARIs on healthcare systems and society. Compared to prior regional surveillance efforts, our findings offer three key advances. First, the 26-month post-pandemic observation window captured a sustained 17-month MP epidemic (August 2023–December 2024), a duration far exceeding the typical 3–6 months seasonal peaks reported in Hebei [2019–2023 (5)] and Zhongshan [2019–2023 (4)], providing novel empirical evidence of extended MP circulation following the relaxation of NPIs. Second, our fine-grained age stratification [vs. the commonly used <1, 1–<3, and 3–<5 years groupings in prior work (11,24)] revealed a stepwise five-fold increase in MP positivity from 6.81% in infants to 35.34% in 4–<5 years, a gradient not fully quantified in earlier northern Chinese cohorts. Third, with a sample size nearly twice that of comparable single-center studies in Shanghai [n=8,942 (6)] and Shijiazhuang [n=9,127 (5)], our analysis had sufficient power to confirm statistically and clinically meaningful age-related pathogen distribution patterns, even after Bonferroni correction for multiple comparisons.
This study contains multiple key limitations that constrain the generalizability and causal interpretation of results. First, all participants were recruited from a single tertiary women and children’s hospital, introducing hospital-based selection bias and referral-center bias; the cohort only reflects hospitalized children with sufficiently severe ARIs to require inpatient care, and findings cannot be generalized to the full population of children residing in Qingdao, China or used to estimate community-level ARI incidence. Second, no uninfected control group or community outpatient comparison group was included to benchmark pathogen background circulation in the general pediatric population. Third, testing was limited to a panel of six predefined respiratory pathogens, leaving other common infectious agents undetected. Fourth, multiple admission episodes from identical children separated by more than 7 days were retained as independent analytical units, which may create non-independent repeated observations and mild clustering effects. Fifth, post-pandemic shifts in parental healthcare-seeking behavior altered hospital admission volumes over the 26-month study period, which may confound monthly pathogen detection proportions. Sixth, only throat swabs instead of nasopharyngeal specimens were collected for PCR testing, potentially reducing analytical sensitivity for RSV and other respiratory viruses. Finally, positive PCR results merely confirm the presence of pathogen nucleic acid in upper respiratory specimens and cannot establish causal links between detected microbes and the child’s acute respiratory illness, nor can monthly hospitalization-based positivity rates be interpreted as true population infection incidence. Future multicenter studies incorporating community outpatient cohorts and expanded pathogen panels are needed to overcome these constraints.
Conclusions
RSV and MP were the most frequently detected pathogens among the six tested targets in hospitalized children aged <5 years with ARIs in Qingdao, China. Infants aged <1 year exhibited the highest RSV PCR positivity rate, while MP PCR positivity rose progressively with age. Months with maximal RSV and MP detection proportions showed visually separated temporal maxima. FluA and FluB maintained low epidemic intensity throughout the study period, while HRV circulated continuously year-round.
Supplementary
The article’s supplementary files as
Acknowledgments
We would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Review Committee of Qingdao Women and Children’s Hospital (No. QFELL-YJ-2026-49; approval date: 2026-02-06). Informed consent was waived in this retrospective study.
Footnotes
Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0509/rc
Funding: This study was funded by the Qingdao Key Medical and Health Discipline Project (supported by Qingdao Health Commission).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0509/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0509/dss
References
- 1. Walker CLF, Rudan I, Liu L, et al. Global burden of childhood pneumonia and diarrhoea. Lancet 2013;381:1405-16.. 10.1016/S0140-6736(13)60222-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Zhu G, Xu D, Zhang Y, et al. Epidemiological characteristics of four common respiratory viral infections in children. Virol J 2021;18:10.. 10.1186/s12985-020-01475-y [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Meskill SD, O'Bryant SC. Respiratory Virus Co-infection in Acute Respiratory Infections in Children. Curr Infect Dis Rep 2020;22:3.. 10.1007/s11908-020-0711-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Zhan M, Xia HH, Liu J, et al. The epidemiological characteristics of seven pathogens causing acute respiratory infections in hospitalized children in Zhongshan City from 2019 to 2023. Chinese Journal of Nosocomiology 2025;35:1829-34. [Google Scholar]
- 5. Cao BB, Zhai Y, Duan SX. Epidemiological characteristics of respiratory syncytial virus in children in Hebei from 2019 to 2023. Chinese Journal of Microbiology and Immunology 2024;44:142-8. [Google Scholar]
- 6. Fang Y, Wang J, Cheng AN, et al. Analysis of Pathogen Distribution and Epidemiological Characteristics of Pediatric Acute Respiratory Infection Hospitalization Cases in a Single Shanghai Center in 2023. Chinese Journal of Microbiology and Immunology 2025;45:817-25. [Google Scholar]
- 7.Jiang ZF, Wang TY, Shen KL, et al. Zhu Futang's Practical Pediatrics. 9th ed. Beijing: People's Medical Publishing House; 2022:1639.
- 8. Ashrafi-Asgarabad A, Bokaie S, Razmyar J, et al. The burden of lower respiratory infections and their underlying etiologies in the Middle East and North Africa region, 1990-2019: results from the Global Burden of Disease Study 2019. BMC Pulm Med 2023;23:2.. 10.1186/s12890-022-02301-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Wang Y, Chen JL, Feng WJ, et al. Global, high-SDI countries and China: Trends in the burden of lower respiratory tract infection diseases among children under 5 years old from 1990 to 2019. Chinese Journal of Public Health 2024;40:840-8. [Google Scholar]
- 10. Collaro AJ, McElrea MS, Marchant JM, et al. The effect of early childhood respiratory infections and pneumonia on lifelong lung function: a systematic review. Lancet Child Adolesc Health 2023;7:429-40.. 10.1016/S2352-4642(23)00030-5 [DOI] [PubMed] [Google Scholar]
- 11. Li ZJ, Zhang HY, Ren LL, et al. Etiological and epidemiological features of acute respiratory infections in China. Nat Commun 2021;12:5026.. 10.1038/s41467-021-25120-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Chen K, Jia R, Li L, et al. The aetiology of community associated pneumonia in children in Nanjing, China and aetiological patterns associated with age and season. BMC Public Health 2015;15:113.. 10.1186/s12889-015-1422-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Cui AL, Xia BC, Zhu Z, et al. Epidemiological characteristics of human respiratory syncytial virus among acute respiratory infection cases in 16 provinces of China from 2009 to 2023. Zhonghua Yu Fang Yi Xue Za Zhi 2024;58:945-51. [Article in Chinese].. 10.3760/cma.j.cn112150-20231213-00440 [DOI] [PubMed] [Google Scholar]
- 14. Hall CB, Weinberg GA, Blumkin AK, et al. Respiratory syncytial virus-associated hospitalizations among children less than 24 months of age. Pediatrics 2013;132:e341-8.. 10.1542/peds.2013-0303 [DOI] [PubMed] [Google Scholar]
- 15. Li Y, Wang X, Blau DM, et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: a systematic analysis. Lancet 2022;399:2047-64.. 10.1016/S0140-6736(22)00478-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Shi T, McAllister DA, O'Brien KL, et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study. Lancet 2017;390:946-58.. 10.1016/S0140-6736(17)30938-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Zhu Q, Gong L, Hou S, et al. Expert consensus on the response to mycoplasma pneumoniae infection outbreaks. Chinese Journal of Disease Control & Prevention 2025;29:844-9. [Google Scholar]
- 18. Siew JX, Seah XFV, Chew YR, et al. Epidemiology of Adenovirus Infections and Outcomes of Cidofovir Treatment in Severely Ill Children. Pediatr Infect Dis J 2020;39:907-13.. 10.1097/INF.0000000000002726 [DOI] [PubMed] [Google Scholar]
- 19. Fage C, Hénaut M, Carbonneau J, et al. Influenza A(H1N1)pdm09 Virus but Not Respiratory Syncytial Virus Interferes with SARS-CoV-2 Replication during Sequential Infections in Human Nasal Epithelial Cells. Viruses 2022;14:395.. 10.3390/v14020395 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Wang J, Wang R, Xie Z. Rethinking Paediatric Respiratory Infections: The Role of Mixed Pathogen Infections. Rev Med Virol 2025;35:e70021.. 10.1002/rmv.70021 [DOI] [PubMed] [Google Scholar]
- 21. Guo L, Deng S, Sun S, et al. Respiratory syncytial virus seasonality, transmission zones, and implications for seasonal prevention strategy in China: a systematic analysis. Lancet Glob Health 2024;12:e1005-16.. 10.1016/S2214-109X(24)00090-1 [DOI] [PubMed] [Google Scholar]
- 22. Chen J, Zhang J, Lu Z, et al. Mycoplasma pneumoniae among Chinese Outpatient Children with Mild Respiratory Tract Infections during the Coronavirus Disease 2019 Pandemic. Microbiol Spectr 2022;10:e0155021.. 10.1128/spectrum.01550-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Yan C, Xue GH, Zhao HQ, et al. Current status of Mycoplasma pneumoniae infection in China. World J Pediatr 2024;20:1-4.. 10.1007/s12519-023-00783-x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Chinese Preventive Medicine Association . Expert consensus on the passive immunization prevention of infant respiratory syncytial virus infection in China. Zhonghua Liu Xing Bing Xue Za Zhi 2024;45:1-12. [Epub ahead of print].. 10.3760/cma.j.cn112338-20240826-00527 [DOI] [PubMed] [Google Scholar]
- 25. Moline HL, Tannis A, Toepfer AP, et al. Early Estimate of Nirsevimab Effectiveness for Prevention of Respiratory Syncytial Virus-Associated Hospitalization Among Infants Entering Their First Respiratory Syncytial Virus Season - New Vaccine Surveillance Network, October 2023-February 2024. MMWR Morb Mortal Wkly Rep 2024;73:209-14.. 10.15585/mmwr.mm7309a4 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Jiang Z, Li S, Zhu C, et al. Mycoplasma pneumoniae Infections: Pathogenesis and Vaccine Development. Pathogens 2021;10:119.. 10.3390/pathogens10020119 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Zhang HQ, Lu YP, Zhang J. Research progress of Mycoplasma pneumoniae vaccine. Journal of Clinical Pediatrics 2022;40:634-40. [Google Scholar]
- 28. Chinese National Immunization Program Technical Working Group: Influenza Vaccine Working Group . China Influenza Vaccine Vaccination Technical Guidelines (2025-2026). Chinese Journal of Epidemiology 2025;46:2097-107. [Google Scholar]