Analysis of epidemic trend of respiratory pathogens in children after long-term pathogen isolation

Study population

The study cohort comprised 8,720 pediatric patients (aged 0–13 years) receiving clinical care in the First People’s Hospital of Tianshui during the period from April 2023 to March 2024 (Data S1). A total of 9,670 respiratory specimens were collected, with 7,670 allocated to viral detection (including Mycoplasma pneumoniae categorization) and 2,000 to bacterial nucleic acid analysis. Study data were derived predominantly from hospitalized individuals with clinically significant respiratory infections, excluding those with mild or latent presentations. Clinical data were documented in the hospital information system by the pediatricians, encompassing gender, age, physical signs, diagnosis, and diagnostic test results. Specimens were collected and transported to the laboratory by certified nursing staff. Specimens underwent laboratory processing with confirmatory testing as clinically indicated. The laboratory maintained CNAS-accredited quality management systems with rigorous protocol adherence in quality control and evaluation. Written informed consent forms were obtained from all participants before enrollment: A complete description of the research purpose, process, potential risks and benefits, privacy measures (such as data anonymization), the right to voluntary participation, and opt-out mechanisms at any time. For participants with limited educational backgrounds, a trained physician would provide a verbal explanation, followed by a relative witnessing and signing upon confirming comprehension. The Ethics Committee oversaw the study’s implementation and conducted annual reviews of its ethical compliance.

Definitions

1. Patients with ARIs must meet at least one of the inclusion criteria as follows:

(1) Fever (axillary temperature ≥37.2);

(2) White blood cell counts were normal or low, and the proportion of lymphocytes was elevated;

(3) White blood cell counts and neutrophil ratio increased;

(4) Cough, sore throat, wheeze, expectoration, chest pain, moist/dry rales;

2. Lower respiratory tract infections (LRTI) cases were defined by clinical diagnoses of bronchitis, of pneumonia (including lobar pneumonia), emphysema, pulmonary infections, respiratory distress syndrome, and respiratory failure.

3. In this study, co-infection refers to simultaneous infection of greater than or equal to two pathogens. Pathogens detected include: influenza virus A (IFV-A), influenza virus B (IFV-B), respiratory syncytial virus (RSV), human rhinovirus (HRV), human adenovirus (HAdV), Mycoplasma pneumoniae (MP), Staphylococcus aureus (SA), Klebsiella pneumoniae (KP), Legionella pneumophila (LP), Haemophilus influenzae (HI), Pseudomonas aeruginosa (PA), Streptococcus pneumoniae (SP).

Statistics and ethics

The statistical software was SPSS Statistics 23 (IBM Corp., Armonk, NY, USA). Statistical analyses comprised binary logistic regression, Poisson regression and negative binomial regression. Select a regression model based on whether the mean was approximates the variance. Binary logistic regression used the Hosmer-Lemeshow test (HL) to determine the fit degree of the model, p > 0.05, as the inclusion criteria. Poisson regression (PR) and negative binomial regression (NBR) used omnibus test (OT) to determine whether the independent variable had a significant effect on the dependent variable, and p < 0.05 was used as the inclusion criterion.

This study was approved by the Ethics Review Committee of the First People’s Hospital of Tianshui and all participants gave a signed informed consent. The IRB approval number was 2023-07.

Tianshui is located in the northern hemisphere, temperate zone, the terrain is a valley, the environmental data during this period from Tianshui Meteorological Bureau.

Experimental method

In this study, the detection method was QRT-PCR, the detection reagent came from Shengxiang Biotechnology Co., LTD (Jinzhou, China). Reagent batch number: respiratory pathogen nucleic acid (IFV-A, IFV-B, RSV, HRV, HAdV, MP) 23026-2, respiratory pathogenic bacterial nucleic acid (SA, KP, LP, HI, PA, SP) 23002. China Food and Drug Administration (CFDA), State instrument registration number: 20213400256 and 20223400597. Nucleic acid extractor was NT-9600 of Shengxiang Biotechnology Co., LTD (Jinzhou, China). The amplification instrument was the MA-6000 from Yari Biotechnology Co., LTD. Patient samples 300 µl were detected. Reaction system included PCR reaction solution 43.5 µl, enzyme mixture 1.5 µl, patient sample 5 µl (after extraction). PCR reaction conditions included an reverse transcription at 50 °C for 30 min, initial denaturation at 95 °C for 1 min, followed by the 45 cycles of denaturation at 95 °C for 15 s, annealing, extension and fluorescence detection and at 60 °C for 30 s a final extension at 72 °C for 7 min. The minimum detection limits of pathogens in this study were shown in Table 1. All pathogen detection in this study was completed in the laboratory of the Laboratory of Tianshui First People’s Hospital, which passed the CNAS laboratory quality management system certification, which is derived from ISO15189. The experimental instrument has been calibrated and its performance verified, the experimental method has been verified, the experimental reagent has been verified by the batch number, and the personnel have been trained and have a job certificate. The set temperature of the laboratory was 22 °C, the humidity was 35% RH, and the laboratory was disinfected twice a week. Each experiment contained 1 negative quality control and 1 weak positive quality control. The results of each experiment were evaluated, and specimens with poor amplification curves and other possible inaccuracies were re-examined. The CT value less than 38 was considered as the positive criterion.

Analysis of factors related to the number of pathogens infected

Table 2 summarizes meteorological parameters of the Tianshui region. The Tianshui region exhibits a temperate climate characterized by three-month meteorological seasons. with spring occurring from March to May. The data presented in this table comprehensively outlines the environmental characteristics of the Tianshui area for readers. Figure 1 demonstrates the epidemiological patterns of positive detection rates for various respiratory pathogens over time: IFV-A, IFV-B, MP and HAdV all have obvious one-month infection peaks in winter; The detection rate of RSV remains relatively low in summer, while HRV exhibits no significant seasonal variation. The infection level of HRV remained relatively stable throughout the year. The typical high-incidence periods of bacterial pathogens are June, November and January. The distribution of infections exhibits marked seasonal variation. Table 3 included positive detection data, environmental factors, age, gender and other variables for statistical analysis, revealing that IFV-A and IFV-B infections peaked in winter, while KP infections were more prevalent in summer. The expected infection counts attributable to viral pathogens are the highest in the 1–6 age group, followed by the <1-year old age group, and the lowest in the >6-year age group. The expected infection counts for bacterial pathogens decline with age, whereas those for MP increase with age. In terms of gender differences, the expected number of men infected with KP, HI and PA was significantly higher than that of women.

Pathogens and age

The data in Table 4 and Fig. 2 indicated that the male-to-female ratio among patients undergoing respiratory pathogen testing was 1.45:1 (n = 5, 729:3,941), with similar positive ratios of various pathogens between males and females. With increasing age, the RSV infection rate declined from 52.35% to 6.06%, while HRV rates slightly decreased. In contrast, IFV-A, IFV-B, HAdV and MP rates gradually increased, whereas SA, KP and PA rates decreased progressively. The infection rate of COVID-19 among children in this area was extremely low, with a positive rate of 3.4% (total n = 449). The results indicated that HI and SP detection rates were relatively high across all patient groups. By comparing the procalcitonin (PCT) results of the patients, it was found that 12.3% of the patients positive for HI had elevated PCT, and 12.8% of the patients positive for SP had elevated PCT. Therefore, most HI and SP might be parasitic bacteria.

Co-infection

To investigate pathogen co-infection patterns, this study conducted a statistical analysis of 950 patients with 12 pathogens simultaneously detected, and performed pairwise examination of pathogens in the cases involving co-infections of more than two pathogens. The data were shown in Fig. 3. The results showed that the co-infection between HI and SP with other pathogens was the most prevalent, primarily due to the large number of their parasitic infections. In this study, to determine the relationship of co-infection among pathogenic organisms, these two pathogenic bacteria were excluded. Among the pathogens co-infected with IFV-A, MP accounted for the highest proportion at 18.96% (11/58). The IFV-B was MP 9.76% (4/11), RSV was SA 16.07% (45/280), HAdV was MP 13.64% (12/88), HRV was RSV 24.78% (28/113), MP was SA 8.45% (12/142), SA was RSV 30.41% (45/148), KP was RSV 27.63% (21/76), PA was KP 29.03% (9/31), HI was RSV 21.58% (112/519), and SP was RSV 27.94% (197/705). The co-infection patterns among 950 pathogen test results were analyzed using statistical software (Table 5). The data revealed that RSV demonstrated the strongest co-infection potential, followed by IFV-B, MP and SA. The co-infection risk between IFV-B and RSV was the highest, with an estimated coefficient of 20.651 (95% CI [2.797–152.468]). Notably, PA and KP exhibited the ability to inhibit mutual co-infection. SP could inhibit co-infection with HI, SA and KP. This table displayed the predicted data only when the pathogen was present in the respiratory tract. Its pathogenicity and the impact of its parasitic state on other pathogens could not be inferred from this table.

Lower respiratory pathogens

Table 6 showed the infection rates of various pathogens in children with LRTI. Analysis of infection rate of pathogens (Table 4) revealed that although RSV infection rates decreased with age, it remains the primary pathogen of LRTI in children under 6 years old. MP was the primary pathogen causing LRTI in children aged over six years, with its infection rate exceeding that of the overall infection rate. The infection rates of IFV-B and HAdV in LRTI were lower than the overall infection rate across all ages. The infection rate of bacterial pathogens in LRTI was slightly higher than the overall infection rate at all ages. With increasing age, the bacterial pathogen rate in LRTI declined, while the infection rates of IFV-B and HAdV gradually increased, and HRV gradually decreased. HI and SP data differed greatly from actual infection rates (refer to PCT results) and were listed here for reference.