ABSTRACT
Respiratory syncytial virus (RSV) is a leading cause of acute respiratory tract infections (ARTI) in infants and young children. Co-infection with other respiratory pathogens is common in RSV infection. However, the associations of co-infection with disease severity and their potential interactions remain poorly understood, especially in light of evolving epidemiological patterns of respiratory pathogens following the COVID-19 pandemic. This multicenter, retrospective study included 2,862 children under 5 years of age and hospitalized with RSV at three pediatric centers in Wuhan, China, between June 2020 and May 2023. We evaluated the associations of co-infecting pathogens, including bacteria, viruses, fungi, and atypical bacteria, with disease severity, length of hospital stay, and inpatient medical expenses. We found that nearly 44% of children with RSV were co-infected with at least one additional pathogen. Co-infection rates fluctuated in correlation with the implementation and relaxation of COVID-19 non-pharmaceutical interventions. While viral and atypical bacterial co-infections did not significantly alter outcomes, bacterial (OR = 2.00, 95% CI: 1.48, 2.70) and fungal (OR = 2.57, 95% CI: 1.23, 5.35) co-infections were strongly associated with increased disease severity. Both types were also linked to longer hospital stays (days, β = 0.63, 95% CI: 0.38, 0.88 for bacterial; β = 2.68, 95% CI: 1.94, 3.43 for fungal) and higher inpatient medical expenses (Chinese Yuan [CNY], β = 2,317.16, 95% CI: 1,534.43, 3,099.88 for bacterial; β = 6,636.55, 95% CI: 4,366, 8,907.10 for fungal). No significant additive interactions were found between bacterial and fungal co-infections. These findings highlight the importance of distinguishing co-infection types in the clinical management of RSV.
IMPORTANCE
This study clarifies the clinical significance of pathogen co-infections in children hospitalized with respiratory syncytial virus (RSV). We demonstrate a critical distinction that co-infections with bacteria or fungi are associated with more severe disease, longer hospitalizations, and higher costs, whereas viral or atypical bacterial co-infections are not. In the context of evolving RSV epidemiology and pathogen co-infections in the post-pandemic era, this finding may help risk-based clinical management when co-infection exists.
KEYWORDS: respiratory syncytial virus, co-infection, disease severity, interaction, multicenter study
INTRODUCTION
Respiratory syncytial virus (RSV) is the most common cause of acute respiratory tract infections (ARTI) and a major driver of hospitalization and morbidity in infants and young children worldwide (1, 2). RSV infection presents with a broad spectrum of manifestations, from mild upper respiratory illness to severe lower respiratory tract disease that may need intensive care and can be fatal (3). This heterogeneity suggests that factors beyond the virus itself, such as host characteristics, seasonality, and co-circulating respiratory pathogens, may significantly influence disease outcomes (4, 5).
Molecular detection technologies, including multiplex polymerase chain reaction (PCR), isothermal nucleic acid amplification tests, and next-generation sequencing, have demonstrated that over one-third of children hospitalized with RSV have co-infections with other respiratory pathogens (6, 7). While RSV is typically seasonal with epidemics usually occurring in the winter months, the COVID-19 pandemic and associated non-pharmaceutical interventions (NPIs) have substantially altered its epidemiology and other respiratory virus co-circulation patterns (8–10). However, comprehensive data on how co-infection profiles have changed in children with RSV infection during and after the pandemic remained limited. Moreover, these co-infections may potentially interact with RSV, possibly leading to altered immune responses and more severe clinical courses (11, 12). However, existing evidence on the impact of co-infections on disease severity is generally limited, often focusing narrowly on bacterial or viral co-infections, without a systematic assessment of fungal or atypical bacterial infections (13–16). Few studies have systematically and simultaneously evaluated the independent effects of the four major pathogen categories, including bacteria, viruses, fungi, and atypical bacteria (specifically Mycoplasma pneumoniae [MP], Chlamydia pneumoniae [CP], and Legionella pneumophila [LP]), within the same pediatric cohort. It also remains unclear whether different co-infection types interact synergistically or antagonistically to modulate disease severity in children with RSV infection.
To address these knowledge gaps, we conducted a large, multicenter, retrospective study to examine trends in the prevalence of different respiratory co-infection patterns (bacterial, viral, fungal, atypical bacterial) among children under 5 years hospitalized with RSV during and after the COVID-19 pandemic and to evaluate the independent and joint associations of each major co-infection type with the risk of severity outcomes.
RESULTS
A total of 10,684 children hospitalized with ARTI were assessed for eligibility. After excluding 68 cases with data errors or contradictions and 7,754 cases who tested negative for RSV, we included a total of 2,862 children hospitalized with RSV infection (Fig. S1). The median age was 23.00 months (interquartile range, IQR: 8.00, 40.08), and 1,162 (40.6%) were girls. Among the enrolled patients, 1,252 (43.75%) tested positive for at least one additional respiratory pathogen. This included 1,072 cases of double infection (RSV plus one other category of pathogen), 170 cases of triple infection (RSV plus two), and 10 cases of quadruple infection (RSV plus three). The prevalence of RSV-bacterial, RSV-viral, RSV-fungal, and RSV-atypical bacterial co-infections was 495 (17.30%), 429 (14.99%), 36 (1.26%), and 482 (16.84%), respectively. The overall co-infection rate declined during the COVID-19 pandemic but increased sharply after the relaxation of NPIs in late 2022 (Fig. 1A). Correlation analysis revealed generally low associations between different types of RSV-respiratory pathogen co-infections. The strongest negative correlation was observed between RSV-bacterial and RSV-atypical bacterial co-infections (Phi = –0.13, P < 0.001; Fig. 1B). The detection rate of any co-infection was significantly higher in children aged ≥24 months compared with those under 24 months of age (47.7% vs 39.9%, P < 0.001), and in girls compared with boys (47.0% vs 41.5%, P = 0.004) (Fig. 1C and D).
Fig 1.
Time trends in co-infection rate (A), grade of correlation between different co-infection types (B), and comparison of co-infection types by age (C) and sex (D). NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.
Table 1 summarizes the baseline characteristics of children with RSV infection stratified by co-infection status. Compared with the RSV mono-infection group, children with bacterial or atypical bacterial co-infections tended to be older. Notably, patients with fungal co-infections had a higher prevalence of preterm birth and more severe clinical manifestations at admission than the other groups. The prevalence of comorbidities, duration of symptoms before admission, and proportion of abnormal chest radiographic findings were comparable across all groups.
TABLE 1.
| All participants | RSV mono-infection | Double co-infections | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| RSV-bacteria | P value | RSV-other viruses | P value | RSV-fungi | P value | RSV-atypical bacterial | P value | |||
| Number of participants | 2,862 | 1,610 | 369 | 285 | 16 | 402 | ||||
| Age, median (IQR), months | 23.00 (8.00, 40.08) | 20.07 (6.03, 39.00) | 30.82 (9.36, 43.00) | <0.001 | 18.00 (7.13, 38.88) | 0.605 | 9.53 (4.00, 27.25) | 0.324 | 30.00 (17.00, 43.00) | <0.001 |
| Age groups, n (%), months | 0.001 | 0.676 | 0.168 | <0.001 | ||||||
| <24 | 1,463 (51.12) | 879 (54.60) | 166 (44.99) | 160 (56.14) | 12 (75.00) | 156 (38.81) | ||||
| ≥24 | 1,399 (48.88) | 731 (45.40) | 203 (55.01) | 125 (43.86) | 4 (25.00) | 246 (61.19) | ||||
| Sex, n (%) | 0.956 | 0.967 | >0.999 | <0.001 | ||||||
| Boys | 1,700 (59.40) | 994 (61.74) | 229 (62.06) | 175 (61.40) | 10 (62.50) | 175 (43.53) | ||||
| Girls | 1,162 (40.60) | 616 (38.26) | 140 (37.94) | 110 (38.60) | 6 (37.50) | 227 (56.47) | ||||
| Residential area, n (%) | <0.001 | 0.001 | 0.336 | 0.444 | ||||||
| Wuhan | 2,404 (84.00) | 1,392 (86.46) | 278 (75.34) | 225 (78.95) | 12 (75.00) | 354 (88.06) | ||||
| Outside Wuhan | 458 (16.00) | 218 (13.54) | 91 (24.66) | 60 (21.05) | 4 (25.00) | 48 (11.94) | ||||
| Preterm birth, n (%) | 217 (7.58) | 108 (6.71) | 30 (8.13) | 0.393 | 32 (11.23) | 0.01 | 4 (25.00) | 0.017 | 24 (5.97) | 0.673 |
| Comorbidities, n (%) | ||||||||||
| Any | 130 (4.54) | 74 (4.60) | 17 (4.61) | >0.999 | 13 (4.56) | >0.999 | 0 (0.00) | 0.783 | 9 (2.24) | 0.047 |
| Respiratory system | 20 (0.70) | 10 (0.62) | 2 (0.54) | >0.999 | 1 (0.35) | 0.896 | 0 (0.00) | >0.999 | 1 (0.25) | 0.598 |
| Congenital heart disease | 53 (1.85) | 30 (1.86) | 6 (1.63) | 0.927 | 5 (1.75) | >0.999 | 0 (0.00) | >0.999 | 6 (1.49) | 0.771 |
| Nervous system | 11 (0.38) | 4 (0.25) | 1 (0.27) | >0.999 | 3 (1.05) | 0.125 | 0 (0.00) | >0.999 | 0 (0.00) | 0.708 |
| Duration of symptoms prior to admission, median (IQR), days | 5.00 (4.00, 7.00) | 5.00 (4.00, 7.00) | 5.00 (3.00, 7.00) | 0.200 | 5.00 (4.00, 8.00) | 0.001 | 4.50 (3.00, 7.25) | 0.860 | 5.00 (4.00, 7.00) | 0.250 |
| Observations at admission, n (%) | ||||||||||
| Poor consciousness/spirit/appetite | 116 (4.05) | 59 (3.66) | 14 (3.79) | <0.001 | 8 (2.81) | 0.032 | 1 (6.25) | 0.861 | 16 (3.98) | 0.704 |
| Wet rales | 1,662 (58.07) | 917 (56.96) | 245 (66.40) | 0.001 | 151 (52.98) | 0.237 | 11 (68.75) | 0.487 | 220 (54.73) | 0.453 |
| Wheezing sound | 1,073 (37.49) | 602 (37.39) | 144 (39.02) | 0.549 | 120 (42.11) | 0.149 | 8 (50.00) | 0.437 | 121 (30.10) | 0.008 |
| Shortness of breath/three concave signs | 289 (10.10) | 149 (9.25) | 49 (13.28) | 0.025 | 34 (11.93) | 0.193 | 3 (18.75) | 0.386 | 28 (6.97) | 0.177 |
| Chest radiological results, n (%) | <0.001 | 0.191 | 0.142 | 0.021 | ||||||
| Normal | 54 (1.89) | 36 (2.24) | 3 (0.81) | 8 (2.81) | 0 (0.00) | 6 (1.49) | ||||
| Bronchitis | 863 (30.15) | 550 (34.16) | 91 (24.66) | 77 (27.02) | 3 (18.75) | 106 (26.37) | ||||
| Bronchopneumonia | 1,882 (65.76) | 1,005 (62.42) | 253 (68.56) | 195 (68.42) | 12 (75.00) | 283 (70.40) | ||||
| Lobar pneumonia | 51 (1.78) | 14 (0.87) | 17 (4.61) | 4 (1.40) | 1 (6.25) | 6 (1.49) | ||||
| Interstitial pneumonia | 12 (0.42) | 5 (0.31) | 5 (1.36) | 1 (0.35) | 0 (0.00) | 1 (0.25) | ||||
| Study site, n (%) | <0.001 | <0.001 | 0.003 | <0.001 | ||||||
| Children’s Hospital | 570 (19.92) | 179 (11.12) | 234 (63.41) | 75 (26.32) | 6 (37.50) | 16 (3.98) | ||||
| Maternal and Child Health Hospital | 615 (21.49) | 292 (18.14) | 98 (26.56) | 79 (27.72) | 1 (6.25) | 98 (24.38) | ||||
| Tongji Hospital | 1,677 (58.60) | 1,139 (70.75) | 37 (10.03) | 131 (45.96) | 9 (56.25) | 288 (71.64) | ||||
| Admission year, n (%) | <0.001 | <0.001 | 0.347 | <0.001 | ||||||
| 2020 | 127 (4.44) | 71 (4.41) | 3 (0.81) | 16 (5.61) | 1 (6.25) | 30 (7.46) | ||||
| 2021 | 1,094 (38.23) | 720 (44.72) | 29 (7.86) | 80 (28.07) | 4 (25.00) | 231 (57.46) | ||||
| 2022 | 320 (11.18) | 236 (14.66) | 14 (3.79) | 24 (8.42) | 2 (12.50) | 33 (8.21) | ||||
| 2023 | 1,321 (46.16) | 583 (36.21) | 323 (87.53) | 165 (57.89) | 9 (56.25) | 108 (26.87) | ||||
RSV, respiratory syncytial virus; IQR, interquartile range; SD, standard deviation; WBC, white blood cell count; NEU, neutrophil count; LYM, lymphocyte count; CRP, C-reactive protein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Glb, globulin; CNY, Chinese Yuan.
Continuous variables were compared using the Mann-Whitney U test, while categorical variables were analyzed using the χ2 or Fisher’s exact test. The RSV mono-infection group was used as the reference for all comparisons.
After adjustment for potential confounders, both RSV-bacterial co-infection (odds ratio [OR] = 2.00, 95% confidence interval [CI]: 1.48, 2.70) and RSV-fungal co-infection (OR = 2.57, 95% CI: 1.23, 5.35) were associated with an increased risk of composite severity outcomes compared to those without these co-infections (Table 2). In contrast, neither RSV-viral (OR = 1.14, 95% CI: 0.86, 1.51) nor RSV-atypical bacterial co-infection (OR = 1.00, 95% CI: 0.75, 1.34) showed a significant association with the composite severity outcome. Similar results were observed for secondary outcomes. Specifically, RSV-bacterial and RSV-fungal co-infections were associated with higher odds of severe infection (OR = 2.34, 95% CI: 1.70, 3.22; and OR = 3.04, 95% CI: 1.43, 6.47), pediatric intensive care unit (PICU) admission (OR = 8.78, 95% CI: 4.99, 15.46; and OR = 5.08, 95% CI: 1.82, 14.21), and oxygen administration (OR = 1.67, 95% CI: 1.21, 2.31; and OR = 2.42, 95% CI: 1.16, 5.07). RSV-bacterial (β = 0.63 days, 95% CI: 0.38, 0.88) and RSV-fungal co-infections (β = 2.68 days, 95% CI: 1.94, 3.43) were also significantly associated with prolonged hospital stays. Higher inpatient medical expenses were observed in cases with RSV-bacterial (β = 2,317.16 CNY, 95% CI: 1,534.43, 3,099.88), RSV-fungal (β = 6,636.55 CNY, 95% CI: 4,366.00, 8,907.10), and RSV-atypical bacterial co-infections (β = 748.12 CNY, 95% CI: 51.62, 1,444.63).
TABLE 2.
| Outcomes | Categories | Number of cases/number at risk | Estimate (95% CI) | |||
|---|---|---|---|---|---|---|
| Model 1 | Model 2 | Model 3 | Model 4 | |||
| Combined severity outcomes, OR | ||||||
| RSV-bacteria | No | 462/2,367 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 131/495 | 1.68 (1.28, 2.22) | 1.94 (1.45, 2.59) | 2.01 (1.49, 2.72) | 2.00 (1.48, 2.70) | |
| RSV-other viruses | No | 498/2,433 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 95/429 | 1.18 (0.91, 1.52) | 1.12 (0.86, 1.47) | 1.12 (0.85, 1.47) | 1.14 (0.86, 1.51) | |
| RSV-fungi | No | 575/2,826 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 18/36 | 3.38 (1.73, 6.59) | 2.92 (1.45, 5.87) | 2.74 (1.33, 5.63) | 2.57 (1.23, 5.35) | |
| RSV-atypical bacteria | No | 518/2,380 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 75/482 | 0.7 (0.53, 0.92) | 0.97 (0.73, 1.29) | 0.97 (0.73, 1.30) | 1.00 (0.75, 1.34) | |
| Diagnosis of severe infection, OR | ||||||
| RSV-bacteria | No | 350/2,367 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 126/495 | 1.85 (1.39, 2.47) | 2.21 (1.63, 2.99) | 2.36 (1.72, 3.24) | 2.34 (1.70, 3.22) | |
| RSV-other virus | No | 394/2,433 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 82/429 | 1.22 (0.93, 1.60) | 1.15 (0.86, 1.54) | 1.16 (0.86, 1.57) | 1.2 (0.89, 1.63) | |
| RSV-fungi | No | 459/2,826 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 17/36 | 4.06 (2.06, 7.97) | 3.51 (1.71, 7.19) | 3.3 (1.57, 6.93) | 3.04 (1.43, 6.47) | |
| RSV-atypical bacteria | No | 422/2,380 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 54/482 | 0.67 (0.49, 0.92) | 0.98 (0.71, 1.36) | 0.99 (0.71, 1.37) | 1.02 (0.73, 1.43) | |
| PICU admission, OR | ||||||
| RSV-bacteria | No | 45/2,367 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 46/495 | 7.69 (4.43, 13.35) | 8.79 (5.03, 15.36) | 9.66 (5.45, 17.12) | 8.78 (4.99, 15.46) | |
| RSV-other virus | No | 71/2,433 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 20/429 | 1.56 (0.93, 2.60) | 1.48 (0.87, 2.51) | 1.48 (0.85, 2.58) | 1.54 (0.86, 2.76) | |
| RSV-fungi | No | 83/2,826 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 8/36 | 8.45 (3.69, 19.33) | 6.97 (2.94, 16.52) | 6.71 (2.65, 17.01) | 5.08 (1.82, 14.21) | |
| RSV-atypical bacteria | No | 80/2,380 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 11/482 | 0.82 (0.43, 1.57) | 1.28 (0.65, 2.5) | 1.35 (0.68, 2.67) | 1.57 (0.77, 3.21) | |
| Oxygen administration, OR | ||||||
| RSV-bacteria | No | 382/2,367 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 99/495 | 1.51 (1.12, 2.04) | 1.69 (1.24, 2.31) | 1.70 (1.23, 2.35) | 1.67 (1.21, 2.31) | |
| RSV-other virus | No | 407/2,433 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 74/429 | 1.12 (0.84, 1.48) | 1.07 (0.8, 1.43) | 1.05 (0.78, 1.42) | 1.07 (0.79, 1.44) | |
| RSV-fungi | No | 466/2,826 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 15/36 | 3.12 (1.59, 6.13) | 2.71 (1.34, 5.47) | 2.56 (1.24, 5.31) | 2.42 (1.16, 5.07) | |
| RSV-atypical bacteria | No | 419/2,380 | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) | 1.00 (Ref.) |
| Yes | 62/482 | 0.72 (0.53, 0.96) | 0.96 (0.70, 1.30) | 0.96 (0.70, 1.31) | 0.98 (0.71, 1.33) | |
| Length of stay, days, β | ||||||
| RSV-bacteria | No | NA | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) |
| Yes | NA | 0.55 (0.30, 0.80) | 0.62 (0.37, 0.87) | 0.65 (0.39, 0.9) | 0.63 (0.38, 0.88) | |
| RSV-other virus | No | NA | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) |
| Yes | NA | 0.25 (0, 0.49) | 0.23 (−0.01, 0.46) | 0.17 (−0.07, 0.4) | 0.19 (−0.05, 0.42) | |
| RSV-fungi | No | NA | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) |
| Yes | NA | 3.16 (2.39, 3.93) | 3.01 (2.25, 3.77) | 2.77 (2.03, 3.52) | 2.68 (1.94, 3.43) | |
| RSV-atypical bacteria | No | NA | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) |
| Yes | NA | −0.05 (−0.29, 0.18) | 0.08 (−0.15, 0.32) | 0.11 (−0.12, 0.34) | 0.14 (−0.09, 0.37) | |
| Inpatient medical expenses, CNY, β | ||||||
| RSV-bacteria | No | NA | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) |
| Yes | NA | 1,996.83 (1,218.33, 2,775.34) | 2,207.02 (1,434.55, 2,979.48) | 2,343.81 (1,558.47, 3,129.15) | 2,317.16 (1,534.43, 3,099.88) | |
| RSV-other virus | No | NA | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) |
| Yes | NA | 376.35 (−358.68, 1,111.39) | 325.02 (−402.95, 1,052.99) | 239.97 (−489.54, 969.49) | 317.55 (−404.29, 1,039.39) | |
| RSV-fungi | No | NA | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) |
| Yes | NA | 7,829.9 (5,513.96, 10,145.84) | 7,458.5 (5,161.75, 9,755.25) | 6,908.21 (4,625.69, 9,190.72) | 6,636.55 (4,366, 8,907.10) | |
| RSV-atypical bacteria | No | NA | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) | 0.00 (Ref.) |
| Yes | NA | 240.52 (−462.89, 943.92) | 625.61 (−80.94, 1,332.15) | 647.4 (−55.95, 1,350.76) | 748.12 (51.62, 1,444.63) | |
RSV, respiratory syncytial virus; OR, odds ratio; CI, confidence interval; NA, not applicable; Ref., reference.
Mixed-effect logistic regression models were used to estimate odds ratio, and linear mixed models were used to estimate β. Model 1 was unadjusted, model 2 was adjusted for age and sex, model 3 was further adjusted for history of preterm birth, comorbidities, duration of symptoms prior to admission, residential area, and admission year, and model 4 was further adjusted for other co-infections.
Children with both bacterial and fungal co-infections had elevated risks of severe infection (OR = 9.39, 95% CI: 1.76, 50.12) and PICU admission (OR = 40.61, 95% CI: 5.33, 309.53) compared to those with neither co-infection (Table 3). However, no significant additive interaction was detected between RSV-bacterial and RSV-fungal co-infections for any of the severity outcomes. The relative excess risk due to interaction (RERI) was 3.20 (95% CI: –2.71, 9.11) for the composite outcome, 4.38 (95% CI: –2.73, 11.49) for severe infection, 26.00 (95% CI: –25.66, 77.66) for PICU admission, and 1.82 (95% CI: –3.69, 7.33) for oxygen administration.
TABLE 3.
Joint associations of RSV-bacterial and RSV-fungal co-infections with primary and secondary outcomesa,b
| Subgroup | Number of cases/number at risk | OR (95% CI) | RERI (95% CI) |
|---|---|---|---|
| Combined severity outcomes | |||
| No RSV-bacterial co-infection | |||
| No RSV-fungal co-infection | 451/2,341 | 1.00 (Ref.) | NA |
| RSV-fungal co-infection | 11/26 | 2.35 (1.00, 5.55) | NA |
| RSV-bacterial co-infection | |||
| No RSV-fungal co-infection | 124/485 | 1.97 (1.45, 2.68) | NA |
| RSV-fungal co-infection | 7/10 | 6.52 (0.92, 46.36) | 3.20 (−2.71, 9.11) |
| Diagnosis of severe infection | |||
| No RSV-bacterial co-infection | |||
| No RSV-fungal co-infection | 340/2,341 | 1.00 (Ref.) | NA |
| RSV-fungal co-infection | 10/26 | 2.71 (1.12, 6.58) | NA |
| RSV-bacterial co-infection | |||
| No RSV-fungal co-infection | 119/485 | 2.30 (1.66, 3.19) | NA |
| RSV-fungal co-infection | 7/10 | 9.39 (1.76, 50.12) | 4.38 (−2.73, 11.49) |
| PICU admission | |||
| No RSV-bacterial co-infection | |||
| No RSV-fungal co-infection | 42/2,341 | 1.00 (Ref.) | NA |
| RSV-fungal co-infection | 3/26 | 4.90 (1.30, 18.44) | NA |
| RSV-bacterial co-infection | |||
| No RSV-fungal co-infection | 41/485 | 8.72 (4.82, 15.76) | NA |
| RSV-fungal co-infection | 5/10 | 40.61 (5.33, 309.53) | 26.00 (−25.66, 77.66) |
| Oxygen administration | |||
| No RSV-bacterial co-infection | |||
| No RSV-fungal co-infection | 373/2,341 | 1.00 (Ref.) | NA |
| RSV-fungal co-infection | 9/26 | 2.15 (0.89, 5.20) | NA |
| RSV-bacterial co-infection | |||
| No RSV-fungal co-infection | 93/485 | 1.64 (1.18, 2.28) | NA |
| RSV-fungal co-infection | 6/10 | 4.61 (0.94, 22.64) | 1.82 (−3.69, 7.33) |
RSV, respiratory syncytial virus; OR, odds ratio; CI, confidence interval; Ref., reference; RERI, relative excess risk due to interaction; NA, not applicable.
Mixed-effect logistic regression models were used to estimate OR and RERI. The models were adjusted for age, sex, history of preterm birth, comorbidities, duration of symptoms prior to admission, residential area, admission year, RSV-viral co-infection, and RSV-atypical bacterial co-infection.
The associations of RSV-bacterial (Fig. 2A) and RSV-fungal co-infections (Fig. 2B) with the primary outcome were consistent across all subgroups (all P-interaction > 0.05). Sensitivity analyses reaffirmed the association between RSV-bacterial co-infection and the primary outcome (Table S3). However, after excluding children with triple or quadruple co-infections (OR = 1.82, 95% CI: 0.61, 5.49) or adjusting for testing items in the sub-cohort analysis (OR = 2.41; 95% CI: 0.86, 6.61), the association for RSV-fungal co-infection was attenuated and became non-significant, likely due to the reduced sample size.
Fig 2.
Forest plot for the associations of RSV-bacterial (A) and RSV-fungal (B) co-infections with primary outcome in subgroups. OR, odds ratio; CI, confidence interval. Mixed-effect logistic regression models were used to estimate OR and were adjusted for age, sex, history of preterm birth, comorbidities, duration of symptoms prior to admission, residential area, admission year, and other co-infections. To calculate P values for interaction, likelihood ratio tests were conducted by comparing the maximum likelihood χ² values from nested models with and without the multiplicative interaction term between RSV-bacterial or RSV-fungal co-infections and stratification factors. Specifically for panel B, given the limited sample size (n = 36) in the fungal co-infection group and the wide CIs, these results are considered preliminary and should be interpreted with caution.
DISCUSSION
In this multicenter retrospective study of 2,862 children under 5 years hospitalized with RSV, we found that co-infection with other respiratory pathogens was common, affecting 43.75% of the cohort. A significant shift in co-infection rate surrounding the COVID-19 pandemic was observed, with a decline during the period of strict NPIs, followed by a rapid resurgence after their relaxation. We also found that both bacterial and fungal co-infections were independently associated with increased risks of severe outcomes, prolonged hospitalization, and higher inpatient medical expenses. However, no such associations were observed for co-infections with other viruses or atypical bacteria. Furthermore, despite the markedly elevated risk observed in children with both bacterial and fungal co-infections, we did not detect an additive interaction between these two co-infection types on disease severity.
Our study represents one of the few to simultaneously evaluate the associations between four major categories of respiratory pathogen co-infections and clinical outcomes in children hospitalized with RSV. While previous studies have documented the individual impacts of bacterial or viral co-infections, our comprehensive approach provides a broader perspective (13, 14, 17–19). The observed association between bacterial co-infection and increased RSV severity is consistent with a substantial body of literature (6, 13, 17). In contrast, evidence regarding the impact of viral co-infections on disease severity remains conflicting. For instance, a systematic review and meta-analysis reported no significant difference in clinical severity between RSV mono-infection and viral co-infection, except in cases involving human metapneumovirus (19). Conversely, a prospective Belgian study found that children with RSV mono-infection had higher severity scores than those with viral co-infections (18). More recently, a large US population-based surveillance study reported that viral co-detection was not associated with worse outcomes compared to RSV monodetection; co-detection with parainfluenza virus or adenovirus was even associated with lower odds of hospitalization and oxygen use, respectively (14). Within this context of conflicting evidence, our study provides clarifying evidence by demonstrating that, after adjustment for key confounders, including other co-infections, no significant association was observed between RSV-viral co-infection and an increased risk of severe outcomes. It should be noted, however, that our analysis did not differentiate between specific viral types; the overall absence of association may have been influenced by the most commonly co-infected viruses.
Fungal co-infection was relatively uncommon in our cohort, and its relationship with disease severity has not been well characterized in previous studies. Among the limited available literature, only one single-center study reported an association between a higher abundance of Malassezia globosa and a lower risk of hospitalization among infants with RSV infection (20). Our findings demonstrated that RSV-fungal co-infection was associated with the greatest increase in length of stay and inpatient costs, underscoring its clinical relevance. Furthermore, we observed that children with RSV–fungal co-infection tended to be younger, have a history of preterm birth, and present with more severe clinical signs upon admission. These characteristics may provide preliminary evidence to aid in identifying high-risk populations.
Interestingly, RSV-atypical bacterial co-infection was not significantly associated with most severity outcomes, except for a modest increase in inpatient costs. This finding contrasts with a previous, smaller study (n = 182) conducted in younger children (<2 years), which reported that those with MP co-infection had a higher prevalence of fever, flaky opacities on chest X-ray, extrapulmonary manifestations, and longer hospital stays compared to those with RSV bronchiolitis alone (21). However, a more recent study suggested that the proportion of severe pneumonia cases and need for bronchoscopic intervention was lower in children with MP and RSV co-infection than in those with MP mono-infection, implying a potential inhibitory interaction (22). Indeed, our data that included both RSV-negative and RSV-positive participants revealed a negative multiplicative interaction between RSV and atypical bacterial infection (β = −0.513, P = 0.002, data not shown). Although a definitive explanation based on current data remains challenging, our results suggest that atypical bacterial co-infection may not be associated with significantly elevated severity risk in RSV-infected children. The nature of the interaction between RSV and atypical pathogens warrants further investigation.
Beyond dual infections, multiple co-infections (i.e., RSV with two or more other pathogens) are also clinically relevant, as they may indicate a higher microbial burden and potential for synergistic severity mechanisms (23, 24). In the present study, a substantial proportion of co-infected children (180/1,252, 14.4%) had triple or quadruple co-infections. The present study also assessed the possible additive interactions between RSV-bacterial and RSV-fungal co-infections, which are two types independently associated with severe outcomes. The results revealed that the combined effect of RSV-bacterial and RSV-fungal co-infections was similar to the sum of their individual effects, as indicated by a non-significant RERI. This finding of no additive interaction should, however, be interpreted with caution due to the limited number of children with concurrent RSV-bacterial and RSV-fungal co-infections, which reduced the statistical power to detect a significant interaction effect.
Findings of our study should be interpreted with acknowledgment of several limitations. First, due to its retrospective design, the influence of unmeasured confounding factors, such as specific pathogen subtypes or host genetic factors, cannot be entirely ruled out. Moreover, we could not precisely differentiate between co-infections present at admission and superinfections acquired during hospitalization. Second, variations in routine clinical testing protocols across the participating hospitals may have introduced some detection bias in the classification of co-infections, although we attempted to mitigate this by adjusting for the study site as a random effect and performing sensitivity analyses. Furthermore, because more severely ill children were more likely to undergo comprehensive microbiological testing, detection bias could have inflated the observed associations between bacterial and fungal co-infections and clinical severity. However, our sensitivity analysis showed robust results after adjusting for the diagnostic tests ordered, suggesting that the findings were not merely an artifact of differential testing practices. Third, the relatively small number of fungal co-infection cases limited the statistical power and precision of the associated risk estimates, as suggested by the attenuated association after excluding patients with triple or quadruple co-infections. Future studies with larger sample sizes are needed to confirm the association between fungal co-infection and RSV severity. Finally, as our study population was recruited from three major pediatric centers in a single city, the generalizability of our findings to other regions or healthcare settings requires further validation.
In summary, bacterial and fungal co-infections were independently associated with more severe outcomes in children hospitalized with RSV, whereas viral or atypical bacterial co-infections were not. However, we found no evidence of an additive interaction between concurrent bacterial and fungal co-infections. These findings highlight the importance of distinguishing co-infection types in the clinical management of RSV. Future studies are warranted to elucidate the nature of the interaction between RSV and atypical bacteria and to validate the impact of fungal co-infection in larger cohorts.
MATERIALS AND METHODS
Study design and participants
This hospital-based, retrospective study utilized data from three major pediatric medical centers in Wuhan, China, including Tongji Hospital, Wuhan Children’s Hospital, and Maternal and Child Health Hospital of Hubei Province. Participating hospitals provided de-identified data, including diagnostic and procedure codes, billing records, and administrative information. The study included children under 5 years of age who were hospitalized with ARTI between 15 June 2020 and 30 May 2023. We categorized the study duration into two distinct phases based on national health policy: the strict NPIs period (June 2020 to December 2022), characterized by rigorous pandemic control measures, and the relaxation period (January 2023 to May 2023), following the adjustment of COVID-19 management strategies (25). We identified ARTI cases based on the principal discharge ICD-10 codes, with J00–J06 for acute upper respiratory infections, J09–J18 for influenza and pneumonia, and J20–J22 for other acute lower respiratory infections. We focused on ARTI cases with laboratory-confirmed RSV infection.
Laboratory testing and case definition
RSV infection and co-infecting pathogens were identified using a standardized 13-target multiplex real-time PCR panel conducted on nasopharyngeal swabs, nasal flock swabs, nasopharyngeal aspirates, tracheal aspirates, bronchial aspirates, or bronchoalveolar lavage fluid. This single panel simultaneously screened all patients for 11 viral targets (RSV, rhinovirus, bocavirus, parainfluenza virus, coronavirus, influenza A virus, H1N1, H3N2, metapneumovirus, influenza B virus, and adenovirus) and 2 atypical bacterial targets (MP and CP). Beyond this universal screening, additional microbiological investigations were performed based on each hospital’s clinical guidelines or clinician decisions to capture a broader pathogen spectrum. These included conventional methods, such as smear microscopy, bacterial/fungal/mycobacterial cultures, and (1,3)-β-D-glucan (G) and galactomannan (GM) assays, as well as serological antibody detection for five viruses (parainfluenza viruses, RSV, influenza A virus, influenza B virus, and adenovirus) and three atypical pathogens (MP, CP, and LP). These testing methodologies were applied uniformly across the three hospitals. Patients were classified into the co-infection group if any virus (other than RSV) or atypical bacteria was detected, while for bacterial and fungal detections, co-infection was determined by pediatricians through a comprehensive assessment of the pathogen’s pathogenicity and clinical characteristics to distinguish between clinically significant pathogens and potential colonizers (Table S1). Co-infections were categorized as RSV-bacterial, RSV-other viral, RSV-fungal, or RSV-atypical bacterial. These categories were not mutually exclusive, as some children had RSV detected alongside two or more other pathogens.
Covariates data extraction
Data were extracted from electronic health records using a standardized case report form. Demographic variables included age (in months), sex (boys, girls), and residential area (in Wuhan, outside Wuhan). Clinical characteristics encompassed history of preterm birth (no, yes), comorbidities (no, yes), duration of symptoms prior to admission (days), observations at admission (consciousness/spirit/appetite, wet rales, wheezing sound, shortness of breath/three concave signs), chest radiological results (normal, bronchitis, bronchopneumonia, lobar pneumonia, interstitial pneumonia), and hospital admission and discharge dates. Missing values for covariates occurred in less than 0.5% of cases (Table S2) and were managed via mode imputation for categorical variables to maintain the sample size for analysis.
Outcomes
The primary outcome was a composite measure of disease severity, which included diagnosis of severe infection by a pediatrician, PICU admission, or oxygen administration during hospitalization. A diagnosis of severe infection was established if the patient met at least one of the following criteria: poor general condition; refusal to feed or signs of dehydration; impaired consciousness; significantly increased respiratory rate (infants ≥ 70 breaths/min, older children ≥ 50 breaths/min); cyanosis; dyspnea; oxygen saturation <92% at sea level or <90% in high-altitude areas; multi-lobar involvement or >2/3 involvement of a single lobe; pleural effusion; or extrapulmonary complications (26). Secondary outcomes included each component of the primary outcome, length of stay, and inpatient medical expenses.
Statistics analysis
Baseline characteristics were summarized as median (IQR) for continuous variables, and as numbers (percentages) for categorical variables. Only patients with RSV mono-infection or dual co-infections were included in the baseline description to ensure statistical independence. Data were compared across groups using the Mann-Whitney U test for continuous variables and the χ2 test or Fisher’s exact test for categorical variables, with the RSV mono-infection group serving as the reference. We also calculated the annual co-infection rate for each type. Correlations between types of co-infections were assessed using the Phi coefficient (27). Age- and sex-specific co-infection rates were compared using χ2 or Fisher’s exact tests.
In primary analysis, we used a generalized linear mixed model with a logit link function, treating study site as random effects, to estimate ORs and 95% CIs of binary outcomes comparing children with specific co-infection patterns to those without. For continuous outcomes, we used an identity link to estimate mean differences. Model 1 was unadjusted, model 2 was adjusted for age and sex, model 3 was further adjusted for history of preterm birth, comorbidities, duration of symptoms prior to admission, residential area, and admission year, and model 4 was further adjusted for other co-infections. Because both RSV-bacterial co-infection and RSV-fungal co-infection were independently associated with increased risk of disease severity, we calculated the ORs of severity outcomes related to combinations of co-infection status and estimated potential additive interaction as the RERI. The 95% CIs of the RERI were generated by drawing 1,000 bootstrap samples from the estimation data set (28). If there were no additive interaction, the CIs of the RERI would include 0.
We conducted subgroup analyses to test for effect modification by including interaction terms in the fully-adjusted model for the following groups: age (<24 months, and ≥24 months), sex (boys, girls), preterm birth (no, yes), comorbidities (no, yes), and admission year (2020–2022, 2023). We compared the maximum likelihood χ2 from a nested model with and without the multiplicative interaction term. We also performed several sensitivity analyses to examine the robustness of the results. First, we excluded children with preterm birth or with any comorbidities. Second, we excluded children with symptom duration >7 days prior to admission. Third, we excluded children with triple or quadruple co-infections to minimize residual confounding. Fourth, we adjusted for study site as a confounder, rather than treating it as a random effect (29). Last, to mitigate potential detection bias, we conducted an additional analysis in a sub-cohort from Tongji Hospital (n = 1,188) by further adjusting for whether bacterial, fungal, or atypical pathogen tests were performed.
ACKNOWLEDGMENTS
We would like to thank all staff of the Department of Pediatrics of Tongji Hospital, Department of Pediatric Pulmonology of Maternal and Child Health Hospital of Hubei Province, and Department of Respiratory of Wuhan Children's Hospital for data collection.
This study was supported by the Project of the National Health Commission Capacity Building and Continuing Education Center (GWJJZX20251002015), the Investigator-Initiated Studies Program of Merck Sharp & Dohme Corp Grant, the Shanghai Municipal Science and Technology Major Project (ZD2021CY001), and the National Natural Science Foundation of China (82504474). The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Yongjian Huang and Sainan Shu are guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Dong Xu and Zongming Yang contributed equally to the work as co-first authors. Concept and design: Dong Xu, Yongjian Huang, and Sainan Shu; Acquisition, analysis, or interpretation of data: All authors; Drafting of the manuscript: Dong Xu and Zongming Yang; Critical revision of the manuscript for important intellectual content: All authors; Statistical analysis: Dong Xu and Zongming Yang; Obtained funding: Dong Xu and Sainan Shu; Administrative, technical, or material support: Xiuyun Zhou, Wankai Xue, Wenjing Zhang, Rui Shi, Yabin Wu, Yanli Wang, Zenghao Xu, and Xiaoping Luo; Supervision: Yongjian Huang and Sainan Shu.
Contributor Information
Yongjian Huang, Email: yjhuang003@163.com.
Sainan Shu, Email: shusainan@163.com.
Benjamin M. Liu, Children's National Hospital, George Washington University, Washington, DC, USA
ETHICS APPROVAL
The study complied with the Declaration of Helsinki principles and was approved by the Ethics Committee of Tongji Hospital (No. TJ-IRB20221133). Informed consent was waived because anonymous and de-identified secondary data were used for analysis.
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/spectrum.00945-26.
Tables S1 to S3 and Figure S1.
ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.
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Associated Data
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Supplementary Materials
Tables S1 to S3 and Figure S1.