Global Coinfections with Bacteria, Fungi, and Respiratory Viruses in Children with SARS-CoV-2: A Systematic Review and Meta-Analysis

Background: Coinfection with bacteria, fungi, and respiratory viruses has been described as a factor associated with more severe clinical outcomes in children with COVID-19. Such coinfections in children with COVID-19 have been reported to increase morbidity and mortality. Objectives: To identify the type and proportion of coinfections with SARS-CoV-2 and bacteria, fungi, and/or respiratory viruses, and investigate the severity of COVID-19 in children. Methods: For this systematic review and meta-analysis, we searched ProQuest, Medline, Embase, PubMed, CINAHL, Wiley online library, Scopus, and Nature through the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for studies on the incidence of COVID-19 in children with bacterial, fungal, and/or respiratory coinfections, published from 1 December 2019 to 1 October 2022, with English language restriction. Results: Of the 169 papers that were identified, 130 articles were included in the systematic review (57 cohort, 52 case report, and 21 case series studies) and 34 articles (23 cohort, eight case series, and three case report studies) were included in the meta-analysis. Of the 17,588 COVID-19 children who were tested for co-pathogens, bacterial, fungal, and/or respiratory viral coinfections were reported (n = 1633, 9.3%). The median patient age ranged from 1.4 months to 144 months across studies. There was an increased male predominance in pediatric COVID-19 patients diagnosed with bacterial, fungal, and/or viral coinfections in most of the studies (male gender: n = 204, 59.1% compared to female gender: n = 141, 40.9%). The majority of the cases belonged to White (Caucasian) (n = 441, 53.3%), Asian (n = 205, 24.8%), Indian (n = 71, 8.6%), and Black (n = 51, 6.2%) ethnicities. The overall pooled proportions of children with laboratory-confirmed COVID-19 who had bacterial, fungal, and respiratory viral coinfections were 4.73% (95% CI 3.86 to 5.60, n = 445, 34 studies, I2 85%, p < 0.01), 0.98% (95% CI 0.13 to 1.83, n = 17, six studies, I2 49%, p < 0.08), and 5.41% (95% CI 4.48 to 6.34, n = 441, 32 studies, I2 87%, p < 0.01), respectively. Children with COVID-19 in the ICU had higher coinfections compared to ICU and non-ICU patients, as follows: respiratory viral (6.61%, 95% CI 5.06–8.17, I2 = 0% versus 5.31%, 95% CI 4.31–6.30, I2 = 88%) and fungal (1.72%, 95% CI 0.45–2.99, I2 = 0% versus 0.62%, 95% CI 0.00–1.55, I2 = 54%); however, COVID-19 children admitted to the ICU had a lower bacterial coinfection compared to the COVID-19 children in the ICU and non-ICU group (3.02%, 95% CI 1.70–4.34, I2 = 0% versus 4.91%, 95% CI 3.97–5.84, I2 = 87%). The most common identified virus and bacterium in children with COVID-19 were RSV (n = 342, 31.4%) and Mycoplasma pneumonia (n = 120, 23.1%). Conclusion: Children with COVID-19 seem to have distinctly lower rates of bacterial, fungal, and/or respiratory viral coinfections than adults. RSV and Mycoplasma pneumonia were the most common identified virus and bacterium in children infected with SARS-CoV-2. Knowledge of bacterial, fungal, and/or respiratory viral confections has potential diagnostic and treatment implications in COVID-19 children.


Introduction
Although most cases of coronavirus disease 2019  in pediatric populations are mild or asymptomatic [1], the clinical spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children ranges from asymptomatic to life-threatening [2,3]. Similar to adults, coinfection with bacteria, fungi, and respiratory viruses has been described as a factor associated with more severe clinical outcomes in children with COVID-19 [4][5][6][7][8][9][10][11]. Such coinfections have been reported to increase morbidity and mortality, therefore, knowledge of bacterial, fungal, and/or respiratory viral confections has potential diagnostic and treatment implications in children infected with SARS-CoV-2. Many studies have shown that COVID-19 children may develop severe diseases, requiring intensive care admission and/or mechanical ventilation because patients rapidly develop acute respiratory distress syndrome and sepsis, leading to death from multiple organ failure [12][13][14][15][16][17][18][19][20][21][22][23]. SARS-CoV-2 is hypothesized to weaken the bodies of children to bacterial, fungal, and/or respiratory viral coinfections [24], yet the mechanism of coinfection has not been fully established, but represents a threat to the respiratory epithelium favoring bacteremia, fungaemia, and/or viraemia (see Figure 1).
There is a lack of systematic reviews and meta-analyses on the type and frequency of coinfection by bacterial, fungal, and/or respiratory viral infections and associated clinical outcomes among COVID-19 children. We aimed to identify the type and proportion of coinfections with SARS-CoV-2 and bacteria, fungi, and/or respiratory viruses, and investigate the severity of COVID-19 in these patients. Figure 1. Monoinfection with SARS-CoV-2 results in less severe form of COVID-19 and better prognosis. In contrast, SARS-CoV-2 coinfection with bacteria, fungi, and/or respiratory viruses may intensify the severity of COVID-19 and increase the expression of macrophages, T and B defensive cells that may cause the elevation of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, and interleukin-6 in the infected organs, leading to a hyperinflammatory response by recruiting immune cells.

Design
We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA) in conducting this systematic review and meta-analysis [25]. The following electronic databases were searched: PROQUEST, MEDLINE, EMBASE, PUBMED, CINAHL, WILEY ONLINE LIBRARY, SCOPUS, and NATURE with Full Text. We used the following keywords: ("COVID-19" OR "SARS-CoV-2" OR "Severe acute Respiratory Syndrome Coronavirus 2" OR "Coronavirus Disease 2019" OR "2019 novel coronavirus") AND ("children" OR "child" OR "paediatric" OR "pediatric" OR "infant" OR "toddler" OR "adolescent" OR "newborn") AND ("coinfection" OR "co-infection" OR "cocirculation" OR "co-circulation" OR "coinfected" OR "co-infected" OR "co-circulated" OR "mixed" OR "concurrent" OR "concomitant"). The search was limited to papers published in English between 1 December 2019 and 1 October 2022. Based on the title and abstract of each selected article, we selected those discussing and reporting the occurrence of bacterial, fungal, and/or respiratory viral coinfection in children with COVID-19.

Inclusion-Exclusion Criteria
Inclusion criteria were as follows: (1) published case reports, case series, and cohort studies that focused on children infected with SARS-CoV-2 and bacteria, fungi, and/or respiratory viruses; (2) studies of experimental or observational design reporting the incidence of SARS-CoV-2 infection in pediatric patients with other co-pathogens; (3) language restricted to English. The exclusion criteria were as follows: (1) editorials, commentaries, case and animal studies, reviews, and meta-analyses; (2) studies that did not report data on COVID-19 in coinfected patients; (3) studies that never reported details on identified coinfected cases with SARS-CoV-2 infection; (4) studies that reported coinfection in adult COVID-19 patients; (5) studies that reported coinfection in patients with negative SARS-CoV-2 polymerase chain reaction (PCR) tests; (6) duplicate publications.

Data Extraction
Six authors (Saad Alhumaid, Muneera Alabdulqader, Nourah Al Dossary, Zainab Al Alawi, Abdulrahman A. Alnaim, and Koblan M. Al mutared) critically reviewed all of the studies retrieved and selected those judged to be the most relevant. Data were carefully extracted from the relevant research studies independently. Articles were categorized as case report, case series, or cohort studies. The following data were extracted from selected studies: authors; publication year; study location; study design and setting; number of SARS-CoV-2 children tested for co-pathogens; number of coinfected children; age; proportion of male children; patient ethnicity; number of children with bacterial, fungal, and/or respiratory viral coinfections; total organisms identified; antimicrobials prescribed; laboratory techniques for co-pathogen detection; number of children admitted to intensive care unit (ICU), placed on mechanical ventilation, and/or suffered acute respiratory distress syndrome (ARDS); assessment of study risk of bias; and final treatment outcome (survived or died). These data are noted in Table 1.

Quality Assessment
For many selected cohort studies, the Newcastle-Ottawa scale (NOS) was used to assess the risk of bias, a tool which measures quality in the three parameters of selection, comparability, and exposure/outcome, and allocates a maximum of 4, 2, and 3 points, respectively [26]. High-quality studies are scored greater than 7 on this scale, and moderatequality studies between 5 and 7 [26]. Otherwise, quality assessment of the selected case report and case series studies was undertaken based on the modified NOS [27]. Items related to the comparability and adjustment were removed from the NOS, and items which focused on selection and representativeness of cases, and the ascertainment of outcomes and exposure, were kept [27]. Modified NOS consists of five items, each of which requires a yes or no response to indicate whether bias is likely, and these items were applied to singlearm studies [27]. Quality of the study was considered good if all five criteria were met, moderate when four were met, and poor when three or less were met. Quality assessment was performed by six authors (Khalid Al Noaim, Mohammed A. Al Ghamdi, Suha Jafar Albahrani, Abdulaziz A. Alahmari, Sarah Mahmoud Al HajjiMohammed, and Yameen Ali Almatawah) independently, with any disagreement to be resolved by consensus.

Data Analysis
The proportion of confirmed COVID-19 children with bacterial, fungal, and/or respiratory viral coinfection were examined. This proportion was further classified based on initial presentation or during the course of the illness. A random effects DerSimonian-Laird model was used, which produces wider confidence intervals (Cis) than a fixed effect model [28]. Results are illustrated using a forest plot. The Cochran's chi-square (χ 2 ) and the I 2 statistic provided the tools for examining statistical heterogeneity [29]. An I 2 value of >50% suggested significant heterogeneity [30]. To lower the source of heterogeneity, we conducted a subgroup analysis based on children's admission to the ICU. To estimate publication bias, funnel plots and Egger's correlation were used, and a p-value < 0.05 was considered to indicate statistical significance. All p-values were based on two-sided tests and significance was set at a p-value less than 0.05. R version 4.1.0 with the packages finalfit and forestplot was used for all statistical analyses. Figure 1 was created with BioRender.com (agreement no. NX24IV1VNB) (accessed on 14 October 2022).
In fungal coinfected COVID-19 children, subgroup analysis showed almost a threefold increase in the rates between all patients (patients in the ICU and non-ICU group or ICU only group); the ICU only group showed a prevalence of 1.72% (95% CI 0.45 to 2.99, n = 11, three studies, I 2 0%, p = 0.63), while the ICU and non-ICU group showed a prevalence of 0.62% (95% CI 0.00 to 1.55, n = 6, three studies, I 2 54%, p = 0.11), respectively; Figure 4.
However, in the respiratory viral coinfected COVID-19 children, subgroup analysis showed a slight difference in the rates between all patients (patients in the ICU and non-ICU group or ICU only group); the ICU and non-ICU group showed a prevalence of 5.31% (95% CI 4.31 to 6.30, n = 418, 28 studies, I 2 88%, p < 0.01), while the ICU only group showed a prevalence of 6.61% (95% CI 5.06 to 8.17, n = 23, four studies, I 2 0%, p = 0.90), respectively; Figure 5.

Fungal Pathogen Type
Identified

Discussion
This systematic review and meta-analysis included 17,588 laboratory-confirmed COVID-19 children from 130 observational studies to estimate the prevalence of coinfections with bacteria, fungi, and/or respiratory viruses. Children with SARS-CoV-2 infection had the following prevalence of pathogen coinfections: bacterial (4.7%, 95% CI 3.8-5.6), fungal (0.9%, 95% CI 0.1-1.8), and respiratory viral (5.4%, 95% CI 4.4-6.3). COVID-19 children had higher fungal and respiratory viral coinfections in ICU units (1.7%, 95% CI 0.4-2.9 and 6.6%, 95% CI 5-8.1, respectively) than mixed ICU and non-ICU patients. However, bacterial coinfection was lower in children infected with SARS-CoV-2 in ICU group (3%, 95% CI 1.7-4.3). Children with COVID-19 seem to have a distinctly lower susceptibility to bacterial, fungal, and/or respiratory viral coinfections than adults. Our study documents that 4.7% (bacteria), 0.9% (fungal), and 5.4% (viral) of the pediatric COVID-19 population harbor microbiologically confirmed coinfections, which is much lower than the recent systematic review and meta-analysis, including 72 studies, conducted from 1 December 2019 to 31 March 2021, portraying coinfection rates of 15.9% (bacterial), 3.7% (fungal), and 6.6% (viral) in the adult COVID-19 population [141]. Lower rates of bacterial, fungal, and/or respiratory viral coinfection in children with SARS-CoV-2 infection compared to the adult COVID-19 population may have different explanations. Immunologically, children seem to have an immature receptor system, immune-system-specific regulatory mechanisms, and possible cross-protection from other common bacterial, fungal, and viral infections occurring in children [142,143]. A growing body of evidence suggests that children's immune systems can neutralize SARS-CoV-2 because their T cells are relatively naïve and mostly untrained, and thus might have a greater capacity to respond to new viruses and eliminate SARS-CoV-2 before it replicates in large numbers [144][145][146]. Children are also the main reservoir for seasonal coronaviruses, and some researchers have suggested that antibodies for these coronaviruses might confer some protection against SARS-CoV-2 [143,146]. Moreover, children are more protected at the cellular level, as the expression of angiotensin-converting enzyme 2, which is the receptor that SARS-CoV-2 uses for host entry, is less frequently expressed in the epithelial cells of the nasal passages and lungs of younger children [147]. Otherwise, differences can be explained by the numerous different study designs to a large extent, as well as selection bias, consideration of respiratory and extra-respiratory pathogens, microbiological investigations employed, use of culture and non-culture methods, time of specimen collection, exclusion/inclusion of contaminants, climate, temporal variations in microbial epidemiology and the study population itself.
Three previous systematic reviews and meta-analyses reported on bacterial, fungal, and respiratory vial coinfections; however, these studies included mixed populations of adults and children, included a smaller number of studies (with most data for adults and very few pediatric patients), and sensitivity analysis to study the proportion of coinfection in COVID-19 children was not conducted [148][149][150]. To the best of our knowledge, this is the first and largest systematic review and meta-analysis to report exclusively on bacterial, fungal, and respiratory viral coinfection in children with COVID-19, and we pooled evidence from 130 studies, including at least Mycoplasma pneumoniae, Streptococcus pneumoniae, Mycobacterium tuberculosis, Staphylococcus aureus, RSV, rhinovirus, influenza A or B virus, adenovirus, parainfluenza virus, and metapneumovirus due to their virulence and prevalence, in an attempt to avoid measurement bias. Of the 98.6% who had additional respiratory viruses or bacteria detected, we found that the most common identified virus and bacterium in children with COVID-19 were RSV (n = 342, 31.4%) and Mycoplasma pneumonia (n = 120, 23.1%), in line with findings in two previous systematic reviews and meta-analyses, which reported that RSV and Mycoplasma pneumonia were the most commonly isolated co-pathogens in the adult population with SARS-CoV-2 infection [148,150]. RSV and Mycoplasma pneumonia cause acute respiratory tract illness in people of all ages, and all children are infected with RSV by 2 years of age [151], while approximately one-half of patients infected with Mycoplasma pneumonia are <6 years old [school-age years) [152]. RSV is the most common cause of lower respiratory tract infection in children <1 year of age [153], and bronchiolitis (up to 80% of which is caused by RSV) is a leading cause of hospital admission [154] and an important cause of death in infants and young children [155]. Mycoplasma pneumonia is the second most common cause of respiratory tract infections, and upper and lower respiratory tracts may be affected [156]. This pathogen causes a wide spectrum of illness, ranging from asymptomatic to severe community-acquired pneumonia or extrapulmonary manifestations necessitating ICU admission [157,158]. Several countries have reported that there has been a suppression of RSV and Mycoplasma pneumonia circulation, and their typical seasonality, since early 2020 due to the preventive infection control measures and non-pharmaceutical interventions against SARS-CoV-2 [159][160][161][162][163][164]. However, RSV and Mycoplasma pneumonia activity rebounded in early-mid 2021 at a fast pace, as public health restrictions and social distancing regulations were relaxed; higher hospitalization rates were reported, and most of the hospitalized children required ICU admission [165][166][167]. Although two recent studies demonstrated no association between SARS-CoV-2 and RSV coinfection and clinical severity (need or use of supplemental oxygen, ICU admission, mechanical ventilation, and mortality), the evidence was only based on three small studies [167,168]. In contrast, evidence of clinical severity regarding cases coinfected with SARS-CoV-2 and Mycoplasma pneumonia is well-established, and several studies reported such coinfection as being associated with an increase in inpatient mortality, length of hospital stay, and need for mechanical ventilation [69,100,169,170]. In children, both RSV and Mycoplasma pneumonia are similar to SARS-CoV-2; as potential triggers for a cytokine storm, leading to the development of Multisystem Inflammatory Syndrome in Children (MIS-C), they appear to play a role in the pathogenesis, and may contribute to the subsequent clinical severity of COVID-19. The cytokines tumor necrosis factor-alpha, interleukin-8, interleukin-6, and interleukin-1 beta were detected in the airway secretions of children infected with RSV and Mycoplasma pneumonia, which may act as a double whammy of respiratory pathogens and correlate with severe pathogenesis [171][172][173][174]. As coinfection with either the highly contagious RSV or Mycoplasma pneumonia and SARS-CoV-2 can modify the disease course and contribute to severity, and can cause serious compilations in children, especially those with high-risk comorbidities, healthcare workers need to consider RSV or Mycoplasma pneumonia and SARS-CoV-2 coinfection in the differential diagnosis of acute febrile illness in the endemic areas.
It is noteworthy that in the studies where the laboratory techniques for co-pathogen detection were described, a high number of bacterial and viral coinfections in children infected with SARS-CoV-2 included in our review were diagnosed serologically through the detection of immunoglobulins M and/or G. One of the easiest, most convenient, and fastest point-of-care testing to diagnose COVID-19 and other bacterial, fungal, and/or respiratory co-pathogens is by rapid serology tests; however, serology testing has been associated with many false-positive antibody test results for COVID-19 and mixed pathogens [111,175,176]. Therefore, application of serologic laboratory techniques for co-pathogen detection across all studies was likely to reveal an even higher overall coinfection proportion and high rates of anti-infective use for admitted children with SARS-CoV-2 infection to treat documented or presumed bacterial, fungal, and/or respiratory viral coinfections [177][178][179]. In line with previous studies, we identified high anti-infective use in pediatric patients with COVID-19 [177,180,181]. As the prevalence of bacterial, fungal, or respiratory viral coinfections in children with COVID-19 is not high, and anti-infectives likely provide minimal benefit as an empirical treatment, clinicians should prescribe anti-infectives wisely, and only in cases with an objective diagnosis of coinfection, as injudicious use of anti-infectives is associated with unintended consequences, such as adverse events, toxicity, resistance, Clostridioides difficile infections, risk of emergence and transmission of multidrug-resistant organisms, morbidity, and death [182][183][184][185][186][187]. Undoubtedly, coinfection in children with COVID-19 is likely to be an important modifier in the development of these abovementioned unintended consequences; however, the degree to which co-pathogens interact with SARS-CoV-2 remains unclear in many cases, and even where we know that interactions are occurring, the mechanisms are often poorly defined [188,189].
The combined pooled prevalence for fungal coinfections reported in our review in COVID-19 children is very low (0.98%). In general, very low numbers of fungal species, out of thousands of fungi, are pathogenic [190], and fungal infections in children, other than those caused by Candida species, are uncommon [191]. This can be explained by the strong natural immunity towards fungi in healthy children, and almost every invasive fungal infection that occurs in children is opportunistic [192]. In line with previous studies, all children infected with SARS-CoV-2 who were coinfected with fungi had recognized risk factors for fungaemia, such as use of central lines, malignancy, renal failure, mechanical ventilation, immunosuppression, neutropenia, solid organ transplant recipients, and use of broad-spectrum parenteral antibiotics and corticosteroids [193,194]. Fungal infections in children can be curbed by early diagnosis and timely treatment with the optimal prescription of antifungals based on culture and susceptibility tests, along with adopting appropriate hygienic and sanitization measures [195,196].

Limitations of the Study
We acknowledge that our study is not without some limitations. First, while all of the evidence discussed was based on many cohorts and case series, and some case reports, many of these were small and performed in single centers, and not necessarily generalizable to children infected with SARS-CoV-2 who had bacterial, fungal, or respiratory viral coinfections. Second, almost all studies included in this review were retrospective in design, except seven prospective studies, which could have introduced potential reporting bias due to reliance on obtaining illness histories regarding the identified pediatric cases with COVID-19 and coinfection from household members or contacts and clinical case records. Third, to asses factors associated with the clinical severity in children infected with SARS-CoV-2 who have coinfections, a larger cohort of patients is needed. Last, the study was not registered in Prospero, an international prospective register of systematic reviews, as this might have added extra work and the merit was mostly limited to the avoidance of duplication.

Conclusions
Children with COVID-19 seem to have distinctly lower rates of bacterial, fungal, and/or respiratory viral coinfections than adults. RSV and Mycoplasma pneumonia were the most common identified virus and bacterium in children infected with SARS-CoV-2. Knowledge of bacterial, fungal, and/or respiratory viral confections has potential diagnostic and treatment implications in COVID-19 children.