Altered respiratory virome and serum cytokine profile associated with recurrent respiratory tract infections in children

Recurrent acute respiratory tract infections (ARTIs) affect a large population, yet the specific decisive factors are largely unknown. Here we study a population of 4407 children diagnosed with ARTI, comparing respiratory virome and serum cytokine profiles associated with multiple ARTIs and single ARTI during a six-year period. The relative abundance of Propionibacterium phages is significantly elevated in multiple ARTIs compared to single ARTI group. Serum levels of TIMP-1 and PDGF-BB are markedly increased in multiple ARTIs compared to single-ARTI and non-ARTI controls, making these two cytokines potential predictors for multiple ARTIs. The presence of Propionibacterium phages is associated with higher levels of TIMP-1 and PDGF-BB. Receiver operating characteristic (ROC) curve analyses show that the combination of TIMP-1, PDGF-BB and Propionibacterium phages could be a strong predictor for multiple ARTIs. These findings indicate that respiratory microbe homeostasis and specific cytokines are associated with the onset of multiple ARTIs over time.


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Software and code
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Data collection
The clinical data were collected from the medical records of all patients. The NGS data were collected using the BGI-Seq500 platform (BGI, China). The expression data of cytokines were collected using Mepix (InnoScan 300 Microarray Scanner).

Data analysis
The raw NGS data was filtered by SOAPnuke software; Human host sequences were subtracted by SNAP software; and reads were assembled into contigs by Minimo9 and IDBA-UD. All statistical tests were performed using the SPSS19.0 or R package vegan 2.4-4; heatmap was obtained Origin Pro 2017, other graphs were generated by Origin Pro 2017 or GraphPad Prism 6; ROC curve and logistic regression analysis were obtained by SPSS19.0.
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All the sequencing data excluding human sequences were deposited in the CNSA (https://db.cngb.org/cnsa/) of CNGBdb (project number CNP0000429) under the accession number CNS0092588-CNS0092799. All the software used in this study are available from open source.

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Sample size
A total of 4407 children with ARTI were recruited in this study and 192 children were found to experience two or more ARTI episodes. Among them, 160 children had two ARTI episodes, 23 had three, 8 had four and one child had five. To investigate the respiratory virome associated with multiple ARTIs, all 32 other children experienced three to five episodes were selected for meteogenomic sequencing, together with 30 randomly selected children experienced two episodes for comparison. One child having two ARTIs was excluded from the final analysis due to failure in subsequent next generation sequencing (NGS), and thus results from a total of 61 multiple-ARTI children were presented. For comparison, 50 children who experienced only one ARTI episode during this study period (known as the single-ARTI group) and matched the multiple-ARTIs group with respect to age, gender, clinical presentations and laboratory tests were selected. Two of these were excluded from the final analysis due to failure in subsequent NGS, and thus results from a total number of 48 subjects were presented. For cytokine assay, a total of 212 ARTI serum samples (from the same individuals that used for respiratory virome study) including 48 from single ARTI children, 164 from multiple ARTIs children, and 20 healthy controls (non-ARTI group) were initially used for cytokine profile measurement. Five multiple-ARTI samples were excluded due to poor quality of cytokine assay, and thus results from 207 ARTI samples were presented. The sample size in each group were comparable with each other and exceed all previously studies. The results clearly support the conclusions.
Data exclusions One child having two ARTIs was excluded from the final analysis due to failure in subsequent next generation sequencing (NGS), and thus results from a total of 61 multiple-ARTI children were presented. Two of 50 children with one ARTI were excluded from the final analysis due to failure in subsequent NGS, and thus results from a total number of 48 subjects were presented. For the cytokine assay, five multiple-ARTI samples were excluded due to poor quality , and thus results from 207 ARTI samples were presented. No data were excluded from the subsequent analyses.

Replication
In this study, we characterized the respiratory virome and cytokine levels in children with multiple ARTIs. For metagenomic analyses and virome, we used a well-established method that has been widely used, it is not involved in the repeated experiments. For the determination of cytokine levels, we used a proteomic chip-based cytokine assay, and each immune mediator was measured in quadruplicates on the chips.
Randomization We investigated the respiratory virome and immune profiles of single and multiple ARTIs among children from 2009 to 2015. The groups were allocated based on the times of ARTI episodes (from one to five times) during this study period. We included all involved children in the groups of three to five ARTIs. For the group of two ARTI episodes, 30 children were selected for compatibility with other groups (3 to 5 ARTI episodes). In addition, 50 children with one ARTI were randomly selected, and the number was matched the multiple-ARTIs group with respect to age, gender, clinical presentations and laboratory tests . Furthermore, 20 healthy children were included. We think our grouping was convincing.