Viral reactivation in the lungs of patients with severe pneumonia is associated with increased mortality, a multicenter, retrospective study

Viral reactivation is widespread in patients with severe pneumonia, yet the landscape of viral reactivation in the lungs is not well‐known. This study aims to assess the landscape and clinical features of viral reactivation in the early onset of severe pneumonia in ICU patients. The clinical data from 97 patients were collected retrospectively from the intensive care units of five teaching hospitals between June 2018 and July 2021. Metagenomic next‐generation sequencing (mNGS) of the bronchoalveolar lavage fluid (BALF) was performed at the onset of severe pneumonia. Cytomegalovirus (CMV), herpes simplex virus‐1 (HSV‐1), and Epstein‐Barr virus (EBV) were the most common reactivated viruses in the lower respiratory tract of patients with severe pneumonia. After adjusting for the risk of confounding and competition of age, sex, sequential organ failure assessment, acute physiology chronic health assessment II and immunosuppression status, viral reactivation resulted in an overall 2.052‐fold increase in 28‐day all‐cause mortality (95% CI: 1.004–4.194). This study showed that CMV, HSV‐1, and EBV were the most common reactivated viruses in the lungs of patients with severe pneumonia. The existence of viral reactivations was associated with an increased risk of mortality. The simultaneous reactivation of multiple viruses needs to be considered in the design of clinical trials.


| INTRODUCTION
Severe pneumonia is one of the leading causes of death in the intensive care unit (ICU) and is often accompanied by the reactivation of cytomegalovirus (CMV), 1 herpes simplex virus (HSV), 2 and Epstein-Barr virus (EBV) during the disease treatment. CMV or EBV reactivation was always assessed using serum samples, [3][4][5] while HSV-1 reactivation was detected through bronchoalveolar lavage fluid (BALF). 6 There has also been a recent widespread viral reactivation in patients with severe pneumonia who have been transferred to the ICU due to the severe Coronavirus Disease 2019 (COVID-19). [7][8][9] Reactivation of CMV, HSV-1, and EBV is associated with poor prognosis. [10][11][12] Metagenomic next-generation sequencing (mNGS) is not routinely used for unbiasedly detecting pathogenic microorganisms. 13,14 In this study, the BALF of ICU patients with severe pneumonia was analyzed by mNGS. The results indicated that CMV, HSV-1, and EBV were the most commonly reactivated

| Data collection and mNGS assay of BALF
For all included patients, the demographic data such as gender, age, days of ICU stay, days of mechanical ventilation, days from admission to mNGS testing, community-acquired pneumonia, immunosuppressive status, and patient outcomes on day 28 were recorded. To assess the disease severity, the acute physiology and chronic health evaluation II (APACHE II) and sequential organ failure assessment (SOFA) were calculated when the patients were admitted to the ICU and on the day of mNGS testing.
Low-speed centrifugation (1500g for 20 min) was performed to remove human cells in the samples. Samples were then homogenized using bead beating, followed by DNA extraction using the IngeniGen DNA Extraction Kit (IngeniGen XMK Biotechnologies Inc). The DNA libraries were prepared using the IngeniGen DNA Library Prep Kit following the manufacturer's protocols. Briefly, the DNA was fragmented, and the Illuminacompatible adapters were simultaneously added to the fragmented DNA by a tagmented enzyme. The library was purified using magnetic beads and then amplified after 15 PCR cycles. Samples were then homogenized using bead beating, followed by RNA extraction using the IngeniGen RNA Extraction Kit (IngeniGen XMK Biotechnologies, Inc). The RNA libraries were constructed using the IngeniGen XMKbio RNA-seq Library Prep Kit (IngeniGen XMK Biotechnologies, Inc). Briefly, DNase was used to remove residual human DNA, and RNA was fragmented, followed by double-strand cDNA synthesis, end-repair, dA-tailing, and adapter ligation. Sequencing was performed on Illumina MiniSeq (Illumina) using 2 × 75 bp chemistry for 78 samples and Illumina NextSeq (Illumina) using 1 × 100 bp chemistry for 19 samples. A negative control was included in each run to detect background contaminants, and internal control was added to each sample to monitor the entire process. Data analysis was performed as follows: First, the fastp software (https://github.com/OpenGene/fastp) was used to filter raw sequencing data to remove adapters and low-quality sequences, and then the bowtie2 software (http:// bowtie-bio.sourceforge.net/bowtie2/index.shtml) aligned the sequences to the human reference genome (GRCh38) to remove host sequences. 15,16 The processing results of reads are provided in the Supporting Information. The remaining sequences were classified and annotated using the Kraken2 (https://ccb.jhu.edu/ software/kraken2/) software, and the results were visualized using the pavian tool (http://ccb.jhu.edu/software/pavian/). 17 Pavian outputs from Kraken2 results are provided in the Supporting Information. The raw data of mNGS is available from the Genome Sequence Archive for Human (HRA002995).

| Patient characteristics
In this study, the participants were classified based on the presence of CMV, HSV-1, and EBV. The nonreactivation and viral reactivation groups included 51 and 46 patients, respectively ( Table 1). The two groups of patients were not significantly different in terms of age, There was no statistically significant difference between the SOFA and APACHE II at admission or at mNGS testing, which suggested that the two groups of patients had similar severity.
However, 24 of the patients (52.2%) in the reactivation group had an immunosuppressed state, which is significantly higher than 12 of the patients (23.5%) in the nonreactivation group (p = 0.006).

| Twenty-eight-day all-cause mortality
Survival curves were plotted to observe the survival status of the patients in several groups (Figure 2A

| DISCUSSION
In this study, we performed mNGS assays on BALF and showed

| CONCLUSION
This study showed that CMV, HSV-1, and EBV were the most commonly reactivated viruses in the lungs of patients with severe pneumonia. The existence of viral reactivation was associated with an increased risk of mortality. This simultaneous reactivation of multiple viruses needs to be considered in the design of clinical trials.

CONFLICT OF INTEREST
The authors declare no conflict of interest.