Study population and design
Sepsis patients hospitalized in the ICU at Peking Union Medical College Hospital between February 2017 and July 2020 were assessed in this prospective study. Informed consent was obtained from all the patients involved. This study was approved by the Ethics Committee of Peking Union Medical College Hospital and registered at chictr.org.cn (identifier ChiCTR-ROC-17010750).
Inclusion criteria were as follows: (1) age >18 years; (2) ICU stay ≥48 h; and (3) diagnosis of sepsis. Sepsis was defined as life-threatening organ dysfunction resulting from host immune imbalance caused by infection. Infection was diagnosed by an intensivist and met the systemic inflammatory response syndrome criteria. Organ dysfunction was defined as Sequential Organ Failure Assessment (SOFA) score ≥2 as a result of the infection1. The exclusion criteria were: (1) immunosuppression caused by organ or hematopoietic stem cell transplantation; (2) HIV infection; (3) immunosuppressive treatment prior to admission, that is, prednisone 0.5 mg/kg/day at least 2 weeks before the study, and tumor necrosis factor antagonist, methotrexate, or chemotherapy for cancer within 4 weeks of the study; (4) treatment with antiviral drugs active against CMV, such as ganciclovir or valganciclovir before admission; and (5) pregnancy or lactation.
Patients enrolled in this study underwent a thorough physical examination and all necessary tests and laboratory work were completed to determine the site of infection and the causative pathogen, and to eliminate bacterial colonization. For patients suspected of infection at multiple sites, two intensivists were required to confirm the infection foci. If no agreement could be reached, a senior intensivist was consulted for clarification.
The infection foci were confirmed according to the following conditions. Pneumonia was diagnosed clinically as new or progressive pulmonary infiltrates caused by infection with at least two manifestations as follows: fever >38℃ or hypothermia <36℃; leukocytosis (>12 000 cells/mm3) or leukopenia (<4000 cells/mm3); and presence of newly purulent tracheal secretions and hypoxia. Bloodstream infection was defined as symptoms of infection combined with the presence of a typical or atypical pathogen in blood culture12. Abdominal infection was diagnosed as a new or progressive manifestation of peritonitis, such as abdominal tenderness or rebound pain, accompanied by increased ascites and/or changes in its nature13. Skin and soft-tissue infection was diagnosed as soft-tissue infection accompanied by signs and symptoms of systemic toxicity14. Other infections included mediastinal infection and biliary or urinary tract infection, which have been described previously15.
The pathogens were isolated from multiple sites. Blood, body fluid, and tissue specimens obtained strictly in accordance with aseptic procedures from normally sterile sites were cultured. Blood were sampled simultaneously from two different parts at least. For collecting the sputum samples, patients were tracheal intubated. The aspirated samples met the standard for lower-tract samples of >25 white blood cells and <10 epithelial cells per low-power field of view. The tissue specimen was sampled by the surgeon for culture then the incisions were fully sterilized. Urine samples were required of midstream and obtained after placing or changing the catheter.
CMV infection was defined as detection of serum CMV DNA ≥500 copies/mL by real-time quantitative polymerase chain reaction (PCR), with or without clinical manifestations16. CMV negative conversion was defined as retesting of serum CMV DNA <500 copies/mL in patients with CMV infection during ICU/inpatient treatment17. Persistently positive CMV was defined as >500 copies/mL of serum CMV DNA. On the basis of the above definition, sepsis patients were first divided into CMV-infected and non-CMV-infected groups. CMV-infected sepsis patients were further divided into CMV-DNA negative conversion and CMV-DNA persistently positive groups.
Follow-up included length of ICU stay and in-hospital stay, ICU and in-hospital mortality, and 28-day mortality after enrollment. There was no standard treatment for critical illness, and all treatment options and drug choices were determined by the clinicians.
Clinical and laboratory evaluation
All patients underwent a comprehensive clinical evaluation on the day of admission to the ICU. Age, sex, underlying disease, and important infectious and biochemical indicators (Procalcitonin, creatinine, albumin and bilirubin) were all included. CMV-DNA levels were determined as well as Acute Physiology and Chronic Health Evaluation II (APACHE II) 18 and SOFA scores19. Life-sustaining treatments (need for mechanical ventilation, vasopressors or renal replacement therapy) for ≥24 h were recorded based on clinical evaluation and recent recommendations20. In accordance with the Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-Organ Transplantation21 by the Transplantation Society International CMV Consensus Group in 2018, all patients with CMV infection received intravenous infusion of 5 mg/kg ganciclovir q12h and serum CMV-DNA levels were reviewed during treatment.
The CMV-DNA Diagnostic Kit (Qiagen, Valencia, CA, USA) was used to detect the CMV-DNA level in plasma (per 100 uL) by real-time quantitative PCR using Therma-Base Taqman technology in the Roche Light Cycler 480 detection system. CMV DNA was expressed in copies/mL with a threshold of 500.
Immunological laboratory examination
Blood samples were collected from each sepsis patient on the day of ICU admission for flow cytometry (Epics XL; Beckman Coulter, Brea, CA, USA) and rate nephelometry (Array 360; Beckman Coulter) to analyze the immunophenotyping of blood lymphocyte subsets and serum levels of complement factor (C)3, C4, immunoglobulin (Ig)A, IgG and IgM, as previously described22. Peripheral blood mononuclear cells were isolated and stained with combinations of different fluorescent monoclonal antibodies, followed by flow cytometric analysis (Three-Color EPICS-XL; Beckman Coulter) to detect T cells (CD3+), CD4+ T-cell subsets (CD4+CD3+ and CD28+CD4+), CD8+ T-cell subsets (CD8+CD3+, CD28+CD8+), B cells (CD19+) and NK cells (CD3–CD16+CD56+).
All analyses were performed using SPSS for Windows version 24.0 (IBM Corp., Armonk, NY, USA). Kolmogorov–Smirnov test was used to examine the cumulative distribution functions of the samples. For normally distributed continuous variables, we used Student’s t test and analysis of variance for comparisons between two or three groups. For nonparametric variables, we chose the Mann–Whitney U test. The chi-square or Fisher exact test was chosen, as appropriate, for comparison of categorical variables. P values associated with “equal variances not assumed” were reported for variables that violated the homogeneity of variance assumption. Univariate and multivariate logistic regression analyses were performed to identify the immune parameters for prediction of CMV infection and CMV negative conversion, and the results were expressed as Wald index, P value and odds ratio (OR) with 95% confidence intervals (CIs). Variables that showed P<0.05 in univariate analysis were included in the multivariate regression analysis model, and principal component analysis was applied to adjust for the possible multicollinearity among independent variables. Variables with a variance inflation factor (VIF) >5 were considered collinear. The discriminatory ability of immune parameters for predicting 28-day mortality in sepsis patients with CMV infection was determined by receiver operating characteristic (ROC) curve analysis. The reliabilities and consistencies of diagnostic tests were assessed by calculating their sensitivity, specificity, and positive and negative predictive values. Kaplan–Meier survival analysis was used to construct survival curves, and comparisons of survival distributions were based on the log-rank test. All tests performed were two-tailed, with P<0.05 considered to be statistically significant.