背景：感染是风湿免疫病患者常见的并发症之一，影响着治疗和预后。受疾病本身导致的免疫功能障碍及应用糖皮质激素/免疫抑制剂的影响，风湿免疫病患者发生活动性巨细胞病毒（Cytomegalovirus，CMV）感染的风险增加。CMV特异性细胞介导免疫（Cell-Mediated Immunity，CMI）对于控制CMV感染至关重要，然而现有研究多针对移植受者和人类免疫缺陷病毒感染者。当前国际上对风湿免疫病人群中CMV感染的认识不足，探究此类人群临床特征、转归及CMV特异性CMI对风湿免疫病患者CMV感染的风险分层和制定管理策略具有重要意义。

目的：

探讨系统性红斑狼疮（Systemic Lupus Erythematosus，SLE）合并活动性CMV感染患者的临床特征及预后。

探索风湿免疫病患者不同CMV感染状态下CMV抗原特异性多细胞因子水平的差异，筛选具有潜在鉴别价值的细胞因子及其组合。

评估QuantiFERON‐CMV（QF-CMV）方法在预测风湿免疫病患者活动性CMV感染中的临床应用价值。

方法：

第一部分：系统性红斑狼疮合并活动性巨细胞病毒感染的临床特征及预后分析

采用回顾性研究设计。纳入2016年6月至2022年12月期间在北京协和医院住院且确诊为活动性CMV感染的SLE患者。查阅病历资料，提取患者的临床特征、实验室数据及预后信息。采用卡方检验评估血浆CMV DNA病毒载量与pp65抗原血症之间的一致性。采用Logistics回归模型分析SLE患者发生CMV病的相关因素，并通过COX比例风险模型分析该人群3个月内全因死亡和CMV复发的相关因素。

第二部分：巨细胞病毒抗原特异性多细胞因子免疫应答鉴别风湿免疫病患者不同巨细胞病毒感染状态的探索性研究

采用病例对照研究设计。纳入2023年3月至2023年8月期间在于北京协和医院住院的风湿免疫病合并潜伏或活动性CMV感染（包括CMV病和亚临床CMV感染）的患者。使用模拟人类CMV蛋白的肽混合物刺激患者全血样本，通过Luminex平台检测血浆中γ干扰素（Interferon-γ，IFN-γ）、肿瘤坏死因子-α（Tumor Necrosis Factor-α，TNF-α）、白细胞介素-2（Interleukin-2，IL-2）、白细胞介素-4、白细胞介素-6、白细胞介素-10、白细胞介素-17和CXC趋化因子配体2的水平，并进行组间比较，以筛选具有潜在鉴别价值的细胞因子及其组合。

第三部分：QuantiFERON‐CMV预测风湿免疫病患者活动性巨细胞病毒感染发病风险的前瞻性研究

采用前瞻性观察性研究设计。纳入2023年3月至2023年8月期间在北京协和医院住院的风湿免疫病合并潜伏性CMV感染的患者。使用QF-CMV（基于酶联免疫吸附试验的IFN-γ释放试验）量化CMV特异性CMI。同时，收集患者的基线人口统计学特征、临床信息及实验室数据，并对患者进行为期1年的随访，记录活动性CMV感染的发生情况。最后，评估QF-CMV不同阈值对风湿免疫病患者活动性CMV感染的预测价值。

结果：

第一部分：系统性红斑狼疮合并活动性巨细胞病毒感染的临床特征及预后分析

本研究共纳入206例患者，在123例在抗病毒治疗前同时或间隔不超过72小时检测了血浆CMV DNA病毒载量和pp65抗原血症，且间隔期内未调整治疗方案的患者中，血浆CMV DNA病毒载量与pp65抗原血症之间的的一致性不佳（Kappa = -0.304，P < 0.001）。血浆CMV DNA病毒载量≥ 1,600拷贝/mL[比值比（Odds Ratio，OR） 4.411，95% CI 1.871 - 10.402，P = 0.001]、大剂量激素治疗（OR 2.155，95% CI 1.071 - 4.334，P = 0.031）及丙氨酸氨基转移酶升高（OR 3.409，95% CI 1.563 - 7.435，P = 0.002）是诊断CMV病的重要线索。CMV脏器受累[风险比（Hazard Ratio，HR） 47.222，95% CI 5.621 - 396.689，P < 0.001]、SLE受累系统数目（HR 1.794，95% CI 1.029 - 3.128，P = 0.039）和超敏C反应蛋白（Hypersensitive C-Reactive Protein，hsCRP）升高（HR 5.767，95% CI 1.190 - 27.943，P = 0.030）是此类人群3个月全因死亡的独立相关因素。此外，CMV脏器受累（HR 3.404，95% CI 1.074 - 10.793，P = 0.037）也是此类人群3个月内CMV复发的独立相关因素。

第二部分：巨细胞病毒抗原特异性多细胞因子免疫应答鉴别风湿免疫病患者不同巨细胞病毒感染状态的探索性研究

本研究共纳入60例风湿免疫病患者（主要为SLE），其中活动性CMV感染和潜伏性CMV感染各30例，前者包括CMV病16例和亚临床CMV感染14例。潜伏性CMV感染组合并严重淋巴细胞减少症的比例显著低于其他组（P < 0.001）。CMV病组特异性IFN-γ、TNF-α、IL-2水平显著低于潜伏性CMV感染组，P值分别为0.002、0.012、0.007。TNF-α、IFN-γ和IL-2可较好地区分活动性CMV感染和潜伏性CMV感染，曲线下面积分别为0.729、0.708和0.713。TNF-α、IFN-γ与严重淋巴细胞减少症联合应用时AUC值增加至0.854。IFN-γ、TNF-α和IL-2也可较好地区分CMV病和潜伏性CMV感染，AUC值分别为0.806、0.765和0.765。IFN-γ、TNF-α与严重淋巴细胞减少症联合应用时曲线下面积增加至0.935。

第三部分：QuantiFERON‐CMV预测风湿免疫病患者活动性巨细胞病毒感染发病风险的前瞻性研究

178例风湿免疫病患者中有13例（7.3%）在1年内发生活动性CMV感染，中位发病时间为14天。当QF-CMV阈值设定为0.2 IU/mL时，阴性预测值为94.4%。然而，QF-CMV阳性与阴性患者（包括无反应和不确定结果）的1年活动性CMV感染发生率无显著差异（5.6% vs. 11.1%，HR 1.961，95% CI 0.602 - 6.394，Log-rank P = 0.217）。进一步提高阈值至0.5 IU/mL时，此时阴性预测值升至97.4%，QF-CMV阳性患者的1年活动性CMV感染发生率显著低于QF-CMV阴性患者（2.6% vs. 11.0%，HR 4.361，95% CI 1.460 - 13.030，Log-rank P = 0.036）。亚组分析结果显示，在血淋巴细胞计数> 1.0×10⁹/L的患者中QF-CMV检测的最佳阈值为0.1 IU/mL，而在血淋巴细胞计数< 1.0×10⁹/L的患者中QF-CMV检测的最佳阈值为0.5 IU/mL。

结论：

SLE患者中，血浆CMV DNA病毒载量对CMV病的诊疗价值可能更高；CMV脏器受累、SLE多系统受累和hsCRP升高是3个月全因死亡的独立相关因素；CMV脏器受累患者3个月内CMV复发风险更高。

外周血生物标志物（CMV抗原特异性IFN-γ、TNF-α水平联合严重淋巴细胞减少症）可能有助于区分风湿免疫病（尤其是SLE）人群中CMV感染的不同状态。

采用QF-CMV方法评估风湿免疫病患者CMV抗原特异性CMI或能预测风湿免疫病患者活动性CMV感染发病风险，淋巴细胞减少可能影响QF-CMV阈值的选择。

Background: Infection is one of the common complications in patients with rheumatic diseases, which affects treatment and prognosis. Patients with rheumatic diseases are at high risk for active cytomegalovirus (CMV) infection, due to the application of glucocorticoids and immunosuppressants as well as the disease itself. CMV-specific cell-mediated immunity (CMI) is essential for the control of CMV infection; however, most published studies in this field have focused on transplant recipients and human immunodeficiency virus-infected patients. There remains a lack of understanding of CMV infection in patients with rheumatic diseases. Investigating the characteristics, outcomes, and CMV-specific CMI in this population is critical for the risk stratification and management strategy of CMV infection in patients with rheumatic diseases.

Objectives: 

To investigate the clinical features and outcomes of systemic lupus erythematosus (SLE) complicated by active CMV infection.

To explore differences in CMV antigen-specific multiple cytokine secretion profiles and to screen for potential diagnostic cytokine-based biomarkers (alone or in combination) that differentiate CMV infection status in patients with rheumatic diseases.

To evaluate the utility of QuantiFERON‐CMV (QF-CMV) as a predictor of active CMV infection risk in patients with rheumatic diseases.

Methods:

Part 1: Clinical features and prognosis of systemic lupus erythematosus complicated by active cytomegalovirus infection: A retrospective cohort study

The medical records of hospitalized SLE patients with active CMV infection at Peking Union Medical College Hospital from June 2016 to December 2022 were retrospectively reviewed. The consistency between plasma CMV DNA viral load and pp65 antigenemia was analyzed using the chi-square test. Related factors for CMV disease in patients with SLE complicated by active CMV infection were analyzed using logistic regression. Cox hazards regression analysis was used to determine predictors for all-cause mortality and CMV recurrence within a 3-month follow-up period.

Part 2: Cytomegalovirus antigen-specific multi-cytokine immune responses in patients with rheumatic diseases under different cytomegalovirus infection statuses: A Case-Control Study

Patients with rheumatic diseases complicated by latent or active CMV infection at Peking Union Medical College Hospital from March 2023 to August 2023 were selected. Whole blood was stimulated with a peptide cocktail that simulated human CMV proteins. The levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-4, interleukin-6, interleukin-10, interleukin-17, and CXC chemokine ligand 2 in the supernatant were measured using Luminex assays. The receiver operating characteristic curve was used to evaluate the diagnostic accuracy of cytokine-based biomarkers, alone or in combination, for distinguishing different CMV infection statuses. Part 3: Cytomegalovirus antigen-specific T-cell responses predict the risk of active cytomegalovirus infection in patients with rheumatic diseases: A prospective study

Patients with rheumatic diseases and latent CMV infection who were hospitalized at Peking Union Medical College Hospital from March 2023 to August 2023 were prospectively enrolled. CMV-specific CMI was quantified using the QF-CMV assay, an IFN-γ release assay based on enzyme-linked immunosorbent assay. Baseline characteristics, clinical information, and laboratory data were collected. Patients were followed for 1 year or until death. The diagnostic accuracy of the QF-CMV assay at different positivity thresholds for IFN-γ production in predicting the development of the study outcome was estimated.

Results:

Part 1: Clinical features and prognosis of systemic lupus erythematosus complicated by active cytomegalovirus infection: A retrospective cohort study

A total of 206 patients were enrolled in this study. Among the 123 patients who were simultaneously tested for both plasma CMV DNA viral load and pp65 antigenemia within a 72-hour interval, the consistency between the two methods was poor (Kappa = −0.304, P < 0.001). Plasma CMV DNA viral load ≥ 1,600 copies/mL [odds ratio (OR) 4.411, 95% CI 1.871 - 10.402, P = 0.001], high-dose glucocorticoid therapy (OR 2.155, 95% CI 1.071 - 4.334, P = 0.031), and elevated alanine transaminase (OR 3.409, 95% CI 1.563 - 7.435, P = 0.002) were significant clinical indicators of CMV disease. CMV organ involvement [hazard ratio (HR) 47.222, 95% CI 5.621 - 396.689, P < 0.001], SLE multi-system involvement (HR 1.794, 95% CI 1.029 - 3.128, P = 0.039), and elevated hypersensitive C-reactive protein (hsCRP) (HR 5.767, 95% CI 1.190 - 27.943, P = 0.030) were identified as independent related factors for 3-month all-cause mortality. Additionally, CMV organ involvement (HR 3.404, 95% CI 1.074 - 10.793, P = 0.037) was found to be an independent related factor for CMV recurrence within 3 months.

Part 2: Cytomegalovirus antigen-specific multi-cytokine immune responses in patients with rheumatic diseases under different cytomegalovirus infection statuses: A Case-Control Study

A total of 60 patients with rheumatic diseases (particularly SLE) in our center were included, with 30 cases each in each of the active CMV infection and latent CMV infection groups. The proportion of patients with severe lymphopenia was lowest in the latent CMV infection group (P < 0.001). The levels of CMV antigen-specific IFN-γ, TNF-α and IL-2 were significantly lower in CMV disease patients compared to latent CMV infection patients (P = 0.002, 0.012, and 0.007, respectively). CMV antigen-specific IFN-γ, TNF-α levels and severe lymphopenian together provided the best discriminatory performance for distinguishing between latent and either active CMV infection [areas under the curve (AUC) = 0.854] or CMV disease (AUC = 0.935).

Part 3: Cytomegalovirus antigen-specific T-cell responses predict the risk of active cytomegalovirus infection in patients with rheumatic diseases: A prospective study

Thirteen of 178 (7.3%) patients with rheumatic diseases developed active CMV infection, with a median onset time of 14 days. Using an IFN-γ ≥ 0.2 IU/ml test threshold, the negative predictive value for active CMV infection was 94.4%, with no differences in 1-year CMV infection rates between patients with positive or negative (nonreactive or indeterminate) results (5.6% vs. 11.1%, HR 1.961, 95% CI 0.602 - 6.394, Log-rank P = 0.217). By elevating the IFN-γ threshold to 0.5 IU/mL, the negative predictive value improved to 97.4%. Furthermore, the 1-year incidence of active CMV infection was significantly lower in assay-positive compared to assay-negative patients (2.6% vs. 11%，HR 4.361, 95% CI 1.460 - 13.030, Log-rank P = 0.036). The subgroup analysis results showed that the optimal threshold of QF-CMV detection was 0.1 IU/mL in patients with lymphocyte counts > 1.0×10⁹/L, while the optimal threshold of QF-CMV detection was 0.5 IU/mL in patients with a lymphocyte counts < 1.0×10⁹/L.

Conclusions:

In SLE patients, plasma CMV DNA viral load appears to have a higher diagnostic value for CMV disease. CMV organ involvement, SLE multi-system involvement, and elevated hsCRP are independent correlates of 3-month all-cause mortality. In addition, CMV organ involvement is an independent correlate of 3-month CMV recurrence.

Peripheral blood biomarkers (the combination of CMV antigen-specific IFN-γ, TNF-α levels and severe lymphopenia) may have the potential to diferentiate different statues of CMV infection in patients with rheumatic diseases (especially SLE).

An assessment of CMV‐specific immunity using the QF-CMV assay may predict the risk of active CMV infection in patients with rheumatic diseases. Lymphocytopenia may affect the selection of the optimal threshold.