DataSheet_1_Assessment of Serum Macrophage Migration Inhibitory Factor (MIF) as an Early Diagnostic Marker of Leptospirosis.docx

The search for valuable early diagnostic markers for leptospirosis is ongoing. The aim of the present study was to evaluate the diagnostic value of macrophage migration inhibitory factor (MIF) for leptospirosis. MIF is an immunoregulatory cytokine secreted by a variety of cell types involved in immune response and the pathogenesis of various diseases. It was previously described as a severity predictor of diseases. Samples of 142 leptospirosis cases, 101 other febrile cases, and 57 healthy controls were studied. The prevalence of leptospirosis was 47.3%. Autumnalis, Australis, and Canicola were the highly prevalent leptospiral serovars with a microscopic agglutination test (MAT) titer in the range 1:80–1:2,560. Enzyme-linked immunosorbent assay (ELISA) of MIF was carried out to measure the serum MIF levels. We found that the serum MIF levels [median, (interquartile range)] were significantly (p < 0.001) elevated in different clinical forms of leptospirosis, such as febrile illness [7.5 ng/ml (5.32–8.97)], pulmonary hemorrhage [13.2 ng/ml (11.77–16.72)], Weil’s syndrome [8.8 ng/ml (7.25–9.95)], and renal failure [8.6 ng/ml (7.18–10.5)], than in healthy controls [0.65n g/ml (0.5–1.1)]. Serum MIF had sensitivity, specificity, positive predictive value, and negative predictive value of 100%, >90%, >90%, and 100%, respectively. Receiver operating characteristic (ROC) analysis revealed that the serum MIF levels between leptospirosis cases and control subjects had an area under the curve (AUC) value of >0.9 (p < 0.0001). In leptospirosis patients, elevation of serum MIF was significantly (p < 0.001) higher in severe cases with organ dysfunction [10 ng/ml (7.8–14.5)] than that in mild febrile cases [7.5 ng/ml (5.32–8.97)], with the difference of 2.5 indicating that serum MIF acts as a predictor of leptospirosis severity. Pearson’s correlation test demonstrated that the serum MIF level was strongly correlated (r = 0.75, p < 0.0001) with disease progression. The median lethal dose (LD50) of leptospiral lipopolysaccharide (LPS) in BALB/c mice was determined to be 20 mg/kg, which gave rise to endotoxemia. Leptospiral LPS triggered the upregulation of MIF expression at 24 h post-infection, which reached the peak level at 24 h post-treatment in THP-1 cells and showed elevated MIF expressions in different tissues of BALB/c mice at the early stage of infection. Taken together, MIF is an early-phase cytokine that could serve as a rapid diagnostic marker for leptospirosis.

对钩端螺旋体病早期诊断标志物的探寻仍在持续进行。本研究的目的是评估巨噬细胞迁移抑制因子（MIF）在钩端螺旋体病诊断中的价值。MIF是一种由多种参与免疫反应和多种疾病发病机制的细胞类型分泌的免疫调节性细胞因子。它先前已被描述为疾病的严重程度预测因子。本研究对142例钩端螺旋体病例、101例其他发热病例和57例健康对照者进行了研究。钩端螺旋体病的患病率为47.3%。Autumnalis、Australis和Canicola是高度流行的钩端螺旋体血清型，其在显微镜凝集试验（MAT）中的滴度范围为1:80–1:2,560。通过酶联免疫吸附测定（ELISA）对MIF进行了检测，以测量血清MIF水平。我们发现，在钩端螺旋体病的不同临床形式中，如发热性疾病[7.5 ng/ml (5.32–8.97)]、肺出血[13.2 ng/ml (11.77–16.72)]、Weil综合症[8.8 ng/ml (7.25–9.95)]和肾功能衰竭[8.6 ng/ml (7.18–10.5)]，血清MIF水平与健康对照者[0.65 ng/ml (0.5–1.1)]相比显著升高（p < 0.001）。血清MIF具有100%的敏感性、>90%的特异性、>90%的阳性预测值和100%的阴性预测值。接受者操作特征（ROC）分析显示，钩端螺旋体病例与对照者的血清MIF水平之间的曲线下面积（AUC）值大于0.9（p < 0.0001）。在钩端螺旋体病患者中，血清MIF水平的升高在伴有器官功能障碍的重症患者[10 ng/ml (7.8–14.5)]中显著高于轻度发热病例[7.5 ng/ml (5.32–8.97)]，差异为2.5，表明血清MIF可以作为钩端螺旋体病严重程度的预测指标。皮尔逊相关系数测试表明，血清MIF水平与疾病进展密切相关（r = 0.75，p < 0.0001）。在BALB/c小鼠中，钩端螺旋体脂多糖（LPS）的中毒剂量（LD50）为20 mg/kg，可导致内毒素血症。钩端螺旋体LPS在感染后24小时内触发MIF表达的上调，在治疗后24小时在THP-1细胞中达到峰值，并在感染早期不同组织中的BALB/c小鼠显示出MIF表达的升高。综上所述，MIF是一种早期阶段的细胞因子，可以作为钩端螺旋体病的快速诊断标志物。