二步法聚合酶链反应检测血液恶性肿瘤患者曲霉菌感染的研究

目的探讨二步法PCR检测曲霉菌的敏感性和特异性及其在血液肿瘤患者侵袭性曲霉菌感染(IFI)中的诊断价值。方法二步法PCR检测有真菌感染高危因素的41例患者外周血中曲霉菌18SrRNA基因,结合CT检查、曲霉菌培养、患者骨髓抑制状况和抗真菌治疗效果,分析二步法PCR的诊断价值。结果多种曲霉菌菌株均能扩增出特异性条带,PCR产物测序结果与曲霉菌18SrRNA基因序列高度同源。41例中16例PCR阳性,25例阴性。阳性组中8例有曲霉菌感染的影像学表现,各有1例从胸腔积液、鼻分泌物培养出曲霉菌,2例血培养阳性。而12例对照者PCR均阴性。PCR阳性组和阴性组患者化疗后WBC数最低值分别为(0·30±0·14)×109/L、(0·50±0·26)×109/L,差异有统计学意义(P=0·0326)。两组患者WBC低于1·0×109/L的持续时间分别为(19·00±8·31)d、(12·69±6·95)d,差异有统计学意义(P=0·0472)。对PCR阳性者拟诊IFI,予以抗真菌治疗后,化疗相关早期死亡仅1例。结论二步法PCR检测对曲霉菌诊断的敏感性和特异性较高,假阳性和假阴性率均较低,结合临床资料,对IFI的早期诊断以及指导临床选择抗真菌治疗有较好的价值。

Detection of Aspergillus infection in blood of malignant hematopoietic tumor patients by two-step polymerase chain reaction

OBJECTIVE: To investigate the sensitivity and specificity of two-step PCR in detection of Aspergillus species in blood from malignant hematopoietic tumor patients. METHODS: Forty-one blood samples from high-risk patients were detected with two-step PCR. The clinical applicability was assessed with analyzing computed tomography, Aspergillus culture, neutropenia and outcome after anti-fungal therapy. RESULTS: Specific band of PCR was obtained from Aspergillus strains and products of PCR had a high homogeneous in sequence with Aspergillus species. Sixteen of 41 patients were PCR positive and controls were all PCR negative. Among PCR positive patients, 4 patients had positive cultures. The lowest WBC count was (0.30 +/- 0.14) x 10(9)/L and (0.50 +/- 0.26) x 10(9)/L respectively and duration with WBC count less than 1.0 x 10(9)/L was (19.00 +/- 8.31) days and (12.69 +/- 6.95) days respectively in the two groups. After antifungal therapy, one of the patients with PCR positive died of chemotherapy-related early death. CONCLUSIONS: The two-step PCR assay allows for highly sensitive and specific detection of Aspergillus pathogens in vitro and in vivo. The detection with PCR has some value in the early diagnosis and helps us to make decision for antifungal therapy.