Azov Andrey G, Koch Jørn, Hamilton-Dutoit Stephen J
Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark.
APMIS. 2005 Sep;113(9):586-93. doi: 10.1111/j.1600-0463.2005.apm_234.x.
Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.
对福尔马林固定石蜡包埋（FFPE）组织中的分枝杆菌感染进行传统组织学诊断，其敏感性较低且特异性欠佳。为改善这一情况，我们开发了巢式聚合酶链反应（PCR）方案，用于检测FFPE切片中分枝杆菌属特异性的65 kDa热休克蛋白（HSP65）序列以及结核分枝杆菌复合群特异性插入序列IS6110。该方案在最终临床诊断为分枝杆菌感染的20例患者的组织上进行了优化。扩增产物通过测序和限制性内切酶消化进行验证。PCR每个反应能够检测低至三个分枝杆菌基因组。检测对结核分枝杆菌复合群和鸟分枝杆菌复合群感染均显示出100%的敏感性和特异性。随后，对第二组26例具有病因不明的坏死性肉芽肿组织学证据的患者的石蜡块进行分析，作为替代组，以在与常规诊断相似的条件下测试该检测方法。其中23个石蜡块含有可扩增的DNA；9个对结核分枝杆菌复合群DNA呈阳性，4个对其他类型的分枝杆菌DNA呈阳性。此外，用NarI消化HSP65扩增产物可区分结核分枝杆菌和鸟分枝杆菌复合群。总之，我们的巢式PCR检测方法可作为检测FFPE组织中分枝杆菌感染的可靠工具。该检测方法操作简单、快速，与先前报道的方案相比，敏感性和特异性有所提高。
