Tötsch M, Böcker W, Brömmelkamp E, Fille M, Kreczy A, Ofner D, Schmid K W, Dockhorn-Dworniczak B
Department of Pathology, University of Münster, Germany.
J Pathol. 1996 Feb;178(2):221-6. doi: 10.1002/(SICI)1096-9896(199602)178:2<221::AID-PATH441>3.0.CO;2-W.
Diagnosis of mycobacterial infection is made by assessment of characteristic histological features, staining of acid-fast bacilli, or agar culture. Recent advances in molecular biology have provided alternative approaches for the detection of mycobacteria, but only limited data are available dealing with the comparative evaluation of these methods. In order to determine the diagnostic applicability of polymerase chain reaction (PCR)-based assays, 20 formalin-fixed and paraffin-embedded lymph nodes with bacille Calmette-Guérin (BCG) lymphadenitis were investigated which in Löwenstein Jensen agar culture were either positive or negative (ten cases each); ten lymph nodes with non-specific lymphadenitis served as negative controls. Ziehl-Neelsen staining as well as three different PCR assays (including nested PCR), amplifying a specific sequence of the Mycobacterium tuberculosis complex or sequences of the 65 kD antigen gene, were performed. Positive culture was only obtained from lymph nodes which had been surgically removed within 20 weeks after vaccination (P < 0.001). In contrast to microscopic examination, which yielded no more information than agar culture, PCR detection of mycobacterial DNA was unrelated to culture findings. Combined use of different assays, as well as DNA extraction from at least three paraffin sections from each specimen, resulted in the detection of mycobacterial DNA in all lymph nodes with amplifiable DNA (18 out of 20 cases). Controls remained consistently negative. Thus, the combined use of different PCR assays is proposed as a rapid and sensitive technique for the detection of mycobacterial DNA in formalin-fixed and paraffin-embedded tissue.
分枝杆菌感染的诊断通过评估特征性组织学特征、抗酸杆菌染色或琼脂培养来进行。分子生物学的最新进展为分枝杆菌的检测提供了替代方法，但关于这些方法的比较评估仅有有限的数据。为了确定基于聚合酶链反应（PCR）的检测方法的诊断适用性，对20例福尔马林固定石蜡包埋的卡介苗（BCG）淋巴结炎淋巴结进行了研究，这些淋巴结在罗氏培养基上培养结果为阳性或阴性（各10例）；10例非特异性淋巴结炎淋巴结作为阴性对照。进行了萋-尼染色以及三种不同的PCR检测方法（包括巢式PCR），扩增结核分枝杆菌复合群的特定序列或65kD抗原基因的序列。仅从接种疫苗后20周内手术切除的淋巴结中获得了阳性培养结果（P<0.001）。与显微镜检查相比，显微镜检查提供的信息并不比琼脂培养更多，而分枝杆菌DNA的PCR检测与培养结果无关。联合使用不同的检测方法，以及从每个标本至少三个石蜡切片中提取DNA，使得在所有具有可扩增DNA的淋巴结中（20例中的18例）检测到了分枝杆菌DNA。对照始终为阴性。因此，建议联合使用不同的PCR检测方法作为一种快速、灵敏的技术，用于检测福尔马林固定石蜡包埋组织中的分枝杆菌DNA。
