Jethwa H S, Schmitz J L, Dallabetta G, Behets F, Hoffman I, Hamilton H, Lule G, Cohen M, Folds J D
Clinical Microbiology-Immunology Laboratories, University of North Carolina Hospitals, Chapel Hill 27514.
J Clin Microbiol. 1995 Jan;33(1):180-3. doi: 10.1128/jcm.33.1.180-183.1995.
We compared the ability of direct immunofluorescent staining (DFA) and the PCR to detect Treponema pallidum in specimens from patients with genital ulcer disease. Touch preparations from 156 patients with genital lesions were fixed in acetone and stained with a fluorescein-labeled monoclonal antibody specific for the 37-kDa antigen of T. pallidum. After microscopic examination, the smear was removed from the slide with a swab. DNA was extracted with phenol-chloroform and precipitated with isopropanol. Ten microliters of the extracted DNA was amplified by PCR using primers for the gene encoding the 47-kDa protein of T. pallidum and hybridized to an internal probe. Twenty-two of 156 specimens were positive for T. pallidum by DFA and PCR, while 127 were negative by both methods, yielding a concordance of 95.5% (kappa = 0.84). Four specimens were positive by PCR and negative by DFA, while three specimens were negative by PCR and positive by DFA. The DFA-negative, PCR-positive specimens may have resulted from the presence of large numbers of leukocytes on the slides, obscuring visualization of treponemes. The DFA-positive, PCR-negative results were not due to inhibition of the PCR since purified T. pallidum DNA was amplified when added to aliquots of these specimens. Negative results in these specimens were most likely due to inefficient recovery of their DNA. These data suggest that DFA and PCR are equivalent methods for detection of T. pallidum on touch preparations of genital lesions. Further refinements of the PCR assay are necessary for it to significantly improve the detection of T. pallidum in genital lesions.
我们比较了直接免疫荧光染色（DFA）和聚合酶链反应（PCR）检测生殖器溃疡疾病患者标本中梅毒螺旋体的能力。对156例生殖器病变患者的触片用丙酮固定，并用针对梅毒螺旋体37 kDa抗原的荧光素标记单克隆抗体进行染色。显微镜检查后，用拭子从载玻片上取下涂片。用苯酚-氯仿提取DNA，并用异丙醇沉淀。用针对梅毒螺旋体47 kDa蛋白编码基因的引物通过PCR扩增10微升提取的DNA，并与内部探针杂交。156份标本中有22份通过DFA和PCR检测梅毒螺旋体呈阳性，而127份通过两种方法检测均为阴性，一致性为95.5%（kappa = 0.84）。4份标本PCR检测呈阳性而DFA检测呈阴性，3份标本PCR检测呈阴性而DFA检测呈阳性。DFA阴性、PCR阳性的标本可能是由于载玻片上存在大量白细胞，掩盖了梅毒螺旋体的观察。DFA阳性、PCR阴性的结果不是由于PCR受到抑制，因为将纯化的梅毒螺旋体DNA加入这些标本的等分试样中时可以被扩增。这些标本中的阴性结果很可能是由于其DNA回收效率低下。这些数据表明，DFA和PCR是检测生殖器病变触片上梅毒螺旋体的等效方法。PCR检测方法需要进一步改进，才能显著提高生殖器病变中梅毒螺旋体的检测率。
