Department of Infectious Diseases and Pulmonary Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa City, Saitama, Japan.
Diagn Microbiol Infect Dis. 2013 Feb;75(2):155-9. doi: 10.1016/j.diagmicrobio.2012.10.015. Epub 2012 Dec 4.
The aim of this study was to assess the efficacy of nested polymerase chain reaction (PCR) and the loop-mediated isothermal amplification (LAMP) assay, which were developed to detect and identify toxoplasma parasites in human cerebrospinal fluid (CSF). Nested PCR was performed using primers generated by Dr. L.D. Sibley to target the 18S rDNA instead of the conventionally used primers which target the B1 gene. We also designed Toxoplasma gondii-specific LAMP primers targeting both genes. In vitro detection sensitivity was evaluated using 10-fold serially diluted genomic DNA purified from RH tachyzoites, and clinical sensitivity and specificity were evaluated using clinical CSF samples from 16 patients with toxoplasmic encephalitis (TE) and from 12 patients with other diseases. The 18S rDNA nested PCR showed the highest detection sensitivity limit with a minimum of 1.0 × 10(-8) ng/μL. However, sensitivity and specificity of nested PCR with clinical specimens were 50% and 100%, respectively. The sensitivity of molecular diagnosis of TE is not sufficient; therefore, patients clinically suspected of having TE should be treated promptly. Our molecular diagnostic tool would restrictively facilitate a definitive diagnosis of TE at an early stage in approximately 50% of patients.
本研究旨在评估巢式聚合酶链反应（PCR）和环介导等温扩增（LAMP）检测方法的功效，这两种方法是为了检测和鉴定人脑脊液中的弓形虫寄生虫而开发的。巢式 PCR 使用由 L.D. Sibley 博士生成的引物针对 18S rDNA 而不是传统的针对 B1 基因的引物进行。我们还设计了针对两种基因的弓形虫特异性 LAMP 引物。通过从 RH 速殖子中纯化的 10 倍系列稀释基因组 DNA 评估体外检测灵敏度，使用来自 16 例弓形虫脑炎（TE）患者和 12 例其他疾病患者的临床 CSF 样本评估临床敏感性和特异性。18S rDNA 巢式 PCR 的检测灵敏度最高，最小可达 1.0×10(-8)ng/μL。然而，巢式 PCR 对临床标本的敏感性和特异性分别为 50%和 100%。TE 的分子诊断敏感性不足；因此，临床上怀疑患有 TE 的患者应及时进行治疗。我们的分子诊断工具将在大约 50%的患者中限制地有助于在早期阶段明确诊断 TE。
