Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
PLoS Negl Trop Dis. 2012;6(7):e1689. doi: 10.1371/journal.pntd.0001689. Epub 2012 Jul 3.
The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens.
METHODS/PRINCIPAL FINDINGS: Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results.
CONCLUSIONS/SIGNIFICANCE: These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen's buffy coat to increase the sensitivity of PCR analysis.
恰加斯病的实验室诊断具有挑战性，因为不同诊断测试的有用性将取决于疾病的阶段。血清学是慢性期患者的首选方法，而聚合酶链反应（PCR）可成功用于诊断急性和先天性病例。在这里，我们使用三种 TaqMan PCR 检测方法的组合来检测临床标本中的 T. cruzi DNA。
方法/主要发现：纳入分析的是从 320 份 EDTA 血液标本、18 份心脏组织标本、6 份脐带血标本、2 份皮肤组织标本和 3 份脑脊液标本中提取的 DNA。对于血液标本，分析了全血和白细胞层。这些标本来自美国的患者，他们怀疑通过器官移植、接触三锥虫或实验室事故接触 T. cruzi，以及怀疑有 Chagas 病再激活的免疫抑制患者。实时 PCR 成功地诊断了 20 名患者的急性和 Chagas 病再激活，包括 1 例器官传播感染和 1 例先天性病例。与全血分析相比，6 例患者的 EDTA 血液白细胞层分析导致更快的诊断。三种实时 PCR 检测方法对 94%的标本产生了相同的结果。不一致结果的主要原因是检测方法的灵敏度不同，但两种实时 PCR 检测方法也产生了 4 个假阳性结果。
结论/意义：这些数据强烈表明，至少应结合两种具有不同性能的 PCR 检测方法以提高准确性。该评估还突出了从血液标本白细胞层提取 DNA 以提高 PCR 分析灵敏度的益处。
