Direct Molecular Detection and Genotyping of Borrelia burgdorferi from Whole Blood of Patients with Early Lyme Disease
Direct Molecular Detection and Genotyping of Borrelia burgdorferi from Whole Blood of Patients with Early Lyme Disease
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DOI：
10.1371/journal.pone.0036825
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发表时间：
2012-05-08
期刊：
影响因子：
3.7
通讯作者：
Aucott, John N.
中科院分区：
文献类型：
--
作者：
Eshoo, Mark W.;Crowder, Christopher C.;Aucott, John N.
由于循环中的伯氏疏螺旋体DNA含量低，早期莱姆病的血液直接分子检测可能不敏感。为了应对这一挑战，我们开发了一种灵敏的策略来检测和基因型B。直接从初次患者访视期间收集的全血中分离伯氏螺旋体。该策略通过采用1.25 mL全血提高了灵敏度，这是在多位点PCR和电喷雾电离质谱检测试验之前对疏螺旋体DNA的整个标本提取物进行的新型预富集。我们评估了21名流行区患者在初次就诊或抗生素治疗3周后随访时采集的血液，这些患者在初次就诊时经医生诊断为游走性红斑（EM）且两级血清学阳性。该DNA分析的结果显示检测到B。21例患者中13例（62%）为伯氏菌感染。在大多数情况下，新的检测方法也提供了B。burgdorferi基因型。我们的直接检测试验与初始医生访视血清学的综合结果导致21例患者中有19例（90%）在初始访视时检测到早期莱姆病。在21例中的5例中，我们证明了检测B的能力。在莱姆病早期，直接从血清转化前的全血标本中分离出莱姆病螺旋体。
Direct molecular tests in blood for early Lyme disease can be insensitive due to low amount of circulating Borrelia burgdorferi DNA. To address this challenge, we have developed a sensitive strategy to both detect and genotype B. burgdorferi directly from whole blood collected during the initial patient visit. This strategy improved sensitivity by employing 1.25 mL of whole blood, a novel pre-enrichment of the entire specimen extract for Borrelia DNA prior to a multi-locus PCR and electrospray ionization mass spectrometry detection assay. We evaluated the assay on blood collected at the initial presentation from 21 endemic area patients who had both physician-diagnosed erythema migrans (EM) and positive two-tiered serology either at the initial visit or at a follow-up visit after three weeks of antibiotic therapy. Results of this DNA analysis showed detection of B. burgdorferi in 13 of 21 patients (62%). In most cases the new assay also provided the B. burgdorferi genotype. The combined results of our direct detection assay with initial physician visit serology resulted in the detection of early Lyme disease in 19 of 21 (90%) of patients at the initial visit. In 5 of 21 cases we demonstrate the ability to detect B. burgdorferi in early Lyme disease directly from whole blood specimens prior to seroconversion.