Oxford Vaccine Centre, University of Oxford, Oxford, UK.
Ann Clin Microbiol Antimicrob. 2010 Apr 19;9:14. doi: 10.1186/1476-0711-9-14.
Salmonella Typhi causes an estimated 21 million new cases of typhoid fever and 216,000 deaths every year. Blood culture is currently the gold standard for diagnosis of typhoid fever, but it is time-consuming and takes several days for isolation and identification of causative organisms. It is then too late to initiate proper antibiotic therapy. Serological tests have very low sensitivity and specificity, and no practical value in endemic areas. As early diagnosis of the disease and prompt treatment are essential for optimal management, especially in children, a rapid sensitive detection method for typhoid fever is urgently needed. Although PCR is sensitive and rapid, initial research indicated similar sensitivity to blood culture and lower specificity. We developed a fast and highly sensitive blood culture PCR method for detection of Salmonella Typhi, allowing same-day initiation of treatment after accurate diagnosis of typhoid.
An ox bile tryptone soy broth was optimized for blood culture, which allows the complete lysis of blood cells to release intracellular bacteria without inhibiting the growth of Salmonella Typhi. Using the optimised broth Salmonella Typhi bacteria in artificial blood samples were enriched in blood culture and then detected by a PCR targeting the fliC-d gene of Salmonella Typhi.
Tests demonstrated that 2.4% ox bile in blood culture not only lyzes blood cells completely within 1.5 hours so that the intracellular bacteria could be released, but also has no inhibiting effect on the growth of Salmonella Typhi. Three hour enrichment of Salmonella Typhi in tryptone soya broth containing 2.4% ox bile could increase the bacterial number from 0.75 CFU per millilitre of blood which is similar to clinical typhoid samples to the level which regular PCR can detect. The whole blood culture PCR assay takes less than 8 hours to complete rather than several days for conventional blood culture.
This novel blood culture PCR method is superior in speed and sensitivity to both conventional blood culture and PCR assays. Its use in clinical diagnosis may allow early detection of the causative organism and facilitate initiation of prompt treatment among patients with typhoid fever.
伤寒沙门氏菌每年估计导致 2100 万例新的伤寒病例和 21.6 万人死亡。血液培养目前是诊断伤寒的金标准，但它耗时且需要几天时间才能分离和鉴定病原体。此时，开始适当的抗生素治疗为时已晚。血清学检测的敏感性和特异性非常低，在流行地区没有实际价值。由于早期诊断疾病和及时治疗对于最佳管理至关重要，特别是在儿童中，因此迫切需要一种快速敏感的伤寒检测方法。虽然 PCR 具有敏感性和快速性，但初步研究表明其与血液培养的敏感性相似，但特异性较低。我们开发了一种快速、高度敏感的血液培养 PCR 方法来检测伤寒沙门氏菌，允许在准确诊断伤寒后当天开始治疗。
优化了一种牛胆胰蛋白胨大豆肉汤用于血液培养，该肉汤可以完全裂解血细胞以释放细胞内细菌，而不会抑制伤寒沙门氏菌的生长。使用优化的肉汤，在人工血液样本中富集伤寒沙门氏菌细菌，然后通过针对伤寒沙门氏菌 fliC-d 基因的 PCR 进行检测。
测试表明，血液培养中的 2.4%牛胆不仅可以在 1.5 小时内完全裂解血细胞，使细胞内细菌得以释放，而且对伤寒沙门氏菌的生长没有抑制作用。在含有 2.4%牛胆的胰蛋白胨大豆肉汤中，3 小时富集伤寒沙门氏菌可将细菌数量从每毫升血液中的 0.75 CFU 增加到常规 PCR 可检测到的水平。整个血液培养 PCR 检测不到 8 小时即可完成，而不是常规血液培养需要数天时间。
这种新型血液培养 PCR 方法在速度和灵敏度方面均优于常规血液培养和 PCR 检测。在临床诊断中的应用可能有助于早期检测病原体，并促进伤寒患者的及时治疗。
