Habets Marrit N, Cremers Amelieke J H, Bos Martine P, Savelkoul Paul, Eleveld Marc J, Meis Jacques F, Hermans Peter W M, Melchers Willem J, de Jonge Marien I, Diavatopoulos Dimitri A
Laboratory of Pediatric Infectious Diseases, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
Microbiome, Amsterdam, The Netherlands.
J Med Microbiol. 2016 Feb;65(2):129-136. doi: 10.1099/jmm.0.000204. Epub 2015 Dec 1.
Streptococcus pneumoniae is responsible for an estimated 1.6 million deaths worldwide every year. While rapid detection and timely treatment with appropriate antibiotics is preferred, this is often difficult due to the amount of time that detection with blood cultures takes. In this study, a novel quantitative PCR assay for the detection of Streptococcus pneumoniae was developed. To identify novel targets, we analysed the pneumococcal genome for unique, repetitive DNA sequences. This approach identified comX, which is conserved and present in duplicate copies in Streptococcus pneumoniae but not in other bacterial species. Comparison with lytA, the current 'gold standard' for detection by quantitative PCR, demonstrated an analytic specificity of 100% for both assays on a panel of 10 pneumococcal and 18 non-pneumococcal isolates, but a reduction of 3.5 quantitation cycle values (± 0.23 sem), resulting in an increased analytical detection rate of comX. We validated our assay on DNA extracted from the serum of 30 bacteraemic patients who were blood culture positive for Streptococcus pneumoniae and 51 serum samples that were culture positive for other bacteria. This resulted in a similar clinical sensitivity between the comX and lytA assays (47%) and in a diagnostic specificity of 98.2 and 100% for the lytA and comX assays, respectively. In conclusion, we have developed a novel quantitative PCR assay with increased analytical sensitivity for the detection of Streptococcus pneumoniae, which may be used to develop a rapid bedside test for the direct detection of Streptococcus pneumoniae in clinical specimens.
肺炎链球菌每年在全球范围内估计导致160万人死亡。虽然快速检测并及时使用适当的抗生素进行治疗是首选，但由于血培养检测所需的时间，这往往很难实现。在本研究中，开发了一种用于检测肺炎链球菌的新型定量PCR检测方法。为了确定新的靶点，我们分析了肺炎球菌基因组中的独特重复DNA序列。这种方法确定了comX，它在肺炎链球菌中是保守的且有两个拷贝，但在其他细菌物种中不存在。与当前定量PCR检测的“金标准”lytA相比，在一组10株肺炎球菌和18株非肺炎球菌分离株上，两种检测方法的分析特异性均为100%，但comX的定量循环值降低了3.5个（±0.23标准误），从而提高了分析检测率。我们在从30例血培养肺炎链球菌阳性的菌血症患者血清中提取的DNA以及51例培养出其他细菌阳性的血清样本上验证了我们的检测方法。这导致comX和lytA检测方法的临床敏感性相似（47%），lytA和comX检测方法的诊断特异性分别为98.2%和100%。总之，我们开发了一种用于检测肺炎链球菌的新型定量PCR检测方法，其分析敏感性有所提高，可用于开发一种直接检测临床标本中肺炎链球菌的快速床边检测方法。
