Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center of Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Int J Med Microbiol. 2011 Apr;301(4):303-9. doi: 10.1016/j.ijmm.2010.11.004. Epub 2011 Jan 26.
Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.
自从流感嗜血杆菌（Hi）血清型 b 疫苗实施以来，其他血清型和非分型菌株作为 Hi 疾病的病因变得更为重要。需要一种快速准确的方法来检测所有的 Hi，而不论其包膜状态如何。我们开发了 2 种实时 PCR（rt-PCR）检测方法来检测蛋白 D 基因（hpd）的特定区域。这两种 hpd 检测方法对 Hi 的检测都非常特异和敏感。在代表 21 种细菌的 63 种非 Hi 分离株中，没有一种被 hpd #1 检测到，只有 2 种 H. aphrophilus 分离株中的一种被 hpd #3 检测到。hpd #1 和 #3 检测分别检测到 97%（229/237）和 99%（234/237）的 Hi 分离株，与先前针对 ompP2 或 bexA 开发的 rt-PCR 相比，这两种方法对可分型和不可分型 Hi 分离株的检测都更具优势。我们在蒙古乌兰巴托的脑膜炎监测中收集的脑脊液标本上评估了这些 rt-PCR 检测的诊断敏感性和特异性。通过传统方法（培养和乳胶凝集）、先前开发的 rt-PCR 检测和新的 hpd 检测确定了 111 例疑似脑膜炎病例的病因（脑膜炎奈瑟菌、Hi 和肺炎链球菌）。与其他经典方法相比，rt-PCR 检测对脑膜炎病原体的检测更敏感，将检测率从 50%（56/111）提高到 75%（83/111）。hpd #3 检测到了一种非 b Hi，这是 bexA 检测和其他方法遗漏的。一种能够检测可分型和不可分型 Hi 的敏感 rt-PCR 检测方法是改善 Hi 疾病监测的有用工具，尤其是在 Hib 疫苗引入之后。
