中国浙江省呼吸道感染患儿流感嗜血杆菌的流行病学分析及一步多重 PCR 快速检测。
Epidemiological analysis and rapid detection by one-step multiplex PCR assay of Haemophilus influenzae in children with respiratory tract infections in Zhejiang Province, China.
机构信息
Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, 310031, Zhejiang, China.
Department of Basic Medicine, Hangzhou Medical College, Hangzhou, 310058, Zhejiang, China.
出版信息
BMC Infect Dis. 2018 Aug 22;18(1):414. doi: 10.1186/s12879-018-3295-2.
BACKGROUND
Haemophilus influenzae (H. influenzae) is one of the most important pathogenic bacteria causing respiratory tract infection diseases in children. There are two main types of H. influenzae, encapsulated H. influenzae and nontypeable H. influenzae (NTHi). Serotype b of H. influenzae (Hib) used to be the main epidemic type of H. influenzae, causing the invasive infection. However, the epidemiology of invasive H. influenzae disease has changed substantially, and most invasive diseases are now caused by NTHi and other serotypes of H. influenzae. The aim of this study was to determine the main epidemic strains of H. influenzae in Zhejiang Province in China, and establish a one-step multiplex PCR assay to distinguish H. influenzae from other bacteria associated with respiratory tract infections, and distinguish encapsulated H. influenzae from NTHi.
METHOD
In this study, bacterial culture and serum agglutination testing were used to determine the most prevalent serotype of H. influenzae, and the results have served as a gold standard for clinical diagnosis. We also designed a one-step multiplex PCR assay using several kinds of standard strains of respiratory tract infection bacteria, to examine the stability, specificity, and detection limit of the PCR assays. We then used 1514 nasopharyngeal secretion (NPS) samples collected from children with respiratory tract infections to verify the specificity and sensitivity of the PCR assay.
RESULTS
The bacterial culture and serum agglutination test results showed that the positive rates of H. influenzae and encapsulated H. influenzae were 18.49 and 1.18%, respectively. The PCR results showed that the detection limit of the multiplex PCR assay was 1.89 × 10 copies /μL, the ompP6 positive rate was 19.35%, and the bexA positive rate was 1.32%. The sensitivity and specificity of the multiplex PCR were 100 and 99.86%, respectively.
CONCLUSIONS
According to our study, the most prevalent H. influenzae subtype in Zhejiang Province was NTHi, account for 93.57%; the one-step multiplex PCR assay we established can be used as the differential detection of clinical H. influenzae strains, replacing routine bacterial culture and serum agglutination testing.
背景
流感嗜血杆菌（Haemophilus influenzae，H. influenzae）是引起儿童呼吸道感染性疾病的最重要病原菌之一。H. influenzae 有两种主要类型，即有荚膜的流感嗜血杆菌（encapsulated H. influenzae）和无荚膜的流感嗜血杆菌（nontypeable H. influenzae，NTHi）。H. influenzae 血清型 b（Hib）曾是 H. influenzae 的主要流行型，引起侵袭性感染。然而，侵袭性 H. influenzae 疾病的流行病学已发生显著变化，大多数侵袭性疾病现在由 NTHi 和 H. influenzae 的其他血清型引起。本研究旨在确定中国浙江省 H. influenzae 的主要流行株，并建立一步法多重 PCR 检测方法，以区分与呼吸道感染相关的其他细菌，并区分有荚膜的流感嗜血杆菌和 NTHi。
方法
本研究采用细菌培养和血清凝集试验确定 H. influenzae 最常见的血清型，作为临床诊断的金标准。我们还设计了一种一步法多重 PCR 检测方法，使用几种呼吸道感染细菌的标准菌株，以检验 PCR 检测方法的稳定性、特异性和检测限。然后，我们使用 1514 份来自呼吸道感染患儿的鼻咽分泌物（nasopharyngeal secretion，NPS）样本，验证 PCR 检测方法的特异性和敏感性。
结果
细菌培养和血清凝集试验结果显示，H. influenzae 和有荚膜的流感嗜血杆菌的阳性率分别为 18.49%和 1.18%。PCR 结果显示，多重 PCR 检测方法的检测限为 1.89×10 拷贝/μL，ompP6 阳性率为 19.35%，bexA 阳性率为 1.32%。多重 PCR 的灵敏度和特异性分别为 100%和 99.86%。
结论
根据我们的研究，浙江省最常见的 H. influenzae 亚型是 NTHi，占 93.57%；我们建立的一步法多重 PCR 检测方法可用于临床 H. influenzae 菌株的鉴别检测，取代常规细菌培养和血清凝集试验。
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