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

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... 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 3 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.