Identification of Streptococcus pneumoniae and Haemophilus influenzae in culture-negative middle ear fluids from children with acute otitis media by combination of multiplex PCR and multi-locus sequencing typing

Objective: Streptococcus pneumoniae (Spn) and Haemophilus influenzae (Hflu) are major etiologic pathogens for acute otitis media (AOM). However, when Spn and Hflu strains are not identified by traditional culture methods, use of alternative PCR-based diagnosis becomes critical. This study aimed to develop a combined molecular method to accurately detect these otopathogens. Methods: Middle ear fluid (MEF) samples were collected by tympanocentesis from children with AOM to isolate Spn and Hflu by standard culture procedures. Multiplex PCR (mPCR) and multi-locus sequence typing (MLST) techniques were used to detect Spn and Hflu in culture-negative MEF samples. Results: We found 20 Spn or Hflu culture-positive MEF samples that were mPCR-positive and typeable by MLST. The sequences of the housekeeping genes and the MLST allelic profiles obtained from Spn or Hflu culture isolates matched exactly MEF samples that were tested directly without culture isolation. Of 63 MEF samples that were culture-negative for Spn, 38% (24/63) were mPCR-positive for Spn. Of 50 MEF samples that were culture-negative for Hflu, 24% (12/50) were mPCR-positive for Hflu. Among these culture-negative but mPCR-positive MEF samples, 25% (6/24) and 25% (3/12) were typeable by MIST for Spn and Hflu, respectively. Conclusions: MEF samples may be analyzed with mPCR and MIST directly without culture isolation and the addition of mPCR and MIST may accurately identify Spn and Hflu in MEF of children with AOM when bacterial culture is negative. (c) 2010 Elsevier Ireland Ltd. All rights reserved.