Two-Step PCR-Based Assay for Identification of Bacterial Etiology of Otitis Media with Effusion in Infected Lebanese Children

Abstract

ABSTRACT We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included Haemophilus , Streptococcus , and Moraxella ( Branhamella ) catarrhalis , which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was ≥2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an ∼370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) a Haemophilus -specific probe (probe RDR125) as a primer along with DG74, (ii) a Streptococcus -specific primer (primer STR1; designed by us) along with DG74, and (iii) an M. catarrhalis -specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive for Haemophilus , 3 (8.6%) were positive for Streptococcus , and 10 (28.6%) were positive for M. catarrhalis . Ten samples (28.6%) exhibited a mixed infection and were positive for both Haemophilus and M. catarrhalis . Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.