MOLECULAR ANALYSIS OF BACTERIAL PATHOGENS IN OTITIS-MEDIA WITH EFFUSION

Objective.-To determine if the polymerase chain reaction (PCR) can det ect bacterial DNA in pediatric middle ear effusions that are sterile b y standard cultural methods. Design.-Single-center, blinded, comparati ve study of diagnostic assays. The PCR-based detection systems for Mor axella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoni ae were designed and validated using a battery of DNAs obtained from c ultured bacteria. Chronic middle ear effusion specimens were collected and comparatively analyzed by culture and the PCR. Setting.-Tertiary care pediatric hospital. Patients.-A total of 97 middle ear effusions were collected from pediatric outpatients at Children's Hospital of Pi ttsburgh (Pal during myringotomy and tube placement for chronic otitis media with effusion (duration >3 months). All patients had failed mul tiple courses of antimicrobial therapy and were diagnosed by a combina tion of validated otoscopy and tympanograms. Main Outcome Measure.-Dif ferences in the percentage of positive test results between PCR-based assays and culture for M catarrhalis, H influenzae, and S pneumoniae. Results.-Of the 97 specimens of otitis media with effusion, 28 (28.9%) tested positive by both culture and PCR for M catarrhalis, H influenz ae, or S pneumoniae, An additional 47 specimens (48%) were PCR positiv e/culture negative for these three bacterial species. Thus, 75 (77.3%) of the 97 specimens tested PCR positive for one or more of the three test organisms. The minimum number of bacterial genomic equivalents pr esent in the average culture-negative ear was estimated to be greater than 10(4) based on dilutional experiments. Conclusions.-The PCR-based assay systems can detect the presence of bacterial DNA in a significa nt percentage of culturally sterile middle ear effusions. While this f inding is not proof of an active bacterial infectious process, the lar ge number of bacterial genomic equivalents present in the ears is sugg estive of an active process.