Molecular analysis of bacterial pathogens in otitis media with effusion.
OBJECTIVE
To determine if the polymerase chain reaction (PCR) can detect bacterial DNA in pediatric middle ear effusions that are sterile by standard cultural methods.


METHODS
Single-center, blinded, comparative study of diagnostic assays. The PCR-based detection systems for Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae were designed and validated using a battery of DNAs obtained from cultured bacteria. Chronic middle ear effusion specimens were collected and comparatively analyzed by culture and the PCR.


METHODS
Tertiary care pediatric hospital.


METHODS
A total of 97 middle ear effusions were collected from pediatric outpatients at Children's Hospital of Pittsburgh (Pa) during myringotomy and tube placement for chronic otitis media with effusion (duration > 3 months). All patients had failed multiple courses of antimicrobial therapy and were diagnosed by a combination of validated otoscopy and tympanograms.


METHODS
Differences 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 influenzae, or S pneumoniae. An additional 47 specimens (48%) were PCR positive/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 present 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 significant percentage of culturally sterile middle ear effusions. While this finding is not proof of an active bacterial infectious process, the large number of bacterial genomic equivalents present in the ears is suggestive of an active process.
Post
J C
JC
Department of Otolaryngology, University of Pittsburgh, PA 15261, USA.
Preston
R A
RA
Aul
J J
JJ
Larkins-Pettigrew
M
M
Rydquist-White
J
J
Anderson
K W
KW
Wadowsky
R M
RM
Reagan
D R
DR
Walker
E S
ES
Kingsley
L A
LA
Magit
A E
AE
Ehrlich
G D
GD
D001431:Bacteriological Techniques; D002648:Child; D002675:Child, Preschool; D002908:Chronic Disease; D004269:DNA, Bacterial / Q000032:analysis*; D006192:Haemophilus Infections / Q000175:diagnosis; D006193:Haemophilus influenzae / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D001936:Moraxella catarrhalis / Q000235:genetics / Q000302:isolation & purification*; D016870:Neisseriaceae Infections / Q000175:diagnosis; D015345:Oligonucleotide Probes; D010034:Otitis Media with Effusion / Q000382:microbiology*; D011008:Pneumococcal Infections / Q000175:diagnosis; D016133:Polymerase Chain Reaction; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes