BLINDED MULTIPLEX PCR ANALYSES OF MIDDLE-EAR AND NASOPHARYNGEAL FLUIDS FROM CHINCHILLA MODELS OF SINGLE-PATHOGEN-INDUCED AND MIXED-PATHOGEN-INDUCED OTITIS-MEDIA

Multiplex PCR analyses for both bacterial and viral pathogens were con ducted in a blinded manner on 33 archival specimens, of known culture status, procured from chinchilla models of both single- and mixed-path ogen-induced otitis media and from a pediatric patient. These specimen s had been maintained at -70 degrees C for up to 6 years. Experimental specimens evaluated included middle-ear effusions, nasopharyngeal lav age fluids and middle-ear lavage fluids from animals which were immuno logically naive, sham-immunized or actively immunized with nontypeable Haemophilus influenzae antigens. Sampling times used ranged from the day of bacterial or viral challenge to 42 days after challenge. Initia l PCR analyses of the 33 specimens matched the traditional culture dat a in 24 instances (73%), correctly identifying nontypeable H. influenz ae, Moraxella catarrhalis, Streptococcus pneumoniae, or adenovirus as the causative agent. A PCR-positive signal for the microbe(s) inoculat ed was also obtained in four animal model specimens (12%) which were c ulture negative. One of two culture-negative human effusions was also PCR positive. Thus, overall, results obtained by blinded PCR were 85% concordant with traditional culture methods or correctly indicated the specific pathogen introduced in four specimens that were sterile. In no instance was a false-positive signal obtained for any of the five e tiologic agents being evaluated. We conclude that the multiplex PCR an alyses are rapid and accurate methodologies when they are used to retr ospectively evaluate diverse archival specimens of limited volume from experimental models of otitis media.