Detection of Respiratory Viruses in the Middle Ear Fluids of Children With Acute Otitis Media by Multiplex Reverse Transcription: Polymerase Chain Reaction Assay

The association between respiratory viral infections and acute otitis media (AOM) in infants and young children has been well-established.1, 2 A variety of viruses, especially respiratory viruses, have been detected in the middle ear fluid (MEF) of children with AOM. The commonly detected viruses include: respiratory syncytial virus (RSV); parainfluenza virus types 1, 2 and 3; influenza types A and B; adenovirus; rhinovirus; and enterovirus.2–6 Detection of viruses in the MEF has been limited by the small volume of the MEF sample, the relatively low sensitivity of tissue culture for viruses and the unavailability of rapid antigen detection for multiple viruses. The development of rapid molecular techniques, such as polymerase chain reaction assays, has allowed for detection of small numbers of microbial genomes in clinical specimens. Recently polymerase chain reaction techniques were developed for detection of multiple common respiratory bacteria and viruses in the MEF.7, 8 In this study we evaluated the usefulness of a commercially available multiplex reverse transcription-polymerase chain reaction assay (RT-PCR) in detection of seven common respiratory viruses in the MEF of children with AOM. Materials and methods. Infants and children with AOM were enrolled in various antibiotic trials between 1989 and 1993, as previously reported.9, 10 MEFs were collected by tympanocentesis at the time of diagnosis of AOM and after 2 to 5 days of antibiotic therapy. The collected MEF was diluted in 1 ml of PBS and aliquoted for tissue culture for viruses, RSV antigen detection by enzyme immunoassay (EIA) and mediator assays. The original dilution factors of the MEF were from 4 to 300 (median, 11). The MEF specimens tested in the present study were samples remaining from previous studies that had been processed, further diluted, frozen and thawed many times. Nasal wash specimens and viral serologic titers were also concurrently evaluated on the same patients in the previous studies. The multiplex RT-PCR-enzyme hybridization assay (Hexaplex; Prodesse, Waukesha, WI) was used to test for seven viruses in the MEF: RSV-A; RSV-B; parainfluenza viruses 1, 2 and 3; and influenza A and B.11 The tests were performed and results were interpreted independently, without knowledge of viral culture and/or RSV-EIA results. The normal test volume required was 280 μl. Specimens with smaller volumes were tested without further dilution. The test was performed according to the manufacturer's instructions. Multiplex RT-PCR results were compared with previously available viral culture and/or RSV-EIA results of the same specimens. When there were discrepancies between culture, or RSV-EIA results and RT-PCR results, we used virologic results from other specimens collected from the same patient to help resolve the discrepancies and to verify RT-PCR true positive samples. Results. A total of 65 MEF specimens from 40 patients were included. Of these 19 were viral culture-positive and 46 were viral culture-negative (Table 1). Multiplex RT-PCR detected the same virus in 18 of the 19 culture-positive samples: parainfluenza type 3 (n = 8); RSV (n = 6); influenza B (n = 3); and parainfluenza type 1 (n = 1). In addition a second virus was also detected in 5 of the18 positive samples (3 RSV, 1 parainfluenza 3 and 1 influenza B). A MEF that was positive for influenza B by culture was found to be RT-PCR-negative, and this result was considered a RT-PCR false negative. The original dilution of this specimen was 43, and the volume available for the RT-PCR test was 285 μl.TABLE 1: Comparison between multiplex RT-PCR and viral culture and multiplex RT-PCR and RSV-EIA resultsOf the 46 culture-negative samples, multiplex RT-PCR showed 29 MEF samples to be positive for 37 viruses: RSV (n = 19); influenza A (n = 7); parainfluenza type 3 (n = 6); parainfluenza type 1 (n = 4); and influenza A (n = 1). Overall RT-PCR detected 42 more viruses than viral culture (see Table 1, Footnotes * and †). We verified these 42 RT-PCR- positive/culture-negative results by comparing multiplex RT-PCR results with RSV-EIA on the same MEF sample and/or culture results from the other MEF or NW specimens collected from the same patient, or viral serologic data. For example, if a MEF was negative by viral culture and positive for RSV by multiplex RT-PCR, and the patient grew RSV from a nasal wash specimen or a subsequent culture a few days later, then the RT-PCR result was considered a true positive. Of the additional viruses detected in 5 of 18 culture-positive samples (Table 1, Footnote *), 3 were considered to be RT-PCR true positives because other samples from the same patients were positive for the same virus. Of 37 viruses detected in 29 culture-negative samples (Table 1, Footnote †), 16 were considered RT-PCR true positives. Therefore 19 of 42 (45%) viruses detected only by RT-PCR were verified as true positives. Three culture-negative/RT-PCR- negative samples were positive for RSV by RSV-EIA (see Table 1, Footnote ‡); these samples were considered false negative by both culture and RT-PCR. To compare sensitivity of viral culture vs. RT-PCR, we added all viral positive results by culture, RSV-EIA or RT-PCR. Overall 64 viruses were detected: 19 by culture (18 of these were also detected by PCR); 42 by RT-PCR only; and 3 by RSV-EIA only (culture- and RT-PCR-negative). Of these, viral culture detected 19 viruses, and PCR detected 60 viruses. Thus the sensitivity of viral culture was 30%, and the sensitivity of RT-PCR was 94%. Specificity could not be calculated because we could not identify false positive results. RSV-EIA results were available for 41 of the 65 samples (Table 1). RSV-EIA was positive in 8 samples; RT-PCR was positive for RSV in 5 of these. Three samples that were RT-PCR-negative for RSV were RT-PCR-positive for other viruses (Table 1, Footnote §): parainfluenza type 1 (n = 2); and parainfluenza type 3 (n = 1). Viral culture was negative in these 3 samples, thus the discrepant results between RT-PCR and RSV-EIA could not be resolved in this setting. In 33 samples negative for RSV-EIA, RT-PCR was positive for RSV in 12 samples. Of these, 7 (58%) were confirmed to be true positives by positive RSV culture or serology. Overall 20 samples were RSV-positive by either method: 8 EIA-positive samples (5 of which were RT-PCR-positive) and 12 EIA-negative/RT-PCR-positive samples. Of these EIA detected RSV in 8 samples, and RT-PCR detected RSV in 17 samples. The sensitivity of EIA was 40%, whereas the sensitivity of RT-PCR was 85%. Discussion. It is well-known that viruses, especially respiratory viruses, play an important role in the pathogenesis of AOM. A proportion of children with AOM have viruses in their MEF and nasopharyngeal secretions at the time of the initial AOM diagnosis. However, the viral diagnostic methods commonly used, such as tissue culture and rapid viral antigen detection, are likely to underdetect viruses. Studies of AOM that include bacterial and viral studies by conventional methods yield no infectious agents in the MEF in 18 to 25% of cases.3, 9 This study is the first to directly compare a multiplex RT-PCR assay with tissue culture techniques for detection of common respiratory viruses in the MEF. When discrepancies between tissue culture results and multiplex RT-PCR results occurred, we used the results of viral culture and/or rapid viral antigen detection in other specimens such as MEF collected from the contralateral side, MEF collected a few days later, nasal wash specimens and/or viral serologic results from the same patients to verify true positive results. We found that RT-PCR substantially increased the detection of viruses, compared with viral culture, or RSV-EIA (for RSV detection). Almost one-half of the positive results obtained by RT-PCR only were verified to be true positives. The design of our study did not allow the results of the other half of the samples to be identified as either true or false positives, so RT-PCR specificity could not be calculated. The specimens tested were stored specimens that had been processed, diluted and handled multiple times. Despite these limitations the multiplex RT-PCR assay substantially increased the rate of detection of viruses in the MEF samples, compared with detection by tissue culture and/or rapid viral antigen detection assay. Recent studies with this commercial multiplex PCR assay have demonstrated that almost all Hexaplex-positive, tissue culture-negative or EIA-negative samples are true positives (S Kehl, personal communication). This multiplex RT-PCR assay has a reported positive predictive value of 90 to 95% for influenza and 92% for RSV, with negative predictive values of 99.4 to 99.6%. Pitkaranta et al.8 recently report the use of the RT-PCR assay in detection of 3 specific viruses, rhinovirus, RSV and coronavirus, in the MEF and nasopharyngeal secretions of 92 children with AOM. They found that 75% of the children were infected with these viruses and that 48% had the viruses in the MEF. Overall bacteria and or viruses were detected in the MEF of 84% of the children. In their study viral culture or rapid viral antigen detection was not used for comparison, and they did not include RT-PCR for detection of other common respiratory viruses, such as parainfluenza viruses or influenza viruses. That study and ours suggest that viruses are present in the MEF of children with AOM more often than reported previously. Although the multiplex RT-PCR assay detects only viral RNA, not live viruses, viruses were grown from cultures of the same MEFs in many cases we studied. Because viruses require intracellular replication, this finding suggests that active viral infection occurs in the middle ear in a substantial proportion of cases of AOM. Common viruses causing upper respiratory tract infections in children include RSV (types A and B), parainfluenza viruses (types 1, 2 and 3), influenza (types A and B), coronavirus and numerous serotypes of rhinovirus, adenovirus and enterovirus.12 Among these we have found RSV, parainfluenza and influenza viruses to be the most common viruses that invade the middle ear during AOM.6 Because the volume of the MEF is limited and a wide variety of viruses can cause AOM, it is technically difficult to use the RT-PCR technique for detection of all viruses. The multiplex RT-PCR assay for the seven common viruses tested in this study seems to be a helpful tool for sensitive and rapid detection of common viruses in the MEF. However, there is evidence that rhinovirus and coronavirus can also play important roles in AOM.2, 8, 13 The use of an improved multiplex PCR assay that includes these viruses, or the use of more than one assay to detect all the most common viruses in AOM, would provide more complete information regarding viral infection in AOM than has been obtainable in the past. Such information could lead to a better understanding of the role of viruses in the pathogenesis and clinical outcome of AOM and provide the basis for development of new vaccines and reduction of antibiotic use in treatment of AOM. Acknowledgments. This study was supported in part by National Institutes of Health Research Grant R01 DC 02620. We thank Drs. Weimin Hua and Jiang Fan for technical support and Dr. Constance D. Baldwin for reviewing and editing the manuscript.