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POLYMERASE CHAIN REACTION ASSAY FOR DETECTING CHLAMYDIA PNEUMONIAE IN MIDDLE EAR FLUID OF CHILDREN WITH OTITIS MEDIA WITH EFFUSION

1999; Lippincott Williams & Wilkins; Volume: 18; Issue: 10 Linguagem: Inglês

10.1097/00006454-199910000-00021

1532-0987

Jussi Jero, Hannu Alakärppä, Anni Virolainen, Pekka Saikku, P. Karma,

Respiratory viral infections research

By definition otitis media with effusion (OME) refers to the presence of middle ear effusion (MEE) behind an intact tympanic membrane without acute signs or symptoms. Despite intensive study, its etiopathogenesis is poorly understood. In earlier studies OME was considered a sterile process, whereas in more recent publications major bacteria of acute otitis media (AOM) have been cultured from some MEEs of OME.1 However, even in these studies the microbiology of OME in general has been distinctly different from that of AOM, and in the majority of MEE specimens only nonpathogens have been cultured or the MEEs have been bacteriologically sterile. Chlamydia pneumoniae is a common cause of pneumonia, bronchitis and sinusitis.2 It has also been recovered by tympanocentesis in 8% of 101 children with AOM in USA,3 and also in OME patients in Japan.4, 5C. pneumoniae has also been detected in MEE of one patient during a local C. pneumoniae epidemic in Sweden6 and from MEEs of 1 AOM patient and 5 OME patients in Denmark.7 On the contrary Goo et al.8 did not find evidence of chlamydiae even by polymerase chain reaction in the MEEs of American OME patients. The reason for this difference, whether it is geographic, morphologic or random, is unclear. The purpose of our study was to investigate the possible role of C. pneumoniae in OME in Finland by using PCR to detect chlamydial DNA in the MEE samples of children with OME. Material and methods. Altogether 123 children in consecutive order coming to the Department of Otolaryngology, Helsinki University Central Hospital for tympanostomy tube insertion because of OME, were enrolled in the study in 1993-1994. The age range was 7 months to 12 years (median, 2 years, 5 months). The child was considered to have OME if MEE persisted for at least 1 month behind an intact tympanic membrane and there were no signs or symptoms of AOM or upper respiratory tract infection. Children who had purulent MEE or had received antimicrobials within the prior week were not included. All MEE samples were obtained during tympanostomy under general anesthesia and under the control of an operating microscope without sterilizing the ear canal. In bilateral cases only the MEE of the right ear was collected. The samples were cultured within 12 h; and the isolated bacteria were identified by standard methods. The DNA purification was done with the commercial QIAamp blood kit (Qiagen, Hilden, Germany). Briefly, 100 μl of the MEE sample were mixed with 100 μl of phosphate-buffered saline, 25 μl of QIAGEN protease and 200 μl of AL buffer were added, and the mixture was incubated at 70°C for 10 min. The procedure was continued according the instructions of the kit. Purified DNA was stored at −20°C. The oligonucleotide primers used, SC5 (5′TGCCTGT(AG)GGGAA(TC)CC (AT)(GT)CTGA(AT)CCA3′ and SCH4(5′GTCGAAAAGA(AT) AGTC(TA)CC(GA) TAGAAA′), were purchased from the Institute of Biotechnology, Helsinki, Finland. The primers amplify a 145-bp sequence at the 5′ end of the C. pneumoniae omp1 gene.9 Results. Of the 123 MEE specimens 53% were mucoid, 44% were mucopurulent and 3% were serous. The persistence of MEE varied from 1 to 12 (median, 3.5) months; in 90% of the cases MEE had persisted for 2 months or longer. Bacteria were cultured in 55 (45%) of the 123 MEE samples and major pathogens of AOM (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis) in 40 (32%) samples. S. pneumoniae was found in 14 (11%);, nontypable H. influenzae in 18 (15%) and M. catarrhalis in 8 (6%) samples. Staphylococcus aureus was found in 4 (3%) samples, coagulase-negative Staphylococcus in 7 (6%), viridans streptococci in 1 (1%), diphtheroids in 2 (2%) and acinetobacteria in 1 sample (1%). Eight of the MEEs contained more than 1 bacterial strain. All 123 MEE samples of 123 children were C. pneumoniae PCR-negative regardless of the age of the children and the persistence of MEE. Discussion. Our results in detecting C. pneumoniae from MEEs in OME patients are in accordance with the results of Goo et al.8 but differ from the Japanese, Swedish and Danish reports.4-7 The reason for negative results could be the younger age of our study children compared with those studies. Ogawa et al.4 reported 6 C. pneumoniae-positive patients, of whom 4 were adults and only 2 were children (one 7-year-old and one 5-year-old child). In the Swedish study the mean age of the sick children was 6.4 years; correspondingly in the Danish study it was 6.6 years.6, 7 In the study of American children the age range varied between 6 months and 12 years, and the samples, like ours, were C. pneumoniae-negative.8 Another reason for our negative results could be that there was no epidemic of C. pneumoniae disease at the time of sample collection during the present study. During an epidemic in a military garrison in Finland, C. pneumoniae was cultured once from MEE of a military trainee who was rehospitalized with sinusitis 7 weeks after C. pneumoniae pneumonia and who also had acute otitis media.9 None of our children had received antibiotics within 1 week before the study, and none of the children had received erythromycin or other macrolide antibiotics during the preceding month. Therefore we believe that our PCR results are not affected by previous antimicrobial treatments. The PCR method we used has been reliable for detecting C. pneumoniae in sputum specimens, except in cases with high serum C. pneumoniae antibody titers.10 We have also successfully used the same DNA purification method (the Qiagen commercial kit) for the isolation of S. pneumoniae DNA from MEEs of OME patients.11 The use of this kit removes the effect of polymerase inhibitors like heme or urea in the DNA purification, which minimizes possible false negative findings in the present study. We did not culture C. pneumoniae of the MEE specimens in our OME patients and therefore were not able to compare the sensitivity and specificity of PCR to culture. According to our study C. pneumoniae does not play a significant role in the pathogenesis of OME during the normal respiratory illness season, at least not in young children. Acknowledgments. We thank Teija Jaakkola for her excellent technical assistance. This study was financially supported by the Orion Research Foundation. Jussi Jero, M.D. Hannu Alakärppä, M.D. Anni Virolainen, M.D. Pekka Saikku, M.D. Pekka Karma, M.D. Department of Otolaryngology; Helsinki University Central Hospital; Helsinki (JJ, PK) National Public Health Institute; Oulu (HA, PS) Haartman Institute; Helsinki (AV) Finland