Masaru Kunimoto

Detection of Haemophilus influenzae in middle ear of otitis media with effusion by polymerase chain reaction

Otitis media with effusion (OME) is one of the major causes of hearing loss in childhood. The pathogenesis still remains unclear, though it is closely related to acute otitis media with bacterial infections. It is known that Haemophilus influenzae is one of the most common bacteria isolated from middle ear effusions (MEEs). Recently, in vitro DNA amplification by polymerase chain reaction (PCR) is a new technology that has considerable implication for diagnosis of viral and bacterial infections because of its potentially precise specificity and sensitivity. In the present experiment polymerase chain reaction (PCR) was applied to the detection of DNA genome of H. influenzae contained in middle ear effusions. By Southern blot hybridization, two characteristic bands for H. influenzae DNA were detected at 273 b.p. and 550 b.p. position in 15 of 27 MEEs. However, no organism was cultured by conventional methods. Our results indicate the PCR technique is more specific and sensitive in detection of bacteria in middle ear effusion of OME, compared with conventional methods. It strongly suggests more involvement of the bacteria, especially H. influenzae, in OME onset.

Analysis of Viral Infection in Patients with IgA Nephropathy

We investigated viral infections in the tonsils, pharynx and renal tissues of patients with IgA nephropathy using cell culture, polymerase chain reaction (PCR) and immuno-fluorescent techniques, and measured antibody titers against numerous types of viruses. Neutralization tests found no significant inhibition of growth of adenovirus-1, 2, 3, 4, 5, 6, 7, 11 or 19, Coxsackie virus-A7, A9, A16, B1, B2, B3, B4, B5 or B6, or RS virus. Swabs of the oral cavity of patients with IgA nephropathy were cultured with Hel cells, MDCK cells, FL cells, BHK-21 cells and RD-18S cells. No cytopathic effect was detected in any of these cell cultures. We failed to detect the presence of herpes simplex virus-1 and -2, varicella-zoster virus, cytomegalovirus and Epstein-Barr virus (EBV)-1 and -2 in tonsils, renal tissues and mouthwashings from patients with IgA nephropathy. On the other hand, EBV alone was detected with the PCR technique, in mouthwashings from 6 out of 14 patients with IgA nephropathy (42%). Immunohistological and serological analyses were done to examine the relationship between EBV and IgA nephropathy. No evidence was obtained that EBV-infected B lymphocytes were producing IgA. It seems unlikely that the viral infections examined in this study play a significant role in the pathogenesis of IgA nephropathy.