Anni Virolainen

Detection of Rhinovirus, Respiratory Syncytial Virus, and Coronavirus Infections in Acute Otitis Media by Reverse Transcriptase Polymerase Chain Reaction

Objective. To determine the frequencies of human rhinovirus (HRV), respiratory syncytial virus (RSV), and coronavirus (HCV) infection in children with acute otitis media (AOM). Methods. Middle ear fluids (MEF) collected by tympanocentesis and nasopharyngeal aspirates (NPA) at the time of the AOM diagnosis were examined by reverse transcriptase polymerase chain reaction for HRV, RSV, and HCV RNA. Patients. Ninety-two children aged 3 months to 7 years during a 1-year period. Results. Virus RNA was detected in a total of 69 children (75%) and in 44 MEF samples (48%) and 57 NPA samples (62%) at the time of AOM diagnosis. HRV RNA was detected in both MEF and NPA in 18 (20%), in MEF alone in 4 (4%), and in NPA alone in 10 (11%). RSV was detected in both MEF and NPA in 12 (13%), in MEF alone in 5 (5%), and in NPA alone in 9 (10%). HCV RNA was detected in both MEF and NPA in 5 (5%), in MEF alone in 2 (2%), and in NPA alone in 9 (10%). Dual viral infections were detected in 5% of children. HRV and RSV were detected simultaneously in 2 MEF samples and in 2 NPA samples; RSV and HCV were detected in 1 NPA sample. Bacterial pathogens were detected in 56 (62%) MEF from 91 children. Viral RNA was detected in 20 (57%) MEF of 35 bacteria-negative and in 25 (45%) of 56 bacteria-positive MEF samples. No important differences in the risk of treatment failure, relapse, or occurrence of late secretory otitis media were noted between children with virus-positive and virus-negative MEF aspirates. Conclusion. These findings highlight the importance of common respiratory viruses, particularly HRV and RSV, in predisposing to and causing AOM in young children.

Polymerase chain reaction-based detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion.

Abstract Objectives: To study the association of human rhinovirus (HRV), respiratory syncytial virus (RSV), and human coronavirus infections in children aged 6 months to 12 years with otitis media with effusion (OME). To determine how long HRV RNA can be detected after HRV infection. Methods: Middle ear effusion (MEE) samples collected at the time of tympanostomy tube placement from 100 children with OME were examined. Viral RNA was detected by reverse-transcriptase polymerase chain reaction. For HRV the results were compared with virus isolation in cell culture. In vitro studies of the persistence of HRV infectivity and RNA were conducted by combining ~105 median cell culture infectious doses of HRV with pooled MEE at 37°C and assaying serial samples for 12 weeks. Results: Virus RNA was detected in 30 children. HRV was detected by reverse-transcriptase polymerase chain reaction in 19 children with OME and by virus isolation in 5 children. RSV RNA was found in 8 and HCV in 3 children with OME. No dual viral infection was found. Bacterial pathogens were isolated from 35 MEE samples and were associated with viral RNA in 11 cases, most often with HRV (9 cases). Under in vitro conditions, HRV culture positivity declined rapidly (<2 days), but RNA was detectable for up to 8 weeks. Conclusions: These results suggest that virus infection, particularly HRV infection, either alone or concurrent with bacteria, is present in a larger percentage of children with OME than previously suspected. It remains to be determined how often the presence of viral RNA in MEE represents persistent RNA, ongoing viral replication, or recurrent infection. (J Pediatr 1998;133:390-4)

Human antibodies to pneumococcal surface protein A in health and disease.

Background. Diseases caused by Streptococcus pneumoniae have a high impact in young children whose ability to mount antibodies to capsular polysaccharides is impaired. Pneumococcal surface protein A (PspA) is a potential vaccine candidate for this age group. Methods. We used Western blot analysis and enzyme immunoassay to study human sera of healthy adults from Alabama (n = 20) and from Finland (n = 21), healthy children from Finland (n = 20) and ill children from Finland, those with pneumococcal invasive infection (n = 26) and those with nonpneumococcal invasive infection (n = 26). Results. Human antibodies to PspA exhibited strong cross‐reactivity among different pneumococcal strains. The geometric mean titer of IgG antibody to PspA in sera from 21 healthy adults was 4040, from ten 3‐year‐old healthy children 1080 and from ten 2‐month‐old healthy children 1650. The geometric mean titer of PspA antibody of acute phase sera of children with invasive pneumococcal disease was 140, significantly (P < 0.001) lower than the respective value, 1020, for children with infection caused by other bacteria. Conclusions. We demonstrate for the first time the existence of antibodies to PspA in human sera in health and disease. The findings in ill children suggest that antibodies to PspA might play a role in protection against pneumococcal disease.

Circulating antibody secreting cell response to parenteral pneumococcal vaccines as an indicator of a salivary IgA antibody response

This study assessed the mucosal immune response in healthy adult volunteers immunized parenterally with either pneumococcal polysaccharide (N = 8) or pneumococcal polysaccharide-protein conjugate (N = 10) vaccine with an aim to evaluate the relevance of antibody secreting cell (ASC) response after parenteral vaccination. An ASC response to the four types of capsular polysaccharide tested was observed in all vaccinees 7-9 days after immunization. IgA was the predominant class in the ASC response, and IgG the next common, with very few IgM ASCs. The IgA/IgG ratio in the ASC response was higher after immunization with the polysaccharide than the conjugate vaccine. Antibodies of the IgA class were frequently seen in the saliva already before immunization; especially to serotypes 14 and 19F. A twofold increase of the type specific secretory IgA antibodies in saliva was found in eight of the 16 instances in which the specific IgA ASC response was > 100 ASC per 10(6) cells and in only one of the 52 instances with fewer ASCs. We conclude that the ASC response in the peripheral blood is a useful parameter of the antibody response to pneumococcal vaccines and a good indicator of a secretory IgA response in the saliva.

PCR Assay for Detecting Streptococcus pneumoniae in the Middle Ear of Children with Otitis Media with Effusion

We compared a newly developed pneumococcal polymerase chain reaction (PCR) for Streptococcus pneumoniae (Pnc) to bacterial culture in 123 middle ear effusion (MEE) samples of 123 children with otitis media with effusion (OME). For the pneumococcal PCR assay, DNA of MEE samples was purified by a QIAamp blood kit. The outer primers used amplified a 348 basepair region of the pneumolysin gene, and the inner a 208. Pnc was cultured in 14 (11%) and pneumolysin PCR was positive in 57 (46%) of the 123 MEE samples. All the culture positive samples were also PCR-positive. Both the samples with culturable Pnc and with positive pneumolysin PCR increased with shorter duration of OME and a greater number of acute otitis media during the preceding 6 months. In conclusion, pneumolysin PCR suggests pneumococcal involvement in MEE even in OMEs with no evidence of Pnc in culture, and thus offers a good diagnostic tool when a more accurate and sensitive pneumococcal diagnosis is needed.

Comparison of serum antibodies to pneumolysin with those to pneumococcal capsular polysaccharides in children with acute otitis media

BACKGROUND Streptococcus pneumoniae is a major bacterial pathogens in acute otitis media. Pneumolysin is a species-specific protein toxin produced intracellularly by all clinically relevant pneumococcal strains, and antibodies to pneumolysin should therefore represent pneumococcal involvement in the disease, regardless of the serotype. METHODS Antibodies to pneumococcal pneumolysin and capsular polysaccharides were measured by enzyme immunoassay in acute and convalescent sera of 121 children with acute otitis media. A pneumococcal otitis episode was defined by a positive middle ear fluid culture and/or pneumolysin PCR. RESULTS Median age of the 10 children who developed a seroconversion response to pneumolysin was 1 year 8 months, and of the 21 children responding to polysaccharides it was 2 years 9 months. Eight of the 10 seroconversion responses to pneumolysin were of IgA class alone, whereas 17 of the 21 polysaccharide responses were of IgG class alone or IgG together with IgM and/or IgA. Of the 41 children with a pneumococcal otitis episode, 13 (39%) showed a seroconversion response, 3 (7%) to pneumolysin and 11 (27%) to capsular polysaccharides. The children with a pneumococcal otitis episode had lower titers of acute phase IgG to the capsular polysaccharide pool of S. pneumoniae (containing types 6B, 14, 19F and 23F), as compared with the titers in children with otitis caused by other pathogens and pneumococci only in the nasopharynx or not found at all (P = 0.04). CONCLUSIONS Serum antibodies to pneumolysin can be detected at an earlier age than those to the capsular polysaccharides. However, a seroconversion is rare and therefore of no diagnostic value. The presence of serum IgG to the pneumococcal capsular polysaccharides seems beneficial in the prevention of pneumococcal otitis.

Prognosis of acute otitis media. Factors associated with poor outcome

Factors associated with poor outcome of acute otitis media (AOM) were analysed in 131 children aged 1/4 to 7 1/2 (median 2 1/2) years. After AOM, altogether 37 (28%) of the children had poor outcome: 15 children (12%) clinical failure (unimprovement or worsening of pre-treatment signs and symptoms within 2 weeks of onset of therapy) and 31 (24%) persistent middle ear effusion (MEE) > or = 1 month post-treatment. Of the different variables studied in multivariate analysis, age < 2 years (p < 0.01), history of allergic skin or respiratory symptoms (p = 0.02), > or = 6 h duration of pre-treatment earache (p = 0.01) and B. catarrhalis in MEE (p = 0.05) were associated with clinical failure. Children with previous adenotomy or unilateral AOM had no failures. Persistence of MEE at 1 month was associated with age < 2 years (p = 0.05), otitis proneness (p = 0.03), bilaterality of AOM (p < 0.01) and S. pneumoniae in MEE (p = 0.01) in univariate but not in multivariate analysis.

Antibodies to Pneumolysin and Pneumococcal Capsular Polysaccharides in Middle Ear Fluid of Children with Acute Otitis Media

Antibodies to pneumococcal pneumolysin and capsular polysaccharides were measured by enzyme immunoassay in 169 acute phase middle ear fluid samples of 116 children with acute otitis media. Antibodies to pneumococcal pneumolysin were detected in 84% and to capsular polysaccharides in 50% of the MEF samples. The Ig class detected most often was IgA to both types of pneumococcal antigens, and it was present in MEF even with non-detectable levels of serum IgA of the same specificity. 59% of the MEF samples positive for IgA to pneumolysin were also positive for secretory component of the same specificity, and 53% of IgA to capsular polysaccharide pool (containing serotypes 6B, 14, 19F, and 23F), respectively. This suggests both leakage of specific IgA from serum to the middle ear and local production of it. In contrast, specific IgG was detected in MEF only with concomitant IgG in serum. Antibodies to pneumolysin occurred in no relation to bacterial findings in MEF. On the contrary, IgG class antibodies to capsular polysaccharides, most likely serum-derived, were detected less often in MEF samples positive for pneumococcus than for other bacteria.

An outbreak of pneumonia associated with S-pneumoniae at a military training facility in Finland in 2006

Streptococcus pneumoniae is a well‐known cause of community‐acquired bacterial pneumonia. The purpose of this study was to assess the cause and extent of the outbreak of pneumonia which occurred among military recruits following a 1‐week hard encampment in Finland. We also assessed the carriage rate and molecular characteristics of the S. pneumoniae isolates. All pneumococcal isolates were studied for antibiotic susceptibility, serotyped, genotyped by multilocus sequence typing (MLST), and the presence of pneumococcal rlrA pilus islet was detected. The genotype results defined by MLST corresponded with the serotype results. S. pneumoniae serotype 7F, ST2331, seemed to be associated with an outbreak of pneumonia and nasopharyngeal carriage among 43 military recruits. Of the 43 military recruits, five (12%) were hospitalized with pneumonia and two (40%) of them were positive for S. pneumoniae serotype 7F, ST2331 by blood culture. Eighteen (42%) of the 43 men were found to be positive for S. pneumoniae by nasopharyngeal culture, and nine (50%) of them carried pneumococcal serotype 7F, ST2331. The outbreak strain covered 55% of all the pneumococcal findings. Outbreaks of invasive pneumococcal disease seem to occur in a crowded environment such as a military training facility even among previously healthy young men.

Clinical outcome of acute pneumococcal otitis media and serum antibody responses to pneumococcal pneumolysin and polysaccharides in children.

Serum antibody responses to pneumococcal antigens and their relationship to the clinical outcome were determined in a prospective study of 121 children with acute otitis media (AOM). Pneumococcus positive children with a pneumolysin response more often had a recurrence and middle ear effusion (MEE) after 1 month than did the non-responders ( p =0.005 and p =0.04, respectively). All the children who responded to pneumolysin also had clinically strong symptoms and signs of AOM. Children who responded to pneumococcal polysaccharides developed otitis media with effusion within a 6-month follow-up period more often than did the non-responders ( p =0.005). The results of this study suggest that children with pneumococcal AOM and an antibody response to the intracellular pneumococcal protein pneumolysin behave clinically differently from children with an antibody response to polysaccharides.