PCR is a rapid, reproducible method for nucleic acid detection. However, this technology displays significant deficiencies when applied in clinical microbiology. This work’s aim was to improve current diagnostics and provide sensitive and quantitative real-time PCRs. Paper I describes the development of a sensitive and specific quantitative real-time PCR for the detection of Streptococcus pneumoniae, based on the Spn9802 DNA fragment. Applied to nasopharyngeal aspirates from 166 pneumonia patients, Spn9802 PCR had a sensitivity of 94% and a specificity of 98%. In Paper II the performance of a ply gene PCR for identification of pneumococcal lower respiratory tract infection (LRTI) was evaluated on bronchoalveloar lavage fluids. At the detection limit 103 genome copies/mL, 89% sensitivity but only 43% specificity was achieved. Paper III shows that S. pneumoniae DNA is detectable in plasma from acutely febrile patients. Sensitivities were low (26-42%) for detection of pneumococcal pneumonia, for bacteraemic pneumococcal pneumonia they were 60-70%. Paper IV describes evaluation of four PCR targets for Haemophilus influenzae detection. A real-time PCR based on the P6 gene was developed and applied to 166 CAP patients, using cut-off of 104 genome copies/mL the assay had a sensitivity of 97% and a specificity of 96%. In paper V, the two real-time PCRs presented in papers I and IV were combined with a PCR for detection of Neisseriae meningitidis. The analytical sensitivity of this multiplex real-time PCR was not affected by using a mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae) in single tubes. Applied to 156 LRTI patients, this PCR had sensitivities over 90% for S. pneumoniae and H. influenzae, and specificities of 89% and 96%, respectively. In conclusion, real-time PCR assays are useful for the diagnosis of S. pneumoniae and H. influenzae. They enable detection after antibiotic installation, and quantification increases the etiological specificity of pneumonia.

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第20623号 / 社医博第81号 / 社新制||医||9(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 木原 正博, 教授 中川 一路, 教授 平家 俊男 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

IV. ABSTRACT The human metapneumovirus is a novel paramyxovirus associated with acute respiratory infections in children, adults, elderly and immunocompromised individuals. It has a worldwide distribution and the prevalence range between 1.5% to 25% in individuals with respiratory infections. Based on phylogenetic analysis 2 distinct genetic groups (A and B) that are sub-divided into four subgroups (A1, A2, B1 and B2) have been shown to circulate. Until recently, there was no information on the molecular epidemiology and the clinical characteristics of the hMPV in Africa, including South Africa, a region with a high prevalence of paediatric human immunodeficiency virus type-1 (HIV) infection. The molecular epidemiology and clinical characteristics of the hMPV in South Africa was investigated over a three period (2000-2002) in children hospitalized with lower respiratory tract infection. The children were part of a cohort participating in a phase 3 clinical trial investigating the efficacy of a 9-valent-pneumocococcal protein-polysaccharide conjugate vaccine (PCV). The objectives of the study were: i. to investigate the molecular epidemiology of hMPV in South Africa; ii. characterize the burden of hMPV disease and determine the clinical features of hMPV-LRTI in children infected and not infected by HIV; iii. probe the role of Streptococcus pneumoniae in the pathogenesis of hMPV-LRTI. The overall prevalence of hMPV in children hospitalized with lower respiratory tract infections (LRTI) was 7.4%. The mean age of children with hMPV associated LRTI (hMPV-LRTI) in South Africa was 13.3 months (range 1.4-49.2 months), with HIV infected children being older than children not infected with HIV (mean [range] 17.6 [4.5-44.3] vs. 12.3 [1.4-49.2] months; P=0.007). The incidence of hMPV-LRTI was 5.0 (95%C.I.3.3-7.5) fold greater in HIV infected children (incidence rate: 2 504 [95%C.I. 1 683-3 577] per 100 000) than in HIV uninfected children (incidence rate: 505 [95%C.I. 409-618] per 100 000, P<0.0001). Human metapneumovirus was identified less frequently than RSV but more commonly than other studied respiratory viruses. The double-blind PCV-9 vs. placebo controlled trial was used to probe the role of pneumococcal co-infections contributing to the pathogenesis of severe hMPV-LRTI. The incidence of hospitalization for hMPV-LRTI was reduced by 46% (95%, CI, 25-63; P=0.0002) in PCV-9 vaccinees compared to placebo recipients. This inferred that coinfection with Streptococcus pneumoniae was integral to the pathogenesis of hMPV-LRTI requiring hospitalization. Both groups of the hMPV circulated during the three year period including concurrent circulation of multiple subtypes of the virus. There was a transition from group B to group A subtype virus as the dominant circulating virus over sequential years. Sequence analysis of the two attachment glycoproteins (F and G), showed the F gene protein to be highly conserved, in contrast the attachment protein gene (G protein) was highly variable particularly in the extracellular domain between lineages. Repeat hMPV-LRTI by either homologous or heterologous strains within 3 months of each other suggested that natural infection did not confer complete immunity to hMPV. The present study demonstrated that hMPV is a leading pathogen associated with LRTI among children in Africa and indicated that occult pneumococcal co-infections’ were integral in the pathogenesis of hMPV-LRTI requiring hospitalization. Additionally, this is the first study to have characterized the molecular epidemiology of hMPV in Africa and provides insight as to issues that may exist regarding the design of an hMPV vaccine.

Despite advances in health promotion through efforts to reduce tobacco smoking, tobacco-related health conditions have continued to be significant. Exposure to secondhand smoke has been identified as a health risk also in addition to infant health risks related to maternal smoking. In contrast, breastfeeding has been found to promote infant health and is strongly encouraged. Despite literature supporting both of these statements, the combined effects of both breastfeeding and maternal smoking on infant wellbeing have not been delineated. Otitis media represents a common health problem among infants and young children. Tobacco exposure has been shown to increase its incidence while breastfeeding has been shown to reduce its occurrence. In the current study, a consecutive sample of all infants less than 5 years of age with otitis media and breastfed for at least 6 months was collected from a busy urban clinic for analysis. A survey tool was administered to those meeting study criteria. Primary analysis examined the odds ratio of developing otitis media among breastfed infants between those whose mothers smoked tobacco and those whose mothers did not. As a result, the association between the protective effects of breastfeeding and the detrimental effects of maternal smoking was evaluated in relation to the development of otitis media. Secondary variables including demographics, family history, past medical and birth history, and secondhand smoke exposure were also assessed. Results failed to demonstrate a significant difference in otitis media between the 2 cohorts in this study, and of the secondary variables, only cranio-facial deformities and/or a family history of these conditions resulted in higher otitis media occurrence. Further study with larger populations with higher tobacco use rates may offer additional insights into this matter.

Background: Humanmilk oligosaccharides (HMOs) support and concurrently shape the neonatal immune system through various mechanisms. Thereby, they may contribute to lower incidence of infections in infants. However, there is limited evidence on the role of individual HMOs in the risk of otitis media (OM), as well as lower and upper respiratory tract infections (LRTI and URTI, respectively) in children up to 2 years. Objective: To investigate whether individual HMO concentrations measured at 6 weeks of lactation were associated with risk of OM, LRTI or URTI up to 2 years in breastfed infants. Associations with OM, LRTI and URTI were determined for the most prominent human milk oligosaccharides including 13 neutral, partly isomeric structures (trioses up to hexaoses), two acidic trioses, and lactose. Design: HMO measurements and physician reported data on infections were available from human milk samples collected at 6 weeks postpartum (n = 667). Associations of HMOs with infections were assessed in crude and adjusted models using modified Poisson regression. Results: Absolute concentrations (median [min, max], in g/L) of 2′-fucosyllactose (2′-FL) tended (p = 0.04) to be lower, while lacto-N-tetraose (LNT) was higher in the milk for infants with OM in the 1st year of life (p = 0.0046). In the milk of secretor mothers, LNT was significantly higher in the milk for infants with OM (RR [95% CI]: 0.98 [0.15, 2.60]) compared to infants without OM (RR [95% CI]: 0.76 [0.14, 2.90]) at 1 year (p = 0.0019). No statistically significant milk group differences and associations were observed for OM, LRTI, and URTI (p > 0.0031). Conclusion: Our findings suggest that neither prominent neutral individual HMOs (ranging from 2′-FL to LNDFHs) nor acidic human milk sialyllactoses or lactose are significantly associated with a reduced or increased risk of infections in infants up to 2 years of age. Further research is needed to determine whether specific HMOs could potentially reduce the incidence or alleviate the course of distinct infections in early life.

<p><strong>Background</strong>: Human rhinovirus/enterovirus (HRV/ENT) infections are commonly identified in children with acute respiratory infections (ARIs), but data on their clinical severity remains limited. We compared the clinical severity of HRV/ENT to respiratory syncytial virus (RSV), influenza A/B (FLU) and other common respiratory virus in children.</p> <p><strong>Methods</strong>: Retrospective study of children with ARIs and confirmed single positive viral infections on mid-turbinate swabs by molecular assays. Outcome measures included hospital admission and, for inpatients, a composite end-point consisting of intensive care admission, hospitalization greater than 5 days, oxygen requirements or death.</p> <p><strong>Results</strong>: A total of 116 HRV/ENT, 102 RSV, 99 FLU and 64 other common respiratory viruses were identified. Children with single HRV/ENT infections presented with significantly higher rates of underlying immunosuppressive conditions compared to those with RSV (37.9% vs 13.6%; p</p> <p><strong>Conclusions</strong>: Children with HRV/ENT had a more severe clinical course than those with RSV and FLUA/B infections and often had significant comorbidities. These findings emphasize the importance of considering HRV/ENT infection in children presenting with severe acute respiratory tract infections.</p> / Master of Science (MSc)