No abstract

The aim of the present study was to develop a broad-range PCR based on bacterial 16S rDNA for use in the routine diagnostic clinical microbiology service. The optimization and validation of the assay for use on clinical specimens from normally sterile sites is described, and preliminary results are reported on the use of the assay in the clinical diagnosis of bacterial infection in 382 paediatric specimens over a 2-year period. These results are compared to those obtained by standard culture techniques and show increased diagnosis of bacterial infection when both culture and PCR are used together; 16S rDNA PCR provided the sole evidence of pathogenic infection in 71 cases. Key stages in the assay development and potential pitfalls of the technique are highlighted and the improvement the assay offers in the diagnosis of infection in the paediatric setting is discussed.

BackgroundIntrauterine infection may play a role in preterm delivery due to spontaneous preterm labor (PTL) and preterm prolonged rupture of membranes (PPROM). Because bacteria previously associated with preterm delivery are often difficult to culture, a molecular biology approach was used to identify bacterial DNA in placenta and fetal membranes.Methodology/Principal findingsWe used broad-range 16S rDNA PCR and species-specific, real-time assays to amplify bacterial DNA from fetal membranes and placenta. 74 women were recruited to the following groups: PPROM <32 weeks (n = 26; 11 caesarean); PTL with intact membranes <32 weeks (n = 19; all vaginal birth); indicated preterm delivery <32 weeks (n = 8; all caesarean); term (n = 21; 11 caesarean). 50% (5/10) of term vaginal deliveries were positive for bacterial DNA. However, little spread was observed through tissues and species diversity was restricted. Minimal bacteria were detected in term elective section or indicated preterm deliveries. Bacterial prevalence was significantly increased in samples from PTL with intact membranes [89% (17/19) versus 50% (5/10) in term vaginal delivery p = 0.03] and PPROM (CS) [55% (6/11) versus 0% (0/11) in term elective CS, p = 0.01]. In addition, bacterial spread and diversity was greater in the preterm groups with 68% (13/19) PTL group having 3 or more positive samples and over 60% (12/19) showing two or more bacterial species (versus 20% (2/10) in term vaginal deliveries). Blood monocytes from women with PTL with intact membranes and PPROM who were 16S bacterial positive showed greater level of immune paresis (p = 0.03). A positive PCR result was associated with histological chorioamnionitis in preterm deliveries.Conclusion/SignificanceBacteria are found in both preterm and term fetal membranes. A greater spread and diversity of bacterial species were found in tissues of women who had very preterm births. It is unclear to what extent the greater bacterial prevalence observed in all vaginal delivery groups reflects bacterial contamination or colonization of membranes during labor. Bacteria positive preterm tissues are associated with histological chorioamnionitis and a pronounced maternal immune paresis.

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These data suggest that QFT-IT may not provide a determinate test result in a substantial proportion of children in a tertiary care setting due to the combination of young age and primary and acquired immune deficiencies.