Detection of bacteria in sterile body fluids

Poster (1998, October)

Purpose of the study: Development of a polymerase chain reaction (PCR) method for the detection of any bacterial DNA in synovial, cerebrospinal, pleural, and peritoneal fluids. Methods: Most of bacterial ... [more ▼]

Purpose of the study: Development of a polymerase chain reaction (PCR) method for the detection of any bacterial DNA in synovial, cerebrospinal, pleural, and peritoneal fluids. Methods: Most of bacterial species possess highly conserved, multicopy 16S ribosomal RNA genes, which can be hybridized with a single set of oligonucleotide primers. Samples were processed by an extraction protocol using proteinase K, and subjected to PCR amplification using two universal bacterial primers: RW01 and DG74; then the PCR products were detected by ethidium bromide gel electrophoresis. Study: Synovial, cerebrospinal, pleural and peritoneal fluids, obtained from 32 patients were analyzed by Gram stain, culture and PCR. Results: 1. The limit of detection, determined by analyzing successive dilutions of Staphylococcus aureus and Escherichia coli cultures in sterile water, was: 1.105 cfu/100µl. 2. We obtained 9 positive samples by culture: - 7 synovial fluids: S. agalactiae, S. viridans, coagulase negative Staphylococcus (2), S. epidermidis (2), and S. aureus. - 2 pleural fluids: S. pyogenes and Enterobacter aerogenes. All were PCR positive. 3. We tested 23 culture negative samples. All were negative by PCR. Conclusion: PCR presents some interesting features: 1. Only small amount of sample is necessary (100µl). 2. The duration of the test is shorter than 8 hours. 3. PCR provides similar or eventually greater sensitivity than culture and an excellent specificity (no false-positive and no false-negative results actually observed). [less ▲]