Detection of bacteria in sterile body fluids

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).