Background: Culture remains the gold standard in the diagnosis of
bacterial infection, but molecular biological techniques have yielded
promising results. In this study, we validated a combined polymerase chain
reaction and reverse line blot hybridization protocol for identifying
Methods: Samples were obtained from seventy-six patients undergoing
orthopaedic surgery for various aseptic and septic indications. The diagnosis
of infection was based on a review of all available clinical and culture data.
In addition to routine culture for aerobic and anaerobic growth, samples were
analyzed with a broad-range 16S rRNA polymerase chain reaction and subsequent
reverse line blot hybridization with use of twenty-eight group, genus, and
species-specific oligonucleotide probes.
Results: An infection was diagnosed on the basis of patient data in
thirty-one patients. All but one of the patients with a clinical diagnosis of
infection had a positive result of the polymerase chain reaction-reverse line
blot hybridization. Five of the forty-five patients in whom an infection was
not suspected on the basis of patient data had at least one positive result of
the polymerase chain reaction-reverse line blot hybridization. Cultures
demonstrated microorganisms in twenty-five patients with an infection and in
two patients in whom an infection was not suspected on the basis of the
patient data. Staphylococcus aureus was the most common organism
grown on culture. The species identified by the polymerase chain
reaction-reverse line blot hybridization was in full accordance with that
grown on culture in all but one patient.
Conclusions: Polymerase chain reaction-reverse line blot
hybridization performed well in detecting and identifying the various
bacterial species and was more sensitive than routine culture. It identified
Staphylococcus aureus as the most frequently found microorganism.
Five patients in whom an infection was not suspected on the basis of the
patient data had a positive result of the polymerase chain reaction, which may
have been caused by contamination of the samples. However, three of these
patients had aseptic loosening of a total hip prosthesis, suggesting the
presence of a low-grade bacterial infection that remained undetected by the
culture but was detected by the polymerase chain reaction-reverse line blot
Level of Evidence: Diagnostic Level III. See Instructions
to Authors for a complete description of levels of evidence.