Polymerase Chain Reaction molecular diagnostic technology for monitoring chronic osteomyelitis

Abstract

BackgroundOsteomyelitis is a devastating condition whose treatment relies on the detection of bacteria. The current standard of microbiology culture may not be adequate. Molecular biology based diagnostic procedures for detecting bacteria in orthopaedic infections was previously established, but has not been applied to the setting of chronic osteomyelitis. We aim to determine the applicability of molecular diagnostic procedures for monitoring chronic osteomyelitis, and to evaluate if these procedures are superior to standard culture methods of osteomyelitis detection.MethodsA rabbit experimental model of chronic osteomyelitis was used; infection was induced in the proximal, medial aspect of the tibia with Staphylococcus aureus at titers ranging from 1 × 102 to 1 × 106 colony forming units. At 28 days post-infection, animals were sacrificed, and the tibias were examined radiographically, harvested, and assayed for the presence of bacteria. Two bacterial detection methods were used: (1) standard microbiological culturing, and (2) polymerase chain reaction (PCR) based diagnostic method to detect bacterial genomic DNA.ResultsThe molecular diagnostic method was highly sensitive and accurate, and detected low titer infections that were undetected by radiographic and microbiological methods. By using two sets of PCR primers, one for a universal bacterial gene (16S rRNA) and one for a species-specific gene (nuc), the molecular protocol allowed both the detection and speciation of the bacterial infection.ConclusionsThe use of the PCR-based method was effective for high-sensitivity detection and identification of bacteria associated with chronic osteomyelitis in a rabbit model. Our findings illustrate the applicability of PCR for monitoring chronic osteomyelitis, which may be useful for improved detection of osteomyelitis organisms in humans.

Antimicrobial activity of mesenchymal stem cells against Staphylococcus aureus

“…One method used in the past to determine the number of viable microbial cells in culture was based on quantitation of ATP, but with inconsistent results. Our lab actually pioneered the 16S RNA-based RT-PCR method for detecting joint infection [54] and recently adopted it for osteomyelitis [55]. However, this method detects all bacteria, including non-viable bacteria, potentially giving false positives.…”

Development of a large animal rabbit model for chronic periprosthetic joint infection

“… 39 Radiography showed that by day 28, 100% of the animals in the experimental group suffered bone infection as defined by increased radiolucency around the implant, which is a hallmark of PJI. 40 - 42 Histological analysis of explanted bone from the inoculated group revealed fibrous tissue formation within the bone tunnel that stained positively on Gram’s stain in serial sections, whereas healthy bone formation was observed in the aseptic screw group. 32 The positive Gram’s stain was coincident with particles characteristic of bacteria in size.…”

The Polymerase Chain Reaction

“…1. Ethidium bromide (EtBr) can be used for the staining of amplified DNA product [31,32]. It has UV absorbance maxima at 300 and 360 nm, and an emission maximum at 590 nm, and being a DNA intercalator, EtBr inserts itself between the base pairs in the double helix.…”