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Real-time polymerase chain reaction test may accelerate diagnosing joint infection
Researchers detected bacteria like S. aureus in synovial fluid 5 hours after RT PCR tests.
The need for accurate and specific tests to diagnose joint infections spawned promising research into the effectiveness of real-time polymerase chain reaction for testing synovial fluid samples. This may eventually yield a test orthopedic surgeons can use to quickly diagnose some of the nearly 3% of hips and knees they see annually that are potentially infected, researchers said.
A clinical trial related to the sampling analysis is under way with about half of the required 60 patients already enrolled, according to Rocky S. Tuan, PhD. “The application is for septic arthritis in general and periprosthetic infection in particular,� he said.
The reverse transcriptase (RT) polymerase chain reaction (PCR) test Tuan and colleagues studied produced no false negatives, could detect all viable bacteria and yielded results in about 5 hours.
Research was conducted at Tuan’s laboratory at the Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal & Skin Diseases, National Institutes of Health (NIH).
“We were able to quantify directly the number of viable bacteria, which could lead us to determine and optimize treatment duration,� said researcher Patrick Birmingham, MD.
Tuan told Orthopedics Today, “The challenge is currently significant because when you have a situation when you suspect infection and you need to decide your treatment protocol, the decision is whether to proceed with the orthopedic procedure or treat the infection first.�
He estimated a cost savings of about $25,000 per case if infection can be ruled out in this fashion before proceeding with joint revision surgery.
Study of mRNA
The method was used to analyze messenger RNA (mRNA) in the synovial fluid of knees and hips, which researchers hoped would be more sensitive at detecting septic arthritis than synovial fluid cultures. “Cultures are really not a good gold standard,� Birmingham said.
Results of cultures take from 2 to 3 days to receive and rates of false positives and negatives are high, approaching 50%, he said. “Delay in decision making has clearly been associated with poor outcomes.�
Tuan agreed: “It complicates the clinical decision making and as a result sometimes you make a wrong decision.�
Accurate, fast
Investigators chose PCR testing because earlier research showed it produced fairly accurate bacterial counts within 24 hours. However, regular PCR was overly sensitive and detected dead and viable bacteria equally well. So researchers used polymerase enzyme-based RT quantitative PCR to target just the viability gene to detect the presence of bacteria in simulated septic arthritis synovial fluids and determine whether any bacteria they found were alive or not.
“It’s quantitative and we can actually measure the active bacterial load,� Tuan said.
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Deep wound infection associated with knee prosthesis. Note formation of drainage sinus. |
Lateral radiograph showing periprosthetic infection and loosening of femoral prosthetic component. Images: Birmingham P |
Samples identified
Researchers inoculated synovial fluid samples from 12 patients’ uninfected joints with bacteria, including Escherichia coli, Staphylococcus aureus, and methicillin-resistant S. aureus, and they used uninoculated samples as controls.
Researchers then analyzed each sample for type of infection using the test. They also treated separate samples with antibiotics to simulate patients who may be on antibiotic therapy prior to surgery.
“All samples were correctly identified with strong statistical significance without any false positives,� Birmingham said. Detection of S. epidermis was most sensitive at 10-5 .
The amount of mRNA decreased exponentially in antibiotic-treated samples, he said.
While regular PCR, which is based on DNA, cannot detect differences between antibiotic-treated and untreated samples, “With RT PCR we are able to document a concomitant decrease in the mRNA signal that is a function of antibiotics treatment,� Tuan said. Eventually, clinicians may be able to use that approach to gauge how well the antibiotics they give their patients are working.
The instrumentation to run such tests, although costly, is now available in labs that test for HIV or Lymes disease, Tuan said. “All you really need is the specimen and target genes. That is what we are focusing on — if these target genes are good reporters — so far, so good.�
For more information:
- Patrick Birmingham, MD, is chief orthopedic resident, The George Washington University Medical Center. He can be reached at 2300 Eye St. NW, Washington DC 20037. He has no direct financial interest in any products or companies mentioned in this article.
- Rocky S. Tuan, PhD, is chief of Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal & Skin Diseases, He can be reached at NIH Building 50, Room 1523, 50 South Drive, MSC 8022, Bethesda, MD 20892; 301-451-6854; e-mail: tuanr@mail.nih.gov. He has no direct financial interest in any products or companies mentioned in this article.
Reference:
- Birmingham P, Helm J, Manner PA, Tuan RS. Assessing joint infection by rapid detection of live bacteria via real time polymerase chain reaction. #101. Presented at the American Academy of Orthopaedic Surgeons 74th Annual Meeting. Feb. 14-18, 2007. San Diego.