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Novel genetic approach may improve ability to detect infection in arthroplasty

New method diagnoses infection by identifying specific genetic activity in responding white blood cells.

Issue: May 2005

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--- Carl Deirmengian

WASHINGTON — When it comes to managing painful joint arthroplasty, ambiguous test results for infection very often leave surgeons short on diagnostic confidence. They can miss one infection in five when looking at a knee replacement, for example, a recent study showed.

Now a novel molecular genetic approach to diagnosing infection offers the potential to change all that. In a new study researchers found that by examining synovial fluid white blood cells (WBC) using a genetic profiling process, they can distinguish whether inflammation in a total joint arthroplasty comes from septic or aseptic causes. That would go a long way toward helping to determine what treatment, if any, to apply.

Lead researcher Carl Deirmengian, MD, won the Mark Coventry Award for the Best Basic Science Paper from The Knee Society for this work. The study takes a significant step forward in efforts to detect infection not only in joint arthroplasty inflammation but in many other areas, too.

“This is one of the most exciting things I’ve seen in a long time,� said Arlen D. Hanssen, MD, of the Mayo Clinic, in introducing Deirmengian before his presentation at the recent American Academy of Orthopaedic Surgeons 72nd Annual Meeting. “It really highlights the importance of all this technology we keep hearing about.�

Cure for frustration

This study could potentially solve a couple of very frustrating problems for surgeons as follows: (1) false positive results in infection tests, presumably arising from contaminated cultures; and, (2) false negatives, caused by presumptive antibiotics, formation of biofilm or possibly from some organisms that “are very particular and don’t want to grow in the agar we have,� said Deirmengian, a resident at the University of Pennsylvania Health System in Philadelphia.

Deirmengian and his colleagues have changed the traditional views of WBC and their connection to infection. “We been concentrating on quantity for a long time – we count the white blood cells,� but his new process suggests the real differences lie in the gene expression of WBC.

The study centered on certain WBC (neutrophils) in the synovial fluid taken from inflamed joints. While researchers historically have considered neutrophils uninteresting cells with a one-size-fits-all inflammatory response, this study found that neutrophils actually modulate their genetic response depending on the cause of infection.

Do neutrophils differ?

Researchers aspirated synovial fluid from patients with either acute Staphyloccocus aureus infection or with gout of the knee. They chose a single bacterial species to minimize the theoretical gene expression differences between samples classified as infection. They chose gout as a model for aseptic inflammation because it would make it easier to establish a confident diagnosis without concerns over an underlying infection, said Deirmengian, who is also with 3B Orthopaedics in Philadelphia.

He and his colleagues found that neutrophils from S. aureus-infected knees showed “a dramatically different gene expression profile when compared to neutrophils from acute gout.�

The researchers collected the fluid over 15 months from the knees of seven patients with S. aureus infection and five patients with acute gout. They included only patients with S. aureus infection who had fever, acute arthritis, more than one positive S. aureus culture, high CRP values (average, 22; range, 1.9-34), high ESR values (average, 90; range 39-125), and an elevated synovial WBC count (average, 90,000 cells/mm; range 27,000-183000; average differential 93% neutrophils).

The researchers took five samples from infected total knee arthroplasties (TKAs) and two samples from infected native knees. All patients with gout showed monosodium urate crystals, elevated synovial fluid WBC count (average, 10,000 range, 800-16,000, average differential 90% neutrophils) and clinical resolution without antibiotics, the researchers wrote in their summary paper. All gout samples came from native knees.

The researchers developed a new methodology for isolating the synovial WBC RNA, and analyzed the results using the Affymetrix U133a GeneChip. That allowed them to study thousands of genes simultaneously and produce a “snapshot� of a cell’s genomic expression. They found a unique gene expression “signature,� associated with each diagnosis. The neutrophils in the synovial fluid of knees with acute S. aureus infection could be distinguished from those with acute gout “by nature of a unique gene expression ‘signature.’ �Researchers identified 1615 genes as being significantly different (P<.05) between the groups, and 124 of the genes associated with P values between 0.0000001 and 0.0001. “This represents a dramatic difference between groups,� the researchers wrote. The presence of antibiotics at the time of aspiration did not impede the ability to diagnose infection.

The genes making up the unique signature in the infection group come from a variety of functional families, including defense genes.

In basic terms, one could take the whole human genome and ask, “What percent of the genes are ‘defense genes?’ �Deirmengian said. “Then I can look at the group of genes I found to be up-regulated in this project, and say, what percentage of these are defense genes? I found that 27% were defense genes, whereas only 10% of the genes in the human genome are defense genes,� Deirmengian said. The P value associated with such an overabundance of defense genes: 1 × 10-30. That means the gene expression “signature� observed in the neutrophils from infected knees makes biologic sense, Deirmengian added.

The bottom line: All neutrophils may look morphologically similar, but genetically they respond very differently. Those differences in neutrophil gene expression“ … may be used to develop simple laboratory tests that distinguish the causes of inflammation in total joint arthroplasty,� Deirmengian explained.

Deirmengian concluded by noting that the GeneChip remains too impractical for everyday analysis but researchers could use the data from the GeneChip studies to help choose specific markers that are a diagnostic for infection. “These markers can be applied in a simpler, cost-effective format that is amenable to widespread clinical use,� he said, adding, “They could be detected in the synovial fluid by rapid ELISA testing in a standard clinical laboratory, or tested in the pathology laboratory by histology.�

Still, any widespread use will require further research and remains at least a couple of years away, he said.

For more information:

• Deirmengian C, Lonner JH, Booth RE. WBC gene expression: a novel approach toward the study and diagnosis of infection. Presented at The Knee Society Specialty Day. Feb. 26, 2005. Washington.