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Silkworm larvae plasma could provide a promising biomarker for reimplantation
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Pouya Alijanipour
Javad Parvizi
One of the major issues facing orthopedic surgeons who treat patients with periprosthetic joint infection is to determine the optimal timing for reimplantation following a resection arthroplasty. Currently, at least in North America, patients with periprosthetic joint infection are treated with antibiotics for 6 weeks following a resection arthroplasty followed by 2 weeks of a “drug holiday” and reimplantation is performed at that point unless there are “gross” signs of infection.
It is known that C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) may still be elevated at 8 weeks following resection arthroplasty, and hence, these serums markers cannot be used as a metric for timing the reimplantation. In fact, studies have demonstrated that the level of ESR and CRP at the time of reimplantation is not predictive of further failure. Another issue is that these blood markers, especially ESR, may take 3 months or longer to return to normal. Thus, waiting for complete normalization of the serum markers may subject the patient to a prolonged period of disability and pain.
Limitations of current methods
At our institution, we prefer to observe a decline in the level of ESR and CRP during the antibiotic treatment and do not wait for complete normalization of them after resection arthroplasty. Measurement of synovial white cell count and neutrophil percentage, even if one is able to obtain adequate fluid from the joint with a spacer in place, for such analysis is also not helpful in this circumstance as the optimal “threshold” for these parameters has not been determined. Culture of the joint aspirate prior to surgery is also not useful as patients are still under the effect of systemic antibiotic therapy and also eluding antibiotics from the spacer adversely affects the ability of the culture to isolate the infecting organism.
Currently, there is little to no science to guide us regarding the optimal timing of a reimplantation. Some authorities in Europe and the rest of the world believe that 6 weeks of antibiotic treatment is too excessive, and patients may be reimplanted much earlier. On the other hand, there are those who delay reimplantation in patients with “severe” infection believing that waiting longer is likely to improve eventual outcome. The International Consensus on Periprosthetic Joint Infection recommends that reimplantation may be attempted as early as 2 weeks. It is important to know that patients with a spacer in place have severely compromised function that prevents them from working, or even performing activities of daily living. In addition, these patients frequently develop mechanical problems with their spacer, necessitating multiple visits to the office and/or emergency room, elevating the associated economic and psychological burden of the disease. The question that arises is then what, if any, metric can be used to determine the optimal timing of reimplantation. We were fortunate to be the recipient of a generous funding from the Orthopaedic Research and Education Foundation that has allowed us to examine this issue.
Indications of ongoing research
This ongoing study has revealed a potential serum biomarker that may have a promising role. A natural biochemical phenomenon called prophenoloxidase (PPO) cascade may be able to provide a method that has been missing in our set of diagnostic tools for PJI. The PPO cascade is part of the innate defense mechanism of various invertebrates such as silkworm, hornworm, housefly, flesh fly, beetle and crabs. This mechanism exists in plasma, hemolymph and cuticle of the invertebrates and their larvae. It consists of a series of biochemical reactions that are initiated by the presence of peptidoglycan (PG, which constitutes 70% and 20% of the cell walls of gram positive and gram negative bacteria, respectively), and beta-glucan (BG, the major component of fungal cell walls). The cascade consists of a chain of enzymatic reactions including phenoloxidase and serine proteases that will finally lead to the production of quinone derivatives. Polymerization of quinone derivatives will lead to the formation of melanin polymers, which are the action molecules of this innate defense mechanism and act by surrounding the pathogens and damaged tissues.
Silkworm larvae plasma (SLP) reagent, prepared from the plasma fraction of silkworm body fluid, can detect PG or BG using this phenomenon. Production of dark pigment of melanin alters the color and light transmittance (or absorbance) of the solutions that contain PG or BG. The intensity of color change and the time of reaction can be measured and are indicative of presence and concentration of target molecules (PG or BG), respectively. The test is technically simple and highly sensitive, being able to detect PG or BG with concentrations as low as 50 pg/mL to 100 pg/mL in samples from blood, cerebrospinal fluid and wound drainage fluid in gastrointestinal surgery. The human body is not able to produce PG or BG indicating that, theoretically, the test is totally specific for infectious processes in anatomic sites that are physiologically sterile. Nonetheless, SLP test and other similar PPO-based methods are not able to typify the pathogenic bacteria or fungi but they can be helpful in ruling in or ruling out infectious causes of failed prostheses, allowing the orthopedic surgeon to strategize the treatment plan correctly. These tests might also be able to determine the optimal timing for re-implantation surgery in patients who undergo staged exchange arthroplasty. Moreover, the test has the potential to be employed as a point-of-care tool for outpatient or intraoperative settings.
Our initial experiments on several samples of synovial fluid in patients with confirmed PJI and patients who underwent primary total joint arthroplasty have shown that SLP reagent is highly sensitive and specific for detection of bacterial remnants in serum. All of the infected samples that were tested were positive and all of the non-infected samples were negative in several experiments. We are in the process of further refining this test for this particular application.