Prosthetic Joint Infection Diagnosis Using Broad-Range PCR of Biofilms Dislodged from Knee and Hip Arthroplasty Surfaces Using Sonication

Periprosthetic tissue and/or synovial fluid PCR has been previously studied for prosthetic joint infection (PJI) diagnosis; however, few studies have assessed the utility of PCR on biofilms dislodged from the surface of explanted arthroplasties using vortexing and sonication (i.e., sonicate fluid PCR). We compared sonicate fluid 16S rRNA gene real-time PCR and sequencing to culture of synovial fluid, tissue, and sonicate fluid for the microbiologic diagnosis of PJI. PCR sequences generating mixed chromatograms were decatenated using RipSeq Mixed. We studied sonicate fluids from 135 and 231 subjects with PJI and aseptic failure, respectively. Synovial fluid, tissue, and sonicate fluid culture and sonicate fluid PCR had similar sensitivities (64.7, 70.4, 72.6, and 70.4%, respectively; P > 0.05) and specificities (96.9, 98.7, 98.3, and 97.8%, respectively; P > 0.05). Combining sonicate fluid culture and PCR, the sensitivity was higher (78.5%, P < 0.05) than those of individual tests, with similar specificity (97.0%). Thirteen subjects had positive sonicate fluid culture but negative PCR, and 11 had negative sonicate fluid culture but positive PCR (among which 7 had prior use of antimicrobials). Broad-range PCR and culture of sonicate fluid have equivalent performance for PJI diagnosis.     

Prosthetic Joint Infection: A Major Threat to Successful Total Joint Arthroplasty

Total joint arthroplasty (TJA) is one of the most common and reliable orthopaedic procedures that has significantly improved the quality of life of patients with degenerative joint diseases. Following the increase in the ageing population, availability of trained orthopaedic surgeons and advances in implantation procedures, demand for TJA both globally and in India is significantly increasing. Though TJA is one of the most cost-successful orthopaedic procedures, prosthetic joint infection (PJI) is one of the major complications of joint arthroplasty. Accurate diagnosis of PJI is challenging. Since total hip and knee arthroplasties comprises the majority of TJAs, this review focuses on the current understanding of incidence, risk factors, pathogenesis, causative microorganisms, diagnosis, treatment and prevention of PJI related to these two procedures.     

Improved Diagnosis of Periprosthetic Joint Infection by Multiplex PCR of Sonication Fluid from Removed Implants

The microbiological diagnosis of periprosthetic joint infection (PJI) is crucial for successful antimicrobial treatment. Cultures have limited sensitivity, especially in patients receiving antibiotics. We evaluated the value of multiplex PCR for detection of microbial DNA in sonication fluid from removed orthopedic prostheses. Cases of PJI in which the prosthesis (or part of it) was removed were prospectively included. The removed implant was sonicated, and the resulting sonication fluid was cultured and subjected to multiplex PCR. Of 37 PJI cases (17 hip prostheses, 14 knee prostheses, 4 shoulder prostheses, 1 elbow prosthesis, and 1 ankle prosthesis), pathogens were identified in periprosthetic tissue in 24 (65%) cases, in sonication fluid in 23 (62%) cases, and by multiplex PCR in 29 (78%) cases. The pathogen was detected in 5 cases in sonication fluid only (Propionibacterium acnes in all cases; none of these patients had previously received antibiotics) and in 11 cases by multiplex PCR only (all of these patients had previously received antibiotics). After exclusion of 8 cases caused by P. acnes or Corynebacterium species, which cannot be detected due to the absence of specific primers in the PCR kit, sonication cultures were positive in 17 cases and multiplex PCR sonication cultures were positive in 29 cases (59% versus 100%, respectively; P < 0.01). Among 19 cases (51%) receiving antibiotics, multiplex PCR was positive in all 19 (100%), whereas sonication cultures grew the organism in 8 (42%) (P < 0.01). Multiplex PCR of sonication fluid is a promising test for diagnosis of PJI, particularly in patients who previously received antibiotics. With modified primer sets, multiplex PCR has the potential for further improvement of the diagnosis of PJI.Modern medicine has developed a variety of artificial devices to assist in the performance of physiological functions as short-term devices (e.g., catheters and fracture fixation hardware) or permanent devices (e.g., artificial cardiac valves, pacemakers, and prosthetic joints). Orthopedic devices are used for the treatment of degenerative joint disease (osteoarthritis) and bone fracture (10). They are increasingly implanted in the growing population of the elderly. The risk of periprosthetic joint infections (PJI) is additionally increasing due to longer resident time of the implant in the body, which can become infected from a distant infectious focus by hematogenous route at any time after implantation (29). Thus, the number of PJI is expected to increase steadily in the next decades.The identification of the infecting microorganism is crucial for successful treatment of PJI. Currently, cultures of synovial fluid and intraoperative periprosthetic tissue represent the standard method for diagnosing PJI. With newer techniques, such as sonication of removed implants, the sensitivity has been significantly increased (18, 22). However, in a significant proportion of patients with PJI, the infecting microorganism remains unknown, particularly when patients had previously received antimicrobial treatment (1, 22). Molecular methods may improve the diagnosis for PJI due to high sensitivity and culture independence.Several researchers have evaluated the role of PCR in the diagnosis of osteoarticular infections, including septic arthritis (8, 14, 15) and PJI (1a, 4, 5, 12, 17, 25). The value of PCR was mainly investigated in synovial fluid or periprosthetic tissue specimens, whereas sonication fluid was evaluated only recently (18, 26). The limitations of these studies are the use of a specific PCR, which is typically able to detect only a single microorganism, or the use of a broad-range (16S ribosomal DNA [rDNA]) PCR, which can detect previously unknown organisms but has lower sensitivity and specificity than specific PCR, requires subsequent sequencing for bacterial identification, and fails to detect mixed infections.Herein, we investigated a novel approach for diagnosis of PJI combining two complementary diagnostic methods, namely, sonication of removed implants and multiplex real-time PCR of the resulting sonication fluid. The removed prostheses were sonicated as previously described (18, 22), and the resulting sonication fluid was cultured and subjected to molecular detection with a real-time multiplex PCR test (SeptiFast; Roche Diagnostics, Basel, Switzerland). This PCR kit was designed for detection and identification of the most common bacterial and fungal pathogenic species in blood (Table ​(Table1)1) (2, 11, 13, 16, 28) and has not yet been investigated for the diagnosis of PJI. We hypothesized that the combined approach of sonication and multiplex PCR of sonication fluid will improve the sensitivity and specificity for diagnosing PJI.

TABLE 1.

Microorganisms that can be detected by multiplex PCR (SeptiFast)

PCR-Based Diagnosis of Prosthetic Joint Infection

We performed a meta-analysis to evaluate use of PCR assays for diagnosis of prosthetic joint infection (PJI). The pooled sensitivity and specificity were 0.86 (95% confidence interval [CI], 0.77 to 0.92) and 0.91 (CI, 0.81 to 0.96), respectively. Subgroup analyses showed that use of tissue samples may improve sensitivity, and quantitative PCR and sonication of prostheses fluid may improve specificity. The results showed that PCR is reliable and accurate for detection of PJI.     

Sonication of Explanted Prosthesis Combined with Incubation in BD Bactec Bottles for Pathogen-Based Diagnosis of Prosthetic Joint Infection

Prosthetic joint infection (PJI) is a rare but refractory complication of arthroplasty. Accurate identification of pathogens is a key step for successful treatment of PJI, which remains a challenge for clinicians and laboratory workers. We designed a combined culture method with sonication of implants and incubation in a BD Bactec system to improve the effectiveness of pathogen diagnosis in PJI. The aims of this study were to investigate the diagnostic accuracy of sonicate fluid cultures in the BD Bactec system and to compare the results with those of synovial fluid cultures in the BD Bactec system. The prosthetic components removed were sonicated in Ringer''s solution, and then sonicate fluid was incubated in Bactec bottles for 5 days. Synovial fluid was incubated in Bactec bottles for 5 days as a control. Synovial fluid cultures with Bactec bottles and sonicate fluid cultures with Bactec bottles showed sensitivities of 64% and 88%, respectively (P = 0.009), with specificities of 98% and 87% (P = 0.032), respectively. Sonicate fluid cultures with Bactec bottles were more sensitive than synovial fluid cultures with Bactec bottles regardless of whether antimicrobial agents were used within 14 days before surgery (81% versus 52%; P = 0.031) or not (93% versus 72%; P = 0.031). Sonication of explanted prostheses followed by incubation of the resulting sonicate fluid in Bactec bottles detected many more pathogens than did synovial fluid cultures with Bactec bottles. This method is also effective in cases with antibiotic treatment before surgery.     

Microbiologic Diagnosis of Prosthetic Shoulder Infection by Use of Implant Sonication

We recently described a sonication technique for the diagnosis of prosthetic knee and hip infections. We compared periprosthetic tissue culture to implant sonication followed by sonicate fluid culture for the diagnosis of prosthetic shoulder infection. One hundred thirty-six patients undergoing arthroplasty revision or resection were studied; 33 had definite prosthetic shoulder infections and 2 had probable prosthetic shoulder infections. Sonicate fluid culture was more sensitive than periprosthetic tissue culture for the detection of definite prosthetic shoulder infection (66.7 and 54.5%, respectively; P = 0.046). The specificities were similar (98.0% and 95.1%, respectively; P = 0.26). Propionibacterium acnes was the commonest species detected among culture-positive definite prosthetic shoulder infection cases by periprosthetic tissue culture (38.9%) and sonicate fluid culture (40.9%). All subjects from whom P. acnes was isolated from sonicate fluid were male. We conclude that sonicate fluid culture is useful for the diagnosis of prosthetic shoulder infection.The frequency of shoulder replacement surgery is increasing (1). The incidence of prosthetic shoulder infection varies from 0.4 to 15.4% (6, 7). When an infection is present, the infection requires unique medical and surgical management, rendering an accurate diagnosis critical. However, since patients with prosthetic shoulder infection often present with stiffness and/or pain alone (7), the achievement of an accurate diagnosis is challenging.Periprosthetic tissue has been the specimen cultured for the microbiologic diagnosis of prosthetic shoulder infection. Specificity is an issue, as microorganisms (e.g., Propionibacterium and Staphylococcus spp.) can be contaminants, and the number of microorganisms in tissue is small. As a result, it has been suggested that multiple samples be obtained; for prosthetic hips and knees, it is recommended that five or six periprosthetic tissue specimens be cultured (2). No such data are available for shoulder implants.We recently clinically validated a sonication technique that is used to sample biofilm bacteria on the surface of removed hip and knee implants placed in solid containers. We demonstrated that the culture of samples obtained by sonication of the implant was more sensitive than the culture of periprosthetic tissue for the diagnosis of prosthetic hip and knee infections (22). The poor sensitivity of the latter likely relates to the presence of bacteria in biofilms on the prosthesis surface, a site not well sampled when periprosthetic tissue samples for culture are obtained. No data on the accuracy of sonication for the diagnosis of prosthetic shoulder infection are available.The proportion of patients with shoulder infections due to Propionibacterium acnes is significantly greater than the proportion of patients with lower limb infections due to P. acnes (12). Sperling et al. reported that Propionibacterium spp. account for 16% of prosthetic shoulder infections (16). Franta et al. reported that among 31/282 patients (11%) with unsatisfactory shoulder arthroplasties, positive intraoperative cultures were found in 23 at the time of revision surgery, with the most common organisms isolated being coagulase-negative Staphylococcus spp., followed by P. acnes (11). Cheung et al. reported the results of reimplantation of glenoid components following removal and allogeneic bone grafting in seven patients; specimens from two patients demonstrated the growth of P. acnes (5). These two patients had continuing pain and radiographic evidence of glenoid component loosening and subsequently underwent repeat revision surgery, whereas the remaining patients did well and did not require repeat revision surgery (5), suggesting a role for P. acnes in pain and component loosening. Accordingly, the accurate detection of a Propionibacterium spp. is paramount in the diagnosis of prosthetic shoulder infection.The purpose of the present study was to compare implant sonication to periprosthetic tissue culture for the diagnosis of prosthetic shoulder infection. We also evaluated immunofluorescence microscopy and PCR analysis of sonicate fluid to detect prosthetic shoulder infection caused by the two most frequently associated microorganisms. Finally, we compared patient characteristics associated with Propionibacterium prosthetic shoulder infection versus those associated with non-Propionibacterium prosthetic shoulder infection.(This work was presented in part at the 16th Annual European Congress of Clinical Microbiology and Infectious Diseases, April 2006, Nice, France, and the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, September 2007, Chicago, IL.)     

Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection

We previously showed that culture of samples obtained by prosthesis vortexing and sonication was more sensitive than tissue culture for prosthetic joint infection (PJI) diagnosis. Despite improved sensitivity, culture-negative cases remained; furthermore, culture has a long turnaround time. We designed a genus-/group-specific rapid PCR assay panel targeting PJI bacteria and applied it to samples obtained by vortexing and sonicating explanted hip and knee prostheses, and we compared the results to those with sonicate fluid and periprosthetic tissue culture obtained at revision or resection arthroplasty. We studied 434 subjects with knee (n = 272) or hip (n = 162) prostheses; using a standardized definition, 144 had PJI. Sensitivities of tissue culture, of sonicate fluid culture, and of PCR were 70.1, 72.9, and 77.1%, respectively. Specificities were 97.9, 98.3, and 97.9%, respectively. Sonicate fluid PCR was more sensitive than tissue culture (P = 0.04). PCR of prosthesis sonication samples is more sensitive than tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection and provides same-day PJI diagnosis with definition of microbiology. The high assay specificity suggests that typical PJI bacteria may not cause aseptic implant failure.     

Laboratory Diagnosis of Prosthetic Joint Infection, Part I

Prosthetic joint infection (PJI), although a rare complication of primary or revision arthroplasty, is reported more frequently as the number patients undergoing arthroplasty increases. Accurate diagnosis of PJI is essential for adequate management and outcome. Although multiple tests have been applied, in some cases, differentiation of PJI from aseptic loosening of the prosthesis remains a challenge. Here, we review the current diagnostic laboratory modalities used for the diagnosis PJI. In Part I of this two-part article, components of the preoperative evaluation of the patient and the histology of the intraoperative evaluation is discussed.     

Laboratory Diagnosis of Prosthetic Joint Infection, Part II

Prosthetic joint infection (PJI), although a rare complication of primary or revision arthroplasty, is reported more frequently as the number patients undergoing arthroplasty increases. Accurate diagnosis of PJI is essential for adequate management and outcome. Although multiple tests have been applied, in some cases, differentiation of PJI from aseptic loosening of the prosthesis remains a challenge. Here, we review the current diagnostic laboratory modalities used for the diagnosis PJI. In Part I of this two-part article, components of the preoperative evaluation of the patient and the histology of the intraoperative evaluation were discussed. Part II of the article discusses the remaining components of the intraoperative evaluation, including periprosthetic tissue and sonicate fluid cultures. In addition, recent investigational approaches for the diagnosis of PJI, antimicrobial susceptibility testing, and management of PJIs are reviewed.     

Multiplex Antibody Detection for Noninvasive Genus-Level Diagnosis of Prosthetic Joint Infection

We developed and evaluated a multiplex antibody detection-based immunoassay for the diagnosis of prosthetic joint infections (PJIs). Sixteen protein antigens from three Staphylococcus species (Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis) (8 antigens), Streptococcus agalactiae (4 antigens), and Propionibacterium acnes (4 antigens) were selected by comparative immunoproteomics using serum samples from PJI cases versus controls. A bead-based multiplex immunoassay that measured serum IgG against purified, recombinant forms of each of the 16 antigens was developed. We conducted a prospective study to evaluate the performance of the assay. A PJI was defined by the presence of a sinus tract and/or positive intraoperative sample cultures (at least one sample yielding a virulent organism or at least two samples yielding the same organism). A total of 455 consecutive patients undergoing revision or resection arthroplasty (hip, 66.3%; knee, 29.7%; shoulder, 4%) at two French reference centers for the management of PJI were included: 176 patients (38.7%) were infected and 279 (61.3%) were not. About 60% of the infections involved at least one of the species targeted by the assay. The sensitivity/specificity values were 72.3%/80.7% for targeted staphylococci, 75%/92.6% for S. agalactiae, and 38.5%/84.8% for P. acnes. The assay was more sensitive for infections occurring >3 months after arthroplasty and for patients with an elevated C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). However, it detected 64.3% and 58.3% of targeted staphylococcal infections associated with normal CRP and ESR values, respectively. This new multiplex immunoassay approach is a novel noninvasive tool to evaluate patients suspected of having PJIs and provides information complementary to that from inflammatory marker values.     

核医学影像鉴别诊断人工关节置换术后感染与松动

临床人工关节置换术后,若处理不当,有较高的感染和假体松动风险,两者特点不一,治疗方案也存有较大差异,鉴别诊断中,核医学影像作用明确,假体松动在18FDG-PET显像中以局部骨-假体界面对放射性摄取的增加为表现,表明反应性炎症可诱导炎性肉芽肿形成,引发假体、骨界面松动;感染以骨-假体放射性弥漫性聚集及对软组织产生侵犯影响。相较其它检查手段诊断,18FDG-PET显像正确率居更高水平,可有效的鉴别诊断方法。     

Diagnosis of Prosthetic Joint Infection by Use of PCR-Electrospray Ionization Mass Spectrometry

We compared PCR-electrospray ionization mass spectrometry (PCR-ESI/MS) to culture using sonicate fluid from 431 subjects with explanted knee (n = 270) or hip (n = 161) prostheses. Of these, 152 and 279 subjects had prosthetic joint infection (PJI) and aseptic failure, respectively. The sensitivities for detecting PJI were 77.6% for PCR-ESI/MS and 69.7% for culture (P = 0.0105). The specificities were 93.5 and 99.3%, respectively (P = 0.0002).     

Improved Diagnosis of Orthopedic Implant-Associated Infection by Inoculation of Sonication Fluid into Blood Culture Bottles

Sonication improved the diagnosis of orthopedic implant-associated infections (OIAI). We investigated the diagnostic performance of sonication fluid inoculated into blood culture bottles in comparison with that of intraoperative tissue and sonication fluid cultures. Consecutive patients with removed orthopedic hardware were prospectively included and classified as having OIAI or aseptic failure (AF) according to standardized criteria. The diagnostic procedure included the collection of five intraoperative tissue cultures and sonication of the removed device, followed by conventional culture of the sonication fluid. Cultures were incubated for 7 days (aerobic) or 14 days (anaerobic). In addition, 10 ml of sonication fluid was inoculated into each aerobic and anaerobic BacT/Alert FAN blood culture bottle and incubated in the automated blood culture system for 5 days. Of 75 included patients, 39 had OIAI and 36 AF. The sensitivity of sonication fluid inoculated into blood culture bottles (100%) was higher than that of conventional sonication fluid (87%; P = 0.05) or intraoperative tissue cultures (59%; P < 0.01). Previous antibiotic therapy reduced the culture sensitivity of conventional sonication fluid to 77% and that of intraoperative tissue to 55%, while it remained 100% for blood culture-inoculated sonication fluid. The time to positivity was shorter in blood culture-inoculated sonication fluid, with detection of 72% of microorganisms after 1 day of incubation, than for intraoperative tissue and conventional sonication fluid cultures, with detection of 18% and 28% of microorganisms, respectively. In conclusion, compared to conventional sonication fluid and intraoperative tissue cultures, sonication fluid inoculated into blood culture bottles improved the diagnosis of OIAI and considerably reduced the time to culture positivity.     

Sonication of Antibiotic Loaded Cement Spacers: A Valuable Technique for Detection of Infection Persistence in Two-stage Revision for Infected Joint Arthroplasty

We evaluated the diagnostic utility of sonication of antibiotic loaded cement spacers comparing with periprosthetic tissue cultures for the detection of persisting infection in 14 patients undergoing staged procedures. Sonication improved microbial detection of intraoperative cultures from 14.2% to 28.5% (P = 0.481). Routine sonication of spacers is recommended.     

Concentration of Sonication Fluid through Centrifugation Is Superior to Membrane Filtration for Microbial Diagnosis of Orthopedic Implant-Associated Infection

Microbial identification of orthopedic implant-associated infections using sonication fluid (SF) submitted to a concentration step by membrane filtration (SMF) was compared with the standard centrifugation (SC) method. Among 33 retrieved infected implants, sonication identified microorganisms in 26 (78.8%). The sensitivity of SC was higher than that of SMF (78.8% versus 30.3%; P < 0.001).     

Preoperative Aspiration Culture for Preoperative Diagnosis of Infection in Total Hip or Knee Arthroplasty

This meta-analysis evaluated preoperative aspiration culture for diagnosing prosthetic joint infection (PJI) in total hip arthroplasty (THA) and total knee arthroplasty (TKA). The pooled sensitivity and specificity were 0.72 (95% confidence interval, 0.65 to 0.78) and 0.95 (0.93 to 0.97), respectively. Subgroup analyses revealed nonsignificant worse diagnostic performance for THA than for TKA (sensitivity, 0.70 versus 0.78; specificity, 0.94 versus 0.96). Preoperative aspiration culture has moderate to high sensitivity and very high specificity for diagnosing PJI.     

Detection of Prosthetic Joint Infection by Use of PCR-Electrospray Ionization Mass Spectrometry Applied to Synovial Fluid

PCR coupled with electrospray ionization mass spectrometry applied to synovial fluid specimens had an 81% sensitivity and a 95% specificity for the diagnosis of prosthetic joint infection.     

In Vitro Effect of Ultrasound on Bacteria and Suggested Protocol for Sonication and Diagnosis of Prosthetic Infections

Sonication of implants has been shown to be a promising method for diagnosis of prosthetic infections due to its improved sensitivity, simplicity, and low cost. The aim of the present study was to evaluate the effects of ultrasound performed under different conditions regarding temperature, duration, and composition of sonication tubes on bacterial species often associated with prosthetic infections. We found that ultrasound had an inhibitory effect on bacteria, of which gram-negative bacteria, in particular Escherichia coli, were almost eradicated after 5 min of sonication at 35°C. Gram-positive bacteria were found to be resistant to the effect of ultrasound. Four factors were important for the inhibitory effect of sonication: the type of microorganism, the temperature of the sonication buffer, the duration of exposure to ultrasound (minutes), and the material and composition of the sonication tube in which sonication is performed. On the basis of the results from the present study, we propose a protocol for sonication and recovery of bacteria associated with biofilm on infected implants prior to conventional culture. From the present protocol, we recommend sonication for 7 min at 22°C at the maximum effect which permits survival of gram-negative bacteria.In the United States, 250,000 hip and 400,000 knee replacements are performed annually, and the numbers are expected to double within the next 2 decades due to an increasing number of elderly persons (9). Prosthetic joint infection is second to aseptic failure as a cause of joint replacement and is associated with a substantial morbidity rate and cost (20). The levels of risk for infection within 2 years after insertion are less than 1% and 2% for hip and knee implants, respectively, compared with 5 to 40% after revision surgery (20). The main sources of infection are contamination from the skin flora of patients or hospital staff (12).Diagnosis of prosthetic infections is still a challenge, as clinical signs and a laboratory investigation, including microbiological findings, do not always distinguish aseptic loosening from loosening due to infection (6, 8, 27). Microbiological investigation of implants is complicated due to the sizes of the prosthetic components and problems associated with conventional culture, i.e., presence of intracellular bacteria, microorganisms in biofilm, and small-colony variants of bacteria (1, 5, 9, 14), the last of which may be induced after treatment with gentamicin (26).Due to its simplicity and low cost, sonication appears to be the most promising method among the newer techniques for diagnosis of infected implants (2, 4, 10, 15, 17, 19, 21, 23). Several studies have shown that sonication of implants improves sensitivity compared with that obtained with periprosthetic-tissue culture (2, 8, 19, 24).In medicine, ultrasound transducers are used for diagnosis of abnormalities and fetal, abdominal, and heart diseases. Ultrasound has been applied in microbiology for sonication and inactivation of bacteria in food processing (13). However, the effect of ultrasound has mainly been studied for food- and water-associated bacteria, and data are lacking for clinically important species, such as staphylococci, enterococci, Haemophilus influenzae, and Pseudomonas aeruginosa (3, 13).The aim of the present study was to evaluate the effects of temperature, duration, composition of the sonication buffer, and material in the sonication tube during sonication of bacteria often associated with prosthetic infections. On the basis of the results from these experiments, we propose a protocol for sonication of biofilm on extracted implants prior to conventional culture.