Naomi Kobayashi

Diagnosis of Periprosthetic Infection

Periprosthetic infections are rare, but there is evidence to suggest that their frequency may be underestimated. No single laboratory test has perfect sensitivity and specificity for diagnosing infection. Most tests have better specificity when they are performed for patients in whom infection is suspected clinically rather than when they are used as screening tests. Screening test results that may suggest the possibility of infection include elevation of the erythrocyte sedimentation rate and/or serum C-reactive protein level more than three months after an arthroplasty. Most serologic tests are difficult to interpret when the patient has an underlying inflammatory arthropathy. Cultures of aspirated joint fluid can be especially helpful for patients who have symptoms suggestive of infection, but their results are best interpreted two weeks after administration of antibiotics has been discontinued. Joint fluid cell counts may also be helpful, but Gram stains of joint fluid have poor sensitivity and specificity. Criteria for diagnosing infection on the basis of frozen sections of implant membranes have not yet been standardized, but in many laboratories more than five neutrophils per high-power field in five or more fields (excluding surface fibrin) has been found to be suggestive of infection. Most polymerase chain reactions that detect the universal 16S rRNA bacterial gene have problems with false-positive results, but combining a universal polymerase chain reaction with subsequent bacterial sequencing can help improve specificity. Polymerase chain reactions can detect necrotic bacteria, so the clinical importance of positive results of this analysis in the absence of other features of infection remains to be determined.

Molecular Identification of Bacteria from Aseptically Loose Implants

AbstractPolymerase chain reaction (PCR) assays have been used to detect bacteria adherent to failed orthopaedic implants, but some PCR assays have had problems with probable false-positive results. We used a combination of a Staphylococcus species-specific PCR and a universal PCR followed by DNA sequencing to identify bacteria on implants retrieved from 52 patients (92 implants) at revision arthroplasty. We addressed two questions in this study: (1) Is this method able to show the existence of bacterial DNA on presumed aseptic loosed implants?; and (2) What proportion of presumed aseptic or culture-negative implants was positive for bacterial DNA by PCR? Fourteen implants (15%) were believed infected, whereas 74 implants (85%) were believed aseptic. Each implant was sonicated and the resulting solution was submitted for dual real-time PCR assay and culture. All implants believed aseptically loose were culture-negative, but nine of the 74 (12%) had bacterial DNA by PCR; two (2.7%) were PCR-positive and also showed histologic findings suggestive of infection. Uniquely developed PCR and bacterial sequencing assays showed bacterial DNA on 12% of implants removed for presumed aseptic loosening. Additional studies are needed to determine the clinical importance of bacterial DNA detected by PCR but not by conventional culture. Level of Evidence: Level III, diagnostic study. See the Guidelines for Authors for a complete description of levels of evidence.

Brief ultrasonication improves detection of biofilm-formative bacteria around a metal implant.

Biofilms are complex microenvironments produced by microorganisms on surfaces. Ultrasonication disrupts biofilms and may make the microorganism or its DNA available for detection. We determined whether ultrasonication could affect our ability to detect bacteria adherent to a metal substrate. A biofilm-formative Staphylococcus aureus strain was used for an in vitro implant infection model (biofilm-formative condition). We used quantitative culture and real time-polymerase chain reaction to determine the influence of different durations of ultrasound on bacterial adherence and viability. Sonication for 1 minute increased the yield of bacteria. Sonication longer than 5 minutes led to fewer bacterial colonies by conventional culture but not by polymerase chain reaction. This suggests short periods of sonication help release bacteria from the metal substrate by disrupting the biofilm, but longer periods of sonication lyse bacteria prohibiting their detection in microbiologic cultures. A relatively short duration of sonication may be desirable for maximizing detection of biofilm-formative bacteria around implants by culture or polymerase chain reaction.

Perivascular and Diffuse Lymphocytic Inflammation are not Specific for Failed Metal-on-metal Hip Implants

BackgroundSeveral studies suggest that histologic findings from tissues obtained at revision arthroplasty for failed metal-on-metal (MOM) total hip implants may reflect an immune reaction to particles or ions in some patients. However, only a limited number of cases without MOM implants were reported as controls in those studies.Questions/purposesThe purpose of this study is to better define the extent and distribution of morphologic features attributed to an immune reaction in tissues sampled at revision surgery for failed nonMOM THA.Patients and MethodsAs part of a multicenter, prospective study, we reviewed 612 capsular and interface tissues obtained from 130 patients at revision THA. The samples were selected from periacetabular regions (154 samples from 103 patients), femoral implant/cement-bone interface (154 samples from 79 patients), and from areas of the joint capsule that had an intraoperative gross appearance suggesting the possibility of either infection or metallosis (256 samples from 129 patients). All patients had more than one sample obtained. The extent and distribution of lymphocytes and plasma cells, acute inflammation, and visible particles of debris were graded using criteria similar to those described to grade inflammation around failed MOM implants.ResultsWe identified perivascular lymphocytes in 111 biopsy samples taken from 65 (50%) of 130 patients, and in 87 specimens from 57 (53%) of 107 patients thought to have aseptic loosening. Diffusely distributed lymphocytes were identified in 86 (66%) of 130 patients, and in 66 (62%) of the 107 hips with aseptic loosening, although few had the highest grade of inflammation. Increasing extent of diffuse and perivascular lymphocytes correlated with increasing extent of metal particles.ConclusionsMild lymphocytic inflammation, diffuse and especially perivascular, is common in tissues around failed nonMOM implants. Although extensive inflammation in an inflammatory pseudotumor pattern is rare, it does occur in some cases of failed metal-polyethylene hip arthroplasties. The importance of inflammation is unknown, but the extent of diffuse inflammation shows a positive correlation with metal debris, so it could reflect a reaction to particles or ions in some patients.

Diagnosis of periprosthetic joint infection

Liaison: Benjamin Zmistowski BS Leaders: Craig Della Valle MD (US), Thomas W Bauer MD (US), Konstantinos N. Malizos MD, PhD (International) Delegates: Abbas Alavi MD, Hani Bedair MD, Robert E Booth MD, Peter Choong MD, Carl Deirmengian MD, Garth D Ehrlich PhD, Anil Gambir MD, Ronald Huang MD, Yair Kissin MD, Hideo Kobayashi MD, Naomi Kobayashi MD, Veit Krenn MD, Drago Lorenzo MD, SB Marston MD, Geert Meermans MD, Javier Perez MD, JJ Ploegmakers MD, Aaron Rosenberg MD, C Simpfendorfer MD, Peter Thomas MD, Stephan Tohtz MD, Jorge A Villafuerte MD, Peter Wahl MD, Frank-Christiaan Wagenaar MD, Eivind Witzo MD

A molecular gram stain using broad range PCR and pyrosequencing technology: a potentially useful tool for diagnosing orthopaedic infections.

The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the “molecular Gram stain” were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.

Use of F-18 Fluoride Pet to Differentiate Septic From Aseptic Loosening in Total Hip Arthroplasty Patients

Purpose: The preoperative differentiation of aseptic and septic loosening following a total hip arthroplasty (THA) remains a challenging issue for clinicians to which several molecular imaging techniques have been applied. In our current study, we used F-18 fluoride positron emission tomography (PET) to evaluate THA cases with stable, septic or septic loosened implants to assess the possibility of differentiating these clinical settings using a novel uptake-type classification approach. Materials and Methods: A total of 65 joints were enrolled in this prospective study comprising 27 asymptomatic stable hips (control group), 11 painful hips conservatively treated after THA due to a suspicion of loosening, and 27 painful hips surgically treated after THA. PET imaging was classified into 3 types according to the uptake pattern. The maximum standardized uptake value (SUVmax) was then measured for each joint. A final diagnosis was made via tissue examinations of surgically treated cases, and by serological and radiographic findings in conservatively treated cases. Results: There were significant differences found between the SUVmax values for the aseptic and septic loosening THA cases. In the diagnosis of infection with type 3 pattern, the sensitivity and specificity were measured at 0.95 and 0.98 for all cases, and 0.95 and 0.88 for surgically treated cases, respectively. Conclusions: The results of our current study demonstrate that F-18 fluoride PET has considerable potential as a method for differentiating septic from aseptic loosening following a THA. The type classification of the uptake pattern can be performed relatively simply, and quantifications using the SUVmax values can then provide an objective evaluation.

New application of 18F-fluoride PET for the detection of bone remodeling in early-stage osteoarthritis of the hip.

PurposeRecent studies have reported the acceleration of subchondral bone remodeling during the development of osteoarthritis (OA). However, it is not possible to evaluate such molecular abnormalities using conventional radiographic techniques. We have applied 18F-fluoride PET to the analysis of painful or dysplastic hips at various stages of OA and then compared this with radiographic findings and clinical findings. MethodsA consecutive series of 65 joints from 48 patients (average age, 40 years; range, 19–72 years) with a hip joint complaint or radiographic dysplastic hip were enrolled in this study. Twenty-five contralateral joints without any evidence of OA or clinical symptoms were assigned as a normal control group. Radiographic evaluations were performed on the basis of Kellgren and Lawrence grade and the minimum joint space. Clinical evaluations were performed using the grading scale for pain severity and the SUVmax was measured for each joint. The association between SUVmax and the radiographic or clinical findings was evaluated. Results18F-fluoride PET shows a significantly higher uptake value for progressive-stage OA cases than for early-stage cases and also shows a significantly higher uptake value in cases with severe pain. Even in early-OA-stage patients who do not show joint space narrowing on a plain x-ray, cases with severe pain show a significantly higher uptake value. Conclusions18F-fluoride PET has great potential as an imaging method for diagnosing early-stage OA without any marked radiographic changes.

Simultaneous intraoperative detection of methicillin-resistant Staphylococcus and pan-bacterial infection during revision surgery: use of simple DNA release by ultrasonication and real-time polymerase chain reaction.

BACKGROUND Periprosthetic infection is one of the most serious complications of arthroplasty, and low-grade infections are particularly difficult to diagnose with use of conventional culture methods. Real-time polymerase chain reaction is a potentially viable way to overcome this detection problem as it is a more rapid and sensitive technique. In the current study, we used intraoperative polymerase chain reaction identification combined with a simple DNA-release method with ultrasonication to diagnose periprosthetic infections during revision surgery. METHODS Thirty revision arthroplasty procedures were included in this prospective study. Surgical specimens were obtained intraoperatively, treated with ultrasonication, and then analyzed with real-time polymerase chain reaction. Methicillin-resistant Staphylococcus-specific polymerase chain reaction and 16S rRNA gene universal polymerase chain reaction were performed simultaneously to facilitate both specific and broad-range detection. Specimens obtained from the same sites were also analyzed with microbiologic culture and histopathological evaluation. RESULTS The specific polymerase chain reaction revealed methicillin-resistant Staphylococcus infection in specimens from six of the thirty operations analyzed in the present study, and the 16S rRNA gene universal polymerase chain reaction analysis was positive for specimens from thirteen operations. Conventional cultures revealed six methicillin-resistant Staphylococcus infections, two Staphylococcus aureus infections, one infection with another Staphylococcus species, and two Streptococcus infections. The sensitivity of the polymerase chain reaction method was 0.87 and the specificity was 0.8 when compared with the combined results of microbiologic culture and histopathological evaluation. CONCLUSIONS The ultrasonication method that we developed for accelerated DNA sample preparation as a replacement for conventional extraction made possible the potential intraoperative identification of periprosthetic infection during revision surgery. The simultaneous detection of methicillin-resistant Staphylococcus and broad-range bacterial infections would be invaluable for the informed selection of antibiotics and also for the formulation of the subsequent treatment strategy (a one-stage or two-stage revision) for the patient.

Rapid and sensitive detection of methicillin-resistant Staphylococcus periprosthetic infections using real-time polymerase chain reaction.

The aim of this study was to validate the accuracy, sensitivity, and specificity of a methicillin-resistant Staphylococcus (MRS) real-time polymerase chain reaction (PCR) assay in clinical periprosthetic infection cases. A total of 36 cases of revision arthroplasty were enrolled in this prospective study, and the primer and probe set of a methicillin-resistant Staphylococcus aureus detection kit were used for the specific detection of the MecA gene with a LightCycler system. The specimens were also tested in microbiologic cultures and histopathologic evaluations. Of the 36 cases tested, 14 were found to be PCR positive for MRS infection. Of these 14 cases, however, only 8 were also found to be MRS infected using the culture method, whereas 3 were culture negative and 3 samples showed growth of another organism. The accuracy, sensitivity, and specificity were 0.83, 1.00, and 0.79, respectively. We conclude that the use of this approach will improve the diagnosis of MRS having a direct impact in the management of cases of periprosthetic infections.