Potential value of a rapid syndromic multiplex PCR for the diagnosis of native and prosthetic joint infections: a real-world evidence study.

Introduction: The BIOFIRE Joint Infection (JI) Panel is a diagnostic tool that uses multiplex-PCR testing to detect microorganisms in synovial fluid specimens from patients suspected of having septic arthritis (SA) on native joints or prosthetic joint infections (PJIs). Methods: A study was conducted across 34 clinical sites in 19 European and Middle Eastern countries from March 2021 to June 2022 to assess the effectiveness of the BIOFIRE JI Panel. Results: A total of 1527 samples were collected from patients suspected of SA or PJI, with an overall agreement of 88.4 % and 85 % respectively between the JI Panel and synovial fluid cultures (SFCs). The JI Panel detected more positive samples and microorganisms than SFC, with a notable difference on Staphylococcus aureus, Streptococcus species, Enterococcus faecalis, Kingella kingae, Neisseria gonorrhoeae, and anaerobic bacteria. The study found that the BIOFIRE JI Panel has a high utility in the real-world clinical setting for suspected SA and PJI, providing diagnostic results in approximately 1 h. The user experience was positive, implying a potential benefit of rapidity of results' turnover in optimising patient management strategies. Conclusion: The study suggests that the BIOFIRE JI Panel could potentially optimise patient management and antimicrobial therapy, thus highlighting its importance in the clinical setting.

Outcomes in orthopedic device infections due to Streptococcus agalactiae: a retrospective cohort study.

BACKGROUND: Group B streptococci (Streptococcus agalactiae) (GBS) is a rare cause of prosthetic joint infection (PJI) occurring in patients with comorbidities and seems to be associated with a poor outcome. Depiction of GBS PJI is scarce in the literature. METHODS: A retrospective survey in 2 referral centers for bone joint infections was done Patients with a history of PJI associated with GBS between 2014 and 2019 were included. A descriptive analysis of treatment failure was done. Risk factors of treatment failure were assessed. RESULTS: We included 61 patients. Among them, 41 had monomicrobial (67%) infections. The median duration of follow-up was 2 years (interquartile range 2.35) Hypertension, obesity, and diabetes mellitus were the most reported comorbidities (49%, 50%, and 36% respectively). Death was observed in 6 individuals (10%) during the initial management. The rate of success was 63% (26/41). Removal of the material was not associated with remission (p = 0.5). We did not find a specific antibiotic regimen associated with a better outcome. CONCLUSION: The results show that S. agalactiae PJIs are associated with high rates of comorbidities and a high treatment failure rate with no optimal treatment so far.

A prospective multicentre evaluation of BioFire® Joint Infection Panel for the rapid microbiological documentation of acute arthritis.

OBJECTIVES: To assess the performance of the rapid syndromic BioFire® Joint Infection Panel (BF-JIP) to detect bacterial and fungal pathogens, as well as antibiotic resistance genes, directly in synovial fluid specimens collected from patients with acute arthritis. METHODS: The study was conducted in six French bacteriological laboratories. To assess the performances of BF-JIP, results were compared with those of synovial fluid 14-day culture and, in case of discrepancy, with those of complementary molecular methods and intraoperative samples. A total of 308 synovial fluid specimens were tested after collection from 308 adults and children presenting with clinical and biological suspicion of acute arthritis; patients presenting with acute periprosthetic joint infection were included according to the European Bone and Joint Infection Society 2021 criteria. RESULTS: Only one specimen failed (no result). On the basis of the consolidated data, the BF-JIP was concordant with the 14-day culture in 280 (91.2%) of the 307 specimens finally included in the study. The positive percentage agreement was 84.9% (95% CI, 78.8-89.8%) and the negative percentage agreement was 100% (95% CI, 97.2-100%). The positive predictive value was extremely high (100%; 95% CI, 97.6-100%), whereas the negative predictive value was lower (82.6%; 95% CI, 75.7-88.2%), partially explained by the missing target species in the panel. DISCUSSION: The BF-JIP showed high performances to detect pathogens involved in acute arthritis.

Accuracy of the GeneXpert® MRSA/SA SSTI test to diagnose methicillin-resistant Staphylococcus spp. infection in bone fixation and fusion and management of infected non-unions.

INTRODUCTION: The GeneXpert® MRSA/SA SSTI (Methicillin Resistant Staphylococcus aureus / S. aureus skin and soft tissue infection) PCR test allows early detection of methicillin resistance in staphylococci. This test was developed for skin infections and has been evaluated for prosthetic joint infections but, to our knowledge, has not been evaluated for hardware infections outside of arthroplasties. Furthermore, we conducted a retrospective study in patients with non-prosthetic osteosynthesis hardware aiming: 1) to identify the diagnostic values of the PCR test compared to conventional cultures and the resulting rate of appropriate antibiotic therapy. 2) to identify the rate of false negative (FN) results, 3) to identify and compare the rates of failure of infectious treatment (FN versus others) 4) to search for risk factors for FN of the PCR test. HYPOTHESIS: The PCR test allowed early and appropriate targeting of antibiotic therapy. MATERIAL AND METHODS: The results of PCR tests and conventional cultures for osteoarticular infections of non-prosthetic hardware over four years (2012-2016) were compared to identify the diagnostic values of using the results of conventional culture as a reference and the rate of appropriate antibiotic therapies. Infectious management failures between the results of the FN group and the others were compared, and variables associated with a FN of the PCR test were identified. RESULTS: The analysis of 419 PCR tests allowed us to establish a sensitivity of 42.86%, a specificity of 96.82%, a positive predictive value of 60% and a negative predictive value of 93.83%. Using the results of the PCR test for the targeting of postoperative antibiotic therapy, it was suitable for staphylococcal coverage in 90.94% (381/419). The rates of patients for whom infectious treatment failed were not significantly different between the FN group and the other patients (20.8% versus 17.7%, respectively; Hazard Ratio = 1.12 (95%CI 0.47-2.69, p = 0.79)). A skin opening during the initial trauma (p = 0.005) and a polymicrobial infection were significantly associated with a risk of FN from the PCR test (p < 0.001). CONCLUSION: The PCR test makes it possible to reduce the duration of empirical broad-spectrum antibiotic therapy during the treatment of an infection of osteosynthesis hardware but causes a lack of antibiotic coverage in 9.06% of cases. LEVEL OF EVIDENCE: III; Diagnostic case control study.

Relationship between COVID-19 and ICU-Acquired Bloodstream Infections Related to Multidrug-Resistant Bacteria.

A bloodstream infection (BSI) is a severe ICU-acquired infection. A growing proportion is caused by multidrug-resistant bacteria (MDRB). COVID-19 was reported to be associated with a high rate of secondary infections. However, there is a lack of data on the relationship between COVID-19 and the incidence of MDRB ICU-acquired BSI. The aim of this study was to evaluate the relationship between COVID-19 and ICU-acquired BSI related to MDRB. This retrospective study was conducted in a single-center ICU during a one-year period. All adult patients admitted for more than 48 h were included. The cumulative incidence of ICU-acquired BSI related to MDRB was estimated using the Kalbfleisch and Prentice method. The association of COVID-19 status with the risk of ICU-acquired BSI related to MDRB was assessed using cause-specific Cox's proportional hazard model. Among the 1320 patients included in the analysis, 497 (37.65%) had COVID-19. ICU-acquired BSI related to MDRB occurred in 50 patients (36 COVID patients (7%) and 14 non-COVID patients (1.6%)). Extended-spectrum beta-lactamase Enterobacteriacae (46%) and carbapenem-resistant Acinetobacter baumannii (30%) were the most commonly isolated MDRB. COVID-19 was significantly associated with a higher risk of MDRB ICU-acquired BSI (adjusted cHR 2.65 (1.25 to 5.59) for the whole study period). However, this relationship was only significant for the period starting at day 15 after ICU admission. ICU-acquired BSI related to MDRB was significantly associated with ICU mortality (HR (95%CI) 1.73 (1-3)), although COVID-19 had no significant impact on this association (p het 0.94). COVID-19 is significantly associated with an increased risk of ICU-acquired BSI related to MDRB, mainly during the period starting at day 15 after ICU admission.

An unusual case of native aortic endocarditis due to Corynebacterium pseudodiphtheriticum.

Corynebacterium pseudodiphtheriticum, a Gram-positive rod belonging the oropharynx microbiota, is usually described in pulmonary infections, especially in immunocompromised patients. This paper describes a rare case of native aortic infectious endocarditis (IE) and reviews the literature on similar cases. A 62-year-old man with rheumatic fever since childhood was hospitalized for surgical treatment of a febrile IE due to C. diphtheriticum with a large vegetation (15.8 X 8.3 mm). MALDI-TOF-MS from strain isolated in positive blood cultures identified C. pseudodiphtheriticum (2.34), and 16S rRNA sequencing from the valve sample confirmed the identification. The summary of 25 cases shows that the outcome of an IE due to C. pseudodiphtheriticum is bad. The review of the literature shows that this agent found in blood cultures in a cardiovascular context deserves to be explored meticulously because an unfavorable prognosis is frequent.

High Frequency of Specific Polysaccharide Antibody Deficiency in Adults With Unexplained, Recurrent and/or Severe Infections With Encapsulated Bacteria.

BACKGROUND: Primary immunodeficiencies (PIDs) in adults are mainly revealed by recurrent and/or severe bacterial infections. The objective of this study was to evaluate a systematic research strategy of PIDs in adults with unexplained bacterial infections, with a special focus on specific polysaccharide antibody deficiency (SPAD). METHODS: In this prospective multicenter study, inclusion criteria were recurrent benign upper and lower respiratory tract infections (RTIs) for at least two years (group 1), at least one upper or lower RTI requiring hospitalization (group 2), and/or at least one invasive infection documented with encapsulated bacteria (group 3). Main exclusion criteria were all local and general conditions that could explain infections. If no PID diagnosis was made, response to polysaccharide antigens was assessed using a pneumococcal polysaccharide vaccine. RESULTS: From March 2015 to March 2020, 118 patients were included (37 males, median age of 41 years): 73, 17, and 28 in groups 1, 2, and 3, respectively. Forty-seven PIDs were diagnosed, giving an estimated frequency of 39.8% (95% confidence interval [CI] [30.4, 48.8]). SPAD was the most frequent diagnosis by far (n = 37/47, 78.7%), and was made in 23, 5, and 9 patients from groups 1 to 3, respectively. All SPAD patients received conjugate vaccines and, according to their infectious history, were on surveillance or treated with preventive antibiotics (n = 6) and/or with immunoglobulins replacement therapy (n = 10), the latter being dramatically efficient in all cases. CONCLUSIONS: Considering its high prevalence among adults with unexplained recurrent and/or severe bacterial infections, SPAD should be screened in those patients. CLINICAL TRIALS REGISTRATION: NCT02972281.

Evolution of antibiotic susceptibility profiles of staphylococci from osteoarticular infections: A 10-year retrospective study.

BACKGROUND: Knowledge of the antibiotic susceptibility profiles of the bacteria responsible for osteoarticular infections is crucial for choosing the appropriate empirical antibiotic regimen. Wide use of broad spectrum antibiotics in these infections may have lead to selection of resistant bacteria. The aim of our study was to answer to these questions: (1) Did the bacterial pathogens isolated from osteoarticular infections (OAIs) and their antibiotic susceptibility profile change over the 10-year period in our University Hospital, particularly for Staphylococcus aureus and Coagulase negative staphylococci? (2) Are the antibiotics used for post-operative antibiotic therapy still effective against staphylococci involved in OAIs? (3) Are the antibiotics used for documented therapy still effective against staphylococci involved in OAIs? HYPOTHESIS: We hypothetise that bacterial epidemiology and antibiotic resistance rates have changed little thanks to a reasoned prescription of antibiotics in our Center. MATERIALS AND METHODS: We performed a retrospective study describing the antibiotic susceptibility profile of bacteria isolated from osteoarticular infections over 10years in our University Hospital, with a focus on the Staphylococcus genus. RESULTS: A total of 3474 staphylococci were included (2373 coagulase negative staphylococci and 1101 S. aureus), 34.8% (1207/3469) of which were resistant to methicillin. Antibiotic susceptibility profiles remained quite stable between 2010 and 2019, except for rifampicin (14.1% (45/318) versus 5.7% (23/401), p=0.0001) and fluoroquinolones (35.3% (109/309) versus 20.3% (81/399), p=0.000008) for which resistance rates significantly decreased even among methicillin-resistant strains. DISCUSSION: In spite of wide use of antibiotics in orthopaedic units, overall resistance rates did not increase over the last 10years. The prescription of these molecules in combination regimens guided by the antibiotic susceptibility patterns performed on reliable samples and on the basis of multidisciplinary discussions may explain these results. LEVEL OF EVIDENCE: IV, retrospective study.

Apyretic pulmonary oedema revealing Cardiobacterium hominis endocarditis: Case report and review of literature.

Cardiobacterium hominis is a member of the HACEK group of bacteria, responsible for infective endocarditis, mainly in patients with damaged or prosthetic valves. The low virulence of this organism can explain the insidious presentation and subacute or chronic progression of C. hominis infective endocarditis. Here, a 41-year-old man with a past history of surgery for a Waldhausen type aortic coarctation was hospitalised with dyspnea and chest pains revealing an acute pulmonary oedema, without fever. Transesophageal echocardiography indicated a 20 mm vegetation on biscuspid aortic valve. Six sets of blood culture were positive with Cardiobacterium hominis. In case of lack of fever, the diagnosis of infectious endocarditis is difficult because other symptoms are non-specific and biological markers of inflammatory syndrome are quiet or non-existent. This is the first case of C. hominis infectious endocarditis with a clinical presentation of acute pulmonary oedema in the literature. We report here an apyretic pulmonary oedema revealing C. hominis endocarditis and a review of the literature on apyretic infective endocarditis due to C. hominis.

Short communication: dog bite and back pain.

Capnocytophaga canimorsus is a Gram-negative rod commensal of oral cavity of dogs and cats. It can cause sepsis, septic shock, endocarditis, and meningitis associated with bites or licking wounds, especially in immunocompromised patients. Herein, we report a case of C. canimorsus spondylodiscitis linked to a dog bite in a previously healthy patient and review the literature on this pathogen.

The benefits of systematic intraoperative sampling during lower limb arthroplasties due to sequelae from prior osteoarticular infections: A retrospective study of 92 cases.

INTRODUCTION: Osteoarticular infections (OAIs) of native joints lead to cartilage damage which may require subsequent arthroplasty. There is no consensus on systematic intraoperative microbiological sampling when performing an arthroplasty on a native joint with a history of OAI. We carried out a retrospective study to: (1) identify the frequency of the persistence of the microorganism(s) involved during the initial, presumed cured OAI, when performing an arthroplasty for sequelae of osteoarthritis, (2) to find an association between the length of time between the OAI and arthroplasty, and the recurrence of bacterial infection, (3) to assess the influence of the presence of hardware on the risk of infectious recurrence. HYPOTHESIS: Systematic sampling is justified during a subsequent arthroplasty after an OAI, even after a prolonged period. MATERIAL AND METHOD: This single-center, retrospective descriptive study included all patients whose indication for arthroplasty resulted from osteoarthritis, osteitis or bacterial osteomyelitis of a native joint, or in the aftermath of an infection post osteosynthesis. All patients were considered to have recovered from the initial infection at the time of the arthroplasty. Between 2008 and 2019, 92 patients were included in the study, with an average age of 56.5years (range: 21-97years). OAI occurred at a mean age of 35years (range: 1-84years). The average time from OAI to implantation was 15years (range: 1-65years). The bacteria most frequently found in the initial OAI was Staphylococcus aureus, involved in 35.8% of cases (n=33/92). RESULTS: The intraoperative samples came back positive in 17% of cases (n=16/92), including 9 positive for the same bacteria as the OAI (56%, n=9/16). For these 16 cases, the time between the OAI and the arthroplasty was 1year for 5 patients, between 1 and 15years for 5 patients and greater than 15years for 6 patients. For 3 positive patients, the information on the initial microorganism was not known and 4 patients were positive for a bacterium different from the initial one. The time from the initial OAI to the arthroplasty was not associated with positive results (p=0.38). There was no significant difference between a positive culture at the time of arthroplasty and the initial type of OAI [native joint versus presence of hardware and/or open fracture (p=0.41)]. CONCLUSION: The results of this work suggest there is value in microbiological sampling when performing an arthroplasty on a previously infected joint, regardless of the duration of the infection. LEVEL OF EVIDENCE: IV; retrospective study.

Evaluation of the new BL-RED (ß-Lactamase Rapid Electrochemical Detection) test in positive blood culture broths.

The rapid detection of extended-spectrum ß-lactamase Enterobacterales (ESBL-E) in a positive blood culture is important in order to initiate an appropriate antibiotic therapy and thus decrease mortality. We evaluated the new BL-RED (ß-Lactamase Rapid Electrochemical Detection) test in 100 positive blood culture broths to detect (in ten minutes) the presence or absence of ESBL-E. The BL-RED test appears to be an easy, rapid and reliable test to detect the presence of ESBL directly in Gram negative bacilli-positive blood culture broths, with good performances (sensibility =97.3%, specificity =90.5%, predictive positive value =85.7% and predictive negative value =98.3%). This test could be useful for therapeutic decisions and adjustments of sepsis empirical antibiotic therapy, particularly in wards where the ecology is unfavorable, such as in intensive care units.

Fully oral targeted antibiotic therapy for Gram-positive cocci-related periprosthetic joint infections: a real-life before and after study.

BACKGROUND: The optimal length of the intravenous antibiotic treatment of periprosthetic joint infections (PJIs) generally ranges from one to six weeks and is a matter of debate. Most antibiotics active against Gram-positive cocci (GPC) exhibit both high oral bioavailability and bone diffusion. Thus, early oral therapy may be a reasonable option in GPC-related PJIs. METHODS: A 2 year before and after monocentric study that aimed to compare two antibiotic strategies. Empirical intravenous postoperative antibiotic treatment was followed by 7 to 10 days of intravenous targeted therapy ('before' group) or by full orally targeted antibiotic treatment ('after' group). The primary outcome was a treatment failure during follow-up. RESULTS: A total of 93 patients were analysed, 43 and 50 in the before and the after groups, respectively. Both groups were comparable in terms of surgical procedures, comorbidities, microbiological documentation and infection site. Antibiotics prescribed to our patients had high oral bioavailability and bone diffusion with rifampicin/fluoroquinolone combinations being the most frequent antibiotic regimens. Both hospital stay and intravenous antibiotic treatment mean durations were shorter in the before group than in the after group [15.0 versus 11.0 days; (P < 0.01) and 13.0 versus 7.0 days; P < 0.001, respectively]. The remission rate assessed after at least a year of follow-up was comparable in the before and the after groups (hazard ratio = 0.70; 95% CI 0.30-1.58). CONCLUSIONS: Full oral targeted antibiotic therapy using a drug regimen with high oral bioavailability and good bone diffusion is an option for the treatment of patients with GPC-related PJIs.

Challenging Methicillin Resistance Detection in Bone and Joint Infections: Focus on the MRSA/SA SSTI® Strategy.

The genus Staphylococcus is the main causative agent of bone and joint infections (BJI) in which outcomes are impacted by both effective surgical and appropriate antimicrobial management. In this context, methicillin resistance (MR) detection is a microbiological challenge to optimize the anti-staphylococcal drug coverage and to secure the surgical procedure. During the last decade, molecular tools have been developed to rapidly detect bacterial-resistant strains in clinical samples. The GeneXpert MRSA/SA SSTI® assay (Cepheid, Sunnyvale, CA, USA) is a real-time PCR method aimed at detecting methicillin-resistant Staphylococcus aureus (MRSA) in skin and soft tissues infections. In the literature, this test has been reported to be diverted from its original purpose to be evaluated in surgical samples. Within the current review, we update the GeneXpert MRSA/SA SSTI® assay performance in staphylococcal species determination (i.e., S. aureus vs. coagulase-negative species) together with MR genotype detection, when performed in osteoarticular infections.

Does the alpha-defensin lateral flow test conserve its diagnostic properties in a larger population of chronic complex periprosthetic infections? Enlargement to 112 tests, from 42 tests in a preliminary study, in a reference center.

BACKGROUND: Diagnosis of periprosthetic infection (PPI) is crucial for management of bone and joint infection. The preoperative gold-standard is joint aspiration, providing results after 2-14 days' culture, with non-negligible false negative rates due to the fragility of certain micro-organisms and/or prior antibiotic treatment. The Synovasure™ alpha-defensin lateral flow test (Zimmer, Warsaw, IN, USA) contributes within minutes to joint fluid diagnosis of almost all infectious agents, including in case of concomitant antibiotic therapy. Validity remains controversial, notably in complex microbiological situations: multi-operated patients, diagnostic doubt despite iterative sterile culture, long-course antibiotic therapy. We extended a prospective study reported in 2018, to determine whether the test maintained diagnostic value in a larger population, assessing 1) negative (NPV) and positive (PPV) predictive value, and 2) sensitivity and specificity. HYPOTHESIS: Synovasure™ maintains NPV above 95% in a broader population of microbiologically complex suspected PPI. MATERIAL AND METHODS: Synovasure™'s performance was assessed between October 2015 and October 2019 in 106 patients (112 tests) in complex diagnostic situations: 37 discordant cultures (discordant findings between 2 samples), 65 cases with clinically or biologically suspected infection but iterative sterile culture, 10 emergencies (requiring surgery, precluding antibiotic window, or mechanical failure in suspected infection), including 5 with ongoing antibiotic therapy for infection in another organ. Six tests were repeated in the same patient and same joint at >6 months' interval for strong clinical suspicion of infection. The main endpoint was the MSIS score (MusculoSkeletal Infection Society, 2018). RESULTS: NPV was 98.8%, PPV 72.4%, sensitivity 95.5% and specificity 91%. Prevalence of infection was 19.6%. Only 1 of the 22 infected patients had negative Synovasure™ tests, compared to 81 of the 84 non-infected patients. CONCLUSION: Synovasure™ is a reliable novel diagnostic test, contributing mainly to ruling out infection thanks to its strong NPV. The cost imposes sparing use, but medico-economic assessment would be worthwhile. LEVEL OF EVIDENCE: III; prospective of diagnostic performance.

Diagnostic accuracy of the BJI InoPlex™ (Diaxonhit) immunoassay on blood samples for periprosthetic joint infection in complex microbiological situations. Preliminary results of 24 cases in a French Reference Center for Complex Bone and Joint Infection (CRIOAC).

BACKGROUND: While joint aspiration is the benchmark for diagnosing periprosthetic joint infections (PJI), the results can be flawed because certain bacteria are difficult to culture, the patient is on concurrent antibiotic therapy or in some cases, repeated joint aspirations confer conflicting results. The BJI InoPlex™ (Diaxonhit) is a multiplex ELISA (Enzyme Linked Immunosorbent Assay) that measures the immune response (presence of specific IgG) to certain bacterial species from three families: Staphylococcus (8 antigens) epidermidis, aureus and lugdunensis, Streptococcus B (4 antigens) and Cutibacterium acnes (4 antigens). This assay is done with peripherally collected blood. However, there are few published studies about this assay, especially if the microbiological diagnosis is in doubt in cases of suspected chronic PJI. This led us to conduct a retrospective study in a French tertiary care center to determine 1) the sensitivity and specificity of the BJI InoPlex™, 2) its positive (PPV) and negative predictive value (NPV) and 3) what causes diagnostic errors. HYPOTHESIS: The BJI InoPlex has a sensitivity/specificity and PPV/NPV above 75%. MATERIALS AND METHODS: The BJI InoPlex was used 24 times on 24 patients between January 2016 and January 2017 in scenarios where the microbiological diagnosis was difficult: 1 with on-going antibiotic therapy, 13 conflicting repeat joint aspirations, 10 negative cultures with history of infection and/or clinical evidence of a PJI. The series consisted of 11 hip arthroplasty and 13 knee arthroplasty cases. The results of the BJI InoPlex test were compared to the MusculoSkeletal Infection Society (MSIS) the criteria for a joint infection. RESULTS: For the bacterial species covered by the test, the sensitivity of the BJI InoPlex for diagnosing a chronic PJI based on the 2018 MSIS criteria was 50%, the specificity was 56%, the PPV was 36% and the NPV was 69%. DISCUSSION: While innovative, minimally invasive, and rapid (results in a few hours), the BJI InoPlex does not provide an effective diagnosis of chronic PJI in complex microbiological situations. In this study, we used the test in the most difficult situations possible and on a small number of patients, which may explain why the results were not as good as in other studies. Its current performance and cost mean there is no role for it in our algorithm for treating patients with a suspected PJI, contrary to other biomarkers. Its spectrum must include other bacterial strains involved in chronic PJI. Knowledge of the specific infectious agent increases its diagnostic value, it could be used to monitor the outcome of a PJI, although other studies would be needed to support this use. LEVEL OF EVIDENCE: IV-Retrospective diagnostic study.