Identification of prosthetic joint infections with 16S amplicon metagenomic sequencing - comparison with standard cultivation approach.

Prosthetic joint infections (PJIs) are commonly diagnosed via culture-based methods, which may miss hard-to-grow pathogens. This study contrasts amplicon metagenomic sequencing (16S AS) with traditional culture techniques for enhanced clinical decision-making. We analyzed sonicate fluid from 27 patients undergoing revision arthroplasty using both methods, emphasizing the distinction between contaminants and true positives. Our findings show moderate agreement between the two methods, with a Cohen's kappa of 0.490, varying across bacterial genera (Cohen's kappa -0.059 to 1). The sensitivity of 16S AS compared to culture was 81% (95% CI, 68% to 94%). Sequencing revealed greater microbial diversity, including anaerobic genera like Anaerococcus and Citrobacter. Interestingly, several culture-negative PJI samples showed diverse bacteria via 16S AS. Despite rigorous controls and algorithms to eliminate contaminants, confirming bacteria presence with 16S AS remains a challenge. This highlights the need for improved PJI diagnostic methods, while also pointing out the limitations of next-generation sequencing (NGS) as a clinical diagnostic tool.

Accuracy of Allplex SARS-CoV-2 assay amplification curve analysis for the detection of SARS-CoV-2 variant Alpha.

Aim: To evaluate the accuracy of two PCR-based techniques for detecting SARS-CoV-2 variant Alpha (B.1.1.7). Materials & methods: A multicenter prospective cohort with 1137 positive specimens from Slovenia was studied. A mutation-based assay (rTEST-COVID-19 qPCR B.1.1.7 assay) and amplification curve pattern analysis of the Allplex SARS-CoV-2 assay were compared with whole-genome sequencing. Results: SARS-CoV-2 variant Alpha was detected in 155 samples (13.6%). Sensitivity and specificity were 98.1 and 98.0%, respectively, for the rTEST-COVID-19 qPCR B.1.1.7 assay and 97.4 and 97.5%, respectively, for amplification curve pattern analysis. Conclusion: The good analytical performance of both methods was confirmed for the preliminary identification of SARS-CoV-2 variant Alpha. This cost-effective principle for screening SARS-CoV-2 populations is also applicable to other emerging variants and may help to conserve some whole-genome sequencing resources.

Cutibacterium acnes clonal complexes display various growth rates in blood culture vials used for diagnosing orthopedic device-related infections.

OBJECTIVES: Blood culture bottles (BCBs) are commonly used for the diagnosis of infections associated with orthopedic devices. Although Cutibacterium acnes is an important pathogen in orthopedics, relatively little is known about its growth characteristics in BCBs. This prompted us to analyze the influence of bacterial genotype and clinical significance on time-to-detection (TTD) in BCBs. METHODS: We reviewed 59 cases of orthopedic device-related infections in which at least one intraoperative specimen yielded a pure C. acnes culture from anaerobic BCBs (BD Bactec Lytic/10 Anaerobic/F; Lytic-Ana) and/or solid media. A strain was considered infectant if the same genotype was present in two or more intraoperative samples. From these cases, we isolated a total of 72 unique C. acnes strains belonging to four multilocus sequence type clonal complexes (CCs): CC18, CC28, CC36 and CC53. Growth rate and TTD in Lytic-Ana BCB were studied under experimental conditions (inoculation of standard inoculum) and in clinical samples (inoculation of periprosthetic tissue samples). RESULTS: Median TTD values were shorter for CC53 compared to other CCs under experimental conditions (69 vs. 103 h; p < 0.001) and from clinical specimens (70 vs. 200 h; p = 0.02). Infectant strains had a shorter median TTD compared to contaminant strains in a clinical situation, while the difference was not observed under experimental conditions. CONCLUSIONS: The detection dynamics of C. acnes in Lytic-Ana BCBs were associated with genotype. Thus, TTD not only reflects the bacterial load in clinical samples, but may also reflect the intrinsic properties of the clonal complex of C. acnes.

Detection of bacteria with molecular methods in prosthetic joint infection: sonication fluid better than periprosthetic tissue.

Background and purpose - The correct diagnosis of prosthetic joint infection (PJI) can be difficult because bacteria form a biofilm on the surface of the implant. The sensitivity of culture from sonication fluid is better than that from periprosthetic tissue, but no comparison studies using molecular methods on a large scale have been performed. We assessed whether periprosthetic tissue or sonication fluid should be used for molecular analysis. Patients and methods - Implant and tissue samples were retrieved from 87 patients who underwent revision operation of total knee or total hip arthroplasty. Both sample types were analyzed using broad-range (BR-) PCR targeting the 16S rRNA gene. The results were evaluated based on the definition of periprosthetic joint infection from the Workgroup of the Musculoskeletal Infection Society. Results - PJI was diagnosed in 29 patients, whereas aseptic failure was diagnosed in 58 patients. Analysis of sonication fluid using BR-PCR detected bacteria in 27 patients, whereas analysis of periprosthetic tissue by BR-PCR detected bacteria in 22 patients. In 6 of 7 patients in whom BR-PCR analysis of periprosthetic tissue was negative, low-virulence bacteria were present. The sensitivity and specificity values for periprosthetic tissue were 76% and 93%, respectively, and the sensitivity and specificity values for sonication fluid were 95% and 97%. Interpretation - Our results suggest that sonication fluid may be a more appropriate sample than periprosthetic tissue for BR-PCR analysis in patients with PJI. However, further investigation is required to improve detection of bacteria in patients with so-called aseptic failure.

Identification of the same species in at least two intra-operative samples for prosthetic joint infection diagnostics yields the best results with broad-range polymerase chain reaction.

PURPOSE: Prosthetic joint infection (PJI) is a devastating complication of total joint arthroplasty. No single laboratory test has perfect sensitivity and specificity; however, culture of periprosthetic tissue is the standard method for PJI diagnosis. Interpretation of positive culture results in PJI diagnostics can be difficult due to the possibility of contamination with microorganisms originating from skin micro flora. Criteria have been established to aid in distinguishing pathogen from contaminant for culture results. A similar criterion has not however been established for polymerase chain reaction (PCR) analysis, which is in part responsible for confusion about the reliability of PCR for PJI diagnostics. The aim of our study was to establish a criterion for interpretation of broad range (BR) PCR results in PJI diagnostics. METHODS: Samples of periprosthetic tissue were retrieved from 100 patients with joint prosthesis failure and analysed with BR-PCR. The results of BR-PCR were evaluated based on the number of samples of periprosthetic tissue with the same bacterial species. RESULTS: The sensitivity (87.5%) of BR-PCR was highest if the same species was present in at least one sample, although this criterion also resulted in the lowest specificity (92.1%). The sensitivity decreased (83.2%), although without a statistically significant difference, if the same species was present in two or more samples but, at the same time, specificity increased (100%), with a statistically significant difference. CONCLUSIONS: For diagnostics of PJI with BR-PCR the criterion of the same bacterial species in at least two specimens of periprosthetic tissue from the same patient should be used for interpretation of positive results.

Comparison of molecular and culture method in diagnosis of prosthetic joint infection.

Diagnosis of prosthetic joint infection with culture technique can be problematic since the causative agent(s) are not possible to cultivate in all cases. Molecular methods had been evaluated in many studies but their inclusion in routine diagnostics is still controversial. The purpose of our prospective study was to compare the diagnostic accuracy of broad-range (BR)-PCR and culture technique. Intraoperative samples of periprosthetic tissue were retrieved in 67 patients undergoing revision arthroplasty. Samples were analyzed with culture technique, immunohistochemistry and BR 16S rRNA gene PCR. Bacteria in PCR-positive samples were identified using two different methods: direct sequencing of PCR products and specific TaqMan assays. In 63 cases, full concordance was found between BR-PCR and culture technique. Specific TaqMan assays failed to identify bacteria in four culture- and BR-PCR-positive cases and therefore had a lower sensitivity in comparison with BR-PCR. Molecular methods detected bacteria with the same accuracy as culture; however, identification of bacteria was inferior to culture. Further development of species-recognition techniques is required to improve identification of causative microorganisms.