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Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_19 | Pages 73 - 73
22 Nov 2024
DEVELOPMENT AND VALIDATION OF A MULTIPLEX PCR FOR THE DETECTION OF THE MOST COMMON PATHOGENS IN PERIPROSTHETIC JOINT INFECTIONS
Erbeznik A Smrdel KS Kišek TC Cvitković-Špik V Triglav T Vodicar PM
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Aim

The aim of this study was to develop an in-house multiplex PCR real-time assay on the LightCycler 480 system (Roche, Basel, Switzerland) with the aim of rapid detection of common pathogens in prosthetic joint infections (PJI), followed by validation on clinical samples (sonication fluid and tissue biopsies) routinely collected for PJI diagnosis.

Methods

Using the PrimerQuest and CLC WorkBench tool, we designed six primer sets with specific fluorescently labelled TaqMan probes for the nuc gene in different Staphylococcus species (S. aureus, S. epidermidis, S. capitis, S. lugdunensis, S. hominis, S. haemolyticus). In addition, primers previously developed by Renz et al. (2022) for C. acnes were integrated into our assay with internal control of isolation, leading to the development of specific mPCR assay with seven included targets. Analytical sensitivity and specificity were evaluated using reference bacterial strains. To determine the assay's limit of detection (LOD), we conducted serial dilutions of eluates containing known concentrations of bacterial DNA copies/µl. The overall LOD in spiked clinical samples, including sample preparation and DNA isolation on MagnaPure24, was measured through 10-fold serial dilutions (from 109 to 10-1 CFU/ml) including additional dilutions of 5000, 500, 50 and 5 CFU/ml.

Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_19 | Pages 75 - 75
22 Nov 2024
CAN MOLECULAR TECHNIQUES BECOME THE NEW “GOLD STANDARD” IN DIAGNOSTICS OF PERIPROSTHETIC JOINT INFECTIONS?
Erbeznik A Šturm AC Smrdel KS Triglav T Cvitković-Špik V Kišek TC Kocjancic B Pompe B Dolinar D Mavcic B Mercun A Kolar M Avsec K Papst L Vodicar PM
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Aim

We prospectively evaluated four different microbiological tools for diagnostics of prosthetic joint infections (PJI), and assessed their impact on the categorization of infection according to EBJIS guidelines. We compared culture, in-house real-time mPCR for S. aureus, S. lugdunensis, S. hominis, S. epidermidis, S. capitis, S. haemolyticus, C. acnes (mPCR), broad-spectrum PCR (Molzym) with 16S rRNA V3-V4 amplicon Sanger sequencing (16S PCR), and 16S rRNA V3-V4 amplicon next-generation sequencing (16S NGS) on MiSeq (Ilumina).

Methods

A total of 341 samples (sonication fluid, tissue biopsy, synovial fluid) were collected from 32 patients with suspected PJI who underwent 56 revision surgeries at the Orthopaedic Centre University Hospital Ljubljana, between 2022 and 2024. Samples were processed using standard protocols for routine culture, followed by DNA isolation using the MagnaPure24 (Roche). All samples were tested with mPCR, and an additional ≥4 samples from each revision (244 in total) were subjected to further metagenomic analysis. Culture results were considered positive if the same microorganism was detected in ≥2 samples, ≥50 CFU/ml were present in the sonication fluid, or ≥1 sample was positive for a more virulent microorganism or if the patient had received antibiotic treatment.

Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_19 | Pages 74 - 74
22 Nov 2024
16S RRNA V3-V4 AMPLICON NEXT-GENERATION SEQUENCING IN THE DIAGNOSIS OF PERIPROSTHETIC JOINT INFECTIONS
Erbeznik A Šturm AC Smrdel KS Triglav T Kocjancic B Pompe B Dolinar D Mavcic B Mercun A Kolar M Avsec K Papst L Vodicar PM
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Aim

To date, no ultimate diagnostic gold standard for prosthetic joint infections (PJI) has been established. In recent years, next generation sequencing (NGS) has emerged as a promising new tool, especially in culture-negative samples. In this prospective study, we performed metagenomic analysis using 16S rRNA V3-V4 amplicon NGS in samples from patients with suspected PJI.

Methods

A total of 257 (187 culture-negative (CN) and 70 culture-positive (CP)) prospectively collected tissues and sonication fluid from 32 patients (56 revisions) were included. 16S rRNA V3-V4 amplicons were sequenced using Illumina's MiSeq (California, USA) followed by bioinformatic analysis using nf-core/ampliseq pipeline.

Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_17 | Pages 73 - 73
24 Nov 2023
GENOME-BASED ANALYSIS OF VIRULENCE FACTORS OF THE GENUS CUTIBACTERIUM ISOLATED FROM IMPLANT-ASSOCIATED INFECTIONS IN SLOVENIA
Erbeznik A Šturm AC Smrdel KS Triglav T Vodicar PM
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Aim

The aim of our study was to analyze putative genes for virulence factors of Cutibacterium isolates obtained from implant-associated infections.

Methods

We analyzed 64 isolates of Cutibacterium spp. (C. acnes (53/64), C. avidum (6/64), C. granulosum (4/64), C. namnetense (1/64)) using NextSeq 550 (Illumina, San Diego, CA, USA) and performed genomic analysis of 24 genes associated with virulence factors (VFs) of C. acnes previously reported in the literature. Most isolates were obtained from implant-associated infections (IAI) between 2012–2021 at the Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana. Additionally, we included the first C. namnetense isolated in our laboratory from surgical site infection.

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