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Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_22 | Pages 81 - 81
1 Dec 2017
Azamgarhi T Shah A Palanivel S Mack D Cleaver L Warren S
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Introduction

Fungi are a rare and devastating cause of Periprosthetic Joint Infection (PJI). Diagnosis and treatment is a challenge as there are currently no specific guidelines. A recently published review identified 75 case reports of fungal PJI.

Aim

The aim is to describe our experience of treating fungal PJI since 2011 within the Bone Infection Unit at our institution.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_23 | Pages 32 - 32
1 Dec 2016
Cleaver L Gorton R Gandy M Palanivel S Mack D Warren S
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Aim

Diagnosing Orthopaedic infection is limited by the sensitivity of culture methods. Next generation sequencing (NGS) offers an alternative approach for detection of microorganisms from clinical specimens. However, the low ratio of pathogen DNA to human DNA often inhibits detection of microorganisms from specimens. Depletion of human DNA may enhance the detection of microbial DNA1. Our aim was to compare four DNA extraction methods for the recovery of microbial DNA from orthopaedic samples for NGS.

Method

Simulated samples; pooled culture negative sample matrix was spiked with known concentrations of microorganisms, each panel consisting of 7 samples. Broth culture was performed on simulated samples for comparison with NGS*.

DNA Extraction; total nucleic acid extraction was performed on an automated extraction platform** using the viral NA assay. Modifications included: (1) mechanical lysis (glass beads), (2) lysis of human cells (saponin 0.025%), turbo DNase treatment and (3) mechanical lysis and addition of MspJI enzyme post-extraction for methylated DNA digestion.

Detection of human and microbial DNA; human endogenous (HE) gene rtPCR*** was utilised following manufacturer's recommendations. Microbial DNA was detected using SYBR green 16s ribosomal RNA rtPCR with high resolution melt-curve analysis****.