Methods and materials for prosthetic joint infection detection and diagnosis

Abstract

A biomarker for detecting implant related infection and a method of detecting a biomarker in a sample, said biomarker including at least part of the cnt operon and / or staphylopine. The method is suitable for diagnosing infection of a prosthetic joint and said method including the step of detecting the presence of at least part of the cnt operon and / or staphylopine in said sample.

Description

[0001]Methods and Materials for Prosthetic Joint Infection Detection and Diagnosis The present invention relates to methods for detecting and / or diagnosing implant related infections, in particular but not exclusively, the infection of prosthetic joints in humans and animals, as well as products and materials used in the diagnosis of the same Although the following description refers exclusively to the detectionof infection in synovial fluid, the skil led person will appreciate thatthe methods and techniques involved can be applied to samples taken from other fluids and tissues. Joint replacement surgery is common, particularly in countries with aging populations. For example, in the UK over eighty thousand hip and ninety thousand knee replacements are conducted each year. In some patients, these joints become infected with bacteria which can colonise prosthetic surfaces and establish chronic infections. These are not usually treatable with antibiotics and require additional invasive and costly surgical revision. With >9500 revisions required per year in the UK and the average hip revision costing approximately £50,000 this results in £100 million in costs per annum. A group of bacteria known as the ‘Coagulase-negative Staphylococci’ (CoNS) are the most common cause of infection and are isolated fromapproximately 50% of prosthetic joint infections (PJI). CoNS are common members of our skin microbiome (acting as a source of infecting organisms). Specifically diagnosing infection of a joint is a major challenge because joints can become inflamed due to non-infective reasons (e.g. gout), or loose from non-infected reasons (aseptic loosening) which can sometimes be managed without more extensive surgery. CoNS infection is the main cause of PJI and diagnosing infection is very difficult because recovery of CoNS from a wound swab is often not specific, in that it is unclear if the organism detected was present in the joint or picked up from layers of the skin during sampling.Current diagnostics of infection are based on host biomarkers (e.g.Calprotectin) which are over-expressed during infection, these are however not very specific and there is a major need for a sensitive and specific test that reports the presence of a pathogen from within a joint when infection is suspected. At present, a specific biomarker confirming joint infection by staphylococci is unavailable. It is therefore an aim of the present invention to provide a method ofdetecting and / or diagnosing joint infection, specifically PJI, inhumans and animals that addresses the abovementioned problems. It is a further aim of the present invention to provide apparatus and materials to detect and / or diagnose joint infection, specifically PJI in humans and animals that addresses the abovementioned problems. In a first aspect of the invention there is provided at least one biomarker for detecting implant related infection, said at least one biomarker including at least part of the cnt operon and / or staphylopine. In a second aspect of the invention there is provided a method of detecting a biomarker in a sample, said detection suitable for diagnosing infection of a prosthetic joint by at least one Coagulase Negative Staphylococci (CoNS) strain, said method including the stepof detecting the presence of at least part of the cnt operon and / orstaphylopine in said sample. The skilled person will appreciate that the cnt operon is conservedacross CoNS and Staphylococcus aureus , which are responsible for mostcases of prosthetic joint infection, and therefore a diagnostic test based on this biomarker is of excellent clinical value.Typically cntE (staphylopine export protein) is included in at least partof the cnt operon detected. Further typically the sample is from and / or includes the joint synovial fluid. As such, the method provides a positive predictive value in that expression of staphylopine is induced within synovial fluid and therefore detection indicated the presence of CoNS within the joint where infection is suspected. In a preferred embodiment of the invention the detection of at leastpart of the cnt operon includes subjecting the sample to quantitativepolymerase chain reaction (qPCR) analysis. Preferably the analysis is reverse-transcription quantitative real-time PCR (RT-qPCR). Typicallythe qRT-PCR analysis detects the presence of at least part of cntEgene in the cnt cluster. In a preferred embodiment staphylopine is detected using chromatographic techniques. Typically the compound is detected using affinity chromatography. Further typically the techniques to detect the Cnt proteins include immunochromatographic assay and / or enzyme-linked immunosorbent assays (ELISA). In a third aspect of the invention at least part of the cnt operon and / or staphylopine is used as a biomarker for identifying and / or diagnosing joint infection. Preferably the joint is a joint that has undergone repair and / or is a prosthetic joint or part thereof. Typically any one or any combination of RT-qPCR, protein assays and / or staphylopine detection provides a diagnostic test. Further typically the diagnostic test identifies the group of bacteria known as the ‘Coagulase-Negative Staphylococci’ (CoNS), this may also beapplicable to prosthetic joint infection caused by Staphylococcus aureus .In a fourth aspect of the invention there is provided a diagnostic kit for CoNS infection including RT-qPCR, protein assays and / or staphylopine detection agents. Specific embodiments of the invention are now described with reference to the following figures wherein: Figures 1a and 1b show a TraDIS plot file showing reduced mutantdensity across cnt operon and cntE respectively;Figures 2a and 2b show expression data heatmaps for cntE ;Figure 3 shows the RT-qPCR analysis of cntE expression in S.epidermidis 846 and RP62A in response to infected synovial fluid;Figures 4a and 4b show plots from RNASeq analysis (analysed using the Degust platform) when only infected synovial fluid samples were used; Figure 5 shows S. epidermidis 846 ΔcntE growth, where the mutant was isolated from the TraDIS library; and Figures 6a and 6b show gels indicating the conservation of the cnt operon. Prosthetic joint infection (PJI) is a major healthcare burden with an ageing population requiring increased numbers of joint replacements. Over a million people in the UK have a replacement joint with >160,000 primary replacements performed annually. Infection is a major complication often necessitating revision with >9500 revisions required per year in the UK. With the average hip revision costing approximately £50,000 this results in >£100 million in costs per annum. Revision is required post infection as antibiotic treatment is ineffective due to bacteria forming resistant biofilms on prosthetics. Given the severity of surgery and costs, differentiating infection from non-infective causes is critical but there are no accurate diagnostictests in routing use. Most cases of PJI are attributed to Staphylococcus ,as these organisms are common skin residents which can inhabit follicles making antisepsis difficult, they commonly contaminate diagnostic samples. A specific biomarker confirming joint infection by staphylococci is unavailable. In developing a diagnostic tool for PJI we have establish a laboratory model of PJI in which we grow bacteria in donated synovial fluid. We have used this model to investigate the potential biomarkers of joint infection by Coagulase Negative Staphylococci (CoNS). We have used a combination of RNASeq and a large transposon mutant library to identify biomarkers of PJI (Figures 1-3). A Staphylococcus epidermidis transposon mutant library was screened to identify genes essential for growth in human synovial fluid (Figure 1). We also exposed two strains of Staphylococcus epidermidis to humansynovial fluid to identify genes which are upregulated in response to synovial fluid (Figure 2). Combining these two datasets to identify genes which were upregulated upon exposure and essential for growth resulted in a small sub-set of genes with potential to be used as a diagnostic biomarker.Of the genes identified using these techniques, the cnt operon,encoding the biosynthesis and utilisation of the metallophorestaphylopine, was found to be entirely upregulated (Figure 3,4).Importantly, the cnt genes were only over-expressed when CoNS weregrown in synovial fluid from infected patients (filtered to remove any bacteria) – suggesting expression is induced as zinc is depleted as a specific response not seen when CoNS were grown in uninfectedsynovial fluid (or in other conditions including as a biofi lm in growthmedia). Given the ability of the human host to limit zinc in response to infection, and the previous identification of calprotectin as a possible host biomarker, staphylopine offers a novel option as a bacterial biomarker for diagnosis of PJI. We have confirmed the conservationand presence of the cnt operon in strains of Staphylococcus epidermidis,capitis , warneri , lugdunensis and aureus isolated from PJI, suggesting thatthe use of staphylopine as a marker of infection has broadapplicability to the most common causes of PJI.Current diagnosis of infection relies on culture of a causative pathogen. For those organisms not part of the normal skin flora isolation can have good predictive power. However, there is often diagnostic uncertainty about the significance of isolation of CoNS as the distinction between isolation of a causative pathogen and contaminating skin commensal organisms can be unclear. This is a major problem as CoNS are the major cause of PJI and clinicalsymptoms of infection are often unspecific. As a result, there hasbeen significant effort put into trying to develop novel bacterial biomarkers of infection. Our results which have identified specific behaviours of CoNS in synovial fluid allows us to propose a novelbiomarker to identify prosthetic joint infection by CoNS using a set ofgenes and their products which are only expressed in the presence of infection.We have validated the cnt operon as a novel biomarker of prostheticjoint infection and determined that RT-qPCR, protein assays and / or staphylopine detection is effective as a diagnostic. RT-qPCR is found to be effective and thus developed into a nucleic acid-based test, there is proof of principle for doing this with existing tests produced that either identify pathogen DNA, or host gene expression as biomarkers In addition, protein or staphylopine detection is also found to be effective and practical, thus we have explored additional possibilities for diagnostic tool development. Due to the slow-growing nature of CoNS, they often do not elicit a high immune response, evading detection by current host based diagnostic methods such as calprotectin and alpha-defensin. A diagnostic tool which could identify these infections at an earlier time point would therefore open the possibility of less severe treatments, offering a benefit both in terms of recovery time for the patient and economically for healthcare providers. Given that the cnt operon is conserved across CoNS and Staphylococcusaureus, which are responsible for most cases of prosthetic jointinfection, the diagnostic tests based on this biomarker are far superior to current methods. This is based on providing a positive predictive value in that expression of staphylopine is induced within synovial fluid and therefore detection indicates the presence of CoNS within the joint where infection is suspected. In combination with host infection biomarkers such as calprotectin this a test can be developed with both high positive and negative predictive values. Directdetection of staphylopine, either at the RNA level, molecule itself orprotein export is achieved using existing methodology and would not require a high level of specialist equipment and training as currently required for sequencing techniques. Figure 1a shows a TraDIS plot file showing reduced mutant density across cnt operon.The cnt operon is responsible for the production and uti lisation ofthe metallophore staphylopine, previously linked to zincacquisition in S. aureus.We chose to focus on cntE (staphylopine export protein) in figure 1b due to extracellular portions which may be easier to identify using aprotein-based diagnostic test (e.g. ELISA or lateral flow)Figures 2a and 2b show RNA sequence data heatmaps for cntE .Figure 3a shows the RT-qPCR analysis of cntE expression in S.epidermidis 846 and RP62A in response to infected versus non-infectedsynovial fluid (separate fluid sample from those used for RNASeq).Figures 4a and 4b show plots from RNASeq analysis (Degust) whenonly infected SF samples were used. MDS plot – measures difference between samples (checks if treatment vs control are more different than treatment vs treatment. Heatmap – log fold change in expression on exposure to SF vs control. CPM – cntE counts per million sequence reads. The right hand side – different way to visualise log fold change in expression of individual genes,BLJHFILP_01759 and SERP2367 – both cntE .We have also looked for cntE in a selection of Staphylococcus isolatesfrom PJI cases and found that:• Present in all Staphylococcus epidermidis strains checked (7strains) •Not present in Staphylococcus haemolyt icus• Present in all other non-aureus staphylococci in ourcollection isolated from PJI (one representative of each in collection, but all appear to be correctly assigned speciesbased on MLST): •S. lugdunensis , S. capitis, S. warneri• cnt operon identif ied in S. aureus.Figure 5 shows S. epidermidis 846 ΔcntE growth, where the mutant was isolated from the TraDIS library. Growth is compared to wild type showing a large reduction in cfu. Both experiments biological triplicate. TraDIS experiments: A transposon mutant library was produced in Staphylococcus epidermidis strain 15TB0846 (referred to as “846”), isolated from a healthy skin swab. The library contained approximately 50,000 unique mutants. Human synovial fluid obtained as diagnostic excess through the Norwich Research Park Biorepository. Fluid was diluted 1:1 in PBS containing 0.1% glucose (mimicking the concentration found in vivo), and filter sterilised. The transposon mutant pool was grown in either the processed human synovial fluid, or Müller Hinton medium, in two independent replicates. DNA was extracted from the mutant pools, prepared for transposon- directed sequencing, and sequenced using Illumina technology. The reads were mapped to the genome assembly for S. epidermidis 15TB0846 using the BioTraDIS pipeline, and the associated comparison tool was used to identify differences in essential genes between the control and test samples. Cutoffs of 1 log2 fold change and a q value <0.05 were used. RNASeq experiments: Two strains of Staphylococcus epidermidis were used, RP62A and 15TB0846 (“846”). Cells from an overnight culture were diluted 1 / 200 and grown to OD6 0 0 0.2-0.3. The cells were centrifuged and the harvested cell pellet resuspended in 0.5 mL either processed synovial fluid, or Müller Hinton medium as a control. These were incubated at 37 °C for 30 minutes. Cells were washed in PBS and resuspended in Zymo DNA / RNAShield in BeadBashing tubes. The cells were broken using beadbeating, then RNA was harvested using the Zymo Quick- RNA Fungal / Bacterial Miniprep kit according to manufacturer’s instructions. RNA quality was assessed using nanodrop (A260 / A280 >1.8) and Agilent Tapestation (RIN >6). The samples were shipped to Azenta for sequencing. Data was analysed using Galaxy, with HISAT2 used to map the reads to the reference RP62A genome and assembled 846 genome, featurecounts used to count the number of mapped reads per gene, and DESeq2 to assess which genes were differentially expressed uponexposure to synovial fluid versus the medium control. Cutoffs usedwere as for the TraDIS experiment.

Claims

Claims 1. A biomarker for detecting implant related infection, said biomarker including at least part of the cnt operon and / or staphylopine.

2. A method of detecting a biomarker in a sample, said detection suitable for diagnosing infection of a prosthetic joint and said method including the step of detecting the presence of at least part of the cnt operon and / or staphylopine in said sample.

3. A method according to claim 2 wherein said detection is suitable for diagnosing infection at least one Coagulase Negative Staphylococci (CoNS) strain.

4. A method according to claim 3 wherein cntE (staphylopine export protein) is included in at least part of the cnt operon detected.

5. A method according to claims 2-4 wherein the sample is from and / or includes the joint synovial fluid.

6. A method according to claims 2-5 wherein the detection of at least part of the cnt operon includes subjecting the sample to quantitative polymerase chain reaction (qPCR) analysis.

7. A method according to claim 6 wherein the analysis is reverse- transcription quantitative real-time PCR (RT-qPCR).

8. A method according to claim 7 wherein the qRT-PCR analysis detects the presence of at least part of cntE gene in the cnt cluster.

9. A method according to any preceding claim wherein staphylopine is detected using chromatographic techniques.

10. A method according to claim 9 wherein the compound is detected using affinity chromatography.

11. A method according to any preceding claim wherein said method is also applicable to prosthetic joint infection caused by Staphylococcusaureus.

12. A diagnostic kit for detection of CoNS infection including RT- qPCR, protein assays and / or staphylopine detection agents.

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