Masters, Thao

Background: Although cellularity is traditionally assessed morphologically, deep sequencing approaches being used for microorganism detection may be able to provide information about cellularity. We hypothesized that cellularity predicted using CIBERSORTx (Stanford University), a transcriptomic-based cellular deconvolution tool, would differentiate between infectious and non-infectious arthroplasty failure. Methods: CIBERSORTx-derived cellularity profiles of 93 sonicate fluid samples, including 53 from subjects who underwent failed arthroplasties due to periprosthetic joint infection (PJI) (abbreviated for the purpose of this study as PJIF) and 40 from subjects who had undergone non-infectious arthroplasty failure (abbreviated NIAF) that had been subjected to bulk RNA sequencing were evaluated. Results: Samples from PJIF and NIAF subjects were differentially clustered by principal component analysis based on the cellularity profile. Twelve of the 22 individual predicted cellular fractions were differentially expressed in the PJIF cases compared with the NIAF cases, including increased predicted neutrophils (mean and standard error, 9.73% ± 1.06% and 0.81% ± 0.60%), activated mast cells (17.12% ± 1.51% and 4.11% ± 0.44%), and eosinophils (1.96% ± 0.37% and 0.42% ± 0.21%), and decreased predicted M0 macrophages (21.33% ± 1.51% and 39.75% ± 2.45%), M2 macrophages (3.56% ± 0.52% and 8.70% ± 1.08%), and regulatory T cells (1.57% ± 0.23% and 3.20% ± 0.34%). The predicted total granulocyte fraction was elevated in the PJIF cases (32.97% ± 2.13% and 11.76% ± 1.61%), and the samples from the NIAF cases had elevated predicted total macrophage and monocyte (34.71% ± 1.71% and 55.34% ± 2.37%) and total B cell fractions (5.89% ± 0.30% and 8.62% ± 0.86%). Receiver operating characteristic curve analysis identified predicted total granulocytes, neutrophils, and activated mast cells as highly able to differentiate between the PJIF cases and the NIAF cases. Within the PJIF cases, the total granulocyte, total macrophage and monocyte, M0 macrophage, and M2 macrophage fractions were differentially expressed in Staphylococcus aureus compared with Staphylococcus epidermidis -associated samples. Within the NIAF cases, the predicted total B cell, naïve B cell, plasma cell, and M2 macrophage fractions were differentially expressed among different causes of failure. Conclusions: CIBERSORTx can predict the cellularity of sonicate fluid using transcriptomic data, allowing for the evaluation of the underlying immune response during the PJIF and NIAF cases, without a need to phenotypically assess cell composition.

AbstractAlthough Streptococcus agalactiae periprosthetic joint infection (PJI) is not as prevalent as staphylococcal PJI, invasive S. agalactiae infection is not uncommon. Here, RNA‐seq was used to perform transcriptomic analysis of S. agalactiae PJI using fluid derived from sonication of explanted arthroplasties of subjects with S. agalactiae PJI, with results compared to those of S. agalactiae strain NEM316 grown in vitro. A total of 227 genes with outlier expression were found (164 upregulated and 63 downregulated) between PJI sonicate fluid and in vitro conditions. Functional enrichment analysis showed genes involved in mobilome and inorganic ion transport and metabolism to be most enriched. Genes involved in nickel, copper, and zinc transport, were upregulated. Among known virulence factors, cyl operon genes, encoding β‐hemolysin/cytolysin, were consistently highly expressed in PJI versus in vitro. The data presented provide insight into S. agalactiae PJI pathogenesis and may be a resource for identification of novel PJI therapeutics or vaccines against invasive S. agalactiae infections.

Although Streptococcus agalactiae periprosthetic joint infection (PJI) is not as prevalent as staphylococcal PJI, invasive S. agalactiae infection has recently increased in incidence. Here, RNA-Seq was used to perform transcriptomic analysis of S. agalactiae PJI using fluid derived from sonication of explanted arthroplasties of subjects with S. agalactiae PJI, with results compared to those of S. agalactiae strain NEM316 grown in vitro. 227 genes with outlier expression were found (164 up-regulated and 63 down-regulated) between PJI sonicate fluid and in vitro conditions. Functional enrichment analysis showed genes involved in mobilome and inorganic ion transport and metabolism to be most enriched. Genes involved in nickel, copper, and zinc transport, were upregulated. Among known virulence factors, cyl operon genes, encoding beta-hemolysin/cytolysin, were consistently highly expressed in PJI versus in vitro. The data presented provide insight into S. agalactiae PJI pathogenesis and may be a useful resource for the identification of novel PJI therapeutics or vaccines against invasive S. agalactiae infections.

Abstract Whole genome sequencing is emerging as a promising tool for the untargeted detection of a broad range of microbial species for diagnosis and analysis. However, it is logistically challenging to perform the multistep process from sample preparation to DNA amplification to sequencing and analysis within a short turnaround time. To address this challenge, we developed a digital microfluidic device for rapid whole genome amplification of low-abundance bacterial DNA and compared results with conventional in-tube DNA amplification. In this work, we chose Corynebacterium glutamicum DNA as a bacterial target for method development and optimization, as it is not a common contaminant. Sequencing was performed in a hand-held Oxford Nanopore Technologies MinION sequencer. Our results show that using an in-tube amplification approach, at least 1 pg starting DNA is needed to reach the amount required for successful sequencing within 2 h. While using a digital microfluidic device, it is possible to amplify as low as 10 fg of C. glutamicum DNA (equivalent to the amount of DNA within a single bacterial cell) within 2 h and to identify the target bacterium within 30 min of MinION sequencing100× lower than the detection limit of an in-tube amplification approach. We demonstrate the detection of C. glutamicum DNA in a mock community DNA sample and characterize the limit of bacterial detection in the presence of human cells. This approach can be used to identify microbes with minute amounts of genetic material in samples depleted of human cells within 3 h.

Abstract Background Periprosthetic joint infection (PJI), a devastating complication of total joint replacement, is of incompletely understood pathogenesis and may sometimes be challenging to clinically distinguish from other causes of arthroplasty failure. Methods We characterized human gene expression in 93 specimens derived from surfaces of resected arthroplasties, comparing transcriptomes of subjects with infection- versus non-infection-associated arthroplasty failure. Results Differential gene expression analysis confirmed the association of 28 previously investigated biomarkers with PJI- bactericidal/permeability increasing protein (BPI), cathelicidin antimicrobial peptide (CAMP), chemokines CCL3, CCL4, and CXCL2, colony stimulating factor 2 receptor (CSF2RB), colony stimulating factor 3 (CSF3), alpha-defensin (DEFA4), receptor CD64B, intercellular adhesion molecule 1 (ICAM1), IFNG, IL13RA2, IL17D, IL1A, IL1B, IL1RN, IL2RA, IL2RG, IL5RA, IL6, IL8, lipopolysaccharide binding protein (LBP), lipocalin (LCN2), lactate dehydrogenase C (LDHC), lactotransferrin (LTF), matrix metallopeptidase 3 (MMP3), peptidase inhibitor 3 (PI3), and vascular endothelial growth factor A (VEGFA), as well as identified three novel molecules with diagnostic potential for detection of PJI- chemokine CCL20, coagulation factor VII (F7), B cell receptor FCRL4. Comparative analysis of infections caused by staphylococcal versus non-staphylococcal and Staphylococcus aureus versus Staphylococcus epidermidis showed significant elevated expression of IL13, IL17D, and metalloprotease protein MMP3 in staphylocococcal infections, and increased expression of IL1B, IL8, and platelet factor PF4V1 in S. aureus infections. Pathway analysis of over-presented genes suggested activation of host immune response and cellular maintenance and repair functions in response to invasion of infectious agents. Conclusion Our study provides new potential targets for diagnosis of PJI and targets for differentiation of PJI-associated infectious agents. Disclosures Matthew P. Abdel, MD, Dr. Abdel receives royalties from Stryker on certain hip and knee products, and is a paid consultant for Stryker. (Consultant) Robin Patel, MD, Accelerate Diagnostics (Grant/Research Support)CD Diagnostics (Grant/Research Support)Contrafect (Grant/Research Support)Curetis (Consultant)GenMark Diagnostics (Consultant)Heraeus Medical (Consultant)Hutchison Biofilm Medical Solutions (Grant/Research Support)Merck (Grant/Research Support)Next Gen Diagnostics (Consultant)PathoQuest (Consultant)Qvella (Consultant)Samsung (Other Financial or Material Support, Dr. Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued.)Selux Dx (Consultant)Shionogi (Grant/Research Support)Specific Technologies (Consultant)