Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review

Background: There is growing interest in the potential utility of real-time polymerase chain reaction (PCR) in diagnosing bloodstream infection by detecting pathogen deoxyribonucleic acid (DNA) in blood samples within a few hours. SeptiFast (Roche Diagnostics GmBH, Mannheim, Germany) is a multipathogen probe-based system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection. As background to this study, we report a systematic review of Phase III diagnostic accuracy studies of SeptiFast, which reveals uncertainty about its likely clinical utility based on widespread evidence of deficiencies in study design and reporting with a high risk of bias. 

Objective: Determine the accuracy of SeptiFast real-time PCR for the detection of health-care-associated bloodstream infection, against standard microbiological culture. 

Design: Prospective multicentre Phase III clinical diagnostic accuracy study using the standards for the reporting of diagnostic accuracy studies criteria. 

Setting: Critical care departments within NHS hospitals in the north-west of England. 

Participants: Adult patients requiring blood culture (BC) when developing new signs of systemic inflammation. 

Main outcome measures: SeptiFast real-time PCR results at species/genus level compared with microbiological culture in association with independent adjudication of infection. Metrics of diagnostic accuracy were derived including sensitivity, specificity, likelihood ratios and predictive values, with their 95% confidence intervals (CIs). Latent class analysis was used to explore the diagnostic performance of culture as a reference standard. 

Results: Of 1006 new patient episodes of systemic inflammation in 853 patients, 922 (92%) met the inclusion criteria and provided sufficient information for analysis. Index test assay failure occurred on 69 (7%) occasions. Adult patients had been exposed to a median of 8 days (interquartile range 4–16 days) of hospital care, had high levels of organ support activities and recent antibiotic exposure. SeptiFast real-time PCR, when compared with culture-proven bloodstream infection at species/genus level, had better specificity (85.8%, 95% CI 83.3% to 88.1%) than sensitivity (50%, 95% CI 39.1% to 60.8%). When compared with pooled diagnostic metrics derived from our systematic review, our clinical study revealed lower test accuracy of SeptiFast real-time PCR, mainly as a result of low diagnostic sensitivity. There was a low prevalence of BC-proven pathogens in these patients (9.2%, 95% CI 7.4% to 11.2%) such that the post-test probabilities of both a positive (26.3%, 95% CI 19.8% to 33.7%) and a negative SeptiFast test (5.6%, 95% CI 4.1% to 7.4%) indicate the potential limitations of this technology in the diagnosis of bloodstream infection. However, latent class analysis indicates that BC has a low sensitivity, questioning its relevance as a reference test in this setting. Using this analysis approach, the sensitivity of the SeptiFast test was low but also appeared significantly better than BC. Blood samples identified as positive by either culture or SeptiFast real-time PCR were associated with a high probability (> 95%) of infection, indicating higher diagnostic rule-in utility than was apparent using conventional analyses of diagnostic accuracy. 

Conclusion: SeptiFast real-time PCR on blood samples may have rapid rule-in utility for the diagnosis of health-care-associated bloodstream infection but the lack of sensitivity is a significant limiting factor. Innovations aimed at improved diagnostic sensitivity of real-time PCR in this setting are urgently required. Future work recommendations include technology developments to improve the efficiency of pathogen DNA extraction and the capacity to detect a much broader range of pathogens and drug resistance genes and the application of new statistical approaches able to more reliably assess test performance in situation where the reference standard (e.g. blood culture in the setting of high antimicrobial use) is prone to error.

Strategies for detection and quantification of cysteine cathepsins-evolution from bench to bedside

The cysteine cathepsins are a family of closely related thiol proteases, normally found in the endosomal and lysosomal compartments of cells. A growing body of evidence has clearly linked the dysregulated activity of these proteases with many diseases and pathological conditions, offering therapeutic, prognostic and diagnostic potential. However, these proteases are synthesised as inactive precursors and once activated, are controlled by factors such as pH and presence of endogenous inhibitors, meaning that overall protein and activity levels do not necessarily correlate. In order to fully appreciate the role and potential of these proteases, tools are required that can detect and quantify overall cathepsin activity. Two main strategies have evolved; synthetic substrates and protease-labelling with affinity-binding probes (or activity-based probes). This review examines recent innovations in these approaches as the field moves towards developing tools that could ultimately be used in patients for diagnostic or prognostic applications.

Sparse multi-carrier index keying OFDM with index separation over correlated sub-carriers

In this paper, we propose a sparse multi-carrier index keying (MCIK) method for orthogonal frequency division multiplexing (OFDM) system, which uses the indices of sparse sub-carriers to transmit the data, and improve the performance
of signal detection in highly correlated sub-carriers. Although a receiver is able to exploit a power gain with precoding in OFDM, the sensitivity of the signal detection is usually high as the orthogonality is not retained in highly dispersive
environments. To overcome this, we focus on developing the trade-off between the sparsity of the MCIK, correlation, and performances, analyzing the average probability of the error propagation imposed by incorrect index detection over highly correlated sub-carriers. In asymptotic cases, we are able to see how sparsity of MCIK should be designed in order to perform superior to the classical OFDM system. Based on this feature, sparse MCIK based OFDM is a better choice for low detection errors in highly correlated sub-carriers.

Endoscopic tri-modal imaging improves detection of gastric intestinal metaplasia among a high-risk patient population in Singapore

BACKGROUND: Detection of pre-neoplastic gastric mucosal changes and early gastric cancer (EGC) by white-light endoscopy (WLE) is often difficult. In this study we investigated whether combined autofluorescence imaging (AFI) and narrow band imaging (NBI) can improve detection of pre-neoplastic lesions and early gastric cancer in high-risk patients.

PATIENTS AND METHODS: Chinese patients who were 50-years-old or above with dyspepsia were examined by both high-resolution WLE and combined AFI followed by NBI (AFI-NBI), consecutively in a prospective randomized cross-over setting, by two experienced endoscopists. The primary outcome was diagnostic ability of the two methods for patients with pre-neoplastic lesions such as intestinal metaplasia (IM) and mucosal atrophy.

RESULTS: Sixty-five patients were recruited. One patient with large advanced gastric cancer was found and excluded from the analysis. Among the remaining 64 patients, 38 (59%) had IM; of these, 26 (68%) were correctly identified by AFI-NBI (sensitivity 68%, specificity 23%) and only 13 (34%) by WLE (sensitivity 34%, specificity 65%). AFI-NBI detected more patients with IM than did WLE (p=0.011). Thirty-one patients (48%) had mucosal atrophy. Ten patients (32%) were identified by AFI-NBI (sensitivity 32%, specificity 79%) and four patients (13%) by WLE (sensitivity 13%, specificity 88%) (p=0.100). No dysplasia or EGC was found.

CONCLUSION: AFI-NBI identified significantly more patients with IM than did WLE. Our result warrants further studies to define the role of combined AFI-NBI endoscopy for detection of precancerous conditions.

Development of a Novel Assay for the Detection of Active Neutrophil Elastase in Patients with Chronic Obstructive Pulmonary Disease

Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including chronic obstructive pulmonary disease (COPD). However, it’s detection and quantification in biological samples is confounded by a lack of reliable and robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex clinical samples containing multiple proteolytic and hydrolytic enzymes which have the ability to hydrolyse the substrate, thereby resulting in an over-estimation of the target protease. Furthermore, ELISA systems measure total protease levels which can be a mixture of latent, active and protease-inhibitor complexes. Therefore, we have developed a novel immunoassay (ProteaseTag™ Active NE Immunoassay) which is selective and specific for the capture of active NE in sputum and Bronchoalveolar Lavage (BAL) in patients with COPD. The objective of this study was to clinically validate ProteaseTag™ Active NE Ultra Immunoassay for the detection of NE in sputum from COPD patients. 20 matched sputum sol samples were collected from 10 COPD patients (M=6, F=4; 73 ± 6 years) during stable and exacerbation phases. Samples were assayed for NE activity utilising both ProteaseTag™ Active NE Ultra Immunoassay and a fluorogenic substrate-based kinetic activity assay. Both assays detected elevated levels of NE in the majority of patients (n=7) during an exacerbation (mean=217.2 μg/ml ±296.6) compared to their stable phase (mean=92.37 μg/ml ±259.8). However, statistical analysis did not show this difference to be significant (p=0.07, ProteaseTag™ Active NE Ultra Immunoassay; p=0.06 kinetic assay), most likely due to the low study number. A highly significant correlation was found between the 2 assay types (p≤0.0001, r=0.996). NE as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. ProteaseTag™ Active NE Immunoassay specifically measures only active NE in clinical samples, is quick and easy to use (< 3 hours) and has no dependency on a kinetic readout. ProteaseTag™ technology is currently being transferred to a lateral flow device for use at Point of Care.

CGRP-cleavage enzyme detection using a biotinylated hydroxymate affinity probe

Introduction: Neuropeptides contribute to the pathophysiology of peripheral inflammation and a neurogenic component has been described for many inflammatory diseases, including periodontitis. Neuropeptides are susceptible to cleavage by peptidases and therefore the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies by our research group suggested that levels of the neuropeptide calcitonin gene-related peptide (CGRP) may be regulated by peptidases present in gingival crevicular fluid (GCF). Objectives: The aim of this work was to purify and partially characterize the GCF enzyme responsible for CGRP degradation using a biotinylated hydroxymate affinity probe (based on the P1-P4 amino acid sequence of the observed cleavage site) which we previously showed to inhibit CGRP degradation. Methods: Pooled healthy and pooled periodontitis GCF samples were subject to a pre-clear step with magnetic streptavadin beads. Healthy and diseased samples were incubated with the biotinylated hydroxymate probe (20 uM) after which biotinylated proteins were purified from the sample using magnetic streptavadin beads. Bound proteins were subjected to SDS-PAGE and western blotting. Biotin incorporated proteins were disclosed using a streptavadin horse radish peroxidase conjugate. Results: A band was disclosed in the periodontitis pooled sample at a molecular weight of approximately 60 kDa. The band was absent in the pooled healthy samples. As expected, when periodontitis samples were pre-boiled to denature proteins before the addition of the hydroxymate probe, no biotin incorporated band was present. Conclusions: This work demonstrates the purification and disclosure of a protein found specifically in periodontitis which binds to the specific biotinylated hydroxymate affinity probe based on the cleavage site of CGRP only when in its native form. We intend to scale up the sample size thus allowing the identification of the putative CGRP degrading peptidase using MALDI-mass spectrometry.
Funded by an IADR/GlaxoSmithKline Innovation in Oral Care Award

Selective Multi-Carrier Index Keying OFDM: Error Propagation Rate with Moment Generating Function

We propose a new selective multi-carrier index keying in orthogonal frequency division multiplexing (OFDM) systems that opportunistically modulate both a small subset of sub-carriers and their indices. Particularly, we investigate the performance enhancement in two cases of error propagation sensitive and compromised deviceto-device (D2D) communications. For the performance evaluation, we focus on analyzing the error propagation probability (EPP) introducing the exact and upper bound expressions on the detection error probability, in the presence of both imperfect and perfect detection of active multi-carrier indices. The average EPP results in closedform are generalized for various fading distribution using the moment generating function, and our numerical results clearly show that the proposed approach is desirable for reliable and energy-efficient D2D applications.

The application of a novel, cell permeable activity-based probe for the detection of cysteine cathepsins.

Cysteine cathepsins, such as cathepsin S (CTSS), are implicated in the pathology of a wide range of diseases and are of potential utility as diagnostic and prognostic biomarkers. In previous work, we demonstrated the potency and efficiency of a biotinylated diazomethylketone (DMK)-based activity-based probe (ABP), biotin-PEG-LVG-DMK, for disclosure of recombinant CTSS and CTSS in cell lysates. However, the limited cell permeability of both the biotin and spacer groups restricted detection of CTSS to cell lysates. The synthesis and characterisation of a cell permeable ABP to report on intracellular CTSS activity is reported. The ABP, Z-PraVG-DMK, a modified peptidyl diazomethylketone, was based on the N-terminus of human cystatin motif (Leu-Val-Gly). The leucine residue was substituted for the alkyne-bearing proparcylglycine to facilitate conjugation of an azide-tagged reporter group using click chemistry, following irreversible inhibition of CTSS. When incubated with viable Human Embryonic Kidney 293 cells, Z-PraVG-DMK permitted disclosure of CTSS activity following cell lysis and rhodamine azide conjugation, by employing standard click chemistry protocols. Furthermore, the fluorescent tag facilitated direct detection of CTSS using in-gel fluorescent scanning, obviating the necessity for downstream biotin-streptavidin conjugation and detection procedures.