Development of a rapid detection system for the foot and mouth disease virus

The total syntheses of BocAL(Z)QAMC (i) and BocAL(Boc)Q(Trt)AMC (ii) have been accomplished. Synthesis of compound (i) was achieved by initially coupling the amino acid adjacent to the fluorophore, AMC, using selenocarboxylate / azide amidation coupling conditions, followed by the dipeptide coupling on to the third amino acid in the sequence Q using the coupling reagent HATU, under optimised conditions.1 Synthesis of compound (ii) was achieved using automated peptide synthesis of the protected tripeptide sequence and the final coupling to the fluorophore; AMC was achieved using HATU, under optimised conditions. The integrity of compound (i) and its fragment Q-AMC was found to be compromised during purification steps and storage, by spontaneous decomposition resulting in the premature, non-enzyme assisted cleavage of the fluorophore, AMC. This decomposition was minimised by the addition of the bulky trityl protecting group, therefore compound (ii) was used for biochemical testing. Successful deprotection of compound (ii) was accomplished prior to the enzymatic assay, with the target enzyme 3Cpro. Importantly, the proof of concept has been gained through evidence of enzyme assisted breakdown of the detection probe by fluorescence measurements. Also the deprotected version of compound (ii) was found to show selectivity towards the target enzyme FMDV 3Cpro over other enzymes potentially also present in clinical samples. These enzymes include chymotrypsin, thrombin and trypsin in FMDV clinical samples and the carefully selected TEV protease due to its reported cleavage after amino acid Q in peptide sequences.2 However, problems with the stability of the detection probe after deprotection were still apparent, resulting in a very short shelf-life of the probe. Further work is to be done in the stability of the probe but this project has proved 3Cpro recognises and processes shorter peptide fragments, previously reported not to be the case

Synthesis and initial evaluation of a novel fluorophore for selective FMDV 3C Protease detection

The development and evaluation of a Boc-AL(Boc)Q(Trt)-AMC fluorophore to detect 3C Protease, produced by Foot and Mouth Disease Virus (FMDV) is reported, with a view to a potential use as a rapid screen for FMDV infected livestock The peptide-linked conjugate fluorophore is evaluated in vitro for sensitivity, specificity, stability and rapidity and shows statistically significant increases in fluorescence when exposed to physiologically relevant concentrations of 3C Protease and selectivity when compared with other common proteases likely to be located, typically in the absence of FMDV. The stability of deprotected Boc-AL(Boc)Q(Trt)-AMC is reported as a limitation of this probe

The development of a fluorescent / colorimetric marker spray for the rapid detection of MRSA / MSSA

Methicillin-resistant 'staphylococcus aureus' (MRSA) is commonly referred to as a 'super bug', presenting a major problem within the health care environment, as this highly infectious disease requires expensive antibiotic treatment and a lengthier stay in a hospital, which further puts pressure on health-care based resources. The main reason for the spread of this infectious agent, is that there exists no rapid detection system that can be used on a large scale directly on surfaces / equipment / staff / patients at the point of care, to highlight infected areas. Deep cleaning procedures are currently systematic; however rapid detection method could allow targeting of these specific areas and thereby reduce the risk of infection spreading. A similar issue occurs with undetected methicillin sensitive 'staphylococcus aureus' (MSSA), which can be just as infectious as MRSA, even if more easily treated. This project involves the proof of concept and development of a non-lab based, rapid detection system. By improving on a coumarin dye system first reported in the 1970s, based on compound Butoxycarbonyl - Valine - Proline - Arginine - Methyl Coumarin (Boc-Val-Pro-Arg-Methyl Coumarin), The concept of this system is based on staphylothrombin's biological action, it recognises a specific tripeptide sequence and cleaves after the arginine residue releasing a free fluorophore, detected via a fluorescent shift. Modification to improve on this system involves the replacement of the poorly sensitive, low extinction coefficient fluorophore, methyl coumarin, with the opposite, a highly sensitive chromophore with a higher extinction coefficient that on release is accompanied with a visual colour change, promoting the desired rapid qualitative result. Reported are the chromophores tested for their suitability in our proposed dye system. Results suggest Rhodamine 110 is the most suitable and synthetic work was attempted following a convergent synthetic plan after successful isolation of the tripeptide sequence, not previously reported. Following successful synthesis of the tripeptide - chromophore conjugate, problems associated with the most suitable chromophore, Rhodamine 110 were highlighted and other approaches have been reported in order to eliminate these problems and ultimately develop the proposed system

Development of an in situ culture-free screening test for the rapid detection of Staphylococcus aureus within healthcare environments.

This article reports the development of a novel fluorometric indicator which shows a rapid response when exposed to coagulase positive Staphylococcus aureus (SA) bacteria (including methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) bacteria). The test is robust and will detect a wide variety of SA strains and there is no significant fluorescence response observed for other species of bacteria commonly found in clinical specimens, including other Staphylococcus bacteria. This research forms the basis of a prototype SA testing kit for the rapid detection of SA within hospital and healthcare environments as an economical prescreen or alternative to the current PCR based testing methodology. Rapid identification of SA carriers will allow hospital infection control teams to be pre-emptive and could significantly reduce the incidence of hospital acquired infections involving this organism

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)