MICROBIAL TECHNOLOGY Dr Katie Hopkins illustrated how to provide timely sequencing for local diagnosis.
rigorous study design, large statistical input and, importantly, a real degree of patient involvement. Professor Dark described in great detail the ADAPT trial. A somewhat tortuous acronym standing for biomArker-guided Duration of Antibiotic treatment in hospitalised PaTients with sepsis. This large trial is designed to provide a definitive answer to the question does a treatment protocol based on serial monitoring of C-reactive protein (CRP) or procalcitonin (PCT) safely allow reduction in duration of antibiotic therapy in hospitalised patients with sepsis? It is worth pointing out that the drive to reduce excessive antibiotic usage is not solely due to justifiable concerns about the development of antibiotic resistance, but the actual harm that antibiotic use can cause. This is particularly so in neonates where concern over neonatal sepsis leads to what all neonatologists agree is a huge over-prescribing of antibiotics, leading to very common and sometimes very serious deleterious effects on the patient. It is sobering to reflect that the use of the CRP test to guide the diagnosis of sepsis has been in widespread use for approximately 40 years, but there is still no good evidence to show its benefit, principally owing to the small size of the trials that have taken place. In the ADAPT study the total sample size of 2760 patients is being enrolled, and it’s a tribute both to those who are running the trial and to all the ICU staff and patients who are working on the trial that it managed to keep ticking over during the COVID pandemic, and is now back on
track to its predicted recruitment rate, and should finally provide a definitive answer on the value of these two tests.
Direct identification and sequencing
In the next talk Adela Alcolea-Medina, Clinical Scientist with ViaPath at St Thomas’ Hospital, London described her work using quite a different technology – Oxford Nanopore (ONT) sequencing for the direct detection and identification of viruses in respiratory clinical samples. Next-generation sequencing (NGS) allows thousands to billions of DNA fragments to be simultaneously and independently sequenced. One great advantage of NGS in clinical microbiology is that it is ‘agnostic’ and should allow for an unbiased approach to the detection of pathogens.
Published rapid methods for clinical metagenomics-only sequence DNA or RNA have long turnaround times to provide clinical reports. Although Library preparations for combined RNA and DNA sequencing have been published, these take days to provide results, mainly owing to the huge quantity of human DNA present in clinical samples, which outnumber pathogen DNA by a factor of thousands if not more. She described a method for depleting human DNA
while limiting centrifugation or the use of chemicals and a library preparation that can sequence RNA and DNA organisms at the same time. The benefits of a rapid unified, unbiased method for detecting and characterising DNA and RNA organisms are obvious and has become something of a ‘holy grail’ in recent years. It should remove the need for multiple samples or sample splitting the different tests and hopefully to exclude the need for targeted PCR. In addition, it has the potential to identify unexpected novel and emerging pathogens. The workflow she described was simple and the first results were obtained seven hours after receipt of the specimen, and a 24-hour workflow generated full genome sequences of pathogens. The technique had been successfully applied to respiratory specimens, and several different viruses have been identified with results correlating well with conventional tests. In the last couple of months, the focus has switched, for obvious reasons, to testing skin swabs from patients presenting with a blistering rash, and a number of cases of monkeypox had been detected. Interestingly, in a patient with suspected monkeypox, varicellar-zoster virus (VZV) and not monkeypox was detected. The gene coverage obtained was a 100%, which gave a high degree of confidence in the result and obviously made a huge difference to patient management. Although the attractions of the technology are obvious, it is likely that the need to establish rigid quality control (QC) criteria rather than cost will determine how quickly it is introduced into the diagnostic laboratory.
Phenotypic versus genotypic sensitivity testing After a break, the next talk was given by Dr Katie Hopkins, Lead Clinical Scientist, Antimicrobial Resistance & Mechanisms Service, UK Health Security Agency at Colindale, who delivered a comprehensive overview of the different technologies used for antimicrobial resistance and also the challenge of selecting which ones to use within a diagnostic laboratory. Phenotypic methods are commonly used and while slower, with inconsistencies in reproducibility, they do provide a quick method of detecting resistance in organisms, even if the
The CRP test to guide the diagnosis of sepsis has been in widespread use for approximately 40 years, but there is still no good evidence to show its benefit
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