Extraction and sensitive detection of toxins A and B from the human pathogen Clostridium difficile in 40 seconds using microwave-accelerated metal-enhanced fluorescence.

Clostridium difficile is the primary cause of antibiotic associated diarrhea in humans and is a significant cause of morbidity and mortality. Thus the rapid and accurate identification of this pathogen in clinical samples, such as feces, is a key step in reducing the devastating impact of this disease. The bacterium produces two toxins, A and B, which are thought to be responsible for the majority of the pathology associated with the disease, although the relative contribution of each is currently a subject of debate. For this reason we have developed a rapid detection assay based on microwave-accelerated metal-enhanced fluorescence which is capable of detecting the presence of 10 bacteria in unprocessed human feces within 40 seconds. These promising results suggest that this prototype biosensor has the potential to be developed into a rapid, point of care, real time diagnostic assay for C. difficile

Ultra-Fast and Sensitive Detection of Non-Typhoidal Salmonella Using Microwave-Accelerated Metal-Enhanced Fluorescence (“MAMEF”)

Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1∶1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids)

Current and future trends in the laboratory diagnosis of sexually transmitted infections

Sexually transmitted infections (STIs) continue to exert a considerable public health and social burden globally, particularly for developing countries. Due to the high prevalence of asymptomatic infections and the limitations of symptom-based (syndromic) diagnosis, confirmation of infection using laboratory tools is essential to choose the most appropriate course of treatment and to screen at-risk groups. Numerous laboratory tests and platforms have been developed for gonorrhea, chlamydia, syphilis, trichomoniasis, genital mycoplasmas, herpesviruses, and human papillomavirus. Point-of-care testing is now a possibility, and microfluidic and high-throughput omics technologies promise to revolutionize the diagnosis of STIs. The scope of this paper is to provide an updated overview of the current laboratory diagnostic tools for these infections, highlighting their advantages, limitations, and point-of-care adaptability. The diagnostic applicability of the latest molecular and biochemical approaches is also discussed

Applications of Microwave Energy in Medicine

Microwaves are a highly utilized electromagnetic wave, used across a range of industries including food processing, communications, in the development of novel medical treatments and biosensor diagnostics. Microwaves have known thermal interactions and theorized non-thermal interactions with living matter; however, there is significant debate as to the mechanisms of action behind these interactions and the potential benefits and limitations of their use. This review summarizes the current knowledge surrounding the implementation of microwave technologies within the medical industry.</jats:p

Rapid, point-of-care microwave lysis and electrochemical detection of Clostridioides difficile directly from stool samples

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen’s two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile

Lab-on-a-chip platforms for pathogen analysis

Infectious diseases caused by pathogenic microorganisms are a big burden in developed and developing countries. The emergence and rapid global spread of virus and antimicrobial resistant bacteria is a significant threat to patients, healthcare systems and the economy of countries. Early pathogen detection is often hampered by low concentrations present in complex matrices such as food and body fluids.Microfluidic technologies offer new and improved approaches for detection of pathogens on the microscale. Here, two microfluidic platforms for pathogen sorting and molecular identification were investigated: (1) inertial focusing and (2) microscale immiscible filtration. Inertial focusing in two serpentine channel designs etched in glass at different depths was evaluated with different microparticles, bacteria and blood. The shallow design allowed 2.2-fold concentration of Escherichia coli O157 cells, whereas the deep design accomplished recovery of 54% E. coli O157 depleted from 97% red blood cells in 0.81% haematocrit at flowrates of 0.7 mL min-1.A lab-on-a-chip platform based on microscale immiscible filtration was investigated for capture and detection of nucleic acids and bacteria. For nucleic acids, oligo (dT) functionalised magnetic beads or silica paramagnetic particles in GuHCl were used to capture genomic RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and genomic DNA from Neisseria gonorrhoeae, respectively. On-chip amplification and detection were performed via colorimetric loop-mediated isothermal amplification (LAMP). Results showed sensitive and specific detection of targeted nucleic acids (470 RNA copies mL-1 and 5 × 104 DNA copies mL-1) with no cross-reactivity to other RNAs and DNAs tested. The whole workflow was integrated in a single device and time from sample-in to answer-out was within 1h. The platform only required power for a heat source and showed potential for point of care diagnostics in resource-limited settings. For bacteria detection, anti-E. coli O157 functionalised magnetic beads were used to capture cells with > 90% efficiency and on-chip fluorescence in situ hybridisation and a staining assay were explored for bacteria identification.A wide variety of microfluidic approaches for pathogen analysis have been devised in the literature with different advantages and drawbacks. Careful evaluation based on their purpose, integrated steps and end user is critical. Input from stakeholders right from the start of a project and throughout is vital to success. The platforms investigated herein have potential for applications such as sample preparation, pathogen concentration and specific molecular detection of E. coli O157, N. gonorrhoeae DNA, and SARS-CoV-2 RNA. With further development and clinical validation, the widespread use of these systems could facilitate early diagnosis of infectious diseases, allowing timely management of outbreaks and treatment and slowing the incidence of antimicrobial resistance

Health service implications of the introduction of STI point-of-care testing in Australia

Chlamydia trachomatis (CT) and Neisseria gonorrhoea (NG) are curable sexually transmissible infections (STIs) which represent a significant public health burden, particularly in young Australia Aboriginal people in remote communities. Prompt testing and treatment is fundamental to STI control yet distance to urban laboratories is a significant barrier. CT/NG point-of-care (POC) testing offers an ideal solution, but until recently had been unavailable. This thesis aims to identify settings where POC testing could be beneficial, benefits and barriers to implementation and health service staff acceptability. The thesis was based on two sets of qualitative interviews. The first occurred with 18 Australian key informants with remote, sexual health and laboratory expertise, and generated three discrete studies. The second occurred with 16 trained nurses and Aboriginal health workers (AHWs) from the first 12 remote primary care services internationally to use GeneXpert CT/NG POC testing and resulted in study four. Study one focused on settings where the technology would have greatest benefit, with remote Aboriginal communities most commonly identified, as well as juvenile justice and outreach services to highly mobile or marginalised populations. In study two, informants identified the benefits of POC use for clinical practice including improved management of STIs- more timely and targeted treatment, earlier commencement of partner notification, and reduced effort associated with client recall, but noted it will result in changes to the STI management pathway, and policy and clinical guidelines may need to be altered. Study three focused on the public health implications; with the key perceived benefit being STI control, and barriers including the potential to negatively impact on disease notification and NG antibiotic sensitivity surveillance. In study four, most nurses and AHWs found the test easy to use and useful, and reported improved management of STIs consistent with the key informant’s perceived benefits. In conclusion, this thesis has provided information to inform implementation of CT/NG POC testing, including selection of appropriate settings, and the need to review clinical guidelines and establish systems to avoid adverse impact on public health surveillance. Importantly the research demonstrated the new technology was highly acceptable to staff working in remote primary care

Clinical Management of Complicated Urinary Tract Infection

Complicated urinary tract infections (cUTIs) are a major cause of hospital admissions and are associated with significant morbidity and health care costs. Knowledge of baseline risk of urinary tract infection can help clinicians make informed diagnostic and therapeutic decisions. Prevalence rates of UTI vary by age, gender, race, and other predisposing risk factors. In this regard, this book provides comprehensive information on etiology, epidemiology, immunology, pathology, pathogenic mechanisms, symptomatology, investigation and management of urinary tract infection. Chapters cover common problems in urinary tract infection and put emphasis on the importance of making a correct clinical decision and choosing the appropriate therapeutic approach. Topics are organized to address all of the major complicated conditions frequently seen in urinary tract infection. The authors have paid particular attention to urological problems like the outcome of patients with vesicoureteric reflux, the factors affecting renal scarring, obstructive uropathy, voiding dysfunction and catheter associated problems. This book will be indispensable for all professionals involved in the medical care of patients with urinary tract infection

Pathogenicity & a bedside real-time detection assay for clostridium difficile in the faeces of hospitalized patients

Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is the commonest cause of hospital acquired infection in the UK. The organism initiates infection through spore formation and attachment, germination in the gut and then the production of two potent cytotoxins; toxins A and B. While the contribution of toxins A and B to infection is beyond dispute the relative importance of each toxin is a subject of debate. Thus diagnostic assays capable of rapidly detecting the presence of both toxins are needed. To develop such an assay we first characterised the structure of C. difficile spores to better understand their role in pathogenicity and adherence to organic and inorganic surfaces. Following attachment the spore germinates and the resulting vegetative bacteria express toxins. To facilitate the development of an assay capable of detecting both toxins, we employed a bioinformatics based approach which identified highly conserved nucleotide sequences within regions of each toxin which we hypothesised were under strict selective pressure. The specificity of the probes identified was confirmed using a panel of 58 clinical C. difficile isolates, related Clostridium isolates, non-related species and human gut metagenomic DNA samples. Selected probes were incorporated into a metal enhanced fluorescent assay platform and their ability to detect the organism in various organic backgrounds was determined. We were able to detect as few as 10 bacteria in 500 μl of human faecal material within 40 seconds, suggesting that this approach has the potential to be developed into a commercial assay. To support the development of this assay we sought to develop an insect infection model using the worm Manduca sexta. Our inability to initiate infection, inspite of the fact that bioinformatic analysis revealed the presence of genes with homology to known insect virulence factors, suggests that C. difficile may have potential evolutionary association to invertebrates