Optimisation of a propidium monoazide based method to determine the viability of microbes in faecal slurries for transplantation

© 2018 Elsevier BV. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (December 2018) in accordance with the publisher’s archiving policyThe efficacy of faecal microbiota transplantation (FMT) as a therapeutic intervention may depend on the viability of the microorganisms in faecal slurries (FS) prepared from donor stool. However, determining the viability of these organisms is challenging. Most microorganisms in stool are refractory to culture using standard techniques, and culture-independent PCR-based methods derive signal from both viable and non-viable cells. Propidium monoazide (PMA) treatment has been shown to be effective in preventing PCR amplification of DNA from non-viable bacteria in a range of contexts. However, this methodology can be sensitive to factors such as bacterial load and sample turbidity. We describe the optimisation of a PMA treatment methodology for FS that restricts quantitative PCR-based bacterial enumeration to viable cells. When applied to concentrated FS (10–25% stool content), PMA treatment at 100 μM concentration was ineffective in preventing DNA amplification from heat-killed cells. Efficacy was not significantly improved by doubling the PMA concentration. However, PMA treatment efficacy was improved markedly following 10-fold sample dilution, and was found to be optimal at 100-fold dilution. Substantial reductions in viable bacterial load could be observed following both freeze-thaw and heat-treatment of FS. This method successfully prevented DNA amplification of heat-killed Pseudomonas and Staphylococcus spiked into stool and could reliably determine the proportion of live bacteria and viable E. coli counts present in fresh and heat-treated stool. With appropriate sample dilution, PMA treatment excluded >97% of non-viable cells from amplification in all assays, without significantly affecting the amplification of DNA from viable cells. This method can be applied to optimise sample processing of FMT donor material, and to characterise bacterial viability within faecal samples more widely

Case report: Identification of intra-laboratory blood culture contamination with Staphylococcus aureus by whole genome sequencing

© 2019 Elsevier Inc. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (February 2019) in accordance with the publisher’s archiving policyStaphylococcus aureus in blood cultures is rarely considered a contaminant. We report a case of intra-laboratory contamination between blood culture bottles which was confirmed by whole genome sequencing, highlighting the importance of molecular analysis in the clinical laboratory setting

Preventing empirical antibiotic treatment failure in migrant populations: screening by infection risk, not ethnic background

Multidrug-resistant organisms (MDROs) are a major international health threat. In many low and middle-income countries poorly regulated antibiotic use, limited surveillance, and inadequate sanitation give rise to high rates of antibiotic resistance. A resulting reliance on last-line antibiotic options further contributes to the emergence of MDROs. The potential for these pathogens to spread across international borders is a matter of considerable concern. However, this problem is commonly framed as primarily a threat to the health security of countries where resistance is not yet endemic. In fact, it is little acknowledged that those at greatest risk from antibiotic treatment failure are individuals who move from regions of high MDRO prevalence to settings where standard empirical treatment options remain largely effective. In this perspective, we highlight the poor treatment outcomes for disseminated bacterial infections in individuals who have moved from settings in which MDROs are common to those where MDROs are currently less common. We discuss MDRO screening strategies that could avoid stigmatizing vulnerable populations by focusing on future risk of disseminated infection, rather than past risk of acquisition. In practical terms, this means screening individuals before childbirth, immunosuppressive treatments, major surgery, or other events associated with disseminated infection risk, rather than prioritizing screening for individuals from regions with high carriage rates. We argue that such measures would reduce antibiotic treatment failure and improve outcomes while protecting migrant populations from the divisive consequences of targeted screening programs

Bacterial viability in faecal transplants: Which bacteria survive?Research in context

Background: The therapeutic potential of faecal microbiota transplantation (FMT) is under investigation for a range of inflammatory conditions. While mechanisms of benefit are poorly understood, most models rely on the viability of transplanted microbes. We hypothesised that protocols commonly used in the preparation of faecal transplants will substantially reduce the number, diversity and functional potential of viable microbes. Methods: Stools from eight screened donors were processed under strict anaerobic conditions, in ambient air, and freeze-thawed. Propidium monoazide (PMA) sample treatment was combined with quantitative PCR, 16S rRNA gene amplicon sequencing and short-chain fatty acid (SCFA) analysis to define the viable microbiota composition and functional potential. Findings: Approximately 50% of bacterial content of stool processed immediately under strict anaerobic conditions was non-viable. Homogenisation in ambient air or freeze-thaw reduced viability to 19% and 23% respectively. Processing of samples in ambient air resulted in up to 12-fold reductions in the abundance of important commensal taxa, including the highly butyrogenic species Faecalibacterium prausnitzii, Subdoligranulum variable, and Eubacterium hallii. The adverse impact of atmospheric oxygen exposure on the capacity of the transplanted microbiota to support SCFA biosynthesis was demonstrated by significantly reduced butyrate and acetate production by faecal slurries processed in ambient air. In contrast, while reducing overall levels of viable bacteria, freeze-thaw did not significantly alter viable microbiota composition. Interpretation: The practice of preparing material for faecal transplantation in ambient air profoundly affects viable microbial content, disproportionately reducing the abundance of anaerobic commensals and the capacity for biosynthesis of important anti-inflammatory metabolites. Fund: This work was supported by the South Australian Health and Medical Research Institute. LP is supported by a scholarship from the Flinders Foundation. GR is supported by a Matthew Flinders Research Fellowship. Keywords: Bacterial viability, Fecal microbiota transplantation, qPCR, Propidium monoazid

A longitudinal cohort study of watch and wait in complete clinical responders after chemo-radiotherapy for localised rectal cancer:study protocol

BACKGROUND: Rectal Cancer is a common malignancy. The current treatment approach for patients with locally advanced rectal cancer involves neoadjuvant chemoradiotherapy followed by surgical resection of the rectum. The resection can lead to complications and long-term consequences. A clinical complete response is observed in some patients after chemoradiotherapy. A number of recent studies have shown that patients can be observed safely after completing chemoradiotherapy (without surgery), provided clinical complete response has been achieved. In this approach, resection is reserved for cases of regrowth. This is called the watch and wait approach. This approach potentially avoids unnecessary surgical resection of the rectum and the resulting complications. In this study, we will prospectively investigate this approach. METHODS: Adult patients with a diagnosis of rectal cancer planned to receive neoadjuvant long course chemoradiotherapy (± subsequent combination chemotherapy) will be consented into the study prior to commencing treatment. After completing the chemoradiotherapy (± subsequent combination chemotherapy), based on the clinical response, subjects will be allocated to one of the following arms: subjects who achieved a clinical complete response will be allocated to the watch and wait arm and others to the standard management arm (which includes resection). The aim of the study is to determine the rate of local failure and other safety and efficacy outcomes in the watch and wait arm. Patient reported outcome measures and the use of biomarkers as part of the clinical monitoring will be studied in both arms of the study. DISCUSSION: This study will prospectively investigate the safety of the watch and wait approach. We will investigate predictive biomarkers (molecular biomarkers and imaging biomarkers) and patient reported outcome measures in the study population and the cost effectiveness of the watch and wait approach. This study will also help evaluate a defined monitoring schedule for patients managed with the watch and wait approach. This protocol covers the first two years of follow up, we are planning a subsequent study which covers year 3–5 follow up for the study population. Trial registration. Name of the registry: Australia and New Zealand Clinical Trials Registry (ANZCTR). Trial registration number: Trial ID: ACTRN12619000207112 Registered 13 February 2019,https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=376810 SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12885-022-09304-x

Targeted reduction of airborne viral transmission risk in long-term residential aged care

COVID-19 has demonstrated the devastating consequences of the rapid spread of an airborne virus in residential aged care. We report the use of CO2-based ventilation assessment to empirically identify potential ‘super-spreader’ zones within an aged care facility, and determine the efficacy of rapidly implemented, inexpensive, risk reduction measures

Prevention of SARS-CoV-2 (COVID-19) transmission in residential aged care using ultraviolet light (PETRA): a two-arm crossover randomised controlled trial protocol

Background: SARS-CoV-2 poses a considerable threat to those living in residential aged care facilities (RACF). RACF COVID-19 outbreaks have been characterised by the rapid spread of infection and high rates of severe disease and associated mortality. Despite a growing body of evidence supporting airborne transmission of SARS-CoV-2, current infection control measures in RACF including hand hygiene, social distancing, and sterilisation of surfaces, focus on contact and droplet transmission. Germicidal ultraviolet (GUV) light has been used widely to prevent airborne pathogen transmission. Our aim is to investigate the efficacy of GUV technology in reducing the risk of SARS-CoV-2 infection in RACF. Methods: A multicentre, two-arm double-crossover, randomised controlled trial will be conducted to determine the efficacy of GUV devices to reduce respiratory viral transmission in RACF, as an adjunct to existing infection control measures. The study will be conducted in partnership with three aged care providers in metropolitan and regional South Australia. RACF will be separated into paired within-site zones, then randomised to intervention order (GUV or control). The initial 6-week period will be followed by a 2-week washout before crossover to the second 6-week period. After accounting for estimated within-zone and within-facility correlations of infection, and baseline infection rates (10 per 100 person-days), a sample size of n = 8 zones (n = 40 residents/zone) will provide 89% power to detect a 50% reduction in symptomatic infection rate. The primary outcome will be the incidence rate ratio of combined symptomatic respiratory infections for intervention versus control. Secondary outcomes include incidence rates of hospitalisation for complications associated with respiratory infection; respiratory virus detection in facility air and fomite samples; rates of laboratory confirmed respiratory illnesses and genomic characteristics. Discussion: Measures that can be deployed rapidly into RACF, that avoid the requirement for changes in resident and staff behaviour, and that are effective in reducing the risk of airborne SARS-CoV-2 transmission, would provide considerable benefit in safeguarding a highly vulnerable population. In addition, such measures might substantially reduce rates of other respiratory viruses, which contribute considerably to resident morbidity and mortality. Trial registration Australian and New Zealand Clinical Trials Registry ACTRN12621000567820 (registered on 14th May, 2021).</p

Australian consensus statements for the regulation, production and use of faecal microbiota transplantation in clinical practice

Objective: Faecal microbiota transplantation (FMT) has proved to be an extremely effective treatment for recurrent Clostridioides difficile infection, and there is interest in its potential application in other gastrointestinal and systemic diseases. However, the recent death and episode of septicaemia following FMT highlights the need for further appraisal and guidelines on donor evaluation, production standards, treatment facilities and acceptable clinical indications. Design: For these consensus statements, a 24-member multidisciplinary working group voted online and then convened in-person, using a modified Delphi approach to formulate and refine a series of recommendations based on best evidence and expert opinion. Invitations to participate were directed to Australian experts, with an international delegate assisting the development. The following issues regarding the use of FMT in clinical practice were addressed: donor selection and screening, clinical indications, requirements of FMT centres and future directions. Evidence was rated using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) system. Results: Consensus was reached on 27 statements to provide guidance on best practice in FMT. These include: (1) minimum standards for donor screening with recommended clinical selection criteria, blood and stool testing; (2) accepted routes of administration; (3) clinical indications; (4) minimum standards for FMT production and requirements for treatment facilities acknowledging distinction between single-site centres (eg, hospital-based) and stool banks; and (5) recommendations on future research and product development. Conclusions: These FMT consensus statements provide comprehensive recommendations around the production and use of FMT in clinical practice with relevance to clinicians, researchers and policy makers