High-Moisture Extrusion of a Dietary Protein Blend Impairs In Vitro Digestion and Delays In Vivo Postprandial Plasma Amino Acid Availability in Humans.

BACKGROUND: Industrial processing can alter the structural complexity of dietary proteins and, potentially, their digestion and absorption upon ingestion. High-moisture extrusion (HME), a common processing method used to produce meat alternative products, affects in vitro digestion, but human data are lacking. We hypothesized that HME of a mycoprotein/pea protein blend would impair in vitro digestion and in vivo postprandial plasma amino acid availability. METHODS: In Study A, 9 healthy volunteers completed 2 experimental trials in a randomized, double-blind, crossover design. Participants consumed a beverage containing 25 g protein from a "dry" blend (CON) of mycoprotein/pea protein (39%/61%) or an HME content-matched blend (EXT). Arterialized venous blood samples were collected in the postabsorptive state and regularly over a 5-h postprandial period to assess plasma amino acid concentrations. In Study B, in vitro digestibility of the 2 beverages were assessed using bicinchoninic acid assay and optical fluorescence microscopy at baseline and during and following gastric and intestinal digestion using the INFOGEST model of digestion. RESULTS: Protein ingestion increased plasma total, essential (EAA), and branched-chain amino acid (BCAA) concentrations (time effect, P < 0.0001) but more rapidly and to a greater magnitude in the CON compared with the EXT condition (condition × time interaction, P < 0.0001). This resulted in greater plasma availability of EAA and BCAA concentrations during the early postprandial period (0-150 min). These data were corroborated by the in vitro approach, which showed greater protein availability in the CON (2150 ± 129 mg/mL) compared with the EXT (590 ± 41 mg/mL) condition during the gastric phase. Fluorescence microscopy revealed clear structural differences between the 2 conditions. CONCLUSIONS: These data demonstrate that HME delays in vivo plasma amino acid availability following ingestion of a mycoprotein/pea protein blend. This is likely due to impaired gastric phase digestion as a result of HME-induced aggregate formation in the pea protein. This trial was registered at clinicaltrials.gov as NCT05584358.

Investigating the Human Intestinal DNA Virome and Predicting Disease-Associated Virus-Host Interactions in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Understanding how the human virome, and which of its constituents, contributes to health or disease states is reliant on obtaining comprehensive virome profiles. By combining DNA viromes from isolated virus-like particles (VLPs) and whole metagenomes from the same faecal sample of a small cohort of healthy individuals and patients with severe myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), we have obtained a more inclusive profile of the human intestinal DNA virome. Key features are the identification of a core virome comprising tailed phages of the class Caudoviricetes, and a greater diversity of DNA viruses including extracellular phages and integrated prophages. Using an in silico approach, we predicted interactions between members of the Anaerotruncus genus and unique viruses present in ME/CFS microbiomes. This study therefore provides a framework and rationale for studies of larger cohorts of patients to further investigate disease-associated interactions between the intestinal virome and the bacteriome.

The Proteome of Extracellular Vesicles Produced by the Human Gut Bacteria Bacteroides thetaiotaomicron In Vivo Is Influenced by Environmental and Host-Derived Factors.

Bacterial extracellular vesicles (BEVs) released from both Gram-negative and Gram-positive bacteria provide an effective means of communication and trafficking of cell signaling molecules. In the gastrointestinal tract (GIT) BEVs produced by members of the intestinal microbiota can impact host health by mediating microbe-host cell interactions. A major unresolved question, however, is what factors influence the composition of BEV proteins and whether the host influences protein packaging into BEVs and secretion into the GIT. To address this, we have analyzed the proteome of BEVs produced by the major human gut symbiont Bacteroides thetaiotaomicron both in vitro and in vivo in the murine GIT in order to identify proteins specifically enriched in BEVs produced in vivo. We identified 113 proteins enriched in BEVs produced in vivo, the majority (62/113) of which accumulated in BEVs in the absence of any changes in their expression by the parental cells. Among these selectively enriched proteins, we identified dipeptidyl peptidases and an asparaginase and confirmed their increased activity in BEVs produced in vivo. We also showed that intact BEVs are capable of degrading bile acids via a bile salt hydrolase. Collectively these findings provide additional evidence for the dynamic interplay of host-microbe interactions in the GIT and the existence of an active mechanism to drive and enrich a selected group of proteins for secretion into BEVs in the GIT. IMPORTANCE The gastrointestinal tract (GIT) harbors a complex community of microbes termed the microbiota that plays a role in maintaining the host's health and wellbeing. How this comes about and the nature of microbe-host cell interactions in the GIT is still unclear. Recently, nanosized vesicles naturally produced by bacterial constituents of the microbiota have been shown to influence responses of different host cells although the molecular basis and identity of vesicle-born bacterial proteins that mediate these interactions is unclear. We show here that bacterial extracellular vesicles (BEVs) produced by the human symbiont Bacteroides thetaiotaomicron in the GIT are enriched in a set of proteins and enzymes, including dipeptidyl peptidases, an asparaginase and a bile salt hydrolase that can influence host cell biosynthetic pathways. Our results provide new insights into the molecular basis of microbiota-host interactions that are central to maintaining GIT homeostasis and health.

Desulfovibrio diazotrophicus sp. nov., a sulfate-reducing bacterium from the human gut capable of nitrogen fixation.

Sulfate-reducing bacteria (SRB) are widespread in human guts, yet their expansion has been linked to colonic diseases. We report the isolation, sequencing and physiological characterization of strain QI0027T , a novel SRB species belonging to the class Desulfovibrionia. Metagenomic sequencing of stool samples from 45 Chinese individuals, and comparison with 1690 Desulfovibrionaceae metagenome-assembled genomes recovered from humans of diverse geographic locations, revealed the presence of QI0027T in 22 further individuals. QI0027T encoded nitrogen fixation genes and based on the acetylene reduction assay, actively fixed nitrogen. Transcriptomics revealed that QI0027T overexpressed 42 genes in nitrogen-limiting conditions compared to cultures supplemented with ammonia, including genes encoding nitrogenases, a urea uptake system and the urease complex. Reanalyses of 835 public stool metatranscriptomes showed that nitrogenase genes from Desulfovibrio bacteria were expressed in six samples suggesting that nitrogen fixation might be active in the gut environment. Although frequently thought of as a nutrient-rich environment, nitrogen fixation can occur in the human gut. Animals are often nitrogen limited and have evolved diverse strategies to capture biologically active nitrogen, ranging from amino acid transporters to stable associations with beneficial microbes that provide fixed nitrogen. QI0027T is the first Desulfovibrio human isolate for which nitrogen fixation has been demonstrated, suggesting that some sulfate-reducing bacteria could also play a role in the availability of nitrogen in the gut.

Results of a multicentre UK-wide retrospective study evaluating the efficacy of brentuximab vedotin in relapsed, refractory classical Hodgkin lymphoma in the transplant naive setting.

Relapsed or refractory classical Hodgkin lymphoma (cHL) is associated with a poor outcome when standard chemotherapy fails. Brentuximab vedotin (BV) is an anti-CD30 monoclonal antibody-drug conjugate licensed for use at relapse after autologous stem cell transplant (ASCT) or following two prior therapies in those unsuitable for ASCT. There are limited data assessing the ability of BV to enable curative SCT. We performed a UK-wide retrospective study of 99 SCT-naïve relapsed/refractory cHL. All had received 2 prior lines and were deemed fit for transplant but had an insufficient remission to proceed. The median age was 32 years. Most had nodular sclerosis subtype, Eastern Cooperative Oncology Group performance status 0-1 and advanced stage disease. The median progression-free survival (PFS) was 5·6 months and median overall survival (OS) was 37·2 months. The overall response rate was 56% (29% complete response; 27% partial response). 61% reached SCT: 34% immediately post-BV and 27% following an inadequate BV response but were salvaged and underwent deferred SCT. Patients consolidated with SCT had a superior PFS and OS to those not receiving SCT (P < 0·001). BV is an effective, non-toxic bridge to immediate SCT in 34% and deferred SCT in 27%. 39% never reached SCT with a PFS of 3·0 months, demonstrating the unmet need to improve outcomes in those unsuitable for SCT post-BV.

Development and validation of a LC-MS/MS assay for quantitation of plasma citrulline for application to animal models of the acute radiation syndrome across multiple species.

The potential risk of a radiological catastrophe highlights the need for identifying and validating potential biomarkers that accurately predict radiation-induced organ damage. A key target organ that is acutely sensitive to the effects of irradiation is the gastrointestinal (GI) tract, referred to as the GI acute radiation syndrome (GI-ARS). Recently, citrulline has been identified as a potential circulating biomarker for radiation-induced GI damage. Prior to biologically validating citrulline as a biomarker for radiation-induced GI injury, there is the important task of developing and validating a quantitation assay for citrulline detection within the radiation animal models used for biomarker validation. Herein, we describe the analytical development and validation of citrulline detection using a liquid chromatography tandem mass spectrometry assay that incorporates stable-label isotope internal standards. Analytical validation for specificity, linearity, lower limit of quantitation, accuracy, intra- and interday precision, extraction recovery, matrix effects, and stability was performed under sample collection and storage conditions according to the Guidance for Industry, Bioanalytical Methods Validation issued by the US Food and Drug Administration. In addition, the method was biologically validated using plasma from well-characterized mouse, minipig, and nonhuman primate GI-ARS models. The results demonstrated that circulating citrulline can be confidently quantified from plasma. Additionally, circulating citrulline displayed a time-dependent response for radiological doses covering GI-ARS across multiple species.

Source reservoir controls on the size, frequency, and composition of large-scale volcanic eruptions.

Large-scale, explosive volcanic eruptions are one of the Earth's most hazardous natural phenomena. We demonstrate that their size, frequency, and composition can be explained by processes in long-lived, high-crystallinity source reservoirs that control the episodic creation of large volumes of eruptible silicic magma and its delivery to the subvolcanic chamber where it is stored before eruption. Melt percolates upward through the reservoir and accumulates a large volume of low-crystallinity silicic magma which remains trapped until buoyancy causes magma-driven fractures to propagate into the overlying crust, allowing rapid magma transfer from the reservoir into the chamber. Ongoing melt percolation in the reservoir accumulates a new magma layer and the process repeats. Our results suggest that buoyancy, rather than crystallinity, is the key control on magma delivery from the source reservoir. They identify an optimum reservoir size for the largest silicic eruptions that is consistent with data from natural systems and explain why larger magnitude eruptions are not observed on Earth.

Comparison of Antibacterial Activity of Phytochemicals against Common Foodborne Pathogens and Potential for Selection of Resistance.

Antimicrobial resistance is now commonly observed in bacterial isolates from multiple settings, compromising the efficacy of current antimicrobial agents. Therefore, there is an urgent requirement for efficacious novel antimicrobials to be used as therapeutics, prophylactically or as preservatives. One promising source of novel antimicrobial chemicals is phytochemicals, which are secondary metabolites produced by plants for numerous purposes, including antimicrobial defence. In this report, we compare the bioactivity of a range of phytochemical compounds, testing their ability to directly inhibit growth or to potentiate other antimicrobials against Salmonella enterica Typhimurium, Pseudomonas aeruginosa, Listeria monocytogenes, and Staphylococcus aureus. We found that nine compounds displayed consistent bioactivity either as direct antimicrobials or as potentiators. Thymol at 0.5 mg/mL showed the greatest antimicrobial effect and significantly reduced the growth of all species, reducing viable cell populations by 66.8%, 43.2%, 29.5%, and 70.2% against S. enterica Typhimurium, S. aureus, P. aeruginosa, and L. monocytogenes, respectively. Selection of mutants with decreased susceptibility to thymol was possible for three of the pathogens, at a calculated rate of 3.77 × 10-8, and characterisation of S. enterica Typhimurium mutants showed a low-level MDR phenotype due to over-expression of the major efflux system AcrAB-TolC. These data show that phytochemicals can have strong antimicrobial activity, but emergence of resistance should be evaluated in any further development.

Comparison of three-weekly and six-weekly pembrolizumab United Kingdom prescribing practice for advanced and resected melanoma.

BACKGROUND: Pembrolizumab is approved for the treatment of advanced and resected melanoma and was originally licensed as a three-weekly infusion (Q3W). In April 2019, a six-weekly infusion schedule (Q6W) was also approved. We retrospectively reviewed pembrolizumab prescribing for patients with melanoma across multiple United Kingdom (UK) centres to compare the safety and efficacy of Q6W with Q3W in real-world clinical practice. METHODS: Case notes for melanoma patients treated with pembrolizumab between April 2019 and August 2020 at eight UK centres were reviewed. Prespecified baseline characteristics of the Q3W and Q6W cohorts were compared, as well as toxicity and efficacy outcomes. Prescribers were surveyed about their prescribing practice. RESULTS: Two hundred seventy-seven patients were included: 116 commenced Q3W and 161 commenced Q6W pembrolizumab. The proportion of Q6W prescriptions varied by the centre (range 32-88%). Patient factors associated with an increased likelihood of receiving Q3W over Q6W were preexisting autoimmune comorbidity (odds ratio [OR] 0.33; 95% confidence interval [CI] 0.12-0.82) and treatment for advanced (versus resected) disease (OR 0.54; 95%CI 0.33-0.90). Toxicity outcomes were broadly similar for Q6W and Q3W: 14.9% versus 15.5% ≥ grade 3 Common Terminology Criteria for Adverse Events. Estimated 12-month recurrence-free survival for adjuvantly treated patients was 78.9% for Q6W and 74.2% for Q3W (hazard ratio [HR] 0.93; 95%CI 0.50-1.73). Estimated 12-month progression-free survival for advanced patients was 41.8% for Q6W and 55.9% for Q3W (HR 1.21, 95%CI 0.67-2.18). CONCLUSIONS: Q6W is an appropriate option for administering pembrolizumab, given the opportunity to reduce the health service resource burden.

Differential effects of cyclosporin and etanercept treatment on various pathologic parameters in a murine model of irradiation-induced mucositis.

BACKGROUND: Radiation therapy is the most prescribed treatment for many oncologic indications. One of its common side effects is mucositis with hallmark apoptosis in the intestinal crypt and diarrhea. OBJECTIVE: We investigated the potential beneficial effects of etanercept and cyclosporin treatment during radiation exposure. The effects of these drugs on intestinal apoptosis, long-term weight loss, diarrhea severity, and survival were examined. METHODS: For acute observation studies, animals pretreated with phosphate buffer saline (PBS) vehicle, either etanercept, or cyclosporin were challenged with either 1 Gy or 13 Gy irradiation and sacrificed 6 hours later. The animals' small intestines were then harvested for histologic analysis. For chronic survival studies, 14.5 Gy irradiation was applied. Etanercept or cyclosporin treatments were given 15 minutes before the irradiation, followed by daily administration. RESULTS: At 6 hours postirradiation the maximum apoptotic index observed in the small intestine was ∼25% for both 1 Gy and 13 Gy irradiation. Etanercept and cyclosporin pretreatment had no effect on the irradiation-induced apoptosis. During chronic observation, the rate of weight loss was similar in all test groups. At 7 days postirradiation, the weight loss in phosphate buffered saline-treated control, etanercept, and cyclosporin groups reached a maximum at 19%, 24%, and 31.8%, respectively. The weight lost in the cyclosporin group was significantly higher than in the control group. Neither treatment reduced the severity of diarrhea, but cyclosporin increased the survival rate. Sixty percent of cyclosporin-treated animals survived compared with 27% in the PBS-treated control group and 47% in the etanercept-treated group. Serum tumor necrosis factor-α levels, a biomarker for both etanercept's mechanism of action and treatment efficacy, was inhibited by etanercept throughout the study, but cyclosporin only showed an inhibitory effect at 48 hours postirradiation. CONCLUSIONS: Our study demonstrates that cyclosporin increases the survival rate of irradiated animals without affecting parameters such as intestinal histology, weight loss, and diarrhea severity.

Extracellular vesicles produced by the human commensal gut bacterium Bacteroides thetaiotaomicron affect host immune pathways in a cell-type specific manner that are altered in inflammatory bowel disease.

The gastrointestinal (GI) tract harbours a complex microbial community, which contributes to its homeostasis. A disrupted microbiome can cause GI-related diseases, including inflammatory bowel disease (IBD), therefore identifying host-microbe interactions is crucial for better understanding gut health. Bacterial extracellular vesicles (BEVs), released into the gut lumen, can cross the mucus layer and access underlying immune cells. To study BEV-host interactions, we examined the influence of BEVs generated by the gut commensal bacterium, Bacteroides thetaiotaomicron, on host immune cells. Single-cell RNA sequencing data and host-microbe protein-protein interaction networks were used to predict the effect of BEVs on dendritic cells, macrophages and monocytes focusing on the Toll-like receptor (TLR) pathway. We identified biological processes affected in each immune cell type and cell-type specific processes including myeloid cell differentiation. TLR pathway analysis highlighted that BEV targets differ among cells and between the same cells in healthy versus disease (ulcerative colitis) conditions. The in silico findings were validated in BEV-monocyte co-cultures demonstrating the requirement for TLR4 and Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) in BEV-elicited NF-kB activation. This study demonstrates that both cell-type and health status influence BEV-host communication. The results and the pipeline could facilitate BEV-based therapies for the treatment of IBD.

In vivo effects of immunomodulators in a murine model of Fluorouracil-induced mucositis.

BACKGROUND: Fluorouracil (5-FU) is a pyrimidine analogue used as a cancer treatment. Its toxic side effects, including mucositis, are reported to occur in 40% of the treated patients. Because of the inflammatory component of mucositis, we explored the possibility of modulating this condition with an immunomodulatory agent and a tumor necrosis factor-α inhibitor. OBJECTIVE: The aim of this study was to evaluate the effect of 2 immunosuppressive agents, etanercept and cyclosporine, in a murine model of 5-FU-induced mucositis. METHODS: To study the short-term effects of 5-FU on mucositis, cyclosporine and etanercept were administered to mice after an injection of 5-FU. The animals (n = 8) were euthanized at 6 hours post-challenge. Hematoxylin and eosin-stained histologic sections of the small intestine were examined for signs of apoptosis. To further examine the potential of cyclosporine in the treatment of 5-FU-induced mucositis in a longer duration, the animals (N = 15) were given 2 challenges of 5-FU within 6 hours. All mice were dosed daily until day 9 with either cyclosporine (100 mg/kg) or phosphate-buffered saline (PBS). RESULTS: Six hours after 5-FU challenge, 25 mg/kg etanercept and 50 mg/kg cyclosporine had no effect on 5-FU-induced apoptosis (P > 0.05). However, 100 mg/kg cyclosporine significantly reduced the cumulative level of apoptosis >41.6% of the intestinal crypt surface (P < 0.05). During long-term observation, all mice began to lose weight at a rate of approximately 0.8 g/day after 5-FU exposure. The rates of weight loss and survival were not affected by cyclosporine treatment. The diarrhea onset began on day 4 with 46.7% of the PBS-treated mice showing signs of diarrhea compared with 53.3% in the cyclosporine group. The diarrhea score for both groups plateaued on day 7, with a cumulative score of 41 for the PBS group and 50 for the cyclosporine group. Cyclosporine treatment did not affect the diarrhea onset day or severity compared with the PBS-treated group (P > 0.05). CONCLUSIONS: Our data indicated that etanercept is not a suitable treatment for 5-FU-induced mucositis. Despite decreased apoptosis in the gut, cyclosporine did not affect the severity of the diarrhea or survival. Therefore, we concluded that cyclosporine treatment was only effective in mediating the short-term apoptotic events in the intestines but has no long-term effect on the animals' survival and diarrhea.

The Natural History of Acute Radiation-induced H-ARS and Concomitant Multi-organ Injury in the Non-human Primate: The MCART Experience.

ABSTRACT: The dose response relationship and corresponding values for mid-lethal dose and slope are used to define the dose- and time-dependent parameters of the hematopoietic acute radiation syndrome. The characteristic time course of mortality, morbidity, and secondary endpoints are well defined. The concomitant comorbidities, potential mortality, and other multi-organ injuries that are similarly dose- and time-dependent are less defined. Determination of the natural history or pathophysiology associated with the lethal hematopoietic acute radiation syndrome is a significant gap in knowledge, especially when considered in the context of a nuclear weapon scenario. In this regard, the exposure is likely ill-defined, heterogenous, and nonuniform. These conditions forecast sparing of bone marrow and increased survival from the acute radiation syndrome consequent to threshold doses for the delayed effects of acute radiation exposure due to marrow sparing, medical management, and use of approved medical countermeasures. The intent herein is to provide a composite natural history of the pathophysiology concomitant with the evolution of the potentially lethal hematopoietic acute radiation syndrome derived from studies that focused on total body irradiation and partial body irradiation with bone marrow sparing. The marked differential in estimated LD50/60 from 7.5 Gy to 10.88 Gy for the total body irradiation and partial body irradiation with 5% bone marrow sparing models, respectively, provided a clear distinction between the attendant multiple organ injury and natural history of the two models that included medical management. Total body irradiation was focused on equivalent LD50/60 exposures. The 10 Gy and 11 Gy partial body with 5% bone marrow sparing exposures bracketed the LD50/60 (10.88 Gy). The incidence, progression, and duration of multiple organ injury was described for each exposure protocol within the hematopoietic acute radiation syndrome. The higher threshold doses for the partial body irradiation with bone marrow sparing protocol induced a marked degree of multiple organ injury to include lethal gastrointestinal acute radiation syndrome, prolonged crypt loss and mucosal damage, immune suppression, acute kidney injury, body weight loss, and added clinical comorbidities that defined a complex timeline of organ injury through the acute hematopoietic acute radiation syndrome. The natural history of the acute radiation syndrome presents a 60-d time segment of multi-organ sequelae that is concomitant with the latent period or time to onset of the evolving multi-organ injury of the delayed effects of acute radiation exposure.

The Origin of Plasma-Derived Bacterial Extracellular Vesicles in Healthy Individuals and Patients with Inflammatory Bowel Disease: A Pilot Study.

The gastrointestinal tract harbors the gut microbiota, structural alterations of which (dysbiosis) are linked with an increase in gut permeability ("leaky gut"), enabling luminal antigens and bacterial products such as nanosized bacterial extracellular vesicles (BEVs) to access the circulatory system. Blood-derived BEVs contain various cargoes and may be useful biomarkers for diagnosis and monitoring of disease status and relapse in conditions such as inflammatory bowel disease (IBD). To progress this concept, we developed a rapid, cost-effective protocol to isolate BEV-associated DNA and used 16S rRNA gene sequencing to identify bacterial origins of the blood microbiome of healthy individuals and patients with Crohn's disease and ulcerative colitis. The 16S rRNA gene sequencing successfully identified the origin of plasma-derived BEV DNA. The analysis showed that the blood microbiota richness, diversity, or composition in IBD, healthy control, and protocol control groups were not significantly distinct, highlighting the issue of 'kit-ome' contamination in low-biomass studies. Our pilot study provides the basis for undertaking larger studies to determine the potential use of blood microbiota profiling as a diagnostic aid in IBD.

Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome.

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as "viral dark matter". However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating "rare" members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.

Validation of the Decontamination of a Specialist Transport System for Patients with High Consequence Infectious Diseases.

When transferring highly infective patients to specialist hospitals, safe systems of work minimise the risk to healthcare staff. The EpiShuttle is a patient transport system that was developed to fit into an air ambulance. A validated decontamination procedure is required before the system can be adopted in the UK. Hydrogen peroxide (H2O2) vapour fumigation may offer better penetration of the inaccessible parts than the liquid disinfectant wiping that is currently suggested. To validate this, an EpiShuttle was fumigated in a sealed test chamber. Commercial bacterial spore indicators (BIs), alongside organic liquid suspensions and dried surface samples of MS2 bacteriophage (a safe virus surrogate), were placed in and around the EpiShuttle, for the purpose of evaluation. The complete kill of all of the BIs in the five test runs demonstrated the efficacy of the fumigation cycle. The log reduction of the MS2 that was dried on the coupons ranged from 2.66 to 4.50, but the log reduction of the MS2 that was in the organic liquids only ranged from 0.07 to 1.90, confirming the results of previous work. Fumigation with H2O2 alone may offer insufficient inactivation of viruses in liquid droplets, therefore a combination of fumigation and disinfectant surface wiping was proposed. Initial fumigation reducing contamination with minimal intervention allows disinfectant wipe cleaning to be completed more safely, with a second fumigation step inactivating the residual pathogens.