Iron Is Critical for Mucosal-Associated Invariant T Cell Metabolism and Effector Functions.

Mucosal-Associated Invariant T (MAIT) cells are a population of innate T cells that play a critical role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells can rapidly respond via both TCR-dependent and -independent mechanisms, resulting in robust cytokine production. The metabolic and nutritional requirements for optimal MAIT cell effector responses are still emerging. Iron is an important micronutrient and is essential for cellular fitness, in particular cellular metabolism. Iron is also critical for many pathogenic microbes, including those that activate MAIT cells. However, iron has not been investigated with respect to MAIT cell metabolic or functional responses. In this study, we show that human MAIT cells require exogenous iron, transported via CD71 for optimal metabolic activity in MAIT cells, including their production of ATP. We demonstrate that restricting iron availability by either chelating environmental iron or blocking CD71 on MAIT cells results in impaired cytokine production and proliferation. These data collectively highlight the importance of a CD71-iron axis for human MAIT cell metabolism and functionality, an axis that may have implications in conditions where iron availability is limited.

Yeast mannan rich fraction positively influences microbiome uniformity, productivity associated taxa, and lay performance.

BACKGROUND: Alternatives to antibiotic as growth promoters in agriculture, such as supplemental prebiotics, are required to maintain healthy and high performing animals without directly contributing to antimicrobial resistance bioburden. While the gut microbiota of broiler hens has been well established and successfully correlated to performance, to our knowledge, a study has yet to be completed on the effect of prebiotic supplementation on correlating the mature laying hen productivity and microbiota. This study focused on establishing the impact of a yeast derived prebiotic, mannan rich fraction (MRF), on the cecal microbiota of late laying hens. This study benefitted from large sample sizes so intra- and intergroup variation effects could be statistically accounted for. RESULTS: Taxonomic richness was significantly greater at all taxonomic ranks and taxonomic evenness was significantly lower for all taxonomic ranks in MRF-supplemented birds (P < 0.005). Use of principal coordinate analyses and principal component analyses found significant variation between treatment groups. When assessed for compositional uniformity (an indicator of flock health), microbiota in MRF-supplemented birds was more uniform than control birds at the species level. From a food safety and animal welfare perspective, Campylobacter jejuni was significantly lower in abundance in MRF-supplemented birds. In this study, species associated with high weight gain (an anticorrelator of performance in laying hens) were significantly lower in abundance in laying hens while health-correlated butyrate and propionate producing species were significantly greater in abundance in MRF-supplemented birds. CONCLUSIONS: The use of prebiotics may be a key factor in controlling the microbiota balance limiting agri-food chain pathogen persistence and in promoting uniformity. In previous studies, increased α- and ß-diversity indices were determinants of pathogen mitigation and performance. MRF-supplemented birds in this study established greater α- and ß-diversity indices in post-peak laying hens, greater compositional uniformity across samples, a lower pathogenic bioburden and a greater abundance of correlators of performance.

Extreme Symptom Burden for Patients With COVID-19 at the End of Life; Extrapolation of Knowledge Gained to Achieve Sustained Comfort and Dignity for all Patients in Their Last Days of Life1.

BACKGROUND: We describe two complex cases in the setting of COVID-19 at the End of Life, to enhance learning for all patients. CASE PRESENTATION: Maintenance of sustained comfort in two cases required multiple drugs, specifically selected for symptoms that necessitated three separate pumps delivering continuous 24-hour subcutaneous infusion. CASE MANAGEMENT: Management of sustained comfort included opioid, midazolam, anti-secretory, diclofenac for intractable temperature, phenobarbital for extreme agitation, in one, where seizure activity was present, while insomnia, was a prominent feature of another. Management of Akatasia was also required. CASE OUTCOME: Attention to each individual patient's rapidly evolving symptoms, during the dying phase, with a thorough differential diagnosis, wa s vitally important in the context of a 'Good Death'. This was achieved in both cases, reflected by evidence at the bedside of comfort and a minimum need for 'as required' drugs in the last days of life. CONCLUSIONS: COVID-19 being a new illness, we need to prospectively study the symptom burden/clustering at End of Life and learn from management of this new disease for other illnesses also. Further research is required to develop protocols on; when does Midazolam dose reach tolerance and when should an alternative drug such as phenobarbital for sustained Gamma-Aminobutyric Acid effects be initiated; examine the optimal approach to sustained temperature control; be cognisant of extrapyramidal side effects of drugs used at End of Life and consider looking at a lack of need for 'as required' drugs in the last days of life as an outcome measure of sustained comfort.

Education and Training Adaptations for Health Workers during the COVID-19 Pandemic: A Scoping Review of Lessons Learned and Innovations.

Introduction: The COVID-19 pandemic has considerably impacted the clinical education and training of health workers globally, causing severe disruptions to learning environments in healthcare facilities and limiting the acquisition of new clinical skills. Consequently, urgent adaptation measures, including simulation training and e-learning, have been implemented to mitigate the adverse effects of clinical education. This scoping review aims to assess the impact of COVID-19 on medical education and training, examine the implemented adaptation measures, and evaluate their effectiveness in improving health workers' education and training during the pandemic. Methods: Employing the PRISMA-ScR framework and Arksey and O'Malley's methodological guidance, we conducted a scoping review, systematically searching PubMed, medRxiv, Google, and DuckDuckGo databases to account for the grey literature. The search included studies published between 1 December 2019 and 13 October 2021, yielding 10,323 results. Of these, 88 studies focused on health worker education and training during the pandemic. Results: Our review incorporated 31,268 participants, including physicians, medical trainees, nurses, paramedics, students, and health educators. Most studies (71/88, 81%) were conducted in high-income and lower-middle-income countries. The pandemic's effects on health workers' clinical skills and abilities have necessitated training period extensions in some cases. We identified several positive outcomes from the implementation of simulation training and e-learning as adaptation strategies, such as enhanced technical and clinical performance, increased confidence and comfort, and an expanded global educational outreach. Conclusions: Despite challenges like insufficient practical experience, limited interpersonal interaction opportunities, and internet connectivity issues, simulation training, e-learning, and virtual training have proven effective in improving clinical education and training during the COVID-19 pandemic. Further research is required to bolster preparedness for future pandemics or similar situations.

Acinetobacter baumannii from grass: novel but non-resistant clones.

Acinetobacter baumannii is one the most worrisome nosocomial pathogens, which has long been considered almost mainly as a hospital-associated bacterium. There have been some studies about animal and environmental isolates over the last decade. However, little effort has been made to determine if this pathogen dwells in the grass. Here, we aim to determine the evolutionary relationships and antibiotic resistance of clones of A. baumannii sampled from grass to the major human international clones and animal clones. Two hundred and forty genomes were considered in total from four different sources for this study. Our core and accessory genomic epidemiology analyses showed that grass isolates cluster in seven groups well differentiated from one another and from the major human and animal isolates. Furthermore, we found new sequence types under both multilocus sequence typing schemes: two under the Pasteur scheme and seven for the Oxford scheme. The grass isolates contained fewer antibiotic-resistance genes and were not resistant to the antibiotics tested. Our results demonstrate that these novel clones appear to have limited antibiotic resistance potential. Given our findings, we propose that genomic epidemiology and surveillance of A. baumannii should go beyond the hospital settings and consider the environment in an explicit One Health approach.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Differential impact of swine, bovine and poultry manure on the microbiome and resistome of agricultural grassland.

Land spreading of animal manure is an essential process in agriculture. Despite the importance of grassland in global food security the potential of the grass phyllosphere as a reservoir of antimicrobial resistance (AMR) is unknown. Additionally, the comparative risk associated with different manure sources is unclear. Due to the One Health nature of AMR there is an urgent need to fully understand the risk associated with AMR at the agriculture - environmental nexus. We performed a grassland field study to assess and compare the relative and temporal impact of bovine, swine and poultry manure application on the grass phyllosphere and soil microbiome and resistome over a period of four months, using 16S rRNA amplicon sequencing and high-throughput quantitative PCR (HT-qPCR). The soil and grass phyllosphere contained a diverse range of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). Manure treatment was found to introduce ARGs belonging to clinically important antimicrobial classes, such as aminoglycoside and sulphonamide into grass and soil. Temporal analysis of ARGs and MGEs associated with manure treatment indicated ARGs patterns were similar across the different manure types in the manure treated soil and grass phyllosphere. Manure treatment resulted in the enrichment in members of the indigenous microbiota and the introduction of manure associated bacteria, with this impact extending past the recommended six-week exclusion period. However, these bacteria were in low relative abundance and manure treatment was not found to significantly impact the overall composition of the microbiome or resistome. This provides evidence that the current guidelines facilitate reduction of biological risk to livestock. Additionally, in soil and grass samples MGEs correlated with ARGs from clinically important antimicrobial classes, indicating the key role MGEs play in horizontal gene transfer in agricultural grassland. These results demonstrate the role of the grass phyllosphere as an under-studied sink of AMR.

Trends in obstetric anal sphincter injury in spontaneous vaginal delivery versus operative vaginal delivery over a 10-year period: Lessons learned.

OBJECTIVE: To evaluate trends in the incidence of women with obstetric anal sphincter injuries (OASIS) over a 10-year period comparing spontaneous vaginal delivery (SVD) and operative vaginal delivery (OVD). METHODS: A retrospective study was performed in which all women who underwent vaginal delivery over a 10-year period (n = 86 242, 2009-2018) at the Rotunda Hospital were reviewed. The overall incidence of OASIS was compared with incidence rates stratified by parity and type of vaginal birth. RESULTS: The 10-year vaginal delivery rate was 69% (n = 59 187) where 24 580 women (42%) were primiparous and 34 607 women (58%) were multiparous. SVD rate was 74% and OVD rate was 26%. The overall incidence of OASIS was 2.9%. The incidence of OASIS in OVD was 5.5% and the incidence in SVD was 2%. Of 498 multipara who sustained OASIS, 366 (73%) had an SVD without episiotomy compared with 14 (3%) who had an episiotomy. There was a significant reduction of OASIS over the 10-year period in primipara who had an OVD but no reduction in the other groups. CONCLUSION: The primiparous OVD group had a significant reduction of OASIS. Continued education around perineal protection and episiotomy at SVD could positively impact further reduction in OASIS, particularly in the SVD groups.

First Peoples' knowledge leads scientists to reveal 'fairy circles' and termite linyji are linked in Australia.

In the past, when scientists encountered and studied 'new' environmental phenomena, they rarely considered the existing knowledge of First Peoples (also known as Indigenous or Aboriginal people). The scientific debate over the regularly spaced bare patches (so-called fairy circles) in arid grasslands of Australian deserts is a case in point. Previous researchers used remote sensing, numerical modelling, aerial images and field observations to propose that fairy circles arise from plant self-organization. Here we present Australian Aboriginal art and narratives, and soil excavation data, that suggest these regularly spaced, bare and hard circles in grasslands are pavement nests occupied by Drepanotermes harvester termites. These circles, called linyji (Manyjilyjarra language) or mingkirri (Warlpiri language), have been used by Aboriginal people in their food economies and for other domestic and sacred purposes across generations. Knowledge of the linyji has been encoded in demonstration and oral transmission, ritual art and ceremony and other media. While the exact origins of the bare circles are unclear, being buried in deep time and Jukurrpa, termites need to be incorporated as key players in a larger system of interactions between soil, water and grass. Ecologically transformative feedbacks across millennia of land use and manipulation by Aboriginal people must be accounted for. We argue that the co-production of knowledge can both improve the care and management of those systems and support intergenerational learning within and across diverse cultures.

Comparison of soil and grass microbiomes and resistomes reveals grass as a greater antimicrobial resistance reservoir than soil.

Grasslands cover a large proportion of global agricultural landmass used to feed herbivores and ruminants and link the environment to the food chain via animals onto humans. However, most scientific studies of antimicrobial resistance and microbiomes at the environmental - animal nexus have focused on soil or vegetables rather than grasslands. Based on previous microbiome phyllosphere-soil studies we hypothesised that the microbiome and resistomes across soil and grass would have a core of shared taxa and antimicrobial resistance genes (ARGs), but that in addition each would also have a minority of unique signatures. Our data indicated grass contained a wider variety and higher relative abundance of ARGs and mobile genetic elements (MGEs) than soil with or without slurry amendments. The microbiomes of soil and grass were similar in content but varied in the composition proportionality. While there were commonalities across many of the ARGs present in soil and on grass their correlations with MGEs and bacteria differed, suggesting a source other than soil is also relevant for the resistome of grass. The variations in the relative abundances of ARGs in soil and on grass also indicated that either the MGEs or the bacteria carrying the ARGs comprised a higher relative abundance on grass than in soil. We conclude that while soil may be a source of some of these genes it cannot be the source for all ARGs and MGEs. Our data identifies grass as a more diverse and abundant reservoir of ARGs and MGEs in the environment than soil, which is significant to human and animal health when viewed in the context of grazing food animals.

Effect of Mannan-rich fraction supplementation on commercial broiler intestinum tenue and cecum microbiota.

BACKGROUND: The broiler gastrointestinal microbiome is a potent flock performance modulator yet may also serve as a reservoir for pathogen entry into the food chain. The goal of this project was to characterise the effect of mannan rich fraction (MRF) supplementation on microbiome diversity and composition of the intestinum tenue and cecum of commercial broilers. This study also aimed to address some of the intrinsic biases that exist in microbiome studies which arise due to the extensive disparity in 16S rRNA gene copy numbers between bacterial species and due to large intersample variation. RESULTS: We observed a divergent yet rich microbiome structure between different anatomical sites and observed the explicit effect MRF supplementation had on community structure, diversity, and pathogen modulation. Birds supplemented with MRF displayed significantly higher species richness in the cecum and significantly different bacterial community composition in each gastrointestinal (GI) tract section. Supplemented birds had lower levels of the zoonotic pathogens Escherichia coli and Clostridioides difficile across all three intestinum tenue sites highlighting the potential of MRF supplementation in maintaining food chain integrity. Higher levels of probiotic genera (eg. Lactobacillus and Blautia) were also noted in the MRF supplemented birds. Following MRF supplementation, the cecum displayed higher relative abundances of both short chain fatty acid (SFCA) synthesising bacteria and SCFA concentrations. CONCLUSIONS: Mannan rich fraction addition has been observed to reduce the bioburden of pathogens in broilers and to promote greater intestinal tract microbial biodiversity. This study is the first, to our knowledge, to investigate the effect of mannan-rich fraction supplementation on the microbiome associated with different GI tract anatomical geographies. In addition to this novelty, this study also exploited machine learning and biostatistical techniques to correct the intrinsic biases associated with microbiome community studies to enable a more robust understanding of community structure.

Comparative genomics and pangenomics of vancomycin-resistant and susceptible Enterococcus faecium from Irish hospitals.

Introduction. Enterococcus faecium has emerged as an important nosocomial pathogen, which is increasingly difficult to treat due to the genetic acquisition of vancomycin resistance. Ireland has a recalcitrant vancomycin-resistant bloodstream infection rate compared to other developed countries.Hypothesis/Gap statement. Vancomycin resistance rates persist amongst E. faecium isolates from Irish hospitals. The evolutionary genomics governing these trends have not been fully elucidated.Methodology. A set of 28 vancomycin-resistant isolates was sequenced to construct a dataset alongside 61 other publicly available Irish genomes. This dataset was extensively analysed using in silico methodologies (comparative genomics, pangenomics, phylogenetics, genotypics and comparative functional analyses) to uncover distinct evolutionary, coevolutionary and clinically relevant population trends.Results. These results suggest that a stable (in terms of genome size, GC% and number of genes), yet genetically diverse population (in terms of gene content) of E. faecium persists in Ireland with acquired resistance arising via plasmid acquisition (vanA) or, to a lesser extent, chromosomal recombination (vanB). Population analysis revealed five clusters with one cluster partitioned into four clades which transcend isolation dates. Pangenomic and recombination analyses revealed an open (whole genome and chromosomal specific) pangenome illustrating a rampant evolutionary pattern. Comparative resistomics and virulomics uncovered distinct chromosomal and mobilomal propensity for multidrug resistance, widespread chromosomal point-mutation-mediated resistance and chromosomally harboured arsenals of virulence factors. Interestingly, a potential difference in biofilm formation strategies was highlighted by coevolutionary analysis, suggesting differential biofilm genotypes between vanA and vanB isolates.Conclusions. These results highlight the evolutionary history of Irish E. faecium isolates and may provide insight into underlying infection dynamics in a clinical setting. Due to the apparent ease of vancomycin resistance acquisition over time, susceptible E. faecium should be concurrently reduced in Irish hospitals to mitigate potential resistant infections.

Shooting hoops: globetrotting plasmids spreading more than just antimicrobial resistance genes across One Health.

Our study provides novel insights into the global nature of antimicrobial resistance (AMR) plasmids across the food chain. We provide compelling evidence of the globetrotting nature of AMR plasmids and the need for surveillance to sequence plasmids with a template of analyses for others to expand these data. The AMR plasmids analysed were detected in 63 countries and in samples from humans, animals and the environment. They contained a combination of known and novel AMR genes, metal resistance genes, virulence factors, phage and replicon types.

Metagenomic and HT-qPCR analysis reveal the microbiome and resistome in pig slurry under storage, composting, and anaerobic digestion.

Direct application of pig slurry to agricultural land, as a means of nutrient recycling, introduces pathogens, antibiotic resistant bacteria, or genes, to the environment. With global environmental sustainability policies mandating a reduction in synthetic fertilisation and a commitment to a circular economy it is imperative to find effective on-farm treatments of slurry that maximises its fertilisation value and minimises risk to health and the environment. We assessed and compared the effect of storage, composting, and anaerobic digestion (AD) on pig slurry microbiome, resistome and nutrient content. Shotgun metagenomic sequencing and HT-qPCR arrays were implemented to understand the dynamics across the treatments. Our results identified that each treatment methods have advantages and disadvantages in removal pollutants or increasing nutrients. The data suggests that storage and composting are optimal for the removal of human pathogens and anaerobic digestion for the reduction in antibiotic resistance (AMR) genes and mobile genetic elements. The nitrogen content is increased in storage and AD, while reduced in composting. Thus, depending on the requirement for increased or reduced nitrogen the optimum treatment varies. Combining the results indicates that composting provides the greatest gain by reducing risk to human health and the environment. Network analysis revealed reducing Proteobacteria and Bacteroidetes while increasing Firmicutes will reduce the AMR content. KEGG analysis identified no significant change in the pathways across all treatments. This novel study provides a data driven decision tree to determine the optimal treatment for best practice to minimise pathogen, AMR and excess or increasing nutrient transfer from slurry to environment.

Global protein responses of multidrug resistance plasmid-containing Escherichia coli to ampicillin, cefotaxime, imipenem and ciprofloxacin.

OBJECTIVES: This study compared the proteomics of Escherichia coli containing the multidrug resistance plasmid pEK499 under antimicrobial stress and with no antimicrobial. METHODS: We utilised mass spectrometry-based proteomics to compare the proteomes of the bacteria and plasmid under antimicrobial stress and no antimicrobial. RESULTS: Our analysis identified statistically significant differentially abundant (SSDA) proteins common to groups exposed to the ß-lactam antimicrobials but not ciprofloxacin, indicating a ß-lactam stress response to exposure from this class of drugs, irrespective of ß-lactam resistance or susceptibility. Data arising from comparisons of the proteomes of ciprofloxacin-treated E. coli and controls detected an increase in the relative abundance of proteins associated with ribosomes, translation, the TCA cycle and several proteins associated with detoxification, and a decrease in the relative abundance of proteins associated with the stress response, including oxidative stress. We identified changes in proteins associated with persister formation in the presence of ciprofloxacin but not the ß-lactams. The plasmid proteome differed across each treatment and did not follow the pattern of antimicrobial-antimicrobial resistance (AMR) protein associations: a relative increase was observed in the amount of CTX-M-15 in the presence of cefotaxime and ciprofloxacin, but not the other ß-lactams, suggesting regulation of CTX-M-15 protein production. CONCLUSION: The proteomic data from this study provided novel insights into the proteins produced from the chromosome and plasmid under different antimicrobial stresses. These data also identified novel proteins not previously associated with AMR or antimicrobial responses in pathogens, which may well represent potential targets of AMR inhibition.

Tracing Antibiotic Resistance Genes along the Irrigation Water Chain to Chive: Does Tap or Surface Water Make a Difference?

Irrigation water is well known as potential source of pathogens in fresh produce. However, its role in transferring antibiotic resistance determinants is less well investigated. Therefore, we analyzed the contribution of surface and tap water to the resistome of overhead-irrigated chive plants. Field-grown chive was irrigated with either surface water (R-system) or tap water (D-system), from planting to harvest. Water along the two irrigation chains as well as the respective plants were repeatedly sampled and screened for 264 antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), using high-capacity qPCR. Differentially abundant (DA) ARGs were determined by comparing the two systems. On R-chive, ß-lactam ARGs, multidrug-resistance (MDR) determinants, and MGEs were most abundant, while D-chive featured DA ARGs from the vancomycin class. Diversity and number of DA ARGs was the highest on young chives, strongly diminished at harvest, and increased again at the end of shelf life. Most ARGs highly enriched on R- compared to D-chive were also enriched in R- compared to D-sprinkler water, indicating that water played a major role in ARG enrichment. Of note, blaKPC was detected at high levels in surface water and chive. We conclude that water quality significantly affects the resistome of the irrigated produce.

Investigation into the effect of mannan-rich fraction supplementation on the metagenome of broiler chickens.

Antibiotic resistance is regarded as one of the most serious threats to human health worldwide. The rapid increase in resistance rates has been attributed to the extensive use of antibiotics since they became commercially available. The use of antibiotics as growth promotors has been banned in numerous regions for this reason. Mannan-rich fraction (MRF) has been reported to show similar growth-promoting effects to antibiotics. We investigated the effect of MRF on the microbial community, resistome and metabolic pathways within the caecum of commercial broilers at two different timepoints within the growth of the broiler, day 27 and day 34. The data indicated an overall increase in health and economic gain for the producer with the addition of MRF to the diet of the broilers. The only significant difference across the microbial composition of the samples was in the richness of the microbial communities across all samples. While all samples harboured resistance genes conferring resistance to the same classes of antibiotics, there was significant variation in the antimicrobial resistance gene richness across time and treatment and across combinations of time and treatment. The taxa with positive correlation comprised Bacilli and Clostridia. The negative correlation taxa were also dominated by Bacilli, specifically the Streptococcus genera. The KEGG-pathway analysis identified an age-related change in the metabolism pathway abundances of the caecal microflora. We suggest that the MRF-related increases in health and weight gain in the broilers may be associated with changes in the metabolism of the microbiomes rather than the microbial composition. The resistome variations across samples were correlated with specific genera. These data may be used to further enhance the development of feed supplements to reduce the presence of antibiotic resistance genes (ARGs) within poultry. While the ARGs of greatest concern to human or animal health were not detected in this study, it has identified the potential to reduce the presence of ARGs by the increase in specific genera.

Longitudinal analysis of client appointment adherence under Universal Test and Treat strategy: A stepped-wedge trial.

OBJECTIVES: Universal Test and Treat (UTT) strategies are being adopted across sub-Saharan Africa based on clinical benefits to morbidity and mortality and to attain targets of the Joint United Nations Programme on HIV/AIDS (UNAIDS). Universal Test and Treat is expected to change the client population at clinics, due to more asymptomatic HIV clients initiating antiretroviral therapy (ART). We assessed the impact of UTT on client appointment adherence at 14 government-managed health facilities in Eswatini's public sector health system. METHODS: We assessed the impact of UTT on client adherence to appointment schedules from 2014 to 2017 in a stepped-wedge trial. Repeated measures analysis was used to assess adherence to each scheduled appointment (primary definition: presenting for care within 7 days after the scheduled appointment), adjusting for time, age, sex, stage, marital status, ART status and facility. RESULTS: Among 3354 clients (62.1% female; 57.4% < 35 years), a median (interquartile range) of 10 (6-15) appointments were scheduled during follow-up. In a multivariable-adjusted model, appointment adherence was significantly greater in clients who were female [odds ratio (OR) = 1.38, 95% confidence interval (CI): 1.25-1.52], older (e.g. 40 to < 50 years vs. < 20 years; OR = 1.45, 95% CI: 1.00-2.09), married (OR = 1.31, 95% CI: 1.19-1.44), had lower WHO stage at study enrolment (1-2 vs. 3-4: OR = 1.26, 95% CI: 1.13-1.41), and were currently on ART (OR = 3.55, 95% CI: 2.62-4.82). However, UTT strategy was not significantly associated with client adherence to scheduled appointments (OR = 1.02, 95% CI: 0.72-1.45). CONCLUSIONS: Despite transitioning to UTT, there was no change in visit adherence, a reassuring finding given the large volume of clients currently being initiated at earlier stages of HIV.