Directed evolution of bacteriophages: thwarted by prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, where we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing revealed that the observed host-range expansion was due to a Casadabanvirus prophage originating from a lysogenic host that was only included in the first three rounds of the experiment. This prophage could infect five of eight bacterial hosts initially used, allowing it to persist and proliferate until the termination of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment, but despite being subjected to directed evolution conditions, it does not appear to have evolved. This work highlights the impact of prophages in directed evolution experiments and the importance of genetically verifying output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine the presence of prophage.IMPORTANCEDirected evolution is a common strategy for evolving phages to expand the host range, often targeting pathogenic strains of bacteria. In this study, we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophages are active players in directed evolution and can contribute to observation of host-range expansion. Since prophages are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving the experimental design of future directed evolution studies.

Viral adaptations to alternative hosts in the honey bee pathogen Paenibacillus larvae.

Bacteriophages (phages) that are intended to be used to treat bacterial infections are often improved using genetic engineering or experimental evolution. A protocol called "Appelmans" utilizes evolution in microtiter plates to promote the evolution of phages that can infect nonpermissive hosts. We tested a modification of the Appelmans protocol using the honey bee pathogen, Paenibacillus larvae. Three phages evolved together on four P. larvae strains following the standard Appelmans protocol and a modified version to ensure high phage diversity throughout ten rounds of passaging. The host range of 360 plaques were characterized and six new phage lysis patterns were identified. These new phage lysis patterns included plaque formation on previously nonpermissive, phage-resistant isolates that were used to identify phage types. The modified protocol did not drastically change the rate or number of new phage types observed but did prevent the phage population from being dominated by one phage that tended to rapidly raise in frequency. These findings showed how a minor modification of the Appelmans protocol influenced the development of phages for phage therapy. The method also provided improved phages for the treatment of bacterial infections in honey bees.

Less than full-time training, barriers and future needs. A UK-wide online survey of doctors in training.

BACKGROUND: The the current views of less-than full-time (LTFT) training by both LTFT and full-time (FT) doctors in training, with regards to health and well-being, clinical and non-clinical opportunities, in addition to associated future workforce implications and challenges, are uncertain. METHODS: We performed a cross-sectional study of UK-based doctors in training via an online questionnaire, designed and piloted by the Royal College of Physicians Edinburgh (RCPE) Trainees and Members Committee. Design was informed by prior investigation into LTFT training undertaken amongst similar populations by RCPE in 2019. The survey was distributed from May to September 2023 via email to trainees across the United Kingdom and was open to all specialties and training grades. The survey focused on lived experience of both LTFT and FT training, as well as perceived challenges for the future workforce. RESULTS: There were 648 responses from doctors in training across the United Kingdom, with an even spread of LTFT and FT trainees. Most responses (53%) were from trainees in medical specialties. Most LTFT trainees plan to train at this pace for more than 5 years. LTFT was commonly linked to improved work-life balance and well-being, with FT training perceived to feature high stress and burnout. Concerns around LTFT relate to administrative errors, reduced training opportunities and colleague prejudice. Trainees are concerned about LTFT leading to rota gaps and incoherent workforce planning around training numbers. CONCLUSION: With ever-increasing popularity in LTFT training, flexible and robust long-term workforce planning is necessary for the system to adapt to a new normal and improve retention of doctors in training.

Carrion use by a reptile is influenced by season, habitat and competition with an apex mammalian scavenger.

Scavenging on carrion is critical and often fiercely competitive for a range of vertebrate species, from native apex predators to invasive species and even reptiles. Within Australia, a notable reptilian scavenger is the lace monitor (Varanus varius). In this study, we quantified lace monitor activity at carcasses and compared their use of the resource to common co-occurring predators that also scavenge; the invasive red fox (Vulpes vulpes) and a native apex predator, the dingo (Canis dingo). To do so, we deployed 80 macropod carcasses equally across seasons (summer and winter) and habitats (open and closed canopy) in a temperate bioregion and monitored vertebrate scavenging with camera traps. Lace monitor activity (visitation at carcass sites inclusive of both non-scavenging and scavenging events) was 1.67 times higher in summer than in winter, but it did not differ across closed and open habitats. Monitor activity occurred earlier after carcass deployment at sites deployed in summer than winter (1.47-fold earlier), and at carcasses in open than closed habitats (0.22-fold earlier). Lace monitors initially discovered carcass sites faster in summer than winter and before both red foxes and dingoes in summer. The species was active diurnally in both summer and winter, differing from the red fox, which was strictly a nocturnal scavenger and the dingo, which was significantly more active at night across both seasons. Finally, we found that lace monitor activity at carcass sites decreased slightly with higher rates of activity for dingoes (0.04-fold decrease as dingo activity increased), but not with red fox activity. Our results have implications for understanding lace monitor foraging and scavenging and highlight the value of monitoring carcasses to provide important insights into the behaviour of varanid lizards that scavenge.

Bacteriophage resistance evolution in a honey bee pathogen.

Honey bee (Apis mellifera) larvae are susceptible to the bacterial pathogen Paenibacillus larvae, which causes severe damage to bee colonies. Antibiotic treatment requires veterinary supervision in the United States, is not used in many parts of the world, perpetuates problems associated with antibiotic resistance, and can necessitate residual testing in bee products. There is interest in using bacteriophages to treat infected colonies (bacteriophage therapy) and several trials are promising. Nevertheless, the safety of using biological agents in the environment must be scrutinized. In this study we analyzed the ability of P. larvae to evolve resistance to several different bacteriophages. We found that bacteriophage resistance is rapidly developed in culture but often results in growth defects. Mutations in the bacteriophage-resistant isolates are concentrated in genes encoding potential surface receptors. Testing one of these isolates in bee larvae, we found it to have reduced virulence compared to the parental P. larvae strain. We also found that bacteriophages are likely able to counteract resistance evolution. This work suggests that while bacteriophage-resistance may arise, its impact will likely be mitigated by reduced pathogenicity and secondary bacteriophage mutations that overcome resistance.

Directed evolution of bacteriophages: impacts of prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods is to propagate phage on multiple bacterial hosts, pool the lysate, and repeat the propagation process until phage(s) can form plaques on the target host(s). In theory, this propagation process produces a phage lysate that contains input phages and their evolved phage progeny. However, in practice, this phage lysate can also include prophages originating from bacterial hosts. Here we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, in which we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing analysis revealed that this observed host-range expansion was due to a Casadabanvirus prophage that originated from one of the Appelmans hosts. Host-range analysis of the prophage showed that it could infect five of eight bacterial hosts initially used, allowing it to proliferate and persist through the end of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment. This work highlights the impact of prophages in directed evolution experiments and the importance of incorporating sequencing data in analyses to verify output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine presence of prophage. IMPORTANCE: Directed evolution is a common strategy for evolving phages to expand host range, often targeting pathogenic strains of bacteria. In this study we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophage are active players in directed evolution and can contribute to observation of host-range expansion. Since prophage are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving experimental design of future directed evolution studies.

Wombat burrows are hotspots for small vertebrates in a landscape subject to gigafire.

Ecosystem engineers modify their environment and influence the availability of resources for other organisms. Burrowing species, a subset of allogenic engineers, are gaining recognition as ecological facilitators. Burrows created by these species provide habitat for a diverse array of other organisms. Following disturbances, burrows could also serve as ecological refuges, thereby enhancing ecological resistance to disturbance events. We explored the ecological role of Common Wombat (Vombatus ursinus) burrows using camera traps in forests of southeastern Australia. We compared animal activity at paired sites with and without burrows, from the same fire severity class and habitat. We examined how animal activity at Common Wombat burrows was affected by the 2019-20 Black Summer bushfires in Australia. We predicted that burrows would serve as hotspots for animal activity and as refuges in burned areas. The activity of several species including Bush Rat (Rattus fuscipes), Agile Antechinus (Antechinus agilis), Lace Monitor (Varanus varius), Painted Button-quail (Turnix varius), and Grey Shrike-thrush (Colluricincla harmonica) increased at sites where Common Wombat burrows were present, while other species avoided burrows. Species that were more active at burrows tended to be smaller mammal and bird species that are vulnerable to predation, whereas species that avoided burrows tended to be larger mammals that might compete with Common Wombat for resources. Species composition differed between sites with and without burrows, and burrow sites had higher native mammal species richness. The association of several species with burrows persisted or strengthened in areas that burned during the 2019-20 Black Summer bushfires, suggesting that Common Wombat burrows may act as ecological refuges for animals following severe wildfire. Our findings have relevance for understanding how animals survive, persist, and recover following extreme wildfire events.

Flooding, season and habitat interact to drive changes in vertebrate scavenging and carcass persistence rates.

Scavenging dynamics are influenced by many abiotic and biotic factors, but there is little knowledge of how scavengers respond to extreme weather events. As carrion is a major driver of the organisation and structure of food webs within ecological communities, understanding the response of scavengers to extreme weather events is critical in a world that is increasingly subject to climate change. In this study, vertebrate scavenging and carcass persistence rates were quantified in the Simpson Desert of central Australia; a system that experiences major fluctuations and extremes in weather conditions. Specifically, a total of 80 adult red kangaroo (Osphranter rufus) carcasses were placed on the landscape and monitored using remote sensor cameras. This included 40 carcasses monitored before and then 40 carcasses monitored after a major flooding event. The carcasses were monitored equally before and after the flood across different seasons (warm and cool) and in dune and interdune habitats. Overall, a total of 8124 scavenging events for 97,976 visitation minutes were recorded for 11 vertebrate species within 30 days of carcass placement pre- and post-flood. Vertebrate scavenging increased post-flood in the warm season, especially by corvids which quadrupled their scavenging events during this time. There was little difference in carcass persistence between habitats, but carcasses persisted 5.3-fold longer post-flood in warm seasons despite increased vertebrate scavenging. The results demonstrate that a flood event can influence scavenging dynamics and suggest a need to further understand how seasons, habitats and extreme weather events can drive changes in carrion-based food webs.

Spatial and temporal analysis of HIV clinical outcomes in Florida reveals counties with persistent racial and ethnic disparities during 2012-2019.

BACKGROUND: Racial/ethnic disparities in the HIV care continuum have been well documented in the US, with especially striking inequalities in viral suppression rates between White and Black persons with HIV (PWH). The South is considered an epicenter of the HIV epidemic in the US, with the largest population of PWH living in Florida. It is unclear whether any disparities in viral suppression or immune reconstitution-a clinical outcome highly correlated with overall prognosis-have changed over time or are homogenous geographically. In this analysis, we 1) investigate longitudinal trends in viral suppression and immune reconstitution among PWH in Florida, 2) examine the impact of socio-ecological factors on the association between race/ethnicity and clinical outcomes, 3) explore spatial and temporal variations in disparities in clinical outcomes. METHODS: Data were obtained from the Florida Department of Health for 42,369 PWH enrolled in the Ryan White program during 2008-2020. We linked the data to county-level socio-ecological variables available from County Health Rankings. GEE models were fit to assess the effect of race/ethnicity on immune reconstitution and viral suppression longitudinally. Poisson Bayesian hierarchical models were fit to analyze geographic variations in racial/ethnic disparities while adjusting for socio-ecological factors. RESULTS: Proportions of PWH who experienced viral suppression and immune reconstitution rose by 60% and 45%, respectively, from 2008-2020. Odds of immune reconstitution and viral suppression were significantly higher among White [odds ratio =2.34, 95% credible interval=2.14-2.56; 1.95 (1.85-2.05)], and Hispanic [1.70 (1.54-1.87); 2.18(2.07-2.31)] PWH, compared with Black PWH. These findings remained unchanged after accounting for socio-ecological factors. Rural and urban counties in north-central Florida saw the largest racial/ethnic disparities. CONCLUSIONS: There is persistent, spatially heterogeneous, racial/ethnic disparity in HIV clinical outcomes in Florida. This disparity could not be explained by socio-ecological factors, suggesting that further research on modifiable factors that can improve HIV outcomes among Black and Hispanic PWH in Florida is needed.

Depression and Anxiety Symptoms and Treatment Utilization, and Associated HIV Outcomes among Adults with HIV in Rural Florida.

Persons living with HIV (PLWH) and depression or anxiety in the rural South may have suboptimal HIV outcomes. We sought to examine the proportion of PLWH from rural Florida with symptoms of depression or anxiety, the proportion who received depression or anxiety treatment, and the relationship between untreated and treated symptoms of depression or anxiety and HIV outcomes. Cross-sectional survey data collected between 2014 and 2018 were analyzed. Among 187 PLWH residing in rural Florida (median age 49 years, 61.5%, male 45.5% Black), 127 (67.9%) met criteria for symptoms of depression and/or anxiety. Among these 127 participants, 60 (47.2%) were not on depression or anxiety treatment. Participants with untreated symptoms of depression and anxiety (OR 3.2, 95% CI 1.2-9.2, p = 0.03) and treated depression and anxiety with uncontrolled symptoms (OR 1.4, 95% CI 0.5-4.0, p = 0.52) were more likely to have viral non-suppression compared to those without depression or anxiety in an unadjusted bivariate analysis. Only the association between untreated symptoms of depression and anxiety and viral non-suppression was statistically significant, and when adjusting for social and structural confounders the association was attenuated and was no longer statistically significant. This suggests that social and structural barriers impact both mental health and HIV outcomes. Our findings support the need for increased mental health services and resources that address the social and structural barriers to care for PLWH in the rural South.

Scavenging with invasive species.

Carrion acts as a hotspot of animal activity within many ecosystems globally, attracting scavengers that rely on this food source. However, many scavengers are invasive species whose impacts on scavenging food webs and ecosystem processes linked to decomposition are poorly understood. Here, we use Australia as a case study to review the extent of scavenging by invasive species that have colonised the continent since European settlement, identify the factors that influence their use of carcasses, and highlight the lesser-known ecological effects of invasive scavengers. From 44 published studies we identified six invasive species from 48 vertebrates and four main groups of arthropods (beetles, flies, ants and wasps) that scavenge. Invasive red foxes (Vulpes vulpes), domestic dogs (Canis familiaris), feral pigs (Sus scrofa), black rats (Rattus rattus) and feral cats (Felis catus) were ranked as highly common vertebrate scavengers. Invasive European wasps (Vespula germanica) are also common scavengers where they occur. We found that the diversity of native vertebrate scavengers is lower when the proportion of invasive scavengers is higher. We highlight that the presence of large (apex) native vertebrate scavengers can decrease rates of scavenging by invasive species, but that invasive scavengers can monopolise carcass resources, outcompete native scavengers, predate other species around carcass resources and even facilitate invasion meltdowns that affect other species and ecological processes including altered decomposition rates and nutrient cycling. Such effects are likely to be widespread where invasive scavengers occur and suggest a need to determine whether excessive or readily available carcass loads are facilitating or exacerbating the impacts of invasive species on ecosystems globally.

The Impact of HIV-Related Stigma on Racial/Ethnic Disparities in Retention in HIV Care Among Adults Living with HIV in Florida.

BACKGROUND: Our study examines the effects of distinct HIV stigma subtypes on retention in care and racial-ethnic differences among persons with HIV (PWH). METHODS: Using Florida Medical Monitoring Project 2015-2017 data, we analyzed patients' clinical and behavioral characteristics. We analyzed 89,889 PWH in Florida (50.0% non-Hispanic Blacks, 20.8% Hispanics, 29.2% non-Hispanic whites). HIV stigma subtypes, negative self-image, anticipated stigma, personalized stigma, and retention in care were examined with logistic regressions. RESULTS: People with high negative self-image and anticipated stigma were less likely to be retained (CI: 0.84-0.92; 0.47-0.53). The association between HIV-related stigma subtypes and retention in care differed between Black, White, and Hispanic participants. Negative self-image was associated with higher retention rates among Hispanics (CI: 5.64-9.26) and Whites (CI: 1.04-1.27), while low retention rates among Blacks (0.617-0.686). The likelihood of staying in care was lower across all racial-ethnic groups when the anticipated stigma was high or moderate. In contrast, personalized stigma increased retention across all racial-ethnic groups. CONCLUSION: Results showed that distinct types of HIV stigma differentially impact retention, and these associations differ by race and ethnicity. Future interventions should address the effect HIV stigma subtypes have on racially minoritized PWH retention.

Network typologies predict future molecular linkages in the network of HIV transmission.

OBJECTIVE: HIV molecular transmission network typologies have previously demonstrated associations to transmission risk; however, few studies have evaluated their predictive potential in anticipating future transmission events. To assess this, we tested multiple models on statewide surveillance data from the Florida Department of Health. DESIGN: This was a retrospective, observational cohort study examining the incidence of new HIV molecular linkages within the existing molecular network of persons with HIV (PWH) in Florida. METHODS: HIV-1 molecular transmission clusters were reconstructed for PWH diagnosed in Florida from 2006 to 2017 using the HIV-TRAnsmission Cluster Engine (HIV-TRACE). A suite of machine-learning models designed to predict linkage to a new diagnosis were internally and temporally externally validated using a variety of demographic, clinical, and network-derived parameters. RESULTS: Of the 9897 individuals who received a genotype within 12 months of diagnosis during 2012-2017, 2611 (26.4%) were molecularly linked to another case within 1 year at 1.5% genetic distance. The best performing model, trained on two years of data, was high performing (area under the receiving operating curve = 0.96, sensitivity = 0.91, and specificity = 0.90) and included the following variables: age group, exposure group, node degree, betweenness, transitivity, and neighborhood. CONCLUSIONS: In the molecular network of HIV transmission in Florida, individuals' network position and connectivity predicted future molecular linkages. Machine-learned models using network typologies performed superior to models using individual data alone. These models can be used to more precisely identify subpopulations for intervention.

Does the Relationship Between HIV Stigma Subtypes and Viral Suppression Differ by Age?: A Stratified Analysis of Data from the Florida Medical Monitoring Project.

HIV-related stigma is recognized as a top barrier to achieve viral suppression in the United States, but data describing who is most affected by HIV stigma is limited. The study sought to (1) identify the relationships between HIV-related stigma and unsuppressed viral load and (2) examine whether the association between HIV stigma subtypes and unsuppressed viral load differ by age group (i.e., 18-34, 35-49, and 50+ years-old) using surveillance data from the Florida Medical Monitoring Project (n = 1195). Most participants were 50+ years-old (55%), male (71%), and Black (51%). Enacted stigma was significantly associated with unsuppressed viral loads among the 18-34-year-old age group (OR 1.68, CI 1.09-2.60). After adjusting for potential confounders, only enacted stigma was independently associated with unsuppressed viral load in the 18-34-year-old age group. Results highlight the need for targeted interventions to reduce enacted stigma among younger persons with HIV to achieve viral suppression.

Reply to Vorland et al. Mixed Random and Nonrandom Allocation, and Group Randomization Have Been Mislabeled and Misanalysed, Necessitating Reanalysis. Comment on "Conner et al. KiwiC for Vitality: Results of a Randomized Placebo-Controlled Trial Testing the Effects of Kiwifruit or Vitamin C Tablets on Vitality in Adults with Low Vitamin C Levels. Nutrients 2020, 12, 2898".

We have read the Comment article by Vorland et al. [...].

Molecular Epidemiology of HIV-1 Subtype B Infection across Florida Reveals Few Large Superclusters with Metropolitan Origin.

Florida is considered an epicenter of HIV in the United States. The U.S. federal plan for Ending the HIV Epidemic (EHE) within 10 years prioritizes seven of Florida's 67 counties for intervention. We applied molecular epidemiology methods to characterize the HIV infection networks in the state and infer whether the results support the EHE. HIV sequences (N = 34,446) and associated clinical/demographic metadata of diagnosed people with HIV (PWH), during 2007 to 2017, were retrieved from the Florida Department of Health. HIV genetic networks were investigated using MicrobeTrace. Associates of clustering were identified through boosted logistic regression. Assortative trait mixing was also assessed. Bayesian phylogeographic methods were applied to evaluate evidence of imported HIV-1 lineages and illustrate spatiotemporal flows within Florida. We identified nine large clusters spanning all seven EHE counties but little evidence of external introductions, suggesting-in the absence of undersampling-an epidemic that evolved independently from the rest of the country or other external influences. Clusters were highly assortative by geography. Most of the sampled infections (82%) did not cluster with others in the state using standard molecular surveillance methods despite satisfactory sequence sampling in the state. The odds of being unclustered were higher among PWH in rural regions, and depending on demographics. A significant number of unclustered sequences were observed in counties omitted from EHE. The large number of missing sequence links may impact timely detection of emerging transmission clusters and ultimately hinder the success of EHE in Florida. Molecular epidemiology may help better understand infection dynamics at the population level and underlying disparities in disease transmission among subpopulations; however, there is also a continuous need to conduct ethical discussions to avoid possible harm of advanced methodologies to vulnerable groups, especially in the context of HIV stigmatization. IMPORTANCE The large number of missing phylogenetic linkages in rural Florida counties and among women and Black persons with HIV may impact timely detection of ongoing and emerging transmission clusters and ultimately hinder the success of epidemic elimination goals in Florida.

Correction: Conner et al. KiwiC for Vitality: Results of a Placebo-Controlled Trial Testing the Effects of Kiwifruit or Vitamin C Tablets on Vitality in Adults with Low Vitamin C Levels. Nutrients 2020, 12, 2898.

In the original publication title [...].

Marijuana Use and Health Outcomes in Persons Living With HIV: Protocol for the Marijuana Associated Planning and Long-term Effects (MAPLE) Longitudinal Cohort Study.

BACKGROUND: Marijuana use is common in persons with HIV, but there is limited evidence of its relationship with potential health benefits or harms. OBJECTIVE: The Marijuana Associated Planning and Long-term Effects (MAPLE) study was designed to evaluate the impact of marijuana use on HIV-related health outcomes, cognitive function, and systemic inflammation. METHODS: The MAPLE study is a longitudinal cohort study of participants living with HIV who were recruited from 3 locations in Florida and were either current marijuana users or never regular marijuana users. At enrollment, participants completed questionnaires that included detailed marijuana use assessments, underwent interviewer-administered neurocognitive assessments, and provided blood and urine samples. Ongoing follow-ups included brief telephone assessments (every 3 months), detailed questionnaires (annually), repeated blood and urine samples (2 years), and linkage to medical records and statewide HIV surveillance data. Supplemental measures related to intracellular RNA, COVID-19, Alzheimer disease, and the gut microbiome were added after study initiation. RESULTS: The MAPLE study completed enrollment of 333 persons between 2018 and 2021. The majority of participants in the sample were ≥50 years of age (200/333, 60.1%), male (181/333, 54.4%), cisgender men (173/329, 52.6%), non-Hispanic Black (221/333, 66.4%), and self-reported marijuana users (260/333, 78.1%). Participant follow-up was completed in 2022, with annual updates to HIV surveillance data through at least 2027. CONCLUSIONS: The MAPLE study is the largest cohort specifically designed to understand the use of marijuana and its effects on HIV-related outcomes. The study population has significant diversity across age, sex, gender, and race. The data will help clinicians and public health officials to better understand patterns of marijuana use associated with both positive and negative health outcomes, and may inform recommendations for future clinical trials related to medical marijuana and HIV. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/37153.