A descriptive analysis of dengue in Peace Corps Volunteers, 2000-2019.

BACKGROUND: Peace Corps Volunteers (PCVs) are a unique expatriate population at risk for dengue. Previous studies examined travelers or lacked demographic information about expatriates. We examined dengue incidence among PCVs before and after deployment of an electronic medical record (EMR) to assess temporal and demographic factors. METHODS: Dengue cases within Peace Corps' Epidemiologic Surveillance System from 2000 to 2019 were identified using a standard case definition, and two timeframes were compared: pre-EMR 2000-2015 and post-EMR 2016-2019. RESULTS: Annual infections occurred in a roughly 3-year cyclic pattern from 2007 to 2019. Incidence rate decreased from 1.35 cases per 100 dengue Volunteer-years (95% CI 1.28-1.41) in 2000-2015 to 1.25 cases (95% CI 1.10-1.41) in 2016-2019. Among PCVs who served from 2016 to 2019, the majority of infections occurred in females and 20-29 year olds, and 7% were medically evacuated. Among PCVs who served from 2015 to 2019, 21% were hospitalized in-country. CONCLUSIONS: Among PCVs, a non-significant decrease in dengue incidence occurred from 2000-2015 to 2016-2019. Annual infection rates peaked every three years, offering opportunities for targeted prevention efforts. Dengue infection in PCVs appears to mimic the overall demographic of Peace Corps. Expatriates like PCVs are at an increased risk for dengue infection compared to short-term travelers.

Natural or Synthetic RNA Delivery: A Stoichiometric Comparison of Extracellular Vesicles and Synthetic Nanoparticles.

RNA therapeutics have high potential that is yet to be fully realized, largely due to challenges involved in the appropriate delivery to target cells. Extracellular vesicles (EVs) are lipid bound nanoparticles released by cells of all types and possess numerous features that may help overcome this hurdle and have emerged as a promising RNA delivery vehicle candidate. Despite extensive research into the engineering of EVs for RNA delivery, it remains unclear how the intrinsic RNA delivery efficiency of EVs compares to currently used synthetic RNA delivery vehicles. Using a novel CRISPR/Cas9-based RNA transfer reporter system, we compared the delivery efficiency of EVs to clinically approved state-of-the-art DLin-MC3-DMA lipid nanoparticles and several in vitro transfection reagents. We found that EVs delivered RNA several orders of magnitude more efficiently than these synthetic systems. This finding supports the continued research into EVs as potential RNA delivery vehicles.

Publisher Correction: A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.

Drug Delivery with Extracellular Vesicles: From Imagination to Innovation.

Extracellular vesicles are nanoparticles produced by cells. They are composed of cellular membrane with associated membrane proteins that surrounds an aqueous core containing soluble molecules such as proteins and nucleic acids, like miRNA and mRNA. They are important in many physiological and pathological processes as they can transfer biological molecules from producer cells to acceptor cells. Preparation of the niche for cancer metastasis, stimulation of tissue regeneration and orchestration of the immune response are examples of the diverse processes in which extracellular vesicles have been implicated. As a result, these vesicles have formed a source of inspiration for many scientific fields. They could be used, for example, as liquid biopsies in diagnostics, as therapeutics in regenerative medicine, or as drug delivery vehicles for transport of medicines. In this Account, we focus on drug delivery applications. As we learn more and more about these vesicles, the complexity increases. What originally appeared to be a relatively uniform population of cellular vesicles is increasingly subdivided into different subsets. Cells make various distinct vesicle types whose physicochemical aspects and composition is influenced by parental cell type, cellular activation state, local microenvironment, biogenesis pathway, and intracellular cargo sorting routes. It has proven difficult to assess the effects of changes in production protocol on the characteristics of the cell-derived vesicle population. On top of that, each isolation method for vesicles necessarily enriches certain vesicle classes and subpopulations while depleting others. Also, each method is associated with a varying degree of vesicle purity and concomitant coisolation of nonvesicular material. What emerges is a staggering heterogeneity. This constitutes one of the main challenges of the field as small changes in production and isolation protocols may have large impact on the vesicle characteristics and on subsequent vesicle activity. We try to meet this challenge by careful experimental design and development of tools that enable robust readouts. By engineering the surface and cargo of extracellular vesicles through chemical and biological techniques, favorable characteristics can be enforced while unfavorable qualities can be overruled or masked. This is coupled to the precise evaluation of the interaction of extracellular vesicles with cells to determine the extracellular vesicle uptake routes and intracellular routing. Sensitive reporter assays enable reproducible analysis of functional delivery. This systematic evaluation and optimization of extracellular vesicles improves our insight into the critical determinants of extracellular vesicle activity and should improve translation into clinical application of engineered extracellular vesicles as a new class of drug delivery systems.

An assessment of household water quality among Peace Corps volunteers in Guatemala.

BACKGROUND: Gastrointestinal (GI) illness is the most commonly reported health concern among Peace Corps Volunteers (PCVs) serving in Guatemala. This project identified water types and treatment and storage practices used by PCVs and measured select water quality parameters in their household water. METHODS: A survey about water types and practices was conducted of PCVs in Guatemala. The water type most frequently consumed in the household ("primary drinking water") and other water types present in the household ("secondary water") were tested for free chlorine residual (FCR) and for the presence of Escherichia coli and total coliforms. A negative binomial regression model was used to analyze data on incidence of self-reported GI illness. RESULTS: Tambo (commercially purified water in a 5-gal bottle) was the water type most frequently (64%) reported as primary drinking water in 39 PCV households. Most (74%) PCVs reported drinking water other than primary drinking water ≥1 day per week; the incidence rate of GI illness per PCV per month was significantly lower among PCVs who reported never consuming water other than primary drinking water compared to those who did (0.4 and 1.6 GI illnesses per PCV per month, respectively) (p < 0.05). E. coli was not detected in any primary drinking water sample, but was detected in 35% of secondary water samples. Total coliforms were detected in more than two-thirds of primary drinking water and secondary water samples. Nearly all water samples had an FCR of < 0.2 mg/L. CONCLUSIONS: Consuming primary drinking water exclusively likely contributes to reducing the rate of GI illness among PCVs. However, most PCVs reported drinking multiple water types, which may include contaminated secondary water types in the household. All water intended for consumption, including secondary sources within and outside the household, should be properly treated and safely stored.

Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking.

Extracellular vesicles (EVs) are increasingly being recognized as mediators of intercellular signaling via the delivery of effector molecules. Interestingly, certain types of EVs are also capable of inducing therapeutic responses. For these reasons, the therapeutic potential of EVs is a topic of intense research, both in the context of drug delivery and regenerative medicine. However, to fully utilize EVs for therapeutic purposes, an improved understanding of the mechanisms by which they function would be highly advantageous. Here, the current state of knowledge regarding the cellular uptake and trafficking of EVs is reviewed, along with a consideration of how these pathways potentially influence the functions of therapeutic EVs. Furthermore, the natural cell-targeting abilities, biodistribution profiles, and pharmacokinetics of exogenously administered EVs, along with the components responsible for these features are discussed. An overview of the potential clinical applications and preclinical examples of their successful use is also provided. Finally, examples of EV modifications that have successfully been employed to improve their therapeutic characteristics receive a particular focus. We suggest that, in addition to investigation of EV cell targeting and routes of uptake, future research into the routes of intracellular trafficking in recipient cells is required to optimally utilize EVs for therapeutic purposes.

Diagnosis of COPD and clinical course in patients with unrecognized airflow limitation.

Chronic obstructive pulmonary disease (COPD) is frequently under-recognized and underdiagnosed. To determine the natural history of recognized and unrecognized COPD, we studied the rate of diagnosis, health care utilization, and mortality in patients with airflow limitation (AFL). Three hundred forty-seven outpatients at the Cincinnati Veterans Administration Medical Center performed spirometry and completed a respiratory questionnaire. Patients were followed for a minimum of 30 months and medical records were reviewed for COPD diagnosis, mortality, respiratory-related health care utilization, comorbidities, and respiratory medications. Three hundred twenty-five of 347 (94%) patients performed technically adequate spirometry and completed questionnaires. When AFL was defined by fixed ratio (FR, forced expiratory volume in 1 second [FEV(1)]/forced vital capacity [FVC] < 0.7), patients with AFL and a diagnosis of COPD had a higher annual mortality rate (7.1% ± 2% versus 2.4% ± 0.8%, P = 0.01), more hospitalizations per year (0.2 ± 0.06 versus 0.04 ± 0.01, P < 0.001 mean ± standard error of the mean), increased respiratory symptoms (12.0 ± 0.9 versus 7.2 ± 0.6, P < 0.0001), and higher Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage compared with undiagnosed patients. Ninety-two of 137 patients with AFL (67%) had unrecognized AFL; 16 (17%) of the 92 were subsequently diagnosed. When AFL was defined by the lower limit of normal (LLN, FEV(1)/FVC < LLN), 67 of 103 patients (65%) had unrecognized AFL; 12 (18%) of the 67 were subsequently diagnosed. Patients with AFL defined by FR who were subsequently diagnosed had more emergency department visits per year (0.33 ± 0.11 versus 0.11 ± 0.05, P = 0.009), increased respiratory symptoms (10.2 ± 1.6 versus 6.5 ± 0.7, P < 0.05), and higher GOLD stage, but similar mortality and hospitalizations compared with the persistently undiagnosed patients. The annual rate of documented COPD diagnosis was 7% for both FR and LLN definitions. Patients with AFL and a diagnosis of COPD have more severe disease, higher health care utilization, and mortality than undiagnosed patients. The annual rate of COPD diagnosis is 7% among individuals with unrecognized AFL. Worse AFL, increased respiratory symptoms, and ED visits are associated with a subsequent COPD diagnosis in individuals with unrecognized AFL.

High prevalence of chronic obstructive pulmonary disease among veterans in the urban midwest.

Although chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality within the Veterans Health care Administration, its prevalence and recognition are not known. We measured airflow limitation and diagnosed COPD at the Cincinnati Veteran's Administration Medical Center. Participants were 326 outpatients who performed spirometry and completed questionnaires. Health care-provider-diagnosis and self-diagnosis of COPD were compared with COPD defined by forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) < 0.7 (fixed ratio) and (FEV1/FVC)/lower limit of normal (LLN) < 1.0. COPD prevalence was 43% (95% confidence interval: 36.9, 48.1) by fixed ratio and 33% (95% confidence interval: 27.2, 36.8) by LLN. Eighteen percent of the patients had health care-provider-recorded and 23% had self-reported diagnoses of COPD. Positive predictive values for the diagnosis of COPD were 79% and 64% for healthcare providers versus 68% and 62% for patients; negative predictive values were 64% and 74% for healthcare providers versus 64% and 76% for patients (fixed ratio and LLN, respectively). COPD prevalence is higher among Cincinnati veterans than among general U.S. population. COPD is under-recognized by both health care providers and veterans.