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BackgroundRabies is a viral disease of animals and people causing fatal encephalomyelitis if left untreated. Although effective pre- and post-exposure vaccines exist, they are not widely available in many endemic countries within Africa. Since many individuals in these countries remain at risk of infection, post-exposure healthcare-seeking behaviors are crucial in preventing infection and warrant examination.MethodologyA rabies knowledge, attitudes, and practices survey was conducted at 24 geographically diverse sites in Uganda during 2013 to capture information on knowledge concerning the disease, response to potential exposure events, and vaccination practices. Characteristics of the surveyed population and of the canine-bite victim sub-population were described. Post-exposure healthcare-seeking behaviors of canine-bite victims were examined and compared to the related healthcare-seeking attitudes of non-bite victim respondents. Wealth scores were calculated for each household, rabies knowledge was scored for each non-bitten survey respondent, and rabies exposure risk was scored for each bite victim. Logistic regression was used to determine the independent associations between different variables and healthcare-seeking behaviors among canine-bite victims as well as attitudes of non-bitten study respondents.ResultsA total of 798 households were interviewed, capturing 100 canine-bite victims and a bite incidence of 2.3 per 100 person-years. Over half of bite victims actively sought medical treatment (56%), though very few received rabies post-exposure prophylaxis (3%). Bite victims who did not know or report the closest location where PEP could be received were less likely to seek medical care (p = 0.05). Respondents who did not report having been bitten by a dog with higher knowledge scores were more likely to respond that they would both seek medical care (p = 0.00) and receive PEP (p = 0.06) after a potential rabies exposure event.ConclusionsThere was varying discordance between what respondents who did not report having been bitten by a dog said they would do if bitten by a dog when compared to the behaviors exhibited by canine-bite victims captured in the KAP survey. Bite victims seldom elected to wash their wound or receive PEP. Having lower rabies knowledge was a barrier to theoretically seeking care and receiving PEP among not bitten respondents, indicating a need for effective and robust educational programs in the country.

Many applications have been developed for electronic data collection. However, offline field navigation tools incorporating secure electronic data capture and field visit tracking are currently scarce. We created an R-Shiny application, HTrack (Household Tracking), for use on encrypted Android devices in the field. The application was implemented in the Communities Organized to Prevent Arboviruses (COPA) project, a study beginning in 2018 to better understand arboviral disease incidence in 38 communities in Puerto Rico. The application was used to navigate to randomly selected structures and capture visit outcomes after conducting multiple visits for participant recruitment. It also served as a bridge to an alternate software, Epi Info, to collect participant-level questionnaire data. This application successfully captured each visit outcome and improved the logistics of field level activities for the COPA project, eliminating the use of paper maps for navigation. We show the development of HTrack and comment on the limitations and strengths of this application and further improvements.

Rocky Mountain spotted fever (RMSF) transmitted by the brown dog tick (Rhipicephalus sanguineus sensu lato) has emerged as a significant public health risk on American Indian reservations in eastern Arizona. During 2003-2012, more than 250 RMSF cases and 19 deaths were documented among Arizona's American Indian population. The high case fatality rate makes community-level interventions aimed at rapid and sustained reduction of ticks urgent. Beginning in 2012, a two year pilot integrated tick prevention campaign called the RMSF Rodeo was launched in a ∼ 600-home tribal community with high rates of RMSF. During year one, long-acting tick collars were placed on all dogs in the community, environmental acaricides were applied to yards monthly, and animal care practices such as spay and neuter and proper tethering procedures were encouraged. Tick levels, indicated by visible inspection of dogs, tick traps and homeowner reports were used to monitor tick presence and evaluate the efficacy of interventions throughout the project. By the end of year one,

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.

Targeted gene integration via precise homologous recombination (HR)-based gene editing has the potential to correct genetic diseases. AAV (adeno-associated virus) can mediate nuclease-free gene integration at a disease-causing locus. Therapeutic application of AAV gene integration requires quantitative molecular characterization of the edited sequence that overcome technical obstacles such as excess episomal vector genomes and lengthy homology arms. Here we describe a novel molecular methodology that utilizes quantitative next-generation sequencing to characterize AAV-mediated targeted insertion and detects the presence of unintended mutations. The methods described here quantify targeted insertion and query the entirety of the target locus for the presence of insertions, deletions, single nucleotide variants (SNVs) and integration of viral components such as inverted terminal repeats (ITR). Using a humanized liver murine model, we demonstrate that hematopoietic stem-cell derived AAVHSC15 mediates in vivo targeted gene integration into human chromosome 12 at the PAH (phenylalanine hydroxylase) locus at 6% frequency, with no sign of co-incident random mutations at or above a lower limit of detection of 0.5% and no ITR sequences at the integration sites. Furthermore, analysis of heterozygous variants across the targeted locus using the methods described shows a pattern of strand cross-over, supportive of an HR mechanism of gene integration with similar efficiencies across two different haplotypes. Rapid advances in the application of AAV-mediated nuclease-free target integration, or gene editing, as a new therapeutic modality requires precise understanding of the efficiency and the nature of the changes being introduced to the target genome at the molecular level. This work provides a framework to be applied to homologous recombination gene editing platforms for assessment of introduced and natural sequence variation across a target site.