Guanabenz repurposed as an antiparasitic with activity against acute and latent toxoplasmosis

Toxoplasma gondii is a protozoan parasite that persists as a chronic infection. Toxoplasma evades immunity by forming tissue cysts, which reactivate to cause life-threatening disease during immune suppression. There is an urgent need to identify drugs capable of targeting these latent tissue cysts, which tend to form in the brain. We previously showed that translational control is critical during infections with both replicative and latent forms of Toxoplasma. Here we report that guanabenz, an FDA-approved drug that interferes with translational control, has antiparasitic activity against replicative stages of Toxoplasma and the related apicomplexan parasite Plasmodium falciparum (a malaria agent). We also found that inhibition of translational control interfered with tissue cyst biology in vitro. Toxoplasma bradyzoites present in these abnormal cysts were diminished and misconfigured, surrounded by empty space not seen in normal cysts. These findings prompted analysis of the efficacy of guanabenz in vivo by using established mouse models of acute and chronic toxoplasmosis. In addition to protecting mice from lethal doses of Toxoplasma, guanabenz has a remarkable ability to reduce the number of brain cysts in chronically infected mice. Our findings suggest that guanabenz can be repurposed into an effective antiparasitic with a unique ability to reduce tissue cysts in the brain

Predicting Functional and Regulatory Divergence of a Drug Resistance Transporter Gene in the Human Malaria Parasite

Background: The paradigm of resistance evolution to chemotherapeutic agents is that a key coding mutation in a specific gene drives resistance to a particular drug. In the case of resistance to the anti-malarial drug chloroquine (CQ), a specific mutation in the transporter pfcrt is associated with resistance. Here, we apply a series of analytical steps to gene expression data from our lab and leverage 3 independent datasets to identify pfcrt-interacting genes. Resulting networks provide insights into pfcrt’s biological functions and regulation, as well as the divergent phenotypic effects of its allelic variants in different genetic backgrounds. Results: To identify pfcrt-interacting genes, we analyze pfcrt co-expression networks in 2 phenotypic states - CQ-resistant (CQR) and CQ-sensitive (CQS) recombinant progeny clones - using a computational approach that prioritizes gene interactions into functional and regulatory relationships. For both phenotypic states, pfcrt co-expressed gene sets are associated with hemoglobin metabolism, consistent with CQ’s expected mode of action. To predict the drivers of co-expression divergence, we integrate topological relationships in the co-expression networks with available high confidence protein-protein interaction data. This analysis identifies 3 transcriptional regulators from the ApiAP2 family and histone acetylation as potential mediators of these divergences. We validate the predicted divergences in DNA mismatch repair and histone acetylation by measuring the effects of small molecule inhibitors in recombinant progeny clones combined with quantitative trait locus (QTL) mapping. Conclusions: This work demonstrates the utility of differential co-expression viewed in a network framework to uncover functional and regulatory divergence in phenotypically distinct parasites. pfcrt-associated co-expression in the CQ resistant progeny highlights CQR-specific gene relationships and possible targeted intervention strategies. The approaches outlined here can be readily generalized to other parasite populations and drug resistances

Fidelity and Adherence to a Liquefied Petroleum Gas Stove and Fuel Intervention during Gestation: The Multi-Country Household Air Pollution Intervention Network (HAPIN) Randomized Controlled Trial

BACKGROUND: Clean cookstove interventions can theoretically reduce exposure to household air pollution and benefit health, but this requires near-exclusive use of these types of stoves with the simultaneous disuse of traditional stoves. Previous cookstove trials have reported low adoption of new stoves and/or extensive continued traditional stove use. METHODS: The Household Air Pollution Intervention Network (HAPIN) trial randomized 3195 pregnant women in Guatemala, India, Peru, and Rwanda to either a liquefied petroleum gas (LPG) stove and fuel intervention (n = 1590) or to a control (n = 1605). The intervention consisted of an LPG stove and two initial cylinders of LPG, free fuel refills delivered to the home, and regular behavioral messaging. We assessed intervention fidelity (delivery of the intervention as intended) and adherence (intervention use) through to the end of gestation, as relevant to the first primary health outcome of the trial: infant birth weight. Fidelity and adherence were evaluated using stove and fuel delivery records, questionnaires, visual observations, and temperature-logging stove use monitors (SUMs). RESULTS: 1585 women received the intervention at a median (interquartile range) of 8.0 (5.0-15.0) days post-randomization and had a gestational age of 17.9 (15.4-20.6) weeks. Over 96% reported cooking exclusively with LPG at two follow-up visits during pregnancy. Less than 4% reported ever running out of LPG. Complete abandonment of traditional stove cooking was observed in over 67% of the intervention households. Of the intervention households, 31.4% removed their traditional stoves upon receipt of the intervention; among those who retained traditional stoves, the majority did not use them: traditional stove use was detected via SUMs on a median (interquartile range) of 0.0% (0.0%, 1.6%) of follow-up days (median follow-up = 134 days). CONCLUSIONS: The fidelity of the HAPIN intervention, as measured by stove installation, timely ongoing fuel deliveries, and behavioral reinforcement as needed, was high. Exclusive use of the intervention during pregnancy was also high

Designing a comprehensive behaviour change intervention to promote and monitor exclusive use of liquefied petroleum gas stoves for the Household Air Pollution Intervention Network (HAPIN) trial.

INTRODUCTION: Increasing use of cleaner fuels, such as liquefied petroleum gas (LPG), and abandonment of solid fuels is key to reducing household air pollution and realising potential health improvements in low-income countries. However, achieving exclusive LPG use in households unaccustomed to this type of fuel, used in combination with a new stove technology, requires substantial behaviour change. We conducted theory-grounded formative research to identify contextual factors influencing cooking fuel choice to guide the development of behavioural strategies for the Household Air Pollution Intervention Network (HAPIN) trial. The HAPIN trial will assess the impact of exclusive LPG use on air pollution exposure and health of pregnant women, older adult women, and infants under 1 year of age in Guatemala, India, Peru, and Rwanda. METHODS: Using the Capability, Opportunity, Motivation-Behaviour (COM-B) framework and Behaviour Change Wheel (BCW) to guide formative research, we conducted in-depth interviews, focus group discussions, observations, key informant interviews and pilot studies to identify key influencers of cooking behaviours in the four countries. We used these findings to develop behavioural strategies likely to achieve exclusive LPG use in the HAPIN trial. RESULTS: We identified nine potential influencers of exclusive LPG use, including perceived disadvantages of solid fuels, family preferences, cookware, traditional foods, non-food-related cooking, heating needs, LPG awareness, safety and cost and availability of fuel. Mapping formative findings onto the theoretical frameworks, behavioural strategies for achieving exclusive LPG use in each research site included free fuel deliveries, locally acceptable stoves and equipment, hands-on training and printed materials and videos emphasising relevant messages. In the HAPIN trial, we will monitor and reinforce exclusive LPG use through temperature data loggers, LPG fuel delivery tracking, in-home observations and behavioural reinforcement visits. CONCLUSION: Our formative research and behavioural strategies can inform the development, implementation, monitoring and evaluation of theory-informed strategies to promote exclusive LPG use in future stove programmes and research studies. TRIAL REGISTRATION NUMBER: NCT02944682, Pre-results

Resources and Geographic Access to Care for Severe Pediatric Pneumonia in Four Resource-limited Settings

Rationale: Pneumonia is the leading cause of death in children worldwide. Identifying and appropriately managing severe pneumonia in a timely manner improves outcomes. Little is known about the readiness of healthcare facilities to manage severe pediatric pneumonia in low-resource settings. Objectives: As part of the HAPIN (Household Air Pollution Intervention Network) trial, we sought to identify healthcare facilities that were adequately resourced to manage severe pediatric pneumonia in Jalapa, Guatemala (J-GUA); Puno, Peru (P-PER); Kayonza, Rwanda (K-RWA); and Tamil Nadu, India (T-IND). We conducted a facility-based survey of available infrastructure, staff, equipment, and medical consumables. Facilities were georeferenced, and a road network analysis was performed. Measurements and Main Results: Of the 350 healthcare facilities surveyed, 13% had adequate resources to manage severe pneumonia, 37% had pulse oximeters, and 44% had supplemental oxygen. Mean (±SD) travel time to an adequately resourced facility was 41 ± 19 minutes in J-GUA, 99 ± 64 minutes in P-PER, 40 ± 19 minutes in K-RWA, and 31 ± 19 minutes in T-IND. Expanding pulse oximetry coverage to all facilities reduced travel time by 44% in J-GUA, 29% in P-PER, 29% in K-RWA, and 11% in T-IND (all P < 0.001). Conclusions: Most healthcare facilities in low-resource settings of the HAPIN study area were inadequately resourced to care for severe pediatric pneumonia. Early identification of cases and timely referral is paramount. The provision of pulse oximeters to all health facilities may be an effective approach to identify cases earlier and refer them for care and in a timely manner

LPG stove and fuel intervention among pregnant women reduce fine particle air pollution exposures in three countries: Pilot results from the HAPIN trial

The Household Air Pollution Intervention Network trial is a multi-country study on the effects of a liquefied petroleum gas (LPG) stove and fuel distribution intervention on women's and children's health. There is limited data on exposure reductions achieved by switching from solid to clean cooking fuels in rural settings across multiple countries. As formative research in 2017, we recruited pregnant women and characterized the impact of the intervention on personal exposures and kitchen levels of fine particulate matter (PM2.5) in Guatemala, India, and Rwanda. Forty pregnant women were enrolled in each site. We measured cooking area concentrations of and personal exposures to PM2.5 for 24 or 48 h using gravimetric-based PM2.5 samplers at baseline and two follow-ups over two months after delivery of an LPG cookstove and free fuel supply. Mixed models were used to estimate PM2.5 reductions. Median kitchen PM2.5 concentrations were 296 μg/m3 at baseline (interquartile range, IQR: 158-507), 24 μg/m3 at first follow-up (IQR: 18-37), and 23 μg/m3 at second follow-up (IQR: 14-37). Median personal exposures to PM2.5 were 134 μg/m3 at baseline (IQR: 71-224), 35 μg/m3 at first follow-up (IQR: 23-51), and 32 μg/m3 at second follow-up (IQR: 23-47). Overall, the LPG intervention was associated with a 92% (95% confidence interval (CI): 90-94%) reduction in kitchen PM2.5 concentrations and a 74% (95% CI: 70-79%) reduction in personal PM2.5 exposures. Results were similar for each site. CONCLUSIONS: The intervention was associated with substantial reductions in kitchen and personal PM2.5 overall and in all sites. Results suggest LPG interventions in these rural settings may lower exposures to the WHO annual interim target-1 of 35 μg/m3. The range of exposure contrasts falls on steep sections of estimated exposure-response curves for birthweight, blood pressure, and acute lower respiratory infections, implying potentially important health benefits when transitioning from solid fuels to LPG

Facing the Realities of Pragmatic Design Choices in Environmental Health Studies: Experiences from the Household Air Pollution Intervention Network Trial

Objective: Household Air Pollution Intervention Network (HAPIN) investigators tested a complex, non-pharmacological intervention in four low- and middle-income countries as a strategy to mitigate household air pollution and improve health outcomes across the lifespan. Intervention households received a liquefied petroleum gas (LPG) stove, continuous fuel delivery and regular behavioral reinforcements for 18 months, whereas controls were asked to continue with usual cooking practices. While HAPIN was designed as an explanatory trial to test the efficacy of the intervention on four primary outcomes, it introduced several pragmatic aspects in its design and conduct that resemble real-life conditions. We surveyed HAPIN investigators and asked them to rank what aspects of the design and conduct they considered were more pragmatic than explanatory. Methods: We used the revised Pragmatic Explanatory Continuum Indicator Summary (PRECIS-2) to survey investigators on the degree of pragmatism in nine domains of trial design and conduct using a five-point Likert rank scale from very explanatory (1) to very pragmatic (5). We invited 103 investigators. Participants were given educational material on PRECIS-2, including presentations, papers and examples that described the use and implementation of PRECIS-2. Results: Thirty-five investigators (mean age 42 years, 51% female) participated in the survey. Overall, only 17% ranked all domains as very explanatory, with an average (±SD) rank of 3.2 ± 1.4 across domains. Fewer than 20% of investigators ranked eligibility, recruitment or setting as very explanatory. In contrast, ≥50% of investigators ranked the trial organization, delivery and adherence of the intervention and follow-up as very/rather explanatory whereas ≤17% ranked them as rather/very pragmatic. Finally, &lt;25% of investigators ranked the relevance of outcomes to participants and analysis as very/rather explanatory whereas ≥50% ranked then as rather/very pragmatic. In-country partners were more likely to rank domains as pragmatic when compared to investigators working in central coordination (average rank 3.2 vs. 2.8, respectively; Wilcoxon rank-sum p &lt; 0.001). Conclusion: HAPIN investigators did not consider their efficacy trial to be rather/very explanatory and reported that some aspects of the design and conduct were executed under real-world conditions; however, they also did not consider the trial to be overly pragmatic. Our analysis underscores the importance of using standardized tools such as PRECIS-2 to guide early discussions among investigators in the design of environmental health trials attempting to measure efficacy.</jats:p

The landscape of inherited and de novo copy number variants in a plasmodium falciparum genetic cross

<p>Abstract</p> <p>Background</p> <p>Copy number is a major source of genome variation with important evolutionary implications. Consequently, it is essential to determine copy number variant (CNV) behavior, distributions and frequencies across genomes to understand their origins in both evolutionary and generational time frames. We use comparative genomic hybridization (CGH) microarray and the resolution provided by a segregating population of cloned progeny lines of the malaria parasite, <it>Plasmodium falciparum</it>, to identify and analyze the inheritance of 170 genome-wide CNVs.</p> <p>Results</p> <p>We describe CNVs in progeny clones derived from both Mendelian (i.e. inherited) and non-Mendelian mechanisms. Forty-five CNVs were present in the parent lines and segregated in the progeny population. Furthermore, extensive variation that did not conform to strict Mendelian inheritance patterns was observed. 124 CNVs were called in one or more progeny but in neither parent: we observed CNVs in more than one progeny clone that were not identified in either parent, located more frequently in the telomeric-subtelomeric regions of chromosomes and singleton <it>de novo </it>CNVs distributed evenly throughout the genome. Linkage analysis of CNVs revealed dynamic copy number fluctuations and suggested mechanisms that could have generated them. Five of 12 previously identified expression quantitative trait loci (eQTL) hotspots coincide with CNVs, demonstrating the potential for broad influence of CNV on the transcriptional program and phenotypic variation.</p> <p>Conclusions</p> <p>CNVs are a significant source of segregating and <it>de novo </it>genome variation involving hundreds of genes. Examination of progeny genome segments provides a framework to assess the extent and possible origins of CNVs. This segregating genetic system reveals the breadth, distribution and dynamics of CNVs in a surprisingly plastic parasite genome, providing a new perspective on the sources of diversity in parasite populations.</p