Juvenile sparids (Rhabdosargus holubi) consistently select structurally dense vegetated habitat in nursery seascapes.

Seagrass habitats provide structural complexity in coastal estuarine and marine environments, which offer fish optimal foraging grounds and refuge from predation. However, seagrasses are some of the most threatened ecosystems globally, with anthropogenic activities such as population growth and environmental degradation leading to the fragmentation, thinning, and loss of these habitats. Rhabdosargus holubi is one of only a few vegetation-associated marine fish species in South African estuaries. Although field studies have shown a strong association with seagrass over other aquatic vegetation for the juveniles of this species, habitat choice has never been empirically tested. Here, we used artificial vegetation units to test habitat choice (different structural complexities) for this species. We also tested whether habitat choice is influenced by a predatory threat, with fish preferentially selecting dense habitat in the presence of a predator and whether this effect may be more apparent in smaller individuals. We found that R. holubi significantly prefer greater structural complexity over less complex habitats, in both the absence and presence of a predator and for both small and large juveniles, showing that R. holubi actively choose more complex structures and are attracted to the structure per se irrespective of the threat of predation. This study highlights the importance of dense seagrass as nursery areas for this species and demonstrates how the loss of these habitats could impact the nursery function of estuaries.

Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated.

BACKGROUND: Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. METHODS: DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation. RESULTS: Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. CONCLUSION: Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

The use of internal heart rate loggers in determining cardiac breakpoints of fish.

As marine environments are influenced by global warming there is a need to thoroughly understand the relationship between physiological limits and temperature in fish. One quick screening method of a physiological thermal tipping point is the temperature at which maximum heart rate (ƒHmax) can no longer scale predictably with warming and is referred to as the Arrhenius break temperature (TAB). The use of this method has been successful for freshwater fish by using external electrodes to detect an electrocardiogram (ECG), however, the properties of this equipment pose challenges in salt water when evaluating marine fish. To overcome these challenges, this study aimed to explore the potential use of implantable heart rate loggers to quantify the TAB of Chrysoblephus laticeps, a marine Sparid, following the ECG method protocols where ƒHmax is monitored over an acute warming event and the TAB is subsequently identified using a piece-wise linear regression model. Of the nine experimental fish, only five (56%) returned accurate ƒHmax data. The TAB of successful trials was identified each time and ranged from 18.09 to 20.10 °C. This study therefore provides evidence that implantable heart rate loggers can estimate TAB of fish which can be applied to many marine species.

Monoallelic expression in melanoma.

BACKGROUND: Monoallelic expression (MAE) is a frequent genomic phenomenon in normal tissues, however its role in cancer is yet to be fully understood. MAE is defined as the expression of a gene that is restricted to one allele in the presence of a diploid heterozygous genome. Constitutive MAE occurs for imprinted genes, odorant receptors and random X inactivation. Several studies in normal tissues have showed MAE in approximately 5-20% of the cases. However, little information exists on the MAE rate in cancer. In this study we assessed the presence and rate of MAE in melanoma. The genetic basis of melanoma has been studied in depth over the past decades, leading to the identification of mutations/genetic alterations responsible for melanoma development. METHODS: To examine the role of MAE in melanoma we used 15 melanoma cell lines and compared their RNA-seq data with genotyping data obtained by the parental TIL (tumor infiltrating lymphocytes). Genotyping was performed using the Illumina HumanOmni1 beadchip. The RNA-seq library preparation and sequencing was performed using the Illumina TruSeq Stranded Total RNA Human Kit and subsequently sequenced using a HiSeq 2500 according to manufacturer's guidelines. By comparing genotyping data with RNA-seq data, we identified SNPs in which DNA genotypes were heterozygous and corresponding RNA genotypes were homozygous. All homozygous DNA genotypes were removed prior to the analysis. To confirm the validity to detect MAE, we examined heterozygous DNA genotypes from X chromosome of female samples as well as for imprinted and olfactory receptor genes and confirmed MAE. RESULTS: MAE was detected in all 15 cell lines although to a different rate. When looking at the B-allele frequencies we found a preferential pattern of complete monoallelic expression rather then differential monoallelic expression across the 15 melanoma cell lines. As some samples showed high differences in the homozygous and heterozygous call rate, we looked at the single chromosomes and showed that MAE may be explained by underlying large copy number imbalances in some instances. Interestingly these regions included genes known to play a role in melanoma initiation and progression. Nevertheless, some chromosome regions showed MAE without CN imbalances suggesting that additional mechanisms (including epigenetic silencing) may explain MAE in melanoma. CONCLUSION: The biological implications of MAE are yet to be realized. Nevertheless, our findings suggest that MAE is a common phenomenon in melanoma cell lines. Further analyses are currently being undertaken to evaluate whether MAE is gene/pathway specific and to understand whether MAE can be employed by cancers to achieve a more aggressive phenotype.

Submicroscopic Plasmodium prevalence in relation to malaria incidence in 20 villages in western Cambodia.

BACKGROUND: Cambodia has seen a marked reduction in the incidence of Plasmodium falciparum over the past decade without a corresponding decline in Plasmodium vivax incidence. It is unknown to what extent local transmission is sustained by a chain of clinical and sub-clinical infections or by continued re-introduction via migration. Using an ultrasensitive molecular technique, 20 villages in western Cambodia were surveyed to detect the low season prevalence of P. falciparum and P. vivax and local treatment records were reviewed. METHODS: During March to May 2015 cross-sectional surveys were conducted in 20 villages in Battambang, western Cambodia. Demographic and epidemiological data and venous blood samples were collected from 50 randomly selected adult volunteers in each village. Blood was tested for Plasmodium infections by rapid diagnostic test (RDT), microscopy and high volume (0.5 ml packed red blood cell) quantitative polymerase chain reaction (uPCR). Positive samples were analysed by nested PCR to determine the Plasmodium species. Malaria case records were collected from the Provincial Health Department and village malaria workers to determine incidence and migration status. RESULTS: Among the 1000 participants, 91 (9.1%) were positive for any Plasmodium infection by uPCR, seven (0.7%) by microscopy, and two (0.2%) by RDT. uPCR P. vivax prevalence was 6.6%, P. falciparum 0.7%, and undetermined Plasmodium species 1.8%. Being male (adjusted OR 2.0; 95% CI 1.2-3.4); being a young adult <30 years (aOR 2.1; 95% CI 1.3-3.4); recent forest travel (aOR 2.8; 95% CI 1.6-4.8); and, a history of malaria (aOR 5.2; 95% CI 2.5-10.7) were independent risk factors for parasitaemia. Of the clinical malaria cases diagnosed by village malaria workers, 43.9% (297/634) and 38.4% (201/523) were among migrants in 2013 and in 2014, respectively. Plasmodium vivax prevalence determined by uPCR significantly correlated with vivax malaria incidences in both 2014 and 2015 (p = 0.001 and 0.002, respectively), whereas no relationship was observed in falciparum malaria (p = 0.36 and p = 0.59, respectively). DISCUSSION: There was heterogeneity in the malaria parasite reservoir between villages, and Plasmodium prevalence correlated with subsequent malaria incidence. The association was attributable chiefly to P. vivax infections, which were nine-fold more prevalent than P. falciparum infections. In the absence of a radical cure with 8-aminoquinolines, P. vivax transmission will continue even as P. falciparum prevalence declines. Migration was associated with over a third of incident cases of clinical malaria. Trial registration clinicaltrials.gov (NCT01872702). Registered 4 June 2013.

Community engagement and population coverage in mass anti-malarial administrations: a systematic literature review.

BACKGROUND: Mass anti-malarial administration has been proposed as a key component of the malaria elimination strategy in South East Asia. The success of this approach depends on the local malaria epidemiology, nature of the anti-malarial regimen and population coverage. Community engagement is used to promote population coverage but little research has systematically analysed its impact. This systematic review examines population coverage and community engagement in programmes of mass anti-malarial drug administration. METHODS: This review builds on a previous review that identified 3049 articles describing mass anti-malarial administrations published between 1913 and 2011. Further search and application of a set of criteria conducted in the current review resulted in 51 articles that were retained for analysis. These 51 papers described the population coverage and/or community engagement in mass anti-malarial administrations. Population coverage was quantitatively assessed and a thematic analysis was conducted on the community engagement activities. RESULTS: The studies were conducted in 26 countries: in diverse healthcare and social contexts where various anti-malarial regimens under varied study designs were administered. Twenty-eight articles reported only population coverage; 12 described only community engagement activities; and 11 community engagement and population coverage. Average population coverage was 83% but methods of calculating coverage were frequently unclear or inconsistent. Community engagement activities included providing health education and incentives, using community structures (e.g. existing hierarchies or health infrastructure), mobilizing human resources, and collaborating with government at some level (e.g. ministries of health). Community engagement was often a process involving various activities throughout the duration of the intervention. CONCLUSION: The mean population coverage was over 80% but incomplete reporting of calculation methods limits conclusions and comparisons between studies. Various community engagement activities and approaches were described, but many articles contained limited or no details. Other factors relevant to population coverage, such as the social, cultural and study context were scarcely reported. Further research is needed to understand the factors that influence population coverage and adherence in mass anti-malarial administrations and the role community engagement activities and approaches play in satisfactory participation.

Neoadjuvant cemiplimab for resectable hepatocellular carcinoma: a single-arm, open-label, phase 2 trial.

BACKGROUND: Surgical resection of early stage hepatocellular carcinoma is standard clinical practice; however, most tumours recur despite surgery, and no perioperative intervention has shown a survival benefit. Neoadjuvant immunotherapy has induced pathological responses in multiple tumour types and might decrease the risk of postoperative recurrence in hepatocellular carcinoma. We aimed to evaluate the clinical activity of neoadjuvant cemiplimab (an anti-PD-1) in patients with resectable hepatocellular carcinoma. METHODS: For this single-arm, open-label, phase 2 trial, patients with resectable hepatocellular carcinoma (stage Ib, II, and IIIb) were enrolled and received two cycles of neoadjuvant cemiplimab 350 mg intravenously every 3 weeks followed by surgical resection. Eligible patients were aged 18 years or older, had confirmed resectable hepatocellular carcinoma, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate liver function. Patients were excluded if they had metastatic disease, if the surgery was not expected to be curative, if they had a known additional malignancy requiring active treatment, or if they required systemic steroid treatment or any other immunosuppressive therapy. After resection, patients received an additional eight cycles of cemiplimab 350 mg intravenously every 3 weeks in the adjuvant setting. The primary endpoint was significant tumour necrosis on pathological examination (defined as >70% necrosis of the resected tumour). Secondary endpoints included delay of surgery, the proportion of patients with an overall response, change in CD8+ T-cell density, and adverse events. Tumour necrosis and response were analysed in all patients who received at least one dose of cemiplimab and completed surgical resection; safety and other endpoints were analysed in the intention-to-treat population. Patients underwent pre-treatment biopsies and blood collection throughout treatment. This trial is registered with ClinicalTrials.gov (NCT03916627, Cohort B) and is ongoing. FINDINGS: Between Aug 5, 2019, and Nov 25, 2020, 21 patients were enrolled. All patients received neoadjuvant cemiplimab, and 20 patients underwent successful resection. Of the 20 patients with resected tumours, four (20%) had significant tumour necrosis. Three (15%) of 20 patients had a partial response, and all other patients maintained stable disease. 20 (95%) patients had a treatment-emergent adverse event of any grade during the neoadjuvant treatment period. The most common adverse events of any grade were increased aspartate aminotransferase (in four patients), increased blood creatine phosphokinase (in three), constipation (in three), and fatigue (in three). Seven patients had grade 3 adverse events, including increased blood creatine phosphokinase (in two patients) and hypoalbuminaemia (in one). No grade 4 or 5 events were observed. One patient developed pneumonitis, which led to a delay in surgery by 2 weeks. INTERPRETATION: This report is, to our knowledge, the largest clinical trial of a neoadjuvant anti-PD-1 monotherapy reported to date in hepatocellular carcinoma. The observed pathological responses to cemiplimab in this cohort support the design of larger trials to identify the optimal treatment duration and definitively establish the clinical benefit of preoperative PD-1 blockade in patients with hepatocellular carcinoma. FUNDING: Regeneron Pharmaceuticals.

Capturing patient-reported and quality of life outcomes with use of shorter regimens for drug-resistant tuberculosis: mixed-methods substudy protocol, TB PRACTECAL-PRO.

INTRODUCTION: People living with multidrug-resistant tuberculosis currently have few options for effective treatment and cure. Regimens that are available are toxic, may involve injections and take up to 2 years to complete treatment, with success rates as low as 50%. The TB-PRACTECAL trial is evaluating shorter, more tolerable regimens of oral drugs; we detail the substudy within this trial, PRACTECAL-PRO, which aims to evaluate patient experiences and perspectives on treatment, to understand outcomes more fully. METHODS AND ANALYSIS: We are conducting a mixed-methods evaluation within both investigational and standard of care arms within the TB-PRACTECAL trial, using sequential quality of life (QoL) surveys and in-depth interviews. Data collection involves the Short Form 12 (SF-12) and St George's Respiratory Questionnaire (SGRQ), collected at up to four fixed timepoints, from baseline, to up to 12 months later. Healthy volunteers will be surveyed to establish locally relevant controls. We will also purposively sample participants for qualitative data collection and analysis, to provide rich explanation of QoL scores. The study will be implemented in all six TB-PRACTECAL study sites in Uzbekistan, South Africa and Belarus. QoL surveys will be scored and analysed according to SF-12 and SGRQ developers' manuals. Differences between scores at baseline and later timepoints will be evaluated as well as graphical exploration of group score trajectories of investigational and standard of care arms. ETHICS AND DISSEMINATION: Ethics approval was obtained from the Médecins Sans Frontières Ethics Review Board. Local ethics approval has been obtained in Uzbekistan, Belarus and South Africa. Results of the substudy will be shared with local health authorities, the WHO and submitted for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT03942354; Pre-results.

Human sample authentication in biomedical research: comparison of two platforms.

Samples used in biomedical research are often collected over years, in some cases from subjects that may have died and thus cannot be retrieved in any way. The value of these samples is priceless. Sample misidentification or mix-up are unfortunately common problems in biomedical research and can eventually result in the publication of incorrect data. Here we have compared the Fluidigm SNPtrace and the Agena iPLEX Sample ID panels for the authentication of human genomic DNA samples. We have tested 14 pure samples and simulated their cross-contamination at different percentages (2%, 5%, 10%, 25% and 50%). For both panels, we report call rate, allele intensity/probability score, performance in distinguishing pure samples and contaminated samples at different percentages, and sex typing. We show that both panels are reliable and efficient methods for sample authentication and we highlight their advantages and disadvantages. We believe that the data provided here is useful for sample authentication especially in biorepositories and core facility settings.

Thermal tolerance, safety margins and vulnerability of coastal species: Projected impact of climate change induced cold water variability in a temperate African region.

Anthropogenic induced climate change is predicted to increase the thermal variability in coastal waters, which can have strong physiological effects on individuals and populations of marine ectotherms. The magnitude and direction of these thermal effects varies depending on species, life stage, biogeography, habitat and season. This study aimed to compare the thermal tolerance of a range of juvenile fish and adult macro-invertebrates from intertidal and estuarine habitats in a warm-temperate, thermally variable region on the south-east coast of South Africa. Seasonal variability in thermal tolerance was compared between species, taxonomic groups, biogeographical distribution and habitat affinity and related to existing and projected water temperature data to gauge the local vulnerability of each species. Critical thermal maximum (CTmax), critical thermal minimum (CTmin), thermal breadths and scopes, and the thermal safety margins of each species were quantified. The greatest differences in thermal tolerance patterns were based on taxonomy, with macro-invertebrates having broader thermal tolerance compared to fish, with the exception of the Cape sea urchin, in both summer and winter. Relatively narrow lower breadths in tolerance and safety margin values for transient juvenile sub-tropical and temperate fish species from the intertidal rocky low-shore habitat were observed in both summer and winter. This indicates that these fish species and the Cape sea urchin may be more vulnerable to projected increases in cold temperature (upwelling in summer) than warm temperature variability in this warm-temperate region if they are unable to seek thermal habitat refuge.

Optimising recruitment to a late-phase tuberculosis clinical trial: a qualitative study exploring patient and practitioner experiences in Uzbekistan.

BACKGROUND: Addressing the global burden of multidrug-resistant tuberculosis (MDR-TB) requires identification of shorter, less toxic treatment regimens. Médecins Sans Frontières (MSF) is currently conducting a phase II/III randomised controlled clinical trial, to find more effective, shorter and tolerable treatments for people with MDR-TB. Recruitment to the trial in Uzbekistan has been slower than expected; we aimed to study patient and health worker experiences of the trial, examining potential factors perceived to impede and facilitate trial recruitment, as well as general perceptions of clinical research in this context. METHODS: We conducted a qualitative study using maximum variation, purposive sampling of participants. We carried out in-depth interviews (IDIs) and focus group discussions (FGDs) guided by semi-structured topic guides. In December 2019 and January 2020, 26 interviews were conducted with patients, Ministry of Health (MoH) and MSF staff and trial health workers, to explore challenges and barriers to patient recruitment as well as perceptions of the trial and research in general. Preliminary findings from the interviews informed three subsequent focus group discussions held with patients, nurses and counsellors. Focus groups adopted a person-centred design, brainstorming potential solutions to problems and barriers. Interviews and FGDs were audio recorded, translated and transcribed verbatim. Thematic analysis, drawing on constant comparison, was used to analyse the data. RESULTS: Health system contexts may compete with new approaches especially when legislative health regulations or policy around treatment is ingrained in staff beliefs, perceptions and practice, which can undermine clinical trial recruitment. Trust plays a significant role in how patients engage with the trial. Decision-making processes are dynamic and associated with relationship to diagnosis, assimilation of information, previous knowledge or experience and influence of peers and close relations. CONCLUSIONS: This qualitative analysis highlights ways in which insights developed together with patients and healthcare workers might inform approaches towards improved recruitment into trials, with the overall objective of delivering evidence for better treatments.

Different drivers, common mechanism; the distribution of a reef fish is restricted by local-scale oxygen and temperature constraints on aerobic metabolism.

The distributions of ectothermic marine organisms are limited to temperature ranges and oxygen conditions that support aerobic respiration, quantified within the metabolic index (ϕ) as the ratio of oxygen supply to metabolic oxygen demand. However, the utility of ϕ at local scales and across heterogenous environments is unknown; yet, these scales are often where actionable management decisions are made. Here, we test if ϕ can delimit the entire distribution of marine organisms when calibrated across an appropriate temperature range and at local scales (~10 km) using the endemic reef fish, Chrysoblephus laticeps, which is found in the highly heterogenous temperature and oxygen environment along the South African coastal zone, as a model species. In laboratory experiments, we find a bidirectional (at 12°C) hypoxia tolerance response across the temperature range tested (8 to 24°C), permitting a piecewise calibration of ϕ. We then project this calibrated ϕ model through temperature and oxygen data from a high spatial resolution (11 to 13 km) ocean model for the periods 2005 to 2009 and 2095 to 2099 to quantify various magnitudes of ϕ across space and time paired with complementary C. laticeps occurrence points. Using random forest species distribution models, we quantify a critical ϕ value of 2.78 below which C. laticeps cannot persist and predict current and future distributions of C. laticeps in line with already observed distribution shifts of other South African marine species. Overall, we find that C. laticeps' distribution is limited by increasing temperatures towards its warm edge but by low oxygen availability towards its cool edge, which is captured within ϕ at fine scales and across heterogenous oxygen and temperature combinations. Our results support the application of ϕ for generating local- and regional-scale predictions of climate change effects on organisms that can inform local conservation management decisions.

Performance of Ultrasensitive Rapid Diagnostic Tests for Detecting Asymptomatic Plasmodium falciparum.

Proposed interventions for eliminating drug-resistant Plasmodium falciparum malaria include the targeting of asymptomatic carriers through screening and treatment. We report on the diagnostic performance of the recently developed ultrasensitive rapid diagnostic test (uRDT) compared with screening with conventional RDTs (cRDT) and polymerase chain reaction (PCR) under field conditions in Cambodia in a total of 2,729 individuals. The P. falciparum positivity by quantitative PCR (qPCR) was 3.8% (26/678) in those screened during active case detection and 0.5% (10/2,051) in the cross-sectional survey. Compared with qPCR, the sensitivity of the uRDTs was 53.8% (95% CI: 33.4-73.4%) when used in active case detection and 60.0% (95% CI: 26.2-87.8%) in the cross-sectional survey. The uRDTs did not show a significant improvement in diagnostic performance over cRDTs when used for active case detection and for a malaria prevalence survey in the context of this low-transmission setting.

Implantation, orientation and validation of a commercially produced heart-rate logger for use in a perciform teleost fish.

Quantifying how the heart rate of ectothermic organisms responds to environmental conditions (e.g. water temperature) is important information to quantify their sensitivity to environmental change. Heart rate studies have typically been conducted in lab environments where fish are confined. However, commercially available implantable heart rate biologgers provide the opportunity to study free-swimming fish. Our study aimed to determine the applicability of an implantable device, typically used on fusiform-shaped fish (e.g. salmonids), for a perciform fish where morphology and anatomy prevent ventral incisions normally used on fusiform-shaped fish. We found that ventrolateral incisions allowed placement near the heart, but efficacy of the loggers was sensitive to their orientation and the positioning of the electrodes. Electrocardiogram detection, signal strength and subsequent heart rate readings were strongly influenced by logger orientation with a significant effect on the quality and quantity of heart rate recordings. We provide details on the surgical procedures and orientation to guide future heart rate biologger studies on perciform-shaped fish.

Exploitation may influence the climate resilience of fish populations through removing high performance metabolic phenotypes.

Physiological rates and processes underpin the relationships between ectothermic organisms, such as fish, and their environment. The response and persistence of fish populations in an increasingly variable ocean is dependent on the distribution and diversity of physiological phenotypes. Growing evidence suggests that fisheries exploitation can selectively target certain physiological and behavioural phenotypes, which may shift exploited populations to altered physiological states. Here we test if commercial fisheries have the potential to do this in a "natural laboratory" along the South African coast. We compare metabolic traits of exploited and protected populations of the fish species, Chrysoblephus laticeps, which is a major component of the South African hook and line fishery. We find that high-performance aerobic scope phenotypes are reduced in the fished population. The most likely mechanism for this finding is a positive relationship between aerobic scope and capture vulnerability in passive-gear fisheries. Our results further highlight the selective nature of capture-fisheries and suggest that exploitation has the capacity to alter climate responses of fish populations on a physiological level. Our finding also implicates how Marine Protected Areas, through harbouring individuals with a greater diversity of physiological traits, may provide greater fish response diversity to environmental variability.

Forest Goers and Multidrug-Resistant Malaria in Cambodia: An Ethnographic Study.

Multidrug-resistant Plasmodium falciparum malaria on the Cambodia-Thailand border is associated with working in forested areas. Beyond broad recognition of "forest-going" as a risk factor for malaria, little is known about different forest-going populations in this region. In Oddar Meanchey Province in northwestern Cambodia, qualitative ethnographic research was conducted to gain an in-depth understanding of how different populations, mobility and livelihood patterns, and activities within the forest intersect with potentiate malaria risk and impact on the effectiveness of malaria control and elimination strategies. We found that most forest-going in this area is associated with obtaining precious woods, particularly Siamese rosewood. In the past, at-risk populations included large groups of temporary migrants. As timber supplies have declined, so have these large migrant groups. However, groups of local residents continue to go to the forest and are staying for longer. Most forest-goers had experienced multiple episodes of malaria and were well informed about malaria risk. However, economic realities mean that local residents continue to pursue forest-based livelihoods. Severe constraints of available vector control methods mean that forest-goers have limited capacity to prevent vector exposure. As forest-goers access the forest using many different entry and exit points, border screening and treatment interventions will not be feasible. Once in the forest, groups often converge in the same areas; therefore, interventions targeting the mosquito population may have a potential role. Ultimately, a multisectoral approach as well as innovative and flexible malaria control strategies will be required if malaria elimination efforts are to be successful.

Evaluation of Methods for the Extraction of Microbial DNA From Vaginal Swabs Used for Microbiome Studies.

Background: The composition of the microbiome in human body sites plays an important role in health. The vaginal environment is colonized by several species of bacteria that have a major influence on reproductive health. The advancement of sequencing technologies has made the assessment of the composition of the microbiota possible through microbial DNA extraction and sequencing. Therefore, it is of a paramount importance to select a sensitive and reproducible DNA extraction method, that facilitates isolation of microbial DNA with a sufficient quantity and purity, from microbial species living in the vaginal environment. Here, we have evaluated four different DNA extraction protocols from self-collected vaginal swabs. Methods: Five healthy female volunteers were enrolled in the study. Each donor was asked to self-collect 4 samples using Copan ESwab. DNA was extracted using Qiagen DNeasy kit and three modified protocols of the MoBio PowerSoil kit ("DNeasy PowerSoil" after acquisition from Qiagen). DNA quantity and integrity was checked through Nanodrop and LabChip GX. DNA was further tested through quantitative real-time PCR (qPCR) and 16S sequencing. Vaginal microbiota diversities were determined using MiSeq-Illumina high-throughput sequencing of bacterial 16S rDNA V1-V3 fingerprint. Sequencing data were analyzed using QIIME pipeline. Results: Qiagen DNeasy protocol resulted in the highest DNA yield as compared to the modified protocols of MoBio Powersoil kit. The size of the DNA extracted using each protocol was ~40 kb. Qiagen DNeasy protocol gave the highest Genomic Quality Score (average ± standard deviation: 4.24 ± 0.36), followed by the different MoBio Powersoil protocols. A substantial variability in microbial DNA abundance was found across the protocols. The vaginal microbiota of the healthy volunteers was dominated by Lactobacillus species. MoBio Powersoil kit provided a significantly higher alpha diversity as compared to the Qiagen DNeasy kit, while beta diversity measures did not reveal any significant cluster changes, except when the Bray-Curtis method was applied. Conclusion: We were able to isolate microbial DNA from the vaginal swabs. Qiagen DNeasy method gave the highest DNA yield and quality but was not optimal in detecting microbial diversity. The modified MoBio PowerSoil protocols showed higher microbial diversities as compared to the standard protocol.

Reflections on a Community Engagement Strategy for Mass Antimalarial Drug Administration in Cambodia.

Mass drug administration (MDA) to interrupt malaria transmission requires the participation of entire communities. As part of a clinical trial in western Cambodia, four villages received MDA in 2015-2016. Before approaching study communities, a collaboration was established with the local health authorities, village leaders, and village malaria workers. Formative research guided the development of engagement strategies. In each village, a team of volunteers was formed to explain MDA to their neighbors and provide support during implementation. Public mobilization events featuring drama and music were used to introduce MDA. Villages comprised groups with different levels of understanding and interests; therefore, multiple tailored engagement strategies were required. The main challenges were explaining malaria transmission, managing perceptions of drug side effects, and reaching mobile populations. It was important that local leaders took a central role in community engagement. Coverage during each round of MDA averaged 84%, which met the target for the trial.

Impact of Enhanced Health Interventions for United States-Bound Refugees: Evaluating Best Practices in Migration Health.

With an unprecedented number of displaced persons worldwide, strategies for improving the health of migrating populations are critical. United States-bound refugees undergo a required overseas medical examination to identify inadmissible conditions (e.g., tuberculosis) 2-6 months before resettlement, but it is limited in scope and may miss important, preventable infectious, chronic, or nutritional causes of morbidity. We sought to evaluate the feasibility and health impact of diagnosis and management of such conditions before travel. We offered voluntary testing for intestinal parasites, anemia, and hepatitis B virus infection, to U.S.-bound refugees from three Thailand-Burma border camps. Treatment and preventive measures (e.g., anemia and parasite treatment, vaccination) were initiated before resettlement. United States refugee health partners received overseas results and provided post-arrival medical examination findings. During July 9, 2012 to November 29, 2013, 2,004 refugees aged 0.5-89 years enrolled. Among 463 participants screened for seven intestinal parasites overseas and after arrival, helminthic infections decreased from 67% to 12%. Among 118 with positive Strongyloides-specific antibody responses, the median fluorescent intensity decreased by an average of 81% after treatment. The prevalence of moderate-to-severe anemia (hemoglobin < 10 g/dL) was halved from 14% at baseline to 7% at departure (McNemar P = 0.001). All 191 (10%) hepatitis B-infected participants received counseling and evaluation; uninfected participants were offered vaccination. This evaluation demonstrates that targeted screening, treatment, and prevention services can be conducted during the migration process to improve the health of refugees before resettlement. With more than 250 million migrants globally, this model may offer insights into healthier migration strategies.

Induction of tissue factor expression and release as microparticles in ECV304 cell line by Chlamydia pneumoniae infection.

The association between Chlamydia pneumoniae (C. pneumoniae) infection and the onset and progression of atherosclerosis has become apparent recently. Moreover, increased expression of tissue factor (TF) as a result of C. pneumoniae infection has been previously demonstrated. We have examined the expression of TF on the surface of endothelial cells and the release of TF-containing cell-derived microparticles, over seven days. Additionally, using cells expressing a procoagulantly active EGFP-TF hybrid protein, we examined the kinetics of TF trafficking on the cells and incorporation into shed microparticles. Finally, in an attempt to associate this with the activation of NFkappaB, we used a luciferase reporter to measure the duration of the activation of this transcription factor. TF-containing microparticles were released within 24h of infection and continued for up to 7 days. Moreover, the initial release of TF containing microparticles was associated with NFkappaB activation and was suppressed on inclusion of an NFkappaB inhibitor, pyrrolidinedithiocarbamate ammonium. Moreover, persistent dissemination of TF-containing microparticles at later stages of infection was associated with the release of the infective C. pneumoniae elementary bodies. The released procoagulant, cellular microparticles are known to be strongly atherogenic and therefore we suggest a mechanism for the involvement of C. pneumoniae in the onset and progression of vascular disease.