timsTOF HT Improves Protein Identification and Quantitative Reproducibility for Deep Unbiased Plasma Protein Biomarker Discovery.

Mass spectrometry (MS) is a valuable tool for plasma proteome profiling and disease biomarker discovery. However, wide-ranging plasma protein concentrations, along with technical and biological variabilities, present significant challenges for deep and reproducible protein quantitation. Here, we evaluated the qualitative and quantitative performance of timsTOF HT and timsTOF Pro 2 mass spectrometers for analysis of neat plasma samples (unfractionated) and plasma samples processed using the Proteograph Product Suite (Proteograph) that enables robust deep proteomics sampling prior to mass spectrometry. Samples were evaluated across a wide range of peptide loading masses and liquid chromatography (LC) gradients. We observed up to a 76% increase in total plasma peptide precursors identified and a >2-fold boost in quantifiable plasma peptide precursors (CV < 20%) with timsTOF HT compared to Pro 2. Additionally, approximately 4.5 fold more plasma peptide precursors were detected by both timsTOF HT and timsTOF Pro 2 in the Proteograph analyzed plasma vs neat plasma. In an exploratory analysis of 20 late-stage lung cancer and 20 control plasma samples with the Proteograph, which were expected to exhibit distinct proteomes, an approximate 50% increase in total and statistically significant plasma peptide precursors (q < 0.05) was observed with timsTOF HT compared to Pro 2. Our data demonstrate the superior performance of timsTOF HT for identifying and quantifying differences between biologically diverse samples, allowing for improved disease biomarker discovery in large cohort studies. Moreover, researchers can leverage data sets from this study to optimize their liquid chromatography-mass spectrometry (LC-MS) workflows for plasma protein profiling and biomarker discovery. (ProteomeXchange identifier: PXD047854 and PXD047839).

Metagenomic shotgun sequencing reveals host species as an important driver of virome composition in mosquitoes.

High-throughput nucleic acid sequencing has greatly accelerated the discovery of viruses in the environment. Mosquitoes, because of their public health importance, are among those organisms whose viromes are being intensively characterized. Despite the deluge of sequence information, our understanding of the major drivers influencing the ecology of mosquito viromes remains limited. Using methods to increase the relative proportion of microbial RNA coupled with RNA-seq we characterize RNA viruses and other symbionts of three mosquito species collected along a rural to urban habitat gradient in Thailand. The full factorial study design allows us to explicitly investigate the relative importance of host species and habitat in structuring viral communities. We found that the pattern of virus presence was defined primarily by host species rather than by geographic locations or habitats. Our result suggests that insect-associated viruses display relatively narrow host ranges but are capable of spreading through a mosquito population at the geographical scale of our study. We also detected various single-celled and multicellular microorganisms such as bacteria, alveolates, fungi, and nematodes. Our study emphasizes the importance of including ecological information in viromic studies in order to gain further insights into viral ecology in systems where host specificity is driving both viral ecology and evolution.

Clinical Utility of a Blood-based Protein Assay on Diagnostic Colonoscopy Referrals for Elevated-risk Colorectal Cancer Patients in Primary Care.

BACKGROUND: Colonoscopies are effective in finding early stage colorectal cancer (CRC), which when found in a timely manner, dramatically improve survival rates. A significant number of at-risk patients are still not screened. We investigated the utility of a blood-based protein assay to assess for CRC in patients with elevated risk on the quality of preventive care delivered by board-certified primary care physicians (PCPs) in the United States. METHODS: We report on the results of a 3-part, longitudinal, randomized controlled trial. Part 1 assessed physicians' ability to identify simulated patients at risk for CRC and found PCPs missed colonoscopy referrals for high-risk patients ~40% of the time. Part 2 randomized PCPs into control and intervention arms and demonstrated that a novel blood-based protein assay increased referral rates for a diagnostic colonoscopy when caring for simulated patients. Part 3, reported herein, compares real-world colonoscopy rates of actual patients cared for by control versus intervention physicians. Part 3 was executed to confirm whether the use of the assay demonstrated the same utility in their real world, high-risk patients as found in part 2 using simulated patients. RESULTS: In the simulations, physicians with access to the assay were significantly more likely to order diagnostic colonoscopies. Similarly, in real-world practice, patients were also more likely to be referred for a diagnostic colonoscopy (odds ratio, 4.57; 95% confidence interval, 1.19-17.57). CONCLUSIONS: An increase in CRC risk, as indicated by the assay in simulated and real-life patients, was associated with a higher likelihood of appropriate patients being referred to diagnostic colonoscopy.

Using virtual standardized patients to accurately assess information gathering skills in medical students.

Introduction: Practicing a medical history using standardized patients is an essential component of medical school curricula. Recent advances in technology now allow for newer approaches for practicing and assessing communication skills. We describe herein a virtual standardized patient (VSP) system that allows students to practice their history taking skills and receive immediate feedback. Methods: Our VSPs consist of artificially intelligent, emotionally responsive 3D characters which communicate with students using natural language. The system categorizes the input questions according to specific domains and summarizes the encounter. Automated assessment by the computer was compared to manual assessment by trained raters to assess accuracy of the grading system. Results: Twenty dialogs chosen randomly from 102 total encounters were analyzed by three human and one computer rater. Overall scores calculated by the computer were not different than those provided by the human raters, and overall accuracy of the computer system was 87%, compared with 90% for human raters. Inter-rater reliability was high across 19 of 21 categories. Conclusions: We have developed a virtual standardized patient system that can understand, respond, categorize, and assess student performance in gathering information during a typical medical history, thus enabling students to practice their history-taking skills and receive immediate feedback.

Operationalizing One Health Employing Social-Ecological Systems Theory: Lessons From the Greater Mekong Sub-region.

The idea of the interdependency of the health of humans, animals, and ecosystems emerged from the interplay of theory and concepts from medicine, public health and ecology among leading thinkers in these fields during the last century. The rationale for One Health and its focus on the "human, animal, and environmental interface" stems from this legacy and points to transdisciplinary, ecological and complex systems approaches as central to One Health practice. Demonstration of One Health's efficacy, its wider adoption and continual improvement require explicit operational criteria and evaluation metrics on this basis. Social-Ecological Systems Theory with its unique conception of resilience (SESR) currently offers the most well-developed framework for understanding these approaches and development of performance standards. This paper describes operational criteria for One Health developed accordingly, including a protocol currently being tested for vector borne disease interventions. Wider adoption of One Health is most likely to occur as One Health practitioners gain an increasing familiarity with ecological and complex systems concepts in practice employing a transdisciplinary process. Two areas in which this inevitably will be required for significant further progress, and where the beginnings of a foundation for building upon exist, include: (1) Emerging and re-emerging zoonotic diseases, and (2) successful implementation of the United Nations (UN) Sustainable Development Goals (SDGs). The former includes the challenge of stemming the threat of new microbial pathogens, anti-microbial resistant variants of existing pathogens, as well as resurgence of malaria and other recalcitrant diseases. The applicability of SESR in this regard is illustrated with two case examples from the Greater Mekong Subregion, Avian Influenza (H5N1) and Liver Fluke (Opisthorchis viverrini). Each is shown to represent a science and policy challenge suggestive of an avoidable social-ecological system pathology that similarly has challenged sustainable development. Thus, SESR framing arguably is highly applicable to the SDGs, which, to a large extent, require consideration of human-animal-environmental health linkages. Further elaboration of these One Health operational criteria and metrics could contribute to the achievement of many of the SDGs.

Vector-borne disease and climate change adaptation in African dryland social-ecological systems.

BACKGROUND: Drylands, which are among the biosphere's most naturally limiting and environmentally variable ecosystems, constitute three-quarters of the African continent. As a result, environmental sustainability and human development along with vector-borne disease (VBD) control historically have been especially challenging in Africa, particularly in the sub-Saharan and Sahelian drylands. Here, the VBD burden, food insecurity, environmental degradation, and social vulnerability are particularly severe. Changing climate can exacerbate the legion of environmental health threats in Africa, the social dimensions of which are now part of the international development agenda. Accordingly, the need to better understand the dynamics and complex coupling of populations and environments as exemplified by drylands is increasingly recognized as critical to the design of more sustainable interventions. MAIN BODY: This scoping review examines the challenge of vector-borne disease control in drylands with a focus on Africa, and the dramatic, ongoing environmental and social changes taking place. Dryland societies persisted and even flourished in the past despite changing climates, extreme and unpredictable weather, and marginal conditions for agriculture. Yet intrusive forces largely out of the control of traditional dryland societies, along with the negative impacts of globalization, have contributed to the erosion of dryland's cultural and natural resources. This has led to the loss of resilience underlying the adaptive capacity formerly widely exhibited among dryland societies. A growing body of evidence from studies of environmental and natural resource management demonstrates how, in light of dryland system's inherent complexity, these factors and top-down interventions can impede sustainable development and vector-borne disease control. Strengthening adaptive capacity through community-based, participatory methods that build on local knowledge and are tailored to local ecological conditions, hold the best promise of reversing current trends. CONCLUSIONS: A significant opportunity exists to simultaneously address the increasing threat of vector-borne diseases and climate change through methods aimed at strengthening adaptive capacity. The integrative framework and methods based on social-ecological systems and resilience theory offers a novel set of tools that allow multiple threats and sources of vulnerability to be addressed in combination. Integration of recent advances in vector borne disease ecology and wider deployment of these tools could help reverse the negative social and environmental trends currently seen in African drylands.

Transdisciplinary and social-ecological health frameworks-Novel approaches to emerging parasitic and vector-borne diseases.

Ecosystem Health, Conservation Medicine, EcoHealth, One Health, Planetary Health and GeoHealth are inter-related disciplines that underpin a shared understanding of the functional prerequisites of health, sustainable vitality and wellbeing. All of these are based on recognition that health interconnects species across the planet, and they offer ways to more effectively tackle complex real-world challenges. Herein we present a bibliometric analysis to document usage of a subset of such terms by journals over time. We also provide examples of parasitic and vector-borne diseases, including malaria, toxoplasmosis, baylisascariasis, and Lyme disease. These and many other diseases have persisted, emerged or re-emerged, and caused great harm to human and animal populations in developed and low income, biodiverse nations around the world, largely because of societal drivers that undermined natural processes of disease prevention and control, which had developed through co-evolution over millennia. Shortcomings in addressing drivers has arisen from a lack or coordinated efforts among researchers, health stewards, societies at large, and governments. Fortunately, specialists collaborating under transdisciplinary and socio-ecological health umbrellas are increasingly integrating established and new techniques for disease modeling, prediction, diagnosis, treatment, control, and prevention. Such approaches often emphasize conservation of biodiversity for health protection, and they provide novel opportunities to increase the efficiency and probability of success.

A large-scale and robust dynamic MRM study of colorectal cancer biomarkers.

Over the past 20 years, mass spectrometry (MS) has emerged as a dynamic tool for proteomics biomarker discovery. However, published MS biomarker candidates often do not translate to the clinic, failing during attempts at independent replication. The cause can be shortcomings in study design, sample quality, assay quantitation, and/or quality/process control. To address these shortcomings, we developed an MS workflow in accordance with Tier 2 measurement requirements for targeted peptides, defined by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) "fit-for-purpose" approach, using dynamic multiple reaction monitoring (dMRM), which measures specific peptide transitions during predefined retention time (RT) windows. We describe the development of a robust multipex dMRM assay measuring 641 proteotypic peptides from 392 colorectal cancer (CRC) related proteins, and the procedures to track and handle sample processing and instrument variation over a four-month study, during which the assay measured blood samples from 1045 patients with CRC symptoms. After data collection, transitions were filtered by signal quality metrics before entering receiver operating characteristic (ROC) analysis. The results demonstrated CRC signal carried by 127 proteins in the symptomatic population. The workflow might be further developed to build Tier 1 assays for clinical tests identifying symptomatic individuals at elevated risk of CRC. SIGNIFICANCE: We developed a dMRM MS method with the rigor of a Tier 2 assay as defined by the CPTAC 'fit for purpose approach' [1]. Using quality and process control procedures, the assay was used to quantify 641 proteotypic peptides representing 392 CRC-related proteins in plasma from 1045 CRC-symptomatic patients. To our knowledge, this is the largest MRM method applied to the largest study to date. The results showed that 127 of the proteins carried univariate CRC signal in the symptomatic population. This large number of single biomarkers bodes well for future development of multivariate classifiers to distinguish CRC in the symptomatic population.

Thinking beyond Opisthorchis viverrini for risk of cholangiocarcinoma in the lower Mekong region: a systematic review and meta-analysis.

BACKGROUND: Cholangiocarcinoma (CCA) is a fatal bile duct cancer associated with infection by the liver fluke, Opisthorchis viverrini, in the lower Mekong region. Numerous public health interventions have focused on reducing exposure to O. viverrini, but incidence of CCA in the region remains high. While this may indicate the inefficacy of public health interventions due to complex social and cultural factors, it may further indicate other risk factors or interactions with the parasite are important in pathogenesis of CCA. This systematic review aims to provide a comprehensive analysis of described risk factors for CCA in addition to O. viverrini to guide future integrative interventions. MAIN BODY: We searched five international and seven Thai research databases to identify studies relevant to risk factors for CCA in the lower Mekong region. Selected studies were assessed for risk of bias and quality in terms of study design, population, CCA diagnostic methods, and statistical methods. The final 18 included studies reported numerous risk factors which were grouped into behaviors, socioeconomics, diet, genetics, gender, immune response, other infections, and treatment for O. viverrini. Seventeen risk factors were reported by two or more studies and were assessed with random effects models during meta-analysis. This meta-analysis indicates that the combination of alcohol and smoking (OR = 11.1, 95% CI: 5.63-21.92, P <  0.0001) is most significantly associated with increased risk for CCA and is an even greater risk factor than O. viverrini exposure. This analysis also suggests that family history of cancer, consumption of raw cyprinoid fish, consumption of high nitrate foods, and praziquantel treatment are associated with significantly increased risk. These risk factors may have complex relationships with the host, parasite, or pathogenesis of CCA, and many of these risk factors were found to interact with each other in one or more studies. CONCLUSIONS: Our findings suggest that a complex variety of risk factors in addition to O. viverrini infection should be addressed in future public health interventions to reduce CCA in affected regions. In particular, smoking and alcohol use, dietary patterns, and socioeconomic factors should be considered when developing intervention programs to reduce CCA.

Analytical validation of a novel multiplex test for detection of advanced adenoma and colorectal cancer in symptomatic patients.

Early detection of colorectal cancer (CRC) is key to reducing associated mortality. Despite the importance of early detection, approximately 40% of individuals in the United States between the ages of 50-75 have never been screened for CRC. The low compliance with colonoscopy and fecal-based screening may be addressed with a non-invasive alternative such as a blood-based test. We describe here the analytical validation of a multiplexed blood-based assay that measures the plasma concentrations of 15 proteins to assess advanced adenoma (AA) and CRC risk in symptomatic patients. The test was developed on an electrochemiluminescent immunoassay platform employing four multi-marker panels, to be implemented in the clinic as a laboratory developed test (LDT). Under the Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) regulations, a United States-based clinical laboratory utilizing an LDT must establish performance characteristics relating to analytical validity prior to releasing patient test results. This report describes a series of studies demonstrating the precision, accuracy, analytical sensitivity, and analytical specificity for each of the 15 assays, as required by CLIA/CAP. In addition, the report describes studies characterizing each of the assays' dynamic range, parallelism, tolerance to common interfering substances, spike recovery, and stability to sample freeze-thaw cycles. Upon completion of the analytical characterization, a clinical accuracy study was performed to evaluate concordance of AA and CRC classifier model calls using the analytical method intended for use in the clinic. Of 434 symptomatic patient samples tested, the percent agreement with original CRC and AA calls was 87% and 92% respectively. All studies followed CLSI guidelines and met the regulatory requirements for implementation of a new LDT. The results provide the analytical evidence to support the implementation of the novel multi-marker test as a clinical test for evaluating CRC and AA risk in symptomatic individuals.

Mosquito vector-associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod-borne diseases.

Vector-borne diseases are a major health burden, yet factors affecting their spread are only partially understood. For example, microbial symbionts can impact mosquito reproduction, survival, and vectorial capacity, and hence affect disease transmission. Nonetheless, current knowledge of mosquito-associated microbial communities is limited. To characterize the bacterial and eukaryotic microbial communities of multiple vector species collected from different habitat types in disease endemic areas, we employed next-generation 454 pyrosequencing of 16S and 18S rRNA amplicon libraries, also known as metabarcoding. We investigated pooled whole adult mosquitoes of three medically important vectors, Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus, collected from different habitats across central Thailand where we previously characterized mosquito diversity. Our results indicate that diversity within the mosquito microbiota is low, with the majority of microbes assigned to one or a few taxa. Two of the most common eukaryotic and bacterial genera recovered (Ascogregarina and Wolbachia, respectively) are known mosquito endosymbionts with potentially parasitic and long evolutionary relationships with their hosts. Patterns of microbial composition and diversity appeared to differ by both vector species and habitat for a given species, although high variability between samples suggests a strong stochastic element to microbiota assembly. In general, our findings suggest that multiple factors, such as habitat condition and mosquito species identity, may influence overall microbial community composition, and thus provide a basis for further investigations into the interactions between vectors, their microbial communities, and human-impacted landscapes that may ultimately affect vector-borne disease risk.

Discovery and validation of a colorectal cancer classifier in a new blood test with improved performance for high-risk subjects.

BACKGROUND: The aim was to improve upon an existing blood-based colorectal cancer (CRC) test directed to high-risk symptomatic patients, by developing a new CRC classifier to be used with a new test embodiment. The new test uses a robust assay format-electrochemiluminescence immunoassays-to quantify protein concentrations. The aim was achieved by building and validating a CRC classifier using concentration measures from a large sample set representing a true intent-to-test (ITT) symptomatic population. METHODS: 4435 patient samples were drawn from the Endoscopy II sample set. Samples were collected at seven hospitals across Denmark between 2010 and 2012 from subjects with symptoms of colorectal neoplasia. Colonoscopies revealed the presence or absence of CRC. 27 blood plasma proteins were selected as candidate biomarkers based on previous studies. Multiplexed electrochemiluminescence assays were used to measure the concentrations of these 27 proteins in all 4435 samples. 3066 patients were randomly assigned to the Discovery set, in which machine learning was used to build candidate classifiers. Some classifiers were refined by allowing up to a 25% indeterminate score range. The classifier with the best Discovery set performance was successfully validated in the separate Validation set, consisting of 1336 samples. RESULTS: The final classifier was a logistic regression using ten predictors: eight proteins (A1AG, CEA, CO9, DPPIV, MIF, PKM2, SAA, TFRC), age, and gender. In validation, the indeterminate rate of the new panel was 23.2%, sensitivity/specificity was 0.80/0.83, PPV was 36.5%, and NPV was 97.1%. CONCLUSIONS: The validated classifier serves as the basis of a new blood-based CRC test for symptomatic patients. The improved performance, resulting from robust concentration measures across a large sample set mirroring the ITT population, renders the new test the best available for this population. Results from a test using this classifier can help assess symptomatic patients' CRC risk, increase their colonoscopy compliance, and manage next steps in their care.

Developing a Conversational Virtual Standardized Patient to Enable Students to Practice History-Taking Skills.

INTRODUCTION: Although traditional virtual patient simulations are designed to teach and assess clinical reasoning skills, few employ conversational dialogue with the patients. The virtual standardized patients (VSPs) described herein represent standardized patients that students interview using natural language. Students take histories and develop differential diagnoses of the VSPs as much as they would with standardized or actual patients. The student-VSP interactions are recorded, creating a comprehensive record of questions and the order in which they were asked, which can be analyzed to assess information-gathering skills. Students document the encounter in an electronic medical record created for the VSPs. METHODS: The VSP was developed by integrating a dialogue management system (ChatScript) with emotionally responsive 3D characters created in a high-fidelity game engine (Unity). The system was tested with medical students at the Ohio State University College of Medicine. Students are able to take a history of a VSP, develop a differential diagnosis, and document the encounter in the electronic medical record. RESULTS: Accuracy of the VSP responses ranged from 79% to 86%, depending on the complexity of the case, type of history obtained, and skill of the student. Students were able to accurately develop an appropriate differential diagnosis on the basis of the information provided by the patient during the encounter. CONCLUSIONS: The VSP enables students to practice their history-taking skills before encounters with standardized or actual patients. Future developments will focus on creating an assessment module that will automatically analyze VSP sessions and provide immediate student feedback.

Fish sharing as a risk factor for Opisthorchis viverrini infection: evidence from two villages in north-eastern Thailand.

BACKGROUND: Foodborne trematodiasis (FBT) is a significant global health problem, with the liver flukes Opisthorchis viverrini, O. felineus, and Clonorchis sinensis contributing to half of the global burden of FBT. North-eastern Thailand where O. viverrini is endemic and un-cooked fish dishes remain an integral part of the food culture has the highest reported incidence of opisthorchiasis, including associated cholangiocarcinoma. Both food sharing and eating practices are potentially important factors in FTB, suggesting an important role for the social ecology of disease transmission in these rural communities. METHODS: Two rural Thai-Lao villages that were part of a 12-village project in Northeastern Thailand were selected for detailed investigation of O. viverrini infection risk associated with sharing of raw fish dishes among households. The project included screening individuals for infection and cholangiocarcinoma, a household questionnaire, and offering treatment options for positive individuals. Social network mapping was used to construct raw fish dish-sharing networks and create a proxy variable capturing variability in the degree of food sharing (DFS), measured as the number of different households with which each household shared fish dishes. Measures of associations between DFS, O. viverrini infection, the frequency of raw fish consumption, and the number of raw fish dishes consumed were generated using binary logistic regression, proportional odds ordinal logistic regression, and Poisson regression. RESULTS: The results showed that the probability that a household has members infected with O. viverrini increased by ~7% (P < 0.01) for each additional household included in its network. Moreover, the frequency and number of types of raw fish dishes consumed increased significantly as the DFS increased. Of the two villages, that with the highest infection prevalence (48% versus 34.6%) had significantly higher social connectivity overall (P < 0.001). CONCLUSIONS: Our findings suggest that the social ecology of human settlements may be key to understanding the transmission dynamics of some FBT. In the case of O. viverrini in Thai-Lao communities, for which food sharing is a traditional practice supporting social cohesion, food sharing network mapping should be incorporated into community-based interventions. These should encourage fish dish preparation methods that minimize infection risk by targeting households with high DFS values.

Role of socio-cultural and economic factors in cyprinid fish distribution networks and consumption in Lawa Lake region, Northeast Thailand: Novel perspectives on Opisthorchis viverrini transmission dynamics.

Opisthorchis viverrini (Ov) is a fish-borne parasite endemic in parts of Lao PDR, Cambodia, southern Vietnam and Northeast Thailand (Isaan) where an estimated 10 million people are infected. Human Ov infection, associated with hepatobiliary complications, including cholangiocarcinoma (CCA), occurs when infected fish are consumed raw or undercooked, a longstanding cultural tradition in the region. This mixed- methods descriptive study was carried out in Isaan villages around Lawa Lake, Khon Kaen Province, known for their Ov endemicity. Focus group discussions (FGDs) and in depth interviews (IDIs) were used to explore socio-cultural determinants underlying raw fish consumption practices, and global positioning system (GPS) devices to map local fish distribution networks. Qualitative data affirmed major socio-cultural and dietary lifestyle transitions occurring consequent on recent decades of modernization policies and practices, but also the persistence of Isaan traditional raw-fish eating practices and incorrect beliefs about infection risk avoidance. Fish traders/middlemen purchase most of the catch at the lakeshore and play the dominant role in district market fish distribution networks, at least for the larger and less likely infected, fish species. The lower economic value of the small potentially-infected cyprinid fish means local fishermen typically distribute them free, or sell cheaply, to family and friends, effectively concentrating infection risk in already highly Ov infected villages. Our study confirmed the persistence of traditional Isaan raw-fish meal practices, despite major ongoing socio-cultural lifestyle transitions and decades of Ov infection health education programs. We contend that diffuse socio-cultural drivers underpin this practice, including its role as a valued cultural identity marker. A "fish economics" factor was also evident in the concentration of more likely infected fish back into local villages due to their low economic value at district market level. The complexity of factors supporting "risky" fish-eating traditions in Isaan underscores the importance of integrated liver fluke infection control strategies to draw on transdisciplinary knowledge beyond biomedicine and also embrace participatory protocols for engaging communities in developing, implementing and evaluating interventions.

Balancing biomedical and ecological perspectives in research framing of liver fluke and cholangiocarcinoma in NE Thailand.

This review examines the association of Asian liver flukes and cholangiocarcinoma (CCA) from the standpoint of two contrasting research perspectives: that aligned with the biomedical model predominantly employed to date; and, that aligned with ecological (and evolutionary) thinking increasingly being used to frame research questions that address this association in Northeast Thailand. An examination of the assumptions that underlie most of this research, requisite of evidence-based health research, shows how a broadened research frame that incorporates 'ecologic' perspectives provides alternatives to the prevailing scientific interpretations and public narrative. A more balanced and integrative research approach that combines elements of the biomedical model and ecologic models of health is suggested to overcome the limited progress toward the reduction of liver fluke infection prevalence and CCA incidence in this region. Similarly, this approach presents an opportunity to further enhance collaborative research programs involving Parasitology and the complementary fields in the health sciences.

Seasonal and Spatial Environmental Influence on Opisthorchis viverrini Intermediate Hosts, Abundance, and Distribution: Insights on Transmission Dynamics and Sustainable Control.

BACKGROUND: Opisthorchis viverrini (Ov) is a complex-life-cycle trematode affecting 10 million people in SEA (Southeast Asia). Human infection occurs when infected cyprinid fish are consumed raw or undercooked. Ov requires three hosts and presents two free-living parasitic stages. As a consequence Ov transmission and infection in intermediate and human hosts are strongly mediated by environmental factors and understanding how environmental variability influences intermediate host abundance is critical. The objectives of this study were 1) to document water parameters, intermediate hosts abundance and infection spatio-temporal variation, 2) to assess their causal relationships and identify windows of transmission risk. METHODOLOGY/PRINCIPAL FINDINGS: Fish and snails were collected monthly for one year at 12 sites in Lawa Lake, an Ov-endemic region of Khon Kaen Province in Northeast Thailand. Physicochemical water parameters [pH, temperature (Tp), dissolved oxygen (DO), Salinity, electrical conductivity (EC), total dissolved solid (TDS), nitrite nitrogen (NO2-N), lead (Pb), total coliform bacteria (TCB) and fecal coliform bacteria (FCB)] were measured. Multivariate analyses, linear models and kriging were used to characterize water parameter variation and its influence on host abundance and infection prevalence. We found that sampling sites could be grouped in three clusters and discriminated along a nitrogen-salinity gradient where higher levels in the lake's southern region predicted higher Bithynia relative abundance (P<0.05) and lower snail and fish species diversity (P<0.05). Highest Bithynia abundance occurred during rainy season (P<0.001), independently of site influence. Cyprinids were the most abundant fish family and higher cyprinid relative abundance was found in areas with higher Bithynia relative abundance (P<0.05). Ov infection in snails was anecdotal while Ov infection in fish was higher in the southern region (P<0.001) at sites showing high FCB. CONCLUSIONS/SIGNIFICANCE: Our results indicate that water contamination and waterways configuration can influence freshwater communities' assemblages possibly creating ideal conditions for sustained transmission. Sustainable control may require a better appreciation of the system's ecology with wise governance and development planning particularly in the current context of SEA agricultural intensification and landscape modification.

A Plasma-Based Protein Marker Panel for Colorectal Cancer Detection Identified by Multiplex Targeted Mass Spectrometry.

INTRODUCTION: Colorectal cancer (CRC) testing programs reduce mortality; however, approximately 40% of the recommended population who should undergo CRC testing does not. Early colon cancer detection in patient populations ineligible for testing, such as the elderly or those with significant comorbidities, could have clinical benefit. Despite many attempts to identify individual protein markers of this disease, little progress has been made. Targeted mass spectrometry, using multiple reaction monitoring (MRM) technology, enables the simultaneous assessment of groups of candidates for improved detection performance. MATERIALS AND METHODS: A multiplex assay was developed for 187 candidate marker proteins, using 337 peptides monitored through 674 simultaneously measured MRM transitions in a 30-minute liquid chromatography-mass spectrometry analysis of immunodepleted blood plasma. To evaluate the combined candidate marker performance, the present study used 274 individual patient blood plasma samples, 137 with biopsy-confirmed colorectal cancer and 137 age- and gender-matched controls. Using 2 well-matched platforms running 5 days each week, all 274 samples were analyzed in 52 days. RESULTS: Using one half of the data as a discovery set (69 disease cases and 69 control cases), the elastic net feature selection and random forest classifier assembly were used in cross-validation to identify a 15-transition classifier. The mean training receiver operating characteristic area under the curve was 0.82. After final classifier assembly using the entire discovery set, the 136-sample (68 disease cases and 68 control cases) validation set was evaluated. The validation area under the curve was 0.91. At the point of maximum accuracy (84%), the sensitivity was 87% and the specificity was 81%. CONCLUSION: These results have demonstrated the ability of simultaneous assessment of candidate marker proteins using high-multiplex, targeted-mass spectrometry to identify a subset group of CRC markers with significant and meaningful performance.