Validation of an enzyme- linked immunoassay assay for osteocalcin, a marker of bone formation, in dried blood spots

ObjectivesInvestigating factors that contribute to bone loss and accretion across populations in remote settings is challenging, particularly where diagnostic tools are scarce. To mitigate this challenge, we describe validation of a commercial ELISA assay to measure osteocalcin, a biomarker of bone formation, from dried blood spots (DBS).MethodsWe validated the Osteocalcin Human SimpleStep ELISA kit from Abcam (ab1951214) using 158 matched plasma and DBS samples. Passing- Bablok regression analysis assessed the relationships between plasma and DBS osteocalcin concentrations. Dilutional linearity and spike and recovery experiments determined if the DBS matrix interfered with osteocalcin measurement, and intra- and inter- assay coefficients of variation (CVs) were calculated. Limit of detection, analyte stability, and specific forms of osteocalcin measured by the kit were also investigated.ResultsMean plasma osteocalcin value was 218.2 ng/mL (range 64.6- 618.1 ng/mL). Linear relationships existed between plasma and DBS concentrations of osteocalcin, with no apparent bias in plasma vs DBS concentrations. There was no apparent interference of the DBS matrix with measurement of osteocalcin in DBS. Intra- assay CV for DBS was ~8%, while average inter- assay CV was 14.8%. Limit of detection was 0.34- ng/mL. Osteocalcin concentrations were stable in DBS stored at - 28°C and room temperature, but not those stored at 37°C. This ELISA kit detects total osteocalcin.ConclusionsOsteocalcin, a bone formation biomarker, can be measured from DBS. Combined with a previously validated DBS assay for TRACP- 5b, a bone resorption biomarker, these assays have the potential to help researchers disentangle the many factors contributing to bone strength.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162811/2/ajhb23394.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162811/1/ajhb23394_am.pd

Local domestication of lactic acid bacteria via cassava beer fermentation

Cassava beer, or chicha, is typically consumed daily by the indigenous Shuar people of the Ecuadorian Amazon. This traditional beverage made from cassava tuber (Manihot esculenta) is thought to improve nutritional quality and flavor while extending shelf life in a tropical climate. Bacteria responsible for chicha fermentation could be a source of microbes for the human microbiome, but little is known regarding the microbiology of chicha. We investigated bacterial community composition of chicha batches using Illumina high-throughput sequencing. Fermented chicha samples were collected from seven Shuar households in two neighboring villages in the Morona-Santiago region of Ecuador, and the composition of the bacterial communities within each chicha sample was determined by sequencing a region of the 16S ribosomal gene. Members of the genus Lactobacillus dominated all samples. Significantly greater phylogenetic similarity was observed among chicha samples taken within a village than those from different villages. Community composition varied among chicha samples, even those separated by short geographic distances, suggesting that ecological and/or evolutionary processes, including human-mediated factors, may be responsible for creating locally distinct ferments. Our results add to evidence from other fermentation systems suggesting that traditional fermentation may be a form of domestication, providing endemic beneficial inocula for consumers, but additional research is needed to identify the mechanisms and extent of microbial dispersal

Market Integration and Soil-Transmitted Helminth Infection among the Shuar of Amazonian Ecuador

Background Soil-transmitted helminth (STH) infections have many negative health outcomes (e.g., diarrhea, nutritional deficiencies) that can also exacerbate poverty. These infections are generally highest among low-income populations, many of which are also undergoing market integration (MI; increased participation in a market-based economy). Yet the direct impact of MI-related social and environmental changes on STH infection patterns is poorly understood, making it unclear which lifestyle factors should be targeted to better control disease spread. This cross-sectional study examines if household infrastructure associated with greater MI is associated with lower STH burdens among Indigenous Ecuadorian Shuar. Methods Kato-Katz fecal smears were used to determine STH infection status and intensity (n = 620 participants; 308 females, 312 males, aged 6 months—86 years); Ascaris lumbricoides (ascarid) and Trichuris trichiura (whipworm) were the primary infection types detected. Structured interviews assessing lifestyle patterns (e.g., measures of household infrastructure) measured participant MI. Multilevel regression analyses and zero-inflated negative binomial regression models tested associations between MI measures and STH infection status or intensity, controlling for individual and community characteristics. Results Participants residing in more market-integrated households exhibited lower infection rates and intensities than those in less market integrated households. Parasite infection status and T. trichiura infection intensity were lower among participants living in houses with wood floors than those with dirt floors, while individuals using well or piped water from a spring exhibited lower A. lumbricoides infection intensities compared to those using river or stream water. Unexpectedly, latrine type was not significantly related to STH infection status or intensity. These results suggest that sources of exposure differ between the two helminth species. Conclusions This study documents associations between household measures and STH infection among an Indigenous population undergoing rapid MI. These findings can help healthcare programs better target interventions and reduce STH exposure among at-risk populations

Market Integration Predicts Human Gut Microbiome Attributes across a Gradient of Economic Development

Economic development is marked by dramatic increases in the incidence of microbiome-associated diseases, such as autoimmune diseases and metabolic syndromes, but the lifestyle changes that drive alterations in the human microbiome are not known. We measured market integration as a proxy for economically related lifestyle attributes, such as ownership of specific market goods that index degree of market integration and components of traditional and nontraditional (more modern) house structure and infrastructure, and profiled the fecal microbiomes of 213 participants from a contiguous, indigenous Ecuadorian population. Despite relatively modest differences in lifestyle across the population, greater economic development correlated with significantly lower within-host diversity, higher between-host dissimilarity, and a decrease in the relative abundance of the bacterium Prevotella. These microbiome shifts were most strongly associated with more modern housing, followed by reduced ownership of traditional subsistence lifestyle-associated items

Regional variation in Ascaris lumbricoides and Trichuris trichiura infections by age cohort and sex: effects of market integration among the indigenous Shuar of Amazonian Ecuador

Background: Soil-transmitted helminth (STH) infection peaks during childhood and varies by sex. The impact of market integration (MI) (increasing production for and consumption from a market-based economy) on these infection patterns, however, is unclear. In this study, STH infection is examined by sex and age among indigenous Shuar inhabiting two regions of Amazonian Ecuador: (1) the modestly market-integrated Upano Valley (UV) and (2) the more traditional Cross-Cutucú (CC) region. Methods: Kato-Katz fecal smears were examined for parasite presence and infection intensity. Factorial ANOVAs and post hoc simple effects analyses were performed by sex to compare infection intensity between regions and age categories (infant/child, juvenile/adolescent, adult). Results: Significant age and regional differences in Ascaris lumbricoides and Trichuris trichiura infection were detected. Overall, infants/children and juveniles/adolescents displayed higher parasite loads than adults. CC females exhibited higher A. lumbricoides loads than UV females, while the opposite pattern was observed for T. trichiura infection in males. Conclusions: Regional infection patterns varied by sex and parasite species, perhaps due to MI-linked environmental and lifestyle changes. These results have public health implications for the identification of individuals at risk for infection and contribute to ongoing efforts to track changes and alleviate STH infection in indigenous populations undergoing MI

A dried blood spotâ based method to measure levels of tartrateâ resistant acid phosphatase 5b (TRACPâ 5b), a marker of bone resorption

ObjectivesA number of basic questions about bone biology have not been answered, including population differences in bone turnover. In part, this stems from the lack of validated minimally invasive biomarker techniques to measure bone formation and resorption in fieldâ based populationâ level research. The present study addresses this gap by validating a fingerprick dried blood spot (fDBS) assay for tartrateâ resistant acid phosphatase 5b (TRACPâ 5b), a wellâ defined biomarker of bone resorption and osteoclast number.MethodsWe adapted a commercially available enzymeâ linked immunosorbent assay (ELISA) kit from MyBiosource for the quantitative determination of TRACPâ 5b levels in serum and plasma for use with DBS. We used a rigorous process of assay modification and validation, including the use of a matched set of 189 adult plasma, fDBS, and venous DBS (vDBS) samples; parameters evaluated included precision, reliability, and analyte stability.ResultsPlasma and DBS TRACPâ 5b concentrations showed a linear relationship. There were no systematic differences in TRACPâ 5b levels in fDBS and vDBS, indicating no significant differences in TRACPâ 5b distribution between capillary and venous blood. Parallelism and spikeâ andâ recovery results indicated that matrix factors in DBS do not interfere with measurement of TRACPâ 5b levels from DBS using the validated kit. Intraâ and interassay CVs were 5.0% and 12.1%, respectively. DBS samples should preferably be stored frozen but controlled room temperature storage for up to a month may be acceptable.ConclusionsThis DBSâ based ELISA assay adds to the methodological toolkit available to human biologists and will facilitate research on bone turnover in population studies.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149369/1/ajhb23240.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149369/2/ajhb23240_am.pd

A dried blood spot‐based method to measure levels of tartrate‐resistant acid phosphatase 5b (TRACP‐5b), a marker of bone resorption

ObjectivesA number of basic questions about bone biology have not been answered, including population differences in bone turnover. In part, this stems from the lack of validated minimally invasive biomarker techniques to measure bone formation and resorption in fieldâ based populationâ level research. The present study addresses this gap by validating a fingerprick dried blood spot (fDBS) assay for tartrateâ resistant acid phosphatase 5b (TRACPâ 5b), a wellâ defined biomarker of bone resorption and osteoclast number.MethodsWe adapted a commercially available enzymeâ linked immunosorbent assay (ELISA) kit from MyBiosource for the quantitative determination of TRACPâ 5b levels in serum and plasma for use with DBS. We used a rigorous process of assay modification and validation, including the use of a matched set of 189 adult plasma, fDBS, and venous DBS (vDBS) samples; parameters evaluated included precision, reliability, and analyte stability.ResultsPlasma and DBS TRACPâ 5b concentrations showed a linear relationship. There were no systematic differences in TRACPâ 5b levels in fDBS and vDBS, indicating no significant differences in TRACPâ 5b distribution between capillary and venous blood. Parallelism and spikeâ andâ recovery results indicated that matrix factors in DBS do not interfere with measurement of TRACPâ 5b levels from DBS using the validated kit. Intraâ and interassay CVs were 5.0% and 12.1%, respectively. DBS samples should preferably be stored frozen but controlled room temperature storage for up to a month may be acceptable.ConclusionsThis DBSâ based ELISA assay adds to the methodological toolkit available to human biologists and will facilitate research on bone turnover in population studies.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149369/1/ajhb23240.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149369/2/ajhb23240_am.pd

Development and validation of an ELISA for a biomarker of thyroid dysfunction, thyroid peroxidase autoantibodies (TPO-Ab), in dried blood spots

Abstract Background The prevalence of allergic and autoimmune conditions has been steadily increasing in wealthy nations over the past century. One hypothesis put forward to explain this is the Old Friends Hypothesis, which posits that increased hygiene, urbanization, and lifestyle changes have reduced our exposure to parasites and microbes that we co-evolved with, resulting in immune dysregulation. However, research in traditionally living populations, who are exposed to greater parasite and pathogen loads such as those encountered during our evolution, is limited, in part due to a lack of minimally invasive, field-friendly biomarkers of autoimmune disorders. We therefore developed an ELISA to assess positivity for thyroid peroxidase autoantibody (TPO-Ab), an indicator of autoimmune thyroid disease, based on dried blood spot (DBS) samples. Results We used the Accubind anti-thyroid peroxidase test system to screen our validation samples comprising matched fingerprick DBS, venous DBS, and plasma samples from 182 adults. After confirming that we had TPO-Ab-positive individuals in our validation sample (n = 12), we developed an indirect ELISA to measure TPO-Ab levels from one 3-mm DBS punch. The sensitivity and specificity of our assay for DBS samples ranged from 91.7–100% and 98.2–98.8%, respectively, using a cut-off value of ≥ 26 IU/mL. Intra-assay reliability for duplicate quality control DBS punches was 5.2%, while inter-assay reliability ranged from 11.5–24.4% for high, medium, and low DBS controls. Dilutional linearity ranged from 80 to 120%, and spike and recovery experiments indicated that the DBS matrix does not interfere with the detection of TPO-Ab. TPO-Ab levels remained stable in DBS samples stored at − 28 °C or − 80 °C, but decreased over time in DBS samples kept at 22 °C or at 37 °C. Conclusions We developed an in-house, kit-independent indirect ELISA assay to determine individuals’ TPO-Ab positivity based on dried blood spots, representing a cost-effective method with potential applications in a range of research settings.http://deepblue.lib.umich.edu/bitstream/2027.42/173958/1/40101_2020_Article_228.pd

Physical growth of the shuar: Height, Weight, and BMI references for an indigenous amazonian population

Information concerning physical growth among small-scale populations remains limited, yet such data are critical to local health efforts and to foster basic understandings of human life history and variation in childhood development. Using a large dataset and robust modeling methods, this study aims to describe growth from birth to adulthood among the indigenous Shuar of Amazonian Ecuador. Mixed-longitudinal measures of height, weight, and body mass index (BMI) were collected from Shuar participants (n = 2,463; age: 0-29 years). Centile growth curves and tables were created for each anthropometric variable of interest using Generalized Additive Models for Location, Scale, and Shape (GAMLSS). Pseudo-velocity and Lambda-Mu-Sigma curves were generated to further investigate Shuar patterns of growth and to facilitate comparison with United States Center for Disease Control and Prevention and multinational World Health Organization growth references. The Shuar are small throughout life and exhibit complex patterns of growth that differ substantially from those of international references. Similar to other Amazonians, Shuar growth in weight compares more favorably to references than growth in height, resulting in BMI curves that approximate international medians. Several additional characteristics of Shuar development are noteworthy, including large observed variation in body size early in life, significant infant growth faltering, extended male growth into adulthood, and a markedly early female pubertal growth spurt in height. Phenotypic plasticity and genetic selection in response to local environmental factors may explain many of these patterns. Providing a detailed reference of growth for the Shuar and other Amazonian populations, this study possesses direct clinical application and affords valuable insight into childhood health and the ecology of human growth

Market integration and soil-transmitted helminth infection among the Shuar of Amazonian Ecuador.

BackgroundSoil-transmitted helminth (STH) infections have many negative health outcomes (e.g., diarrhea, nutritional deficiencies) that can also exacerbate poverty. These infections are generally highest among low-income populations, many of which are also undergoing market integration (MI; increased participation in a market-based economy). Yet the direct impact of MI-related social and environmental changes on STH infection patterns is poorly understood, making it unclear which lifestyle factors should be targeted to better control disease spread. This cross-sectional study examines if household infrastructure associated with greater MI is associated with lower STH burdens among Indigenous Ecuadorian Shuar.MethodsKato-Katz fecal smears were used to determine STH infection status and intensity (n = 620 participants; 308 females, 312 males, aged 6 months-86 years); Ascaris lumbricoides (ascarid) and Trichuris trichiura (whipworm) were the primary infection types detected. Structured interviews assessing lifestyle patterns (e.g., measures of household infrastructure) measured participant MI. Multilevel regression analyses and zero-inflated negative binomial regression models tested associations between MI measures and STH infection status or intensity, controlling for individual and community characteristics.ResultsParticipants residing in more market-integrated households exhibited lower infection rates and intensities than those in less market integrated households. Parasite infection status and T. trichiura infection intensity were lower among participants living in houses with wood floors than those with dirt floors, while individuals using well or piped water from a spring exhibited lower A. lumbricoides infection intensities compared to those using river or stream water. Unexpectedly, latrine type was not significantly related to STH infection status or intensity. These results suggest that sources of exposure differ between the two helminth species.ConclusionsThis study documents associations between household measures and STH infection among an Indigenous population undergoing rapid MI. These findings can help healthcare programs better target interventions and reduce STH exposure among at-risk populations