Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies

The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10[superscript 6] reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.National Institutes of Health (U.S.) (1R01HG005975-01)National Science Foundation (U.S.) (award #0743432)National Institutes of Health (U.S.) (grant NIHU54HG004969

Longitudinal Change in Common Impairments in Children with Cerebral Palsy from age 1.5 to 11 years

Purpose: This project aimed to determine if change occurs over time for impairments of balance, range of motion (ROM), endurance, and strength of children with cerebral palsy (CP), by Gross Motor Function Classification System (GMFCS) levels. Methods: Measurements were completed in 77 children at two sessions (T1, T2) on average 5.8 years apart. Mean ages were 2.9 years (SD .9) and 8.7 years (SD 1.1) at T1 and T2, respectively. Results: Significant differences were noted from T1 to T2 for some children (GMFCS levels I, II, and III/IV: balance increased, GMFCS levels I and II: strength increased, and GMFCS levels III/IV and V: ROM decreased). Endurance scores were not different. Endurance scores did not change. Conclusions: Longitudinal changes in most impairments occurred in children with CP. Monitoring and targeted interventions should support each child’s development

Stability of the Gross Motor Function Classification System, Manual Ability Classification System, and Communication Function Classification System.

AIM: To determine the stability of the Gross Motor Function Classification System (GMFCS), Manual Ability Classification System (MACS), and Communication Function Classification System (CFCS) over 1-year and 2-year intervals using a process for consensus classification between parents and therapists. METHOD: Participants were 664 children with cerebral palsy (CP), 18 months to 12 years of age, one of their parents, and 90 therapists. Consensus between parents and therapists on level of function was ≥92% for the GMFCS, MACS, and CFCS. A linearly weighted kappa coefficient of ≥0.75 was the criterion for stability. RESULTS: Kappa coefficients varied from 0.76 to 0.88 for the GMFCS, 0.59 to 0.73 for the MACS, and 0.57 to 0.77 for the CFCS. For children younger than 4 years of age, level of function did not change for 58.2% on the GMFCS, 30.3% on the MACS, and 39.3% on the CFCS. For children 4 years of age or older, level of function did not change for 72.3% on the GMFCS, 49.1% on the MACS, and 55% on the CFCS. INTERPRETATION: The findings support repeated classification of children over time. The kappa coefficients for the GMFCS are attributed to descriptions of levels for each age band. Consensus classification facilitates discussion between parents and professionals that has implications for shared decision-making. WHAT THIS PAPER ADDS: The findings support repeated classification of children over time. Stability was higher for the Gross Motor Function Classification System than the Manual Ability Classification System and Communication Function Classification System. The function of younger children was more likely to be reclassified. Percentage agreement between parents and therapists using consensus classification varied from 92% to 97%. The intraclass correlation coefficient overestimated stability compared with the weighted kappa coefficient

Validity of the Early Activity Scale for Endurance and the 6-Minute Walk Test for Children With Cerebral Palsy.

PURPOSE: This study aimed to describe Early Activity Scale for Endurance (EASE) scores and 6-minute walk test (6MWT) distances of children with cerebral palsy (CP) by functional ability level, sex, and age and to examine the convergent validity of the 2 tests. METHODS: A total of 708 participants with CP, Gross Motor Function Classification System (GMFCS) levels I to V, completed the EASE, and 376 of the study participants (3-12years), GMFCS levels I to III, completed the 6MWT. RESULTS: Children with CP vary in EASE scores and 6MWT distances based on GMFCS level and, to a lesser extent, age. The EASE and the 6MWT demonstrate a statistically significant but low, positive correlation. CONCLUSIONS: Understanding the relationship between these outcomes and GMFCS levels and age assists clinicians in establishing plans of care targeted at improving endurance for activity and functional walking capacity for children with CP

Developmental Trajectories and Reference Percentiles for the 6-Minute Walk Test for Children With Cerebral Palsy.

PURPOSE: The purposes of this study were to document longitudinal developmental trajectories in 6-minute walk test (6MWT) distances and to develop age-specific reference percentiles for children across different Gross Motor Function Classification System (GMFCS) levels. METHODS: A TOTAL OF: 456 children with cerebral palsy ages 3 to 12 years of, GMFCS levels I to III participated. Children\u27s motor function was classified on the GMFCS, and children completed the 6MWT 2 to 5 times in 2 years. RESULTS: Longitudinal developmental trajectories support that 6MWT distances increase with age followed by a tapering, as children approach their functional limit relative to their GMFCS level. Reference percentile graphs were created to monitor change over time. CONCLUSIONS: The 6MWT longitudinal developmental trajectories, reference percentiles, and interpretation of percentile change should assist collaborative and proactive intervention planning relative to functional walking capacity for children with cerebral palsy

Developmental Trajectories and Reference Percentiles for Range of Motion, Endurance, and Muscle Strength of Children With Cerebral Palsy.

BACKGROUND: Children with cerebral palsy (CP) frequently present with secondary impairments in spinal alignment and extremity range of motion, endurance for activity, and muscle strength. Creation of developmental trajectories for these impairments will help guide clinical decision-making. OBJECTIVE: For children in each level of the Gross Motor Function Classification System (GMFCS) this study aimed to: (1) create longitudinal developmental trajectories for range of motion (Spinal Alignment and Range of Motion Measures [SAROMM]), endurance (Early Activity Scale for Endurance [EASE]), and functional strength (Functional Strength Assessment [FSA]); and (2) develop age-specific reference percentiles and amount of change typical over 1 year for these outcomes. DESIGN: This study used a longitudinal cohort design. METHODS: Participants comprised 708 children with CP across GMFCS levels, aged 18 months up to the 12th birthday, and their families. In 2 to 5 assessments every 6 months over 2 years, trained therapists performed the SAROMM and FSA, and parents completed the EASE questionnaire. For children in each GMFCS level, longitudinal trajectories using linear and nonlinear mixed-effects models from all visits, and reference percentiles using quantile regression from the first, 12-month, and 24-month visits were created for each measure. RESULTS: Longitudinal trajectories and percentile graphs for SAROMM, FSA, and EASE were primarily linear, with different performance scores among GMFCS levels. There was much variability in both longitudinal trajectories and percentiles within GMFCS levels. LIMITATIONS: Limitations included a convenience sample and varying numbers of participants assessed at each visit. CONCLUSIONS: The longitudinal trajectories and percentile graphs have application for monitoring how children with CP are performing and changing over time compared with other children with CP. The resources presented allow therapists and families to collaboratively make decisions about intervention activities targeted to children\u27s unique needs

Physical, occupational, and speech therapy for children with cerebral palsy.

AIM: To explore the relationship between rehabilitation therapies and development in children with cerebral palsy (CP). METHOD: We conducted a prospective, longitudinal study involving 656 children with CP (mean age [SD] 6y [2y 8mo] at study entry; 1y 6mo-11y 11mo; 287 females, 369 males), and their parents. Children were assessed two to five times over 2 years by therapists using standardized measures of balance and walking endurance. Parents completed questionnaires on demographics, rehabilitation therapies, and their children\u27s performance in self-care and participation in recreation. Therapists and parents collaboratively classified children\u27s Gross Motor Function Classification System (GMFCS) levels. We created longitudinal graphs for each GMFCS level, depicting change across time using centiles. Using multinomial models, we analyzed the relationship between therapies (amount, focus, family-centeredness, and the extent therapies met children\u27s needs) and whether change in balance, walking endurance, and participation was \u27more than\u27 and \u27less than\u27 the reference of \u27as expected\u27. RESULTS: Children were more likely to progress \u27more than expected\u27 when participating in recreation when therapies were family-centered, met children\u27s needs, and focused on structured play/recreation. A focus on health and well-being was positively associated with participation and self-care. The amount of therapy did not predict outcomes. INTERPRETATION: Therapy services that are family-centered, consider the needs of the child, and focus on structured play/recreational activities and health/well-being may enhance the development of children with CP

Developmental Trajectories for the Early Clinical Assessment of Balance by Gross Motor Function Classification System Level for Children With Cerebral Palsy.

Background: Children with cerebral palsy (CP) characteristically present with impairments in balance. Currently, the pattern and timing of the development of balance ability have not been described for children with CP of varying Gross Motor Function Classification System (GMFCS) levels. Objective: The purpose of this study was to document longitudinal developmental trajectories in a measure of balance, the Early Clinical Assessment of Balance (ECAB) scores, along with age-specific reference percentiles and the amount of change typical over a 1-year period for children within different GMFCS levels. Design: The design was a longitudinal cohort study. Methods: Participants included 708 children with CP, aged 18 months through their 12th birthday, and their families. Children participated in 2 to 5 assessments using the GMFCS and ECAB. Results: Longitudinal trajectories describing the average change in the ECAB score with respect to age were created by fitting separate nonlinear mixed-effect models for children in each GMFCS level. Reference percentiles were constructed using quantile regression of ECAB data from the first visit (baseline) and 12-month and 24-month visits. Using these reference points, the amount of change in percentiles was calculated for all children by subtracting the baseline percentile score from the 12-month percentile score. Children whose percentile changes are within the 80% limits can usually be described as developing as expected for their age and GMFCS levels. Limitations: Limitations of this study included use of a convenience sample, a ceiling effect of the ECAB for some children in GMFCS levels I and II, and the use of both a 12-month and 24-month study protocol that impacted the number of children available for each assessment session. Conclusions: When used appropriately to monitor development and change over time for children with CP, the ECAB longitudinal trajectories, reference percentiles, and the associated change scores presented here should assist therapists and families in collaborative interaction to proactively plan services and interventions relative to balance ability

Factors associated with anemia among women of the reproductive age group in Thatta district: Study protocol

Background: Maternal anemia is a leading public health issue placing pregnant women at higher risk of low birth weight, preterm birth, perinatal mortality, and neonatal mortality. Women in developing countries are at higher risk of anemia which could be either due to micronutrient deficiencies, hemoglobinopathies, infections or other socio-demographic factors. Thus, it is highly essential to explore the factors of anemia among women of the reproductive age group in order to design suitable interventions. The primary objective of this study is to assess the biological and socio-demographic factors that are associated with anemia among the women of the reproductive age group in Thatta district.Methods: An exploratory mixed method study using quantitative and qualitative approaches will be conducted in district Thatta Pakistan from September 2018 to January 2019. In the qualitative phase, data will be collected through focus group discussions and key informant interviews to understand the perceptions of women, their husbands and healthcare providers about anemia. In addition, a quantitative approach using cross-sectional study will be conducted to determine biological and socio-demographic factors associated with anemia. Approximately 150 non-pregnant women and their spouses will be included in the quantitative component of the study. In addition to thematic analysis for the qualitative component, Logistic regression will be done to calculate adjusted Odds ratios with their respective 95% CIs to assess the factors associated with anemia.Discussion: The better understanding of biological, socio-demographic factors and community perceptions of anemia will help us to design strategies and interventions to better address anemia during the reproductive cycle in rural areas of Pakistan. This will help the researchers and policymakers to take the appropriate action accordingly by designing suitable approaches to address the specific type of anemia in the rural population of Pakistan. This will, in turn, reduce the chances of adverse maternal and fetal outcomes associated with anemia