Complementary feeding practices of caregivers of infants with Down syndrome as compared to caregivers of typically developing infants.

Caregiver feeding practices during the complementary feeding period (6 months-2 years) may be particularly important for infants with Down syndrome (DS) as they are at higher risk for later health conditions (e.g., obesity, diabetes) that can be influenced by early feeding practices. However, how well caregivers of infants with DS are meeting infant feeding evidence-based practices is relatively unknown. Caregivers of infants with DS (N = 75) and caregivers of typically developing (TD) infants (N = 66) aged 0-2 years completed an online survey about their infant feeding practices and information sources. Caregiver practices and information sources were statistically compared between groups. Results indicated that there are significant differences in the feeding practices of caregivers of infants with DS when compared to caregivers of TD infants. Caregivers of infants with DS were less likely to meet infant feeding evidence-based practices than caregivers of TD infants. Caregivers of infants with DS were also more concerned about their infant's food intake and later weight status. Some individual feeding practices also significantly differed between groups, with caregivers of infants with DS more likely to meet evidence-based practices of purchasing iron rich foods and avoiding added salt, but less likely to use responsive feeding practices than caregivers of TD infants. Caregivers of infants with DS were also less likely to receive information about how to navigate the complementary feeding period than caregivers of TD infants. Coupled with existing research, the results of the present study suggest that infant feeding evidence-based practices should be reviewed for their appropriateness for this population and additional support for caregivers of infants with DS should be implemented to help them navigate this important period.

An observational study of parental language during play and mealtime in toddlers at variable likelihood for autism.

Parental language input influences child language outcomes but may vary based on certain characteristics. This research examined how parental language differs during two contexts for toddlers at varying likelihood of autism based on their developmental skills. Parental language (quantity, quality, and pragmatic functions) was analyzed during dyadic play and mealtime interactions as a secondary data analysis of observational data from a study of toddlers at elevated and lower likelihood of autism. Child developmental skills and sensory processing were also assessed. Parents used more words per minute, directives, and verbs during play and more adjectives, descriptions, and questions during mealtime. Parental language differed based on child fine motor skills, receptive language, and levels of sensory hyporesponsiveness but not autism likelihood. Overall, this study found that parental language varies based on context and child developmental skills. Future research examining parental language should include pragmatic functions and context across developmental trajectories.

Alterations in the gut microbiome implicate key taxa and metabolic pathways across inflammatory arthritis phenotypes.

Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis.

A call for obesity prevention interventions for young children with intellectual and developmental disabilities.

Health disparities among children with intellectual and developmental disabilities (IDD) are present in early childhood. Yet, this population is underrepresented in health behavior research. In this commentary the authors highlight the need for multi-level physical activity and nutrition research for obesity prevention with a specific focus on young children with Down syndrome, a population at greater risk of developing overweight and obesity compared to typically developing peers. This commentary describes the comorbidities and developmental challenges faced by many children with Down syndrome which may influence weight-related physical activity and nutrition behaviors. Additionally, the authors advocate for involving a multidisciplinary team of experts to inform the adaptation or development of multi-level, theory-driven behavioral interventions to prevent obesity among children with Down syndrome.

Metagenomic assessment of gut microbial communities and risk of severe COVID-19.

BACKGROUND: The gut microbiome is a critical modulator of host immunity and is linked to the immune response to respiratory viral infections. However, few studies have gone beyond describing broad compositional alterations in severe COVID-19, defined as acute respiratory or other organ failure. METHODS: We profiled 127 hospitalized patients with COVID-19 (n = 79 with severe COVID-19 and 48 with moderate) who collectively provided 241 stool samples from April 2020 to May 2021 to identify links between COVID-19 severity and gut microbial taxa, their biochemical pathways, and stool metabolites. RESULTS: Forty-eight species were associated with severe disease after accounting for antibiotic use, age, sex, and various comorbidities. These included significant in-hospital depletions of Fusicatenibacter saccharivorans and Roseburia hominis, each previously linked to post-acute COVID syndrome or "long COVID," suggesting these microbes may serve as early biomarkers for the eventual development of long COVID. A random forest classifier achieved excellent performance when tasked with classifying whether stool was obtained from patients with severe vs. moderate COVID-19, a finding that was externally validated in an independent cohort. Dedicated network analyses demonstrated fragile microbial ecology in severe disease, characterized by fracturing of clusters and reduced negative selection. We also observed shifts in predicted stool metabolite pools, implicating perturbed bile acid metabolism in severe disease. CONCLUSIONS: Here, we show that the gut microbiome differentiates individuals with a more severe disease course after infection with COVID-19 and offer several tractable and biologically plausible mechanisms through which gut microbial communities may influence COVID-19 disease course. Further studies are needed to expand upon these observations to better leverage the gut microbiome as a potential biomarker for disease severity and as a target for therapeutic intervention.

RNA-based amplicon sequencing is ineffective in measuring metabolic activity in environmental microbial communities.

BACKGROUND: Characterization of microbial activity is essential to the understanding of the basic biology of microbial communities, as the function of a microbiome is defined by its biochemically active ("viable") community members. Current sequence-based technologies can rarely differentiate microbial activity, due to their inability to distinguish live and dead sourced DNA. As a result, our understanding of microbial community structures and the potential mechanisms of transmission between humans and our surrounding environments remains incomplete. As a potential solution, 16S rRNA transcript-based amplicon sequencing (16S-RNA-seq) has been proposed as a reliable methodology to characterize the active components of a microbiome, but its efficacy has not been evaluated systematically. Here, we present our work to benchmark RNA-based amplicon sequencing for activity assessment in synthetic and environmentally sourced microbial communities. RESULTS: In synthetic mixtures of living and heat-killed Escherichia coli and Streptococcus sanguinis, 16S-RNA-seq successfully reconstructed the active compositions of the communities. However, in the realistic environmental samples, no significant compositional differences were observed in RNA ("actively transcribed - active") vs. DNA ("whole" communities) spiked with E. coli controls, suggesting that this methodology is not appropriate for activity assessment in complex communities. The results were slightly different when validated in environmental samples of similar origins (i.e., from Boston subway systems), where samples were differentiated both by environment type as well as by library type, though compositional dissimilarities between DNA and RNA samples remained low (Bray-Curtis distance median: 0.34-0.49). To improve the interpretation of 16S-RNA-seq results, we compared our results with previous studies and found that 16S-RNA-seq suggests taxon-wise viability trends (i.e., specific taxa are universally more or less likely to be viable compared to others) in samples of similar origins. CONCLUSIONS: This study provides a comprehensive evaluation of 16S-RNA-seq for viability assessment in synthetic and complex microbial communities. The results found that while 16S-RNA-seq was able to semi-quantify microbial viability in relatively simple communities, it only suggests a taxon-dependent "relative" viability in realistic communities.  Video Abstract.

Parental Decision-Making Around Introducing Complementary Foods: An Integrative Review.

A primary role in infant parenting is feeding, and this role undergoes a significant transition when introducing complementary foods (CF), with important long-term health implications. Understanding the influences on parental decision-making around timing the introduction to CF can help health care providers provide parents with effective support for feeding; however, the factors that influence parental decision-making have not been recently reviewed in the United States. To determine influences and information sources, this integrative review examined the literature from 2012 to 2022. Results indicated that parents are confused and distrustful of inconsistent and changing guidelines around CF introduction. Instead, developmental readiness signs may be a more appropriate way for practitioners and researchers to support parents in appropriate CF introduction. Future work is needed to evaluate interpersonal and societal influences on parental decision-making, as well as to develop culturally sensitive practices to support healthful parental decisions.

Short report: The role of oral hypersensitivity in feeding behaviors of young autistic children.

LAY ABSTRACT: Feeding problems are common among autistic children and are linked to negative health consequences. Therefore, understanding feeding problems and factors that influence these behaviors is important for developing supports for children and families. While certain sensory processing patterns are commonly associated with feeding problems, less is known about the link between sensory processing and feeding behaviors in autism, as well as how parent behaviors and feelings during mealtime differ based on child sensory preferences. This research examined two groups of young autistic children who were reported to be picky eaters by their parents: those with and those without oral hypersensitivity. Children with oral hypersensitivity had more difficulty with food acceptance and their parents reported more negative feelings around feeding their child. However, the two groups of children (oral hypersensitive and not) did not differ in their medical/oral motor symptoms, mealtime behavior, or parent use of strategies at mealtimes. This research supports the need for personalized treatment strategies based on the child's sensory preferences to support both the child and parent in managing mealtimes.

Extending and improving metagenomic taxonomic profiling with uncharacterized species using MetaPhlAn 4.

Metagenomic assembly enables new organism discovery from microbial communities, but it can only capture few abundant organisms from most metagenomes. Here we present MetaPhlAn 4, which integrates information from metagenome assemblies and microbial isolate genomes for more comprehensive metagenomic taxonomic profiling. From a curated collection of 1.01 M prokaryotic reference and metagenome-assembled genomes, we define unique marker genes for 26,970 species-level genome bins, 4,992 of them taxonomically unidentified at the species level. MetaPhlAn 4 explains ~20% more reads in most international human gut microbiomes and >40% in less-characterized environments such as the rumen microbiome and proves more accurate than available alternatives on synthetic evaluations while also reliably quantifying organisms with no cultured isolates. Application of the method to >24,500 metagenomes highlights previously undetected species to be strong biomarkers for host conditions and lifestyles in human and mouse microbiomes and shows that even previously uncharacterized species can be genetically profiled at the resolution of single microbial strains.

Effects of Lifetime Exposures to Environmental Contaminants on the Adult Gut Microbiome.

Emerging experimental evidence indicates that toxicant-induced alterations in gut microbiota composition and activity may affect host homeostasis. However, data from human studies are scarce; to our knowledge, no previous studies have quantified the association of lifetime exposure to environmental chemicals, across multiple time points, with the composition of the adult gut microbiome. Here we studied 124 individuals born in the Faroe Islands in 1986-1987 who were followed approximately every seven years from birth through age 28 years. Organochlorine compounds, including polychlorinated biphenyls (PCBs) and pesticides, perfluoroalkyl substances (PFAS), and mercury (Hg), were measured in cord blood and longitudinally in participants' blood. At age 28, the gut microbiome was assessed using shotgun metagenomic sequencing. Historical contaminant exposures had little direct effect on the adult gut microbiome, while a small number of fastidious anaerobes were weakly linked to recent PFAS/PFOS exposures at age 28. In this cohort, our findings suggest no lasting effects of early life exposures on adult gut microbial composition, but proximal exposures may contribute to gut microbiome alterations. The methods developed and used for this investigation may help in future identification of small but lasting impacts of environmental toxicant exposure on the gut microbiome.

Systematic Review of the Relation Between Feeding Problems and Sensory Processing in Children With Autism Spectrum Disorder.

PURPOSE: Many studies have linked sensory sensitivities to feeding problems in children with autism spectrum disorder (ASD). Despite the importance of sensory processing for a variety of mealtime and eating skills, the specific sensory processes that may impact feeding problems in children with ASD have not been comprehensively reviewed. Thus, the goal of this systematic review was to understand the associations between sensory processing and feeding difficulties in children with ASD. METHOD: This systematic review was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The PubMed, CINAHL Plus with Full Text (EBSCOhost), Scopus (Elsevier), and Embase (Elsevier) databases were searched from their dates of inception through the final search date of April 19, 2022, for English language studies that examined both sensory processing and feeding among children with ASD. Studies were assessed for quality using the Joanna Briggs Institute critical appraisal tools. RESULTS: A total of 27 studies were included. Findings supported the existence of a relationship between sensory processing and feeding problems in children with ASD. Specifically, studies reported that overall scores on sensory processing measures as well as measures of oral sensory processing were frequently associated with feeding problems. CONCLUSIONS: This review supports the development of future feeding interventions focusing on sensory processing given the relationship between sensory processing and feeding problems among children with ASD. Future research should focus on utilizing consistent feeding assessments specific to children with ASD and collect information on medical diagnoses that can impact feeding in order to report on feeding more holistically in this population. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.21453909.

Access to Care for US Children with Co-Occurrence of Autism Spectrum Disorder and Epilepsy.

Epilepsy is a common comorbidity among children with autism spectrum disorder (ASD). There is a lack of understanding of the inequality in access to care for children with co-occurring ASD and epilepsy (ASD-EP). The purpose of this study is to examine key indicators for access to care and care coordination for children with ASD-EP in the US National Survey of Children's Health (NSCH). Data were collected from the 2017-2019 NSCH. Our analytic sample included children with ASD without epilepsy (N = 2150), children with both ASD and epilepsy (N = 143), and children with epilepsy without ASD (N = 711). The dependent variables included important access to care indicators such as having usual sources of care, having adequate coverage, being frustrated in efforts to get service, and receiving care coordination. The independent variables included ASD-EP status, child demographics, and an intellectual disability (ID) diagnosis. Our results show that demographic characteristics such as sex, race, income level, and insurance type affect access to care. Inadequate access to healthcare was significantly higher among female children, children from low-income families, and children with ID. The access barriers among children with ASD-EP were more likely due to the interplay of multiple clinical and individual factors.

Hypocalcemia as a predictor of mortality and transfusion. A scoping review of hypocalcemia in trauma and hemostatic resuscitation.

BACKGROUND: Calcium plays an essential role in physiologic processes, including trauma's "Lethal Diamond." Thus, inadequate serum calcium in trauma patients exacerbates the effects of hemorrhagic shock secondary to traumatic injury and subsequently poorer outcomes compared to those with adequate calcium levels. Evidence to date supports the consideration of calcium derangements when assessing the risk of mortality and the need for blood product transfusion in trauma patients. This review aims to further elucidate the predictive strength of this association for future treatment guidelines and clinical trials. METHODS: Publications were collected on the relationship between i-Ca and the outcomes of traumatic injuries from PubMed, Web of Science, and CINAHL. Manuscripts were reviewed to select for English language studies. Hypocalcemia was defined as i-Ca <1.2 mmol/L. RESULTS: Using PRISMA guidelines, we reviewed 300 studies, 7 of which met our inclusion criteria. Five papers showed an association between hypocalcemia and mortality. CONCLUSIONS: In adult trauma patients, there has been an association seen between hypocalcemia, mortality, and the need for increased blood product transfusions. It is possible we are now seeing an association between low calcium levels prior to blood product administration and an increased risk for mortality and need for transfusion. Hypocalcemia may serve as a biomarker to show these needs. Therefore, hypocalcemia could potentially be used as an independent predictor for multiple transfusions such that ionized calcium measurements could be used predictively, allowing faster administration of blood products.

Metagenomic assessment of gut microbial communities and risk of severe COVID-19.

The gut microbiome is a critical modulator of host immunity and is linked to the immune response to respiratory viral infections. However, few studies have gone beyond describing broad compositional alterations in severe COVID-19, defined as acute respiratory or other organ failure. We profiled 127 hospitalized patients with COVID-19 (n=79 with severe COVID-19 and 48 with moderate) who collectively provided 241 stool samples from April 2020 to May 2021 to identify links between COVID-19 severity and gut microbial taxa, their biochemical pathways, and stool metabolites. 48 species were associated with severe disease after accounting for antibiotic use, age, sex, and various comorbidities. These included significant in-hospital depletions of Fusicatenibacter saccharivorans and Roseburia hominis, each previously linked to post-acute COVID syndrome or "long COVID", suggesting these microbes may serve as early biomarkers for the eventual development of long COVID. A random forest classifier achieved excellent performance when tasked with predicting whether stool was obtained from patients with severe vs. moderate COVID-19. Dedicated network analyses demonstrated fragile microbial ecology in severe disease, characterized by fracturing of clusters and reduced negative selection. We also observed shifts in predicted stool metabolite pools, implicating perturbed bile acid metabolism in severe disease. Here, we show that the gut microbiome differentiates individuals with a more severe disease course after infection with COVID-19 and offer several tractable and biologically plausible mechanisms through which gut microbial communities may influence COVID-19 disease course. Further studies are needed to validate these observations to better leverage the gut microbiome as a potential biomarker for disease severity and as a target for therapeutic intervention.

Discovery of bioactive microbial gene products in inflammatory bowel disease.

Microbial communities and their associated bioactive compounds1-3 are often disrupted in conditions such as the inflammatory bowel diseases (IBD)4. However, even in well-characterized environments (for example, the human gastrointestinal tract), more than one-third of microbial proteins are uncharacterized and often expected to be bioactive5-7. Here we systematically identified more than 340,000 protein families as potentially bioactive with respect to gut inflammation during IBD, about half of which have not to our knowledge been functionally characterized previously on the basis of homology or experiment. To validate prioritized microbial proteins, we used a combination of metagenomics, metatranscriptomics and metaproteomics to provide evidence of bioactivity for a subset of proteins that are involved in host and microbial cell-cell communication in the microbiome; for example, proteins associated with adherence or invasion processes, and extracellular von Willebrand-like factors. Predictions from high-throughput data were validated using targeted experiments that revealed the differential immunogenicity of prioritized Enterobacteriaceae pilins and the contribution of homologues of von Willebrand factors to the formation of Bacteroides biofilms in a manner dependent on mucin levels. This methodology, which we term MetaWIBELE (workflow to identify novel bioactive elements in the microbiome), is generalizable to other environmental communities and human phenotypes. The prioritized results provide thousands of candidate microbial proteins that are likely to interact with the host immune system in IBD, thus expanding our understanding of potentially bioactive gene products in chronic disease states and offering a rational compendium of possible therapeutic compounds and targets.

Health Care Utilization for Privately and Publicly Insured Children During Autism Insurance Reform.

We examined the effects of insurance type on health service utilization among children with autism spectrum disorder (ASD) following autism insurance reform by analyzing the most recent data from the 2019 National Survey of Children's Health. Families with private insurance were less likely to report that their health insurance covered needed services compared to families with public insurance. Privately versus publicly insured children were not significantly different in receiving behavioral or medication treatment, or in parental frustration in efforts to obtain services. However, parents' frustration escalated with increased ASD severity. Findings from this study suggest the need for continuing to improve implementation of health insurance reform legislation and providing adequate ASD-related services for children with private insurance.

Multivariable association discovery in population-scale meta-omics studies.

It is challenging to associate features such as human health outcomes, diet, environmental conditions, or other metadata to microbial community measurements, due in part to their quantitative properties. Microbiome multi-omics are typically noisy, sparse (zero-inflated), high-dimensional, extremely non-normal, and often in the form of count or compositional measurements. Here we introduce an optimized combination of novel and established methodology to assess multivariable association of microbial community features with complex metadata in population-scale observational studies. Our approach, MaAsLin 2 (Microbiome Multivariable Associations with Linear Models), uses generalized linear and mixed models to accommodate a wide variety of modern epidemiological studies, including cross-sectional and longitudinal designs, as well as a variety of data types (e.g., counts and relative abundances) with or without covariates and repeated measurements. To construct this method, we conducted a large-scale evaluation of a broad range of scenarios under which straightforward identification of meta-omics associations can be challenging. These simulation studies reveal that MaAsLin 2's linear model preserves statistical power in the presence of repeated measures and multiple covariates, while accounting for the nuances of meta-omics features and controlling false discovery. We also applied MaAsLin 2 to a microbial multi-omics dataset from the Integrative Human Microbiome (HMP2) project which, in addition to reproducing established results, revealed a unique, integrated landscape of inflammatory bowel diseases (IBD) across multiple time points and omics profiles.

Metatranscriptomics for the Human Microbiome and Microbial Community Functional Profiling.

Shotgun metatranscriptomics (MTX) is an increasingly practical way to survey microbial community gene function and regulation at scale. This review begins by summarizing the motivations for community transcriptomics and the history of the field. We then explore the principles, best practices, and challenges of contemporary MTX workflows: beginning with laboratory methods for isolation and sequencing of community RNA, followed by informatics methods for quantifying RNA features, and finally statistical methods for detecting differential expression in a community context. In thesecond half of the review, we survey important biological findings from the MTX literature, drawing examples from the human microbiome, other (nonhuman) host-associated microbiomes, and the environment. Across these examples, MTX methods prove invaluable for probing microbe-microbe and host-microbe interactions, the dynamics of energy harvest and chemical cycling, and responses to environmental stresses. We conclude with a review of open challenges in the MTX field, including making assays and analyses more robust, accessible, and adaptable to new technologies; deciphering roles for millions of uncharacterized microbial transcripts; and solving applied problems such as biomarker discovery and development of microbial therapeutics.

The Sulfur Microbial Diet and Risk of Colorectal Cancer by Molecular Subtypes and Intratumoral Microbial Species in Adult Men.

INTRODUCTION: We recently described the sulfur microbial diet, a pattern of intake associated with increased gut sulfur-metabolizing bacteria and incidence of distal colorectal cancer (CRC). We assessed whether this risk differed by CRC molecular subtypes or presence of intratumoral microbes involved in CRC pathogenesis (Fusobacterium nucleatum and Bifidobacterium spp.). METHODS: We performed Cox proportional hazards modeling to examine the association between the sulfur microbial diet and incidence of overall and distal CRC by molecular and microbial subtype in the Health Professionals Follow-Up Study (1986-2012). RESULTS: We documented 1,264 incident CRC cases among 48,246 men, approximately 40% of whom had available tissue data. After accounting for multiple hypothesis testing, the relationship between the sulfur microbial diet and CRC incidence did not differ by subtype. However, there was a suggestion of an association by prostaglandin synthase 2 (PTGS2) status with a multivariable adjusted hazard ratio for highest vs lowest tertile of sulfur microbial diet scores of 1.31 (95% confidence interval: 0.99-1.74, Ptrend = 0.07, Pheterogeneity = 0.04) for PTGS2-high CRC. The association of the sulfur microbial diet with distal CRC seemed to differ by the presence of intratumoral Bifidobacterium spp. with an adjusted hazard ratio for highest vs lowest tertile of sulfur microbial diet scores of 1.65 (95% confidence interval: 1.14-2.39, Ptrend = 0.01, Pheterogeneity = 0.03) for Bifidobacterium-negative distal CRC. We observed no apparent heterogeneity by other tested molecular markers. DISCUSSION: Greater long-term adherence to the sulfur microbial diet could be associated with PTGS2-high and Bifidobacterium-negative distal CRC in men. Additional studies are needed to further characterize the role of gut microbial sulfur metabolism and CRC.

Statistical approaches for differential expression analysis in metatranscriptomics.

MOTIVATION: Metatranscriptomics (MTX) has become an increasingly practical way to profile the functional activity of microbial communities in situ. However, MTX remains underutilized due to experimental and computational limitations. The latter are complicated by non-independent changes in both RNA transcript levels and their underlying genomic DNA copies (as microbes simultaneously change their overall abundance in the population and regulate individual transcripts), genetic plasticity (as whole loci are frequently gained and lost in microbial lineages) and measurement compositionality and zero-inflation. Here, we present a systematic evaluation of and recommendations for differential expression (DE) analysis in MTX. RESULTS: We designed and assessed six statistical models for DE discovery in MTX that incorporate different combinations of DNA and RNA normalization and assumptions about the underlying changes of gene copies or species abundance within communities. We evaluated these models on multiple simulated and real multi-omic datasets. Models adjusting transcripts relative to their encoding gene copies as a covariate were significantly more accurate in identifying DE from MTX in both simulated and real datasets. Moreover, we show that when paired DNA measurements (metagenomic data) are not available, models normalizing MTX measurements within-species while also adjusting for total-species RNA balance sensitivity, specificity and interpretability of DE detection, as does filtering likely technical zeros. The efficiency and accuracy of these models pave the way for more effective MTX-based DE discovery in microbial communities. AVAILABILITY AND IMPLEMENTATION: The analysis code and synthetic datasets used in this evaluation are available online at http://huttenhower.sph.harvard.edu/mtx2021. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.