Conclusions: All groups administered enrofloxacin and oregano oil did not develop a drug-resistant phenotype during the 5-day treatment period, as grouped in this trial. In 2022, after Announcement No. 194 of the Ministry of Agriculture and Rural Affairs in China, the antimicrobial resistance (AMR) trend declined, but it did not fundamentally change, presumably due to the impact of environmental pollution caused by the long-term use of antimicrobials.Objectives: The goal of this study was to reveal the gut microbiota of pigs and chickens in central China and the dynamics of antibiotic resistance genes carried by microorganisms. Methods: Free range and factory-farmed Gushi chickens and Huainan pigs were divided into eight groups. Faecal samples were collected from each group, and the metagenomic sequencing method was used to detect each group of samples. Results: The resistance genes showed the following trend, from high to low relative abundance: tetW was the highest, followed by tetW/N/W, then lnuA; and others from high to low were mdtB, lnuC, ANT6-la, ErmB, mdtC, ErmQ, tetBP, vatE, evgS, acrB, cpxA, mefA, Escherichia coli-ampC, tetL, yojl, AcrF and mdtA. All groups administered enrofloxacin and oregano oil did not develop a drug-resistant phenotype during the 5-day treatment period, as grouped in this trial. In 2022, after Announcement No. 194 of the Ministry of Agriculture and Rural Affairs in China, the antimicrobial resistance (AMR) trend declined, but it did not fundamentally change, presumably due to the impact of environmental pollution caused by the long-term use of antimicrobials.Background: Basic data concerning the gut microbiota of the main animal husbandry breeds (pigs and chickens) are scarce in China. The dynamics of gut microbiota (pigs and chickens) in China and antibiotic resistance genes carried by microorganisms in the natural environment are unknown. Methods: Free range and factory-farmed Gushi chickens and Huainan pigs were divided into eight groups. Faecal samples were collected from each group, and the metagenomic sequencing method was used to detect each group of samples. Results: The resistance genes showed the following trend, from high to low relative abundance:  The discovery and widespread use of antimicrobials in the development of human civilization has an indelible meaning . The extHowever, in the long term, AMR has become a major threat to human health in the 21st century. The animal breeding industry is generally believed to be one of the main sources of AMR, which causes resistant bacteria and genes to circulate through the entire chain of animals, food, environment, and humans, and threatens human health. At present, AMR is common in bacteria of animal origin. Multidrug resistant and even pandrug resistant strains are constantly appearing ,14,15,16Irrational use of antimicrobials promotes the emergence of AMR, resulting in changes in the composition and diversity of animal gut microbiota, which adversely affect host health . QuantitThe study of microorganisms has been based mainly on pure culture for the hundreds of years since Antoni van Leeuwenhoek invented the microscope. Among the trillions of microbial species, only 0.1–1% of the species are culturable , which gMetagenomics is a method that was first proposed by Handelman  to studyIn recent years, with the rapid development of sequencing technology and information technology, the use of next-generation sequencing technology to study metagenomics can quickly and accurately obtain a large amount of biological data and rich microbial research information ,25,26. SAccording to the statistics of the World Food and Agriculture Organization, China’s number of live pigs, total pork production and poultry egg production ranks first in the world, and poultry egg production has ranked first in the world for many consecutive years. China has become one of the most important animal husbandry countries. The current understanding of the pig and chicken gut microbiome is limited, due mainly to the lack of comprehensive pig and chicken gut microbial reference gene sets and genome sets, which severely limits the mining and use of metagenomic sequencing data.Fluoroquinolones are important antimicrobials used to treat Salmonella infections in poultry. Studies have shown that high-dose enrofloxacin ) has a good killing effect on Salmonella enterica in chickens, but at the same time, the composition and structure of intestinal flora are greatly affected .E. coli wild-type population [The observed intestinal ENR concentrations were shown to be both theoretically (based on pharmacokinetic and pharmacodynamic principles) and effectively (in vivo measurement) capable of significantly reducing the intestinal pulation .In this study, the characteristics of drug resistance groups, drug-resistant bacteria and ARG contamination levels of pigs and chickens on different farms under different feeding modes were studied to provide a reference for the optimisation of antibiotic use in farms.In this study, ten fresh faeces of Gushi chickens were collected from an intensive farm in Gushi County, Henan Province, P.R. China, on 15 July 2022, and ten fresh faeces of Gushi chickens were collected from a family farm. Ten fresh faeces of Huainan pigs were collected from an intensive Huainan pig farm, and 10 fresh faeces of Huainan pigs were collected from a mountain farm. The above collected subjects had not used antimicrobials in the past 2 months. On 19 July 2022, 10 faeces of the factory-bred Gushi chicken oregano oil group, 10 faeces of the enrofloxacin group (treatment amount), and 10 faeces of the factory-bred Huainan pig oregano oil group and enrofloxacin group (treatment amount) were collected, respectively. All animal-related work was carried out in strict accordance with the “Administrative Regulations on Laboratory Animals” formulated by the Ministry of Agriculture and Rural Affairs of China, and the project was approved by the Animal Ethics and Welfare Committee (AEWC) of Xinyang Agriculture and Forestry University, and then transported to the laboratory for extraction of total nucleic acid from the samples. Total DNA and total RNA were extracted from 80 samples.A.Free-range Gushi chicken without an additive group;B.Factory-bred Gushi chicken oregano oil group (0.25 mL/kg BW);C.Factory-bred Gushi chicken ENR group (5.0 mg/kg BW);D.Free-range Huainan pigs without an additive group;E.Factory-bred Huainan pigs without an additive group;F.Factory-bred Huainan pigs (2021011803). All samples were stored in the sample preservation solution, mixed with pig oregano oil group (0.25 mL/kg BW);G.Factory-bred Huainan pigs ENR Group (5.0 mg/kg BW)H.Factory-bred Gushi chicken without an additive group.Considering the consistency of animal species, age, weight, feeding place, feed and drinking water, the following test scheme was adopted in order to reduce the impact of the test on animal production. The sample size of this experiment was sufficient to reflect the basic distribution situation of the intestinal flora of the animals.Factory or intensive breeding was performed by industrial production in a factory farm in the following manner. First, the scale of factory breeding was large . Second, the hybrid combination or mating line of animals with a consistent body shape, appearance, and balanced growth, were selected. Third, some mechanised and automatic equipment, such as automatic drinking fountains, automatic feeding troughs, and automatic dung cleaning, were used. Fourth, fewer staff, less land occupation, and high labor productivity, occurred. Fifth, scientific operation and management methods were used to organise production, so that production conditions and technological processes operated in accordance with standards and rules, including full price compound feed, modern epidemic prevention measures and all inflow and all outflow production process.The family farm, or mountain farm, operated under the free-range breeding model. The family farm or mountain farm engaged in animal husbandry with an annual production of more than 500 pigs, more than 5000 chickens for broilers, more than 2000 laying hens, or more than 3000 mixed breeding poultry. The animals moved freely. The animals were fed full price compound feed, and wild and green feed. Normal epidemic prevention and expulsion of parasites occurred.The above animals were all adult animals. Pigs were in the fattening stage (5–8 months), and chickens were in the laying stage (19–28 weeks). The ENR group and oregano oil group were administered oregano orally twice a day for 5 days. The water intake of the animals was controlled in the experiment.The raw samples or the extracted nucleic acid samples was transported cold  to the biotech company. The received samples were tested for eligibility. Qualified DNA samples were detected, and library construction and library detection were performed. Illumina NOVASEQ 6000  was used to sequence the qualified library, and the raw data obtained by sequencing were used for later information analysis.Library construction and sequencing yielded a sufficient amount of high-quality nucleic acids. The detection of DNA samples mainly included the analysis of DNA purity and integrity by agarose gel electrophoresis (AGE), and Qubit accurately quantified the DNA concentration.Qualified DNA samples were randomly broken into fragments of approximately 350 bp in length with a Covaris ultrasonic breaker, and the entire library was prepared through the steps of end repair, A-tail addition, sequencing adapter addition, purification, and PCR amplification.After the library was constructed, Qubit 2.0 was used for preliminary quantification, the library was diluted to 2 ng/µL, and then an Agilent 2100  was used to detect the insert size of the library. After the insert size met expectations, the Q-PCR method was used to determine the effective concentration of the library. Accurate quantification (library effective concentration > 3 nM) was performed to ensure library quality.After the library was qualified, the different libraries were pooled according to the requirements of effective concentration and target data volume, and then Illumina NOVASEQ 6000 sequencing was performed.There was a certain proportion of low-quality data in the raw data obtained by sequencing. To ensure the accuracy and reliability of subsequent information analysis results, quality control and host filtering of the raw data were first carried out to obtain valid data.Reads that contained low-quality bases  that exceeded a certain percentage (set to 40 bp by default) were removed.Reads with a certain proportion of N bases (default was 10 bp) were removed.Reads whose overlap with the adapter exceeded a certain threshold (set to 15 bp by default) were removed.Metagenome assembly started from clean data after the quality control of each sample was performed.After preprocessing, clean data were obtained, and MEGAHIT assembly software was used for assembly analysis.The assembly parameters were: presets meta-large.The assembled scaffolds were interrupted from N junctions to obtain sequence fragments without N, called scaftigs .After quality control, the clean data of each sample were compared with the assembled scaftigs of each sample using Bowtie2 Version 2.4.5 software.The alignment parameters were: –end-to-end, –sensitive, -I 200, -X 400.For scaftigs generated by single-sample assembly, fragments below 500 bp were filtered out, and statistical analysis and subsequent gene prediction were performed.Gene prediction started from scaftigs assembled from single samples. MetaGeneMark was used for gene prediction, and the genes predicted from each sample were combined to remove redundancy and construct a gene catalogue. Based on the clean data of each sample, the abundance information of the gene catalogue in each sample was obtained.The basic steps of gene prediction and abundance analysis were as follows.Starting from the scaftigs (≥500 bp) of each sample, MetaGeneMark was used to perform an open reading frame (ORF) prediction, and information of length less than 100 nt was filtered out.The open reading frame (ORF) prediction results assembled by each sample were made de-redundant using CD-HIT software to obtain a nonredundant initial gene catalogue . The default was identity 95%, coverage 90% for clustering, and the longest sequence was selected as the representative sequence.The parameters were: -c 0.95, -G 0, -aS 0.9, -g 1, -d 0.The clean data of each sample were compared with the initial gene catalogue by Bowtie2, and the number of reads of the gene in each sample was calculated.The alignment parameters were: –end-to-end, –sensitive, -I 200, -X 400.The genes that supported the number of reads ≤2 in each sample were filtered out, and the final gene catalogue (unigenes) for subsequent analysis was obtained.r is the number of reads of the gene in the alignment, and L is the length of the gene.Based on the number of reads and gene length in the alignment, the abundance information of each gene in each sample was calculated, and the calculation formula was as follows:Based on the abundance information of each gene in each sample in the gene catalogue, the basic information statistics, core–pan gene analysis, correlation analysis between samples, and Venn diagram analysis of the number of genes were performed.The species annotation information of each gene (Unigene) was obtained from the gene catalogue and compared with the MicroNR library, and the species abundance table of different taxonomic levels was obtained by combining the gene abundance table.Based on the species abundance table and functional abundance table, the abundance clustering analysis, PCA and NMDS dimensionality reduction analysis, ANOSIM analysis and sample clustering analysis were able to be performed. When group information was available, multivariate statistical analysis of Metastat and LEfSe and comparative analysis of metabolic pathways were also able to be performed to explore the differences in species composition and functional composition among samples.The gene catalogue and the Comprehensive Antibiotic Resistance Database (CARD) were used for annotation, the abundance distribution of resistance genes was obtained, and the species attribution and resistance mechanism of these resistance genes were found.The sequencing data preprocessing results are shown in Based on the assembly results, the distribution of the length of scaftigs in each sample was counted and plotted, and the results are shown in The gene catalogue length distribution is shown in Based on the abundance table of genes in each sample, the number of genes in each sample can be obtained. By randomly extracting different numbers of samples, the number of genes between combinations of different numbers of samples can be obtained. Therefore, we constructed and mapped the dilution curve of the core and pan genes, and the results are shown in To examine the number of genes between the specified groups, the coexistence and unique information between different groups were analysed, and a Venn graph, or petals, were drawn. The results are shown in Biological repetition is necessary for biological experiments, and high-throughput sequencing technology is no exception. The correlation between the genetic abundance of the sample is an important indicator for testing the reliability of the experiment and whether the sample is selected. The closer to the correlation coefficient, the higher the similarities of the gene abatement mode between the samples. The results are shown in −5).Genes were compared in various functional databases by DIAMOND software. Unigenes were compared with sequences for alignment p, e-value ≤ 1 × 10Alignment result filtering: for the alignment result of each sequence, the alignment result with evalue ≤ minimum evalue*10 was selected for subsequent analysis.After filtering, since each sequence may have multiple alignment results and obtain multiple different species classification, to ensure its biological significance, the lowest common ancestor (LCA) algorithm (applied to the systematic classification of MEGAN software) was applied.The taxonomic level before a branch represented the species annotation information of the sequence.Based on the LCA annotation results and gene abundance table, the abundance information of each sample at each taxonomic level  was obtained. The abundance of a species in a sample was equal to the sum of gene abundances that the species annotated as the species.Based on the LCA annotation results and the gene abundance table, the number of genes in each sample at each taxonomic level  was obtained. Among the genes of the species, the number of genes whose abundance was not 0 was presented.Based on the abundance table at each taxonomic level (Jiemenae genus and species) and Krona analysis, the relative abundance overview was displayed, the abundance cluster heatmap was displayed, PCA and nonmetric multidimensional scaling (NMDS) dimensionality reduction were analysed, the ANOSIM difference between (within) groups was analysed, and Metastat and LEfSe multivariate statistical analyses of different species between groups were performed.To comprehensively and intuitively display the relative abundance of different classification levels in each sample, we used Krona to display the species annotation results. The results are shown in Studies have shown that the gut microbiota is individual-specific, and the composition of the microbiota of pigs under different feeding conditions and different intestinal parts is different.Based on the relative abundance tables of different classification levels, the top 10 species with the largest relative abundance were selected in each group, the rest of the species were set as Others, and the species annotation results corresponding to each sample in the column chart of relative abundance at different taxonomic levels are shown in Based on the relative abundance tables of different classification levels, the top 35 genera in terms of abundance and their abundance information in each sample were selected to draw a heatmap, and were clustered at the species level to facilitate the display of results and information discovery to determine the species with more aggregation in the sample. The results are shown in At present, principal component analysis (PCA) and nonmetric multidimensional scaling (NMDS) are the main methods of dimension reduction analysis applied to ecological research. Among these methods, PCA is a dimension reduction analysis based on a linear model that applies the method of variance decomposition to reduce the dimension of multidimensional data to extract the most important elements and structures in the data. PCA can extract two coordinate axes that reflect the differences between samples to the greatest extent to reflect the differences of multidimensional data on the two-dimensional coordinate map and then reveal the simple laws in the complex data background. NMDS is a nonlinear model whose purpose is to overcome the shortcomings of linear models and better reflect the nonlinear structure of ecological data. NMDS analysis is applied to reflect the species information contained in the sample in the form of points in the multidimensional space, while the difference between different points is reflected by the distance between points, which can reflect the differences between groups or within groups of samples. Based on the species abundance tables at different classification levels, we conducted PCA and NMDS analysis. If the species composition of the samples was more similar, then the distance between them was closer, as shown in the PCA and NMDS diagrams in Principal coordinate analysis (PCoA) extracts the most important elements and structures from multidimensional data through a series of eigenvalues and eigenvector ordering. We conducted PCoA based on the Bray–Curtis distance and selected the principal coordinate combination with the largest contribution rate for graphic display. If the sample distance was closer, the species composition structure was more similar. Therefore, the samples with high similarity in community structure tended to gather together, and the samples with large differences in community were far apart. The Bray–Curtis distance matrix was obtained based on species abundance tables at different taxonomic levels. The results of the PCoA analysis based on the gate level are shown in To study the similarity of different samples, a clustering tree of the samples was constructed by clustering the samples. The Bray–Curtis distance is the most common distance indicator used in systematic clustering. It is used mainly to describe the similarity between samples, and its size is the main basis for sample classification. Starting from the abundance table of genes in each sample, the Bray–Curtis distance matrix was used to perform cluster analysis among samples, and the clustering results were integrated with the relative abundance of species at the phylum level in each sample for display. The results are shown in To screen the species biomarkers with significant differences between groups, first, the rank sum test method was used to detect the different species between different groups, and linear discriminant analysis (LDA) was used to achieve dimensionality reduction and evaluate the influence of the different species; that is, the LDA score was obtained. The LEfSe analysis results of species with differences between them included three parts, namely, the LDA value distribution histogram, the evolutionary branch diagram (phylogenetic distribution), and the abundance comparison diagram of biomarkers with significant differences between groups. The LDA value distribution map and evolutionary branch map of different species are shown in Resistance genes are ubiquitous in both human intestinal microorganisms and other environmental microorganisms. The abuse of antimicrobials leads to irreversible changes in the microbial community in the human body and the environment, which poses risks to human health and the ecological environment. Therefore, research on resistance genes has received extensive attention from researchers. CARD is a resistance gene database that has emerged in recent years. It has the advantages of comprehensive information, user-friendliness, timely updates and maintenance. The core component of this database is antibiotic resistance ontology (ARO), which integrates information such as sequence, antibiotic resistance, mechanism of action and association between AROs and provides online interfaces between ARO and databases such as Protein Data Bank (PDB) and the National Center for Biotechnology Information (NCBI).Unigenes were compared with the CARD database using the resistance gene identifier (RGI) software provided by the CARD database (v2.0.1) .According to the comparison results of RGI and the abundance information of unigenes, the relative abundances of AROs were calculated.Based on the abundance of ARO, the following were generated: the column diagram of abundance, the heatmap of abundance clustering, the circle diagram of abundance distribution, the analysis of ARO differences between groups, and the species attribution analysis of resistance genes (unigenes annotated to ARO) .Based on the relative abundance table of resistance genes, we calculated the content and percentage of ARO in each sample and screened the top 20 AROs with the largest abundance. The results are shown in To more intuitively observe the abundance ratio of ARO in each sample as a whole, and more intuitively display the overall distribution of ARO abundance, the ARO with the largest abundance of TOP10 was selected to be drawn as an overview circle, as shown in The resistance mechanism of resistance genes in the CARD database was classified, and the following resistance mechanism distribution map was drawn according to the relationship between the action mechanism of these resistance genes and species. The results are shown in The sequencing results clearly showed that the abundance and relative percentage of drug resistance genes in the eight typical samples showed a trend from high to low.tetW, tetW/N/W, lnuA, mdtB, lnuC, ANT6-la, ErmB, mdtC, ErmQ, tetBP, vatE, evgS, acrB, cpxA, mefA, Escherichia coli-ampc, tetL, yojl, AcrF and mdtA.The relative abundance and relative percentage of drug-resistant genes showed the following trend of descending order: All groups administered enrofloxacin and oregano oil did not develop a drug-resistant phenotype during the 5-day treatment period, as grouped in this trial.One of the most important functions of the gut microbiota is to prevent bacterial overgrowth and infection by pathogenic microorganisms, mainly through the production of antibacterial substances, site-occupying protection, and competition for nutrients with pathogenic bacteria.Escherichia coli. In addition, metabolites such as short-chain fatty acids (SCFAs) produced during bacterial metabolism can reduce intestinal pH, thereby inhibiting the growth and reproduction of exogenous bacteria. At the same time, the acidification of the intestinal environment promotes the acceleration of intestinal peristalsis, resulting in exogenous bacteria not interacting with the mucosa, and exogenous bacteria are excreted. The space-occupying protective effect of intestinal flora is to form a biofilm by closely combining with intestinal mucosal epithelial cells, thereby effectively preventing pathogenic bacteria from contacting the intestinal mucosa. Under the limited nutrient intake of the host, intestinal flora, especially symbiotic bacteria, have a natural competitive advantage for these nutrients, inhibiting the growth and reproduction of pathogenic bacteria.Bacteriocins are antimicrobial peptides or complex proteins synthesised by the ribosomes of various Gram-positive and Gram-negative bacteria, and have bacteriostatic and bactericidal effects. For example, the bacteriocin-like substances secreted by lactic acid bacteria have broad-spectrum antibacterial effects and can inhibit the growth and reproduction of Gram-negative bacteria such as The diversity and distribution of drug resistance groups in pig and chicken faeces under different feeding modes (free range and factory feeding) were systematically analysed.Based on the basic situation of the intestinal flora of animals (pig and chicken) in central China, the drug-resistant phenotype presents dual traces of history and current practice. Tetracycline resistance remains the most abundant type of resistance, and this result is consistent with the findings of other Chinese scholars in other provinces ,30.Some factory-farmed pigs and chickens have higher levels of intestinal bacterial resistance than free-range pigs and chickens.In this study, an integrated gene set of gut microbes was constructed using the metagenomic sequencing data of eight groups of swine and chicken gut microbes from different feeding patterns, combined with the gene sets of existing metagenomic sequencing data, which included 237,785 complete genes, of which 5% were unknown, expanding the comprehensiveness and completeness of the swine and chicken gut microbial gene sets. The genus-level dominant species represented by the 35 metagenomic assembled genomes were analysed by software. Using analytical software, we further revealed the diversity of swine and chicken gut microbiota composition and drug resistance phenotypes and identified differences in drug resistance phenotypes.Comparative studies in animal samples provide important links to understanding the global distribution of ARGs and the spread of multidrug-resistant bacteria, resistance exchange networks, and how different habitats and phylogenetic relationships influence the evolutionary dynamics of global antimicrobial resistance.Drug resistance in pathogenic bacteria is a growing threat to global health, and the development and spread of drug resistance in microorganisms is largely attributable to the misuse of antimicrobials.AMR in animals is country-, or even, region-specific. A map of the global distribution of resistance shows resistance hotspots in northeastern China, northeastern India and northern Pakistan, while resistance is just beginning to emerge in central India and central and southern China, Kenya, Uruguay and southern Brazil ,32,33,34There are differences in the intestinal microbiota under different feeding styles . AnimalstetW was the highest relative abundance resistance gene, followed by tetW/N/W, then lnuA; and others from high to low were mdtB, lnuC, ANT6-la, ErmB, mdtC, ErmQ, tetBP, vatE, evgS, acrB, cpxA, mefA, Escherichia coli-ampC, tetL, yojl, AcrF and mdtA.Metagenomic sequencing was conducted to study the relative changes of intestinal microflora and drug resistance genes in pigs and chickens in central China, after treatment amounts of enrofloxacin and oregano oil were added into the feed in factory and free range mode, respectively. The consistency of the trend of intestinal microflora changes and the existence of drug resistance genes were found after treatment, providing reference for rational drug use in the veterinary clinic. The results showed that


Feed enzymes have been widely used with the goal to improve nutrient digestibility and growth performance of pigs. However, recent studies have shown that feed enzymes, especially phytase and xylanase, may provide potential benefits associated with the intestinal health and microbiota of nursery pigs and broiler chickens. Phytase and xylanase are catalyzers for the hydrolysis of phytic acid and β-1,4- xylan bonds, respectively. With a reduction in the antinutritional properties of phytic acid and non-starch polysaccharides (NSP) by the supplementation of these enzymes, there may be a possibility to improve the intestinal health of nursery pigs and broiler chickens. Intestinal health can be a determinant for the overall health and subsequent performance of the animals. Some of the factors affecting the intestinal health of nursery pigs could be related to antinutritional properties from phytic acid and NSP. Thus, this review paper aimed to discuss the nutritional and functional roles associated phytase and xylanase supplementation enhancing the intestinal health and growth of nursery pigs and broiler chickens.This review paper discussed the nutritional and functional roles of phytase and xylanase enhancing the intestinal and growth of nursery pigs and broiler chickens. There are different feed enzymes that are currently supplemented to feeds for nursery pigs and broiler chickens. Phytase and xylanase have been extensively studied showing consistent results especially related to enhancement of nutrient digestibility and growth performance of nursery pigs and broiler chickens. Findings from recent studies raise the hypothesis that phytase and xylanase could play functional roles beyond increasing nutrient digestibility, but also enhancing the intestinal health and positively modulating the intestinal microbiota of nursery pigs and broiler chickens. In conclusion, the supplementation of phytase and xylanase for nursery pigs and broiler chickens reaffirmed the benefits related to enhancement of nutrient digestibility and growth performance, whilst also playing functional roles benefiting the intestinal microbiota and reducing the intestinal oxidative damages. As a result, it could contribute to a reduction in the feed costs by allowing the use of a wider range of feedstuffs without compromising the optimal performance of the animals, as well as the environmental concerns associated with a poor hydrolysis of antinutritional factors present in the diets for swine and poultry. Intestinal health is one of the most discussed contemporary issues in animal nutrition, due to its significance in the overall biological response of the animals. It is usually described as based on a combination of different parameters from different metabolic and physiological reactions that can impact the overall growth and health . AccordiIn the first weeks after weaning and hatching, nursery pigs and broiler chickens start consuming diets with a greater amount of plant-based feedstuffs, which can contain different antinutritional factors and allergenic compounds that can lead to negative impacts on nutrient digestibility, growth performance, and intestinal health ,9,10. CoEnzymes are organic catalysts that accelerate reactions and act on specific substrates and reagents ,17. The Phytase is a feed enzyme that catalyzes the hydrolysis of phytic acid increasing the bioavailability of nutrients, especially phosphorus (P) and consequently leading to benefits on nutrient digestibility and growth performance ,25. ChanBased on the mechanisms of action and benefits of supplemental phytase and xylanase described above, it has been hypothesized that these enzymes could also play functional roles associated with the intestinal health of nursery pigs and broiler chickens. These functional roles could be related with a reduction in oxidative damage products and inflammatory cytokines, and a positive modulation of the intestinal microbiota ,13,15,32Phytate is a mixed salt of phytic acid  present in plant-based feedstuffs and is constantly present in diets of monogastric animals ,34. PhytIn general, around 70% of the P content in cereal grains and oil seeds used in diets for nursery pigs and broiler chickens is present in the form of phytic acid, which is not bioavailable for animal utilization ,42. GeneDuring the digestion process for nursery pigs and broiler chickens, phytic acid is soluble under the acid pH of the gastric phase, which means that it is less susceptible to binding to other nutrients, reducing their bioavailability ,47. As tThe NSP have been described as antinutritional factors for nursery pigs and broiler chickens due to their inability to digest these compounds as a consequence of the absence of NSP degrading enzymes ,12,51. ANon-starch polysaccharides are described as soluble and insoluble due to their structure, solubility, and impacts on the physicochemical properties of the digesta ,56. CereXylan is described as the major class of hemicellulose and one of the main soluble NSP present in plant-based feedstuffs included in the diets of nursery pigs and broiler chickens ,31,59. IAs previously mentioned, soluble NSP, especially xylan, can affect the digesta properties leading to negative impacts not only associated with nutrient digestibility but also on the intestinal health and mucosa-associated microbiota in the jejunum ,64,65. TIn nursery pigs and broiler chickens, AX is poorly digested and can lead to a production of a viscous chyme in the small intestine resulting in an increase in the relative abundance of pathogenic bacteria, inflammatory and immune response, oxidative stress, and impairment of the intestinal barrier function ,64,69. APhytases (myo-inositol hexakiphosphate phosphohydrolase) are a class of phosphatases enzymes responsible for catalyzing the hydrolysis and release of P of phytic acid present in plant-based feedstuffs used in diets for monogastric animals ,50. ThroPhytase is a heat labile enzyme, which means that its optimal activity is more susceptible to be affected at high temperatures usually applied to ensure the quality of some feed mixing processes ,77. SomeAt the same time that the gastric phase of digestion provides better conditions for the hydrolysis of phytic acid, it also provides better conditions for optimal phytase activity ,49. GeneWith the improvement of manufacturing technologies in the last few years, there was an increase in the production and development of new types of phytase ,44,80. TPhytase can be classified based on the location of the carbon where the hydrolysis of the inositol ring will initiate ,82,87. TTheoretically, 6-phytase may express optimal activity at a broader range of pH being more active and resistant than 3-phytase at the small intestine environment ,24,50. HDuring the last few years, the supplementation of 3- and 6-phytase at different levels for swine and poultry at different stages of growth has shown consistent results, ranging from improvements on nutrient digestibility and growth performance to intestinal health parameters and modulation of the intestinal associated microbiota . GeneralThe concept of supplementing phytase beyond traditional levels, also known as “super dosing” phytase, emerged due to economic and environmental concerns associated with high amounts of inorganic P sources in diets of swine and poultry, as well as the quality and availability of the main plant-based feedstuffs, such as corn and soybean meal ,85,194. Inositol can be found in mammalian cells and tissues in the forms of myo-inositol or phosphatidylinositol, and it is considered an important cellular mediator for signal transduction and a regulator of growth metabolism ,196,197.The “extra phosphoric effects” associated with supplementing phytase beyond traditional levels, are well documented and show a wide range of benefits, such as enhancement of nutrient digestibility, growth performance and intestinal health of pigs and broiler chickens ,201,202.Over the last few decades, there has been an increase in the interest in the investigation of the effects of phytase associated with the intestinal health of broiler chickens and nursery pigs . DiffereHelicobacter and Pelomonas populations of the mucosa-associated microbiota to decrease in the jejunum of animals fed with phytase at 2000 FTU/kg feed. The authors attributed these effects to pH alterations possibly caused by a reduction in dietary Ca levels in the diets with phytase, and by the active hydrolysis of the phytate-complexes by phytase increasing the bioavailability of Ca and other minerals that can also affect the gastric and intestinal pH. Additionally, an increase was reported in the jejunal villus height, apparent ileal digestibility of nutrients, and bone health. Although the authors observed positive effects in the modulation of the mucosa-associated microbiota in the jejunum, no effects were observed in the inflammatory and oxidative stress parameters.The authors of  reportedLactobacillus and decreases in Streptococcus populations in the ileum of broiler chickens supplemented with phytase at 5000 FTU/kg feed. The authors explained these effects with alterations in the pH values in the crop, ileum, and caeca and short chain fatty acids production caused by the supplementation of phytase. Phytase supplementation increased the pH values in different gastric and intestinal sections, which may lead to a creation of a more acidic environment favoring beneficial bacteria and leading to a bacteriostatic effect against pathogenic bacteria. Additionally, an increase in the total short chain fatty acids, DL-lactate, and acetic acid in the ileum was reported with the addition of phytase, all of which are considered antioxidants and potential bacterial substrate. According to the authors, these findings coupled with the pH alterations in the crop, ileum, and caeca may be responsible for the positive effects observed in the microbiota of broiler chickens.In another study conducted by , the autThe authors of  reportedIn a study evaluating the effects of a corn-expressed phytase for nursery pigs , reporteAnother possible mechanism of phytase related to intestinal health, may be associated the “extra-phosphoric” effects aimed with the supplementation of high doses of this enzyme. As previously mentioned, these effects are characterized and discussed as a greater bioavailability and uptake of nutrients, and a greater generation of myo-inositol and lower inositol esters ,194,200.In another study , also obEven though different studies reported positive effects on the intestinal health of nursery pigs and broiler chickens by supplementing phytase, there is still no consensus among authors regarding the mechanisms behind these benefits.Xylanases are described as a carbohydrase and classified under the glycosyl hydrolase enzyme family, which means that they catalyze the hydrolysis of glycosidic bonds in complex sugars compounds ,211,212.http://www.cazy.org, accessed on 16 November 2022). The activity of xylanase can be expressed as xylanase unit (XU), where one unit will liberate 1 µmol of reducing sugar measured as xylose equivalents from xylan per minute under standard assay conditions.Endo-xylanases  are described as hydrolyzing β-D-xylosidic bonds of the interior xylan backbone  and exo-Besides optimal temperature and pH conditions, it is important to ensure the presence of the targeted substrate in the diets in order to optimize dietary enzyme activity and enhance its associated benefits. Monogastric animals, especially swine and poultry, do not endogenously secrete the necessary enzymes to hydrolyze xylan , which aCompared to growing and finishing pigs, which also shows potential results supplementing xylanase, nursery pigs have an immature digestive system and capacity as a result of the wide range of stressors affecting them after weaning. As the pigs grow during nursery phase, the levels of NSP in their diets tend to increase since there will be a greater inclusion of plant-based feedstuffs. Recently, more studies are being conducted evaluating the supplementation xylanase for nursery pigs, and they have been showing potential results, especially related to enhancements in nutrient digestibility and intestinal health ,23,29,30The efficacy of xylanase supplementation in swine and poultry diets has been relatively variable in comparison with phytase with some studies reporting improvements in the reduction in digesta viscosity and growth performance as well as positive alterations in intestinal health and microbiota; however, other studies reported no effects . These iThroughout the last few years, there has been an increase in the interest in the investigation of the effects of xylanase related with the intestinal health of broiler chickens and nursery pigs . SeveralThe reduction in digesta viscosity and increase in nutrient digestibility are described as some of the primarily established benefits of xylanase supplementation for pigs and broiler chickens. Most of the studies for broiler chickens showed consistent improvements related to the reduction in jejunal digesta viscosity (=37%), which may account for other benefits reported in each of the respective described studies . For nurThe authors of  reportedMoreover, a reduction was reported in the MDA and PC concentrations in the jejunal mucosa as indicators of reduced oxidative stress. These results are in agreement with  and 213213, wherThe potential mechanism of xylanase associated with the reduction in inflammatory response parameters remains unclear. The authors of  reportedCupriavidus and Megasphera and on the increase in Succinivibrio and Pseudomonas in jejunal mucosa of nursery pigs. The authors of [Succinivibrio with gastrointestinal disorders and loss of intestinal integrity in the colon of humans, and [Lactobacillus and Bifidobacterium, and decreasing Streptococcus and Turicibacter in the ileum digesta; and increasing Bifidobacterium and decreasing Escherichia-shigella in the ileum mucosa of growing pigs. Regarding the effects of xylanase on the intestinal microbiota of broiler chickens [Escherichia coli and an increase in Lacbotobacillus in the cecum, whilst [Rumonococcaceae also in the cecum.According to , the intthors of  found a ans, and  reportedans, and  also obschickens , observe, whilst  reportedAlthough the effects of xylanase positively modulating the intestinal microbiota of nursery pigs and broiler chickens show great potential, further investigation is still desired in order to elucidate the specific mechanisms of action. A positive modulation of the intestinal microbiota even at different levels and sections can contribute to enhance positive effects associated with other parameters and overall biological response. An increase in the RA of selected beneficial bacteria may increase the endogenous secretion of other degrading enzymes and short chain fatty acids, collectively enhancing nutrient digestibility, intestinal health, and growth performance of nursery pigs and broiler chickens. Even showing variable results, the supplementation of xylanase also shows positive results associated with the intestinal health of nursery pigs and broiler chickens. However, there is still no consensus among authors regarding the different mechanisms of action of xylanase among the evaluated parameters, animal species, and inclusion levels.In conclusion, 6-phytase seems to be more effective and to provide a wider range of benefits compared to 3-phytase, especially related to growth performance. The supplementation of 6-phytase for nursery pigs and broiler chickens reaffirmed the positive results related to enhancement of nutrient digestibility, bone health, and growth performance, but also showed functional roles reducing oxidative stress parameters, and positively modulating the intestinal microbiota. Supplementing phytase beyond traditional levels (500 to 1000 FTU/kg feed), so-called “super-dosing” (above 1000 FTU/kg feed), showed a more active hydrolysis of phytic acid providing “extra-phosphoric effects” that were further reflected in improvements in intestinal and bone health, nutrient digestibility, growth performance of nursery pigs and broiler chickens.The supplementation of xylanase can effectively reduce the digesta viscosity of nursery pigs and broiler chickens, which in turn could lead to potential benefits associated with the reduction in immune and oxidative stress parameters, positive modulation of intestinal microbiota, and enhancement of nutrient digestibility. Therefore, the growth performance of the animals can be potentially enhanced by supplementing xylanase.Overall, the supplementation of phytase and xylanase in diets for nursery pigs and broiler chickens reaffirmed their nutritional roles enhancing nutrient digestibility and growth performance, whilst also playing functional roles reducing the oxidative stress response, and possibly through the modulation of the mucosal microbiota in the small intestine. As a result, it could contribute to a reduction in the feed costs by allowing the use of a wider range of feedstuffs without compromising the optimal performance of the animals, as well as the environmental concerns associated with a poor hydrolysis of antinutritional factors present in the diets for nursery pigs and broilers chickens.


Sub-Saharan Africa (SSA) has made major progress in improving access to health care over the past three decades. Despite efforts made toward achieving universal health coverage, the health systems of countries in the sub-region are inundated by a myriad of challenges that have become more virulent amid the COVID-19 pandemic. This paper discusses the health systems challenges and responses in SSA amidst the COVID-19 using the World Health Organization's (WHO) building blocks of health systems functioning. Long-lasting abysmal health system financing and insufficient government investment in SSA pose major challenges to the effective health systems functioning amid the COVID-19 pandemic. This situation also makes it difficult for the health system to meet the demands of the COVID-19 pandemic and at the same time, cater for other essential health services. Countries in SSA must prioritize the reformation of their health systems through effective health system policy development and implementation, human resources development, training, service delivery, governance and regulation, and sustainable health financing. In December 2019, a novel coronavirus (SARS-CoV-2) which causes the coronavirus disease 2019 (COVID-19) was isolated in China . At the Using the health systems building blocks, the key research question of this paper is: What are the delivery, health workforce, health information systems (HIS), access to essential medicines, financing, and leadership/governance challenges and responses toward ending the COVID-19 pandemic in sub-Saharan Africa?Effective health service delivery is central to the achievement of the health-related Sustainable Development Goals (SDGs), which include the delivery of interventions to reduce child mortality, maternal mortality and the burden of HIV/AIDS, tuberculosis and malaria by the end of 2030 . In SSA,There have been some successful responses across the sub-region toward improving healthcare access in terms of the availability of health facilities. Many governments are investing in building more health facilities and specialized centers. For instance, the Ghana government with funds from the private sector put up the first infectious disease isolation and treatment center; Ghana Infectious Disease Centre . This isThe capacity of a nation to deliver adequate health services to its population depends principally on the skills, competence, knowledge, dedication, motivation and deployment of well-trained health professionals . HoweverThere have been some successful responses as well. COVID-19 has, for instance, contributed to improving the skills of health professionals as it has provided opportunities for training and orientation of health professionals in different health service competencies including infection control and prevention protocol, laboratory testing guideline and management of infectious diseases like COVID-19. Regarding the laboratory testing services, the pandemic has contributed to improvements in health technology in SSA. For instance, in Ghana, to deal with lack of laboratory equipment and supplies that delay the COVID-19 testing, Kwame Nkrumah University of Science and Technology (KNUST) and Incas Diagnostics has fast-tracked the urgent development of a rapid diagnostic test kit that is approved by Food and Drug Authority (FDA) of the country for emergency use . A similA health information system constitutes a system designed to manage healthcare data. The HIS include collection, storing, managing , transmitting, and converting the data into information and use of clients' medical records for decision making . CompletDespite the inadequate HIS in SSA even before the pandemic, it is important to indicate some successful responses experienced by SSA regarding the HIS. Some countries in SSA have, for instance, deployed diverse digital information system infrastructures and innovation to increase pandemic communication (daily case updates), contact tracing, tacking vaccination coverage, to effectively manage the COVID-19 and keep the general population informed , 22. To Access to essential health intervention including essential medicines and supplies was already limited in SSA before COVID-19 . Some SSHealth financing is fundamental to the effective functioning of health care systems leading to the achievement of the SDGs, including universal health coverage by 2030 . Health Responding to the inadequacy of financial risk protection in SSA, countries including Ghana, Tanzania, Nigeria, Ethiopia, Kenya, and Rwanda have formulated and implemented national health insurance schemes , 30. DesDespite the shortfall of the health financing and leadership in SSA during the COVID-19 pandemic, it is also worth pointing out some successful responses that are observed during the pandemic. To effectively mobilize the needed resources to manage the pandemic , diverse resource mobilization was explored, including special COVID-19 response taxes, international grants, cooperate organization donations, political parties fundraising and private citizen donations . In NigePoor governance and financial challenges are linked to ineffective integration and delivery of adequate health services in SSA . The leaSome governments across SSA have demonstrated political leadership during COVID-19 by establishing several fiscal policies to improve funding of their health systems. These innovative approaches include effective collection of corporate and business taxes and swopping debt reduction for domestic investment in health systems . For exaLong-lasting abysmal health system financing and insufficient government investment in SSA pose major challenges to the effective health service provision amid the COVID-19 pandemic. This situation also makes it difficult for the health system to meet the demands of the COVID-19 pandemic and at the same time, cater for essential services. Despite the constraints faced, interventions introduced by leaders of the various countries induced some resilience among the populace. Countries in SSA must prioritize the reformation of their health systems through effective health system policy development and implementation, human resources development, training, service delivery, governance and regulation, and sustainable health financing. Future studies could also examine the effectiveness of the responses to COVID-19 in sub-Saharan Africa.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.HA conceived the study. HA, RKD, FIS, and JAE wrote the initial draft of the manuscript. LEB and EET provided critical comments which improved the scientific quality of the manuscript. All authors contributed to a review of the initial manuscript draft, gave consent, and approved the final draft of the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Panax ginseng) is a perennial herbaceous plant belonging to Panax genus of Araliaceae. Ginsenosides are a kind of important compounds in ginseng and minor ginsenosides are secondary metabolic derivatives of ginsenosides. Studies have shown that minor ginsenosides have many pharmacological effects, such as antioxidant, anti-tumor, anti-platelet aggregation, and neuroprotective effects. However, the therapeutic effects of minor ginsenosides are limited due to poor solubility in water, short half-life, and poor targeting accuracy. In recent years, to improve the application efficiency, the research on the nanocrystallization of minor ginsenosides have attracted extensive attention from researchers. This review focuses on the classification, preparation methods, pharmacological effects, and action mechanisms of minor ginsenoside nanoparticles, as well as existing problems and future direction of relevant research, which provides a reference for the in-depth research of minor ginsenoside nanoparticles.Ginseng ( Panax genus of Araliaceae, ginseng is a perennial herbaceous plant with fleshy roots. It is also a traditional precious herb, a tonic that can enhance physical vitality  means that supercritical fluid COs to CO2 . Tao et in vivo. At present, nano-drug carrier technology has been applied to the administration of minor ginsenosides. The commonly used preparation methods of minor ginsenoside nano-drug carrier include film hydration, emulsion solvent evaporation, desolvation and self-assembly.Nano-drug carrier has the advantages of improving the bioavailability, the pharmacokinetics of traditional chemotherapy drugs, and the accuracy of drugs reaching tumor cells. Nano-drug carrier also reduces the toxicity of drugs and prolongs the action time of drugs Film hydration usually refers to dissolving the carrier material and drug in an appropriate organic solvent, removing the solvent by rotary evaporation, forming a film between the drug and the film-forming material, and then hydrating the film to prepare nanoparticles. Emulsion solvent evaporation method refers to taking water-insoluble organic solvents  as the “oil phase”, in which the carrier material is dissolved, and take acetone or methanol as the “aqueous phase” to dissolve the drug into the influent phase, then make the two phases mix evenly . After sDesolvation is commonly used in the preparation of albumin nanoparticles. This method dissolves albumin in water as the aqueous phase, and drugs are generally dissolved in absolute ethanol as the organic phase . The twoThe chemical components of minor ginsenosides are complex and diverse. After decoction and dissolution, it is easy to use the electrostatic interaction between opposite charges, hydrogen bonding, and acid-base complexation to aggregate into self-assembled nanoparticles . Usuallyin vitro by the MTT method. Compared with commercial Shenyi capsule, the results showed that the two drugs had strong inhibitory effects on HepG2 cells and A549 cells. And when the drug concentration was greater than 50 μg/ml, nano-drugs showed better inhibition of tumor cell proliferation than Shenyi capsules. This result shows that Rg3 nanocrystals have good antitumor activity.According to different carrier materials, nano-drug carriers can be classified into organic nanocarriers, inorganic nanocarriers and composite nanocarriers . The phain vivo and reduce toxicity  [mPEG-b-P (Glu-co-Phe)]. Then the minor ginsenoside Rg3 was encapsulated into it to form mPEG-b-P (Glu-co-Phe) nanoparticles (Rg3-NPs). Compared with free Rg3, Rg3-NPs are more cytotoxic to colon cancer cells and can effectively inhibit the proliferation of tumor cells. The mechanism is that nanoparticles enter cells through endocytosis, and there is electrostatic interaction between drugs and polymers. The increase of acidity in the TME destroys the electrostatic interaction, which is conducive to the release of more drugs, enhancing the accumulation of drugs in tumors and achieving the effect of treating tumor cells . One is to use strong acid oxidation, and the other one is to react with amino acid derivatives and aldehydes to add solubilized parts around the outer surface. Functionalized carbon nanotubes can be connected to a variety of active molecules . Luo et 2-hybridized carbon atoms arranged in a two-dimensional honeycomb lattice -2000] to load minor ginsenoside Rg3. The results showed that compared with HACE alone, the composite nanocarrier had higher cell uptake rate and enhanced circulation time in the blood stream. This may be due to the fact that lipid vesicles can bind to the cell surface and their lipid parts can fuse with the cell membrane, thus improving the cell uptake rate. The lipid fraction also provides spatial stability, thereby prolonging circulation in the blood stream. Therefore, composite nanocarriers can become a superior choice for drug delivery.Combining organic nanomaterials with inorganic nanomaterials to prepare composite nanocarriers can make the carrier absorbed adequately to improve curative effect . Lee et Other novel nanocarriers for the delivery of drugs and active ingredients include nanocapsules, liquid crystals, polymer vesicles and nanofibers . Sun et Minor ginsenosides have attracted the attention of researchers due to their various pharmacological activities. However, because of poor water solubility and stability, the efficacy cannot be well guaranteed, which limits the clinical application. Nanocrystallization of minor ginsenosides can well solve this problem. At present, relevant researchers have explored and practiced in the preparation methods, pharmacological effects and mechanism of minor ginsenosides nanocrystallization, which provides a reference for the clinical application of minor ginsenoside nanoparticles.in vitro, and intracorporeal environment is not easy to be detected. So the development of core technology of nano medicine needs to be further broken.Compared with nanocarriers, nanocrystals have the advantages of high drug load, simple preparation methods and not restricted by carrier materials. The preparation difficulty of nanocrystals is closely related to the choice of preparation methods and physicochemical properties of drugs. However, it is still difficult to shorten the preparation time and improve the stability of nanocrystals. The key factors affecting the preparation of nanocrystals and the content of targeted drug delivery need to be further studied. Compared with nanocrystals, nanocarriers can achieve targeted release and protect drugs from early release before reaching the endpoint. But there are also some shortcomings. In the preparation process, the research and development of nanocarrier technology requirements are high, and the preparation process is complex. The process is greatly affected by equipment, operating conditions, raw materials and process parameters. The subtle changes in the manufacturing process can cause large changes in the final product quality, resulting in high uncertainty and poor reproducibility in production. The tissue specificity of the targeted drug is not high, and most experiments are conducted in vivo and pre-clinical safety evaluation and management of minor ginsenoside nano-drugs, in addition, to evaluate the side effects and adverse reactions of minor ginsenoside nano-drugs to patients and promote the clinical application of nano minor ginsenoside drugs. The development and improvement of new dosage forms of minor ginsenosides show great potential in minor ginsenoside drug treatment.In summary, some achievements have been made in the research of minor ginsenoside nanoparticles. However, the research on the targeted release, mechanism and optimization of preparation of minor ginsenoside nanoparticles needs to be further strengthened. It is necessary to further explore the pharmacokinetics


Pediatric esophageal dysphagia (PED) is an infrequent condition that can be determined by a large number of disorders. The etiologic diagnosis is challenging due to overlapping clinical phenotypes and to the absence of pediatric diagnostic guidelines. This review aims to summarize the most relevant causes of ED during childhood, highlight the clinical scenarios of PED presentation and discuss the indications of available diagnostic tools. Available information supports that PED should always be investigated as it can underlie life-threatening conditions , represent the complication of benign disorders  or constitute the manifestation of organic diseases . Therefore, the diagnosis of functional PED should be made only after excluding mucosal, structural, or motility esophageal abnormalities. Several clinical features may contribute to the diagnosis of PED. Among the latter, we identified several clinical key elements, relevant complementary-symptoms and predisposing factors, and organized them in a multi-level, hierarchical, circle diagram able to guide the clinician through the diagnostic work-up of PED. The most appropriate investigational method(s) should be chosen based on the diagnostic hypothesis: esophagogastroduodenoscopy has highest diagnostic yield for mucosal disorders, barium swallow has greater sensitivity in detecting achalasia and structural abnormalities, chest CT/MR inform on the mediastinum, manometry is most sensitive in detecting motility disorders, while pH-MII measures gastroesophageal reflux. Further studies are needed to define the epidemiology of PED, determine the prevalence of individual underlying etiologies, and assess the diagnostic value of investigational methods as to develop a reliable diagnostic algorithm. Dysphagia is defined as a disruption in the swallowing process that can involve any structure of the upper gastrointestinal (UGI) tract from the lips to the lower esophageal sphincter. The anatomical classification distinguishes oropharyngeal dysphagia (OD), in which the oral or pharyngeal phases of the swallowing process are involved, from esophageal dysphagia (ED) in which the underlying cause arises from the esophageal body or from the esophageal sphincters . DespiteEpidemiologic data regarding the prevalence of dysphagia in children are lacking. A nationwide US survey estimates that 0.9% of children, from 3 to 17 years of age, suffer from swallowing disorders with equal distribution between genders and mean age at onset of 8.2 years . While OPediatric ED (PED) can be the clinical manifestation of a wide variety of disorders. The majority of available studies focus on the specific etiologies of ED and, to the best of our knowledge, no guidelines or comprehensive recommendations are available for the diagnosis of PED. Due to the absence of a standardized diagnostic approach, the management of PED is extremely variable, especially as regards the employment of instrumental diagnostic techniques.The aim of this review is to summarize the most relevant causes of PED, highlight the clinical key elements able to guide the diagnostic process, and discuss the available diagnostic tools with their specific indications. We also aim to propose a symptom-based diagnostic approach able to guide clinicians through the diagnostic work-up of PED.We performed a narrative review of the literature as to satisfy the Scale for the Assessment of Narrative Review Articles (SANRA) . The revBased on the revision of the literature, we developed a symptom-based diagnostic diagram aiming to guide the clinician toward the identification of the etiology underlying PED. To graphically represent this process, we coded in HTML-Javascript a dynamic sunburst chart. The diagram represents in a circular graph hierarchical relational data distributed on different levels. Each level represents the detalization of the previous in a centrifugal way.According to the underlying pathogenetic mechanism, esophageal disorders can be classified into mucosal, structural, and motility diseases as well as disorders caused by luminal esophageal obstruction (EO) or extrinsic compression (EC). The main causes of PED are reported in From an epidemiological point of view, mucosal disorders constitute the most relevant cause of PED, with gastroesophageal reflux disease (GERD) and eosinophilic esophagitis (EoE) being the more frequent. The prevalence of GERD in children is 6% with an incidence that progressively increases with age . With a When, after an appropriate evaluation, ED cannot be explained by another condition, functional dysphagia can be considered in the differential diagnosis. While it is the most prevalent esophageal disorder in adults, no data are available regarding its prevalence in the pediatric population . The diaChildren with ED may present with a variety of complaints ranging from a sensation of food getting stuck in the throat or behind the sternum to a proper inability to swallow any kind of food or beverage with associated drooling . In the Infants usually present with non-specific complaints such as feeding difficulties and bolus regurgitation, thus making it more challenging to recognize ED in this age group.Our review of the literature highlighted the following as relevant clinical elements able to guide the diagnostic work-up of PED.While some disorders causing ED can develop at any age , other conditions tend to arise at a typical age . CongeniThe majority of esophageal disorders presenting with dysphagia cause a chronic or intermittent dysphagia with or without a progressive pattern.ED can also present acutely with a sudden inability to swallow solids and/or liquids. Under these circumstances, EO should always be suspected, especially if sialorrhea is present. While FBI mostly concerns infants and toddlers with no underlying esophageal disfunction, food impaction is the most common cause of acute EO in older children and teenagers. The latter is usually the presenting symptom of an esophageal disorder, among which EoE is the most frequent , 26. LesED can be categorized based on the consistency of foods for which the swallowing process is impaired. Orthodox dysphagia is characterized by an impairment in managing solid foods which may later involve liquids, while paradoxical dysphagia presents with a difficulty in managing liquids from symptom onset. The first is a consequence of a mechanical obstruction of the esophagus (either intrinsic or extrinsic), while the latter is secondary to motility disorders , 29.ED may be an isolated symptom or occur in association with other clinical findings.UGI symptoms such as heartburn, epigastric pain, chest pain, and regurgitation are usually reported in GERD , 25. FooRespiratory symptoms such as nocturnal cough and recurrent respiratory infections are also frequently reported in patients with esophageal achalasia due to recurrent aspiration or tracheal compression from the dilated esophagus . Sore thFever in association to odynophagia and chest pain suggest the diagnosis of IE or, in case of a compatible clinical history, may prompt the possibility of FBI complicated with esophageal perforation and acute mediastinitis . MediastSeveral pre-existing conditions may predispose to the onset of dysphagia. Former esophageal surgery, radiation therapy and CI can lead to acquired stenosis and predispose to GERD . With a The diagnostic work-up of PED can include a luminal, mucosal, anatomic, and functional assessment of the esophagus. Several diagnostic methodologies are available, the specific indications and diagnostic rates of which are discussed below. Moreover, pros and cons of the main diagnostic tools are summarized in The methods and the indications for performing EGD in children with ED have been reported by the ESPGHAN-ESGE guidelines .In the context of GERD-related dysphagia, EGD is useful for: (i) defining the obstructive vs. non-obstructive nature of the symptom (stenosis vs. dysmotility); (ii) detect conditions that predispose to GERD ; (iii) exclude conditions mimicking GERD such as EoE , 23. IndEGD plays a diagnostic and therapeutic role in acute EO. Any symptomatic patient with suspected FBI should emergently undergo EGD as well as asymptomatic patients who swallowed sharp FB or button butteries. The endoscopic removal of asymptomatic blunt esophageal FB is also recommended within 24 h from the ingestion .per se and may miss early disease. Abnormalities are present in the 90% of affected children with most common findings consisting of residual food in the esophagus (75%), closed stomach cardia (73%), esophageal enlargement (58%), and mucosal lesions (28%)  . In patins (28%) , 31.EGD may also reveal a narrowing of the esophageal lumen caused by structural lesions or EC. In these circumstances, however, the definite diagnosis usually requires the employment of other diagnostic modalities including CT, MRI, and endoscopic ultrasound .In children with a truly negative endoscopy , radiologic studies and esophageal function tests should be warranted to investigate for subtle structural lesions , EC and motility disorders .Plain chest X-ray has a limited role in the assessment of PED. It can identify radiopaque FBs stuck into the esophagus, exclude the presence of pulmonary issues when fever or respiratory symptoms are present, and screen for pneumomediastinum or gross mediastinal enlargements .Chest CT and MRI are needed to investigate the mediastinum when an esophageal EC is suspected. When coupled with angiography, these methodologies are the gold standard for the diagnosis of vascular anomalies causing dysphagia .Barium swallow (BS) is useful in diagnosing esophageal webs, rings, stenoses, diverticula, tumors and EC , 49. VirpH-metry or dual pH-multichannel intraluminal impedance (pH-MII) are not routinely recommended for the diagnosis of GERD . HoweverHigh-resolution esophageal manometry (HREM) represents the gold standard for the diagnosis of esophageal motility disorders, particularly achalasia , 31, 53.As said above  have been developed to identify, quantify, and monitor the presence of a swallowing problem either independently from the underlying etiology or in specific pathological contexts .The Eating Assessment Tool (EAT-10)  and its The Reflux Symptom Index (RSI)  is a selSeveral scoring systems have been developed for the clinical evaluation of EoE. In adults, the Eosinophilic Esophagitis Activity Index was found to have the best validity and responsiveness in a systematic review: this tool focuses on dysphagia induced by food of different consistencies and on behavioral adaptation in daily life , 61. In The Eckardt score has been specifically developed for esophageal achalasia to establish clinical disease severity at diagnosis and to evaluate treatment efficacy during follow-up , 64, 65.Our review of the literature shows that PED is caused by a heterogeneous group of disorders, among which EoE and GERD are the most frequent, summarizes the diverse clinical scenarios of PED presentation, and highlights the most appropriate investigational method(s) based on the diagnostic hypothesis. Despite available knowledge regarding individual etiologies, this revision also confirms the lack of pediatric epidemiological data and the absence of diagnostic guidelines regarding PED as a symptom.In adults, ED is considered an alarm sign that may underlie the presence of esophageal carcinoma , 68. In Several diagnostic protocols are available for the diagnosis of ED but none has been specifically made for children. No sufficient data are available to develop a diagnostic algorithm for PED or advocate for the superiority of a diagnostic test over another as first-line investigation.We propose a diagnostic approach tailored on the main diagnostic suspect(s) deriving from the clinical evaluation of the child. To this end, we have identified six clinical key elements capable of recognizing patients who require an immediate medical evaluation  or directing toward a diagnosis . As none of abovementioned clinical key elements can be univocally associated to a single etiology, we have also identified relevant complementary symptoms and predisposing factors that, combined to clinical key elements, can support the clinician throughout the diagnostic process. This symptom-based approach is graphically represented in a circular graph with hierarchical relational data distributed on different levels . Once onTo the best of our knowledge, this is the first comprehensive review regarding PED. Large scale studies are needed to define the epidemiology of PED, determine the prevalence of underlying etiologies, and assess the diagnostic value of available investigational methods as to establish the relative risk of disease and develop a reliable diagnostic algorithm. Prospective studies should be conducted, or a machine-learning approach implemented, to validate our symptom-based approach for the etiological definition of PED.GL, PG, and MC wrote the manuscript, prepared tables and figures which all authors reviewed. GL, CS, SG, LL, VPB, and PG performed the literature search and summarized the results. GF and LB performed a critical review of the findings and participated in the preparation of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Professional certification of community health workers (CHWs) is a debated topic. Although intended to promote CHWs, certification may have unintended impacts given the grassroots nature of the workforce. As such, both intended effects and unintended adverse effects should be carefully evaluated. However, there is a lack of published literature describing such effective evaluations with a robust methodology. In this methods paper, we describe a key component of evaluating CHW certification in Massachusetts-the Massachusetts CHW Workforce Survey.Design of the surveys was informed by a program theory framework that delineated both positive and negative potential impacts of Massachusetts CHW certification on CHWs and CHW employers. Using this framework, we developed measures of interest and preliminary CHW and CHW employer surveys. To validate and refine the surveys, we conducted cognitive interviews with CHWs and CHW employers. We then finalized survey tools with input from state and national stakeholders, CHWs, and CHW employers. Our sample consisted of three frames based on where CHWs are most likely to be employed in Massachusetts: acute care hospitals, community-based organizations, and ambulatory care health centers, primarily community health centers and federally qualified health centers. We then undertook extensive outreach efforts to determine whether each organization employed CHWs and to obtain CHW and CHW employer contact information. Our statistical analysis of the data utilized inverse probability score weighting accounting for organizational, site, and individual response.Wave one of the survey was administered in 2016 prior to launch of Massachusetts CHW certification and wave two in 2021. We report descriptive statistics of the three sample frames and response rates of each survey for each wave. Further, we describe select anticipated results related to certification, including outcomes of the program theory framework.The Massachusetts CHW Workforce Survey is the culmination of 5 years of effort to evaluate the impact of CHW certification in Massachusetts. Our comprehensive description of our methodology addresses an important gap in CHW research literature. The rigorous design, administration, and analysis of our surveys ensure our findings are robust, valid, and replicable, which can be leveraged by others evaluating the CHW workforce. A community health worker (CHW) is a public health worker who utilizes their unique understanding of the populations they serve to carry out several roles, such as health education, outreach, and care coordination . Known fAlthough early research of the efficacy of CHWs was hindered by substantial methodological limitations and program implementation problems, a multitude of recent studies using robust methods has demonstrated the impact CHWs have on an array of measures and outcomes –7. For eGiven this wider recognition, professional certification of CHWs has been raised as a means to legitimize their role, produce a clearer definition of scope, and increase opportunities for sustainable financing and reimbursement , 14. HowAlthough certification is intended to champion and promote the CHW workforce, there may be unintentional adverse impacts , 18. OnePopulation surveys of CHWs and CHW employers are an important tool in evaluating the impact of certification; however there is a notable gap in the published literature describing such methods. Additionally, conducting population surveys presents several methodological challenges . PreviouSurvey procedures were reviewed by the University of Massachusetts Chan Medical School (UMass Chan) institutional review board. The project was deemed non-human subjects research. Additionally, responses and contact information are considered confidential information and are maintained behind MDPH and UMass Chan Office of Survey Research firewalls on password protected computers. Any release of aggregate survey data must adhere to standards set by the MDPH Privacy and Data Compliance Office (PDCO).We began development of the survey sample by searching for a comprehensive list of all organizations in the state within each of the following core frames: acute care hospitals ; community-based organizations (CBO frame); and Massachusetts community health centers (CHCs) and federally qualified health centers (FQHCs) . These three core frames do not reflect an exhaustive list of settings in which CHWs are employed; rather, they reflect evaluation priorities in Massachusetts as well as findings from past surveys conducted in Massachusetts that indicate where CHWs are likely to be employed . Additiovia existing state public health records, exhaustive internet search, historical knowledge of CBOs in Massachusetts from a network of key informants, and organization registries kept by the Massachusetts Association of Community Health Workers (MACHW)  , 27. Unln = 7 in wave one, n = 8 in wave two); community-based health clinics focusing on specialty services such as women's health and reproductive health in the health center frame (n = 10 in each wave); and divisions of health departments providing community-based services in the CBO frame (n = 10 in each wave). Although these are not comprehensive, we felt it was nonetheless important to include these additional organizations to reach as many CHWs as possible in Massachusetts.The three core frames are comprised of the following: 43 hospitals in wave one and 42 in wave two, 56 health centers in wave one and 57 in wave two, and 158 CBOs in wave one and 171 in wave two. Unfortunately, expanding to additional frames in the same comprehensive and systematic fashion where CHWs are employed, such as additional community health clinics that are not CHCs or FQHCs, hospitals focusing on special populations, and state and local health departments, was not feasible given time and funding limitations. As such, we incorporated into each frame a small selection of additional organizations that serve similar functions as their assigned frame: urgent care, healthcare networks, pediatric hospitals, and veteran's hospitals in the hospital frame . This includes experts with decades of expertise in CHW-related research ; perspectives gathered from engaging with CHWs and CHW employers across Massachusetts; and input from local, state, and national partners. These efforts resulted in the derivation of several key evaluation questions. However, due to resource limitations, five of these were prioritized to assess the impact of CHW certification in Massachusetts. Using these evaluation questions as a guide and leveraging the expertise of researchers with decades of experience in survey research, design, psychometrics, and administration , we developed measures and corresponding CHW and CHW employer survey questions.Once we developed the preliminary CHW and CHW employer surveys, the UMass Chan Office of Survey Research conducted cognitive interviews with 10 CHWs and 10 CHW employers during which they reviewed survey questions and responses as well as obtained additional information to inform survey design. Interview participants spanned the three organization types and each interview was ~45–50 min in length. Verbal consent was obtained, and participants were notified that they could discontinue at any time.In addition to reviewing the survey tools, the cognitive interviews gathered additional information regarding employment attributes, funding sources, roles and activities, integration into care teams, perceived value of CHW work within the organization, training, and perceived benefits and barriers related to certification. See After integrating the findings of the cognitive interviews into the surveys, we conducted additional cognitive interviews with state and national CHW SMEs to further refine the clarity, appropriateness, and comprehensiveness of survey questions. Additionally, MACHW and CHW Core Consensus Project (C3) reviewed each tool in its entirety and provided feedback. Finally, the CHW survey tool and the CHW employer survey tool were reviewed by a small group of CHWs and CHW employers, several of whom brought both the CHW and CHW employer perspective. The tools were further modified based on this feedback. Although formal testing is ideal in survey development, resource limitations and funding restrictions were a challenge in conducting additional validation. However, leveraging the subject matter expertise of those developing and reviewing the survey, integration of findings from the cognitive interviews, and the tools undergoing review by several members of the target population enhanced the validity and reliability of the surveys.As a result of these efforts, we finalized the survey tools that broadly covered the following domains: (1) Aspects of CHW employment, such as job title, pay, full or part-time employment, and organization information; (2) Role of the CHW within the organization, such as health issues addressed, health promotion activities, linkages to community resources, and work with special populations; (3) Trainings and certification of the CHW, such as receipt of the 80-h core competency training and trainings in specific disease areas; (4) Clinical care team integration, including use of electronic health records and interactions with the care team ; and (5) Certification, such as certification status, perceptions of certification, and ease of certification process. In the 2016 wave, the CHW survey tool was available in English only. In the 2021 wave, the CHW survey tool was translated into Spanish and Portuguese, given that these are the second and third most commonly spoken languages in Massachusetts and were the most frequently reported second languages on wave one of the CHW survey . TranslaThe English CHW employer and CHW survey tools can be found in After collecting publicly available contact information for all organizations and sites within the sample frames, we initiated outreach prior to each wave of the survey launch to ascertain whether the organization and affiliated sites employed CHWs and to obtain the email addresses of CHW employers and CHWs working at the organization. MDPH and UMass Chan interviewing staff contacted each organization in the sample frame using a call script. Given the variety of roles and job titles CHWs have, interviewers used a fact sheet that provided detailed information on the roles, job titles, and responsibilities of CHWs . If duriThere were several important lessons learned during this process. Using fact sheets and definition of CHWs was critical since most contacts did not know what a CHW was. Receptionists at the organization's general number often were unable to provide any information; as such, interviewers found it best to ask for managers responsible for hiring and payroll. Finally, contacting human resources departments, especially in large organizations, often did not prove fruitful. Overall, interviewers found it was best to be flexible and patient, and to talk to anyone who could provide any information. Often it took multiple efforts to obtain CHW and CHW employer contact information at a given organization.via email with a link to the survey. Wave one was administered between June 14 to August 1, 2016, and wave two between April 13 to July 1, 2021.One day prior to launch of the survey, the MDPH Office of CHWs sent an official prenotification email to all CHW employers and CHWs in the sample frame notifying them of the upcoming survey. This step was intended to reinforce legitimacy of the survey and improve response rates when organizations were later contacted directly. Attached to the email was an official letter from the MDPH Office of CHWs. The survey was then administered sites (organizations that confirmed that they did not employ CHWs as a whole were removed from this step); and individual CHW and CHW employer probability of response to the survey.Once survey administration was complete, we reviewed data files for completeness, missingness, and duplication. After completion of this step, we used inverse probability score weighting (IPW) to account for non-response and permit valid population-level comparisons across survey waves. Regardless of the evaluation or research question assessed with these data , IPW is a critical step in addressing bias introduced by non-response and differential response within and between waves of the survey. We generated inverse probability weights using propensity score models with PROC LOGISTIC in SAS 9.4 using a logit link function and Fisher's Scoring method . We ran All three propensity score models included as predictors organization type  and organization size. We determined organization size based on publicly available tax documents from ProPublica, from which we obtained the number of staff employed by the organization, excluding volunteers . We deteIn addition to organization type and size, the models for the site level and CHW and CHW employer level propensity scores included a Neighborhood Stress Score (NSS) variable developed by Ash et al. . This coIn wave one, the response rate was 67% for the employer survey and 63% for the CHW survey. In wave two, the response rate was 62% for the employer survey and 53% for the CHW survey. Organizational characteristics of CHW and CHW employers responding to the survey are included in Key findings will focus on the evaluation questions related to impact of certification, outlined in An exhaustive list of anticipated results is beyond the scope of this paper. Examples of anticipated findings include whether employers are leveraging stable funding sources for CHW positions; if there are shifts in the perceived value of CHWs and CHW certification; whether there are changes in trainings and promotion opportunities available to CHWs; and whether CHWs have become better integrated into care teams. Additionally, we will assess awareness of Massachusetts CHW certification and ease of and attitudes toward obtaining certification in Massachusetts. Finally, we will further analyze shifts in the CHW workforce between survey wave one and wave two. CHW demographics we will analyze include gender, educational attainment, number of years working as a CHW, income, age, race, and ethnicity. However, if any changes are detected, they are likely multifactorial and not necessarily the result of launch of certification, such as natural variation in the workforce and impacts of the Coronavirus Disease-19 (COVID-19) pandemic. Differentiating these factors will become more feasible with additional administrations of the surveys.Using a rigorous, systematic, and replicable approach, the Massachusetts CHW Workforce Survey serves as a critical tool to monitor the impact of CHW certification in Massachusetts and describe a large, growing, and diverse workforce. In conjunction with ongoing outreach and evaluation efforts conducted by the MDPH Office of CHWs and their partners, findings from these surveys will be critical in assessing impacts of certification in Massachusetts. The rigorous and methodological documentation of our survey methods increases feasibility of administering additional waves of the survey to monitor change over time and replicability of findings. Additionally, researchers, governments, and CHW advocates looking to administer their own surveys can utilize our tools and approaches to inform their work in conjunction with tools and resources currently available through the National Association of Community Health workers (NACHW), the Centers for Disease Control and Prevention, and other organizations , 32. GivThe Massachusetts CHW Workforce Survey is not the first large-scale assessment of CHWs and their employers in Massachusetts. In the early 2000s, MDPH administered CHW and CHW employer surveys. Significant outreach efforts prior to the administration of the survey identified 806 CHWs and 155 employers . AdditioThere have also been national efforts to describe and identify needs and changes in the CHW workforce as well as evaluate potential impacts of certification in the United States. In 2007, the Health Resources and Services Administration (HRSA) of the United States Department of Health and Human Services released a report based on findings of the CHW National Workforce Study . This prOur research adds to these surveys and evaluations by providing not only survey questionnaires, but other tools we developed as well as detailed information on the development of our sample frames. Further, our methods are to the best of our knowledge one of the few focused primarily on evaluation of state CHW certification. Regarding the sample frames, systematic and replicable construction of the sample frame is critical to conceptualize and generalize the results of the survey and increase the validity of conclusions drawn between survey waves, unlike non-probability sampling frequently employed by other surveys. Further, unclear definitions of the sample frame in previous studies impact generalizability of findings. Collection of data beyond basic CHW demographics is critical to validly evaluate impact of certification-such as organizational characteristics where CHWs are employed and length of time a CHW has worked. Failure to do so can result in significant confounding and bias study conclusions. In contrast, our well-defined sample frame along with extensive individual and organizational data permits more robust interpretation, control of confounding, and conceptualization of findings, which is especially critical given the diverse settings, job titles, and functions of CHWs.In addition, our implementation of IPW accounts for non-response and differential response within and between survey waves. IPW is a well-documented method of reducing bias in survey research and is essential for drawing robust conclusions from findings compared to unweighted frequencies and percentages often utilized by other surveys. Although no survey analysis method can eliminate bias, our accounting for organizational and community characteristics potentially associated with response probability lessens risk of bias in our findings. The entirety of the process, from survey design, survey administration, construction of sample frames, and statistical analysis (including weighting variables) are replicable in other states and can be leveraged by researchers undertaking evaluations of the CHW workforce. For example, the variables we included in the IPW  and used to construct the sample frame  are largely publicly available. Finally, the forthcoming analysis of our survey results will add to the literature by providing critical information on several important topics related to CHW certification, such as potential adverse and inequitable impacts of certification, CHW integration into healthcare teams, and shifts in CHW employment, including job opportunities, funding, salary, and training.Our development and administration of the survey resulted in important lessons learned that may be useful for organizations interested in undertaking similar efforts. What cannot be understated is the role CHWs, CHW SMEs, advocacy organizations, and networks played in the development and administration of the survey. Designing valid questions that align with the most pressing needs CHWs face, including potential positive and negative impacts of certification, was crucial to ensure that the results are meaningful and actionable. The qualitative findings were an important component of this, as they aided us in identifying additional outcomes of interest from both the CHW and CHW employer perspectives. Additionally, leveraging networks to ensure as many CHWs are reached as possible and increasing response rates was paramount. Further, applying rigorous, statistical analysis of the data, including accounting for confounding and differential responses, is essential to draw valid, population conclusions within and between waves of the survey. Albeit no survey is without limitations, the extensive analytical efforts we undertook facilitate findings that are robust and replicable.There are important limitations of this survey to note. While this survey reached a large sample of CHWs and CHW employers in Massachusetts, previous surveys suggest that the number of CHWs in Massachusetts is higher , 22, 34.Nonetheless, the results of the Massachusetts CHW Workforce Survey will provide critical insight into this diverse and important workforce as well as the impacts of certification. The survey design, sample frame development, survey administration, and statistical analysis is the culmination of over 5 years of effort, none of which would have been possible without our partners, CHW advocates, and CHW networks. Further, our conscientious design of the survey tools, rigorous documentation of sample frames, leveraging of local, state, and national CHW networks in outreach, and rigorous statistical analysis demonstrate how robust, replicable findings can be captured on this critical topic. Organizations interested in undertaking similar efforts can use, modify, and improve upon our tools and methods for their own assessments of the CHW workforce and their employers.There is a notable gap in the literature of studies rigorously evaluating state certification of CHWs. The results of our statistically weighted survey administered in a robustly defined sample frame before and after the launch of Massachusetts CHW certification will answer several important questions. These include whether state certification improves funding, job opportunities, and salaries for CHWs; whether there are potential adverse effects of state certification, such as inequitable access to certification, shifts in workforce demographics, employers adopting requirements for certification, or whether positive impacts are disproportionately benefiting certified CHWs; and whether state certification promotes the integration of CHWs into healthcare teams. Future research should assess similar measures leveraging longitudinal, observational study designs of individual CHWs to further elucidate the impacts of certification. Future research should also assess how CHW certification impacts health outcomes.https://www.mass.gov/how-to/apply-for-access-to-mdph-confidential-records-data.The datasets presented in this article are not readily available because these data are considered confidential by the Massachusetts Department of Public Health Privacy and Data Compliance Office (PDCO). Any data requests must follow processes and requirements of the Massachusetts Department of Public Health and PDCO. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by University of Massachusetts Chan Medical School (UMass Chan) Institutional Review Board. Written informed consent from the patients/participants was not required to participate in this study in accordance with the national legislation and the institutional requirements.VN, GS, WWSU, and BB-C oversaw this research project. GH, TM, CS, EG, EM, CA, and BB-C provided subject matter expertise in survey design. VN, WWSU, P-PL, DB, and BB-C designed the statistical analysis plan. P-PL, DB, and BB-C collected and analyzed survey data. All authors contributed to the writing and editing of this manuscript. All authors contributed to the article and approved the submitted version.