Rumen is an organ for supplying nutrients for the growth and production of bovine, which might function differently under grass-fed and grain-fed regimens considering the association of gene expression, DNA methylation, and microRNA expression. The objective of this study was to explore the potential mechanism influencing rumen function of grass-fed and grain-fed animals. Methylated DNA binding domain sequencing (MBD-Seq) and microRNA-Seq were respectively utilized to detect the DNA methylation and microRNA expression in rumen tissue of grass-fed and grain-fed Angus cattle. Combined analysis revealed that the expression of the differentially expressed genes ADAMTS3 and ENPP3 was correlated with the methylation abundance of the corresponding differentially methylated regions (DMRs) inside these two genes, and these two genes were reported to be respectively involved in biosynthesis and regulation of glycosyltransferase activity; the differentially expressed microRNA bta-mir-122 was predicted to possibly target the differentially expressed genes OCLN and RBM47, potentially affecting the rumen function; the microRNA bta-mir-655 was exclusively detected in grain-fed group; its targets were significantly enriched in insulin and TGF-beta signaling pathways, which might worked together to regulate the function of rumen, resulting in different characteristics between grass-fed and grain-fed cattle. Collectively, our results provided insights into understanding the mechanisms determining rumen function and unraveled the biological basis underlying the economic traits to improve the productivity of animals. Historically, most of beef products were from grass-finished cattle. Since the 1950’s, numerous studies were performed to improve the efficiency of beef production; meanwhile, the beef cattle feedlot industry began to emerge, where high-energy grains were utilized to improve the productivity of cattle. And, consumers have been accustomed to grain-fed beef, considering the flavor and palatability resulted from the diets with large proportion of high-energy grain . During In the field of genetics, epigenetics refers to cellular and physiological phenotypic trait alterations because of external factors that can influence genes expression without changing the DNA sequence . As one Additionally, the gene expression could also be regulated by miRNAs, which could specifically bind to corresponding mRNAs, leading to the mRNA silencing and translation repression. It is known that each miRNA might have many mRNA targets; and each mRNA could also be regulated by more than one miRNA . In mammAs the largest part of the stomach, rumen constitutes the important workplace for feed digestion and microorganism fermentation, which is critical for supplying nutrients for animals’ growth, development and production. Given the emerging roles of miRNAs and DNA methylation in gene regulation, identifying the DNA methylation profile and expression pattern of miRNAs is critical to understand their functions in the process of bovine development. Therefore, we utilized Illumina sequencing technology to explore the miRNA expression pattern and DNA methylation profile in the rumen tissue of Angus cattle raised under grass-fed and grain-fed regimens. Further, we conducted the combined analysis of miRNAs, DNA methylation, and mRNA expression, which was important for further functional study and valuable biomarkers detection. Accordingly, our results would be significant for exploring potential mechanisms that led to differences observed between animals under altered feeding regimens.We conducted the animal experiments according to the NIH guidelines for housing and care of laboratory animals. The Institutional Animal Care and Use Committee (IACUC) of University of Maryland at College Park reviewed and authorized the protocols (permit number R-08-62).The studied Angus cattle were born and raised at the Wye Angus farm. This farm has produced many progenies with similar genetic background. All experimental animals in this study received the same diet until weaning. After weaning, the calves were randomly assigned to one diet (grass-fed or grain-fed) and followed that regimen until slaughter. The grain-fed steers received conventional diet comprised of trace minerals, shelled corn, corn silage and soybean. The grass-fed steers normally ate grazed alfalfa; in winter, we provided bailage for the animals. The grass-fed animals consumed no industrial or agricultural byproducts and did not receive any type of grain. The grain-fed individuals met the market weight around the age of 14 months; grass-fed steers reached the similar weight value with the age of 20 months. After slaughter at the Old Line Custom Meat Company , the rumen samples from all experimental animals were immediately excised at the same location around the cardiac ostium, and then the samples were rinsed and kept at -80°C for subsequent analysis. For MBD-Seq, two animals were contained per experimental group. Based on the MBD-Seq experiment, we respectively added one animal per group in miRNA-Seq; thus, three animals were respectively utilized in grass-fed and grain-fed group for miRNA-Seq analysis.Firstly, we extracted the genomic DNA from the four experimental animals through the Wizard Genomic DNA purification kit , and measured the concentration via the Qubit dsDNA Broad-Range Assay . The genomic DNA from each individual was adjusted to 0.1μg/μl for a final volume of 55 μl and sheared into 300–500 bp fragments. Secondly, MethylCap kit  was employed to obtain the methylated DNA (meDNA). The 141.8 μl of capture reaction mix containing 12 μl of sheared DNA was prepared according to the instruction of the kit. Then, the capture reaction mix (119 μl) was incubated with 1 μl of diluted MethylCap protein at 40 rpm for 2 hours at 4°C. The rest 22.8 μl was utilized as input sample. Next, meDNA Capture beads (coated with GSH) provided by the kit were utilized to capture methylated DNA. The bead pellet combined by the methylated DNA was collected. After washing the bead pellet, we performed the elution of the captured DNA; 150 μl of low, medium, and high concentration elution buffer were sequentially utilized per reaction. All fractions were treated by the MiniElute PCR Purification Kit .We repaired the end of the fragmented methylated DNA by using the NEBNext End Repair Module . Then the 3’ poly “A” was added through DNA Polymerase I, Large (Klenow) Fragment . Next, we ligated a pair of Solexa adaptors (Illumina) to the repaired ends by T4 ligase . The fragments of the ligated products (DNA plus adaptors) from 200 to 500bp were selected from 2% agarose gels and purified by QIA- quick Gel Extraction Kit . Then, the Phusion Hot Start High-Fidelity DNA Polymerase  was used to enrich the purified DNA templates through PCR. After purification of the PCR products , the concentration of the DNA library was measured through the Qubit assay . Finally, the DNA sequencing was performed in the Solexa 1G Genome Analyzer (Illumina) according to the specification provided by the manufacturer.http://support.illumina.com/sequencing/sequencing_software/igenome.html). During this step, according to the indicators generated by FastQC, we trimmed the first 10 bases and the last 5 bases of each read (50 bp) to keep high sequence quality score; and 35 bp tags were then obtained. For data format conversion, SAMtools and BEDtools were applied in our analysis.We evaluated the quality of the raw reads through FastQC, which is a web-based software. It could thoroughly examine the reads and create a detailed quality assurance report containing “per base sequence quality” and “per sequence GC content”, etc. Then, Bowtie was used to align the sequencing reads to the reference genome (Bos_taurus_UMD3.1/bosTau6) , which wFor peaks identification, the Model Based Analysis of ChIP-Seq (MACS) was employed . IdentifThe ChIPpeakAnno package was utilized for the genomic annotation of the previously identified DMRs . This sohttp://www.urogene.org/methprimer/) and shown in http://quma.cdb.riken.jp).After quality evaluation and quantification, equal amounts of genomic DNA from two experimental animals of each group were pooled together, which were then served as the template for the bisulfite conversion. Firstly, 500 ng of each DNA pool was treated by the sodium bisulfite conversion reagents . We randomly selected the DMRs from the bioinformatics analysis results for validation. The PCR primers were designed via MethPrimer , which were corresponding to RNAs from 21 to 30 bp. After purification, the concentration was measured by the Qubit assay . Finally, we sequenced the libraries identified by the 6-bp index through an Illumina HiSeq 2000 sequencer, as described previously [We extracted the total RNA from the six experimental individuals  by Trizol , the RNA was then treated by DNase digestion and purified by Qiagen RNeasy column , as previously described . We checeviously .After quality evaluation of the tags performed by FastQC, all samples were analyzed employing miRDeep* software, which could quantify known and novel miRNAs . This prAfter identification of the differentially expressed miRNAs, the target genes of those miRNAs were predicted by TargetScan , which wqRT-PCR was performed to verify the differentially expressed miRNAs found in the microRNA-Seq analysis on the iCycler iQ PCR system . The qRT-PCR reactions were performed with a QuantiTect SYBR Green PCR Kit  according to the manufacturer’s instructions. We conducted three technical replicates and two independent biological replicates for each product. RPS18 was chosen as the control gene . The priFor the methylome study, we had four experimental samples in total; the alignment levels were 94.92%, 82.98%, 95.54% and 96.10%, respectively . We finaThe binding affinity between grass-fed and grain-fed cattle was shown in The DMR annotation was subsequently performed . The disIn this study, we screened the genes with promoters containing DMRs, using 10,000 bp as the parameter for maximum distance. From the 217 identified DMRs, 21 were located within the promoters, corresponding to 21 different genes. Our previous study explored the transcriptome profiling in grass-fed and grain-fed animals, and 342 DEGs (FDR < 0.1) were detected between the two groups [In order to assess the reliability and accuracy of DMRs detection from MBD-Seq, 5 regions were randomly selected for validation. The calculated methylation levels are shown in the For the differentially expressed miRNA bta-mir-122, the expression level in grain-fed ruminal wall was significantly higher than in grass-fed ruminal wall, and the results of qPCR and RNA-Seq suggested the same direction .To identify the microRNAs involved in bovine rumen function, total RNAs from three biological replicates for each condition were used to construct small RNA libraries. Via high-throughput sequencing, we obtained an average of 15 million reads per sample. The percentage of alignment for all samples exceeded 90% as shown in http://www.targetscan.org/). For bta-mir-122 and bta-mir-655, we respectively found 145 and 749 target genes. To explore the specific functional features shared by the targets of the above miRNA, online software David Bioinformatics Resources 6.7 was used to perform the GO enrichment analysis, mainly regarding the biological processes, cellular components and molecular functions. For the targets of bta-mir-122, the most significant GO terms were: regulation of GTPase activity, regulation of protein signal transduction, intracellular, GTPase activator activity and GTPase regulator activity (−2), which could produce energy and is an critical metabolic pathway employed by a host of organisms. Therefore, this might be an interesting pathway to study in rumen. For the targets of bta-mir-655, the most significant GO terms of the three categories were: regulation of cellular process, regulation of biological process, biological regulation . For bta-mir-655 exclusively expressed in grain-fed animals, we found that 14 of the 749 targets were overlapped with the DEGs. Among the 14 overlapped genes, four genes displayed increased expression in grain-fed group, among which GAS1, ANO1 and NFASC displayed significantly positive correlation with bta-mir-655 (p < 0.05); the other nine genes  exhibited enhanced expression level in grass-fed group, which demonstrated negative correlation with bta-mir-655, but none of them showed significant difference (p > 0.05) .In addition to genetic makeup, age, species, gender and environment, grass and grain ratio of the diet could also contribute to significant difference in the general profile of fatty acid, animals’ growth rate, the content of vitamin A and E in the meat, the antioxidant enzyme concentration in body tissues and lipid depots , 40–41. The miRNAs could negatively regulate gene expression through degrading the target mRNAs or inhibiting the translation . HoweverOur results provided evidence for explaining the molecular mechanisms leading to the differences between grass-fed and grain-fed cattle. For the function of the DEGs containing the DMR and the differentially expressed miRNAs, extensive experimental validation work was still needed. Thus, overexpression and inhibition of our identified DEGs and miRNAs could be considered for the functional validation, which would provide more supportive information for our findings.In this study, we combined DNA methylation and miRNA expression with transcriptome analysis to explore the potential mechanism influencing rumen function of grass-fed and grain-fed animals. We found that the expression of ADAMTS3 and ENPP3 might be altered by the corresponding DMR inside these two genes, through which rumen function of grass-fed and grain-fed cattle could be regulated. For the differentially expressed miRNA bta-mir-122, it might modulate the rumen function by targeting the two DEGs OCLN and RBM47, which were possibly associated with gastrointestinal function. While expanding the scope of future studies with putative genes relevant to bovine growth and meat quality traits, our analysis provided biological insights into the mechanisms regulating rumen function and uncovered the molecular basis underlying the economic traits to enhance the productivity of animals.S1 Fig(TIF)Click here for additional data file.S2 FigThe mean value of log2 (fold-change) for each group was compared in the bar chart. FC means fold-change.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 TableThe threshold of FDR <0.1 was used to call the significant difference.(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLS)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.


The objective of this research was to examine the pathway from public stigma, to perceived stigma, to depression in adolescents via internalized stigma. Adolescents in grade 7 through 9 from a junior high school in Changhua County in Taiwan completed self-administered surveys from March to July in 2018. Adolescents were asked questions regarding depressive symptoms, obesity-related perceived stigma, and internalized stigma. Structural equation modeling was used to fit the pathway model. The pathway was first analyzed with the full sample and then stratified by actual and perceived weight status. Our final analytic sample consisted of 464 adolescents. The pathway model suggested an acceptable model fit. Perceived weight stigma (PWS) was significantly associated with internalized stigma regardless of actual or self-perceived weight status. Internalized stigma was significantly associated with anxiety for both actual (β = 0.186) and self-perceived nonoverweight (non-OW) participants (β = 0.170) but not for overweight (OW) participants . For OW adolescents, perceived weight stigma was associated with anxiety. However, the internalization process did not exist. It may be that the influence of perceived weight stigma is larger than internalized stigma on anxiety. It may also be that the level of internalization was not yet high enough to result in anxiety. Stigma is a fundamental cause of population health inequalities . Indeed,Additionally, weight stigma (or weight bias) among the different minorities seems to be discussed little in the Asian literature . TherefoThe evidence of the relationship between weight stigma and psychological distress among Asians was recently published, though it proved insufficient ,12,13. MThis study aimed to study the relationships between different types of weight stigma and psychological distress  in subgroups with different objectively defined weight status  and non-OW groups), and different self-perceived weight status , in Taiwan junior high school students. We hypothesized that (1) perceived weight stigma would be a significant predictor for the students’ public stigma on weight, weight-related self-stigma, and anxiety; (2) public stigma on weight would be a significant predictor of the students’ anxiety; and (3) weight-related self-stigma would be a significant predictor of the students’ anxiety. We also hypothesized that the aforementioned hypotheses would be in the same direction between students with different objectively defined weight status and those with different self-perceived weight status.All students in a junior high school in Chang-Hua County in Taiwan were approached (n = 699) to see if they were interested in participating in the study. Four hundred and sixty-four students with parental consent were asked to fill out a self-reported cross-sectional survey in school (67%). Consenting participants also agreed to have the survey linked to school records for height and weight measured by school nurses. The study protocol was approved by the human research ethics committee at the National Cheng Kung University Hospital (IRB: A-BR-106-009).Participants completed the information regarding their age, parental education, parental marital status, number of siblings, and whether their parents were immigrants on a background information sheet.The PWS was assessed using 10 dichotomous items (yes scores = 1 and no scores = 0), which have been used in several previous studies on Chinese populations ,9,16. AcThe Weight Bias Internalization Scale (WBIS) was used to assess weight-related self-stigma . The WBIstrongly disagree to 3 = strongly agree) that asks how an individual believes obesity is under the control of a person with obesity. Six items of the BAOP are negatively worded and they were reverse-coded before summing the eight-item scores. Afterward, the summated score added 24 to examine the belief of an individual toward obesity. A higher score indicates stronger beliefs that people with obesity are unable to control their weight. The psychometric properties of the BAOP Chinese version were supported by confirmatory factor analysis  was used to measure public stigma on weight ,19. The  = 0.050 ).We used the Hospital Anxiety Depression Scale (HADS), a frequently translated scale that has been validated in Chinese-speaking populations , to measWeight and height were measured at the student health center by a school nurse every year. BMI was calculated and categorized as obese, overweight, normal, or underweight based on their age and gender using criteria published by Taiwan Ministry of Health and Welfare . For per2 test together with CFI > 0.9, SRMR < 0.08, and RMSEA < 0.08 indicated satisfactory fit.All the analyses were performed using R software, including the lavaan package for conducting multigroup structural equation modeling (SEM). In the multigroup SEM, we used the same model to conduct twice. The model hypothesized that PWS is associated with BAOP, WBIS, and anxiety; BAOP and WBIS are further associated with anxiety. One multigroup SEM used actual weight status to group the participants into OW or non-OW; another multigroup SEM used self-perceived weight status to group the participants into OW or non-OW. For both SEMs, we applied the same fit indices to examine the data–model fit, where a nonsignificant χp = 0.56), BAOP score , and anxiety . The actual OW group as compared with the actual non-OW group had higher scores in PWS  and WBIS . Similar findings were shown for self-perceived weight status groups, except for the significant differences in anxiety: the self-perceived OW group had higher anxiety (13.6 [3.0]) than did the self-perceived non-OW group .The participant characteristics are presented on p = 0.15 for SEM with multigroups on actual weight status; 15.53 [p = 0.11 for SEM with multigroups on self-perceived weight status) together with other fit indices . WBIS was significantly associated with anxiety for both actual (β = 0.186) and self-perceived non-OW participants (β = 0.170) but not for OW participants . PWS was significantly associated with anxiety for both actual (β = 0.178) and self-perceived OW participants (β = 0.170) but not for non-OW participants . Additionally, diverse findings were observed for the relationship between BAOP and anxiety: significant association was found for actual non-OW (β = −0.134) and self-perceived OW participants (β = −0.179), but not for actual OW and self-perceived non-OW participants .The significant differences between OW and non-OW  in their perceived weight stigma and weight-related self-stigma are supported by prior studies ,12. HoweIn line with our hypothesis, we found strong associations between perceived weight stigma and weight-related self-stigma among all subgroups. The strong associations support the theory on stigma internalization: a person who is at risk of stigmatization will endorse and accept the biased conception on his/her condition  after the person is exposed to the unfriendly environment ,24,25,26The associations between public stigma on weight and anxiety found in our study contradict our hypothesis. Specifically, our findings indicated that if an actual non-OW adolescent has less public stigma on obesity, the adolescent will have a higher level of anxiety. However, there was no association between the public stigma and anxiety among actual OW adolescents. In terms of the self-perceived weight status, a self-perceived OW adolescent will have a higher level of anxiety if he/she has less public stigma on obesity. However, there was no association between the public stigma and anxiety among self-perceived non-OW adolescents. A possible explanation of the low level of public stigma on weight leading to anxiety is the characteristics of anxiety. People who have anxiety are afraid of being judged . TherefoThe association between weight-related self-stigma and anxiety was found in actual or perceived non-OW adolescents. This finding echoes prior research that weight-related self-stigma is a problem beyond people with OW and paying attention to weight-related self-stigma in normal-weight people is necessary ,29. HoweThere are some limitations in this study. First, our study design was cross-sectional, which only provides weak evidence in causal effects. That is, the cross-sectional design only determines the associations among our studied variables; however, the temporal associations could not be determined. Although we have prior literature and theory to support the model we proposed, we were unable to ensure the sequence of the different types of weight stigma and anxiety. Therefore, a longitudinal design or a randomized controlled trial to test the causal relationships is needed. Second, all the participants were solely recruited from one Taiwan junior high school through convenience sampling. Hence, our results cannot be generalized to the entire Asian population. For example, both Taiwan and Hong Kong share the same Chinese culture, but studies have suggested the subculture between Taiwan and Hong Kong still needs to be examined . Third, Based on our results, future studies may want to explore whether sex is a moderator in our proposed model. Specifically, one study discussed the overlap of obesity- and sex-related issues by examining a sample of college students, in which students were asked to rank the order of pictures of potential sexual partners . When chIn conclusion, we found that perceived weight stigma was associated with weight-related self-stigma regardless of weight status; perceived weight stigma was associated with anxiety among OW adolescents; and weight-related self-stigma was associated with anxiety among non-OW adolescents. The aforementioned associations were found consistently in actual and self-perceived weight status. Moreover, eating disturbances were associated with emotional distress regardless of the weight status of our participants. Healthcare providers may want to consider that self-perceived weight status shares the same importance as actual weight of an adolescent when providing services to adolescents coping with their perceived weight stigma and weight-related self-stigma.


Hearing loss substantially impacts pediatric development, and early identification improves outcomes. While intervening before school-entry is critical to optimize learning, early-childhood hearing screening practices are highly variable. Conditioned play audiometry (CPA) is the gold standard for preschool hearing screening, but otoacoustic emission (OAE) testing provides objective data that may improve screening outcomes.To compare outcomes of a community-based low-income preschool hearing program before and after implementation of OAE in a single-visit, two-tiered paradigm. We hypothesized that this intervention would reduce referral rates and improve follow-up while maintaining stable rates of diagnosed sensorineural hearing loss.We performed a cohort study of 3257 children screened from July 2014-June 2016. Department of Public Health data were analyzed pre- and post-implementation of second-line OAE testing for children referred on CPA screening with targeted follow-up by DPH staff.Primary outcomes included referral rates, follow-up rates, and diagnosis of sensorineural hearing loss.Demographics, pure-tone pass rates, and incidence of newly-diagnosed permanent hearing loss were similar across years. After intervention, overall pass rates increased from 92% to 95% (P = 0.0014), while only 0.7% remained unable to be tested (P<0.0001). 5% of children were unable to be tested by CPA screening but passed OAE testing, obviating further evaluation. Referral rate decreased from 8% to 5% (P = 0.0014), and follow-up improved from 36% to 91% (P<0.0001). Identification of pathology in children with follow-up increased from 19% to over 50%. Further, disparities in pass rates and ability to test seen in Year 1 were eliminated in Year 2.In a community setting, implementation of second-line OAE screening for CPA referrals reduced referral rates, increased identification of hearing loss, reduced outcome disparities, and improved follow-up rates. This study provides lessons in how to improve outcomes and reduce disparities in early-childhood hearing screening. Hearing loss affects children’s cognitive, emotional, and social development, and educational, societal, and financial outcomes , 2. EarlEarly-childhood screening (EHS) can identify children with congenital hearing impairment lost to follow-up after a failed NHS , as wellConditioned play audiometry (CPA), in which children are conditioned to perform a specific action upon hearing a pure tone, is considered the gold standard method for pediatric hearing screening , 12. HowThe target age range for EHS, 2–5 years, encompasses the range where the ideal hearing screening method is unclear. While AAA guidelines recommend age 3 as the cutoff for CPA screening, children up to age 5 have been shown to be unable to cooperate ; many ofIn this cohort study, we evaluated the outcomes of a community-based low-income preschool hearing-screening program conducted by the San Francisco Department of Public Health (SFDPH). By analyzing their transition from a CPA-based screening protocol to a novel hybrid CPA/OAE two-tiered, single-visit system, we aimed to compare outcomes and disparities of the two screening paradigms.Children attending preschool programs in the 2014–2015 and 2015–2016 school years underwent hearing screening performed by a single audiometrist in the Office of Childhood Hearing (OCH) at the SFDPH. Screening outcomes and demographic information were recorded by the audiometrist. Children who referred on at least one ear or who were unable to be tested for any reason were documented and their parents given notice of the result with the recommendation for additional evaluation by their primary care provider. In the 2014–2015 school year, screening was performed with CPA. The following year, initial screening was performed with CPA, with distortion product (DP) OAE screening as an immediate second screen for those who did not pass by CPA. Details on the screening program and methodology are provided in the Supporting Information.Final audiologic and medical outcomes were documented by the OCH upon: 1) receipt of a completed medical documentation form given to parents upon notification of the screening outcomes; 2) direct verbal contact by an OCH and/or preschool staff member with the family or medical provider. All outcome documentation was stripped of personal health information and transmitted securely to UCSF at the conclusion of each school year for analysis. A complete description of the screening methods and follow-up protocols is included in the supplemental material.Outcomes of screenings were documented as “Pass,” “Refer,” or “Unable to test” (UTT) at the time of screening. In Year 2, all Refer and UTT children were given OAE testing, leading to greater complexity of outcomes: all Refer children by CPA continued to be documented as Refer; all UTT children who were also UTT by OAE testing continued to be documented as UTT; those children who were UTT by CPA but Refer by OAE were counted as Refer; and those children who were UTT by CPA but passed by OAE were counted as Pass.For the purposes of analyses, Refer and UTT children are categorized collectively as “Not Pass” (NP). Additionally, Pass and Refer children are collectively categorized as “able to test” (ATT). This allows separate analysis of two relevant outcomes of the hearing-screening protocol—the ability of the protocol to test the children (ATT vs UTT), as well as the referrals for further medical evaluation generated by the protocol (Pass vs NP).Chi-square and Fisher’s exact tests, as well as logistic regression analysis both for the aggregate analysis and survey data, were performed by contract biostatisticians at the Clinical and Translational Science Institute at UCSF. Analyses were performed using SAS 9.4. This study was determined to be exempt from formal review by the Committee on Human Research at UCSF.1436 children aged 2–6 were screened by CPA alone from July 2014-June 2015 (Year 1) and 1821 screened on a two-tiered single-visit CPA/OAE protocol from July 2015-June 2016 (Year 2). Screening outcomes  demonstrThe demographic profile, including sex, age, primary language, ethnicity, and teacher concern about communication, was compared across years. Compared to Year 1, there were fewer Spanish- and Cantonese-speaking children, and fewer Asian children in Year 2 .Follow-up among referred families improved after implementation of the two-tiered single-visit CPA/OAE protocol . While iWe investigated the final diagnostic outcomes among all children referred through hearing screening . Among tThe overall prevalence of identified SNHL was 0.70 per 1000 screened children in Year 1, compared to 1.65 per 1000 in Year 2 (P = 0.6353). The prevalence of pathology increased from 7.66 per 1000 screened children in Year 1 to 26.91 per 1000 screened children in Year 2 (P<0.0001). Therefore, implementation of second-line OAE screening increased the identification of pathology among all screened children.Comparison of pass and ATT rates from year 1 to Year 2 are presented in Tables In Year 1, children whose primary language was English were more likely to NP compared to Non-English-speaking children; they were all equally likely to be ATT . In YearDue to sample sizes, ethnic groups were clustered for analysis into Caucasian, Hispanic, Asian, and “Other”, which included African American, Native American, Pacific Islander, Multi-Racial, or Other. In Year 1 there was no relationship between ethnicity and hearing screening outcome. In Year 2, there were no differences in ATT rates among ethnicities, and no differences in Pass rates except among Hispanic children, who were less likely than Caucasian children to Pass. From Year 1 to Year 2 there was no significant change in Pass rates by ethnicity .In Year 1, 7 of the 11 NP children with concern for communication impairment were unable to be tested by CPA . ChildrePreschool hearing loss presents a unique challenge. While it is critical to identify children with hearing loss during sensitive developmental periods, it is difficult to effectively screen and efficiently diagnose hearing loss due to variability in behavioral maturity among preschool children.Using a standard CPA-based hearing screening protocol in a community-based low-income network of preschools, we identified a high UTT rate  with loWith implementation of this two-tier, single-visit strategy, the overall referral (NP) rate dropped significantly, from 7.9% to 5.1%, due almost entirely to a large reduction in referrals for children who were unable to be tested by CPA alone. The referral rate in Year 2 was comparable to previous studies, including those utilizing more traditional 2-tier, 2-visit methods , indicatImportantly, the reduction in referral rates did not result in missed pathology. The incidence of confirmed SNHL was 1.65/1000 children in Year 2, a rate comparable to previously reported rates in preschool populations . The ratThe rate of follow up increased substantially, from 36% in year 1 to 91% in year 2. While this improvement in follow-up rates cannot be definitively tied to the intervention due to the ecological nature of the study, it is possible that the reduced number of referrals in Year 2 could have improved the follow-up rate for children who did refer. DPH and preschool staff indicated that in Year 2 they had the same number of staff supporting a smaller number of referrals, which allowed them to focus more attention on tracking individual children’s outcomes. This could have led families to better understanding and support in seeking follow-up care, as well as improved documentation of follow-up. Further improvements in screening protocols, such as clarification of educational materials given to families and use of resources in additional languages, are currently in progress, and analysis after completion of this intervention would provide further insight into follow-up optimization.In Year 1, there were significant disparities in hearing screening outcomes Tables  and 5; iAccording to 2007 guidelines from the Joint Commission on Infant Hearing , full auDisparities in hearing outcomes relating to primary home language and ethnicity were more complex. In Year 1, we found no association between ethnicity and hearing screening outcomes, but did find, in contrast to prior literature, that non-English-speaking children were twice as likely to Pass hearing screens compared to English-speaking children, with no difference in UTT rates. One possible explanation for the language disparity without ethnicity difference may be the “healthy immigrant effect” , in whicThis study is limited in that it is an un-controlled, un-blinded, ecological prospective cohort study of screening practices. Thus, differences seen in Year 2 cannot be fully attributed to the intervention alone. However, consistency in the screened children demographics, childcare centers involved, and screening personnel between the control (pre-implementation of OAE backup screening) and intervention (post-implementation) groups minimize this limitation. Second, formal diagnostic audiograms were not performed in all screened children, making it impossible to calculate the sensitivity and specificity of the different screening paradigms; we cannot be certain whether either method resulted in false negative screens. Third, there are types of hearing loss that may be missed by reducing referrals with OAE testing: mild losses not identified by OAE screening; and auditory neuropathy spectrum disorder, in which cochlear function is intact but proximal signals remain impaired. These may be uncommon, but identification of these entities remains important. Finally, formal operational and cost analysis is beyond the scope of this current study, and findings may vary depending on local circumstances; future investigation may clarify these issues of importance to inform public health policy.The addition of an immediate second-line OAE screen to pure tone screening for preschool children improved both the effectiveness and efficiency of our community-based hearing screening program, and eliminated disparities in ability to test associated with age, language, and communication delay. The reduction in referral volume also corresponded to an improvement in follow-up rates, possibly by improving resource allocation. We therefore present this model as an effective way to provide single-visit, age-appropriate, efficient and effective hearing screening in community preschools.S1 File(DOCX)Click here for additional data file.


Clinical trials are often challenged with issues of recruitment and retention. Little is known concerning general attitudes and preferences toward trial design and willingness to participate among parents of children treated for cancer. Furthermore, willingness to participate in internet-administered psychological interventions remains unexplored. In this study, we examined attitudes and preferences of the population regarding study procedures for a hypothetical trial of an internet-administered psychological intervention. In addition, differences in the response rate between modes of study invitation and willingness to engage in internet-administered interventions were examined.The primary objective of this study was to examine attitudes and preferences toward participating in an internet-administrated psychological intervention. The secondary objective was to examine the response rates and help-seeking behavior among parents of children treated for cancer.A cross-sectional, Web-based survey was conducted with parents of children who had completed cancer treatment. This Web-based survey examined self-reported emotional distress, prior help-seeking and receipt of psychological support, past research participation, attitudes toward research, preferences concerning recruitment procedures, and attitudes toward different types of trial design.21=0.6, P=.45). The majority  of parents responded that they had experienced past emotional distress. Responses indicated high  or somewhat high trust in research , and the majority of parents would accept, or maybe accept, internet-administered psychological support if offered . In addition, responses showed a preference for postal study invitation letters , sent by a researcher  with additional study information provided on the Web via text  and video . Overall, parents responded that trials utilizing a waiting list control, active alternative treatment control, or a patient-preference design were acceptable.Of all the parents invited, 32.0% (112/350) completed the survey, with no difference in response rate between modes of study invitation (χParents of children treated for cancer appear willing to participate in trials examining internet-administered psychological support. Findings of this study will inform the design of a feasibility trial examining internet-administered psychological support for the population. Due to marked treatment advances across the developed world, the overall 5-year survival rate of children diagnosed with cancer is now >80% . HoweverElectronic mental health interventions  may represent a way of increasing access to psychological support  for pareThis study builds upon the previous MRC phase I development  work 1010,23 by The primary study aims were to examine (1) attitudes and preferences toward trial design and (2) willingness to participate in a hypothetical trial of internet-administered psychological intervention for parents of children treated for cancer. Secondary study aims were to investigate (1) overall response rates and differences in the response rate between two modes of study invitation and (2) current and past emotional distress and help-seeking behavior. The following outcomes were examined:Overall response rateNumber of study invitation reminders required to recruitDifferences in the response rate between two modes of study invitation Self-reported current and past emotional distressPrior help-seeking and receipt of psychological supportWillingness to receive internet-administered psychological supportPast experience of research participation and attitudes toward researchAttitudes and preferences concerning the delivery of initial study invitations and presentation of full study informationAttitudes toward participation in different trial designs .A cross-sectional, Web-based, self-report survey was conducted from April 2017 to June 2017 with parents of children treated for cancer and recruited across Sweden.In this survey study, Swedish-speaking parents of children treated for cancer (aged 0-16 years at study commencement) residing in Sweden were eligible. Parents were included if the child had (1) completed successful cancer treatment 3 months to 5 years earlier at study commencement and (2) been treated in 1 of the 6 pediatric oncology units in Sweden. Parents were excluded if the child had a benign tumor.Potential participants were identified using a two-step screening process:Childhood cancer survivors, meeting the inclusion criteria, were identified via the Swedish Childhood Cancer Registry .Children’s personal identification numbers were linked to parents’ personal identification numbers via the SPAR-Registry (“Statens personadressregister” by Swedish acronym) held by the Swedish Tax Agency to obtain parent contact information.Although the SPAR-Registry includes all currently registered residents in Sweden, at the time of the study, it was only possible to access parent information for children aged ≤16 years. Therefore, the current age-span of children, whereby it was possible to identify parents, was restricted to 0-16 years.From identified parents, an in-house computer program was used to randomize to the mode of invitation (letter vs postcard), with a 1:1 allocation, stratified by cohabitation status . Prior to posting invitations, the most up-to-date information concerning whether children were currently living, or deceased, was checked via the telephone by a member of the research group with the Swedish Tax Agency.Parents were sent an invitation either via a postcard  or a letParents were provided full study information via the U-CARE-portal , and infEthical approval was obtained from the Regional Ethical Review Board in Uppsala (DNR: 2015/426/3).P<.05 (two-tailed).Sample size calculation indicated that a minimum of 340 participants would be required to detect a difference of 15% in the response rate between groups , with a power of 0.8 and The following sociodemographic data were collected for parents via the SPAR-Registry: (1) date of birth; (2) gender; and (3) marital status. Sociodemographic data for children were collected via the Swedish Childhood Cancer Registry: (1) cancer diagnosis; (2) date of diagnosis; (3) date of treatment completion; (4) date of birth; and (5) gender.A Web-based survey, consisting of 20 items and written in Swedish, was designed for the study and comprised 4 subsections as follows: (1) sociodemographics (3 items); (2) emotional distress and psychological support (6 items); (3) experience of participation in research and attitudes toward research (2 items); and (4) attitudes toward proposed trial procedures (9 items). The survey was administered on the U-CARE-portal, an internet research platform, designed to support both data collection and the provision of complex eMental Health interventions . Multimen) and percentages (%) or means and SDs. Chi-square tests were used to assess differences between those who responded and those who did not concerning categorical data, and independent-sample t tests were used to assess differences regarding continuous variables (P<.05). Furthermore, chi-square tests were used to assess differences in response rate between parents invited via postcard and those invited via study invitation letter. Descriptive statistics are used to present the responses to the Web-based survey. All data analyses were performed using SPSS statistics for Windows, version 24.8.Study recruitment and flow are reported using an adapted version of the Consolidated Standards of Reporting Trials Statement . DescripThe majority of parents who responded were female, cohabiting, with a mean age of 43.2 years. Children were predominantly male, had experienced leukemia, with a mean current age of 9.3 years, and had finished cancer treatment an average of 2.9 years ago. No significant differences were noted regarding parent or child sociodemographic and clinical variables between those who responded and those who did not .21=0.6, P=.45). Among those who responded, 18.8% (21/112) responded to the first invitation , 48.2% (54/112) responded following one reminder , and 33.0% (37/112) responded after 2 reminders .No difference was noted between the mode of invitation and response rate  were examined. To the best of our knowledge, this is the first survey to examine attitudes and preferences toward the design of and participation in internet-administered psychological intervention trials within the population. Summarizing the main findings, an overall 32.0% (112/350) response rate was yielded, yet no difference was found in the response rate between modes of study invitation. Self-reported current and past emotional distress was common. Internet-administered psychological support, under guidance of a psychologist, was deemed acceptable by the majority of those parents currently reporting emotional distress and of the surveyed population as a whole. Examination of acceptable recruitment methods indicated the most acceptable method of study invitation would be via postal letter, with full study information presented on a study website via text or video and delivered by a researcher. On the whole, parents responded that they would either accept, or maybe accept, participation in each presented hypothetical trial design , with no overall indication of a preferred trial design.It is interesting to note that no association between the mode of invitation and the response rate was detected. As such, this suggests professional illustration may not increase study response rates. This finding is in contrast to research indicating that professionally designed invitation packs can improve recruitment rates . Still, Another noteworthy finding was that parents showed an overall preference for receiving initial study invitations via the post. While postal recruitment from disease registers is a common recruitment strategy , evidencInterestingly, similar percentages of parents responded they would agree, or maybe agree, to participate in trials using waiting list control, alternative active treatment, and patient-preference designs. Furthermore, little difference was found concerning acceptable waiting times for a hypothetical waiting list RCT. As such, the majority responded they would be willing to participate in hypothetical trial designs utilizing randomization procedures. This finding is in contrast to research reporting that a fear of randomization is a common reason for declining trial participation . One potIn line with previous research ,11, a suThis study has several limitations. First, willingness to participate in a hypothetical trial of an internet-administered psychological intervention may not predict actual trial enrollment . Even thAnother limitation pertains to only examining the acceptability of more traditional clinical trial recruitment strategies, for example, postal recruitment and clinician referral. However, evidence suggests Web-based recruitment strategies are effective for internet-administered intervention trials ,46 and fNotwithstanding these limitations, this is to our knowledge the first study to examine attitudes toward clinical trials, preferences regarding study design, and willingness to receive internet-administered psychological support among parents of children previously treated for cancer. The results from this study will have considerable implications for the design of a planned feasibility study , such asClinical trial conduct is time and resource intensive. While research has been performed to examine attitudes and preferences toward clinical trial design and participation ,48,49, l


Supplemental Digital Content is Available in the Text. Women with HIV face an increased risk of human papillomavirus (HPV) acquisition and persistence, cervical intraepithelial neoplasia, and invasive cervical cancer. Our objective was to determine the cost-effectiveness of different cervical cancer screening strategies among women with HIV in South Africa.We modified a mathematical model of HPV infection and cervical disease to reflect coinfection with HIV. The model was calibrated to epidemiologic data from HIV-infected women in South Africa. Clinical and economic data were drawn from in-country data sources. The model was used to project reductions in the lifetime risk of cervical cancer and incremental cost-effectiveness ratios (ICERs) of Pap and HPV DNA screening and management algorithms beginning at HIV diagnosis, at 1-, 2-, or 3-year intervals. Strategies with an ICER below South Africa's 2016 per capita gross domestic product (US$5270) were considered “cost-effective.”HPV testing followed by treatment (test-and-treat) at 2-year intervals was the most effective strategy that was also cost-effective, reducing lifetime cancer risk by 56.6% with an ICER of US$3010 per year of life saved. Other cost-effective strategies included Pap  at 1-, 2-, and 3-year intervals, and HPV test-and-treat at 3-year intervals. Pap (ASCUS+), HPV testing with 16/18 genotyping, and HPV testing with Pap or visual triage of HPV-positive women were less effective and more costly than alternatives.Considering per capita gross domestic product as the benchmark for cost-effectiveness, HPV test-and-treat is optimal in South Africa. At lower cost-effectiveness benchmarks, Pap (HSIL+) would be optimal. Recent guidelines from the American Society for Clinical Oncology recommend HPV testing at the time of HIV diagnosis and subsequently at an interval of every 2–3 years if resources are available; for lower-resource settings, screening for HIV-infected women is recommended twice as often as in the general population.9 In South Africa—where screening guidelines recommend cytology-based screening, with phasing in of HPV testing based on resource availability—the principles of equity, quality, efficiency, and sustainability have been adopted by the Department of Health.10 Yet data on the cost-effectiveness of different screening and management strategies in HIV-infected women are limited.The World Health Organization (WHO) recommends that women with HIV receive cervical cancer screening with HPV testing at least every 3 years if resources are available; for countries with fewer resources, visual inspection with acetic acid (VIA) is an acceptable screening alternative.To inform ongoing discussions about clinical guidelines, optimal resource use, and the integration of women's health interventions with HIV-related care in South Africa and other low-resource settings with a high burden of HIV, we evaluated the cost-effectiveness of different Pap and HPV screening and management algorithms among HIV-infected women in South Africa.12 to reflect the burden of HPV in HIV-infected women in South Africa . Women in the model are infected with HIV at the age of 20 years, approximating the peak HIV incidence in South Africa.13 Based on HIV progression estimates in the absence of ART15 and estimates of CD4+ cell count at HIV presentation,16 we optimistically assumed (as HIV testing and ART access increase) that women would be diagnosed with HIV around the age of 25 years, at a CD4+ cell count of approximately 350 cells/µL. Before HIV acquisition at the age of 20 years, background mortality rates for uninfected women were drawn from the THEMBISA model (version 2.5).13 On HIV infection at the age of 20 years, women face excess mortality rates dependent on CD4+ cell count and rate of HIV progression.14 On HIV detection at the age of 25 years, women are assumed to begin ART immediately and then face excess mortality from HIV based on a relative survival model that considers current age, age at ART initiation, CD4+ category at ART initiation, and duration on ART.7 Excess mortality due to cervical cancer was derived from estimates of 5-year survival in middle-income countries with a low burden of HIV17 multiplied by hazard ratios from cervical cancer mortality in HIV-infected versus uninfected women.18 Time-dependent survival by stage  was derived from cervical cancer survival estimates in HIV-infected women in Botswana.19 After 10 years following cancer treatment, we assumed women no longer faced excess mortality due to cervical cancer.Because of limited data on the interaction between HPV and HIV, we assumed a similar course of HIV infection and progression for all women in the model  and type-specific immunity. To reflect the greater risk of HPV infection and progression (to CIN and cancer) and the decreased likelihood of HPV clearance and CIN regression in HIV-infected women, as well as parameter uncertainty, we modified the previously described natural history model by setting plausible ranges for factors to apply to baseline transition probabilities derived from longitudinal studies, guided by previous work modeling HPV infection in the general population of women in several low- and middle-income countries with a low burden of HIV20 and published hazard and risk ratios in HIV-infected versus HIV-uninfected women.21 The model underwent repeated model simulations in the absence of any intervention, and for each simulation, single random values from a uniform distribution spanning the plausible range for each factor were selected and applied to the relevant baseline probability to create a unique natural history input parameter set. We then computed a goodness-of-fit score by summing the log likelihood of model-projected outcomes for each unique parameter set to represent the quality of fit to epidemiologic data  on age-specific prevalence of oncogenic HPV and the proportion of HPV type-specific infections in CIN3 and cervical cancer among women with HIV in South Africa.24 We selected the 50 top-fitting parameter sets for analyses and calculated the expected value, as well as the range of values, for all outcomes. Model fit to calibration targets is displayed in Figures 1 and 2, Supplemental Digital Content, http://links.lww.com/QAI/B185. Data sources used to inform the factor search space and the ranges for each transition probability value are provided in Table 1, Supplemental Digital Content, http://links.lww.com/QAI/B185. Model validations to CIN2/3 prevalence and cervical cancer incidence in South Africa are presented in Figures 3 and 4, Supplemental Digital Content, http://links.lww.com/QAI/B185.To inform transitions between HPV-related health states, baseline “prior” transition probabilities were estimated from large epidemiologic studies in predominantly HIV-uninfected populations, as previously documented.10 and WHO guidelines for HIV-infected women.8 Strategies included (1) liquid-based cytology with referral to colposcopy for atypical squamous cells of undetermined significance or worse (ASCUS+) [“Pap (ASCUS+)”]; (2) liquid-based cytology with referral to colposcopy for atypical squamous cells cannot rule out high-grade/high-grade squamous intraepithelial lesions or worse (HSIL+) [“Pap (HSIL+)”]; (3) HPV DNA testing with referral to treatment for all HPV-positive women (“HPV test-and-treat”); (4) HPV DNA testing with triage to VIA for all HPV-positive women, and treatment for all women who are both HPV-positive and VIA-positive (“HPV-VIA”); (5) HPV DNA testing with triage to Pap for all HPV-positive women, and referral to colposcopy for all women who are both HPV-positive and ASCUS+ (“HPV-Pap”); and (6) HPV DNA testing followed by HPV16/18 genotyping for HPV-positive women, with referral to treatment for 16/18-positive women and referral to colposcopy for women with other oncogenic types (“HPV16/18 genotyping”). Screening coverage was 70% of the target population. Of those screened, 85% were assumed to receive the strategy of interest , whereas 15% were assumed to receive a separate VIA strategy, to represent the potential availability of VIA followed by treatment for women without access to Pap or HPV-based strategies; we weighted cost and health outcomes accordingly. Screening and management algorithms are displayed in Figures 5–11, Supplemental Digital Content, http://links.lww.com/QAI/B185. All strategies were considered at 1-, 2-, and 3-year intervals.Screening and management algorithms were based on recently drafted guidelines for South Africa33 Compliance with each recommended clinical encounter was 85% relative to the previous visit. Values for screening and treatment variables are displayed in Table For HPV-testing strategies, we assumed provider-collection of samples. For HPV testing followed by either visual or Pap triage, women who were HPV-positive but triage test–negative were assumed to receive repeat HPV testing in 1 year. After colposcopy, women with normal or CIN grade 1 (CIN1) on histology were referred to repeat screening in 1 year; those with CIN2 or higher were referred to treatment. Treatment included cryotherapy for eligible women, and large loop excision of the transformation zone (LLETZ) for women who were determined to be ineligible for cryotherapy. Screening test performance and treatment effectiveness values were based on studies in HIV-infected women.34; costs were estimated from in-country data sources and included direct medical costs , direct nonmedical costs (women's transportation), and women's time  using consumer price indexes and average annual official exchange rates to report findings for global policy makers. Because HPV DNA and HPV genotyping tests have not yet been procured by the government of South Africa, list prices  did not necessarily reflect economies of scale from bulk purchasing. Each month, beginning at HIV diagnosis, women incurred the average monthly outpatient cost per adult on ART and average monthly inpatient cost for individuals on ART with a CD4 count above 350 cells/µL.40 Further details and data sources are provided in the Supplemental Digital Content, http://links.lww.com/QAI/B185.Consistent with guidelines for cost-effectiveness analysis, we adopted a modified societal perspectiveme Table .35–38 Co34 We then calculated incremental cost-effectiveness ratios (ICERs). An ICER is the additional cost of a strategy divided by its additional health benefit, compared with the next most costly strategy after eliminating strategies that are dominated . Across the 50 top-fitting input parameter sets, the ICER was calculated as the ratio of the mean cost divided by the mean health effect. We considered the following possible cost-effectiveness thresholds as a benchmark to indicate strategies that provide good value for money: (1) South Africa's per capita gross domestic product (GDP) (2016 US$5270)41; (2) 50% of South Africa's per capita GDP (2016 US$2640); and (3) the cost per disability-adjusted life-year averted for extending ART eligibility from adult patients with CD4 counts of 350 cells/µL or less to patients with CD4 counts of 500 cells/µL or less—an approximation of the opportunity cost of HIV care in South Africa—as estimated by a recent cost-effectiveness analysis using 12 mathematical models (high estimate for South African models: 2016 US$1190).42Model outcomes included the absolute lifetime risk of cervical cancer incidence and mortality, life expectancy, and lifetime costs; costs and life-years were discounted at an annual rate of 3%.Scenario analyses assessed the impact of alternate parameter values and assumptions on cost-effectiveness results Table .The impact on cervical cancer incidence and mortality, lifetime cost, life expectancy, and ICER for each screening strategy is presented in Table Pap (HSIL+) every 3 years had the lowest ICER [$340 per year of life saved (YLS)], followed by Pap (HSIL+) every 2 years ($900 per YLS), but these strategies also were the least effective. More effective strategies that were also efficient  included HPV test-and-treat every 3 years ($1780 per YLS), Pap (HSIL+) every year ($2420 per YLS), HPV test-and-treat every 2 years ($3010 per YLS), and HPV test-and-treat every year ($8020 per YLS) , Pap testing (HSIL+ threshold) every 1–2 years would be considered optimal but would yield much lower reductions in cervical cancer risk . Optimal strategies under various cost-effectiveness benchmarks were robust across many scenario analyses. However, when the high direct medical cost of the HPV test in the base case was reduced to $35, HPV test-and-treat at 2- or 3-year intervals was the optimal strategy for all benchmarks considered. Furthermore, because of the lower number of required visits for this strategy compared with Pap-based strategies, less favorable assumptions regarding visit compliance improved the relative attractiveness of HPV test-and-treat.41” Yet recent analyses suggest that accepting interventions according to per capita GDP may displace interventions yielding substantial health benefits with less effective interventions.43 In the absence of a formal threshold based on empirical data from South Africa, we considered alternative thresholds, including 50% of per capita GDP and $1190, which represents the ICER (from a recent modeling study in South Africa) of expanding ART coverage from all adults with CD4 counts below 350 cells/µL to those with CD4 counts below 500 cells/µL. In addition to consideration of value for money, it is critical to consider health equity in determining what services should be available.To determine whether an intervention should be included in a package of services, a benchmark of health opportunity costs, or cost-effectiveness threshold, must be established. The WHO Commission on Macroeconomics and Health suggests that interventions with ICERs less than per capita GDP are “very cost-effective” and less than 3 times per capita GDP are “cost-effective.10 The effectiveness of treatment in HIV-infected women is uncertain. One recent randomized trial found that HIV-infected women with CIN2/3 were more likely to experience recurrence after cryotherapy than after LEEP at 12 and 24 months after treatment, as measured by HSIL+ on cytology; another trial in South Africa found that more women had recurrent CIN2+ after cryotherapy than LEEP at 6 months, but by 12 months, the difference was not significant.44 When we assumed all women were treated with LLETZ instead of cryotherapy, HPV test-and-treat every 3 years was optimal at cost-effectiveness thresholds of per capita GDP and 50% of per capita GDP; less frequent screening was needed because of the assumed greater effectiveness of treatment. Although our analysis included the costs associated with potential overtreatment of screen-positive women in HPV test-and-treat strategies, we did not consider the impact of potential adverse reproductive outcomes. Guidelines will need to weigh safety, effectiveness, and logistical considerations  in recommending surgical versus ablative treatments for women with HIV.In South Africa, access to cryotherapy is limited, but recently drafted guidelines rely heavily on this form of treatment, and our analysis assumed that most eligible women would receive cryotherapy.45 found that HPV testing every 1–2 years followed by Pap triage of HPV-positive women was cost-effective in Brazil but did not consider HPV testing with treatment for all HPV-positive women. Lince-Deroche et al35 estimated the cost per case of CIN2+ detected associated with screening in South Africa and found that HPV testing followed by colposcopy of HPV-positive women had the highest cost per case detected, whereas VIA had the lowest; however, this analysis did not consider the cost offsets of prevented cancers.Because of the complexities of modeling HIV and HPV coinfection, few studies have evaluated the cost-effectiveness of cervical cancer screening in HIV-infected women in a low- or middle-income country. Vanni et al46 Although we modeled screening strategies based on WHO recommendations and recently drafted guidelines in South Africa, HPV testing is not widely available in South Africa at this time. Although there are plans for scale-up as resources become available, the delivery of HPV testing and management may need to be refined based on system resources or improved for more efficient delivery. We did not consider reduced screening intervals after a specific number of negative screening tests, nor did we model switching from Pap-based to HPV-based screening or cotesting after the age of 30 years, as US guidelines suggest.47 We did not consider the future impact of HPV vaccination of girls, which was introduced in South Africa in 2014.There are several limitations to this analysis. We did not have data to inform HPV transitions stratified by CD4 count, viral load, or ART status and thus assumed a common trajectory of HIV disease in the modeled cohort. Further research on the time-dependent impact of ART on HPV and cervical cancer transitions will inform future modeling efforts. Furthermore, our calibration to oncogenic HPV prevalence relied on data from the VICAR study of HIV-infected women in Johannesburg, and we thus could not calibrate to the prevalence of HPV for women younger than 20 years before HIV infection, nor can we assume generalizability to HIV-infected women in other parts of the country. However, by achieving good fit to HPV prevalence beginning at the age of 20 years, when screening does not begin until the age of 25 years, we believe the model reflects appropriate clearance versus persistence of earlier HPV infections. Furthermore, our validation exercises examining model-projected cervical cancer incidence relative to registry data for the general population are consistent with hazard ratios gleaned from studies in HIV-infected versus uninfected women.4; however, tremendous disparities in access to screening remain. Our model-based analysis demonstrates that cervical cancer screening is likely to be both effective and very cost-effective in HIV-infected women in South Africa. If sufficient resources are available, HPV testing at 2-year intervals can achieve greater reductions in cancer risk than Pap-based strategies in South Africa through increased detection of precancer and fewer visits per screening episode. As the demand for HPV testing rises and test costs decline, a shift toward HPV-based test-and-treat strategies in South Africa and other low-resource settings with a high burden of HIV may improve access and save lives. Health services research will be necessary to integrate cervical cancer prevention with HIV-related services through community outreach, task-shifting to nonphysicians, and strengthening information systems to track patients48; integrating and improving service delivery for high-risk women is a tremendous opportunity to transform the health care system and reduce health disparities. We present optimal strategies under a range of benchmarks for cost-effectiveness to stimulate action from international donors, advance policy discussions, and inform implementation efforts—so that women who are saved from HIV will not die of cervical cancer.As women live longer due to improved access to life-saving ART, they will likely continue to face an elevated risk of cervical cancer. In South Africa, expanded access to organized cervical cancer screening with cytology has reduced cervical cancer incidence in HIV-infected women


Luojia 1-01 satellite, launched on 2 June 2018, provides a new data source of nighttime light at 130 m resolution and shows potential for mapping urban extent. In this paper, using Luojia 1-01 and VIIRS nighttime light imagery, we compared several methods for extracting urban areas, including Human Settlement Index (HSI), Simple Thresholding Segmentation (STS) and SVM supervised classification. According to the accuracy assessment, the HSI method using LJ1-01 data had the best performance in urban extent extraction, which presented the largest Kappa Coefficient value, 0.834, among all the results. For the urban areas extracted by VIIRS based HSI method, the largest Kappa Coefficient value was 0.772. In contrast, the largest Kappa Coefficient values obtained by STS method were 0.79 and 0.7512 respectively when using LJ1-01 and VIIRS data, while for SVM method the values were 0.7829 and 0.7486 when using Landsat-LJ and Landsat-VIIRS composite data respectively. The experimented results demonstrated that the utilization of nighttime light imagery can largely improve the accuracy of urban extent extraction and LJ1-01 data, with a higher resolution and more abundant spatial information, can lead to better identification results than its predecessors. Over the past half-century, most regions of the world, especially developing countries, have experienced a phase of high-speed urbanization ,2,3. In As an objective and real-time data source, remote sensing satellite images have been widely used in urban area mapping and urbanization monitoring. Currently, coarse resolution images, such as the nighttime light (NTL) data have proven effective in urban extent extraction at regional and global scales ,19,20,21Generally, the urban extent extraction methods using NTL data can be divided into three types, that is, thresholding-based, classification-based and index-based methods ,45,46. TIn June 2018, Luojia 1-01 (LJ1-01), a new generation of NTL remote sensing satellite, was launched in China successfully. It has provided dramatic improvements over its predecessors, in terms of increased spatial resolution (~130 m), high radiometric quantization (14 bits) with a swath of 250 km. This new generation of NTL data source brings new possibilities and insights to the study of urban extent extraction, especially the improvement of image spatial resolution, which may not only increase the accuracy of urban extent mapping but also show more details of urban structure. Furthermore, urban extent extraction using LJ1-01 NTL data can to some extent fill the gaps in the field of NTL remote sensing in local-scale research.The main purpose of this paper was to assess the accuracy of urban extent extraction at the city scale using LJ1-01 nighttime light imagery. For comparison, index-based, thresholding-based and classification-based methods were all adopted in the experiment. Besides, VIIRS DNB imagery were also utilized to reveal the difference between two kinds of NTL data. Finally, the Google Map high-resolution satellite images were used as reference and a visual interpretation method was applied for accuracy assessment.2 and had a population of 10.89 million by the end of 2017 [Wuhan, situated in the central part of China, is the capital city of Hubei Province. Wuhan covers an area of 8494.41 km of 2017 . Due to  of 2017 . Since thttp://59.175.109.173:8888/, accessed in June 2018). Since the radiometric calibration for LJ1-01 imagery is still under improvement, we used digital number (DN) value in this experiment for analysis. The comparison of parameters for DMSP/OLS, NPP/VIIRS and LJ1-01 NTL data are shown in LJ1-01, launched on 2 June 2018, is the first satellite in the Luojia-1 scientific experimental satellite series owned by Wuhan University. It is the first remote sensing satellite focusing on nighttime light in China and also the first low-orbit satellite with earth observation and satellite navigation enhancement functions. LJ1-01 is a 20 kg-level micro-nano satellite, equipped with a high sensitivity night-light camera that has a spectral bandwidth of 0.319 μm. It can obtain high-precision nighttime light imagery with a dynamic range of up to 14 bits at night, with a spatial resolution of 130 meters and a swath of 250 km. It will provide an objective basis for the research of socio-economic parameter estimation, eco-environmental disaster monitoring, major event assessment, public health and other fields, carrying out dynamic monitoring of macroeconomic operations in China and the world. The data is free to download at the High-Resolution Earth Observation System of the Hubei Data and Application Center (https://ngdc.noaa.gov/eog/viirs/download_dnb_composites.html). Since the monthly composite images of June 2018 have not been released yet, the images of May 2018 were selected for the experiment.VIIRS DNB Cloud-Free Composites (version 1) are a suite of average radiance composite images, where the data impacted by stray light, lightning, lunar illumination and cloud-cover have been excluded. The products are produced in 15 arc-second geographic grids, spanning the globe from 75 N latitude to 65 S. In this research, the VIIRS DNB data was utilized for comparison with LJ1-01 NTL data in urban extent extraction. Data were downloaded from the NOAA National Geophysical Data Center (https://earthexplorer.usgs.gov/). Level-2 data products, also called surface reflectance products, provide an estimate of the surface spectral reflectance at a 30-m spatial resolution in the absence of atmospheric scattering or absorption [Landsat 8 Operational Land Imager (OLI) Level-2 data for the study area were obtained from the United States Geological Survey (USGS) website  and each image has a size of 3000 × 3000 pixels. In addition, since land-reflected moon light has not been excluded from the monthly composite products of VIIRS DNB data, it is necessary to remove background noise before use. In this research, all pixel values in VIIRS imagery were subtracted by 0.5 first, then pixels with negative DN values were assigned values of zero.To apply the HSI method developed by Lu et al. , the NDVThe Human Settlement Index, proposed by Lu et al. , is a meFirst, pixels with zero values were masked out and a natural logarithmic transformation was adopted to the LJ1-01 NTL images to maintain the rich details in the nighttime light. Then, the data was normalized as Equation (3):After that, regions with DN values greater than 0 in the NDWI image were regarded as water bodies, the mask was generated and the corresponding regions in the HSI image were removed. Lastly, multiple thresholds  were applied to extract urban areas from the HSI image.The processing of VIIRS DNB data was exactly the same as that of LJ1-01 data mentioned above. The logarithm transformation and normalization algorithm were adopted first, then the HSI was generated, with water bodies removed by NDWI. Lastly, multiple thresholds were applied to extract urban areas from the HSI image.In order to compare with the result of the HSI method in As mentioned in the Introduction section, thresholding-based, classification-based and index-based methods are the three most commonly used methods for urban extent mapping. Thus, in this research, the SVM supervised classification method was also adopted for comparison ,66. FirsIn order to compare the results using different datasets, NTL data was also utilized in this experiment for classification-based urban extent extraction. The resampled LJ1-01 and VIIRS nighttime light imagery was added to Landsat band 1~7 as an additional band respectively and the Landsat-LJ and Landsat-VIIRS composite data were generated. Then the SVM classifier was applied using the same training samples as above. Lastly, the extraction results went for accuracy assessment, which will be presented in A visual interpretation method was utilized to quantify the performance of different urban extent extraction methods presented in First, 800 randomly sampled points in the study area were generated. Then each point was labeled as “urban area” or “non-urban area” by visual interpretation using Google Map satellite images. After that, extracted urban areas obtained in The confusion matrix is a specific table that is often used to describe the accuracy of classification results . GeneralThe accuracy assessment for the performance of different methods are presented in In terms of PA, the HSI method using LJ1-01 and VIIRS data both maintained significantly high PA values when the thresholds were less than 0.7 and they also had similar change trends. In contrast, the PA values of the STS method using LJ1-01 and VIIRS data were relatively low and dropped sharply with increasing thresholds. In terms of UA, all the methods that used LJ1-01 data kept at a relatively high level and the SVM method using Landsat-LJ composite data had the best result. In the matter of OA, LJ1-01 data still had better performance than VIIRS data. The largest OA values for HSI, STS and SVM method when using LJ1-01 data were 0.9313, 0.915 and 0.9213 respectively, all larger than those of VIIRS data used ones.In this research, we compared several commonly used methods for extracting urban areas, including NTL based and non-NTL based methods. The experimental results demonstrated that the adding of NTL to Landsat data can improve the accuracy of urban extent extraction results.Urban areas shown in For a more intuitive comparison of NTL based and non-NTL based methods, urban extent extraction results with the largest KC values for HSI and STS method are presented in In this research, we also compared the performance of different NTL data in urban extent extraction. LJ1-01 NTL and VIIRS DNB images were utilized to contrast two main nighttime light data sources. According to the experiments and analysis above, we have concluded that LJ1-01 NTL data, with a spatial resolution of 130 m, contains more abundant spatial information of urban heterogeneity and can lead to a more accurate result when used in urban extent extraction. However, there are still some limitations in the application of LJ1-01 NTL data, which is worth more efforts in the future.https://eol.jsc.nasa.gov/), which is run by the National Aeronautics and Space Administration (NASA). Compared with other commonly used nighttime light data, the ISS imagery has several advantages, such as higher spatial resolution, three spectral bands in the visible range and variable overpass times [As a new nighttime light imagery source, the Luojia 1-01 satellite was launched in June 2018, which means that only images from June 2018 to now can be obtained for the time being. The lack of multi-temporal images makes LJ1-01 NTL data temporarily unable to be applied to long-term urban dynamic monitoring. However, if LJ1-01 NTL data can be integrated with other nighttime light data which has similar spatial resolution and long-time archived images, such as the International Space Station (ISS) imagery, this problem can be alleviated to a certain extent. The ISS imagery is a kind of multispectral photograph taken by astronauts in space using digital cameras, which has both daytime and nighttime archives . All thess times . It has In this research, we compared different methods of urban extent extraction in Wuhan city, including a Human Settlement Index using both LJ1-01 and VIIRS images, Simple Thresholding Segmentation using both LJ1-01 and VIIRS images and SVM supervised classification using Landsat and NTL composite data. The performance of different methods was assessed by reference samples from visual interpretation of Google Map satellite images. The results demonstrate that the HSI method using LJ1-01 NTL data had the best performance in urban extent extraction, which can not only accurately extract human settlements but also identify luminous bridges and roads at night. In addition, it presented the largest Kappa Coefficient value, 0.834, among all the results when the threshold was 0.65, while other metrics remained at a high level. The relevant findings of this research are as follows:(1) Compared with non-NTL methods, the addition of NTL data can largely improve the accuracy of the extraction results. For SVM classification method, the values of all accuracy assessment metrics have increased in varying degrees after adding LJ1-01 and VIIRS data respectively. By comparing the urban extent extraction results which have the largest KC values for different methods, we can find out that non-NTL based SVM classification method is likely to cause overestimation in urban suburbs, while NTL based methods exhibited better performance according to the accuracy assessment.(2) LJ1-01 NTL data, which contains more spatial details, can lead to better identification results than VIIRS DNB data in urban extent extraction. The urban areas extracted by the VIIRS based HSI method were fragmented with a large number of small patches, while the LJ1-01 based method identified urban land more accurately. The same finding can be obtained from the accuracy assessment. The largest KC value for the VIIRS based method was 0.772, while it increased to 0.834 for the LJ1-01 based method. As for the STS and SVM methods, the LJ1-01 NTL data also presented better performance compared with VIIRS DNB data.(3) LJ1-01, as a new generation of nighttime light imagery, has provided higher spatial resolution, wider radiometric measurement range and richer urban dynamic information over its predecessors. It displays a great potential for urban extent extraction at city scale and is bound to be more widely used in urban mapping. However, the current lack of multi-temporal images temporarily limits its application in urban dynamic monitoring. By integrating LJ1-01 with other high-resolution nighttime light imagery, we may find a solution to this problem. Moreover, the integration of high-resolution nighttime light imagery and other remote sensing data may provide a possibility to map urban extent in different periods and regions and then monitor urban dynamics continuously at the global scale in the future.


Botulinum toxin type A (BoNT-A) is generally considered safe and is widely used to treat a variety of clinical conditions involving muscle hyperactivity and for cosmetic purposes. However, the effects of BoNT-A poisoning (botulism) on brain function are poorly understood.Herein, we investigated brain functions in 9 patients who received illegal cosmetic injections of botulinum and 18 matched controls by combining the analysis methods of regional homogeneity (ReHo) and amplitude of low-frequency fluctuation (ALFF) based on resting-state fMRI. Compared with the controls, the patients with botulism exhibited significantly reduced ReHo values in the left posterior lobe of the cerebellum extending to the right anterior lobe of the cerebellum, as well as in the right anterior lobe of the cerebellum extending to the parahippocampal gyrus and right posterior lobe of the cerebellum. The patients with botulism also showed weakened ALFF values in the right anterior lobe of the cerebellum extending to the left anterior lobe of the cerebellum and right posterior lobe of the cerebellum, as well as in the right anterior lobe of the cerebellum.The results indicate that BoNT-A may modulate cerebral activation in specific areas, which may play roles in both the adverse effects of botulism and the mechanism underlying clinical treatment with BoNT-A. Botulinum toxin type A (BoNT-A) is widely used to treat a variety of clinical conditions characterized by excessive muscle contraction, including dystonia and spasticity , 2. BoNTIncreasing evidence supported by neuroimaging studies in patients indicates that central reorganization occurs following BoNT-A treatment. In previous fMRI studies in patients with focal spasticity, cortical changes occurred after BoNT-A injections, and brain (such as sensorimotor and cerebellar) activity was modulated by BoNT-A –16. MostAlthough BoNT-A is generally considered safe, it may cause serious adverse events after both therapeutic and cosmetic uses . AdverseHerein, we analyzed resting-state fMRI data to evaluate brain function in patients with botulism and controls by applying the regional homogeneity (ReHo) and amplitude of low-frequency fluctuation (ALFF) analysis methods. The ReHo method tests for local correlations in blood oxygen level-dependent (BOLD) time series using Kendall’s coefficient of concordance (KCC) to measure regional synchronizations of temporal changes in BOLD activity . ALFF chThe study was approved by the Independent Ethics Committee of Shanghai Ninth People’s Hospital (No. 2016-44-T1). All patients and healthy controls provided written informed consent using forms approved by the committee. All the investigations were conducted according to principles expressed in the Declaration of Helsinki.Nine female patients with BoNT-A poisoning (botulism) were recruited from the Department of Neurology at Shanghai Ninth People’s Hospital of the Shanghai Jiao Tong University School of Medicine from October 2016 to December 2016. An attending neurologist diagnosed these patients as having botulism after cosmetic BoNT-A injection based on their medical history, clinical manifestations, and various supplementary examinations. Patient demographic and clinical data are shown in All patients had botulism following cosmetic injections with an unlicensed, highly concentrated botulinum preparation. The patients received the BoNT-A injections in beauty shops. Eight patients were from Shanghai city, and one was from Nanjing city. Six subjects received BoNT-A in the bilateral gastrocnemius muscle for lean legs, 2 subjects received BoNT-A in the bilateral ocular region for facial wrinkles, and one subject received BoNT-A in the bilateral masseter muscle for bilateral masseter hypertrophy treatment. The toxin was purchased through illegal channels (such as smuggling) without a license. The concentrations of unlicensed BoNT-A preparations can be 9 times higher than that normally used in the clinic according to one test report of a similar case from a local health inspection office. However, all patients had normal blood oxygen saturation levels. Although some subjects had dyspnea, their blood gas analysis results were within the normal range, even in the most severe stage. The patients underwent MRI exams after significant relief of all their symptoms , and they had no dyspnea, anxiety, depressive symptoms, or other discomfort during the MRI scans. Patient details are provided in The healthy controls were recruited from the Shanghai population, and they were age- and gender-matched (1:2) to the botulism patients. Subjects who had experienced any neurological disorders or received a BoNT-A injection within the past five years were excluded. The healthy controls had a mean age of 29.4±5.5 years (same as the patient group). All subjects were right-handed and had no history of substance abuse. All neurological and psychiatric diseases were excluded based on a clinical examination and structured interviews.1-weighted, 3-dimensional, magnetization-prepared, rapid-acquisition, gradient-echo pulse sequence. The parameters were as follows: repetition time = 2530 ms, echo time = 2.34 ms, inversion time = 1100 ms, flip angle = 7°, number of slices = 192, sagittal orientation, field of view = 256×256 mm2, matrix size = 256×256, and slice thickness = 1 mm, with a 50% gap. Resting-state fMRI images were acquired using a T2*-weighted, gradient-echo, echo-planar imaging pulse sequence with the following parameters: repetition time = 2000 ms, echo time = 30 ms, flip angle = 90°, number of slices = 33, transverse orientation, field of view = 220 × 220 mm2, matrix size = 64 × 64, slice thickness = 3.5 mm, 25% distance factor, with 210 volumes in total. During the fMRI scan, the subjects were instructed to relax, remain still and close their eyes.Functional and structural MRI data were acquired using a 3.0 Tesla Siemens Trio Tim system  with a 12-channel head coil. All subjects’ head movements were minimized with custom-fit foam pads. We obtained the whole-brain anatomical volume using a high-resolution, Thttp://www.fil.ion.ucl.ac.uk/spm/software/spm12) and MATLAB software  on a personal computer. For each participant, the first 10 volumes were discarded to avoid scanner instability and to allow the participants to adapt to the scanner noise. The remaining volumes were corrected for the intravolume acquisition time delay using slice-timing correction and were realigned to the first volume using a six-parameter (rigid body) spatial transformation. After these corrections, we coregistered the high-resolution, T1-weighted image to the mean functional image. The T1 images were then segmented into gray matter, white matter and deformation field images. Next, the images were spatially normalized to the standard Montreal Neurological Institute (MNI) stereotaxic space and resampled to 3 × 3 × 3 mm3. Spurious signals, including the time series of six head motion parameters and signals from the white matter and cerebrospinal fluid, were regressed out using a general linear model, and linear trends were removed from the fMRI data. Finally, spatial smoothing was performed on the functional images using a Gaussian filter .Resting-state fMRI data preprocessing was conducted with statistical parametric mapping software . Participants were excluded if their maximum translation was > 2 mm or if their rotation was > 2° in any direction; no patients or controls exhibited excessive movement.We used Data Processing and Analysis of Brain Imaging (DPABI) v2.0 to conduct ReHo analysis based on unsmoothed data . A tempoAn individual ReHo map was generated by calculating the concordance of the KCC of the time series of a given voxel with those of its 26 nearest neighbors . To elimALFF analysis was conducted on the basis of preprocessed data. For a given voxel, the time series was transformed to the frequency domain using fast Fourier transforms, and the square root of the power spectrum was calculated and averaged across 0.01–0.1 Hz. This averaged square root was referred to as the ALFF. Finally, the ALFF value of each voxel was converted into a standardized z-score by subtracting the mean ALFF value and dividing the standard deviation of the whole-brain ALFF map such that the maps could be compared across subjects , 27.t-tests within a brain mask. To assess the ReHo and ALFF differences across the groups, significant differences in the ReHo and ALFF maps of the 9 patients with botulism and 18 controls were compared using voxelwise two-sample t-tests within a brain mask. Clusterwise familywise error (FWE) corrections were applied to control for multiple comparisons of ReHo and ALFF maps, with a voxelwise uncorrected threshold of p < 0.001 and a clusterwise corrected threshold of p < 0.02 [The ReHo and ALFF maps for each group were obtained by one-sample p < 0.02 . The surThe demographic and clinical data of the patients with botulism are presented in t-test. The difference between groups was revealed by a two-sample t-test  values in the cerebellar anterior and posterior lobes and in the parahippocampal gyrus, and these findings were consistent with the ALFF and ReHo results. The details of the fALFF method and results are presented in the et al. revealed that BoNT-A treatment resulted in partial restoration of connectivity abnormalities in the sensorimotor and primary visual networks in cervical dystonia patients [In humans, the cerebellum is involved in processing sensorimotor, affective and cognitive information –31. In apatients . These fet al. investigated localized brain activation changes in stroke patients treated with BoNT-A for upper-limb spasticity using fMRI and found that BoNT-A transiently lowered modified Ashworth scale scores, and additional clusters transiently emerged bilaterally in the cerebellum, contralesional sensorimotor cortex, and contralesional occipital cortex at 4 weeks after BoNT-A injection [In this study, the patients with botulism showed abnormal activation in the cerebellum, consistent with previous clinical research. For example, cerebellar activation was altered after BoNT-A treatment for upper-limb spasticity , 16. In njection . Our resWe also found abnormal spontaneous brain activity in the parahippocampal gyrus after BoNT-A poisoning. How BoNT-A affects the parahippocampal gyrus remains unclear. In chronic stroke patients suffering from severe hand paralysis with arm spasticity, task-related fMRI prior to BoNT-A treatment showed extensive activation of the bilateral frontoparietal sensorimotor cortical areas, anterior cingulate gyrus, pallidum, thalamus and cerebellum . The parThe fMRI scan was performed when the patients’ symptoms of muscle weakness had nearly disappeared; they participated in normal activities of daily living and did not have anxiety or depression because their symptoms had been significantly relieved and they knew that their symptoms were completely reversible. Therefore, we believe that mild residual muscle weakness would not have a significant impact on the fMRI results. Ultimately, we found that BoNT-A had an influence on spontaneous brain function in the subacute stage (21–64 days after injection of BoNT-A). It could be speculated that the brain function of patients would be more significantly affected in the earlier stage after injection of BoNT-A. Central effects of BoNT-A reported previously were obtained in patients with focal spasticity, and that kind of central effect could not be a direct effect of BoNT-A on the brain and must be secondary to the therapeutic effect on focal spasticity. However, we found a central effect of BoNT-A in previously healthy individuals, and our study supported the idea that BoNT-A could have a direct impact on brain function.Our research revealed abnormal spontaneous brain activity in patients with botulism who were previously healthy, but the study had several limitations. First, the sample size of patients with botulism was small, although patients come to our hospital  from all over the country. Second, this study was not a strictly designed experiment, and there were individual differences among the subjects, such as the injection position and the time from injection to fMRI scanning. However, in our study, all patients with botulism were young and previously healthy when they unexpectedly received a high dose of BoNT-A; thus, these patients offered a unique opportunity to investigate the direct effects of BoNT-A on brain function in healthy individuals. Although we demonstrated that the botulism patients showed abnormal spontaneous brain activity in the CNS, the central mechanism underlying this result was unclear. Because this study was preliminary, the results need further pharmacological and clinical validation.In this study, we determined that patients with botulism showed not only serious adverse effects on the peripheral neuromuscular junction but also abnormal spontaneous brain activity in the cerebellum and parahippocampal gyrus. Our results indicated that BoNT-A may directly modulate cerebral activation, which may be involved in both the adverse effects and the therapeutic mechanisms of BoNT-A. The findings have potential implications for the therapeutic use of botulinum toxin in complex indications in which clinical improvement cannot be explained by local muscle relaxation alone. This possibility especially applies to the emerging use of botulinum toxin in the treatment of mental disorders, such as depression.S1 Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.