In this study, we hypothesized that if the same deficit can be caused by damage to one or another part of a distributed neural system, then voxel-based analyses might miss critical lesion sites because preservation of each site will not be consistently associated with preserved function. The first part of our investigation used voxel-based multiple regression analyses of data from 359 right-handed stroke survivors to identify brain regions where lesion load is associated with picture naming abilities after factoring out variance related to object recognition, semantics and speech articulation so as to focus on deficits arising at the word retrieval level. A highly significant lesion-deficit relationship was identified in left temporal and frontal/premotor regions. Post-hoc analyses showed that damage to either of these sites caused the deficit of interest in less than half the affected patients (76/162 = 47%). After excluding all patients with damage to one or both of the identified regions, our second analysis revealed a new region, in the anterior part of the left putamen, which had not been previously detected because many patients had the deficit of interest after temporal or frontal damage that preserved the left putamen. The results illustrate how (i) false negative results arise when the same deficit can be caused by different lesion sites; (ii) some of the missed effects can be unveiled by adopting an iterative approach that systematically excludes patients with lesions to the areas identified in previous analyses, (iii) statistically significant voxel-based lesion-deficit mappings can be driven by a subset of patients; (iv) focal lesions to the identified regions are needed to determine whether the deficit of interest is the consequence of focal damage or much more extensive damage that includes the identified region; and, finally, (v) univariate voxel-based lesion-deficit mappings cannot, in isolation, be used to predict outcome in other patients. •Post-hoc analyses of results from voxel-based lesion-deficit analyses are reported.•The effects of different lesion sites cancel each other out when all data are pooled.•Removing patients with one lesion site can reveal other critical lesion sites.•Our findings highlight the need for multivariate lesion-deficit analyses. In the last decade and a half, the relationship between brain lesion data and behaviour has typically been assessed using mass-univariate techniques such as voxel-based morphometry VBM;  or voxelPrevious voxel-based lesion-deficit mapping studies have shown that the process of word retrieval depends on temporal regions , frontalTo illustrate how false negative results can arise in univariate voxel-based lesion-deficit analyses, we searched for brain regions where lesion load is associated with word retrieval abilities (as measured by spoken and written picture naming) in hundreds of stroke patients who, collectively, provided a rich sampling of focal or extensive damage to temporal and frontal areas. We then repeated the same analysis, after removing data from patients who had damage to regions identified in the previous step. This increased sensitivity to regions that are associated with the deficit of interest but were not detected in the first analysis because the absence of damage was only rarely associated with preserved function. In addition, we investigated (i) how consistently patients with damage to the identified regions presented with the deficit of interest and (ii) how the results of univariate voxel-based lesion-deficit mapping are influenced by the type of measure (either continuous or binary) used to quantify structural abnormality.Although we illustrate the utility of iteratively repeating the same lesion-deficit analysis, a full understanding of lesion-deficit relationships will require multivariate methods that take into account how the effect of damage to one region depends on that in another. Recent multivariate approaches to lesion-deficit mapping e.g.  include 22.13; (iii) native speakers of English; (iv) right-handed prior to their stroke; and, (v) more than 3 months since stroke. These criteria were met by 359 left-hemisphere stroke patients, aged between 21 and 90 (mean age 59.4). Patients were selected from the Predicting Language Outcome and Recovery After Stroke (PLORAS) database  accordin2.2All 359 patients were assessed with 27 tasks from the Comprehensive Aphasia Test CAT; . The totAs we were primarily interested in identifying brain regions where damage was associated with word retrieval deficits, we only focus on the results of 6 tasks. Two tasks tested the patients’ ability to retrieve and name pictures of objects in two different modalities; these were the spoken and written picture naming tasks with a word retrieval impairment expected to affect performance on both tasks. Additionally, in order to focus on word retrieval deficits, we included four tasks that tested the patient's ability to: (1) articulate (i.e. motor control of speech) using nonword repetition; (2) recognise, process and remember the semantic content of pictures and auditory words using semantic associations and spoken word comprehension; and, (3) control hand movements using letter and word copying. Task details were as follows:2.2.1The CAT spoken picture naming (Spk-PN) task visually presents 24 line drawing pictures of objects , one at a time, with instructions to name them aloud. Articulatory errors  not affecting the perceptual identity of the target were scored as correct. Verbal, phonemic, neologistic and apraxic errors were scored as incorrect. T-scores equal to or below 61 constitute the impaired range.2.2.2The CAT written picture naming (Writt-PN) task visually presents five pictures of objects , one at a time, with instructions to write their names down. Letters in the correct position were given a score of 1 each. Substitutions, omissions and transpositions were given a score of 0. One point was deducted from the total score if one or more letters were added to the target word. T-scores equal to or below 54 constitute the impaired range.2.2.3The CAT nonword repetition (Rep-N) task presents five nonsense words , one at a time, with instructions to repeat them aloud. Immediate correct responses were given a score of 2; incorrect responses were given a score of 0; correct responses after a self-correction or a delay (> 5 s) were given a score of 1. Articulatory errors  not affecting the perceptual identity of the target were scored as correct. Verbal, phonemic, neologistic and apraxic errors were scored as incorrect. T-scores equal to or below 52 constitute the impaired range.2.2.4The CAT writing copy (Writt-Copy) task visually presents letters and words that the participant is prompted to copy: five letters from upper to upper case and five letters from lower to upper case. Additionally, the patient is asked to copy three words using only capital letters. Correct responses were given a score of 1; incorrect responses were given a score of 0. T-scores equal to or below 51 constitute the impaired range.2.2.5The CAT spoken word comprehension (CSpok-W) task involves hearing a word produced by the examiner and selecting the picture among four possible alternatives that best matches the meaning of the heard word. There are a total of fifteen test trials plus a practice one at the beginning. The scoring system for this task was identical to that used in the nonword reading task. T-scores equal to or below 52 constitute the impaired range.2.2.6The CAT semantic associations (Sem-M) task visually presents five pictures of objects simultaneously on each trial. The instructions are to match the picture at the centre  with one of four possible alternatives according to the strongest semantic association . The inclusion of a semantically related distractor  encouraged deeper levels of semantic processing. There are a total of ten test trials plus a practice one at the beginning. Correct responses were given a score of 1; incorrect responses were given a score of 0. T-scores equal to or below 50 constitute the impaired range.2.3T1-weighted high resolution anatomical whole-brain volumes were available for all patients (n = 359). Four different MRI scanners  were used to acquire the structural images: 167 patients were imaged on a 3 T Trio scanner, 130 on a 1.5 T Sonata scanner, 57 on a 1.5 T Avanto scanner, and five on a 3 T Allegra scanner. Each of these T1-weighted images was then submitted to our fully automated lesion identification procedure for lesion detection and delineation (see below for details). This converts a scanner-sensitive raw image into a quantitative assessment of structural abnormality that should be independent of the scanner used. Additionally, the quality of the generated lesion images is evaluated by visually inspecting the results. Three types of lesion identification errors, which might differ from manually drawn lesions, have been detected. First, the lesion extent includes cerebrospinal fluid in enlarged ventricles. Second, cortical atrophy, e.g. around the dorsal parietal lobes can sometimes be included in the lesion image. Third, the automated approach can miss small cortical lesions where there is normal inter-subject variability in sulci. In addition, there are potential errors that arise in both automated and manually defined lesions, particularly in the specification of the border of the lesion which is typically gradual rather than categorical. We did not attempt to correct any of these errors  and they therefore increased “noise” in the analysis, which may have resulted in false negatives but did not result in false positives as confirmed by our post-hoc analyses. However, we did remove an artefact that was identified as a lesion (in the absence of damage) in the brain stem for several of the patients who were scanned on the 1.5 T Avanto scanner.For anatomical images acquired on the 1.5 T Avanto scanner, a 3D magnetization-prepared rapid acquisition gradient-echo (MPRAGE) sequence was used to acquire 176 sagittal slices with a matrix size of 256 × 224, yielding a final spatial resolution of 1 mm isotropic voxels (repetition time/echo time/inversion time = 2730/3.57/1000 ms). For anatomical images acquired on the other three scanners, an optimised 3D modified driven equilibrium Fourier transform (MDEFT) sequence was used to acquire 176 sagittal slices with a matrix size of 256 × 224, yielding a final spatial resolution of 1 mm isotropic voxels: repetition time/echo time/inversion time = 12.24/3.56/530 ms and 7.92/2.48/910 ms at 1.5 T and 3 T, respectively .U value (i.e. > 0.3) has been optimised to obtain the most accurate results from data collected on our scanners. The binary lesion images were also used to investigate how the results of our regression model changed with binary versus continuous (fuzzy) measures of structural abnormality; see All T1-weighted images were converted to 3D lesion images in standard MNI space as described in 2.4We used voxel-based morphometry VBM;  to assesThe most important advantage of utilising the fuzzy lesion images as in  over altIn Analysis 1 , the fuzzy (or binary) lesion images were entered into a multiple regression model with 6 different regressors  identified in Analysis 1. In Analysis 3, we repeated the same procedure again after excluding all patients who had more than 25% damage to the region(s) identified in Analyses 1 and 2. In other words, we continually (and deliberately) biased our patient selection to those who did not have damage to regions identified in previous steps. By definition, each iteration is conducted with smaller and smaller numbers of patients until no further voxels survive FWE-corrected p thresholds (estimated using random field theory as implemented in SPM). In addition, we performed a set of analyses on synthetically-generated null data sets to test the statistical robustness of the core principles upon which our iterative procedure was built. The simulations suggest that the procedure does not inflate the false positive rate. For a more detailed explanation and full disclosure of results, please see The search volume was limited to voxels that were damaged in at least five patients from the group being examined. For this purpose, lesion overlap maps based on the binary lesion images were created for each analysis see A, thresh33.1Using the fuzzy images, our deficit of interest was associated with greater lesion load in two spatially distinct clusters. The first cluster (280 voxels in size) was in the left temporal lobe extending from the posterior middle temporal gyrus into the arcuate fasciculus, temporal stem and anterior superior temporal gyrus. The second cluster (35 voxels in size) was centred on the left ventral premotor cortex extending into the opercular part of the inferior frontal gyrus. For brevity we refer to these regions, henceforth, as the temporal and frontal regions. When the analysis was replicated using the binary instead of the fuzzy lesion images, the same anatomical regions were identified , 2 , and 3  compared to Group 5 .Having assigned the patients to different groups, we compared the incidence and severity of our deficit of interest (written and spoken picture naming) across groups see . In termF(4353) = 15.70, p < 0.001). However, there were no significant differences (p = 1.000) in picture naming scores for those with frontal versus temporal lesions (Groups 1 versus 2).In terms of the severity, the mean spoken and written picture naming scores were significantly worse in patients who had substantial damage to either or both of the identified regions  relative to those who had relative sparing of both regions , even after accounting for the effect of lesion size , it was not the case that damage to each of the identified areas consistently caused the deficit of interest (i.e. lesion-to-deficit mapping). Indeed, there were less patients with, than without, the deficit of interest when only the temporal region or only the frontal region was damaged (Groups 1 and 2); see 3) that extended outside the borders of our temporal region. Likewise, in Group 1, the smallest lesion associated with the deficit of interest was 7.6 cm3 and included the inferior and middle frontal gyrus, and superior parts of the insula  had lesions which were much larger than the regions that reached statistical significance in the voxel-based analysis. We therefore have no evidence to suggest that focal damage to either the temporal or frontal regions (i.e. sparing other surrounding areas) is sufficient to cause the deficit of interest. For example, in Group 2 patients with the deficit of interest (i.e. impaired performance on both the spoken and written picture naming tasks) had very large lesions  that were partially or substantially damaged in 91% (89/98) of the patients with the deficit of interest. We also found that (i) the process of interest was not more or less affected by damage to the temporal region or the frontal region; (ii) damage to either region was associated with the deficit of interest in less than 50% of the patients; and (iii) the lesions in those with word finding difficulties extended beyond the boundaries of the temporal and frontal regions derived from Analysis 1. As our sample of 359 patients did not include any patients with selective damage to one of the identified regions (sparing surrounding areas), our data do not indicate whether focal lesions to either the temporal or frontal regions is sufficient to cause word finding difficulties. Nor have we explained why partial damage to one or both of our regions caused word finding difficulties in some patients but not in others (Group 4).3.2After removing 232 patients who had 26–100% damage to the temporal and/or frontal regions. Analysis 2 found a significant lesion-deficit association in the left anterior putamen  in 13/127 patients in Analysis 2 see , Table 3amus see . The poi3.3The third analysis did not yield any significant voxels  typically involves searching the whole brain for regions where there is a statistically significant association between damage and a deficit of interest in the patient sample being investigated. The current study illustrates some of the limitations of this approach by conducting post-hoc analyses that report (a) the incidence and severity of impairments in those with damage to the identified regions and (b) new regions that are detected when the voxel-based search is repeated after excluding all patients with damage to the regions identified in the preceding analysis. We have defined the deficit of interest as word finding difficulties, however, the point of the paper is not to describe the brain regions where damage causes word finding difficulties but to highlight the challenges in drawing such conclusions.Below we discuss how (i) the results of voxel-based lesion analyses are affected when the same deficit can be caused by different lesion sites (i.e. distributed processing); (ii) highly significant effects can be observed in regions where damage impairs the function of interest in only a small subset of the patients; (iii) damage limited to regions identified by voxel-based lesion analyses may not be sufficient to cause the deficit of interest; and (iv) the pros and cons of conducting voxel-based lesion analyses on binary (indexing the presence or absence of damage) compared to continuous measures of structural abnormality. Finally, (v) we consider the implications of these findings for future lesion-deficit studies.4.1Our voxel-based lesion-deficit mapping analyses identified three different lesions sites associated with the deficit of interest; Analysis 1 yielded two anatomically distinct lesion sites in frontal and temporal regions that have been reported in previous studies of word finding difficulties . Analysi4.2Our findings highlight that statistically significant lesion-deficit associations in group level studies can be driven by only a small subset of patients. For example, although Analysis 1 identified a highly significant relationship between the deficit of interest  and lesion load in two anatomically distinct regions in the frontal and temporal lobes, post-hoc analyses revealed that, from the total sample of 359 patients, only 68 subjects had substantial damage to the frontal region, 23 to the temporal region, and 71 to both the temporal and frontal regions. More importantly, when we calculated the number of patients with substantial damage to either of the regions, we observed that less than half of them  had the deficit of interest see . SimilarThese post-hoc observations allow us to make two points. First, they demonstrate that statistically significant lesion-deficit mappings derived from mass-univariate analyses can be driven by only a small subset of the patients. Second, they constrain the nature of the interpretation that can be drawn. Specifically, we cannot conclude that a region is “necessary” for the lost function when the majority of patients with damage to the region do not have the deficit. Even in those that do have the deficit, we cannot exclude  that the deficit arose because these patients had large lesions that damaged other areas that are needed to support recovery  or continuous measures of structural abnormality . The extent of the effects was, however, larger when we used the continuous rather than binary measures of brain damage, while controlling for all other factors; and, even though the same patient sample was included in the statistical analyses . This result suggests that images that encode the degree of abnormality on a continuous rather than binary scale e.g.  may incrIn the same vein, care needs to be taken when reporting the results of analyses that use continuous measures of structural integrity. This is because significant structure-function relationships can be identified in healthy (undamaged) tissue . This is particularly important to consider given the numerous VBM studies of the undamaged brain that have shown more grey or white matter volume or density in those with higher abilities e.g., . Again t4.5Finally, in this study, we have shown how iterative analyses along with post-hoc tests can help to understand some of the limitations of mass-univariate techniques. Our results showed that removing patients with damage to regions identified in previous analyses improved the sensitivity to additional lesion-deficit associations, which increases the likelihood of successfully detecting other possible lesion sites that might impair a given cognitive function. The essential contribution of the current paper is thus to reveal a problem, that of susceptibility to false negatives, which we have demonstrated by adopting an iterative mass-univariate approach. Determining the optimal way to resolve this issue awaits further work. In particular, it is likely that there will be circumstances where the iterative procedure presented here will not be practical to use, and multivariate methods such as machine learning  will be Another important implication of our results is that they highlight how very large samples of patients who have, collectively, incurred a wide range of focal lesions are needed if we are to identify and pinpoint all the brain regions that are critical to outcome and recovery after stroke. This challenge is not limited to univariate voxel-based analyses but also applies to multivariate techniques. It calls for international collaborations and data sharing. For the time being, however, the results from smaller samples, with detailed post-hoc analyses can be used to: (i) identify regions where damage may sometimes result in a functional impairment; (ii) report the consistency of these effects in the available sample; and (iii) dissociate lesion sites that have effects on different functions.Findings from the current study also indicate that the results from univariate voxel-based lesion-deficit mapping analyses can only be used to make inferences about outcome and recovery after stroke at the population level but cannot be utilised to make inferences at the individual level.5Overall, this study set out to show (i) how false negative results arise when the same deficit can be caused by different lesion sites and (ii) how false negative effects can be recovered using iterative voxel-based lesion analyses. In addition, our post-hoc tests have also demonstrated several other limitations of mass-univariate techniques including: (iii) statistically significant lesion-deficit mappings can be driven by a subset of patients; and (iv) the areas that reach statistical significance in voxel-based lesion-deficit mapping are much smaller than the true extent of the lesion causing the deficit. Although, it might be the case that only the significant part is causing the deficit, this needs to be demonstrated by identifying patients who only have damage to the critical part using a focal lesion approach. Without such a demonstration, we cannot exclude the possibility that voxel-based analyses are only revealing the tip of the iceberg.Finally, future studies using multivariate approaches such as machine learning will be useful to overcome some of the issues illustrated here. However, multivariate techniques will not be enough; explanatory models of neural networks and degenerated pathways are also necessary. A promising avenue of research will be the integration of results from lesion analyses with those from functional imaging and connectivity studies, which will allow us in the future to gain a better understanding of the relationships between structure, function, and outcome in the damaged brain.


The lingual frenum is a fold of mucous membrane connecting the ventral tongue to the floor of the mouth. In general, lingual frenum serves multiple roles; its main function is to support the tongue and aid in limiting its movement in different directions. Any anatomical or functional deficiency of lingual frenum may have an impact on tongue functions based on its severity. Historically, the absence of lingual frenum was linked to multiple genetic and developmental conditions such as infantile hypertrophic pyloric stenosis, non-syndromic ankyloglossia diseases, and Ehlers–Danlos syndromes and was never reported in otherwise healthy individuals.We report the absence of lingual frenum in an otherwise healthy 21-year-old Middle Eastern woman diagnosed during a routine dental examination.To the best of our knowledge, this is the first case to be reported in the literature with similar clinical presentation. Even without a significant impact on tongue movement or speech, it is important for health practitioners to be aware of such conditions and evaluation steps for diagnosis and management. Frenum is a general term frequently used to describe a fold of integument (skin) or mucous membrane that limits the movements of an organ or specific structure . SeveralSimilar to other structures in the human body, anomalies of LF  may have an impact on tongue function and are often linked to systemic, genetic, or developmental conditions . In someOther LF developmental anomalies have been reported in the literature and linked to other genetic conditions and syndromes, such as Ehlers–Danlos syndromes (EDS) and infantile hypertrophic pyloric stenosis (IHPS) , 10. EDSWe report a case of absent LF in an otherwise healthy individual with no associated medical conditions or syndromes.A 21-year-old Middle Eastern woman presented to King Abdulaziz University – Faculty of Dentistry, Jeddah, Saudi Arabia for a routine dental evaluation. Her medical history was significant for hypothyroidism secondary to thyroidectomy procedure performed 7 years ago to treat early thyroid papillary carcinoma. She received postoperative radioactive iodine as part of the treatment protocol. She had been taking thyroxin 100 mg/day since then to manage secondary hypothyroidism and had no significant allergy history. Her dental history was significant for active orthodontic treatment for the past 2 years.An extra-oral examination was noncontributory with no speech impairment. An intra-oral examination was significant for complete absence of LF with normal surrounding oral structures Fig. . In ordeThe human tongue is a muscular organ attached by multiple ligaments to the mandible, hyoid bone, styloid process, and pharynx . It origIn its complete developmental state, the LF is made of a band of connective tissue and covered with mucosa connecting the mid sublingual surface of the tongue to the floor of the mouth . It helpet al. suggested using absence of LF as an early sign to diagnose IHPS due to its high prevalence [IHPS is the most common condition requiring surgical intervention during the first weeks of life , 18. Theevalence , 23. In evalence .COL5A1 or COL5A2 gene), hypermobility (mutation in TNXB gene), vascular (mutations in COL3A1 gene), kyphoscoliosis (mutation in PLOD1 gene), dermatosparaxis (mutations in ADAMTS2 gene), and arthrochalasia type (mutations in COL1A1 or COL1A2 gene) [EDS is a heterogeneous, multi-organ disease that can be potentially life-threatening . The patA2 gene) . DiagnosA2 gene) , 26. HowA2 gene) .Table 1Tet al. reported a series of 12 patients with EDS compared to 154 non-syndromic patients  [et al. conducted a case-control study of patients with EDS (N = 43) matched with controls (N = 86) and included evaluation of their oral frenum [EDS affects the skin, bone, joints, and blood vessels as well as complete absence of LF . EDS clior IHPS) . As nonel frenum . Out of Clinical examination, including the oral cavity, is key in patients’ workup to reach the proper diagnosis and eventual management when suspecting IHPS or EDS. In the current case, detailed history and examination were obtained in order to rule out EDS and IHPS in the absence of LF. All clinical features for EDS or features suspicious for other syndromes were absent other than the absent LF. Considering that the diagnosis of EDS is based on clinical findings, the current case was diagnosed as absence of LF in an otherwise healthy non-syndromic individual. To the best of our knowledge, this is the first case to be reported in the literature with similar clinical presentation. Even without a significant impact on tongue movement or speech, it is important for health practitioners to be aware of such conditions and evaluation steps for diagnosis and management.Absence of LF is commonly associated with EDS and other congenital syndromes. However, this clinical finding could also be reported in otherwise healthy patients. We report a case of a patient with absent LF and no signs of EDS or other congenital or developmental diseases.


TM (ASQ-3) at 12 months. Among participants of (Japan Environment and Children’s Study) JECS, those with available data of cat and dog ownership at six months and data for the ASQ-3 at 12 months were included . Having dogs showed higher percentages of pass in all five domains measured by ASQ-3  compared to those who did not have dogs. Significantly decreased odds ratios (ORs) of developmental delays were observed in association with having dogs in all fix domains . This study suggested that early life dog ownership may reduce the risks of child developmental delays.Contact with companion animals has been suggested to have important roles in enhancing child development. However, studies focused on child development and pet ownership at a very early age are limited. The purpose of the current study was to investigate child development in relation to pet ownership at an early age in a nationwide prospective birth cohort study: the Japan Environment and Children’s Study. Associations between cat and dog ownership at six months and infant development at 12 months of age were examined in this study. Infant development was assessed using the Ages & Stages Questionnaires Childhood is a crucial phase in its contribution to the quality of health, emotional well-being, learning, and behavior across the life span . HoweverTM (ASQ-3) is a developmental screening tool widely used by clinicians, researchers, and intervention programs around the world [The Ages & Stages Questionnaireshe world . The ASQThe purpose of the current study was to investigate child development in relation to pet ownership at an early age. In this study, associations between cat and dog ownership at six months and infant development at 12 months of age were examined using the data from a nationwide prospective birth cohort study; the Japan Environment and Children’s Study (JECS). The JECS is an ongoing nationwide prospective birth cohort study in Japan. JECS study is conducted at 15 regional centers  in Japan. Details of the JECS project have been described elsewhere ,17,18. BThe JECS protocol was approved by the Ministry of the Environment’s Institutional Review Board on Epidemiological Studies and by the Ethics Committees of each participating institution    .Details of the self-administered questionnaire in this study have been described previously ,17. BrieThe ASQ-3 is comprised of 21 age-specific questionnaires for children ages 1 to 66 months to assess children’s progress in five developmental domains . Each of the 5 domains has 6 questions, resulting in 30 items for each age-interval. Each item describes a skill, ability, or behavior to which the parent responds “yes” (10 points), “sometimes” (5), or “not yet” (0). Parents sometimes omit items when they are unsure of how to respond or because they have concerns about their child’s performance of the item. The ASQ-3 scores were not calculated if there were three or more omitted items in a given domain. In the case of one or two omitted items, an adjusted total domain score was calculated by adding the averaged item score either once for one omission or twice for two omissions. Children who may potentially be at-risk for developmental delays at each age-interval are identified by comparing their scores to cutoff scores. The J-ASQ-3 cutoff scores for each domain were; communication = 15.64, gross motor = 21.49, fine motor = 34.50, problem-solving = 27.32, personal-social = 21.73 based on the original ASQ-3 . In thist-test was used to compare the mean value of maternal age, birth weight, and gestational age among those who had cats and dogs and those who did not. Logistic regression models were used to investigate infant developmental delays in association with having cats and dogs. The models were adjusted for maternal and paternal education, maternal smoking during pregnancy, annual household income during pregnancy, maternal mental illness, and duration of breastfeeding based on the previous literature and correlation between these variables and exposure and outcome. Since there were a small number of missing values for each variable, analyses included only those without missing variables. p < 0.05 was considered as statistically significant. Statistical analyses were performed using SPSS version 24 .ASQ-3 scores of each domain were dichotomized based on the cutoff scores of each domain. Chi-squared test was used to determine whether there was a significant difference in the frequency of pass/fail of ASQ-3 scores at 12 months of age each domain in having or not having cats and dogs at 6 months of age. n = 78,868) and comparison of characteristics between those who had cats and dogs. The pet ownership for cats and dogs at six months was 6358 (8.1%) and 11,934 (15.1%), respectively. Prevalence of maternal smoking at the 2nd trimester was higher in those who had cats and dogs. Both maternal and paternal education levels and annual household income were lower in those who had cats and dogs. Prevalence of possible maternal mental illness was higher in those who had cats but not dogs. Duration of breastfeeding was shorter in those who had cats and dogs. In this study, we found that having dogs at six months was associated with decreased risks of infant showing developmental delays at 12 months in five domains of the ASQ-3. However, the same was not observed among those who had cats. This was the first report on early childhood pet ownership and child development based on the large-scale prospective birth cohort study data.n = 318) as well as US national data [The mean scores of the ASQ-3 in this study were relatively lower compared to the recent cohort study  and those who were excluded from the current study due to no data of pet ownership (n = 860) found some significant differences (The strengths of this study are that we used a dataset of large sample size, longitudinal design, and included a variety of different factors. Health influence in association with pet ownership has been discussed in case-control studies and cross-sectional studies ,49; howeferences . Percentferences . This maSome limitations of this study should be noted. First, the child developmental delays were assessed by a parental report. Even though the ASQ-3 is one of the most widely used screening tools for early childhood development, misclassification may have occurred. We have controlled for possible maternal mental illness based on the validated Japanese version of the K6 score  at 12 moThis study suggested that having dogs in early life may possibly reduce the risks of children to have developmental delays. Further longitudinal follow-up studies are necessary to elucidate associations between early life pet ownership and child development.


Pneumonia is the leading infectious cause of death in children under 5 years of age around the globe. In addition to preventing pneumonia, there is a critical need to provide greater access to appropriate and effective treatment. Studies in Asia have evaluated the effectiveness of 3 days of oral amoxicillin for the treatment of fast-breathing pneumonia; however, further evidence is needed to determine if 3 days of oral amoxicillin is also effective for the treatment of chest indrawing pneumonia.This is a double-blind, randomized, non-inferiority trial with the objective to assess the effectiveness of shorter duration amoxicillin dispersible tablet (DT) treatment of chest indrawing childhood pneumonia in a malaria-endemic region of Malawi. The primary objective of this study is to determine whether 3 days of treatment with oral amoxicillin DT in HIV-uninfected Malawian children two to 59 months of age with chest indrawing pneumonia is as effective as 5 days of treatment. The study will enroll 2000 children presenting to Kamuzu Central or Bwaila District Hospitals in Lilongwe, Malawi. Each child will be randomized to either 3 days of amoxicillin DT followed by 2 days of placebo DT or 5 days of amoxicillin DT. Children in the study will be hospitalized for 48 h after enrollment and will have scheduled study visits at Days 2, 4, 6 and 14. Treatment failure by Day 6 is the primary outcome. We hypothesize that the rates of treatment failure will be similar in both arms and that 3 days of treatment will be non-inferior to 5 days of amoxicillin DT for chest indrawing pneumonia using a relative non-inferiority margin of 1.5. This trial was approved by the Western Institutional Review Board and Malawi College of Medicine Research and Ethics Committee.Given the paucity of data from Africa, African-based research is necessary to establish appropriate duration of treatment with amoxicillin DT for chest indrawing childhood pneumonia in malaria-endemic settings in the region. An expanded evidence base will contribute to future iterations of World Health Organization Integrated Management of Childhood Illness guidelines.NCT02678195: Pre-results. Date registered February 9, 2016.The online version of this article (10.1186/s12879-018-3379-z) contains supplementary material, which is available to authorized users. Pneumonia is the leading infectious cause of childhood mortality worldwide . EstimatThe primary objective of this prospective, double-blind, randomized controlled two-arm, non-inferiority trial is to determine whether or not treatment with 3 days of oral amoxicillin in HIV-uninfected children two to 59 months of age with chest indrawing pneumonia in a malaria-endemic region of Malawi is substantively less effective than 5 days of oral amoxicillin treatment. A non-inferiority design was chosen as there was a strong belief that placebo could not be expected to be more beneficial than amoxicillin with respect to the primary outcome of treatment failure by Day 6, but might be only slightly worse than amoxicillin. This study is conducted at Kamuzu Central Hospital (KCH) and Bwaila District Hospital (BDH) in Lilongwe, Malawi Fig. . A 750-bChildren aged two to 59 months with cough or difficulty breathing are recruited by trained hospital staff during routine intake and screening procedures in the hospitals’ outpatient departments, and referred to study staff for information on the study, written informed screening consent and additional screening to determine enrollment eligibility Fig. . ScreeniCaregivers of those eligible children meeting the case definition of chest indrawing pneumonia , 4, 6, and 14. Target dates for follow-up visits are calculated from Day 1, the date of randomization. All visits must occur on the calendar day on which they are initially scheduled or within 24 h afterwards, with the exception of the Day 14 visit, which can occur either 2 days before or after Day 14 and still be considered completed within the visit window. During follow-up visits, study staff review medical history and study drug adherence since the last study visit and perform a physical examination.On hospital discharge and during follow-up study visits, caregivers are given detailed instructions by study staff on timing and administration of the study drug as well as signs and symptoms that should prompt an immediate call to study staff. A 24/7 study phone number is provided to the caregiver. In case of a no-show at scheduled follow-up visits, children are followed-up with home visits by study staff. During the 14 days of follow-up, all children are followed for treatment failure or relapse signs and symptoms by study staff who collect medical history and perform physical exams and monitor study drug adherence at all scheduled and unscheduled visits. Chest radiographs are obtained at the discretion of study clinicians and the local principal investigator makes the final clinical interpretation. Most children failing treatment during Days 1 to 6 or presenting with relapse during Days 7 to 14 are hospitalized and treated with second-line intravenous antibiotics. Once a second-line antibiotic is administered, the child is considered non-adherent to the randomized treatment.For study drug adherence, completing 80% of all scheduled dose administrations is considered to meet treatment completion criteria . If doses are missed due to non-adherence, no study action is taken beyond documenting the missed doses and counseling the caregiver on adherence and study drug administration. If a child vomits within 30 min of a dose, one repeat dose is attempted. If a child vomits within 30 min after three or more scheduled  dose administrations, this is considered a treatment failure and that child is referred to care for a work-up of the vomiting cause and is prescribed a course of second-line antibiotics.The sample size calculation is based on the primary endpoint. Because the treatment failure rate in the 5 day amoxicillin group is anticipated to be within the range of 4% to 12%, a relative non-inferiority margin is chosen as 1.5 times the failure rate in the 5 day amoxicillin group. The specific non-inferiority margin, 50% higher failure rate in the 3 day amoxicillin group compared to the 5 day amoxicillin group, was chosen after extensive discussions among the investigators and with external experts regarding what failure rate might be acceptable to clinicians for the 3 day amoxicillin group compared to the 5 day amoxicillin group, considering the anticipated potential failure rate in the 5 day amoxicillin group and potential for enrollment into the study. Adjusting for two formal interim analyses  GCP E6, regulatory, sponsoring organization, and institutional requirements for the protection of children and confidentiality of personal and health information.2) < 93% in room air by pulse oximetry at baseline, treatment failure among those with moderate malnutrition (mid-upper arm circumference (MUAC) 11.5–13.5 cm), treatment failure among those with very fast breathing for age , and treatment failure by age group.The primary endpoint is the proportion of children failing treatment by Day 6  general and respiratory danger signs, severe acute malnutrition, and HIV infection or exposure. Safety monitoring for this study includes frequent clinical examination at study visits for the first 4 days, outcome assessment and a clinician on call via an emergency hotline for the first 14 days, treatment and tracking of all adverse events, and an external DSMB for review of cumulative safety and study conduct data.Caregivers may feel compelled to enroll in the study in order to receive care for their child within a research setting, which may be perceived as of a higher quality than the standard of care. In order to minimize the risk of coercion, study staff will not be recruiting participants directly. Instead, hospital clinicians will inform caregivers about the study and refer only those who are interested. During the informed consent process, study staff will emphasize that the child will receive medical care whether enrolled in the study or not. Another possible risk involves blood specimen sampling at screening which can cause pain and bruising at or around the blood draw site. To mitigate this risk, all study staff who will be collecting specimens from children in the study will be trained in the appropriate procedures and supervised accordingly. Participation in the study has the potential to compromise care for hospitalized children if study procedures are prioritized above urgent clinical care for acute infections. In order to minimize the possibility that participation in this trial will interfere with medical management, study staff will have the primary responsibility for the clinical management of hospitalized children in accordance with standard procedures. Furthermore, recognizing that some children may not come back for the follow-up visits, we provide for trained study staff to locate children who miss their follow-up appointments and conduct these visits in the home. We also ensure that study staff take the time to educate caregivers on the importance of adhering to the treatment regimen and follow-up.We plan to disseminate study results in peer-reviewed journals and international conferences, targeting those involved in the clinical care of children in low-resource settings as well as those who develop and advise on policies and guidelines in those settings. The trial is registered with ClinicalTrials.gov number NCT02678195.The following discussion outlines our efforts to safely and efficiently conduct a study that maintains the rigors of a double blind randomized controlled trial protocol with the goal of informative and generalizable results applicable to real world, non-study settings in African low-middle income countries.The study was carefully developed and pragmatically designed with inclusion and exclusion criteria to allow as generalizable results as possible without putting children with severe illness at risk. Children enrolled in this study are diagnosed with chest indrawing pneumonia based on WHO IMCI clinical guidance. While microbiological and/or radiological diagnosis may add improved specificity to the clinical diagnosis of pneumonia, the majority of low-resource settings do not have access to this testing, and children are typically diagnosed based on clinical criteria alone. Children with moderate malnutrition and malaria are included, whereas children with severe forms of acute malnutrition, anemia, malaria, or pneumonia are not. We are enrolling children with more severe pneumonia and/or underlying comorbidities  excluded from this chest indrawing pneumonia study into a parallel prospective observational study to provide additional evidence on the standard of care and outcomes for children with pneumonia, and to generate data on the generalizability of this chest indrawing study. Children in the observational study will receive treatment as per standard of care and will be followed by study staff with scheduled visits at Days 2 , 4, 6 and 14. Study staff will conduct a phone follow-up at Day 30 for a final outcome assessment.Placebo and amoxicillin DT identical in appearance, smell, taste, and dispersion properties are used to ensure blinding. Dedicated trained study staff care and follow children enrolled in the trial to assure the protocol and standard operating procedures are followed, data are accurately collected, and the highest level of safety provided. Standardized training, supervision, oversight, and testing are undertaken to ensure quality, consistency, and harmonized trial procedures and implementation. In addition to regular site monitoring visits by Save the Children, a CRO is employed to assist with close external monitoring of the study which includes regular site monitoring visits to assess compliance with human subjects and other research regulations and guidelines, adherence to the study protocol and procedures, quality and accuracy of data collected, and quality of care and child safety.Limitations to this study and potential sources of bias include study drug non-adherence and loss to follow-up. To minimize non-adherence to the study drug, demonstrations of study drug administration are conducted and caregivers are provided with clear verbal, written, and pictogram instructions. Caregivers are also asked about pill counts at every visit to assess their self-reported adherence to the study drug, any other medications they may be giving their child, or any other health care they have sought. To minimize loss to follow-up, caregivers are provided with clear follow-up instructions as well as called the afternoon before their visits to remind them to come the following day. A travel stipend for all follow-up visits is also provided. In addition, children are followed up with home visits upon a missed visit. A potential source of bias may be caregiver/child suspicion of study group (amoxicillin vs placebo). To mitigate caregiver unblinding, in addition to ensuring that the double-blind is maintained, the study drug is tested to confirm amoxicillin and placebo are identical.Other limitations to this study are the clinical diagnosis of pneumonia, rather than a microbiological or radiological one, and the limited follow-up duration of 14 days. All study staff receive rigorous training in the WHO IMCI classification of pneumonia; however, no microbiological or radiological tests are routinely undertaken unless clinically indicated. Of note, this was a pragmatic design decision as in standard clinical care in this setting, microbiological or radiological tests are not typically undertaken unless clinically indicated. In addition, it is possible that we are missing longer-term consequences post-treatment given the short period of follow-up of 14 days.Additional file 1:Appendix 3. Case report forms. (PDF 518 kb)Additional file 2:Appendix 1. Informed consent form for enrollment. (DOC 67 kb)Additional file 3:Appendix 2. Study protocol and statistical analysis plan. (DOC 636 kb)


Most smokers start smoking during their early adolescence, often with the idea that smoking is glamorous. Interventions that harness the broad availability of mobile phones as well as adolescents' interest in their appearance may be a novel way to improve school-based prevention. A recent study conducted in Germany showed promising results. However, the transfer to other cultural contexts, effects on different genders, and implementability remains unknown.In this observational study, we aimed to test the perception and implementability of facial-aging apps to prevent smoking in secondary schools in Brazil in accordance with the theory of planned behavior and with respect to different genders.We used a free facial-aging mobile phone app (“Smokerface”) in three Brazilian secondary schools via a novel method called mirroring. The students’ altered three-dimensional selfies on mobile phones or tablets and images were “mirrored” via a projector in front of their whole grade. Using an anonymous questionnaire, we then measured on a 5-point Likert scale the perceptions of the intervention among 306 Brazilian secondary school students of both genders in the seventh grade (average age 12.97 years). A second questionnaire captured perceptions of medical students who conducted the intervention and its conduction per protocol.The majority of students perceived the intervention as fun , claimed the intervention motivated them not to smoke , and stated that they learned new benefits of not smoking . Only a minority of students disagreed or fully disagreed that they learned new benefits of nonsmoking  or that they themselves were motivated not to smoke . All of the protocol was delivered by volunteer medical students.Our data indicate the potential for facial-aging interventions to reduce smoking prevalence in Brazilian secondary schools in accordance with the theory of planned behavior. Volunteer medical students enjoyed the intervention and are capable of complete implementation per protocol. Smoking is the leading global cause of preventable death, causing nearly 6 million deaths per year worldwide. A 2011 study of the tobacco-related burden in Brazil found that smoking was accountable for 147,072 deaths (403 deaths per day), 157,126 myocardial infarctions, and 63,753 cases of cancer. It generated 2.69 million disability-adjusted life years and cost the Brazilian health system US $7.37 billion in 2011 alone [Most smokers start smoking during their early adolescence, often with the idea that smoking is glamorous, with the associated health consequences too far in the future to imagine. According to the Adolescent Cardiovascular Risk Study, almost 600,000 adolescents smoke regularly in Brazil and most of them tried their first cigarette between 15 and 17 years of age .The earlier a person starts smoking, the higher the chance of becoming a regular smoker and developing associated diseases. As most smokers start smoking during early adolescence, it is imperative to develop, test, and validate tobacco control strategies that focus on this group through an age-appropriate and innovative approach. Most educational interventions for adolescents have focused on increasing awareness of tobacco-induced diseases . These mIn Brazil, a 2015 randomized controlled trial at the Federal University of the State of São Paulo investigating different school-based interventions to reduce the use of various psychotropic substances among 1316 students showed mixed effects for different drugs/settings with study design limitations precluding interpretation .Furthermore, a study on educational interventions among school adolescents analyzed the effectiveness of an educational program on smoking developed by the Brazilian Cancer Institute. The researchers selected 32 random schools from a total of 46 public schools in the city of Pelotas and randomized them to control and intervention schools. The total sample was 2200 students in the 7th and 8th grades (13-14 years old). They used questionnaires before and after interventions and collected urine samples in order to detect nicotine. Although the results showed no change in tobacco use reduction, they improved the students’ knowledge on passive smoking .Despite these studies, data on school-based tobacco prevention interventions conducted remain scarce.Founded in Germany in 2012, Education Against Tobacco is a global network of medical students that aims to provide science-based and age-appropriate preventions to a large number of adolescents and at the same time sensitizes prospective physicians to the importance of delivering smoking cessation advice and engaging themselves in tobacco control activities after their graduation -10. The , we introduced facial-aging mobile apps that alter a person’s selfie (a self-portrait taken with a mobile phone camera) to predict future appearance if that person smokes [In a recent papern smokes . These an smokes ,13. Theyn smokes ,15. In tn smokes  or to imn smokes . In addin smokes . Facial-n smokes ,19-24. In smokes .We recently implemented a facial-aging mobile app (“Smokerface”) in German secondary schools via a method called mirroring . We “mirThe self-concept of appearance, which photoaging interventions harness, is the strongest predictor of self-esteem in adolescents of both genders ,28. In tThis study investigated if effects are different for female/male participants and if the results of our novel facial-aging intervention are reproducible in Brazil, a country where data on tobacco prevention programs remain scarce. Additionally, a process evaluation investigated whether local volunteering medical students are capable of complete intervention implementation.We included a total sample of 306 students in Grade 7 in our cross-sectional study with an average age of 12.97 years  attending three regular public secondary schools in the city of Ponte Nova in southeast Brazil . Informed consent was obtained from the parents. A large majority of participants  reported that they owned a smartphone.The mirroring approach was implemented via local medical students from the Education Against Tobacco nonprofit organization who were attending the Federal University of Ouro Preto in Brazil -9. Two mThe mirroring intervention consists of a 45-minute app-based module in the classroom setting. Mirroring means that the student’s altered 3D selfies on their mobile phones or tablets are “mirrored” via a projector in front of the whole class, for example, sneezing or coughing . In fronIn the first 10 minutes, the displayed face of one student volunteer was used to show the app’s altering features to their peer group, providing an incentive for the rest of the class to try the app.In the following 15 minutes, students were encouraged to try the app on their own device or one of the tablet computers provided for students not owning a mobile phone or without the app. The number of provided tablet computers was calculated so the phase would take up to 12 minutes at the most, factoring in a utilization time of about 4 minutes per student. By this calculation, 25 minutes of the mirroring intervention and 10 provided tablets were sufficient to have every student within a grade of 40 pupils successfully photoaged at least once.This was followed by a 15-minute interactive discussion of the remaining functions of the app: facial changes, quitting via the free Smokerstop app, and impaired growth, strength, and sagginess of women’s breasts. These topics are strictly in line with the explanatory graphics within the app  and 4.In the last 5 minutes of the time in the classroom, the perception of the intervention by students was measured directly after the intervention via 10 items in an anonymous survey on a 5-point Likert scale: (1) one item on change of intentions (“My 3D selfie motivates me not to smoke”), (2) two items on the perceived reactions of the peer group (“My classmates think I look better as a non-smoker” and “The reactions of my classmates motivate me not to smoke”), (3) three items on future app-use and app-sharing , (4) four items addressing global feedback .The medical students filled out a brief process evaluation consisting of six items capturing the complete implementation of the intervention as well as how the medical students perceived its effectiveness when in class.All data were analyzed and illustrated in regards to overall perceptions of the intervention within the whole sample  but alsoWe measured 94.4% (289/306) agreement on the item measuring the increase of motivation not to smoke: 94.4% agreed/fully agreed that their 3D selfie motivates them not to smoke while only 1.6% (5/306) disagreed or strongly disagreed and 4% were not sure . These rThe two items measuring the reactions of the peer group towards the individual selfie showed positive peer pressure to become or to remain a nonsmoker. The majority of students agreed/totally agreed that their classmates prefer them as nonsmokers  and that their classmates’ reaction to the 3D selfie motivates them not to smoke  . The resWe measured more than 70% agreement in all three items measuring intention to reuse or share the Smokerface app. The majority of the students expressed a desire to show the app to other people , would like to have the app on their mobile phones , and planned to try the app on themselves again later on . These results did not vary notably in males versus females.Almost all participants expressed that they perceived the intervention as fun: 99.3% (304/306) agreement, 0.0% (0/306) disagreement, and 0.7% (2/306) neutral . Almost Our process evaluation conducted among all of the six volunteering medical students via a short questionnaire after every classroom visit revealed that 100% of the secondary school students received the mirroring intervention as outlined in the methods section. All of the medical students were able to have empathic communication with the students, regarded the intervention as enjoyable, and said it motivated them to deliver smoking cessation advice to future patients.Mobile apps are used, evaluated, and optimized in smoking cessation settings -52 whileThe theoretical background of the participant-centered mirroring intervention includes increasing perceived self-efficacy of using the app, which has been proven to encourage repetitive use and is associated with the effectiveness of an intervention according to the theory of planned behavior . AccordiThe theory of planned behavior identifies perceived behavioral control as the strongest predictor of smoking onset . To this end, an age-appropriate reason not to smoke was integrated into the student community by both the name of the app, “Smokerface”, and the fact that it was installed on most students’ devices. A majority (89.8%) of the students stated that the app was an appropriate tool to convince peers to quit smoking when asked after the intervention. Also, many students would refer to smokers as “smokerfaces” or stated that they did not want to be a “smokerface,” which is an age-appropriate reason to decline a cigarette if offered by a peer.Both genders agreed in most categories, which is consistent with recent literature suggesting that appearance aspects play a major role for self-esteem in male as well as in female adolescents. While females tend to be more susceptible to appearance aspects in the past, the differences between the two sexes appear to assimilate ,54,55.Still, in this study a larger fraction of female participants agreed that the Smokerface app motivates other people to quit smoking . Furthermore, handing out the questionnaires after the intervention rather than before might have provoked a social desirability bias despite anonymity. In addition, cross-sectional data without a control group or follow-up cannot determine effectiveness. Thus, the authors plan to conduct a randomized trial .The facial-aging intervention was effective in generating an increased motivation to stay away from tobacco in Brazilian adolescents. The predictors measured indicated an even higher prospective effectiveness in southeast Brazil than in Germany (over 90% of agreement in Brazil vs over 60% of agreement in Germany on the items that measured motivation to remain abstinent) in accordance with the theory of planned behavior. Medical students are capable of complete implementation of the intervention. A randomized controlled trial measuring prospective effects in Brazil is planned as a result of this study .


Guidelines dietary pattern scores at 6-months  and 100% reduction of decayed surfaces with increased intakes of Guidelines dietary pattern foods from 6 to 12-month . Suggesting that following the Guideline dietary pattern, which corresponds most closely to current World Health Organization weaning guidelines, at 6 months and an increase in pattern score between 6 and 12 months were protective against ECC development compared to Predominantly breastmilk, Easy-to-prepare foods and Noodles (in soup) and seafood dietary patterns.Dental caries, although preventable, remains one of the most prevalent chronic disease worldwide. Most studies focused on the relationship between sugar intake and caries. However, examining multidimensional dietary patterns is becoming increasingly important. Here, we examined the relationship between dietary patterns from ages 6 to 12 months and early childhood caries (ECC) at age 2 to 3-years. Infant dietary data was collected from caregivers and dietary pattern trajectories from 6 to 12 months derived. Oral examinations were carried out by trained calibrated dentists at ages 2 and 3 years. Associations between dietary pattern and ECC were estimated using generalized estimating equation. We found a 3.9 fold lower prevalence of decayed surfaces among children with high  Among American children 2 to 11 years of age, 42% experienced caries in their primary dentition, making it the most common chronic disease in children with signs that caries prevalence is increasing after decades of decline2. Dental caries is a dynamic process between protective and pathological factors with the major pathological pathway being the bacterial breakdown of fermentable dietary carbohydrates modified by the protective effects of salivary flow and fluoride intake3.Despite being a largely preventable disease, dental caries remains one of the most prevalent chronic disease worldwide4. Despite the seemingly large number of studies examining the relationship between sugars and dental caries, only 4 out of the 55 studies included in the review looked specifically at the effect of dietary sugars in young children less than 3 years old7, with only 1 study examining children under age 1 year8. These studies show conflicting findings, with some showing a positive relationship8 and one showing no relationship between dietary sugars and dental caries in young children6. The impact of early prevention cannot be understated as it has been found that children with caries in their primary teeth are 3 times more likely to have caries in their permanent teeth as adults9.The notion that dietary sugar is a major factor in developing early childhood caries is well-known10. It is possible to miss out on the effects of and interaction with other potentially contributory groups, when examining only single food group or nutrient for its impact on a disease.Individuals do not consume nutrients or foods in isolation and investigating dietary patterns had the advantage of examining the overall diet which include the foods, food group, and nutrients; their combination and variety; and the frequency and quantity which they are habitually consumed11. These patterns such as the Guidelines dietary pattern, and the Western dietary pattern have been associated with childhood and adolescent intelligent quotient13, and obesity14 respectively. The Guidelines dietary pattern corresponds most closely to current established weaning guidelines recommended by the World Health Organization15 and have been described in several studies across different populations19. The Guidelines dietary pattern is characterized by high consumption of rice porridge, fish and meat, fresh fruit and vegetables as core food items throughout the 6 to 12 months period17. Dietary patterns, a representation of the overall food intake pattern, may be associated with the risk of developing caries, although there are currently no known studies which have examined the relationship between dietary patterns and examining their impact on early childhood caries (ECC).Dietary patterns from 6 to 24 months of age are strongly influenced by mothers and other primary caregivers20. This could in part be attributed to cultural differences in infant caregiving, which is often based on tradition rather than recommended guidelines. For example, a third of infants in a previous study had food added to their bottles and more than a third had sweetened beverages via a bottle, practices that increases frequency of exposure to fermentable carbohydrates17.Singapore is a developed country which has 100% water fluoridation and a multi-ethnic Asian population. Although dental caries prevalence is comparable to other developed countries at 12 years old, ECC is disproportionally high at 40% among Singaporean preschool children21. This study is based on the Growing Up in Singapore Towards healthy Outcomes (GUSTO) cohort which is a prospective mother-offspring cohort study that collected longitudinal data on the offspring from birth22 until early childhood (6 years old). This study aims to investigate the relationship between infant dietary patterns at ages 6 months to 1 year and ECC in 2 to 3-year-old children, with the hypothesis that the different dietary patterns of the infants at 6 months to 1 year results in a difference in severity of ECC at 2 to 3 years.With that in mind, examining dietary pattern in young children may provide more information on feeding habits and the effect of dietary factors on the risk of early childhood caries than the examination of sugar intake aloneThe GUSTO cohort is a mother-offspring cohort study that recruited over 1200 pregnant women aged 18–50 years delivering at the National University Hospital (NUH) and KK Women’s and Children’s Hospital (KKH) between June 2009 and September 2010. The participants were of self-identified homogenous ethnic background representing the 3 major ethnicities of Singapore: Chinese, Malay and Indian. Subjects were followed very closely from birth with detailed observations and samples collected to provide insights into the management and prevention of important diseases. The study received ethical approval from the institutional review boards of the National Healthcare Group and Singapore Health services. (DSRB reference D/09/021)(CIRB reference 2009/280/D) Informed consent was obtained from all participants and their legal guardians. Additionally, all experiments were performed in accordance with relevant guidelines and regulations.This is a cohort study utilizing the GUSTO data where the various domains of the GUSTO study examined nested groups for different health parameters including nutrition and oral health. Demographics such as household income, maternal age, maternal education, and birthweight were recorded at the initial recruitment visit.23. Data from either the 24-hr recalls or 1-day record from food diaries were used for dietary analyses to increase the sample size. A correlation (r ranging from 0.123 to 0.820) of the 1-day record with the 2 other days using a subset of infants with complete 3-day food diaries has been previously established21.Infant diet intake data was collected from mothers or caregivers by mailing 3-day food diaries  prior to their 6-, 9-, and 12-months postnatal visits. In cases where diaries were returned incomplete, a 24-hr recall interview by trained personnel was collected during the visit using a 5-stage, multiple-pass interviewing technique10, was used to categorize the foods consumed locally. Among the food groups, the confectionary and sugar sweetened beverages (SSB) groups were selected for analyses in this study. The confectionary food group consist of food such as chocolates, sweets, ice-cream, puddings and jellies while SSB consists of fruit drinks, carbonated soft drinks, sweetened soya milk, traditional drinks and other sweetened drinks like honey mixed with water.Each food item from the records was then assigned to one of the 72 sub-food groups within the 18 food groups based on type of food or similarities on nutrient content, conceptually similar to previous studieset al.12. Further details can be found in Lim et al.21. Briefly, dietary trajectories were empirically constructed by mapping dietary patterns extracted by exploratory factor analysis at 6-, 9- and 12-months. The dietary patterns across the three time-points were examined for their similarity in the types of foods and their loadings to ascertain their suitability to be modelled as trajectories. The mapping of each pattern to a trajectory was based on similar key constituent foods (with high loadings) found in the dietary patterns across the three time-points and this correspondingly determined the name of each trajectory. In this study, 4 dietary pattern trajectories observed in the participants namely: 1) Predominantly breastmilk, 2) Guidelines, 3) Easy-to-prepare foods and 4) Noodles (in soup) and seafood21.Dietary pattern trajectories were calculated using multi-level mixed models generating intercepts and gradients of various patterns as described by Smithers Guidelines dietary pattern intercept score indicates close adherence to the Guidelines dietary pattern at 6 months of age, while a high Guidelines dietary pattern gradient score indicate continued high adherence to the Guidelines dietary pattern from 6 to 12 months of age.The multi-level mixed model generates estimates of intercepts and gradients. The intercept reflects the trajectory score at the start point of each pattern (6-months of age), while the gradient denotes the rate of change in trajectory scores over time from 6-months to 12 months of age. For example, a high Predominantly breastmilk pattern, had more breastmilk than formula milk with feeding of fresh fruits at 9-months, and consumption of bean-curd, ethnic breads and starchy vegetables at 12-months. The Guidelines dietary pattern had high consumption of rice porridge, fish and meat, fresh fruit and vegetables, and followed the currently recommended WHO weaning guidelines16. The infants who followed the Easy-to-prepare pattern had foods such as infant cereals, juices, cakes and biscuits. The Noodles (in soup) and seafood pattern was a uniquely Asian pattern that was characterized by Asian adult foods such as noodles accompanied by eggs, seafood, bean curd and dried preserved fruits21.Infants following the 24. The participants were examined in the supine position using the knee-to-knee technique. Artificial illumination was used and tooth surfaces examined with sterile mouth mirror and blunt explorer. The tooth status was assessed by visual inspection, aided by tactile inspection if necessary after cleaning and drying of the surfaces with sterile gauze25. No radiographs were taken. Potential confounding factors in oral hygiene habits such as night-time bottle feeding, brushing frequency, and fluoride exposure were recorded as a self-administered questionnaire at the age 2 and age 3 oral examination.Oral examinations were carried out by 3 trained calibrated dental professionals at ages 2 and 3 years  using the modified International Caries Detection and Assessment System (ICDAS) criteriaAs there is a lag time for the influence of dietary patterns to manifest into examinable changes in teeth, caries status at 2 to 3-years were examined for association with their dietary patterns profiled at 6 to 12-months.The primary outcome, ECC, was coded in 3 ways and analysed independently as a binary variable (present/absent), number of decayed teeth (dt) and number of decayed surfaces (ds) with white spot/cavitation lesions  II codes 2–6). An initial univariate analysis of food groups most likely to be associated with ECC, namely sugar confectionary and sugar sweetened beverages (SSB) was conducted using a logistic regression. The amount of sugar consumption was estimated from the diet diaries. Furthermore, marginal associations of dietary pattern trajectories with early childhood caries at ages 2 and 3 years were estimated using generalized estimating equation (GEE) with exchangeable working correlation matrix. Negative binomial family distribution with log link function was used. Incidence rate ratio (IRR) for each variable in the model was estimated with the use of Huber-White sandwich robust estimates for standard errors. Interactions between age and dietary trajectories were not included in the models as they were not statistical significant  as shown by Wald test, indicating similar effects of dietary trajectories on ECC at 2 and 3 years. Confounding factors found in the literature such as socio-demographic characteristics, oral hygiene habits, perinatal and postnatal characteristics were adjusted in the analyses.Significance level was set at p-value < 0.05. Statistical software STATA/SE Version 14  was used to carried out all statistical analysis.In total, 776 children, out of the 1,287 children from GUSTO cohort had oral examinations at either 2 or 3 years old. Oral examinations were conducted on 535 children at age 2 years and 721 children at age 3 years. Unweighted prevalence of caries among children aged 24 and 36 months were noted at 17.8% and 42.9%, respectively. There were 363 children (46.8%) with dental examinations who also had infant feeding pattern data collected at the 6, 9, and 12 month postnatal visits . This was supported by the other 2 methods of coding the outcome variable ECC when examined independently. The After adjustment, significantly lower number of decayed tooth surfaces (ds) were found in Indian participants  compared to Chinese and Malay participants. Controlling for ethnicity had little effects on the associations between a Guideline pattern and the dental outcomes. Additionally, ds was found to be higher in subjects who used fluoride toothpaste at 3 years  when compared to subjects who did not.  with an incidence risk ratio (IRR) of 0.26. This translates to an increase of ds by 3.9 folds per unit decrease in Guidelines dietary pattern intercept score. Increased intake of Guidelines dietary pattern food from 6 to 12 months (gradient) was also found to be associated with a lower ds , with an almost 100% reduction in ds. There were no significant associations of the scores of Predominantly breastmilk pattern , Easy-to-prepare pattern , and Noodles and seafood pattern  with the ds . Children with a high Guidelines dietary pattern score at age 6 month and among children who continue to adhere to Guidelines dietary pattern foods between ages 6 and 12 months which is further supported by significantly lower chance of developing caries and having lower number of decayed teeth. No association was observed with the other dietary patterns. This suggests a very strong protective effect of having a high Guidelines dietary pattern trajectory on developing ECC.The GUSTO cohort has provided an opportunity to examine the relationship between multi-ethnic dietary patterns and the caries status of young Asian children. Key findings of this study include significantly lower decayed surfaces observed among children with high 6. Additionally, the amount given can also be very variable from child to child as seen from the large standard deviation of SSB consumption. In this situation, examining the dietary pattern as a whole can provide a better indicator for risk of developing ECC at a later age instead of focusing on 1 or 2 food groups.The study did not find very strong evidence for traditional paradigm of eating sugary foods causing tooth decay. A reason could be due to the early dietary data time points of less than age 12 months when children at that age not being likely to be given large amounts of dietary sugar. This could be seen from the low amount per day of sugary confectionary (<3 g a day). This is in line with one other study examining the diet of children below 1 yearGuidelines dietary pattern trajectory is characterized by increased exposure of the infant to a variety of food groups including fruits, vegetables, whole grains, poultry and fish28. Infants on a healthy “Guidelines” diet are also likely to be the ones consuming low sugary snacks in between meals such as fruits, vegetables, grains, dairy products. These options may replace sugary snack foods such as SSBs, confectionary which are known risk factors for ECC. Accordingly, Nunn et al. found that children aged 2 to 5 years with good eating practices as measured by the Healthy Eating Index (adherence to the Food Guide Pyramid) are less likely to have severe ECC29.The 30, indicating that dentists and dietitians should collaborate and provide advice for healthy diets and healthy teeth. Dietary changes are more difficult once habits are established, suggesting the importance of encouraging caregivers to adhere better to established feeding guidelines early21. Moreover, maternal dietary pattern is a strong predictor of infant dietary patterns11. This suggests that the target of change should be focused on the primary caregiver and family much earlier than the current practice. By understanding the factors that influence dietary patterns and sugar consumption, more targeted and culturally-appropriate support and advice can be provided by health professionals and policy-makers to help improve dietary patterns as early as during pre-pregnancy counselling31. In addition to oral health promotion, this holistic dietary counselling can improve overall health and wellbeing by impacting obesity and other associated diseases14.There appears to be a strong overlap between the foods that are good for general health and the foods that are good for dental health32. With 16% of the population accounting for 78% of the disease in Singapore20, this means that sometimes resources may not be directed at the areas where they can have the biggest impact. Using dietary pattern analysis as described presently, it can serve as a component in early identification of dietary risk contribution to better direct preventive efforts. This may improve reliability and validity over the current single food group analyses4.One of the factors affecting the effectiveness of caries prevention programs have been the early identification of children at high risk of ECC25. Interestingly, the use of fluoride toothpaste at 3 year old was associated with an increase in ECC. Similar to our previous report, this may be a result of an increased use of fluoride toothpaste due to recommendations from dentists after the child had caries in the first place25, a case of reverse causality.As expected, the caries rate was higher at 3 years old when compared to at 2 years old as it is a chronic disease with a cumulative prevalence. The finding that Indian participants had lower rates of caries was similar to a previous study on a larger sample of the GUSTO cohort33; however, the data was longitudinal in nature with good reproducibility in the records, mitigating the possible bias. Strengths of this study include the capture of important time points in the infant dietary pattern and their association with ECC in a large multi-ethnic Asian population cohort. Moreover, the longitudinal nature of the study allows for the influence of dietary patterns, profiled at 6 to 12-months, to manifest into examinable changes in teeth at 2 to 3-years. Additionally, important differences between the different cultures and ethnicities can be examined in a largely homogenous social structure.Acknowledged limitation of our study is the self-reported dietary data that may introduce recall bias or social desirability biasGuidelines dietary pattern at 6 months and an increase in Guidelines dietary pattern score between 6 and 12 months were protective for the development of ECC compared to Predominantly breastmilk, Easy-to-prepare foods and Noodles (in soup) and seafood dietary patterns. Currently, there are no data on the early effects of dietary pattern on caries status in children. Our findings will aid development of a more holistic and earlier targeting dietary guidelines and policies for caries prevention alongside general health as the overall target. This may paradigm-shift our age-old adage of “don’t eat sweets” to “eat healthy food” to protect children’s teeth from cavities.In conclusion, this is the first mother-offspring cohort study that investigated and found an association between dietary patterns and ECC. Following the Supplementary MaterialSupplemental Methods