Moreover, BP achieved a higher impulsiveness score and more frequently had experienced severe problems with close individuals. To our knowledge, this is the first publication presenting results of numerous questionnaires applied simultaneously in patients on clinical group. Several of them suggest the direction of clinical assessment, such as: the age of onset, family psychiatric burdens, history of stressful life events, learning problems, social and job relations. Further studies are necessary to confirm the utility of this approach.Due to current depression prevalence, it is crucial to make the correct diagnosis as soon as possible. The study aimed to identify commonly available, easy to apply, and quick to interpret tools allowing for a differential diagnosis between unipolar and bipolar disorder. The study group includes women with long duration of unipolar  and bipolar  affective disorder. The diagnosis was established according to the DSM criteria using SCID questionnaire. Additional questionnaires were used to differentiate between UP and BP. BP patients had an earlier age of onset, were hospitalized more times, and more often had a family history of psychiatric disorders than UP ( Depressive episodes are a part of both unipolar (UP) and bipolar (BP) affective disorder. About 4.4% of the global population suffers from UP . Major dBipolar disorder has a highly negative impact on a patient’s life. It is evident in many spheres of life, such as relationships with family and friends, employment and difficulty in work, leisure activities, and the quality of life ,4,5. A lMany authors aimed to find the differences in the characteristic of an episode of depression in unipolar and bipolar disorder, such as an earlier age of onset , a more This study aims to identify early markers of the type of depression among biochemical and clinical features readily available to psychiatrists, derived from medical observation and interview. The picture of depression includes both disturbances in the stress axis and disruptions in the circadian rhythm. Therefore, the self-report questionnaires on the chronotype and stress factors preceding the episode were included in the analysis. Compliance with the psychiatrist is essential for controlling the progress of the disease. Thus, the personality traits, especially a high impulsivity level, were also assessed, as it may hinder obtaining therapeutic success. We demonstrated here for the first time the use of numerous questionnaires conducted on one clinical group for better differential diagnosis: The Barratt Impulsiveness Scale, The Coping Orientation to Problems Experienced, Morningness-Evenigness Questionnaires, The Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale, Brief Life Events Questionnaire. Previous studies have used only two or three questionnaires for the same study group. Single tools may not be effective enough or be useful specifically in the sample. The availability of more results for one study group may shed light on the misdiagnosis problem. Only women were included in the study to avoid differences between gender, and because female patients are more prone to experience depressive episodes, especially at the onset of affective disorder, and the severity of symptoms requires more frequent hospitalization ,14,15,16The study group (SG) included 79 women admitted to the hospital due to current depressive episode, aged 18–76 (mean = 42.23 ± 15.06), with a diagnosis of bipolar (N = 43) or unipolar disorder (N = 36) based on DSM-IV criteria. The lifetime diagnosis was established by two psychiatrists based on SCID-I  ,18. The The control group (CG) included 69 healthy women volunteers, aged 22–63 (mean = 42.54 ± 11.25). CG completed the same five self-questionnaires as SG  to facilitate the recruitment. The exclusion criteria were: more than 10 points obtained in BDI, more than 14 in ESS, shifts workers, and a positive personal history of psychiatric symptoms. Blood samples were collected from the control group and patients (in pre- and post-treatment state), after which basic blood tests  were performed.The study was approved by the Bioethics Committee of Poznan University Medical Sciences (decision 758/17 22 June 2017). All study participants were Caucasians of Polish origin and gave written informed consent.p < 0.05 for all analyses. Statistical calculations were made using the STATISTICA 13.3 .The distribution of variables was studied by the Shapiro–Wilk test. Variables with normal distribution were tested using parametric tests: student’s t-test for comparing two groups and ANOVA for several groups. If the results have shown statistically significant differences, Tukey’s post-hock test was used. For variables that did not meet the criteria of a normal distribution for statistical comparisons, non-parametric tests were used between the examined groups: Mann–Whitney U test distributions for two independent groups, Wilcoxon pair order test for two dependent groups, and ANOVA rank Kruskal–Wallis test for several independent groups. Discrete measures were assessed with the chi-square test. To assess the relationship between the analyzed variables, Spearman’s rank correlation coefficient was applied. Two-sided comparison of two means test was used to compare literature data with our results . The significance level was set at Several sociodemographic data, including age, education years, and marital status, were collected from the study and control group . There wPatient’s medical history data, such as the age of onset, duration of hospitalization in the present episode, and the number of hospitalization, were compared between the UP and BP group . Patientp- and z-value < 0.05). There were no statistically significant differences in the following scales: HAMD, MADRS, YMRS, and BDI, between the diagnostic UP vs. BP group in pre- and post-treatment assessment. Based on SCID-I we compared depressive symptoms profile between the diagnoses (p < 0.05). These were hypersomnia (0.00% UP and 17.95% BP), suicidal ideation (50.00% UP and 76.92% BP) and specific suicidal plan (33.33% UP and 53.85% BP). Other symptoms did not differ between the diagnoses, although suicide attempt was near significant higher  in BP.Hamilton’s total score was higher in pre-treatment patients compared to post-treatment ones, in both diagnoses . Seven piagnoses . Three sp < 0.05). We have collected a list of drugs that were administered to each patient upon admission to the hospital and during the hospital stay. The number of medicines taken was compared between the diagnoses, but there were no statistical differences. However, the number of drugs taken was higher in post-treatment patients than in the pre-treatment group . The mosCurrent functioning was measured with the Global Assessment of Functioning scale  in pre- p = 0.0268) in post-treatment (3.02 µlU/mL) than the pre-treatment state (1.74 µlU/mL).Pre-treatment patients in both diagnoses had a higher CRP score, triglyceride, and LDL than controls, whereas HDL and TSH had lower value in the study group . It is wp < 0.05). The results were compared between the diagnoses; higher values for sleep disturbance (HAMD question 4) and sleep deterioration (BDI question 16) were observed in UP than in BP . The results indicate significant differences in the frequency of chronotypes (p = 0.0038). In CG, intermediate chronotype was the most often found, and, similar to SG, the least observed was morning chronotype . In comparison between patients and the control group, we discovered a difference in the frequency of choosing intermediate chronotype  (p = 0.0489 and z = 2.4024) and showed a pattern represented by the whole SG, whereas BP does not vary considerably from CG.Several of the clinical questionnaires  inquire whether the patients have sleep problems . The analysis of the results obtained in the pre- and post-treatment state showed improved sleep quality in all analyzed items  . MoreoveFrom the questionnaires concerning the quality of sleep and sleepiness (PSQI and ESS), we have chosen several sleep variables and compared them between controls and subgroups of patients . Three cp = 0.0191).The ESS results (daytime sleepiness) were similar for BP and control groups, but differed significantly between UP and controls . UP achip < 0.05) was observed between OPCRIT and questionnaires variables (Patients presenting the evening chronotype were more likely to have high ESS scores. There were no differences in sleep variables  when comariables . As is sp = 0.0152). First- and second-order factor level differences were shown on Motor impulsiveness; BP scored substantially higher than UP. The impulsive patients are more likely to suffer from bipolar disorder. Moreover, total BIS-11 score showed positive correlation with the item “medicine-induced sleep” (correlation coefficient 0.5459) and PSQI total score (correlation coefficient 0.6040) in BP. There was no such relationship for UP patients.The Barrat Impulsiveness scale scores are presented in Based on BLEQ, patients experienced a greater number of stressful life events in comparison to the control group . In SG, COPE questionnaire differentiates 15 strategies for coping with stress. Alcohol/Drug Use and Humor were the least frequently reported by patients with affective disorder . The obtp = 0.0038 for BP and p = 0.0074 for UP). Although experienced psychiatrist conducted the medical interviews the results should be treated with some caution. It is challenging to distinguish the side effects of drugs from the disease symptoms, especially in patients that had the disease for many years and take several different medications. Moreover, depressed patients might be more prone to report side effects due to an overall more negative assessment of reality.Participants reported mild side effects, except for one person, who reported moderate symptoms in the pre-treatment state  and post-treatment state (appetite increase). About half of the symptoms differed significantly between pre- and post-treatment state in bipolar disorder . In mostOur study showed that BP patients had an earlier age of onset, had more frequently a family burden of psychiatric disorders, and they required hospitalization more often than UP patients. Moreover, BP patients were more impulsive and more frequently had a suicidal ideation and specific suicidal plan than UP patients. These data were spread in numerous clinical assessment tools. This suggest that not the specific tool is crucial for differential diagnosis, but rather an ensemble of factors investigated. It is crucial that the differential diagnosis between UP and BP depressive episodes is made as soon as possible. Bipolar disorder was reported to have a negative impact on everyday life, including the ability to take care of daily tasks, difficulties in the performance of work-related activities, social adaptation, and problems with relationships , as well as the ability to manage financial responsibilities ,4,5. BipAnother challenge for people with bipolar disorder is the problem of keeping a job. In our research, 75% of bipolar patients have had a job before the onset of disease; later on, only 30% were employed. Bipolar patients handle worse compared to unipolar patients (46.87% have a job). More than half of retired patients belong to this group because of the severity of the mental disorder (not because of age criteria). Morseli et al.  made simOur patients predominantly showed evening chronotype, which is consistent with the previous studies ,45. ThisImpulsiveness is defined as a predisposition to act rapidly without reflection and without assessing such behavior’s negative consequences . ImpulsiEuthymic unipolar ,51 and eIn this work, the history of adverse life experiences: personal illness, close family death, interpersonal problems, and job loss, were associated with mood disorders. Hosang et al.  showed tOur results of the COPE inventory did not differ between the diagnoses. A similar result was found by Engel-Yeger and colleagues , whereasIt is crucial to distinguish whether the depressive episode occurs in the course of UP or BP. The profile of clinical symptoms does not allow to differentiate it during the first episode. Therefore, a detailed medical interview with the patient is essential to determine the time of the onset of the disease, family burdens, stressful life situations, difficulties in social relations, suicidal thoughts, and the ability to keep a job. OPCRIT is a tool that collects all of the above-mentioned variables in one place. However, several questionnaires, which are more detailed, might be helpful in clarifying the severity of symptoms. One of them is the BDI. This utility is used to assess the severity of the depression by inquiring about other related areas, such as suicidal thoughts, sleep, work, appetite, and interest in other people. Since the test has structured answers instead of “yes” or “no” responses, it is possible to evaluate the severity of each of the symptoms. For example, inquiry about suicidal thoughts shows whether the patient is only thinking about it but is unable to attempt suicide, or if he has a plan and is waiting for the right circumstances. Such an approach allows us to select important factors that could be emphasized in order to improve the patient’s condition. Based on this questionnaire, in our study, unipolar patients more often reported sleep deterioration than BP patients. HAMD is a similar, but more objective questionnaire, as it is filled in by the psychiatrist and not by the patient, as in the case of the BDI. Similarly to BDI, HAMD also showed sleep disturbance more often in UP than in BP. Data on sleep patterns, taken from symptom profiles or additional questionnaires, could be beneficial for differentiation diagnosis and treatment. In our study, UP was less similar to controls in terms of chronotypes (MEQ questionnaire) and ESS. In contrast, hypersomnia (OPCRIT) is more likely to be found in BP patients. Another important aspect is the stress situation before relapse. The most frequently stressful situations and their impact on patient’s psychical health are listed in BLEQ, an easy-to-use tool that allows to find out which stressful situations might have caused the relapse of the disease. Our results indicated that BP patients had more often problems with friends , whereas UP patients more frequently experienced death of a close ones.Impulsiveness is an important personality trait in the context of attempting suicide. It has been shown that more impulsive people make such attempts more often. BIS-11 is a proven test that enables the assessment of impulsivity. Our results had shown that impulsive patients are more likely to suffer from bipolar disorder.Our research shows that BP patients are more often unemployed than UP. During the interview with the patient, the doctor should pay attention whether the patient changes jobs frequently or remains unemployed for a long time, as this could help with the correct diagnosis.There are various advantages and disadvantages to those methods; however, the latter is due to the fact that self-assessment tests are burdened with the patient’s subjectivity, and not all language versions are validated for psychiatric use. Our study demonstrates the legitimacy of using the additional tools among available questionnaires. In our opinion, additional questionnaires, especially those focusing on the age of onset, family predispositions, suicide attempts, sleep problems, impulsiveness, and stressful situations before relapse, could help with the differential diagnosis and correct treatment. We have tested only several tools in this study, but we believe that other tests covering these subject areas may also be useful. Considering numerous aspects and questionnaires, the risk of misdiagnosis might be reduced with the help of several tools. Thus, treatment strategy might be better individualized, contributing to a better quality of life for the patient, fewer relapses and a more favorable course of a disorder.The main limitation of this study is a small research group that completed the questionnaire correctly. Our study group included only women because of the differences in the course of disease between genders. Men with bipolar disorder are more prone to substance use disorders ,56,57. I


Yet we still found several species different in abundance between the two groups. For the EC group, the most significantly increased taxa were Firmicutes, Negativicutes, Selenomonadales, Prevotellaceae, Prevotella, and Veillonellaceae, while the most significantly decreased taxa were Proteobacteria, Betaproteobacteria, Neisseriales, Neisseriaceae, and Neisseria. In conclusion, there are significant alterations in abundance of some oral microbiomes between the EC patients and the healthy controls in the studied Chinese participants, which may be meaningful for predicting the development of EC, and the potential roles of these species in EC development deserve further studies.Emerging evidence supports that oral microbiota are associated with health and diseases of the esophagus. How oral microbiota change in Chinese patients with esophageal cancer (EC) is unknown, neither is their biomarker role. For an objective to understand alterations of oral microbiota in Chinese EC patients, we conducted a case-control study including saliva samples from 39 EC patients and 51 healthy volunteers. 16S rDNA genes of V3-V4 variable regions were sequenced to identify taxon. Relationship between oral flora and disease was analyzed according to alpha diversity and beta diversity. Resultantly, the Shannon index ( Each subject signed a written informed consent for the scientific use of his/her saliva sample and clinical data. And we were committed not to reveal out volunteers' data. Our study was approved by the ethics committees of Luoyang Central Hospital affiliated to Zhengzhou University.Participants were asked to rinse their mouths with warm water to eliminate food debris and then waited for 5 min or longer for natural saliva secretion, not speaking or taking in food or water. Each saliva sample was spit into a sterile container and not <3 ml. Every sample was then transferred into another sterile cryopreservation tube, using a sterile syringe, and stored into a −80°C freezer immediately until extracted for DNA.via a KAPA HiFi Hotstart ReadyMix PCR kit. The PCR products were detected by 2% agarose gel electrophoresis and recovered by gel cutting with AxyPrep DNA gel Recovery Kit (AXYGEN company). NanoDrop 2000 spectrophotometer  and 2% agarose gel electrophoresis were then applied for quality control of the library. After that, the amplified genes were sequenced in Illumina Miseq PE250 instrument  with 2 × 250 base pair (bp) paired-end (PE) sequencing. All the DNA extraction, amplification, and sequencing were performed at Realbio Technology .Total DNA was extracted from saliva samples using a PowerSoil DNA isolation kit , in accordance with the manufacturer's instructions. PCR was then performed following a universal primer for 16s rDNA 341F/806R . V3-V4 variable region of each 16s rDNA was amplified PANDAseq . Firstly, the singletons (corresponding to only one sequence of reads) in the spliced long reads are filtered out. USEARCH is then used to cluster reads into the same OTU when their distance-based similarity was >0.97 Edgar, . After chttp://rdp.cme.msu.edu; Wang et al., We randomly extracted the average value of each sample according to the minimum number of sequences matched to OTU when the samples reached sufficient sequencing depth . It was T-test was used for quantitative variables, while Pearson's chi-square test was applied for categorical variables. Sequencing data were analyzed by R software . A p < 0.05 was identified as statistically significant.Clinical data were analyzed by SPSS . Student's Alpha diversity is the analysis of species diversity in a single sample, including Observed species index, Chao1 index, Shannon index, and Simpson index. QIIME software was applied to calculate the value of alpha diversity index and to generate the corresponding rarefaction curves. Alpha diversity indices with significant differences under different conditions were screened by rank sum test (using Wilcoxon test function in R).p < 0.05 was identified as statistically significant. All p-values were adjusted by false discovery rate (FDR) test.Beta diversity analysis was used to compare differences in species diversity among samples. Phylogenetic distances of the system were calculated by weighted UniFrac to compare species community differences among samples, which took the evolutionary distance between species into account. A larger index means a greater difference between samples. Based on the weighted UniFrac analysis, principal coordinate analysis (PCoA) was used to show differences between the samples. The results indicated that if the two samples were close, the species compositions of the two samples were similar. Heatmap clustered samples with similar beta diversity, reflecting similarities between samples. One-way analysis of similarities (ANOSIM) was used to test the significance of the difference between the two groups. Linear discriminant analysis (LDA) effect size (LEfSe) was used to estimate the impact of abundance of each species on the difference effect and to find out the communities or species in different groups that had an impact on the significance difference. LDA threshold was 2. Rank sum test (Wilcoxon test function of R language stats package) was used to analyze the significant differences among different groups in order to find out the species that had significant differences in the division of groups. A Thirty-nine EC patients and 51 healthy controls were employed in this study. Baseline characteristics are shown in p = 0.2) and Simpson diversity index (p = 0.071), alpha diversity showed no significant difference between the EC group and the healthy group , Betaproteobacteria (p <0.001) at the class level, Neisseriales (p < 0.001) at the order level, Neisseriaceae (p < 0.001) at the family level, and Neisseria (p < 0.001) at the genus level, while the most enriched floras were Firmicutes (p = 0.006) at the phylum level, Negativicutes (p = 0.002) at the class level, Selenomonadales (p = 0.002) at the order level, Veillonellaceae (p = 0.002) at the family level, and Prevotella (p = 0.007) at the genus level. The top 20 microbial species with the largest different abundance are shown in We identified 1,298 microbial taxa from 90 samples, among which 1,250 were at the phylum level, 1,173 at the class level, 1,146 at the order level, 1,038 at the family level, and 667 at the genus level. Core microbiomes with 100% sample coverage and significant differences between groups were o groups . Then, wus level . For thePrevotella was enriched while Neisseria was decreased at the genus level in the EC patients, the change of which might be associated with an increased risk of EC.Our study supports that the oral microbiota in EC patients is different with healthy people. The abundance of Neisseria and Prevotella, respectively . Unlike healthy people mentioned above, the abundance of oral Prevotella in the older EC patients, compared with younger healthy controls, was significantly enriched. We propose that Prevotella may be associated with EC development. In addition, there was an age-matched case-control study about oral microbiota, and esophageal squamous cell carcinoma (ESCC) showed an increased relative abundance of Prevotella in ESCC patients  and BE , driven by Prevotella abundance was only significant at the genus level and family level. It was one of core microbiomes that covered all samples. We believe the alteration of oral Prevotella may be a potential predictor of EC. However, we did not identify floras to the species level. A study showed that increased Prevotella nanceiensis was associated with a high risk of ESCC, while increased Prevotella oral taxon 306 was associated with a low risk of ESCC, and increased P. nanceiensis was associated with a low risk of EAC (Peters et al., Prevotella and EC needs further study.In our study, the increase of Proteobacteria, among which the most decreased was Neisseria. Peters et al. (Neisseria, namely, Neisseria sicca and Neisseria flavescens, was associated with a low risk of EAC risk. However, only six patients of our study were diagnosed with EAC and the others were ESCC. A study about the relationship between oral flora and ESCC risk of Chinese patients also showed a decreased abundance of Neisseria in ESCC patients (Chen et al., Neisseria (Liu et al., Nesseria abundance with several studies of Europeans and Americans suggested the different oral microbiome composition of Chinese people, which might be influenced by diet and habits.The most abundance-increased bacteria from the phylum level to the genus level in our study all belonged to s et al.  suggesteProteobacteria and increased abundance of Firmicutes was associated with BE, which was consistent with our findings (Snider et al., Veillonella, a core microbiome, was significantly enriched in EC patients. It belongs to Veillonellaceae, Selenomonadales, Negativicutes, and Firmicutes, which were all the most enriched flora. Increased abundance of Veillonella, such as Veillonella oral taxon 917, was related to a high risk of EAC (Peters et al., In addition, a study showed that decreased abundance of Inspired by these studies, we will study the relationship between oral floras and serum tumor markers, inflammatory factors, and the differences of oral microbiota structures and functions in patients with EC at different stages. From which we may further understand the role of oral floras in the occurrence and development of EC and search for biomarkers for early prediction or prevention of EC and new targets for treatment of EC.Neisseria and Prevotella, Veillonella as potential new biomarkers for EC. It also provides a direction for the study of the relationship between oral flora and EC.Our study for the first time provides the diversity differences of oral microorganisms between EC patients and healthy people in China. Our study provides https://www.ncbi.nlm.nih.gov/sra/PRJNA660092.The original contributions presented in the study are publicly available. This data can be found at: QZ and BW participated in the design of this study and performed the statistical analysis. QZ collected the important background information, carried out the literature search, and drafted the manuscript. YZ and YY participated in the data analysis. BW and HC carried out the manuscript editing and review. All authors provided assistance for data acquisition, read, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present study proposes a new approach for indexing heavy metals ions to examine groundwater quality in North Kurdufan Province, Sudan. The new approach is developed based on the most frequently used methods for indexing heavy metals pollution in water. It is created in order to avoid the weaknesses of the current indexing systems. As per the new indexing approach, heavy metal contamination in water samples is evaluated by two types of indices: the negative evaluation index (NEI) and positive evaluation index (PEI). The water worthiness is assessed based on a pair of indices, NEI and PEI. Water quality increases with the decrease of PEI and NEI values. NEI indicates the contribution of heavy metals with a concentration not exceeding the highest desirable limit ( Groundwater has the advantage of low transmission cost, which makes it a perfect source for water supply, compared with surface water. Over the last two decades, the use of groundwater primarily for irrigation has increased significantly to meet the agricultural and economic development targets in many areas of Sudan, especially in regions far off the Nile system . UnfortuHeavy metals contamination is posing a major problem to the aquatic environment and, therefore, are hazardous towards living beings ,5. Many ds, etc. ,10,11,12Furthermore, the classification task is arduous as water quality does not only depend on the water constituent’s concentration but also on their relative importance in water as well as their toxicity. Therefore, the overall evaluation indices would make the task straightforward. Many researchers have developed different approaches to water quality indices and, understandably, all of them are semi-empirical ,17,18,19Past studies carried out for Kurdufan regions were limited to hydrological, geological, and hydrochemical properties of the confined and unconfined aquifers ,24,25,26k is taken equal to one for all metals in the literature [HPI proposed by Mohan et al. , is usedEquation ; Qi is tEquation  and n isEquation . Mi is ttandards ,28,29. Iion SSMO 02, WorldTamasi and Cini  applied To assess the quality of drinking water, the value of MI can be divided into six classes: very pure (<0.3), purified (0.3–1.0), slightly affected (1.0–2.0), moderately affected (2.0–4.0), strongly affected (4.0–6.0) and seriously affected (>6.0) .PoS index is developed by Tziritis et al.  to evaluEquation .(8)Wi=wS method , the parAccording to the authentic PoS method, the groundwater samples are categorized into six classes indicating to water quality degradation level: minimum (1.0), low (2.0), medium (3.0), high (4.0), very high (5.0) and severe degradation (6.0) . The refterature .The numerator term in Equation  can leadAs already known, a higher value of HPI indicates poor water quality and vice versa. Thus, when calculating the individual quality rating Equation , the Qi Also, the expression of MI and PoS are given in Equations  and 7)..7). The The estimation of MI as in Eqnuation  considerAs per Equations  and 4),,4), if tThere are many rating ranges for HPIs as excellent, perfect, good, poor, and very poor regarding the water quality. However, the classifications of water quality by the aforementioned approaches are neither clear nor sufficient to determine the water quality. In theory and practice, the rating should be flexible depending on the level of influence of the individual concentration of elements as per water quality standard. This issue has been handled in developing the proposed MHEI method.The formulations of the techniques mentioned above have some major shortcomings; we can summarize them in the following points:k = 2 for all metals. A preliminary assessment suggests that some elements are below the detection limit for all samples. Hence, the present study considered six heavy metal ions, namely iron (Fe), manganese (Mn), zinc (Zn), lead (Pb), cadmium (Cd), and chromium (Cr) in assessing groundwater quality. The parameters data obtained are compared with different standards for drinking purposes, including Sudanese Standards and Metrology Organization (SSMO 2002) [The samples are collected from different locations in a populated area of North Kurdufan State. MO 2002) , World HMO 2002) , and theMO 2002) . A globaIn the present study, a new approach for water quality assessment is proposed, which extends the three most popular techniques in heavy metals indexing by the new elements as discussed below;k is considered to be one for all metals. In this study, the (SSMO 2002) standard is considered. Where the specification is not available in (SSMO 2002) standards, the (GB/T14848-1993) standards guidelines are used. If the highest desirable value is not assigned in the standards, the maximum allowable value is used.Supposing that the water sample consists of n number of heavy metal ions, then MHEI can be expressed by Equation .(9)MHEIquations  and 11)(9)MHEI=∑n1 indicates the number of heavy metals whose n2 means the heavy metals with the The new approach considers two indices to estimate MHEI; the negative evaluation index (NEI), and positive evaluation index (PEI) as defined below:If the measured concentration value quations  and 14)Mi of a hThe spatial interpolation is a numerical method that converts attribute database values of a set of locations (points) into a surface map that reflects the spatial change in these values throughout the study area. The spatial interpolation methods, for map generation, are a beneficial tool, which provides a piece of sensitive information for decision-makers. There are several mathematical methods to complete the spatial interpolation process, such as Kriging, Inverse Distance Weighted (IDW), Natural Neighbor, Spline, Trend, etc. However, the Kriging and IDW technique were widely used in environmental studies and the most appropriate interpolation tool to reflect the behavior of the movement of the contaminants inside the aquifer. In this study, we applied the IDW method to generate spatial interpolation maps.IDW is a geo-statistical-based method, depend on the spatial engagement functions for investigation of the water parameters distribution within the study area. ArcGIS 10.3 software with the aid of  function is used to present the results of interpolation.Six heavy metal ions are considered in the present study to assess groundwater quality for drinking purposes. These elements in groundwater samples are assessed and compared against corresponding maximum permissible values of SSMO and WHO ,28 standIron is a natural element of the earth’s crust . It is pPb, Cd, and Cr are toxic elements, and hazardous towards living beings ,31. The The models are applied to six heavy metals ions: Fe, Mn, Zn, Pb, Cd, and Cr to calculate the HPI based on Equations –4), MI , MI 4), Equation ). SSMO [Equation . Table 7The results of HPI  show thaSpecifically, 12 out of 18 samples have their NEI and PEI varying between −81 to −99 and 0.00 to 14.21, respectively, indicating excellent-good water quality. Also, two samples obtained PEI and NEI in the range of 27.73 to 28.84 and −0.03 to −8.2, respectively, signifying good water quality. The four remaining samples have PEI and NEI within the fields of 54.66 to 84.33 and 0.00 to −16.32, respectively, which indicates slightly polluted water.MI and PoS consider the amount of concentration more than its importance in water quality. Also, regarding MI, sample GW09 is classified as seriously contaminated by heavy metals , but only the concentration of iron is higher than the permissible limit. The fact that the concentration of iron exceeds the permissible limit cannot justify the classification by MI and PoS for GW09. The sub negative and positive evaluation indices are the strengths of the proposed MHEI indexing method, which are missing in HPI, MI, and PoS indexing systems.Computed HPI and MHEI show that all groundwater samples from the boreholes are suitable for human consumption. The spatial distribution of NEI and PEI are in complete agreement with the metals spatial distribution . Also, FThe spatial distribution of NEI and PEI are in complete agreement with the metals spatial distribution.The MI and HPI indexing failed to account for the toxicity of elements in the evaluation of groundwater quality. This may explain why some samples were erroneously indexed. The proposed MHEI considered the element concentration as well as the toxicity in the groundwater quality evaluation dprocess to index the metals thoroughly, thus producing relatively better results.The traditional heavy metals indexing techniques, namely MI, HPI, and PoS always take the metal concentration in water sample as a positive pollutant, even when the measured concentration is below the highest desirable limit. However, according to MHEI, heavy metal effect may be measured by a pair of indices, NEI and PEI. Additionally, some major shortcomings in the formulations of conventional indexing methods.This study also proposed a more flexible water quality rating system that is more in sync with the standard guidelines documents.The performance of the MHEI model proposed was strong, promising, and proved useful for evaluating heavy metals pollution levels in groundwater. It also takes care of many deficiencies of the existing approaches.The present study proposed a new method for heavy metals pollution indexing to assess groundwater quality for drinking purposes in North Kurdufan state, Sudan. The proposed method applies two indices in the water quality evaluation process; negative evaluation index (NEI) and positive evaluation index (PEI). The water quality is evaluated based on a pair of indices, NEI, and PEI. NEI and PEI directly reflect water quality: water quality increases with the decrease of PEI and NEI values. NEI indicates the contribution of heavy metals with a concentration not exceeding the highest desirable limit in water sample quality, while vice versa regarding the PEI. NEI varies from 0 to −100 while PEI may vary from 0 to any positive value. Eighteen groundwater samples collected from a confined aquifer have been analyzed. The results of MI, HPI, PoS and MHEI show that all groundwater samples from the boreholes are suitable for human consumption except sample (GW09) was found unsuitable as per MI and PoS methods. The proposed MHEI was compared with HPI, MI, and PoS which are widely used in the literature, and the following conclusions are drawn;The proposed MHEI indexing can provide accurate and reliable information on water quality and serves as a useful tool for sustainable water resource management in the future.


Acute respiratory infection (ARI) and diarrhoea are the leading causes of childhood morbidity and mortality in Ethiopia. Understanding the associations between infant and young child feeding (IYCF) and ARI and diarrhoea can inform IYCF policy interventions and advocacy in Ethiopia. This study aimed to investigate the relationship between IYCF practices and ARI and diarrhoea in Ethiopian children.This study used the Ethiopia Demographic and Health Survey (EDHS) data for the years 2000 (n = 3680), 2005 (n = 3528), 2011 (n = 4037), and 2016 (n = 3861). The association between IYCF practices and (i) ARI and (ii) diarrhoea were investigated using propensity score matching and multivariable logistic regression models. The IYCF practices include early initiation of breastfeeding, exclusive breastfeeding (EBF), predominant breastfeeding, introduction of complementary foods, continued breastfeeding at two years and bottle feeding.Infants and young children who were breastfed within 1-hour of birth and those who were exclusively breastfed had a lower prevalence of ARI. Infants who were exclusively and predominantly breastfed had a lower prevalence of diarrhoea. Early initiation of breastfeeding  and EBF  were associated with lower risk of ARI. Bottle-fed children had higher odds of ARI . Early initiation of breastfeeding and EBF were associated with lower odds of diarrhoea . Infants who were predominantly breastfed were less likely to experience diarrhoea .The recommended best practices for preventing ARI and diarrhoeal diseases in infants and young children namely: the early initiation of breastfeeding, EBF and avoidance of bottle feeding should be institutionalized and scale-up in Ethiopia as part of implementation science approach to cover the know-do-gaps. Acute respiratory infection (ARI) and diarrhoea are the leading causes of childhood morbidity and mortality globally, particularly in low- and middle-income countries (LMICs) –3. In 20Evidence from sub-Saharan African , 13, 15 Based on the World Bank assessment , EthiopiUnderstanding and quantifying the relationship between IYCF practices and ARI and diarrhoea among infants and young children is crucial to health practitioners and policymakers in Ethiopia. This information will help in IYCF policy formulation and advocacy, which can, in turn, play an important role in reducing child morbidity and mortality due to ARI and diarrhoea. This assessment is also important in Ethiopia given the current global health efforts–the United Nation’s Sustainable Development Goals (SDG-3.2: ending preventable deaths of newborns and under-five children by 2030)  and GlobThe study used the Ethiopia Demographic and Health Survey (EDHS) data for the years 2000 (n = 3680), 2005 (n = 3528), 2011 (n = 4037), and 2016 (n = 3861). The EDHS used the household questionnaire to collect information on households, and the women’s questionnaire to collect information on child health and nutrition. The surveys were implemented by the Ethiopia Central Statistical Agency (CSA) and Inner City Fund (ICF) International, and funded by the United States Agency for International Development, and the Government of Ethiopia , 23–25.The EDHS used a two-stage stratified cluster sampling technique to select households (the secondary sampling unit) for inclusion in the survey. In stage one, after the nine administrative units were stratified into 12 urban and 11 rural strata, Enumeration Areas (EAs) were selected proportional to the household size of the cluster. In stage two, a fixed number of households were selected from each EA using the list of households as a sampling frame , 23–25. Ethiopia is the second most populous country (with more than 110 million population) in Africa after Nigeria . The popThe outcome variables were ARI and diarrhoea, measured based on maternal recall of symptoms of cough and shortness of breath, and diarrhoea, respectively . ARI wasThe main exposure variables were IYCF indicators  . IYCF inEIBF was defined as the proportion of children aged 0–23 months who commenced breastfeeding within the first hour of birth.EBF was defined as the proportion of infants 0–5 months of age who were fed no other food or drink, not even water, except breast milk (including milk expressed or from a wet nurse), but allows the infant to receive oral rehydration salt, drops, and syrups .Predominant breastfeeding was defined as the proportion of infants 0–5 months of age who received breast milk (including milk expressed or from a wet nurse) as the predominant source of nourishment, but allows water, water-based drinks, fruit juice, oral rehydration solution, drops, or syrups of vitamins and medicines during the previous day.Introduction of complementary foods  was defined as the proportion of infants 6–8 months of age who received solid, semi-solid or soft foods in the previous 24 hours, during the day and at night.Continued breastfeeding at two years of age was defined as the proportion of children aged 20–23 months who received breast milk during the previous day.Bottle feeding was defined as the proportion of children 0–23 months of age who were fed any liquid (including breast milk) or semi-solid food from a bottle during the previous day.The potential confounders were selected based on previously published studies , 34, 35 Socio-economic factors included mothers' or fathers’ education, maternal employment and household wealth status. Demographic and behavioural factors included maternal age, family size, desire for pregnancy, listening to the radio, reading newspaper/magazine and watching television. Health service factors included ever use of a vaccine, frequency of antenatal care (ANC) visits, place of birth, and timing of first postnatal care (PNC) visit. Community-level factors included a place of residence and region of residence.The study also considered the type of cooking fuel in the analyses of ARI, and the source of drinking water and type of toilet facility in the analyses of diarrhoea as potential effect measure modifiers. This was done to investigate whether the association between IYCF practices and each outcome differed across each stratum for the type of cooking fuel, source of drinking water and sanitation level. This approach is consistent with previously published studies from Africa , 36–38. The source of drinking water and type of toilet facility were classified as ‘improved’ or ‘not improved’, based on the taxonomy of the WHO and UNICEF Joint Monitoring Programme (JMP) for Water and Sanitation  as appliThe initial analysis involved the tabulation of frequencies and percentages of socioeconomic, demographic, health-service and community-level factors over the survey years (2000–2016). Prevalence of ARI and diarrhoea were calculated for each of the exposure variables . The EDHS data from 2000 to 2016 were combined to increase the study power and precision of estimates. Before statistical analyses, all variables were checked for missing properties; nevertheless, there was no evidence of missingness at random.Propensity score matching (PSM) and multivariable logistic regression were used to investigate the associations between IYCF practices and ARI and diarrhoea. Observational studies  are helpful to investigate the association between exposure and outcome variables . HoweverIn observational studies, researchers have indicated that PSM and multivariable logistic regression modelling are ‘best’ used in combination when investigating the association between two variables of interest , 50, 51.stage one, the propensity score was estimated using binary logistic regression by specifying each IYCF indicator to the outcome and background characteristics  as predictors. The survey weight was included as a covariate in the estimation process of the propensity score, consistent with previously published studies Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript is technically sound. The authors have employed sound study design and methodology to investigate well-defined exposures and outcomes. The sample size was large enough and the data analysis techniques was robust and rigorous. I believe the authors have made all the data underlying the findings although some files could not be opened probably due to poor internet connection at which I was located during the review. The entire presentation of the manuscript followed a logical sequence with near perfect use of English. However, I have the following suggestions for the authors to consider:AbstractBackground:Line 34, ‘are the leading causes of’ not ‘are the leading cause of’ are grammatically more correctConclusion:It would better to conclude by stating that recommended best practices for preventing ARI and diarrhea diseases in infants and young children namely; the early initiation of breastfeeding, exclusive breastfeeding and avoidance of bottle feeding should be institutionalized and scale up in Ethiopia as part of implementation science approach to cover the know-do-gaps.Main manuscriptMethods:It would be nice to begin the methods section with brief descriptions of the study designs and settings, although some mentions of Ethiopian settings were made under the introductionEIBF is better defined as ‘commencement of breastfeeding within 1 hour of birth’ as opposed to ‘infants breastfed or put to breast within 1 hour of birth’. While the former indicates something that has started and continued, the later may mean a one-off event that never happen again. I suggest the authors use a phrase that more appropriately described the event. If feasible, commencement of breastfeeding should be replace put to breast in the entire manuscript.Analytical strategyLine 226 – line 254: starting with explanation on RCTs and ending with why PSM approach was used in this study, the entire paragraph may no be necessary for this section. If at all such explanations are needed, some of them should go under study design or more appropriate in the discussion section. I don’t think these detailed descriptions of RCT, use of PSM approach and its advantages is required under data analysis methods. Discussions here should be limited to how the data were analyzed.ResultsThe authors repeatedly described the difference in prevalence of ARI and diarrhea among the exposed and unexposed as significant. However, neither in the narrative nor in table 2 were the associated p-values stated. Rather, would it be safer to modify the narration by removing the word ‘significantly’?I am also wondering if it would add value to the interpretation of the odd ratios of developing outcomes comparing the exposed and unexposed if P-values are included in the results narrative or in the corresponding tables. I noticed some of the confidence intervals included 1 which is a neutral effect.Study weaknessAnother weakness to consider is that description of ‘loose or watery stool’ to define diarrhea might not have objectively capture infants who had diarrhea and who had not as such descriptions are too subjective.Reviewer #2: General comments• The study is relevant to Ethiopia and other countries with low income status. The topic is particularly relevant to Ethiopia where there is very high prevalence of under-five malnutrition and child mortality. It could provide important insight about the situation of IYCF practices and its association with common childhood illnesses (diarrhea and ARI). The findings of the study could be used to inform planning and policy making exercise in the country and the wider region of sub-Saharan Africa.• The article is well but it could benefit from professional language editor.AbstractPage 2, line 46.• The authors wrote “…..exclusively breastfed had a lower prevalence of ARI.” I think using the word incidence is a better than prevalence.• Too many abbreviations in the abstract. It is better to minimize the abbreviations.Introduction• The introduction clearly illustrates the problem of ARI and diarrhea; the magnitude and consequence of inappropriate infant and young child feeding (IYCF) in low income settings. However, the rationale for the study is not clearly stated. I think the authors need to strengthen the rationale for the study. A number of studies have tried to answer this research question, the authors need to elaborate the research gaps addressed by the study.• Overall, I feel that it could be shortened with more emphasis on the rational of the study.• The authors need to include at least recent Ethiopian studies addressing similar research questions using the same dataset . For instance, Nigatu D, et al 2019 assess effect of exclusive breastfeeding cessation on diarrhea and ARI (using EDHS 2011 and 2016) and Amsalu ET, 2019 also assess determinants of ARI( using EDHS 2016).Method• Page 8-9, Possible confoundersImmunization status of a child could be one of the predictors of ARI and diarrhea, but the authors did not consider it. Since, information on child immunization is available in EDHS data, could you explain why it is excluded?Analysis:• Page 10, line 218-19The authors wrote “The initial analysis involved the tabulation of frequencies and percentages of ARI and diarrhoea by each study variable.”What does “each study variable” refers to? Only exposures?• Page 10, line 226. I do not see the relevance of mentioning RCT as a gold standard method. Rather, it could be more relevant to discuss the advantage of PSM over commonly used multivariable model for this data.• The four surveys conducted in different time points (from 2000 to 2016). Are these data fairly similar to be combined, even if no interaction of survey year and… In fact, the authors stated that infant mortality decreased from 97 in the year 2000 to 43 per 1000 in 2019.• How was missing data handled? You may state the EDHS procedures to handle missing data.• I am not an expert in PSM technique and I suggest an expert in the field should review the appropriateness of the analytic procedures followed.Result• The authors should mention the sub-samples of mother-infant pairs included for each exposure.• Although the overall sample is large. Some exposures seems to have limited power. For instance in table 2. Distribution of ARI over the exposure “continued breastfeeding at 2 years”, one of the cells have only 48 children (weighted). Could you also say something about adequacy power?.• It would be more informative if you present  SDC of sub-samples used for analysis and the trend in IYCF and the outcomes over time.Discussion.• In general the discussion address important points but I feel that it can be made more coherent and short.• The authors need to have a more robust discussion on the possible limitations of the study.For instance, misclassification bias could be a source of bias because ARI and diarrhea are not diagnosed by clinicians. It is based on mothers recall. For instance, common cold could be confused with ARI.Besides, it might be challenging for the mother to differentiate between normal bowel habits of children from diarrhea, specifically mild form of diarrhea.Reviewer #3: Comments to authorsThis is an important, interesting and well-written manuscript. I have only minor comments.Introduction-It would be useful, if possible, to have information about the percentage of children vaccinated in Ethiopia.-Line 103: Could it also be due to the replacement of human milk by complementary foods/drinks?-Line 119-120: The EBF of 59.9%; - which age/age-group? It would be useful to add information on continued breastfeeding until 2 y and the percentage never breastfed. Also, it would be useful to know whether/how the socioeconomic factors are associated with the IYCF indicators in this population.MethodsAs this reviewer has not conducted analysis using the propensity score matching approach, my understanding of the method is limited. Therefore, it was useful for me to have an explanation of this method under methods, but I do not know whether this is of general interest. I recommend that a statistician consider this and review the statistical methods.Results-Under “results” it would be useful to have an overview of sample sizes, so it is easier to understand the different «n`s» in e.g. table 2 and 3.-In general, it would be useful to mention/describe the reference groups in more detail. Were the reference groups mixed groups, e.g. in line 311: Those who were «not predominantly breastfed», what were they fed, were some not breastfed?-Table 1. Is it necessary to include «Region of residence» in the table?-Line 297-299: Should findings be reported in «the same direction»?Discussion-The accuracy of the definition of diarrhoea as the passage of three of more liquid stools should be discussed, as it is normal with at least 3 or more liquid stools per day in exclusively breastfed infants during the first months (1). How this may have affected the findings should be discussed.-Is it possible to compare the «effect size» on EIBF and EBF on ARI and diarrhoea with other studies in similar contexts?-In the discussion it is mentioned that reverse causation cannot be excluded as an interpretation of the findings. It is improbable that this can explain the findings of EIBF and ARI/ diarrhoea, although it is relevant for EBF. 1. Moretti E, Rakza T, Mestdagh B, Labreuche J, Turck D. The bowel movement characteristics of exclusively breastfed and exclusively formula fed infants differ during the first three months of life. Acta paediatrica . 2019;108(5):877-81.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  23 Feb 2020February 23 2020Maria Christine Magnus, MPH Academic Editor PLOS ONEDear Dr. Magnus,RE: Manuscript resubmission – [PONE-D-19-33411] Associations between infant and young child feeding practices and acute respiratory infection and diarrhoea in Ethiopia: A propensity score matching approachThank you for the invitation to revise our subject-titled manuscript and for the very constructive comments from the reviewers and editor. A revised manuscript (clean and version with track changes) reflecting the following point-by-point response to the editor and the reviewers’ comments have been submitted for your consideration.Academic EditorGeneral comments1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttp://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdfhttp://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfResponse: The manuscript has been revised to meet PLOS ONE's style requirements.2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.Response: The manuscript has been thoroughly copyedited by co-authors who are native English language speakers.Additional Editor comments1. You should shorten the introduction substantially and focus on briefly explaining the background of the research question and why it is relevant in the Ethiopian context.Response: We appreciate the Editor comment and note that the entire introduction section has been substantially edited as requested by the Editor (Page 4-6).2. Can you please confirm that you included all background characteristics in table 1 in the prediction model to generate the probability of exposure to use in the propensity score matching?Response: Thank you so much for the observation. We can confirm that all background characteristics were included in model to generate the probability of exposure in the propensity score matching. This information has been noted in the revised manuscript . 3. Please clarify whether you included the outcomes in this prediction model to generate the probability of exposure? To the best of my knowledge of propensity score matching, the probability of exposure should be generated using all background characteristics than can be considered as confounders of the exposure and outcome relationship but not the outcome itself.Response: We appreciate the editor’s concern. We can confirm that the propensity score was generated in the model without the inclusion of the outcome variables.4. I struggled to understand why you included some of the background characteristics, including listening radio, reading magazine, watching TC and desire for pregnancy. Please provide a justification.Response: This study forms part of the requirements for the award of Doctor of Philosophy for the first author. Against this background, we note our previously published studies have elucidated determinants of IYCF practices in many countries, including Ethiopia. Those studies found that those variables (among others) were related with IYCF practices . This justification was noted in the original manuscript References1. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and determinants of early initiation of breastfeeding and exclusive breastfeeding in Ethiopia from 2000 to 2016. Int Breastfeed J, 14(1), 40. 2. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and factors associated with complementary feeding practices in Ethiopia from 2005 to 2016. Matern. Child Nutr., e129263. Victor R, Baines SK, Agho KE, Dibley MJ. Factors associated with inappropriate complementary feeding practices among children aged 6–23 months in Tanzania. Matern Child Nutr. 2014;10(4):545-61. 4. Ogbo FA, Ogeleka P, Awosemo AO. Trends and determinants of complementary feeding practices in Tanzania, 2004-2016. Trop Med Health. 2018;46:40. 5. Ogbo FA, Eastwood J, Page A, Efe-Aluta O, Anago-Amanze C, Kadiri EA, et al. The impact of sociodemographic and health-service factors on breast-feeding in sub-Saharan African countries with high diarrhoea mortality. Public Health Nutr. 2017;20(17):3109-19.Reviewer #1Comments to the AuthorThe manuscript is technically sound. The authors have employed sound study design and methodology to investigate well-defined exposures and outcomes. The sample size was large enough and the data analysis techniques was robust and rigorous. I believe the authors have made all the data underlying the findings although some files could not be opened probably due to poor internet connection at which I was located during the review. The entire presentation of the manuscript followed a logical sequence with near perfect use of English. However, I have the following suggestions for the authors to considerResponse: Thank you for the comment. The reviewer’s specific comments are addressed below in this rebuttal.Background: Line 34, ‘are the leading causes of’ not ‘are the leading cause of’ are grammatically more correctResponse: Revision done (line 34).Conclusion: It would better to conclude by stating that recommended best practices for preventing ARI and diarrhea diseases in infants and young children namely; the early initiation of breastfeeding, exclusive breastfeeding and avoidance of bottle feeding should be institutionalized and scale up in Ethiopia as part of implementation science approach to cover the know-do-gaps.Response: Thank you, and the suggestion is now reflected in the revised manuscript (Page 3)It would be nice to begin the methods section with brief descriptions of the study designs and settings, although some mentions of Ethiopian settings were made under the introductionResponse: We appreciate the comment. However, it is challenging to describe the study design, especially that our analyses were based on secondary data. The information on data sources to let potential readers know of the study design used in the surveys is provided in the methods section (Page 6-7).In response to the reviewer comment on the study setting, we incorporated this text in the revised manuscript EIBF is better defined as ‘commencement of breastfeeding within 1 hour of birth’ as opposed to ‘infants breastfed or put to breast within 1 hour of birth’. While the former indicates something that has started and continued, the later may mean a one-off event that never happen again. I suggest the authors use a phrase that more appropriately described the event. If feasible, commencement of breastfeeding should be replace put to breast in the entire manuscript.Response: Point appreciated and now reflected in the entire revised manuscript.Line 226 – line 254: starting with explanation on RCTs and ending with why PSM approach was used in this study, the entire paragraph may no be necessary for this section. If at all such explanations are needed, some of them should go under study design or more appropriate in the discussion section. I don’t think these detailed descriptions of RCT, use of PSM approach and its advantages is required under data analysis methods. Discussions here should be limited to how the data were analyzed.Response: We have revised the text in response to the reviewer comment (page 11 paragraph 02). We note, however, that the description of the PSM is crucial to potential readers who may have limited knowledge of the PSM for a number of reasons:I. The PSM is often term a ‘pseudo-RCT’ as it tries to emulate the role of randomization in RCTs;II. For rarely applied analytical strategies, it is recommended to have a brief explanation of the method; andIII. Our description of the PSM is based on previously published studies that applied a similar method. Please see the following references . Additionally, we believe that the description of the PSM (its relevance and application) is essential to potential readers given that it forms a core part of our research question (and subsequent title). The removal or moving the description of the PSM to the discussion section, or somewhere else may, in fact, defeat the purpose for why we used the PSM as readers may have to go elsewhere to find out what PSM is about. Nonetheless, we would like to defer the final decision on this comment to the Editor, especially that the Editor appears to have good knowledge of the PSM.References:1. Gibson LA, Hernandez Alava M, Kelly MP, Campbell MJ. The effects of breastfeeding on childhood BMI: a propensity score matching approach. Journal of public health . 2017;39(4)2. Grube, M. M., von der Lippe, E., Schlaud, M., & Brettschneider, A.-K. (2015). Does breastfeeding help to reduce the risk of childhood overweight and obesity? A propensity score analysis of data from the KiGGS study. PLoS One, 10(3)3. Talukder, A., Akter, N., & Sazzad Mallick, T. (2019). Exploring Association Between Individuals' Stature and Type 2 Diabetes Status: Propensity Score Analysis. Environ. Health Insights, 13,The authors repeatedly described the difference in prevalence of ARI and diarrhea among the exposed and unexposed as significant. However, neither in the narrative nor in table 2 were the associated p-values stated. Rather, would it be safer to modify the narration by removing the word ‘significantly’?Response: Revision done in response to the reviewer suggestion (line 332-342)I am also wondering if it would add value to the interpretation of the odd ratios of developing outcomes comparing the exposed and unexposed if P-values are included in the results narrative or in the corresponding tables. I noticed some of the confidence intervals included 1 which is a neutral effect.Response: Revision done (Table 3).Another weakness to consider is that description of ‘loose or watery stool’ to define diarrhea might not have objectively capture infants who had diarrhea and who had not as such descriptions are too subjective.Response: Point appreciated and the text has been revised accordingly (line 536-538)Reviewer #2The study is relevant to Ethiopia and other countries with low income status. The topic is particularly relevant to Ethiopia where there is very high prevalence of under-five malnutrition and child mortality. It could provide important insight about the situation of IYCF practices and its association with common childhood illnesses (diarrhea and ARI). The findings of the study could be used to inform planning and policy making exercise in the country and the wider region of sub-Saharan Africa.Response: Thank you for the comment. The reviewer’s concerns are addressed below. The article is well but it could benefit from professional language editor.Response: We thank the reviewer for the comment and note that the manuscript has been copyedited by co-authors whose first language is English.AbstractPage 2, line 46. • The authors wrote “…..exclusively breastfed had a lower prevalence of ARI.” I think using the word incidence is a better than prevalence.Response: We would like to clarify to the reviewer that:1. Prevalence measures the presence of a disease, a condition or a risk factor such as smoking in a particular population over a given time period and is defined as the proportion of a population who have (or had) a specific attribute in a given time period Common types of prevalenceThe types of prevalence are dependent on the timeframe for the estimate:• Point prevalence refers to the proportion of a population that has the disease at a specific time.• Period prevalence refers to the proportion of a population that has the attribute at any point over a period of interest.• Lifetime prevalence refers to the proportion of a population who at some point in life up to the time of assessment has ever had the disease or attribute.2. Incidence is a measure of the likelihood of new cases of disease or injury in a population within a specified period of time, that is;Incidence has two main types:• Incidence proportion (IP): Incidence proportion is the proportion of an initially disease-free population that develops the disease within a specified period of time.• Incidence rate (IR): The incidence rate is a measure of incidence that incorporates time directly into the denominator.In the present study, ARI was measured as “the occurrence of cough accompanied by short and rapid breathing during the two weeks’ period preceding the survey” in accordance with Measure DHS reports, which did not separately categorize new cases from old cases within the 2-weeks interval. Similarly, diarrhoea was defined as the passage of three or more loose or liquid stools per day during the two weeks’ period preceding the survey, consistent with Measure DHS. This information was noted in the original manuscript, Page 07, Paragraph 02. Epidemiologically, we believe that ‘period’ prevalence is the correct measure as it accounts for both new and old cases of ARI and diarrhoea that would have occurred in the two prior to the surveys.Reference1. Kenneth J. Rothman, Sander Greenland, Timothy L. Lash. Chapter 3 - Measures of Occurrence. Modern epidemiology, . 3rd edition ed2008.Too many abbreviations in the abstract. It is better to minimize the abbreviations.Response: Revision done (page 2–3)Introduction The introduction clearly illustrates the problem of ARI and diarrhea; the magnitude and consequence of inappropriate infant and young child feeding (IYCF) in low income settings. However, the rationale for the study is not clearly stated. I think the authors need to strengthen the rationale for the study. A number of studies have tried to answer this research question, the authors need to elaborate the research gaps addressed by the study. Overall, I feel that it could be shortened with more emphasis on the rational of the study.Response: We appreciate the reviewer comment and note that the introduction section has been substantially edited in response to the Editor comment above (Page 4-6)The authors need to include at least recent Ethiopian studies addressing similar research questions using the same dataset . For instance, Nigatu D, et al 2019 assess effect of exclusive breastfeeding cessation on diarrhea and ARI (using EDHS 2011 and 2016) and Amsalu ET, 2019 also assess determinants of ARI ( using EDHS 2016).Response: Studies now included in the revised manuscript as requested by the reviewer (Page 05 and page 09)MethodsPage 8-9, Possible confounders Immunization status of a child could be one of the predictors of ARI and diarrhea, but the authors did not consider it. Since, information on child immunization is available in EDHS data, could you explain why it is excluded?Response: Thank you for the comment and observation. We have incorporated the variable (ever use of vaccine) as a potential confounder in the entire manuscript, including the analyses, results and discussion as appropriate. Page 10, line 218-19 The authors wrote “The initial analysis involved the tabulation of frequencies and percentages of ARI and diarrhoea by each study variable.” What does “each study variable” refers to? Only exposures? Response: The reviewer has eyes for details – thank you for the observation! Revision done now (page 10 paragraph 03)Page 10, line 226. I do not see the relevance of mentioning RCT as a gold standard method. Rather, it could be more relevant to discuss the advantage of PSM over commonly used multivariable model for this data.Response:As noted above, the exposition of PSM approach is essential to potential readers; nonetheless, we have revised the text in response to the reviewer comment (page 11 paragraph 02). Here is our response to Reviewer #1 on this comment: We note, however, that the description of the PSM is crucial to potential readers who may have limited knowledge of the PSM for a number of reasons:IV. The PSM is often term a ‘pseudo-RCT’ as it tries to emulate the role of randomization in RCTs;V. For rarely applied analytical strategies, it is recommended to have a brief explanation of the method; andVI. Our description of the PSM is based on previously published studies that applied a similar method. Please see the following references . Additionally, we believe that the description of the PSM (its relevance and application) is essential to potential readers given that it forms a core part of our research question (and subsequent title). The removal or moving the description of the PSM to the discussion section, or somewhere else may, in fact, defeat the purpose for why we used the PSM as readers may have to go elsewhere to find out what PSM is about. Nonetheless, we would like to defer the final decision on this comment to the Editor, especially that the Editor appears to have good knowledge of the PSM.References:1. Gibson LA, Hernandez Alava M, Kelly MP, Campbell MJ. The effects of breastfeeding on childhood BMI: a propensity score matching approach. Journal of public health . 2017;39(4)2. Grube, M. M., von der Lippe, E., Schlaud, M., & Brettschneider, A.-K. (2015). Does breastfeeding help to reduce the risk of childhood overweight and obesity? A propensity score analysis of data from the KiGGS study. PLoS One, 10(3)3. Talukder, A., Akter, N., & Sazzad Mallick, T. (2019). Exploring Association Between Individuals' Stature and Type 2 Diabetes Status: Propensity Score Analysis. Environ. Health Insights, 13The four surveys conducted in different time points (from 2000 to 2016). Are these data fairly similar to be combined, even if no interaction of survey year and… In fact, the authors stated that infant mortality decreased from 97 in the year 2000 to 43 per 1000 in 2019.Response:We thank the reviewer for the comment. We also think that the reviewer may have good knowledge about the DHS, which are standardised data, collected every 5 years in many low- and middle-income countries. To answer the reviewer question, Yes, the data are relatively similar despite being collected over varied time points. We note that the first and senior authors have both published over 25 articles in international journals using the DHS data in time and space . Our reference to the reduction in infant mortality is to highlight the main issues relating to a possibly lack of key IYCF measures that may be used to further reduce under 5 mortalities in Ethiopia. References 1. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and determinants of early initiation of breastfeeding and exclusive breastfeeding in Ethiopia from 2000 to 2016. Int Breastfeed J, 14(1), 40. 2. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and factors associated with complementary feeding practices in Ethiopia from 2005 to 2016. Matern. Child Nutr., e12926. 3. Ogbo, F. A., Agho, K., Ogeleka, P., Woolfenden, S., Page, A., & Eastwood, J. (2017). Infant feeding practices and diarrhoea in sub-Saharan African countries with high diarrhoea mortality. PLoS One, 12(2), e0171792. 4. Ogbo, F. A., Nguyen, H., Naz, S., Agho, K. E., & Page, A. (2018). The association between infant and young child feeding practices and diarrhoea in Tanzanian children. Trop. Med. Health, 46, 2. 5. Ogbo, F. A., Page, A., Idoko, J., Claudio, F., & Agho, K. E. (2016). Diarrhoea and suboptimal feeding practices in Nigeria: Evidence from the national household surveys. Paediatr. Perinat. Epidemiol., 30(4), 346-355. How was missing data handled? You may state the EDHS procedures to handle missing data.Response: Thank you. In the analysis, we checked for missingness at random (MAR), but none was evident in the data. This information has been noted in the revised manuscript (line 245-246).I am not an expert in PSM technique and I suggest an expert in the field should review the appropriateness of the analytic procedures followed.Response: As noted above, we have some reasons to believe that the Academic Editor has good knowledge of PSM and note that our PSM approach was appropriately conducted, consistent with past studies .References:1. Marinovich M, Regan A, Gissler M, Magnus MC, Håberg S, Padula A, et al. Developing evidence-based recommendations for optimal interpregnancy intervals in high-income countries: Protocol for an international cohort study. BMJ Open. 2018;9.2. Grube, M. M., von der Lippe, E., Schlaud, M., & Brettschneider, A.-K. (2015). Does breastfeeding help to reduce the risk of childhood overweight and obesity? A propensity score analysis of data from the KiGGS study. PLoS One, 10(3), e0122534-e0122534. 3. Talukder, A., Akter, N., & Sazzad Mallick, T. (2019). Exploring Association Between Individuals' Stature and Type 2 Diabetes Status: Propensity Score Analysis. Environ. Health Insights, 13, 1178630219836975. Result • The authors should mention the sub-samples of mother-infant pairs included for each exposure.Response: Revision done (Table 2) Although the overall sample is large. Some exposures seems to have limited power. For instance in table 2. Distribution of ARI over the exposure “continued breastfeeding at 2 years”, one of the cells have only 48 children (weighted). Could you also say something about adequacy power?.Response: We agree the reviewer that some of the exposures have small sample sizes, and this is due to the short age interval used for the definitions of those indicators. The small sample size may account for the large effect sizes and the wide CIs, particularly with the continued breastfeeding at 2-years.It would be more informative if you present  SDC of sub-samplesused for analysis and the trend in IYCF and the outcomes over time.Response: The subsamples for the exposure have been included in the revised Table 2. We agree with the reviewer that the examination of trends of the exposure variables is a crucial information but it is beyond the scope of the present research question and subsequent analyses. Notably, information relating to trends on the exposures have been published elsewhere by the authors .References1. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and determinants of early initiation of breastfeeding and exclusive breastfeeding in Ethiopia from 2000 to 2016. Int Breastfeed J, 14(1), 40. doi:10.1186/s13006-019-0234-92. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and factors associated with complementary feeding practices in Ethiopia from 2005 to 2016. Matern. Child Nutr., e12926. doi:10.1111/mcn.12926Discussion In general the discussion address important points but I feel that it can be made more coherent and short.Response:We thank the reviewer for the comment and note that the discussion section of the manuscript is organized, robust and tailored to the exposure-outcome relationship, especially that some of the observed associations are contrary to some findings in the literature. We offered relevant explanations for all key IYCF indicators. Notably, in page 21, the delineation of the varying relationship between predominant breastfeeding and diarrhoea demonstrate our understanding of the topic and implications. We also note that past studies on the topic have been published by the authors .Reference1. Ogbo, F. A., Agho, K., Ogeleka, P., Woolfenden, S., Page, A., & Eastwood, J. (2017). Infant feeding practices and diarrhoea in sub-Saharan African countries with high diarrhoea mortality. PLoS One, 12(2), e0171792. 2. Ogbo, F. A., Nguyen, H., Naz, S., Agho, K. E., & Page, A. (2018). The association between infant and young child feeding practices and diarrhoea in Tanzanian children. Trop. Med. Health, 46, 2. 3. Ogbo, F. A., Page, A., Idoko, J., Claudio, F., & Agho, K. E. (2016). Diarrhoea and suboptimal feeding practices in Nigeria: Evidence from the national household surveys. Paediatr. Perinat. Epidemiol., 30(4), 346-355.The authors need to have a more robust discussion on the possible limitations of the study. For instance, misclassification bias could be a source of bias because ARI and diarrhea are not diagnosed by clinicians. It is based on mothers recall. For instance, common cold could be confused with ARI. Besides, it might be challenging for the mother to differentiate between normal bowel habits of children from diarrhea, specifically mild form of diarrhea.Response: This information was noted in the original manuscript but has been clarified in the revised manuscript (page 26–27). Reviewer #3This is an important, interesting and well-written manuscript. I have only minor comments.Response: Thank you for the comment. The reviewer’s concerns are addressed below.IntroductionIt would be useful, if possible, to have information about the percentage of children vaccinated in Ethiopia.Response: Done (page 5 paragraph 02) Line 103: Could it also be due to the replacement of human milk by complementary foods/drinks?Response: Revision done (101–102).Line 119-120: The EBF of 59.9%; - which age/age-group? It would be useful to add information on continued breastfeeding until 2 y and the percentage never breastfed. Also, it would be useful to know whether/how the socioeconomic factors are associated with the IYCF indicators in this population.Response: We appreciate the reviewer comment. However, we are constrained by the word limit to fully incorporate additional information relating to continued breastfeeding until 2 years and the percentage of never breastfed as noted by the Academic Editor and Reviewer # 2. We note that information relating to the determinants of breastfeeding and complementary feeding has been published elsewhere by the authors References1. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and determinants of early initiation of breastfeeding and exclusive breastfeeding in Ethiopia from 2000 to 2016. Int Breastfeed J, 14(1), 40. doi:10.1186/s13006-019-0234-92. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and factors associated with complementary feeding practices in Ethiopia from 2005 to 2016. Matern. Child Nutr., e12926. doi:10.1111/mcn.12926MethodsAs this reviewer has not conducted analysis using the propensity score matching approach, my understanding of the method is limited. Therefore, it was useful for me to have an explanation of this method under methods, but I do not know whether this is of general interest. I recommend that a statistician consider this and review the statistical methods.Response: As noted above, we have some reasons to believe that the Academic Editor has good knowledge of PSM and note that our PSM approach was appropriately conducted, consistent with past studies .References:1. Marinovich M, Regan A, Gissler M, Magnus MC, Håberg S, Padula A, et al. Developing evidence-based recommendations for optimal interpregnancy intervals in high-income countries: Protocol for an international cohort study. BMJ Open. 2018;9.2. Grube, M. M., von der Lippe, E., Schlaud, M., & Brettschneider, A.-K. (2015). Does breastfeeding help to reduce the risk of childhood overweight and obesity? A propensity score analysis of data from the KiGGS study. PLoS One, 10(3), e0122534-e0122534. 3. Talukder, A., Akter, N., & Sazzad Mallick, T. (2019). Exploring Association Between Individuals' Stature and Type 2 Diabetes Status: Propensity Score Analysis. Environ. Health Insights, 13, 1178630219836975. Results-Under “results” it would be useful to have an overview of sample sizes, so it is easier to understand the different «n`s» in e.g. table 2 and 3.Response: The information relating to the sample sizes has been incorporated in Table 2 in the revised manuscript.In general, it would be useful to mention/describe the reference groups in more detail. Were the reference groups mixed groups, e.g. in line 311: Those who were «not predominantly breastfed», what were they fed, were some not breastfed?Response: Information relating to the reference groups for each IYCF indicator is now noted in the definitions for assessing IYCF according to the WHO. In relation to the predominant breastfeeding mentioned, infants aged 0–5 months were feed with is now noted in the definition, that is, breast milk, including milk expressed or from a wet nurse, and other fluids such as water and juice (Page 08–09).Table 1. Is it necessary to include «Region of residence» in the table?Response: Yes, it was necessary to include residence based on past studies  The justification is noted in the original manuscript (line 186–187):References1. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and determinants of early initiation of breastfeeding and exclusive breastfeeding in Ethiopia from 2000 to 2016. Int Breastfeed J, 14(1), 40. doi:10.1186/s13006-019-0234-92. Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and factors associated with complementary feeding practices in Ethiopia from 2005 to 2016. Matern. Child Nutr., e12926. doi:10.1111/mcn.12926Line 297-299: Should findings be reported in «the same direction»?Response: The text has been edited in the revised manuscript (Line 327–329)The accuracy of the definition of diarrhoea as the passage of three of more liquid stools should be discussed, as it is normal with at least 3 or more liquid stools per day in exclusively breastfed infants during the first months (1). How this may have affected the findings should be discussed.Response: Point appreciated, and now reflected in the limitation section of the discussion in the revised manuscript :Is it possible to compare the «effect size» on EIBF and EBF on ARI and diarrhoea with other studies in similar contexts?Response: Response: Yes, it is possible to compare the effect sizes of EIBF and EBF and ARI and diarrhoea with other studies in similar contexts. However, we are constrained with words as we draw on Reviewer # 2 comment which indicated that the original discussion may be too lengthy. Additionally, we believe that our discussion of the results is focus and provides explanations for why there may varied results from the analyses, consistent with reporting of epidemiological studies. The comparison of effect sizes of EIBF and EBF and ARI and diarrhoea with other studies in similar contexts may be as an another research question that we can explore in a meta-analysis of observational studies in the future. Thank you for the suggestions.In the discussion it is mentioned that reverse causation cannot be excluded as an interpretation of the findings. It is improbable that this can explain the findings of EIBF and ARI/ diarrhoea, although it is relevant for EBF. Response: We agree with reviewer that reverse causation cannot be excluded as an interpretation of the findings. However, our search of the literature did not find any studies to support our claim. Accordingly, we have revised the text .We thank the reviewers for the valuable comments and time in reading our manuscript.We look forward to your final discussion in due course. Please contact me should you require any further information.Sincerely,Kedir Yimam Ahmed, MPH The Corresponding authorAhmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and determinants of early initiation of breastfeeding and exclusive breastfeeding in Ethiopia from 2000 to 2016. Int Breastfeed J, 14(1), 40. doi:10.1186/s13006-019-0234-9Ahmed, K. Y., Page, A., Arora, A., & Ogbo, F. A. (2019). Trends and factors associated with complementary feeding practices in Ethiopia from 2005 to 2016. Matern. Child Nutr., e12926. doi:10.1111/mcn.12926https://dhsprogram.com/pubs/pdf/DHSG1/Guide_to_DHS_Statistics_DHS-7.pdfCroft, Trevor, N., Aileen, M. J. M., Courtney, K. A., & et al. (2018). Guide to DHS Statistics: DHS-7. Rockville, Maryland, USA Retrieved from Gibson, L. A., Hernandez Alava, M., Kelly, M. P., & Campbell, M. J. (2017). The effects of breastfeeding on childhood BMI: a propensity score matching approach. Journal of public health , 39(4), e152-e160. doi:10.1093/pubmed/fdw093Grube, M. M., von der Lippe, E., Schlaud, M., & Brettschneider, A.-K. (2015). Does breastfeeding help to reduce the risk of childhood overweight and obesity? A propensity score analysis of data from the KiGGS study. PLoS One, 10(3), e0122534-e0122534. doi:10.1371/journal.pone.0122534Ogbo, F. A., Agho, K., Ogeleka, P., Woolfenden, S., Page, A., & Eastwood, J. (2017). Infant feeding practices and diarrhoea in sub-Saharan African countries with high diarrhoea mortality. PLoS One, 12(2), e0171792. doi:10.1371/journal.pone.0171792Ogbo, F. A., Nguyen, H., Naz, S., Agho, K. E., & Page, A. (2018). The association between infant and young child feeding practices and diarrhoea in Tanzanian children. Trop. Med. Health, 46, 2. doi:10.1186/s41182-018-0084-yOgbo, F. A., Page, A., Idoko, J., Claudio, F., & Agho, K. E. (2016). Diarrhoea and suboptimal feeding practices in Nigeria: Evidence from the national household surveys. Paediatr. Perinat. Epidemiol., 30(4), 346-355. doi:10.1111/ppe.12293Talukder, A., Akter, N., & Sazzad Mallick, T. (2019). Exploring Association Between Individuals' Stature and Type 2 Diabetes Status: Propensity Score Analysis. Environ. Health Insights, 13, 1178630219836975. doi:10.1177/1178630219836975AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 13 Mar 2020Associations between infant and young child feeding practices and acute respiratory infection and diarrhoea in Ethiopia: A propensity score matching approachPONE-D-19-33411R1Dear Dr. Ahmed,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: Yes: Salisu Ishaku MohammedReviewer #2: NoReviewer #3: No 17 Mar 2020PONE-D-19-33411R1 Associations between infant and young child feeding practices and acute respiratory infection and diarrhoea in Ethiopia: A propensity score matching approach Dear Dr. Ahmed:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Maria Christine Magnus Academic EditorPLOS ONE