The World Health Organization (WHO) recently changed its guidance for tuberculosis (TB) preventive treatment (TPT) recommending TPT for all pulmonary TB (PTB) exposed household contacts (HHC) to prevent incident TB disease (iTBD), regardless of TB infection (TBI) status. However, this recommendation was conditional as the strength of evidence was not strong. We assessed risk factors for iTBD in recently-exposed adult and pediatric Indian HHC, to determine which HHC subgroups might benefit most from TPT. We prospectively enrolled consenting HHC of adult PTB patients in Pune and Chennai, India. They underwent clinical, microbiologic and radiologic screening for TB disease (TBD) and TBI, at enrollment, 4–6, 12 and 24 months. TBI testing was performed by tuberculin skin test (TST) and Quantiferon®- Gold-in-Tube (QGIT) assay. HHC without baseline TBD were followed for development of iTBI and iTBD. Using mixed-effect Poisson regression, we assessed baseline characteristics including TBI status, and incident TBI (iTBI) using several TST and/or QGIT cut-offs, as potential risk factors for iTBD. Of 1051 HHC enrolled, 42 (4%) with baseline TBD and 12 (1%) with no baseline TBI test available, were excluded. Of the remaining 997 HHC, 707 (71%) had baseline TBI (TST ≥ 5 mm or QGIT ≥ 0.35 IU/ml). Overall, 20 HHC (2%) developed iTBD . HIV infection  and undernutrition  were independently associated with iTBD. iTBD was not associated with age, diabetes mellitus, smoking, alcohol, and baseline TBI, or iTBI, regardless of TST  or QGIT  cut-offs. Given the high overall risk of iTBD among recently exposed HHCs, and the lack of association between TBI status and iTBD, our findings support the new WHO recommendation to offer TPT to all HHC of PTB patients residing in a high TB burden country such as India, and do not suggest any benefit of TBI testing at baseline or during follow-up to risk stratify recently-exposed HHC for TPT. Household contacts (HHC) of pulmonary tuberculosis (PTB) patients are at high risk for acquiring TB infection (TBI) and TB disease (TBD) compared to the general population . ProvidiHowever, in light of the new WHO recommendation, India is currently considering revising their national guidelines. While HIV screening of HHC is recommended in India, screening HHC for other TB risk factors is not incorporated into current national guidelines. Using TST to screen Indian HHC for TBI, as well as screening for a history of diabetes, smoking and/or alcohol abuse, have been considered to prioritize additional HHC for TPT , 15–17. Routinely screening HHC for the risk factors of iTBD prior to recommending TPT would require significant resources for national TB control programs such as India’s. However, there are limited data available about the potential value of additional risk factor screening to inform the national TPT guidelines for TB-exposed HHC –24. AddiWith India’s massive burden of TBD , optimizBetween August 2014 and December 2017, as part of the Cohort for TB Research with Indo-US Medical Partnership (C-TRIUMPH) study , the NatA written informed consent was obtained from the participating adult HHC (≥ 18 years of age) and from the legal guardian if the participating HHC was a child <18 years of age. As per the local IRB norms a written informed assent was sought and obtained from children within the age group of ≥ 8 to < 18 years. The details of this study design and implementation have been previously described –31. Brie2 for HHC ≥ 18 years of age, BMI for age ≤ minus 2 standard deviations for HHC >10 and <18 years of age, and weight-for-age ≤ minus 2 standard deviations for HHC ≤10 years of age [At enrollment, we obtained socio-demographics, psycho-social history  score) , TB conts of age , 34. At At follow up, HHC underwent clinical and laboratory evaluations for signs and symptoms of TBD. TST was performed at baseline and repeated at 4–6, 12, and 24 months, if the prior TST was negative (defined as TST <10 mm). TST  was administered as 0.1 ml (≤ 5 tuberculin units) of Purified Protein Derivative (PPD) intradermally on the flexor aspect of the forearm. The reaction was read 48–72 hours later. The size of the reaction was determined by measuring the induration diameter in millimeters according to standard published methods . SimilarTBI was defined by applying each of the three distinct published TST induration cut-offs: ≥5mm , an induPrevalent or Baseline TBI of unknown duration included:OR a positive IGRA at baseline ORA HHC with a positive TST AND a positive IGRA at baselineA HHC with a positive TST Incident TBI (iTBI) included: Any HHC who was negative for TBD and TBI at baseline, diagnosed to have iTBI using the same criteria that was used at baseline (1a or 1b above) at any subsequent follow-up visit.confirmed TBD if they had a specimen from any source  that was positive by TB culture or GeneXpert/MTB Rif. An HHC was defined as having probable TBD if all specimens were negative by TB culture and GeneXpert, but they had a specimen from any source  that was positive on AFB smear. An HHC was defined as having possible TBD if all specimens were negative by TB culture, GeneXpert and AFB, but they were treated empirically for TB by the RNTCP based on clinical and/or radiologic findings. These same criteria were used to classify HHC as having prevalent TBD (defined as TBD diagnosed at enrollment) or iTBD (defined as TB diagnosed at any follow-up assessments among those who had no TBD at enrollment) [An HHC was defined as having microbiologically ollment) .Our analyses of HHC at baseline, included additional data that was not available for earlier C-TRIUMPH publications , 30. AftNext, the person-time follow up was calculated as the duration between date of enrollment of HHC to last study follow up date for each HHC, who were at risk of the event (iTBD or iTBI) being considered. The iTBD rate was calculated as number of HHC developing iTBD, per 1000 person-years (PY) follow up. To determine if the iTBD rate was dependent on the baseline TBI definition that was applied, iTBD rates for various combinations of TST cut offs  and IGRA cut offs , were compared. To describe the timing of TBD occurrence we calculated the proportion of HHC with TBD that were diagnosed at each study timepoint.To determine if screening for specific risk factors could identify HHC at higher risk for progression to TBD that could be prioritized for TPT, we examined the association between baseline characteristics of HHC (including multiple TST and/or IGRA definitions of baseline TBI) and iTBD, using univariate, multivariable, and mixed-effect Poisson regression analyses. The HHC characteristics found to be associated with iTBD in the univariate analysis were included in the overall model and/or the adult multivariate models, as relevant. Additionally, those HHC characteristics that were not statistically significant in the univariate analysis but known to be the published risk factors for iTBD , 15–17, Fig 1, a total of 1051 HHC from 442 households were enrolled. Forty-two (4%) HHC with TBD at baseline and 12 (1.1%) HHC with no baseline TBI testing available, were excluded. The median time from enrollment of the PTB index case to enrollment of their HHC was 1 day (Interquartile range (IQR): 0 to 8 days). PTB index patients reported an average symptom duration of 1.5 months (IQR: 1.0 to 3.0 months), prior to their TBD diagnosis and treatment initiation. HHC found to have baseline TBD reported an average symptom duration of 7 days (IQR: 3 to 8 days). Our analyses included 997 (95%) HHC with no evidence of baseline TBD and at least one TBI test available.As shown in Table 1, most (68%) HHC were adults ≥18 years of age, 56% were female, 2% were HIV-infected, 70% had a BCG scar, and 4% had past history of TB. Among adult HHC, 9% had DM, 9% were current smoker, 3% prior smokers, and 20% consumed alcohol.As shown in Table 2). The average family size for the HHC was 5 members (IQR: 4 to 6), 69% were urban households and 71% of HHC slept in the same room as the PTB index patient (Table 3).The PTB index patients for these HHC were predominantly male (61%), under the age of 45 years (63%), 92% had microbiologically confirmed TBD, 7% were HIV-infected, 26% had DM, 13% were current smokers, 18% former smokers and 44% consumed alcohol .Of 997 HHC, 908 (91%) had both TST and IGRA performed, 58 (6%) had only TST and 31 (3%) had only IGRA test performed. Seven-hundred-seven (71%) HHC had evidence of TBI with at least one test positive at baseline screening, of whom 301 (43%) demonstrated both a TST ≥5 mm and an IGRA OD ≥0.35 IU/ml. Of 290 (29%) HHC with no evidence of TBI at baseline, 246 (85%) had both tests performed and demonstrated a TST <5 mm and an IGRA OD <0.35 IU/ml while the remaining 44 (15%) HHC had only one of these two tests performed with a negative result (S1 Table show the iTBD rates stratified by different definitions of baseline TBI. The median time from enrollment of the PTB index patient to development of iTBD in the HHC was 5.6 months (Range: 4.4 to 10.4 months) with 13/20 (65%) occurring within 6 months of their baseline screening.Of 997 HHC, 20 (2%) subsequently developed iTBD during the 24-month follow-up period, yielding an estimated overall iTBD rate of 12 per 1000 PY (95% CI = 8 to 19/1000 PY). Of these, 2 HHC were under 6 years of age, yielding an estimated iTBD rate of 15 per 1000 PY (95% CI = 2 to 53/1000 PY). Tables 3). A multivariate model that included HHC characteristics , index patient characteristics ; identified HIV infection in HHC  and undernutrition  to be independently associated with increased risk of iTBD. Interestingly, there was no statistically significant difference in the risk of iTBD between HHC with and without baseline TBI, regardless of TST  or QGIT  cut-offs used to define baseline TBI or any other factors. A similar adult-only model that included DM, smoking and alcohol consumption in HHC as risk factors, both HIV  and undernutrition  were independently associated with iTBD (Data not shown). These two risk factors however only accounted for 8 (40%) of the 20 iTBD cases (2 with HIV-infection and 7 with undernutrition).Baseline characteristics of HHC, including their TBI status, were analyzed to identify risk factors for subsequent TBD that might identify HHC in India that could be prioritized for TPT. The univariate analyses identified only bed sharing with the index PTB patient  as a risk factor for iTBD (S2 Table).We further examined if recent conversion of TB infection (i.e. iTBI) was associated with risk of iTBD by first calculating the rates of TST and IGRA conversion. Two-hundred twenty-one (89.8%) of 246 HHC who were TBI negative for both tests at baseline had at least 1 subsequent follow-up test available. Of these 221, 123 (55.7%) HHC converted their TBI status, with an overall iTBI estimate of 491 per 1000 PY (95% CI 408 to 586/1000 PY). Of 123 HHC with iTBI, 67 (82%) were detected within 6 months of baseline screening. iTBI estimates varied depending on the TBI definition used (S3 Table).Finally, we compared and found that the iTBD estimates were similar for those HHC with and without incident TBI, irrespective of the TST or IGRA cutoffs used to define iTBI. In addition, the iTBD estimates were similar irrespective of whether the definition of iTBI was based on the requirement of both a positive TST and IGRA test (“AND”) or either test alone (“OR”)  were not associated with an increased risk for iTBD. Given the high baseline risk of iTBD in HHC, and the lack of association between TBI status and iTBD, our results support the new WHO guidelines, to offer TPT to all HHC of PTB patients in India.,.This study and our previous publications have demonstrated high (4%) prevalence of baseline TBD at time of initial HHC screening , 31, whiWe found that only baseline HIV and undernutrition were independently associated with iTBD. No other baseline HHC characteristics including age, gender, DM, smoking, alcohol, baseline TBI and iTBI status, were associated with a higher risk of developing iTBD. These findings were confirmed independently in two distinct multivariate analytical models- an overall model including all the age groups, and a model restricted to the adult population which additionally included variables for DM, smoking (pack-years based quantification of tobacco smoking) and alcohol consumption. However, none of these were found to be independent risk factors for iTBD in either of the analytical model. This may be partly explained based on a published literature review stating that importance of these risk factors depends on prevalence of each risk factor and therefore is subject to the variations between regions and countries . FurtherOther studies have reported that the greatest risk for TBD among HHC is within the first 12–24 months of their primary TBI –20, suggAn important limitation of our study was that we could not assess the impact of TPT (INH prophylaxis) in preventing the progression to TBD, because it is not currently recommended for all adult HHC in India. We also could not assess the predictive value of sustained versus transient IGRA conversion, with the risk of progression to TBD in different age groups .In summary, our study supports the new WHO guidelines to rapidly screen all HHC of PTB patients and to offer TPT to all HHC without TBD and do not suggest any clear benefit of TBI testing at baseline or during follow-up to further risk stratify recently-exposed HHC for targeted TPT .S1 TableThis table shows the incidence of TB disease (iTBD) among the household contacts (HHC) of adult pulmonary TB (PTB) patients in India, stratified using different TST  and/or IGRA  cut offs to define the baseline TB infection (TBI) status. The iTBD rates were similar irrespective of the individual test cut off used to define baseline TBI, and irrespective of whether the definition used both a positive TST and IGRA test (“AND”) or either test alone (“OR”).(DOCX)Click here for additional data file.S2 TableThis table shows the incident TB infection (iTBI) rates among household contacts (HHC) of adult pulmonary TB (PTB) patients in India, stratified using different thresholds for TST conversion and/or IGRA conversion to define iTBI status. The rates of iTBI were higher for lower TST/IGRA conversion thresholds as compared to higher TST/IGRA conversion; and the iTBI rates were higher when the iTBI definition used either test positive (“OR”) criteria as compared to both test positive (“AND”) criteria. In addition, changing the cut-off for a positive IGRA from ≥0.35 to ≥ 0.70 IU/ml did not significantly impact the iTBI estimates. However, increasing the induration TST cut-off from ≥ 5 mm to ≥ 10 mm or requiring a ≥ 6 mm increase in induration from previous reading resulted in lower iTBI estimates, regardless of the IGRA cut off used.(DOCX)Click here for additional data file.S3 TableThis table shows the incident TB disease (iTBD) rates among household contacts (HHC) of adult pulmonary TB (PTB) patients in India, stratified by incident TB infection (iTBI) status which was defined using different TST conversion cut offs  along with the IGRA conversion cut off of ≥ 0.35 IU/l. The comparison shows that the iTBD estimates were similar for those HHC with and without iTBI, irrespective of the TST or IGRA cutoffs used to define iTBI. In addition, the iTBD estimates were similar irrespective of whether the definition of iTBI was based on the requirement of both a positive TST and IGRA test (“AND”) or either test alone (“OR”).(DOCX)Click here for additional data file. 13 May 2020PONE-D-20-08293Tuberculosis preventive therapy should be considered for all household contacts of pulmonary tuberculosis patients in IndiaPLOS ONEDear Dr Paradkar,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. 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Reviewer #1: YesReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********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: 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: No**********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: It is a well-designed study that will add the evidence on LTBI treatment. It is also well written and analyzedAbstract: No commentIntroduction: No commentMethods: No commentResults: No commentDiscussion: No commentReference: No commentReviewer #2: This is a well-designed prospective study that has tried to use mixed-effect Poisson regression for inferential data analyzed. The study has also included a high number of HHC to support the WHO recommendation. It is also a timely and relevant study in one of the high TB burden countries, INDIA.General commentsThis study has come up with an interesting and relevant finding that warrants a detailed and a bit extended discussion. Because the author is claiming/recommending TPT for all HHC and thus supporting the WHO recommendation. As compared to the findings, however, the discussion section is a bit brief and shorter. It seems there are other important findings that needs discussion; for example, why age, diabetes mellitus, and alcohol consumption are not related to iTBD? Could the way age (of HHC and index cases) was categorized, smoking and alcohol consumption were classified, and DM patients were presented logical and acceptable? This necessitates to revise the analysis section. There need to be a discussion as to why these are a not a factor for iTBD in this study, as compared to previous studies that verified as these are strong factors that are related to TB diseases, such as studies mentioned in references 2, 14, and 16. This can have importance in terms of prioritizing TPT in some low income countries who could not afford TPT for all HCC. This is a critical issue in need to be investigated. This is because, the authors are arguing and trying to convince strongly that no HHC is to be prioritized, no need to test for TBI prior to TPT provision. This requires answering the following questions.1.Is it really feasible to provide TPT for all HHC considering limited logistics and availability of the newer combinations of drugs for TPT?2.Which HHC to be given a priority?Hence, the authors justification and arguments need to be a bit stronger and discussed in detail so that NTPs will be convinced to provide TPT for HHC without any prioritization and testing using TST or IGRA.Besides, the way authors are listed and narrated, and their affiliation may be revised to align with the PLOS I format. The study could benefit from language revision; a bit longer and vague sentences are noted. The references as inserted in the body of the manuscript and listed in the reference need revision throughout. The formatting in the reference is not consistent and some are  incomplete. It is also worth considering the use of recent references; there are references older than 10 years .At end, the headings in lines 136, 147, 164,251 and 287 need to be revised and well narrated; at least, the author need to avoid the use of abbreviations/acronyms in the headings.Specific comments1. A sentence in Lines 32 and 33 is very important sentence but lacks clarity. In the same sentence, it is narrated that the study determined which HHC group are beneficial from TPT in India and other high TB burden country. Is that the real and specific objective of the study, as it was carried out among the Indian population?2. TBD in line 35 should be fully written as it appeared for the first time.3. A sentence in lines 33-36 could be revised to be narrated clearly and succinctly. As currently written, it is long and difficult to understand.4. Line 59 & 60, the India’s contribution to the global TB incidence could be described in the form of proportion, 28% (2.8 of 10 million) or near to one-third of… Similarly, line 91 need to be revised.5. Who were the children in your study (as related to age category)? Can we define them with reference?6. A sentence in lines 108-110 is not clear and needs revision.7. What was applied, oral or written informed consent? How was the consent of a child requested and obtained?8. What are the specific psycho-social and medical history in lines 116 & 123, and household characteristics in line 132?9. What is/are the reference (s) for the definition in lines 121-123 and 165-174?10. A sentence in lines 126-131 is too long and better be narrated again. For example, presumptive TB cases should be defined, and which microbiological, tissue-based, or radiologically investigations are indicated for which specific signs or/and symptoms detected during the follow up?11. Consider revision for a sentence in lines 141-143, seems two sentences.12. A sentence in lines 148-149 lacks clarity.13. On what basis was the age category made or classified as described in table 1?14. Having TBD, and HHC without baseline TBI test the only exclusion criteria?15. What IQR stands for in Line212-213?16. Line 241-242 is not a result. The author may consider moving to data analysis section of the method.17. The widowed and divorced  are with lower number to be categorized for the univariate and multivariate analysis. Consider categorizing these again.18. What were the criteria to include the HHC characteristic to multivariate analysis? A lot of variables are not considered to be part of the cultivatable analysis, Table 1. For instance, p-value of 0.65 was included while p-value of 0.67 not included in the multivariate analysis. What does the sentence in lines 198-199 mean? These better be aligned with the way real analysis was made.19. Where in the study have you applied the Fisher’s exact test and Wilcoxon rank sum tests? Because these were stated in lines 181-182.20. How was the level of alcohol consumption and smoking level determined? If possible, objectively quantifying the degree of alcohol consumption and smoking is a better option. It seems that a fewer month’s period of smoking and alcohol consumption were lumped up with the heavy alcohol consumption and a longer period smoking. If the level or degree of smoking or alcohol consumption is not objectively defined, the relation or impact these have on the TBD is not well determined21. Why Table 1 and 3 are written separately? Why not the author considers HHC characteristics in one go? The other option is that tables 1-3 could be presented in two forms; first table/s could be committed to the description of the HHC and index cases, and the second table/s could present the result of the univariate and multivariate analysis.22. Check the consistency in the content of sentences in lines 66& 67, and lines 345 &346.23. The sentence in lines 371-373 is critical yet needs revision to make it so clear. The way “resource’ is used makes the sentence a bit confusing.**********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,  8 Jul 2020Dated 8th July, 2020Editor-in-Chief,Plos OneSubject: #PONE-D-20-08293, Response to Reviewers for the manuscript titled, “Tuberculosis preventive therapy should be considered for all household contacts of pulmonary tuberculosis patients in India”. We thank you for the review of our manuscript. We have now revised the draft as per reviewer’s comments and have addressed each comment as below. We believe the changes suggested by the reviewers made the manuscript stronger and hope that you will consider our revised manuscript for publication.Reviewer #1: No commentsResponse: We appreciate the review and agreement with our manuscript.Reviewer #2: This is a well-designed prospective study that has tried to use mixed-effect Poisson regression for inferential data analyzed. The study has also included a high number of HHC to support the WHO recommendation. It is also a timely and relevant study in one of the high TB burden countries, India.Response: We appreciate your review of the manuscript and thank you for highlighting the importance of our findings.1. General commentsa. Comment: This study has come up with an interesting and relevant finding that warrants a detailed and a bit extended discussion. Because the author is claiming/recommending TPT for all HHC and thus supporting the WHO recommendation. As compared to the findings, however, the discussion section is a bit brief and shorter. It seems there are other important findings that needs discussion; for example, why age, diabetes mellitus, and alcohol consumption are not related to iTBD? Response: We have updated the draft to expand the discussion to address the specific issues that the reviewer has raised below. b. Comment: Could the way age (of HHC and index cases) was categorized, smoking and alcohol consumption were classified, and DM patients were presented logical and acceptable? This necessitates to revise the analysis section. There need to be a discussion as to why these are a not a factor for iTBD in this study, as compared to previous studies that verified as these are strong factors that are related to TB diseases, such as studies mentioned in references 2, 14, and 16. Response: We thanks the reviewer for raising this very important issue with respect to the age, smoking, alcohol consumption and diabetes mellitus (DM) variables used in the analysis. i. We chose our age groups of the household contacts (HHC) based on the following: 1) <6 years is the cut-off for current Indian TB guidelines for TPT household contact recommendations in children. 2) 6-12 years was selected to include the older children. 3) 13-17 years represents the adolescents 4) 18-44 years to represent the younger adults and 5) ≥ 45 years was to represent the older adults. Also, we used age as a continuous variable in the Poisson regression analysis and did not find an association between age and iTBD.ii. The data on smoking was collected under three mutually exclusive categories for the smoking variable, namely- current smoker, former smoker and non-smoker. However, there was only one former smoker who developed the outcome of incident TB disease (iTBD), while no current smoker developed iTBD. Therefore, in the original analysis presented in Table 1, we had combined these two categories as ‘Yes’ (any history of smoking) while the reference category being ‘No’ (no history of current or past smoking). Furthermore, there was no change in the inferences even when univariate and multivariate analysis were performed using the two smoking categories separately. Alternatively, as per the reviewer’s recommendation, to assess the impact of degree of smoking objectively and quantitatively, we also calculated the number of pack-years to reclassify the HHC in 3 categories of current smoker, former smoker and non-smoker and again separately performed the univariate and multivariate analysis (adult only model). This reclassification, however, did not change our conclusions in terms of association with iTBD. We have replaced the originally presented smoking variable with new smoking variable which is based on the pack-years analysis, in the revised Table 1. iii. We had also collected the ‘Alcohol Use Disorders Identification Test (AUDIT) score’, to quantitatively assess the alcohol dependence. Alcohol use disorder (AUD) was defined as having an AUDIT score of at least 8 points. However, there was no HHC with AUD who developed the outcome of incident TB disease (iTBD). Therefore, we have used the originally presented alcohol consumption variable in the revised Table 1. iv. Furthermore, we also revised the adult multivariate model replacing the original smoking variable with the new smoking variable described above  however there was no change the in our conclusions in terms of association with iTBD. v. As defined in Table 1, footnote b, the standard definition of DM was used . The prevalence of DM among the at-risk population (adults) was as low as 9% (70 out of 734 adult HHC had DM), while only 1 of these 70 HHC with DM developed iTBD. In conclusion, no statistically significant association was found between any of the aforementioned variables  and iTBD. The lack of association with these risk factors in our study may possibly be due to- a) possible selection bias as this is not a population based study but a clustered HHC analysis, b) those with DM were more likely to be already diagnosed with prevalent TBD and therefore excluded from the analysis, c) Interaction of DM with malnutrition which is evident by DM having a non-significant protective effect due to higher BMI in diabetics, and d) due to the relatively low number of incident cases, we may not have power to identify individual risk factors that have relatively low prevalence in the HHC.c. Comment: This can have importance in terms of prioritizing TPT in some low-income countries who could not afford TPT for all HHC. This is a critical issue in need to be investigated. This is because, the authors are arguing and trying to convince strongly that no HHC is to be prioritized, no need to test for TBI prior to TPT provision. This requires answering the following questions.i. Is it really feasible to provide TPT for all HHC considering limited logistics and availability of the newer combinations of drugs for TPT?ii. Comment: Which HHC to be given a priority? Hence, the authors justification and arguments need to be a bit stronger and discussed in detail so that NTPs will be convinced to provide TPT for HHC without any prioritization and testing using TST or IGRA.Response: We agree with the reviewer’s comment about the importance of these issues. We felt this was a timely study, because the current WHO TPT recommendations and Indian guidelines are considering this as we described in lines 54-56, 66-67. The fact that we found that pre-screening test for identifying incident TB infection (iTBI) was not necessary to determine the eligibility for TB preventive treatment (TPT), makes provision of TPT for all HHC more feasible. The iTBI status itself was comprehensively assessed using all the possible definitions based on different published TST conversion and IGRA conversion cut off values, both in combinations and also by individual test result. However, even after using each of these iTBI definitions, we did not find any impact on iTBD.As noted in the result section and in response to point 1.b. above, our findings suggest that with the exception of malnutrition and HIV positive status in the HHC, no other HHC subgroup is more likely to benefit from TPT than other HHCs. Furthermore, though some of the HHC subgroups like those with malnutrition and HIV infection, may benefit the most from TPT, nevertheless, the iTBD risk in HHC was much higher than what has been reported in the general population in India or South Africa. Therefore, being HHC of PTB patient itself is an important risk factor for iTBD and since our study did not find any predictors of disease progression, all HHC should be given TPT. Lastly, it might be programmatically easier to provide TPT to all HHC in the light of the fact that about 75% of the HHC in our study, meet a high risk criteria of either being a child < 6 years of age or among those ≥ 6 years of age with a positive TBI test or being HIV infected. However, we acknowledge that this is not a feasibility/cost-effectiveness study, so assessing the feasibility is beyond the scope of this analysis. Future feasibility/cost-effectiveness studies can help address this important consideration. To address these critical issues in above points 1.a., 1.b. and 1.c., we have revised the methods section (Lines 121-134), the results section  and the discussion section (Lines 343-410), in the revised manuscript.d. Comment: Besides, the way authors are listed and narrated, and their affiliation may be revised to align with the PLOS I format. Response: The author list and affiliations sequence have been revised and the appropriate symbols are used in the revised manuscript to align with the PLOS One format as per the reviewer’s comment.e. Comment: The study could benefit from language revision; a bit longer and vague sentences are noted. Response: The manuscript has been revised thoroughly to address reviewer’s comment. f. Comment: The references as inserted in the body of the manuscript and listed in the reference need revision throughout. The formatting in the reference is not consistent and some are  incomplete. Response: The citations in the manuscript body and the reference list has been revised thoroughly to address the reviewer’s comment. g. Comment: It is also worth considering the use of recent references; there are references older than 10 years .Response: The original references 17 and 35 have been replaced with a recent reference as suggested by the reviewer. The original references 44 and 48 have been removed since there are more recent supporting references already cited for the relevant lines. The original references 35, 36, 37 are still relevant and form the basis for the TST induration cut offs used in the analysis, therefore, these references are retained in the revised manuscript. h. Comment: At end, the headings in lines 136, 147, 164,251 and 287 need to be revised and well narrated; at least, the author need to avoid the use of abbreviations/acronyms in the headings.Response: The headings in the lines aforementioned by the reviewer are appropriately narrated and the abbreviations have been replaced with full forms, in lines 154, 165, 182, 269, and 305 of the revised manuscript. 2. Specific commentsa. Comment: A sentence in Lines 32 and 33 is very important sentence but lacks clarity. In the same sentence, it is narrated that the study determined which HHC group are beneficial from TPT in India and other high TB burden country. Is that the real and specific objective of the study, as it was carried out among the Indian population?Response: This has been clarified in the introduction by removing the phrase “other high TB burden country”, in lines 32-34 in the revised manuscript. b. Comment: TBD in line 35 should be fully written as it appeared for the first time.Response: In the revised manuscript, TB disease (TBD) is written fully in line 34, where it has been mentioned for the first time. c. Comment: A sentence in lines 33-36 could be revised to be narrated clearly and succinctly. As currently written, it is long and difficult to understand.Response: The original sentence has been simplified in multiple sentences in the lines 34-36 of the revised manuscript, for the purpose of clarity.d. Comment: Line 59 & 60, the India’s contribution to the global TB incidence could be described in the form of proportion, 28% (2.8 of 10 million) or near to one-third of… Similarly, line 91 need to be revised.Response: In the revised manuscript, the global TB incidence is described in the form of proportion in lines 61-62 and the original sentence in line 91 has been deleted. e. Comment: Who were the children in your study (as related to age category)? Can we define them with reference?Response: As mentioned in the point 1.b. above, broadly, HHC < 18 years of age (non-adult HHC), including the younger children, the older children and the adolescents, are referred to as children. In India, ≥ 18 years is legally the age when a minor individual attains adulthood. f. Comment: A sentence in lines 108-110 is not clear and needs revision.Response: The relevant sentence has been simplified for clarity, in lines 110-112 of the revised manuscript.g. Comment: What was applied, oral or written informed consent? How was the consent of a child requested and obtained?Response: The written informed consent was obtained from the participating adult HHC (≥ 18 years of age) and from the legal guardian if the participating HHC was a child < 18 years of age. As per the local IRB norms a written informed assent was sought and obtained from children within the age group of ≥ 8 to < 18 years. This has been clarified in lines 112-117 of the revised manuscript. h. Comment: What are the specific psycho-social and medical history in lines 116 & 123, and household characteristics in line 132?Response: We have clarified the data variables referred to under the psychosocial, medical history and household characteristics, in lines 122-135, 150-151 of the revised manuscript. i. Comment: What is/are the reference (s) for the definition in lines 121-123 and 165-174?Response: The relevant references have been added for the definitions of undernutrition and TBD in lines 136-139 and lines 184-192 of the revised manuscript, respectively. j. Comment: A sentence in lines 126-131 is too long and better be narrated again. For example, presumptive TB cases should be defined, and which microbiological, tissue-based, or radiologically investigations are indicated for which specific signs or/and symptoms detected during the follow up?Response: The sentence has been simplified by splitting in two sentences in lines 142-149 of the revised manuscript. Also, symptoms and corresponding investigations have been clarified as suggested by the reviewer.k. Comment: Consider revision for a sentence in lines 141-143, seems two sentences.Response: The sentence has been simplified by splitting in two separate sentences in lines 160-162 of the revised manuscript.l. Comment: A sentence in lines 148-149 lacks clarity.Response: The sentence has been simplified for clarity in lines 167-168 of the revised manuscript.m. Comment: On what basis was the age category made or classified as described in table 1?Response: As mentioned above in point 1.b., the age groups were classified based on the following: 1) <6 years is the cut-off for current Indian TB guidelines for TPT household contact recommendations in children. 2) 6-12 years was selected to include the older children. 3) 13-17 years represents the adolescents 4) 18-44 years to represent the younger adults and 5) ≥ 45 years was to represent the older adults. n. Comment: Having TBD, and HHC without baseline TBI test the only exclusion criteria?Response: To ensure that we include only those HHC in the analysis who were at risk of TBD during the study follow up, we excluded HHC with TBD diagnosed at baseline. To identify the TBI test (TST and/or IGRA) conversion and its impact on the incident TBD, availability of at least one test result (TST or IGRA) at baseline was essential, therefore the HHC with no baseline TBI test results for both the tests were excluded. There were the only two exclusion criteria applied, as depicted in Figure 1. o. Comment: What IQR stands for in Line 212-213?Response: IQR stands for interquartile range. This full form has been added at the first mention in line 233, in the revised manuscript.p. Comment: Line 241-242 is not a result. The author may consider moving to data analysis section of the method.Response: The sentence has been moved to the data analysis section, in lines 198-200 of the revised manuscript. q. Comment: The widowed and divorced as a sub-category of marital status are with lower number to be categorized for the univariate and multivariate analysis. Consider categorizing these again.Response: The widowed and divorced categories have been merged in Table 1 of the revised manuscript. The original findings still do not change after this re-categorization as there were no HHC from this category who developed iTBD. r. Comment: What were the criteria to include the HHC characteristic to multivariate analysis? A lot of variables are not considered to be part of the multivariable analysis, Table 1. For instance, p-value of 0.65 was included while p-value of 0.67 not included in the multivariate analysis. What does the sentence in lines 198-199 mean? These better be aligned with the way real analysis was made.Response: The sentence in original lines 198-199 means that, those HHC characteristics which were found to be associated with iTBD in the univariate analysis were included in the overall model and/or the adult multivariate models, as relevant. Additionally, those HHC characteristics that were not statistically significant in the univariate analysis but known to be the published risk factors for iTBD, were included in the multivariate model. This has been clarified in lines 215-219 of the revised manuscript.s. Comment: Where in the study have you applied the Fisher’s exact test and Wilcoxon rank sum tests? Because these were stated in lines 181-182.Response: We agree with the reviewer that Fisher’s exact and Wilcoxon rank sum tests were not applied in the final analysis presented. Therefore, the relevant sentence has been deleted.t. Comment: How was the level of alcohol consumption and smoking level determined? If possible, objectively quantifying the degree of alcohol consumption and smoking is a better option. It seems that a fewer month’s period of smoking and alcohol consumption were lumped up with the heavy alcohol consumption and a longer period smoking. If the level or degree of smoking or alcohol consumption is not objectively defined, the relation or impact these have on the TBD is not well determined.Response: Please refer to our detailed response to point 1.b. above, which addresses the impact of objective quantification of smoking and alcohol variables on the association with iTBD.u. Comment: Why Table 1 and 3 are written separately? Why not the author considers HHC characteristics in one go? The other option is that tables 1-3 could be presented in two forms; first table/s could be committed to the description of the HHC and index cases, and the second table/s could present the result of the univariate and multivariate analysis.Response: We appreciate the reviewer’s suggestion to either have tables 1-3 merged or present them as two tables instead of three separate tables, but that would make the tables very lengthy. However, we are happy to revise the tables as per the editor’s preference. v. Comment: Check the consistency in the content of sentences in lines 66& 67, and lines 345 &346.Response: These sentences in lines 66-67 and lines 345-346  are both different. Line 66-67 mentions that revision of Indian guidelines for provision of TPT is currently under consideration, however are not yet revised. Line 345-346 states the current guideline regarding provision of TPT. w. Comment: The sentence in lines 371-373 is critical yet needs revision to make it so clear. The way “resource’ is used makes the sentence a bit confusing.Response: This critical sentence in the discussion section (Lines 408-411) is revised as follows, “In summary, our study supports the new WHO guidelines to rapidly screen all HHC of PTB patients and to offer TPT to all HHC without TBD and do not suggest any clear benefit of TBI testing at baseline or during follow-up to further risk stratify recently-exposed HHC for targeted TPT.” Journal requirementsWhen submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfResponse: The manuscript has been revised and the files have been renamed, to meet the PLOS ONE’s style requirements. 2. Please consider modifying your title to ensure that it is specific, descriptive, concise, and comprehensible to readers outside the field.Response: The title has been revised as follows, “Tuberculosis preventive treatment should be considered for all household contacts of pulmonary tuberculosis patients in India”http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. 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For information about this choice, including consent withdrawal, please see our Privacy Policy.Yes: Zewdu Gashu DememewReviewer #2:  16 Jul 2020PONE-D-20-08293R1 Tuberculosis preventive treatment should be considered for all household contacts of pulmonary tuberculosis patients in India Dear Dr. Paradkar:I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. 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Antimicrobial resistance (AMR) is a phenomenon resulting from the natural evolution of microbes. Nonetheless, human activities accelerate the pace at which microorganisms develop and spread resistance. AMR is a complex and multidimensional problem, threatening not only human and animal health, but also regional, national, and global security, and the economy. Inappropriate use of antibiotics, and poor infection prevention and control strategies are contributing to the emergence and dissemination of AMR. All healthcare providers play an important role in preventing the occurrence and spread of AMR. The organization of healthcare systems, availability of diagnostic testing and appropriate antibiotics, infection prevention and control practices, along with prescribing practices (such as over-the-counter availability of antibiotics) differs markedly between high-income countries and low and middle-income countries (LMICs). These differences may affect the implementation of antibiotic prescribing practices in these settings. The strategy to reduce the global burden of AMR includes, among other aspects, an in-depth modification of the use of existing and future antibiotics in all aspects of medical practice. The Global Alliance for Infections in Surgery has instituted an interdisciplinary working group including healthcare professionals from different countries with different backgrounds to assess the need for implementing education and increasing awareness about correct antibiotic prescribing practices across the surgical pathways. This article discusses aspects specific to LMICs, where pre-existing factors make surgeons’ compliance with best practices even more important. Antibiotics have saved millions of lives over years and have even been used prophylactically for the prevention of infectious diseases. However, we have now reached a crisis where many antibiotics are no longer effective. AMR is a natural phenomenon that happens as microorganisms evolve, however human activities have altered the pace at which microbes develop and spread resistance ,4,5.AMR is one of the major public health concerns of this century , threateThe drivers of AMR encompass not only antibiotic use, misuse, and abuse in human, animal, and environmental sectors, but also the dissemination of resistant microorganisms within and among these sectors, and therefore around the world ,8. HumanIn May 2015, the World Health Assembly endorsed a global action plan to fight AMR. Five strategic objectives were set out: (1) strengthening both understanding and awareness of AMR; (2) improving knowledge through surveillance and research; (3) reducing the incidence of infection; (4) optimizing the use of antibiotic agents; (5) developing the economic case for sustainable investment, taking into consideration the needs of all countries, and increasing investment in new medicines, vaccines, diagnostic tools, and other interventions .AMR is a complex, multifaceted problem that threatens human and animal health, the global economy, national and global security. In line with a One Health approach, healthcare plays an important role in preventing the emergence and spread of AMR. The success of modern medicine, including organ transplantation, cancer therapy, management of prematurity, or a surplus of advanced major surgeries, might not have been possible without effective antibiotics to control bacterial infections.Patients often face multiple risk factors exposing them to AMR. Acute care facilities are incubators and highly susceptible vulnerable populations create an environment which facilitates both the emergence and transmission of resistant organisms, especially in intensive-care units managing surgical cases. Across the surgical pathway, surgical site infections (SSIs) remain a common postoperative complication worldwide despite the use of prophylactic antibiotics and other preventive measures ,12, and Enterococci spp., all the isolated bacteria were resistant to ampicillin. All the isolated Klebsiella spp. were found to be MDR. Staphylococcus aureus isolates were all resistant to oxacillin. The isolation rate of MDR organisms was higher in emergency, males, and dirty wounds in relation to type of surgery, gender and class of surgical wound respectively. A study carried out at the university hospital of Parakou, Benin in 2017 revealed that 41.2% of the microbial germs isolated in the surgical departments during SSIs were MDR [The problem of AMR seems to represent a special challenge in LMICs characterized by the scarcity of biological and epidemiological data, a lower level of awareness and the absence of locally developed guidelines in most sites. The few existing data are alarming. In a hospital based descriptive cross-sectional study conducted on 83 consented postoperative patients with clinical SSIs at Mbarara regional referral hospital in Uganda a very high AMR rate of bacteria isolated was reported . Overallwere MDR .The appropriate use of antibiotics plays an essential role in optimizing clinical practice. Antibiotics may be lifesaving however these are often used inappropriately, especially when they are unnecessary, or when they are administered without taking into account pharmacokinetic principles, or when they are administered for an excessive duration . The misInfections require not only prompt diagnosis and immediate source control, but also adequate antibiotic therapy . InapproAMR containment interventions in healthcare facilities have mostly been implemented in high-income countries. There is a persistent need to intervene in LMICs since the burden of AMR in LMICs is difficult to quantify, because surveillance activities to lead interventions require not only time, but also financial resources. Moreover, a basic background in epidemiology, microbiology, and communicable diseases should be required, in addition to expertise in data management and analysis ,19. In tAs far back as 2005 a publication from South Africa noted a rapid increase in the presence of resistant strains of infectious organisms across the levels of care with, as expected a higher rate among patients in tertiary facilities . Recent In order to describe and understand the existing status of AMR in Africa, in relation to common causes of infections, and antibiotics recommended in treatment guidelines, an interesting review was published in 2017 using a public health focused approach . The revThe drivers of AMR in Lebanon are multifactorial due to the absence of adequate national surveillance system, the lack of regulations controlling the agricultural industry, the low level of public awareness about antibiotics and the poor management of sanitation and waste. The concept of “healthy humans, animals and environment” is not part of any national plan nor is it a priority on the Lebanese government agenda. The concept of “One Health” is non-existent except for few efforts from non-governmental organization. In Lebanon, antibiotics are available over the counter , and selSignificant challenges in LMICs include the availability of antibiotics over-the-counter, the spread of counterfeit medications, uses of antibiotics in animal health and the agricultural sector in the absence of legal boundaries and limited leadership and governance from health policy decision makers. In an era of limited resources, LMICs are facing major hurdles that shift the focus away from this pandemic that is silently expanding in scope and in scale and reaching not only the individual but also the society, the nation and the entire global ecosystem. Challenges to be overcome in facing AMR in LMICs are reported in Furthermore, addressing AMR in LMICs requires strong political will. Continuous human, infrastructural, and operational resources are needed in order to broadly recognize the nature and extent of AMR, and consequently to implement and update regional and national action plans for AMR prevention and containment within national health systems. Action plans require a multidisciplinary partnership among private and public sectors, civil society and non-profit organizations, patients, healthcare professionals, and communities, and multinational pharmaceutical industry and governments, supported by clear international, national, and regional policy frameworks that suspend private interests for public good .In order to preserve the positive externality of antibiotics, that is the benefit of these medications for the future generations, mitigating AMR should start by fostering the correct use of antibiotics within the One Health framework such as restricting access to antibiotics and shifting status to prescription only for use in human health. The role of antibiotics in animal health and the environmental sector is a crucial part of the solution which must be better regulated but this is not in the scope of this review. In healthcare sectors, applying evidence-based infection control and prevention and implementing antibiotic stewardship programs adapted to the limited resources and expertise pitfalls. In this complex setting physicians are faced with major challenges related to inappropriate microbiology results, questionable infection control practice, limited human resources, lack of public awareness and knowledge about AMR. The good news is that there has been substantial progress in some LMICs, particularly in Africa and especially in South Africa, with the establishment of Antibiotic Surveillance and Stewardship programs in a number of private and public hospitals, with resultant changes in prescribing practices, including those of surgeons. Hospital-based pharmacist-led programs have been instrumental in improving basic hygiene practices and reducing sepsis through hand-washing campaigns, despite numerous challenges common in LMIC’s  , and theA major proportion of the antibiotics prescribed within hospitals are for surgical patients. As observed in a recent worldwide cross-sectional survey, although most surveyed surgeons are aware of the problem of AMR, they underestimate this problem in their own hospital . Both poAppropriate use of antibiotics is an important part of any antimicrobial stewardship program (ASP), and it is necessary to decrease the emergence and dissemination of AMR, and—as a consequence—to guarantee both good clinical practice, and optimal standards of care. Nevertheless, antibiotic prescribing practices among surgeons are often inappropriate, and across the surgical pathway, a noteworthy gap exists between the best evidence and clinical practice .Surgical antibiotic prophylaxis (SAP) plays a pivotal role in a perioperative infection prevention approach ,35,36. TAntibiotic therapy has an important role in the management of surgical infections, particularly in critically ill patients who need immediate empiric antibiotic therapy. Both poor antibiotic coverage, and inappropriate regimens represent the factors most strongly associated with unfavourable outcomes . The dosSurgeons play an essential role in prevention and treatment of infections , and theBecause of the lack of reliable and effective surveillance systems, in LMICs all physicians and surgeons can lack updated information on the AMR pattern within their populations they take care of. In particular it occurs in rural settings where the absence of capacity to perform AMR testing imply difficulties to select the correct antibiotic in the absence of an antibiotic sensitivity test, and here guidance from National Antibiotic Guidelines can be useful as an adjunct for appropriate empiric antibiotic selection . As a reWhile the World Health Organization has reviewed the list of available antimicrobials and classified some as “critically important antimicrobials” , assess ASPs have been implemented to optimize antibiotic utilization, reduce the emergence and spread of AMR, and improve patient outcomes. Nonetheless, the best strategies for ASPs are not absolutely established, and are likely to be different based on local routine clinical practice, culture, and policy, and probably due to limited available resources in LMICs. Although ASPs are needed in LMICs, in these countries all the components necessary for their successful implementation are seldom in place. While in high income countries, hospital ASPs typically include a multidisciplinary committee, continuous monitoring of antibiotic use and their resistance patterns and evaluation of intervention outcomes, along with development of evidence based local treatment guidelines and antibiotic formularies. However, these components are either not present at all or exist at a bare minimum level due to the above-mentioned limitations of human and organizational resources, infrastructure, and funding in LMICs .In order to evaluate the effectiveness of ASPs in hospitals in LMICs an interesting systematic review was published in 2018 . The majPrograms to improve antibiotic prescribing practices among surgical patients in South Africa have shown promise. In both the private sector and the public sector institution of various programs have improved prescribing practises and ensured compliance with step-wise antibiotic protocols based on surveillance and antibiotic stewardship. These programs have shown reductions in unnecessary antibiotic use, redundant drugs and unnecessary prolonged treatment durations . Even emSurgical infections largely contribute to the massive problem of infectious diseases worldwide. One recent scoping review revealed that supply-side intervention strategies are the most successful approach to rectify irrational practices and promote prudent prescription of antibiotics; thereby, this tactic has been considered as the principal protector of overuse, misuse, irrational consumption of antibiotics in LMICs . AnotherAlthough the control of AMR cannot be the sole responsibility of healthcare professionals, they have a pivotal role in containing it. Continuing education is essential to enable physicians learning the need for rational prescription practices and the importance of evidence-based prescription. Antibiotic misuse and overuse is a problem observed worldwide, not only in high income countries, but in LMCIs as well. The requisite to formulate, communicate, and adopt rigorous policies for appropriate antibiotic use is more pressing in LMCIs where the greatest levels of abuse are encountered. This is not difficult but requires a compliant mindset change and teamwork, even in rural or low-resourced settings.The surgeon’s role is paramount in preventing and managing infections which often need a prompt source control, and an appropriate antibiotic therapy, being directly responsible for their outcome. Surgeons must improve the quality of surgical care and avoid inappropriate antibiotic prescribing in surgery, with patients at risk of receiving prolonged durations of antibiotics for prophylactic and therapeutic indications.A well-established antibiotic policy would go a long way towards achieving a reduction of inadequate prescribing. Moreover, the issue can be addressed with a reasonable degree of success when prescribing surgeons have adequate relevant knowledge of both the properties of antibiotic agents and the pathogens that are likely to cause infection or resistance profiles to commonly available antibiotics.


ULTRAPETALA1 (ULT1) is a versatile plant-exclusive protein, initially described as a trithorax group (TrxG) factor that regulates transcriptional activation and counteracts polycomb group (PcG) repressor function. As part of TrxG, ULT1 interacts with ARABIDOPSIS TRITHORAX1 (ATX1) to regulate H3K4me3 activation mark deposition. However, our recent studies indicate that ULT1 can also act independently of ATX1. Moreover, the ULT1 ability to interact with transcription factors (TFs) and PcG proteins indicates that it is a versatile protein with other roles. Therefore, in this work we revised recent information about the function of Arabidopsis ULT1 to understand the roles of ULT1 in plant development. Furthermore, we discuss the molecular mechanisms of ULT1, highlighting its epigenetic role, in which ULT1 seems to have characteristics of an epigenetic molecular switch that regulates repression and activation processes via TrxG and PcG complexes. In multicellular organisms, epigenetic regulation plays crucial roles for the correct deployment of developmental programs and for the establishment of cell fates. Epigenetic mechanisms include post-translational histone modifications (PHM) that modulate chromatin structure to regulate gene expression. The trithorax group (TrxG) is an epigenetic protein complex able to regulate transcriptional activation through trimethylation of lysine 4 and 36 of histone H3 (H3K4me3 and H3K36me3) as well as other associated PHMs . TrxG prArabidopsis thaliana (hereafter Arabidopsis), the main HMTs of TrxG that catalyze the H3K4me3 mark are the ARABIDOPSIS TRITHORAX1 (ATX1) and the ARABIDOPSIS TRITHORAX-RELATED 3/SETDOMAIN GROUP 2 (ATXR3/SDG2)  involved in stem cell maintenance, cell fate identity, and cell proliferation and differentiation . The plaR3/SDG2) , althougR3/SDG2) . InteresR3/SDG2) , whose sR3/SDG2) . HoweverR3/SDG2) . MoreoveR3/SDG2)  suggest Oryza sativa, it has been shown that is important for its multimerization  homolog recognizes the 5′-TTCG-3′ sequence  domain protein that also contains a B-box motif , a motifrization . Meanwhirization , and it rization . In ULT rization . The SANrization . Moreoverization  and alsorization . Therefosequence . This sesequence .Volvox carteri, RegA is involved in somatic cell differentiation ; however, several ULT1 sequences have been reported in other species. In this kingdom, ULT1 seems to be a protein exclusive to Angiosperms, since Gymnosperm, Lycophytes or Mosses lack sequences homologous to ULT1. In angiosperms ULT1 is highly conserved in different species of Eudicots, Monocotyledons, and even in Amborellales, considered one of the most basal angiosperms , the lato Poales . Evolutio Poales . The higULT1 loss and gain-of-function mutant plants (APETALA3 (AP3) and AGAMOUS (AG) (WUSCHEL (WUS) expression  , two genOUS (AG) . Howeverpression , a TF thpression . Therefopression , as wellpression , togethepression .ult1 mutant plants have lower levels of H3K4me3 marks on AG and AP3 genes, which are associated with an increase of H3K27me3 PcG mark on these ULT1 targets  ULT1 acts independently of ATX1  are deregulated in ult1 mutants, and among them only a little subset is shared  in both atx1 and ult1 mutants  of Arabidopsis genes are deregulated in  mutants ; althoug mutants , ATX1 is mutants , evidencThe ATX1-independent function of ULT1 raises the question whether ULT1 acts together with other HMTs of the TrxG complex or by a TrxG-independent mechanism or both in different developmental processes. The analysis of ULT1 protein interactions in different developmental contexts could provide evidence compatible with both mechanisms as shown below.SPATULA (SPA) expression, which promotes carpel marginal tissue apical style and stigma tissue formation  has been reported. UIF1 binds to meristem . Given terminacy .These reports have led to suggestions that ULT1 can act as a link between chromatin-remodeling factors and some TFs . Howeverult1 mutants compared with those observed in atx1 mutants  gene, which is activated by TrxG and repressed by PcG  ULT1 is also involved in the regulation of gynoecium patterning, in which it interacts with KAN1 to repress SPT, (4) ULT1 together with EMF1 maintains repressive marks of some genes related to seed development, and (5) ULT1, independently of ATX1, is involved in the root SCN maintenance  ULT1 activates genes related to floral development through its interaction with ATX1, (2) in association with UIF1, ULT1 represses ntenance . The abiult1 mutants, and their genome-wide effects make it difficult to define modes of action of ULT1. However, these reports illustrate four possible ways of action for ULT1: (i) together with TrxG factors, (ii) with PcG factors, (iii) outside of both TrxG/PcG complex, and (iv) in association with TFs. Furthermore, a possible mechanism cannot be ruled out through which ULT1 and TrxG or PcG converge in association with TFs.The complexity of ULT1 interactions, the phenotypes reported for It would be important to study specific ULT1 targets in different developmental and/or tissue-specific stages to analyze the ULT1 involvement on its activation or repression, which could shed light on the role of ULT1 in association with TrxG and PcG complexes, as a molecular epigenetic switch. Therefore, the future challenge is to define whether ULT1 acts by different mechanisms or in a single mechanism that involves all reported interactions. In this regard, additional research is needed to define whether these mechanisms can coexist or are tissue-, cell type-, or loci-specific.DO-A and MPS conceived and wrote the review. AG-A, ERA-B, and BG-P wrote the review. All authors have read and approved this version of the 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.


Thermodynamic reactivity of GGBFS mixed concrete at different curing temperature was applied to evaluate the compressive strength model, and the experimental results were in good agreement with the model. The results show that when GGBFS replacement ratio was increased, there was a delay in compressive strength. Ground granulated blast furnace slag (GGBFS) conventionally has been incorporated with ordinary Portland cement (OPC) owing to reduce the environmental load and enhance the engineering performance. Concrete with GGBFS shows different strength development of normal concrete, but sensitive, to exterior condition. Thus, a precise strength evaluation technique based on a quantitative model like full maturity model is required. Many studies have been performed on strength development of the concrete using equivalent age which is based on the apparent activation energy. In this process, it considers the effect of time and temperature simultaneously. However, the previous models on the apparent activation energy of concrete with mineral admixtures have limitation, and they have not considered the effect of temperature on strength development. In this paper, the apparent activation energy with GGBFS replacement ratio was calculated through several experiments and used to predict the compressive strength of GGBFS concrete. Concrete and mortar specimens with 0.6 water/binder ratio, and 0 to 60% GGBFS replacement were prepared. The apparent activation energy ( However, the concrete with GGBFS exhibits several differences compared to normal concrete in regards to the strength development attributed to a reduction of compressive strength in early age, caused by retardation of the setting. When the GGBFS comes into contact with concrete pore solution, the impermeable acid film surrounds the particles on the surface is destroyed; therefore, the chemical reaction in concrete would start. The delay in initial compressive strength of concrete with GGBFS is owing to insufficient alkalinity of cement paste [aE is required. The present study is aimed to predict the compressive strength development of the concrete with GGBFS by calculating the aE. Ground granulated blast furnace slag (GGBFS) is widely used in various engineering applications to replace the ordinary Portland cement (OPC) ,2,3. Howreaction ,18,19,20 1074-11 . The appnt paste ,6,22,23.nt paste  studied M and h(T) is maturity and maturity function, respectively. T denotes curing temperature over the age of t.Maturity is a function that quantitatively expresses the effect of curing temperature and time on strength development of the concrete. Therefore, the maturity theory by considering curing temperature and time function can be defined as:sM is maturity at age t. T is the average temperature (°C) of the concrete during the time interval, and T0 is reference temperature (−10 °C) [Tk is the reaction rate constant, A is proportionality constant, aE is apparent activation energy (kJ/mol), R is gas constant (8.314 J/mol∙K), and T is the absolute temperature (Kelvin). Thus, the equivalent age (et) can be calculated by the following equation:H(T) is maturity function at different curing temperature (t), whereas, H(rT) represents the maturity function at fixed curing temperature, i.e., 20 °C (293 K). The equivalent age means curing time at standard temperature (20 °C). By considering aE, et can be derived as:T is the average curing temperature of the concrete during a time interval and rT is the absolute or fixed temperature, i.e., 20 °C (293 K).The maturity function is expressed in linear Equation (2) considering temperature and the age , whereas(−10 °C) .(3)kT=AThe strength development analysis using maturity was carried out by considering curing temperature and duration. Other researchers have suggested a hyperbolic regression model (Equation (6)) which gives more accurate predictions compared to an exponential function ,25,26,27S is predicted compressive strength, whereas, uS represents the obtained experimental compressive strength at 28 days of curing. Tk is the reaction rate constant at curing temperature (T). et and t0 represent the equivalent age and the age when compressive development starts , respectively.aE) can help to calculate the reaction rate constant where minimum energy is required to occur the reaction. It has been reported that aE can vary owing to the nature of Portland cement, which has different hydration reaction patterns with the setting process, curing period, and cementitious components [aE is approximately 33.5 to 47.0 kJ/mol at early-age, and approximately 10 to 30 kJ/mol at long-term age [aE of OPC concrete as a function of curing temperature.aE is apparent activation energy by Freisleben-Hansen and Pedersen with temperature parameter, aT is the average curing temperature of concrete during a time interval.The cement reacts with water at an early age resulting hydration reaction. Due to the hydration of cement paste in concrete, it develops strength. The hydration rate is governed by the reaction rate constant of the point when the cement paste and water react ,28. The mponents . Some reterm age ,47,48,49term age  proposedaE can be determined by the composition, powder level, type, amount, and admixture of the cement [aE is changed by W/C ratio.However, Carino pointed out that e cement ,26, and aE. Therefore, ASTM C 1074 [aE. The procedure for determining the aE and the compressive strength prediction procedure, according to ASTM C 1074 is shown in The hydration reaction of Portland cement can be formulated with M C 1074  has suggx-axis) versus the compressive strength (y-axis), the y-intercept of the linear regression line can be obtained from the regression analysis. aE can be calculated by plotting the reciprocal of the curing temperature(x-axis) versus the reciprocal of Tlnk (y-axis) and dividing the gradient of the linear regression line obtained from the regression analysis. The equivalent age can be calculated by aE of GGBFS concrete from Equation (5), and the compressive strength development is analyzed by the calculated equivalent age and Tk from Equation (6).The final setting time of the mortar cured at three different temperatures is measured. The compressive strength of mortar is measured at 2, 4, 8, 16, 32 and 64 times as the final setting time. By plotting the reciprocal of the age ratio. Details of these ratios are shown in 3 specific gravity. Blaine specific surface area of cement was 3230 cm2/g. GGBFS was used according to ASTM C 989 [3 specific gravity. Blaine specific surface area of cement was 4260 cm2/g. The chemical analysis of these materials are given in The cement used in the present study was ordinary Portland cement Type I containing 3.14 g/cmTM C 989  with 2.8The concrete mixture proportion is presented in 3 of concrete mix was prepared for each batch. Cement, GGBFS, fine aggregate and coarse aggregate were mixed in a screw type mixer for 1 min and 30 s. Thereafter, water and superplasticizer were added, and the mixture was mixed for 3 min and 30 s. In order to obtain the mortar, the fresh concrete was passed through a 5 mm sieve, and cast into a mold (200 mm × 100 mm) for penetration resistance test according to ASTM C403 [Firstly, 0.1 mSTM C403 , whereasSTM C403 . All speSTM C403 .In order to measure the mortar setting time, the penetration resistance of the mortar was measured using a standard needle at a regular time interval according to ASTM C 403 . From thThe compressive strength of mortar was measured by 30 t class universal testing machine, and the average value was used by measuring the compressive strength in a triplicate set of specimens at 2, 4, 8, 16, 32 and 64 times for the final setting time . In addiThe initial and final setting time of mortar with GGBFS was measured by a penetration resistance test. The compressive strength of mortar with GGBFS replacement ratio at each curing temperature is shown in Tk was obtained according to the GGBFS replacement ratio. Tk that takes the curing temperature and the replacement ratio as variables. Tk according to the GGBFS replacement ratio at each curing temperature. As the curing temperature increased, the Tk increased in the form of an exponential function and showed a high correlation between 0.87 and 0.99. In addition, the lower the GGBFS replacement ratio at the same curing temperature, the higher the Tk. The Tk of 50% GGBFS mortar is decreased by 75% and 70% at 5 °C and 35 °C curing temperature, respectively compared to OPC. In addition, the reaction rate constant of OPC and 50% GGBFS mortar is decreased by 76% and 81% at 5 °C compared to 35 °C curing temperature, respectively. Therefore, the curing temperature and GGBFS replacement ratio have a complex effect on the Tk.The relationship between age and compressive strength was analyzed, and Tk and plotting the reciprocal of the curing temperature (K), we can represent the Arrhenius plot, as shown in a/RE, and the y-intercept represents the value of ln(A). As the GGBFS replacement rate increases, the gradients of the linear regression line decreases to a negative value.By taking the natural logarithm of the calculated aE results of GGBFS replacement ratio. As the GGBFS replacement ratio increases, the aE increases linearly, which considered as the result of an increase in the minimum energy for the chemical reaction of cement, GGBFS and water. For OPC mortar, aE is estimated to be 33.475 kJ/mol, which is very similar to the proposed value of Freisleben-Hansen and Pederson, i.e., 33.5 kJ/mol [aE, with or without the superplasticizer, is insignificant, i.e., 3 kJ/mol [aE value of 10%, 30% and 50% GGBFS is found to be 37.325 kJ/mol, 41.958 kJ/mol and 45.541 kJ/mol, respectively.5 kJ/mol . Wirkin 3 kJ/mol . In the This study measured the compressive strength of concrete with GGBFS at the age of 3, 7, 14, and 28 days and used the average of compressive strength of three specimens as a result. The results of compressive strength with varying curing temperature and GGBFS replacement ratio are shown in aE. The compressive strength prediction, according to the equivalent age by GGBFS replacement ratio, is shown in 2 = 0.91) is obtained. This proves that the compressive strength of concrete can be predicted in all strength at various curing temperatures and GGBFS replacement ratio.The maturity model applied in this study is the equivalent age model, as shown in Equation (5), and the equivalent age was derived using calculated aE of the proposed OPC, 10%, 30% and 50% of GGBFS are found to be 33.475 kJ/mol, 37.325 kJ/mol, 41.958 kJ/mol and 45.541 kJ/mol, respectively. Therefore, the equivalent age using aE and a high level of accuracy (R2 = 0.91) is obtained.From the above results and discussion, it is found that curing temperature and GGBFS replacement ratio have a significant effect on the development of concrete compressive strength. The addition of GGBFS also reduces the compressive strength of early age. As a result, the apparent activation energy and the compressive strength of concrete are affected by the GGBFS replacement ratio and curing temperature. Therefore, the existing compressive strength prediction model, i.e., Carino for OPC is not suitable for GGBFS concrete. The