There have been no studies from low- or middle-income countries to investigate the long-term impact of perinatal common mental disorders (CMD) on child educational outcomes.To test the hypothesis that exposure to antenatal and postnatal maternal CMD would be associated independently with adverse child educational outcomes in a rural Ethiopian.T1  and from school records at T2 .A population-based birth cohort was established in 2005/2006. Inclusion criteria were: age between 15 and 49 years, ability to speak Amharic, in the third trimester of pregnancy and resident of the health demographic surveillance site. One antenatal and nine postnatal maternal CMD assessments were conducted using a self-reporting questionnaire, validated for the local use. Child educational outcomes were obtained from the mother at T2. Exposure to repeatedly high maternal CMD scores in the preschool period was not associated with absenteeism after adjusting for antenatal and postnatal CMD. Non-enrolment at T1  was significantly but inversely associated with postnatal maternal CMD. There was no association between maternal CMD and child academic achievement or drop-out.Antenatal CMD (risk ratio (RR) = 1.06, 95% CI 1.05–1.07) and postnatal CMD  were significantly associated with child absenteeism at Our findings support the hypothesis of a critical period for exposure to maternal CMD for adverse child outcomes and indicate that programmes to enhance regular school attendance in low-income countries need to address perinatal maternal CMD.None. In HICs there have been studies indicating that the perinatal period represents a critical period for exposure to maternal CMD;7 whereas others argue that recurrent exposure to adversity is more important than exposure at a specific time.8 In LMICs, there have been short-term studies of the impact of ante- and postnatal CMD on infant growth, health and early child development,11 but we are not aware of any that have sought to disaggregate the effect of perinatal CMD from subsequent exposure to maternal CMD. Furthermore, few studies have investigated the impact of maternal CMD beyond infancy, so that little evidence is available to understand the longer-term impact on child educational outcomes.13Women of childbearing age are at increased risk of common mental disorders (CMD),The aim of this study was to investigate the impact of perinatal  and preschool maternal CMD upon educational outcomes of children. We hypothesised that children exposed to maternal antenatal CMD, postnatal CMD and subsequent repeated high maternal CMD score episodes in the pre-school period would have lower school enrolment, lower academic achievement, higher absence and higher drop-out from primary school.T1  and T2 .The study was an extension of a population-based birth cohort, the Child outcomes in relation to Maternal Mental Illness in Ethiopia (C-MaMiE) study. A total of 1065 pregnant women were recruited in 2005–2006 and have been followed up, together with the index child, to date. In the current study, we examined the exposure of maternal CMD measured repeatedly since the inception of the cohort in relation to the birth date of the child. Educational outcomes of children were measured at two time points: 14 in Butajira, Gurage Zone, Southern Nations Nationalities and Peoples' Region of Ethiopia. Butajira is located 135 km south of the capital Addis Ababa, is predominantly rural and is notable for the diversity of ethnicities and languages in the population. The HDSS has nine subdistricts with different ecological zones (low and highlands) and one urban administration in Butajira town. Butajira is densely populated and livelihoods are based on mixed farming of staples, such as maize and false banana, and cash crops, such as khat and chilli peppers.The C-MaMiE cohort was established within the Health and Demographic Surveillance Site (HDSS)15 The official age for school enrolment is 7 years. Primary education lasts for 8 years (age group 7–14 years) with two cycles: basic (grades 1–4) and general education (grades 5–8). Families are expected to cover the costs of school uniforms, food and exercise books; otherwise, education is free for all Ethiopians.16 Except for one regional examination at the completion of grade 8, the academic performance of students is assessed by the class teacher using non-standardised tests. In the first cycle of primary education, children are taught and evaluated by a single teacher following the ‘self-contained class’ concept.16Ethiopia is striving for complete primary education coverage, although national figures from the 2015/2016 academic year indicate that only 85.5% have enrolled currently, with 10.1% drop-out and 6.7% grade repetition.https://doi.org/10.1192/bjo.2019.69.At the inception of the C-MaMiE cohort, a population-based sample of 1065 women was recruited out of 1234 eligible women (86.3%) meeting inclusion criteria of age between 15 and 49 years, ability to communicate in Amharic, a resident of the HDSS and in the third trimester of pregnancy. The women and the child born from the index pregnancy have been assessed repeatedly over time. Ten time-point assessments were conducted starting in pregnancy and at 2, 12, 30, 36, 42, 48, 60, 78 and 102 months of age of the child in the postnatal period, see supplementary Fig. 1 available at 18 All outcomes were measured in relation to academic years rather than the birth date of the children as shown in supplementary Fig. 1. They were measured as follows.T1.Enrolment: each mother was asked whether the child had ever been enrolled in school by T2.Absenteeism: total number of days of absence was obtained from daily school attendance records at T1 we obtained the information from the mother, but for T2 we extracted the information from school records.School drop-out: students who had enrolled at the beginning of the academic year (September) but who had dropped out of school before the end of the academic year (June) were deemed to have dropped out; for students who were absent from school for a period of time during the school year, but who were attending school at the end of the year, the child was classed as absent and not dropped out. Children who drop-out of school can be re-enrolled and, therefore, are at risk of dropping out again in the subsequent academic year. For T2. Teacher assessments of academic achievement are composite and non-standardised, based on continuous assessment of mastery of content, class participation and interaction, conduct, homework, progress over time and school attendance.Academic achievement: the teacher-reported averaged grade point over two semesters of the Ethiopian school year and this was obtained from school records at The primary educational outcomes for this study  were selected based on their contextual relevance, given that (a) key bottlenecks to academic success occur at the stages of school enrolment , regular attendance and retention in school in this setting, and (b) that regular school attendance has been shown to have important socialisation benefits, regardless of the impact on academic achievement.19 The SRQ-20 items ask about the presence or absence of depressive, anxiety and somatic symptoms in the preceding 1 month (answered ‘yes’ or ‘no’). The SRQ-20 has been validated for perinatal women in this rural Ethiopian population.20 Repeated high maternal CMD scores were generated as the count of time points after the 2-month postnatal time point when the woman scored ≥6.This was measured using the World Health Organization 20-item version of the Self-Reporting Questionnaire (SRQ-20) in pregnancy and at all nine postnatal time points until the child was on average 8.5 years.Measures of potential confounding factors were used from the following time points depending on the hypothesis: (a) model 1 : pregnancy time point, (b) model 2 : postnatal 2-month time point, and (c) model 3 : 60 months postnatal time point.21 scale was used to measure stressful life events over the pregnancy  and preceding 6 months for the postnatal assessments.An adapted version of the 12 item List of Threatening ExperiencesSelf-report of the following proxy indicators of socioeconomic status were measured: current roof material, the experience of hunger in the preceding month because of lack of food or money, and the existence of emergency resources in times of crisis.A report of the frequency of paternal alcohol and/or khat use was obtained from the woman.Literacy level of both parents, age of the mother, marital status, birth order and gender of the child were obtained from self-report of the woman.22 height-for-age z-scores were calculated using World Health Organization Anthro software.23Height measures were carried out by trained project data collectors using a stadiometer with a movable headpiece. Using the World Health Organization reference population,To ensure privacy, confidentiality and her preference, all interviews with the women were carried out in the woman's home or surrounding area. The project data collectors had all completed high-school education, and were experienced in conducting interviews and in the use of the study measures. At each time point, they received an additional 3 days of refresher training on the use of newly added instruments. The questionnaires were piloted before commencing data collection and discrepancies in ratings were discussed to ensure that the data collectors had a common understanding.24 on the day of data collection, where possible. Any identifiable information about the respondent was kept securely and separately from the assessment data and a code number was used to ensure confidentiality.Supervisors and a field coordinator monitored the data-collection process and performed quality checks on a random sample of evaluations. Data-entry clerks double entered data with EpiData version 3.125 to examine the association of maternal CMD in model 1 antenatally, and model 2 at 2 months postnatal using SRQ-20 total score, and in model 3 repeated high maternal CMD scores (as previously defined) with educational outcomes. First, we conducted unadjusted logistic regression for school non-enrolment and drop-out (binary outcomes), zero-inflated Poisson regression for absenteeism  and linear regression for academic achievement . We then carried out two stages of multivariable analysis; first a model containing each primary exposure adjusted for all potential confounders identified a priori, and finally we ran a model containing antenatal, postnatal and repeated high CMD scores and all potential confounders at the 60-month time point in the same model. Estimates of associations were presented with their corresponding 95% confidence intervals. The study has been reported according to the STROBE reporting checklist.A hypothesis-driven analysis was conducted using Stata version 12The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human participants were approved by the Institutional Review Board of the College of Health Sciences, Addis Ababa University, Ethiopia (reference number 082/13/psy) and the Research Ethics Committee of King's College London, UK (reference number PNM/13/14-92). Written or verbal informed consent was obtained from each woman for her own and her child's participation in the study as well and to access school records of the child. Verbal consent was witnessed and formally recorded. For the anthropometric assessment assent was obtained from the child. Any woman who presented with high CMD symptoms and suicidal ideation was supported to seek care at the psychiatric unit at Butajira Hospital, with the project covering treatment and transportation costs.T1, when the children were a mean of 6.5 years (s.d. = 0.03), a total of 830 (77.9%) mother–child dyads participated in the study. See T2, when the children were a mean of 8.5 years , 788 (74.0%) participated in the study. Most loss to follow-up occurred early in the cohort as a result of stillbirth (n = 40), neonatal death (n = 35) and post-neonatal infant mortality (n = 48 by 12 months). A further 12 mothers and 23 children died before the age of 6.5 years. A total of 47 women had out-migrated by 6.5 years. Maternal refusal was low (n = 11)  .Fig. 1FThere was no significant difference between those lost to follow-up and those remaining in the study in terms of demographic, socioeconomic and mental health status. Children for whom educational information was not available did not differ significantly on maternal CMD, socioeconomic status or birth order, but were more likely to have been boys and to have been stunted growth. See supplementary Tables 1 and 2.n = 26) also scored high at the antenatal time point and 22.7% (n = 17) scored high at the 2-month postnatal time point.The median score on the SRQ  was 2 (interquartile range (IQR) = 0–4) at the antenatal assessment and 1 (IQR = 0–2) at 2-months postnatal. In terms of repeated high maternal CMD scores from 12 to 102 months: 607 (73.1%) women had no high scores (≥6), 148 (17.8%), scored high at one time point and 75 (9.0%) had a high score at more than one time point. Of the 84 women who had repeated high CMD scores in the preschool period, 34.7% (T1 (2013/14 academic year), 88 children had still not enrolled in school (11.2%), and a further 122 (17.9%) had enrolled but subsequently dropped out. At T2, during the 2014/2015 academic year, 64 children (12.1%) dropped out. The median number of days of absence was 5 (IQR = 2–11), with 82 children (17.6%) recorded as having no absence. The mean grade point was 62.6 out of 100 (s.d. = 9.5).At T1, but there was no association between antenatal or repeated high maternal CMD scores and non-enrolment.Postnatal CMD (at 2 months) was associated inversely with child school non-enrolment  at Antenatal CMD (adjusted risk ratio (RR) = 1.06, 95% CI 1.05–1.07) and postnatal CMD  were independently and significantly associated with absenteeism, but there was no association between repeated high maternal CMD scores and absenteeism in the multivariable model containing antenatal and postnatal CMD. Exposure to maternal CMD at any time point was not associated with either school drop-out or the academic achievement of the child  and 3.TT2. Exposure to repeated high maternal CMD scores in early childhood was not associated with absenteeism after adjusting for antenatal and postnatal CMD. Postnatal CMD was significantly, but inversely, associated with school non-enrolment. There was no association between exposure to maternal CMD at any time point and either academic achievement or school drop-out.In this longitudinal prospective cohort study from a rural population in Ethiopia, antenatal and postnatal maternal CMD were found to have an independent significant association with school absenteeism at To the best of our knowledge, this is the first population-based study from sub-Saharan Africa to have investigated the effect of perinatal CMD beyond infancy and examined the impact on educational outcomes of children. Furthermore, our study is the first of its kind from a LMIC to test the hypothesis that the perinatal period represents a critical period for exposure to maternal mental health problems. Strengths of the study are the population-based, prospective design with high follow-up rates and repeated measures of maternal CMD and confounding factors using culturally validated measures.Nonetheless, there were also limitations. The exposure of ‘repeated high maternal CMD scores’ was generated from high scores at each assessment time point, but did not capture longitudinal exposure to maternal CMD between the time points. Absenteeism was extracted from data collected routinely by schools, which may not be accurate, although we expect that this would not lead to differential classification of students and would just reduce the power to detect an association. A teacher-reported (composite and non-standardised) measure of academic achievement was used. This composite measure may be more ecologically valid and tied to the day-to-day routine of teaching and learning than narrowly focused assessments of content mastery but this approach may increase measurement error. We were not able to adjust for child illness, although we did measure height-for-age which provides a proxy indicator of chronic ill health. We relied on proxy indicators of socioeconomic status. Although the socioeconomic status indicators have been developed for the population under study, they relied on self-report of the women and may not have been sufficiently comprehensive, thus raising the possibility of residual confounding.26 Potential mechanisms were also explored: anxiety in late pregnancy was found to be associated with hypothalamic–pituitary axis function in the child, particularly the waking cortisol level of the child, which is responsible for orchestrating the stress response and has been implicated in development of emotional disorders.27 In HICs, a delayed effect of antenatal maternal CMD on the temperament of child has also been found.28 Our finding of a significant association between antenatal maternal CMD and later school absenteeism could, therefore, be linked with child temperament and child behavioural and emotional disorders. In support of this, we previously found an independent, prospective association between preschool child emotional and behavioural disorder and school absenteeism in an earlier analysis using an expanded C-MaMiE cohort. Further studies are required to elucidate the mechanisms mediating this association.Evidence for the antenatal period as a critical time for exposure to maternal CMD in terms of later child outcomes is accumulating. In a large birth cohort study from the UK, an independent and significant association was observed between maternal anxiety at a gestational age of 32 weeks and an increased risk of child hyperactivity at the age of 4 years.7 and LMICs30 have shown an association between postnatal maternal CMD and child development. This association appears to be mediated by effects of postnatal CMD on the infant–mother relationship, which is predicted to lead to enduring effects on the child.1 Maternal responsiveness to the physical and psychological needs of an infant has a vital role in the development of secure attachment between the mother and infant.31 Maternal postnatal CMD was found to be associated with insecure attachment in a South African community-based study.31 Although adjustment for concurrent maternal sensitivity led to loss of a significant association in the South Africa study, this may reflect that postnatal depression led to changes in the mother–infant relationships that endured even when maternal depression resolved. Insecure attachment may, therefore, play a role in any enduring effect of postnatal CMD on child outcomes such as absenteeism.Our finding of a significant association between postnatal CMD and school absenteeism is supportive of the critical period hypothesis. A number of earlier studies from HICs13 and in high-income settings;6 however, the result was not replicated in our study. Associations with academic performance in these previous studies have been mediated through associations between postnatal CMD and child cognitive development. However, in the Ethiopia C-MaMiE cohort we found no association between maternal CMD and child development.32 A previous study in Ethiopia also failed to replicate findings from Barbados of a specific adverse effect of early-life child malnutrition on academic performance of children in Barbados,12 with postnatal malnutrition no longer associated with later child development after current nutritional status was taken into account. Our null finding may be also because of the limitations of our measure of academic achievement, which was reliant on non-standardised teacher ratings. However, in this low-income setting, the effect of poverty on cognitive development and high maternal non-literacy may overwhelm any effects of maternal CMD. Substitute caregivers may compensate for a mother with depression and, therefore, limit the impact of maternal CMD on the child's learning environment. In contrast, absenteeism is likely to be related to more than cognitive development, also incorporating interpersonal functioning and confidence to separate from the mother, which are affected by security of attachment and may be more strongly associated with exposure to postnatal CMD.11Postpartum maternal CMD was found to predict lower academic performance of children in Barbados33 Furthermore, our finding is at odds with the conceptualisation that child development is influenced by repeated exposure to developmental adversity.1 Our negative finding might have arisen because fewer than 10% of women had high CMD scores at more than one postnatal time point. This indicates that most children could have experienced extended periods of their early childhood when their mother did not have high CMD symptoms. Maternal resilience to mental health problems is an important area of focus for future studies.We did not find any association between exposure to repeated high maternal CMD scores in early childhood and any of the educational outcomes. This is in contrast to studies from Australia, a high-income country, where chronicity of exposure to maternal CMD was found to be associated with higher levels of child emotional and behavioural problems and lower vocabulary scores at later ages.34 and our earlier report of an association between maternal CMD and school drop-out.35 The most likely explanation is that we were underpowered in this current analysis, which was performed on a subsample of the extended C-MaMiE cohort for whom longitudinal data from antenatal exposure onwards were available. Furthermore, for repeated high CMD scores, we dichotomised the SRQ-20 at each time point, which may also have decreased power to detect an effect. We would expect that absenteeism would be on the pathway to drop-out and that we would see similar patterns of association with maternal CMD. The significant but inverse association between postnatal maternal CMD and child school non-enrolment was unexpected, and we do not have an explanation for this finding. It is possible that this association was observed by chance  or was affected by other unmeasured factors. This finding requires replication and further investigation.The lack of association between any measure of maternal CMD  and school drop-out is in contrast to the study from HICsThere is increasing research evidence of the importance of early investment in children to achieve optimal development, education and economic success. Indeed, for optimal child outcomes, the health of the mother is understood to be critical even before she is pregnant, as well as continuing through pregnancy and into the postnatal period. Our study indicates that there also needs to be a focus on maternal mental health, both in pregnancy and the early postnatal period, to optimise child educational outcomes. This has an important bearing on achievement of the Sustainable Development Goal seeking to achieve quality education for all children .36 seek to equip maternal and primary healthcare workers to detect and treat CMD and other priority mental disorders. The Mental health Gap Action Programme intervention guide emphasises the importance of enhancing social support networks and addressing social stressors to address mental health problems, alongside evidence-based interventions. There is accumulating evidence for the efficacy of psychosocial interventions delivered by non-specialists to women with perinatal CMD, although most studies are from well-resourced settings in middle-income settings and none have examined the impact on child educational outcomes.37In a LMIC, regular school attendance is not just the best way to learn, but frequently the only way to learn the taught topics, as there are limited learning resources at home or in the community to compensate for missed classes. For programmes aimed at enhancing regular school attendance, our study indicates that the perinatal mental health of women needs to be optimised. The high rates of health service contact of antenatal women provides an opportunity for intervention. The World Health Organization Mental health Gap Action Programme and other initiatives to task-share mental healthcareIn conclusion, our findings support the hypothesis of a critical period exposure to maternal CMD for adverse child outcomes, although further definitive evidence is required. Our findings indicate that programmes aimed at enhancing regular school attendance need to address maternal CMD from pregnancy onwards.


The epidemiological tipping point ratio (TPR) has been suggested as a useful indicator to monitor the scale-up of antiretroviral treatment (ART) programmes and determine when scale-up is sufficient to control the epidemic. TPR has been defined as the ratio of yearly number of new HIV infections to the yearly number of new ART initiations or to the yearly net increase in the number of people on ART. It has been used to rank the progress of treatment programmes across countries, with the objective of reaching a TPR value under 1. Our study aims to assess if TPR alone can be used as an indicator of ART success across settings by comparing the expected changes in HIV incidence and ART coverage when TPR is maintained constant over time. In particular, we focus on the effect of ART initiation timing (emphasis on ART being initiated early or late during HIV progression) on the interpretation of the TPR.We used a dynamic model of HIV transmission in South Africa representing ART rollout leading to universal treatment in 2017. The model is calibrated to HIV incidence, HIV prevalence and ART coverage in 2012 in South Africa, and 1000 simulations are selected for the base-case scenario. To measure the effect of TPR, we simulate TPR-preserving interventions, maintaining TPR (yearly number of new ART initiations denominator) at the value observed in 2019 (between 0.65 and 1.25) for 15 years. We compare ART coverage and HIV incidence across TPR values and across strategies in which ART access is prioritized differently. In a secondary analysis, we illustrate the sensitivity of new ART initiations to ART retention, and we compare both definitions of the TPR.Our analysis shows that HIV incidence reduction is weakly correlated to TPR: the same reduction in HIV incidence (15%) can be achieved by implementing the same strategy with a wide range of TPR maintained (0.65–1.12). Assuming high retention in ART, TPR-preserving strategies prioritizing early ART initiation yield greater reduction in HIV incidence than strategies where most individuals initiate ART late. High ART coverage is associated with low HIV incidence and it can be reached with a TPR below or equal to one with strategies favoring early ART initiation. Low ART retention over time results in higher HIV incidence even if TPR is maintained low. If ART retention is low, strategies prioritizing late ART initiation are associated with lower HIV incidence than strategies where ART is initiated early. Maintaining a fixed TPR value based on the net increase in people on ART gives higher HIV incidence reduction and requires fast ART scale-up.Our analysis suggests that the TPR is not an adequate indicator of ART programme impact, without information on ART coverage and retention. Achieving early initiation and adherence to treatment to improve ART coverage might be as important as attaining a specific TPR target. Comparisons of TPR in different settings should account for differences in epidemic conditions. This statistic has been suggested as a useful indicator to monitor the scale-up of ART programmes and determine when a programme is likely to have reached a level sufficient to successfully control the HIV epidemic . IntuitiOne problem with the applicability of the TPR as an independent metric comes from the fact that alternative definitions are used in the literature with respect to how the denominator of the ratio is estimated. Even though first mentions of the TPR  suggest There is no clear recommendation on how to use the TPR in monitoring ART programmes. It has been suggested that the TPR should only be derived once ART coverage exceeds 67% . It alsoMathematical models are invaluable tools in evaluating the effectiveness of biomedical interventions for HIV prevention . In thisIn our main analysis, we simulate different treatment strategies, introduced in 2020 to maintain the TPR at the value observed in 2019. These strategies have different ART access priorities based on CD4 count categories . Maintaining the TPR constant over a prolonged period of time enables us to clearly see how the TPR relates to HIV incidence without the relationship being obscured by changes in TPR over time. Our objective is to verify if lower TPR values and if particular strategies are associated with larger reduction in HIV incidence and greater increase in ART coverage. This analysis informs the validity of the TPR as a measure of ART progress by investigating the importance of lowering the TPR value and helps determine the merit in ranking ART progress in different settings by TPR value only. We emphasize the importance of the distribution of ART initiations across different HIV-infected subgroups and demonstrate that it deserves more attention when ART programmes are planned or implemented.22.1We used a dynamic compartmental model of HIV transmission representative of the HIV epidemic among the sexually active population (15–49 years old) in South Africa in the era of ART rollout from the year 2002 onwards see . The mod3).Individuals can either be HIV- or HIV+, and progression towards AIDS is characterized by passage through one acute phase and four subsequent chronic phases distinguished by CD4 count : CD4 <200 eligibility starting in 2002, partial CD4 200–350 eligibility added in 2010, full CD4 <350 eligibility added in 2012, CD4 350–500 eligibility added in 2015, CD4 >500 eligibility added in 2017 see . We assuOn ART, progress towards AIDS is slowed with the mean duration of each phase extended by 100%–200%. Also, treatment dropout or failure can occur. Individuals dropping out can later re-initiate ART, but individuals failing treatment cannot do so.2.3Parameters related to the natural history of HIV  were informed from published studies . Other p2.4In the base-case scenario, all 1000 simulations using calibrated parameter sets  are extended until 2034, assuming that the ART initiation rates remain unchanged. In these calibrated simulations, the TPR is rising over the 15-year period from 2019 to 2034. Three of the base-case simulations see  with simIn the intervention scenarios, TPR-preserving strategies are implemented starting in 2020. To study the link between the TPR and HIV incidence, we increase ART initiation rates to keep the TPR constant over time and measure the effect on incidence of doing so. Maintaining the TPR constant helps highlight this effect that could be obscured by changes in TPR over time.We study different TPR-preserving strategies by increasing the number of ART initiations sufficiently to maintain the TPR value at the level of 2019 (implemented by varying the rates of ART initiation in different CD4 groups). The “CD4<XX first” strategy assumes that all new ART initiations are distributed proportionally between all HIV-positive untreated compartments with CD4<XX, including both diagnosed and undiagnosed. If not sufficient, they are supplemented with individuals from the adjacent CD4 groups. The “CD4<XX priority” strategy assumes that base-case yearly ART initiation rates by CD4 group are maintained. The rest of the ART initiations needed are distributed proportionally between all diagnosed untreated compartments with CD4<XX. If not sufficient, they are supplemented with undiagnosed individuals with CD4<XX individuals followed by infected individuals from the next CD4 groups. We developed those two strategies using CD4 <200, CD4 <350, CD4 <500 and universal  thresholds as restriction criteria, yielding a total of eight strategies described in more detail in Section 7 of These strategies yield higher ART initiation rates than the base-case rates in the first years after the intervention. Over time, as incidence declines, so do the ART initiation rates, thus keeping the TPR constant. Relative to the base case, more HIV+ individuals are treated yearly just after the intervention, but later on, fewer people may be treated yearly, depending on the epidemic parameters of the simulation. Note that this does not entail a decline of ART coverage if few HIV+ individuals drop out of treatment.Motivated by the fact that an HIV cure is not yet available, we assume that each newly infected individual remains infected for life. In contrast, after initiating ART, the treated individuals do not necessarily remain on ART indefinitely. Therefore, we simulate scenarios with different rates of ART dropout to explore the importance of ART retention to the reported results.2.5We estimated the effective TPR value (the ratio of the yearly number of new HIV infections over yearly number of new ART initiations), the HIV incidence (number of yearly new HIV infections per 1000 HIV- persons) and the ART coverage (percent ratio of HIV+ adults on ART to HIV+ adults) for the base-case scenario and the intervention scenarios in which TPR is maintained constant by different TPR-preserving strategies. We also estimated the relative reduction in HIV incidence To evaluate the importance of TPR for the dynamics of the HIV epidemic, we analyzed the correlation between the TPR value maintained at a constant level and the reduction in HIV incidence in 2034 for the eight TPR-preserving strategies that we designed. To facilitate the comparison of different strategies, we have selected three specific simulations , each having the same HIV incidence. We compared the incidence value in 2034 across all strategies for these three simulations. We also highlighted the crucial role of ART coverage in the relation between TPR and HIV incidence.33.1To provide context for estimating impact of TPR-preserving interventions, we first project the evolution of the HIV epidemic over the 2017–2034 period, assuming base case ART initiation rates by CD4 groups are maintained.Notably, a wide range of HIV incidence values are observed for the same TPR value. For instance, a balanced TPR of 1 may correspond to HIV incidence between 9 and 16 infections per 1000 person-years A. Simila3.2Intuitively, we expect that keeping the TPR low will lead to lower HIV incidence. Therefore, it is expected that if the same TPR-preserving strategy is implemented in different settings, then larger HIV incidence reduction will be achieved with low TPR than with balanced and high TPR. We have simulated all 1000 epidemic conditions, assuming that TPR is maintained constant and assuming equal access to ART by all infected individuals , and compared the expected reduction in HIV incidence to base-case simulations B. OveralIn the three settings selected with similar HIV incidence in 2019 and different TPR, we also compared the expected HIV incidence achieved in 2034 with different TPR-preserving strategies . CompariA supplementary analysis was performed using the base-case simulation with balanced TPR in 2019 and applying all ”CD4<XX first” strategies maintaining TPR at 0.9, 1 or 1.1 from 2020 to 2034. Comparison of HIV incidence curves under different interventions, presented in 3.3In Overall, interventions with early ART initiation  is achieved with early ART initiation  while the highest HIV incidence (4.2 per 1000 person-years) is expected when low CD4 groups have preferential access to ART (“CD4 <200 first”). In contrast, if the annual ART dropout rate is increased to 25%, the lowest HIV incidence (7.3 per 1000 person-years) is achieved when low CD4 groups have preferential access to ART (“CD4 <200 first”) while the highest HIV incidence (7.7 per 1000 person-years) is expected with early ART initiation . Clearly, when people do not stay on ART long, it is most beneficial for the population to target those with the highest infectiousness (CD4 <200).3.6We compared the results obtained in the main analysis with another definition of the TPR proposed and used in the literature . We measured the resulting HIV incidence reduction (relatively to base case) in 2024 and the effective average TPR values derived from projected simulations over 2020–2024 under both definitions (TPR and net TPR). We averaged the TPR to account for fluctuations induced by the compensatory mechanism . We simulated interventions in which ART initiation is adjusted to follow a TPR target value in order to characterize the effect of the TPR on HIV incidence reduction, which is among the key goals of ART scale-up strategies such as US National HIV/AIDS Strategy and UNAIDS 90-90-90. We showed that the measure of ART success in terms of HIV incidence reduction only marginally depends on the TPR. The analysis of selected simulations with the same HIV incidence in 2019 shows that larger HIV incidence reduction can be achieved with balanced TPR near 1 compared with low TPR (near 0.84), even when ART coverage is over the proposed 67% interpretation threshold. Similar results are observed in simulated HIV epidemics having the same HIV incidence but distinct epidemic conditions. Our study suggests that epidemic conditions and programme performance (e.g. ART retention) must be taken into account before interpreting the TPR. Thus, comparisons between TPR values across settings should be avoided without additional analyses. Furthermore, the complexity of the interplay between the HIV epidemic and ART scale-up warrants further analysis and modeling to appreciate whether ART programmes are heading towards success or failure.In places where the TPR is maintained constant over time, if HIV incidence decreases, then the rates of new treatment initiations must go down as well. However, this does not necessary lead to lower ART coverage since the coverage level depends on how long HIV-infected patients remain on ART. Our analysis demonstrated that ART coverage can increase even with ART initiation rates going down as long as treated individuals are retained in care for a long time. This suggests that ART retention is more important to the impact of the ART programme than the TPR value itself.We found that the effect of TPR on HIV incidence depends on programme conditions (TPR-preserving strategies) and epidemic conditions . Based on this study, we conclude the TPR would be inadequate as a sole indicator of the success of ART programmes. We demonstrated that the distribution of new ART initiations should be taken into account to understand or predict the long-term trend of the HIV epidemic in a given setting. Strategies of ART scale-up where a substantial proportion of individuals with CD4 >500 or CD4 350-500 initiate treatment are necessary to achieve high ART coverage within 15 years, assuming that ART dropout is kept to a minimum. In this case, our analysis suggests that ART coverage is a better predictor of programme success than the TPR. Therefore, interventions that measure progress in terms of the overall proportion of virally suppressed among those infected with HIV will trace more precisely their impact on the HIV epidemic. One example is the 90-90-90 initiative of UNAIDS which, if achieved, will increase the proportion of all HIV infected individuals who are virally suppressed to 73%.It is unlikely that HIV elimination (less than 1 new infection per 1000 HIV- per year) will be achieved by 2034 if a substantial increase in ART coverage is not reached quickly (none of our simulations reach that target). We found that high ART coverage is associated with low HIV incidence. Thus, very low TPR values resulting in very high ART coverage would be needed to progress towards this objective, with adequate monitoring of ART coverage to validate progress. This result is in line with other recent modeling studies  which foWe found confusion in the literature around the computation of the TPR denominator. The “increase in new patients on ART”  or “numbOur model and its results are comparable to other treatment-as-prevention models, except for our focus on the TPR. Our work stands out as almost no details are available in the literature about this indicator, its validity and its long term effects on the epidemic trends.Our model has several limitations. It is restricted to the South African epidemic in the era of ART, so our analysis could fail to apply to dissimilar settings. To simplify calibration, the model is not stratified by sex, which presupposes that the flow of infection from men to women is equal to that from women to men . The curIn light of our findings, we plan to pursue further analysis of the TPR along two lines of investigation. First, to determine the significance, if any, of the notion of a “tipping point,” we will study the relation between the TPR and the basic reproduction rate in a simplified model. Second, we will proceed with an analysis of TPR trends in scenarios of ART scale-up towards 90-90-90/95-95-95 targets to determine the usefulness of the TPR for measuring ART progress in specific epidemic settings.5In HIV surveillance, the TPR alone is not sufficient to indicate programme impact without further information on ART coverage and the timing of ART initiation modulated by the distribution of ART initiations across CD4 stages. Our results support the idea that reaching a high ART coverage level is required, but not sufficient by itself, for interpreting the TPR correctly due to the importance of adherence to treatment. We noted that there is confusion in the literature about the definition of the TPR. In a supplementary analysis, we showed that TPR interpretation can be very sensitive to this definition.Our analysis suggests that TPR alone should not be used to compare ART programmes across countries. A careful assessment of epidemic conditions, ART coverage and ART dropout should accompany TPR rankings. Promoting early initiation and adherence to treatment to achieve high ART coverage might be as important as demonstrating that a specific TPR target has been met.With the recent recognition that UNAIDS objectives for 2020 are going off track , ART pro


Vanishing gastroschisis (VG) is a severe complication of gastroschisis with a high mortality rate. We report here a case of VG with a favorable outcome after a 3-year follow-up. A 26-year-old primigravida woman was referred to Strasbourg University Hospital because her fetus was diagnosed with an isolated gastroschisis at 13-week gestation. The ultrasound evolution was marked by a progressive closure of the abdominal wall defect from 19-week gestation and the appearance of dilated intra-abdominal loops. The child was born with a closed abdominal wall except a small remnant at the level of the former gastroschisis orifice. Explorative laparotomy revealed extensive midgut atresia with only 50 cm of remaining midgut. A jejunocolic anastomosis was performed. The child is now 3 years old and has a favorable outcome with only 2 nights a week of parenteral nutrition. A total of 39 cases of VG type D from Perrone et al. classification are described in the literature from 1991 to 2019, among which 19 (48.7%) are alive at the time of publication but only 4 cases are described with a long-term follow-up of 3 years or more. This is the fifth case described with a favorable evolution after 3-year follow-up. This could be explained by vascular injury to the developing intestine causing intestinal resorption; a strangulation and necrosis of the midgut by a narrow defect spontaneous closing or volvulus causing infarction, resorption, and closure of the defect [Gastroschisis is an abdominal wall defect resulting in small intestine prolapse into the amniotic fluid without any protective covering membrane and variable degrees of malrotation. It is a rare congenital anomaly, but the incidence of gastroschisis has increased last few years , 2. It ie defect . Perronee defect . Type D e defect  whereas We report here a case of VG with a favorable outcome after a 3-year follow-up.We report the case of a 26-year-old primigravida woman referred to Strasbourg University Hospital because her fetus was diagnosed with an isolated gastroschisis at 13-week gestation . At 19 wAt 35 weeks of gestation, labor occurred spontaneously. A live male infant was delivered by normal vaginal delivery weighing 2560 grams with an APGAR score of 10 at 1 minute. There was no defect on the abdominal anterior wall except a small, grayish-brown paraumbilical remnant attached to a filiform axis crossing the abdominal wall .Abdominal X-ray with contrast product showed the presence of a voluminous blind intestinal loop of 3 cm in diameter and no passage in the colon. Surgical treatment by an explorative laparotomy was performed because of radiographic evidence of bowel obstruction. Exploration found 65 cm of a dilated small intestine downstream of the blind intestinal loop and atresia of the right colon. We found the same fibrous cord connected to the abdominal remnant and to the atresia zone . The remThe evolution of the disease was marked by several sepsis starting points of the central catheter treated by antibiotherapy and catheter change. Oral feeding was progressively increased. At 2 years and 4 months, the parenteral nutrition was only 3 nights a week. Because of recurrent subocclusive episodes and dilation of distal bowel loops on imaging, a surgical treatment was decided. The small intestine was dilated up to 7 cm upstream of the permeable anastomosis: a new end-to-end anastomosis was performed. The small intestine length was 1.5 meters. At the age of 3 years, the boy was on parenteral nutrition only two nights a week. With growth, the child will probably be weaned from enteral nutrition in the months or years to come.VG is a rare complication of gastroschisis usually associated with a high rate of mortality closed to 70% , 13–16. Perrone et al. proposed in 2019 a new classification from the analysis of 53 children with closing gastroschisis . This clA total of 39 cases of VG type D from Perrone et al. classification are described in the literature from 1991 to 2019 , among wIn our report, antenatal ultrasound and fetal magnetic resonance imaging (MRI) failed to predict the remaining small intestine length. It seems difficult to get reliable prognostic factors to determine fetal outcome. Geslin et al. tried to evaluate prenatal ultrasound parameters as prognostic factors for complex and vanishing gastroschisis . They reThe VG is a rare and severe complication of gastroschisis with a high mortality rate due to SGS and to complications related to PN. Nevertheless, some children have a favorable outcome. Signs of closing gastroschisis in prenatal ultrasound should be carefully sought. Thereby, physicians can adapt prenatal counseling and prepare the parents for this complication and the need of multidisciplinary postnatal care .


Prdm9) is specifying hotspots of meiotic recombination but in hybrids between two mouse subspecies Prdm9 controls failure of meiotic chromosome synapsis and hybrid male sterility. We have previously reported that Prdm9-controlled asynapsis and meiotic arrest are conditioned by the inter-subspecific heterozygosity of the hybrid genome and we presumed that the insufficient number of properly repaired PRDM9-dependent DNA double-strand breaks (DSBs) causes asynapsis of chromosomes and meiotic arrest (PR domain containing 9 (c arrest . We now  Proper synapsis of homologous chromosomes is an important meiotic checkpoint preventing germline transfer of harmful genic and chromosomal mutations to the next generations . SynapsiPrdm9 gene, besides determining position of the recombination hotspots, acts as the major hybrid sterility gene in certain hybrids between house mouse subspecies of Mus m. musculus (mouse strain PWD) and Mus m. domesticus  (Prdm9 gene in sterile (PWD x B6)F1 (hereafter PBF1) hybrids (The fter B6) . Disrupt hybrids .Prdm9 gene was ‘humanized’ by substitution of the C2H2 zinc-finger (ZnF) DNA-binding domain for its human ortholog, the humanized PBF1-Hu/PWDPrdm9 meiocytes regained normal meiotic pairing and hybrid males became fertile. This unexpected finding provided direct evidence for the role of PRDM9 ZnF array in the control of hybrid sterility  is known to create DNA inter-strand (ICL) and intra-strand cross-links. In replicating yeast and mammalian somatic cells removal of ICLs results in DNA DSBs, which can be repaired by the nonhomologous end joining or by homologous recombination, the latter being favored in the germline . RemovalTo assess an effect of exogenous DSBs on meiotic pairing in sterile hybrids we treated the adult (4–8 weeks) PBF1 hybrid males with cisPt and with the 5-ethynyl-2’-deoxyuridine (EdU), a nucleoside analog of thymidine  to distiForty hours after cisPt and EdU injection, 84.1 ± 3.3% (mean ±SE) of leptonemas and 49.3 ± 2.2% of zygonemas were EdU-positive, thus being at the S-phase at the time of injection or shortly after that. The EdU-negative leptonemas (15.9%) most likely started their S-phase 20 hr or more after EdU injection, after assumed depletion of free EdU , while ESince the RPA protein was reported to bind ssDNA soon after resection of DSBs in mitotic and meiotic cells , we usedSurprisingly, when zygonemas were split to EdU-positive and negative, only EdU-positive cells showed a significant increase in the cisPt dosage-dependent RPA foci . The dosSince RPA is an ssDNA-binding protein, it could mark other forms of DNA damage beside DSBs ; therefoWhen split according to the EdU phenotype, the EdU-positive zygonemas showed a significant increase of DMC1 foci at all three concentrations , from 22−9, Tukey’s post-hoc test) and 28.71%  (p=7.3 × 10−13), respectively  in EdU-negative than in EdU-positive pachynemas , on averNo sperm was found in the ductus epididymis of the males 30 days after injection. Histological sections showed atrophy of seminiferous tubules caused by the lethal effect of cisPt on proliferating spermatogonia and somatic cells of seminiferous tubules (not shown). Beside the increased frequency of fully synapsed pachynemas, a short-term effect was apparent from the increased relative incidence of late pachynemas. While in untreated control hybrids the late pachynemas represented 2.03% (4/107) of all pachynemas recorded from the meiotic spreads, the frequency increased to 12.12% (28/231) and 13.10% (30/229) after 5 mg/kg and 10 mg/kg cisPt treatment, respectively.PWD/B6Prdm9 heterozygosity . DSBs in these hotspots are difficult to repair or they repair too late, perhaps using sister chromatids as a template , the cells were immunolabeled using a standard protocol with the following antibodies: anti-HORMAD2  and SYCP3 . Secondary antibodies were used at 1:500 dilutions and incubated at room temperature for 60 min; goat anti-rabbit IgG-AlexaFluor568  and goat anti-mouse IgG-AlexaFluor647 . Visualization of EdU-labeled nuclei was done using an EdU in vivo kit (Baseclick) according to the manufacturer’s instructions. The images were acquired and examined in a Nikon Eclipse 400 microscope with a motorized stage control using a Plan Fluor objective, 60x  and captured using a DS-QiMc monochrome CCD camera (Nikon) and the NIS-Elements program (Nikon). To quantify RPA and DMC1 foci in spread nuclei we used ImageJ . Images were processed using the Adobe Photoshop CS software (Adobe Systems). The estimates of the mean asynapsis rate, their standard errors and 95% confidence intervals were based on the Generalized Linear Mixed Model (GLMM) described in the previous paper , these will tend to be repaired by intersister recombination, which isn't productive in driving synapsis. It isn't clear to me why this would be, and the paper doesn't really address this potential mechanism. Nevertheless, the authors show that exogenous DSBs induced by cisplatin, presumably which occur randomly across all chromosome homologs and thus are "symmetric," substantially rescues synapsis and spermatocytes.The manuscript itself is nicely done, and the results are convincing that cisplatin-induced DSBs rescue synapsis in a substantial fraction of hybrid spermatocytes. This would be an interesting addition to the meiosis literature, in that it supports the findings of older papers indicating that exogenous DSBs can, to some extent, substitute for SPO11 DSBs. Regarding its contribution to the hybrid sterility literature, these results strongly support the idea that a DSB deficiency underlies the synapsis defect. However, the manuscript does not provide direct evidence for the "asymmetric DSB" hypothesis; this would require a comparison of intersister vs. interhomolog repair of DSBs either genome-wide or in chromosomal intervals that are particularly susceptible to asynapsis in hybrids. This is an important shortfall of the paper with respect to linking to the "parent" paper, but it isn't clear how this can actually be addressed directly.Essential revisions:1) Add a cogent description (or cartoon figure) of the symmetric DSB model. This concept is confusing and not widely known in the meiosis field. From reading this in isolation, one might erroneously assume that symmetric DSBs are those that occur on both homologs at the same hotspot in the same spermatocyte, whereas asymmetric ones only occur on one homolog. Another interpretation is that symmetric sites are where PRDM9 binds the same locus of both homologs, but only one receives a DSB.2) Propose an explanation for why asymmetric DSBs are primarily repaired via sister chromatid recombination…this implies that such DSBs along chromosome intervals somehow communicate with one another, i.e., that if all/most DSBs are on one homolog, then they are repaired via the sisters, but if the DSBs are distributed equally between homologs, then interhomolog repair is favored. Are there hints from other model organisms in which intersister events can be monitored directly?3) Address the following concerns related to the data in Figure 4:i) the conclusion that cisplatin improves synapsis at "mid-late" pachynema requires accurate substaging of cells, which is not the case based on Figure 4A. The bottom cell is clearly at late pachynema, as shown by the bulging chromosome ends, behaviour of the XY bivalent, and DAPI-staining. But the top and middle cells are at early pachynema (compare the chromosome morphology and DAPI). The issue is magnified by the apparently very high levels of asynapsis in the untreated mice at mid-late pachynema . As the authors and others have found, cells with asynapsis are eliminated at mid pachynema, so I'm not sure how these high levels of asynapsis can be possible at late prophase I. A systematic problem in stage-matching should be considered here.ii) It isn't clear to me why the authors don't also find improved synapsis at early pachynema. Sure, the frequency of asynapsis will be higher at early than at mid-late pachynema because of the midpachytene checkpoint, but shouldn't a difference be observed between the cisplatin-treated and -untreated mice at early pachynema?4) The effect of cisplatin on other aspects of the hybrid sterile phenotype are not presented. The authors state in data not shown that cisplatin causes atrophy due to toxic effect. But couldn't they assay in the short-term whether the number of cells reaching late pachynema after treatment is increased? And how do toxic effects of cisplatin on pachytene substages influence the interpretations in Figure 4?5) EdU treatment.i) To the non-initiated, the expectations and interpretations of the EdU+ and – cells will not be easy to understand. The authors could do a better job of explaining this.ii) Why are DSB counts assayed at zygonema and pachynema but not leptonema? Isn't the latter stage the important one to focus on given the DSB hypothesis?6) Clarify the following: Why is there so much asynapsis in the early pachytenes (assuming correct staging), even with CP treatment? Is it possible that the timing was such that this cohort was derived from cells that weren't in S phase? And so many asynapsed "Mid-late" pachytenes doesn't make sense in terms of checkpoint elimination…perhaps H1t staining was needed? These potential issues with stage-matching and the lack of an objective measure (beyond incidence of full synapsis) with which to ascertain that cisplatin rescues F1 hybrid cells are major concerns. […] The basic idea is that both homologs need to receive about equal numbers of DSBs to promote interhomolog repair.We are sorry that we have not been clear enough when describing the asymmetric hotspot hypothesis. An unequal mean number of DSBs on homologs is not the explanation of our model of hybrid sterility. The proposed mechanism of asymmetric DSB hotspots  is based on the subspecies-specific erasure of PRDM9 binding sites, as demonstrated in the scheme newly added in Figure 1. The asymmetry in sterile F1 hybrids results in unrepairable or hardly repairable DSBs trying to use the homologous chromosome as a template. These unrepaired DSBs and the failure of pairing due to the deficiency of non-sister repaired DSBs results in elimination of meiotic cells.The argument goes that if one homolog gets the majority of DSBs , these will tend to be repaired by intersister recombination, which isn't productive in driving synapsis. It isn't clear to me why this would be, and the paper doesn't really address this potential mechanism. Nevertheless, the authors show that exogenous DSBs induced by cisplatin, presumably which occur randomly across all chromosome homologs and thus are "symmetric," substantially rescues synapsis and spermatocytes.Prdm9 allele-specific DSBs occuring only on one homolog  are present on both homologs, PWD on B6 chromosome and B6 on PWD chromosome . The asymmetry model is based on the subspecies-specific erosion of PRDM9 binding sites due to the meiotic drive . If the majority of DSBs cannot be repaired by recombination (CO or NCO) from the homologous chromosomes and the number of sucessfully repaired DSBs falls below a certain threshold, then the 100% efficiency of homologous chromosome synapsis becomes compromised.We believe that this premise is based on misunderstanding the asymmetric hotspot hypothesis. The The manuscript itself is nicely done, and the results are convincing that cisplatin-induced DSBs rescue synapsis in a substantial fraction of hybrid spermatocytes. This would be an interesting addition to the meiosis literature, in that it supports the findings of older papers indicating that exogenous DSBs can, to some extent, substitute for SPO11 DSBs. Regarding its contribution to the hybrid sterility literature, these results strongly support the idea that a DSB deficiency underlies the synapsis defect. However, the manuscript does not provide direct evidence for the "asymmetric DSB" hypothesis; this would require a comparison of intersister vs. interhomolog repair of DSBs either genome-wide or in chromosomal intervals that are particularly susceptible to asynapsis in hybrids. This is an important shortfall of the paper with respect to linking to the "parent" paper, but it isn't clear how this can actually be addressed directly.We think that the argument is based on misunderstanding the asymmetric hotspot concept. The question we asked was whether, as predicted by the asymmetry hypothesis, exogenous DSBs can improve synapsis of homologous chromosomes in sterile interspecies hybrids by increasing the number of presumably repairable DSBs. The apoptosis of primary spermatocytes can be provoked by persisting unrepaired DSBs, or to be a response to the failure of meiotic synapsis. We newly compared the rates of DMC1 and RPA foci at the pachytene stage in the fertile untreated parents, B6 and PWD, with sterile (PWD x B6)F1 hybrids  to emphasize the large numbers of persisting DSBs in pachynemas of sterile F1 hybrids. The results are summarized in the subsection “CisPt induced DSBs in early meiotic prophase of sterile male hybrids”, second and fourth paragraphs. To our best knowledge, no method is available to provide direct evidence for intersister chromatid repair in mouse/mammalian meiosis.Essential revisions:1) Add a cogent description (or cartoon figure) of the symmetric DSB model. This concept is confusing and not widely known in the meiosis field. From reading this in isolation, one might erroneously assume that symmetric DSBs are those that occur on both homologs at the same hotspot in the same spermatocyte, whereas asymmetric ones only occur on one homolog. Another interpretation is that symmetric sites are where PRDM9 binds the same locus of both homologs, but only one receives a DSB.We thank the reviewers for the comment. We agree that a cogent description of the symmetric DSB model was missing, which might have led to misunderstandings. We added a schematic drawing of a pair of homologous chromosomes with the asymmetric DSBs in the new Figure 1 and supplied the detailed legend.2) Propose an explanation for why asymmetric DSBs are primarily repaired via sister chromatid recombination…this implies that such DSBs along chromosome intervals somehow communicate with one another, i.e., that if all/most DSBs are on one homolog, then they are repaired via the sisters, but if the DSBs are distributed equally between homologs, then interhomolog repair is favored. Are there hints from other model organisms in which intersister events can be monitored directly?In the manuscript we do not propose that the asymmetric DSBs are primarily repaired via sister chromatid recombination. Rather, we think that the asymmetric DSBs primarily remain unrepaired, as mentioned above. Our only reference to sister chromatid recombination is: “DSBs in these hotspots are difficult to repair or they repair too late, perhaps using sister chromatids as a template .” However, we do present direct cytological evidence for the persisting DSBs at the pachytene stage and for asynapsis, both of them being able to arrest the first meiotic division on its own. Contrary to the clearance of DSBs on unpaired parts of X and Y chromosomes or on certain chromosomal translocations where DMC1/RAD51 foci disappear by late pachytene most likely by inter-sister-chromatid repair, the high numbers of RPA and DMC1 foci persist through the pachytene stage in (PWD x B6)F1 hybrid males. The cells are eliminated before the DSBs could have been repaired by inter-sister recombination . It is probable that the sister chromatid recombination also contributes to the reduction of DSB numbers at the pachytene stage (compared with zygonemas), but we do not possess tools to experimentally verify this idea.3) Address the following concerns related to the data in Figure 4:i) the conclusion that cisplatin improves synapsis at "mid-late" pachynema requires accurate substaging of cells, which is not the case based on Figure 4A.The conclusion is based on the quantitative evaluation of 645 pachynemas classified as early, middle and late pachynemas . Figure 5A illustrates the reliability of the method to detect asynapsis but legend does not specify the pachytene stage.The bottom cell is clearly at late pachynema, as shown by the bulging chromosome ends, behaviour of the XY bivalent, and DAPI-staining. But the top and middle cells are at early pachynema (compare the chromosome morphology and DAPI).We agree with the reviewer that the cell is a late pachynema and admit the pachytene stages were not specified in the legend to Figure 5A. To avoid misunderstanding, the pachytene substage of all three cells is now added to the legend.The issue is magnified by the apparently very high levels of asynapsis in the untreated mice at mid-late pachynema . As the authors and others have found, cells with asynapsis are eliminated at mid pachynema, so I'm not sure how these high levels of asynapsis can be possible at late prophase I. A systematic problem in stage-matching should be considered here.Previously, we analysed the ratio of asynaptic cells in PBF1 hybrids . It shows that the cells with four or less univalents can reach mid pachytene and a few of them late pachytene. Pachynemas with > 4 univalents were detected at early pachytene but were missing later. In this manuscript, we deliberately classified pachynemas into two groups, fully synapsed and pachynemas with asynapsis, to quantify the overall effect of cisPt on meiotic synapsis.ii) It isn't clear to me why the authors don't also find improved synapsis at early pachynema. Sure, the frequency of asynapsis will be higher at early than at mid-late pachynema because of the midpachytene checkpoint, but shouldn't a difference be observed between the cisplatin-treated and -untreated mice at early pachynema?We thank the reviewer for the comment. Indeed, we did not expect such a difference between early and mid-late pachynemas and failed to comment on it in the text. The detailed explanation was added to the second paragraph of the subsection “CisPt treatment enhances meiotic synapsis of homologous chromosomes in sterile hybrids”. See also our response to point 6.4) The effect of cisplatin on other aspects of the hybrid sterile phenotype are not presented. The authors state in data not shown that cisplatin causes atrophy due to toxic effect. But couldn't they assay in the short-term whether the number of cells reaching late pachynema after treatment is increased? And how do toxic effects of cisplatin on pachytene substages influence the interpretations in Figure 4?We thank the reviewers for the suggestion to focus on the late pachynemas after treatment. The comparison indeed shows the increase of late pachynemas. The analysis was included as follows “Beside the increased frequency of fully synapsed pachynemas, a short-term effect was apparent from the increased relative incidence of late pachynemas. While in untreated control hybrids the late pachynemas represented 2.03% (4/107) of all pachynemas recorded from the meiotic spreads, the frequency increased to 12.12% (28/231) and 13.10% (30/229) after 5 mg/kg and 10 mg/kg cisPt treatment, respectively”.For the interpretation of the toxic effect of cisPt in Figure 5, see also our answer to Essential revisions No 6.5) EdU treatment.i) To the non-initiated, the expectations and interpretations of the EdU+ and – cells will not be easy to understand. The authors could do a better job of explaining this.We thank the reviewer for comment. The rationale of EdU treatment was explained as follows: “we treated the adult (4-8 weeks) PBF1 hybrid males with cisPt and with 5-ethynyl-2’-deoxyuridine (EdU), a nucleoside analog of thymidine  to distinguish the spermatogenic cells replicating their DNA at the moment of cisPt injection.”We also added an additional sentence of explanation to the first paragraph of the subsection “CisPt treatment enhances meiotic synapsis of homologous chromosomes in sterile hybrids”.ii) Why are DSB counts assayed at zygonema and pachynema but not leptonema? Isn't the latter stage the important one to focus on given the DSB hypothesis?We had two reasons for assaying zygonemas. The spread preparations contain very few spermatocytes at the leptotene stage, and DMC1 and RAD51 foci are more numerous at zygotene than in leptotene . The most probable reason is that the foci accumulate gradually during the leptotene stage and survive for the major part of the zygotene stage.6) Clarify the following: Why is there so much asynapsis in the early pachytenes (assuming correct staging), even with CP treatment? Is it possible that the timing was such that this cohort was derived from cells that weren't in S phase?We thank the reviewers for the comment. The missing explanation was added on as follows: “We assume that the efficiency of repair of cisPt-induced DSBs by meiotic homologous recombination is low and/or that a significant fraction of spermatocytes carrying them do not survive until early pachytene stage. […] This multiple filtering effect thus enhances the apparent efficiency of cisPt monitored as a proportion of fully synapsed pachynemas at mid-late stage.”And so many asynapsed "Mid-late" pachytenes doesn't make sense in terms of checkpoint elimination…perhaps H1t staining was needed?The question was answered above: The ratio of asynaptic cells in PBF1 hybrids is given in Bhattacharyya . It shows that only cells with four or less univalents can reach mid pachytene and a few of them late pachytene. Pachynemas with > 4 univalents were detected only in the early pachytene stage. In this manuscript, we deliberately classified pachynemas as fully synapsed and pachynemas with asynapsis to verify the overall effect of cisPt on meiotic synapsis.These potential issues with stage-matching and the lack of an objective measure (beyond incidence of full synapsis) with which to ascertain that cisplatin rescues F1 hybrid cells are major concerns.We hope the concerns about the stage-matching were dispelled. We checked the effect of exogenous DSBs at the level of synapsis, which was verified. The ratio of the three pachytene substages was added to show the significant increase of late pachynemas after cisPt treatment. See also the answer to point 4.


Mechanistic approaches to modeling the effects of ionizing radiation on cells are on the rise, promising a better understanding of predictions and higher flexibility concerning conditions to be accounted for. In this work we modified and extended a previously published mechanistic model of cell survival after photon irradiation under hypoxia to account for radiosensitization caused by deficiency or inhibition of DNA damage repair enzymes. The model is shown to be capable of describing the survival data of cells with DNA damage repair deficiency, both under norm- and hypoxia. We find that our parameterization of radiosensitization is invariant under change of oxygen status, indicating that the relevant parameters for both mechanisms can be obtained independently and introduced freely to the model to predict their combined effect. Radiation therapy is one of the cornerstones of cancer care, where ~50% of patents receive radiation during the course of disease . RadiobiTo model the response of hypoxic tumor cells to dual treatment, we based our approach on a previously published model of our group . Similar studies ,23. The  studies ,13 assoctin loss  and theytin loss ,25,26. Ital data ,26.Regarding the hypoxic cell population, Carlson et al.  made com context . In lineng rates , the letn et al.  (a functFlanders ) 11]. I. IHRFDSBRSF) that modifies To further extend the model and describe the particular case of hypoxic tumor cells response to dual treatment, radiotherapy, and DDR inhibition, we introduced a so-called radiosensitization factor  exposed to three distinct oxygenation levels  for each of the mutant cell lines, their survival under normoxia was able to be fitted accurately. The numerical values of the parameters can be found in RSF derived for each mutant under normoxia, the survival of the two mutant cell lines under hypoxia could be predicted satisfactorily. However, deviations can be observed at the highest reported doses.Survival data of the CHO cell line and two of its DNA-PK response-deficient mutants (V3 cell line is DNA-PKcs-deficient and xrs-5 cell line is Ku80-deficient) under normoxia and hypoxia were gathered from Cartwright et al.  30]. Th. Th30]. RSFs for the lethality parameters of the isolated lesions  exposed to different concentrations of an ATM inhibitor (ATMi) and irradiated under normoxia and hypoxia . First, Our model provided an excellent description of the survival data of five cell lines and three oxygen levels presented in RSF) for the two DNA-PK response-deficient mutants analyzed. Therefore we decided to introduce the RSF as a free parameter in UNIVERSE, fitted to each mutant cell line. The RSF values found for the two CHO mutants V3 and xrs5 indicate that the probability of an isolated lesion to become a lethal lesion increases through DNA-PK response deficiency in these cell lines by a factor of about 10 and 15, respectively. Even though both RSF factors are related to the functional DNA-PK repair activity, the difference in the RSF of both cell lines might be retraced to the fact, that both are deficient of different enzymes taking part in the DNA-PK response: While xrs-5 cells are deficient of the Ku80 DNA-PK subunit [RSF is coupled to the activity of both proteins. Furthermore, different extents of remaining expression or compensation by other repair proteins might also lead to the observed difference in the RSF values of both mutants. One would have to compare several groups of cell lines with the exact same deficiencies to gain a better understanding of the underlying dependencies. More importantly, we could show that the survival of the mutant cell lines under hypoxia were well predicted by our model by combining the RSF values derived from the normoxic data of each mutant invariant.The idea of increasing the lethality of isolated lesions LE model . They cot et al.  and foun subunit ,31, V3 c subunit ,32. It iRSF for isolated lesions. Further, the values of the derived RSF illustrate the increasing lethality of isolated lesions with increasing drug concentrations, leading to stronger inhibition of the repair, while staying below the value derived for a pathway deficiency. It is also to be noticed that the RSF values for both H460 and H1437 cells are fairly similar. However, more cell lines have to be analyzed to investigate whether this can be extended to a general trend and if the presented method is generally applicable to other repair-inhibiting drugs. Nevertheless, we could show that the RSF values derived from normoxic data can accurately describe the survival in hypoxic conditions by introducing the Since both DNA-PK and ATM are essential molecules for irradiation-induced DNA damage repair, recruited to the DNA damage sites ,34,35, wRSF in our model. Moreover, this modified lethality could stay invariant under change of oxygen supply, while sustaining good predictive capabilities. Thus, the uniqueness of our approach lies in its capability to describe two separate cell response mechanisms in any combination using minimal input parameters, which can be separately derived. Based on this, we believe that our approach has a high potential to implement further cellular mechanisms in order to produce predictions tailored to diverse clinical scenarios.Taken together, we could show that impairment of DNA damage repair, both in repair-deficient cell lines and cells treated with a DDR inhibitor, could be accounted for by a manipulation of the lethality of isolated lesions with an Experimental data used to benchmark the model on survival of genetically DDR-deficient cell lines were taken from . The dat2 atmosphere. Experiments in hypoxic conditions were performed at 0.5 or 1% O2 and 5% CO2 using a custom hypoxic chamber , including an online monitoring controller for O2 and CO2 concentrations . Fifty cells per well in a 96-well format were seeded not more than 16 h before irradiation. Hypoxic irradiation was performed after incubation for 4 h under respective oxygen conditions. Cells were irradiated in the sealed hypoxia chamber with a dose series of photons of 1, 2, 4, or 8 Gy and thereafter incubated under normoxic conditions. The ATM inhibitor was kindly provided by Merck KGaA, and dissolved in DMSO  and diluted in RPMI 1640 medium. The inhibitor was added to H460 and H1437 cells at 100, 200, or 500 nM just before incubation under hypoxia or normoxia started. Controls also contained < 0.1% DMSO. Inhibitors were left in the media for 24 h and then replaced with fresh RPMI 1640 medium and the plates were returned to the incubator for colony formation. After 4 days (A549), 5 days , or 7 days (H1437), plates were imaged by an online microscopy system at 4x magnification . The images were analyzed by the IncuCyte Zoom Software (ver. 2016a)  and colony counts were confirmed by manual curation.For the validation of our model under hypoxic conditions, additional experiments were performed using Renca  and B16-Blue ISG  (B16) cells. Both cell lines were grown in RPMI 1640 Medium  supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% COThe irradiation plan was carried out as a step and shoot intensity-modulated radiotherapy (IMRT) plan, describing the different dose levels as separate target regions. Planning was done with Raystation treatment planning system  based on a CT scan of the hypoxia chamber containing 96-well plates filled with water. Irradiation was performed on a Siemens Artiste (6 MV) .D the applied dose in Gy. The total number of giant loops  is given by [S values obtained from the Monte Carlo algorithm. The lethality parameters can be determined by fitting the result of this routine to survival data.We implemented a Monte Carlo routine in which, at each iteration, the number of total DSB in the nucleus (given by :(3)S= was introduced into the model. The RSF modifies the lethality parameter of isolated damages In order to model the increased cell killing of repair-deficient mutant cell lines or cells exposed to different concentrations of a repair inhibiting drug, a radiosensitization factor  to repair impaired data, while the lethality parameters


The United States National Institute for Occupational Safety and Health (NIOSH) is developing a protocol to assess the containment performance of closed system transfer devices (CSTDs) when used for drug preparation (task 1) and administration (task 2) and published a draft protocol in September 2016. Nine possible surrogates were proposed by NIOSH for use in the testing. The objectives of this study were to: (A) select the most appropriate surrogate; (B) validate the NIOSH protocol using this surrogate; and (C) determine the containment performance of four commercial CSTDs as compared with an open system of needle and syringe using the validated NIOSH protocol.2-Phenoxyethanol (2-POE) was selected as a surrogate based on its water solubility, Henry’s volatility constant, detectability by mass spectrometry, and non-toxicity. Standard analytical validation methods including system suitability, limit of detection (LOD), and limit of quantitation (LOQ) as well as system cleaning validation were performed. The amount of 2-POE released when the CSTDs were manipulated according to two tasks defined by NIOSH was determined using mass spectrometry coupled to thermal desorption and gas chromatography. This approach allows sensitivity of detection below 1 part per billion (ppb). Equashield, Tevadaptor (OnGuard), PhaSeal, and ChemoClave were assessed according to manufacturers’ instructions for use.2-POE was tested and validated for suitability of use within the NIOSH protocol. A simple and efficient cleaning protocol achieved consistently low background values, with an average value, based on 85 measurements, of 0.12 ppb with a 95% confidence interval (CI) of ±0.16 ppb. This gives an LOD for the tests of 0.35 ppb and an LOQ of 0.88 ppb. The Equashield, Tevadaptor (OnGuard), and PhaSeal devices all showed average releases, based on 10 measurements from five tests, that were less than the LOQ (i.e. < 0.88 ppb), while the ChemoClave Vial Shield with Spinning Spiros showed average releases of 2.9±2.3 ppb and 7.5±17.9 ppb for NIOSH tasks 1 and 2 respectively at the 95% confidence level. The open system of needle and syringe showed releases, based on two measurements from a single test, of 4.2±2.2 ppb and 5.1±1.7 ppb for NIOSH tasks 1 and 2 respectively at the 95% confidence level.2-POE proved to be an ideal surrogate for testing of CSTDs using the NIOSH protocol. We propose that a CSTD can be qualified using the NIOSH testing approach if the experimental LOQ is less than 1 ppb and the release values are below the LOQ. Equashield, Tevadaptor (OnGuard), and PhaSeal meet these acceptance criteria and can therefore all be qualified as CSTDs, but the ChemoClave system does not and so would not qualify as a CSTD. Many anti-cancer drugs are potentially therapeutic to patients, but unintended exposure to these agents poses an occupational hazard to healthcare workers ,2. HealtAccording to NIOSH, CSTDs are drug transfer devices that mechanically prevent the transfer of environmental contaminants into the system and the escape of hazardous drug or vapor concentrations outside the system . CSTDs rIn the US, NIOSH developed a draft protocol in 2015 to assess the performance of CSTDs in response to the increasing number of CSTDs in the marketplace and an awareness of scientifically non-robust and non-quantitative CSTD containment studies sponsored by commercial manufacturers . The 201The main difference with the 2016 draft NIOSH protocol is the measurement of released challenge agent within the chamber under non-flow-through conditions, which allows quantitative time weighted average (TWA) data to be obtained. The test requires the use of an appropriate semi-volatile challenge material from the list of nine agents proposed by NIOSH . Air samTandem air samples are collected on sorbent tubes placed within the NIOSH chamber. The air samples are analyzed using the gold standard analytical detection approach of automated thermal desorption gas chromatography mass spectrometry (ATD-GC-MS) and the released material quantified down to sub-ppb levels. Details are in accordance with the published HSE method MDHS104 .The Biopharma Stability Testing Laboratory (BSTL) and the Health and Safety Laboratory (HSL) in the UK provided a response to the NIOSH request for information in January 2016 and this response is in excellent agreement with the latest draft NIOSH proposal ,13.th of February 2018 [The public comment period for the 2016 draft NIOSH protocol was closed on the 28ary 2018 . One of The present study provides a significant advancement toward a complete protocol by providing data for one surrogate, 2-phenoxyethanol (2-POE), one of nine potential candidate surrogates proposed by NIOSH in the draft protocol . The OSHWith the launch of USP800, there is an urgent requirement for pharmacists to have access to scientifically robust test data that enable assessment of CSTD containment efficacy for the different available commercial CSTDs on the market to help inform a purchasing decision .The 2016 draft NIOSH protocol builds on the earlier protocol and uses the same NIOSH chamber. . BSTL haThe 2015 draft NIOSH protocol used a 70%:30% IPA:water mixture as the surrogate, which has since been rejected by NIOSH because it has a very high volatility and vapor breakthrough that is not representative of any of the classes of known hazardous drugs. The difference in behavior is due to the much lower vapor pressures of hazardous drugs ,20. The Parenteral solutions of hazardous drugs are either supplied as lyophilized powder for reconstitution or as a ready-to-administer solution. Administration is performed using an aqueous solution, which means the amount of drug present in the vapor above the liquid is estimated using Henry’s volatility constant and not by the vapor pressure of the pure substance. The Henry’s volatility constant is able to predict the chemical vapor concentration resulting from liquid leakage of hazardous drugs. We have selected 2-POE as the surrogate of choice based on its physicochemical properties, including its Henry’s constant, which is more than two orders of magnitude greater than that of the most volatile hazardous drug, 5-Fluorouracil (when presented as an aqueous solution). Using a 2.5% v/v solution of 2-POE, a liquid release volume as low as 1 μL can be detected using the analytical methods described in this study, which is due to the high sensitivity of detection, less than 1 ppb of release within the chamber. Smaller volumes of 2-POE release in the liquid state will not be detected due to the experimental limit of detection (LOD) and limit of quantitation (LOQ) demonstrated.et al [Vapor pressures of pure active pharmaceutical ingredients as determined by Kiffmeyer et al  are not et al ,20. HazaBSTL and HSL proposed the use of 2.5% v/v 2-POE in water as challenge agent to be used within the 2016 draft NIOSH protocol, the response can be found on the docket . 2-POE i2.5% v/v 2-POE was prepared by addition of 2.5 mL of 2-POE  to 97.5 mL of Milli-Q water , mixed by shaking, and 50 mL aliquoted into 100 mL type I glass vials . 100 mL drug vials  were prepared by adding 50 mL of Milli-Q water, and sealed. The Milli-Q-containing vials were used for all negative control experiments. Thermal desorption (TD) tubes 89 mm long × 6.4 mm outer diameter and packed with 200 mg of Tenax TA  were used. Internal standard was prepared by dissolving d8 toluene  in analytical grade methanol .et al. [A NIOSH chamber was constructed as described in the 2015 draft NIOSH protocol with specific modifications described by Wilkinson et al. . The chaet al.  was usedFor additional detail, please refer to the supplemental materials provided electronically.The assessment of CSTD containment performance was performed in accordance with the 2016 draft NIOSH protocol (11) as follows:A 2.5% v/v POE solution was used as the challenge agentAn air purge of the chamber was performed at a flow rate of 15 L/min for 30 minutes prior to useAn environmental air sample was taken each day prior to testingAir samples were collected at a flow rate of 100 mL/min and for 30 minutes using dual air sampling pump systems connected to Tenax TA TD tubesDual blank chamber measurements were recorded prior to testing of a CSTD to ensure no contamination inside the chamberA robust chamber cleaning procedure was established and used throughout the testingIn the 2016 draft NIOSH protocol, the air sampling is stopped after completion of the NIOSH tasks 1 and 2. However, in the present protocol the pumps continue collecting air for a fixed duration of 30 minutes. NIOSH tasks 1 and 2 were performed as stated in the 2016 draft NIOSH protocol except that the number of tests was increased to five . NegativSorbent tubes were analyzed using automated ATD-GC-MS detection. Analytical conditions and instrument calibration are described fully in the supplemental materials. A TWA sample approach allowed for determination of the mass of POE collected on the sorbent tubes, in nanograms, using a calibration curve of POE . The masData processing, determination of the experimental LOD and LOQ, and data analysis were performed in accordance with the HSE MHDS104 method . The POEi.e. that no 2-POE was detected on any of the system blank tubes analyzed after each test sample. Using the analytical method described, an instrumental LOD of less than 0.1 ng for 2-POE and an experimental LOD of 0.4 ppb were demonstrated.The breakthrough tests performed using sample volumes of 3, 6, and 12 liters confirmed that there was no breakthrough using the air sampling approach described, and furthermore that >99.9% recovery was achieved from the Tenax TA air sampling tubes, meaning that 2-POE is totally desorbed by the analytical procedure. Under the conditions of testing this is well within the upper limits for breakthrough for the Tenax sorbent media. We also demonstrate that no carryover was observed, A total of four commercially available CSTDs that employed either physical barrier  or air filtration technology  were included in our study .A larger number of replicates were used in this study (n = 5) than recommended in the original protocol (n = 4) ,18. WithChamber blank measurements (n = 85) and positive controls  were performed prior to each device test as described in the modified NIOSH protocol. No background contamination was present (data not shown). Additional blank measurements were performed using water as a negative control (n = 2). A typical gas chromatogram signature for 2-POE detection by mass spectrometry is shown in Results for 2-POE concentration in the test chamber for tasks 1 and 2 are presented in The average values obtained for PhaSeal, Equashield, and Tevadaptor (n = 5 each) were below the blank control-corrected LLOQ for each of the two tasks (≤ 0.88 ppb). However, ChemoClave’s Vial Shield with Spinning Spiros produced values that were 5 to 25 times the LOD, and one measurement was 120 times higher than the LOD. The higher values obtained with the ChemoClave device can be attributed to the liquid residue observed on the membranes following disconnection , resultiA strength of the proposed protocol when 2-POE is employed as challenge agent is that devices that produce a clearly observable liquid release give much higher vapor concentrations of 2-POE. This is because the 2-POE released is quickly taken in to the vapor and measured. In contrast, devices that did not appear to produce any liquid release gave either below LOQ or close to LOQ values for 2-POE. The findings suggest that the protocol provides a means to differentiate devices that give a release of liquid from those that do not, and that vapor release is much less significant compared with liquid release in terms of increasing the vapor concentration of 2-POE.An independent evaluation and review of the first draft NIOSH protocol from 2015 identified several limitations . First, i.e. under the conditions ofthe 2015 draft NIOSH protocol [There are other practical concerns associated with the detection devices used in the 2015 draft NIOSH protocol design . Real-tiprotocol . Furtherprotocol ,13. HoweThe TWA approach proposed in the current 2016 draft NIOSH universal protocol does provide a truly quantitative assessment of containment performance of CSTDs ,13. By sThe unified 2016 draft NIOSH protocol employing ATD-GC-MS analysis allows both air filtration and physical barrier CSTDs to be assessed and represents a paradigm shift from the pre-NIOSH protocol and the 2015 draft NIOSH protocol era, when it was not possible to compare CSTDs based on robust scientific data ,13. Withet al demonstrates the difficulties in cleaning the NIOSH chamber in between releases when TEU is used as challenge agent in the NIOSH testing [The change in challenge agent from IPA to 2-POE presents a more representative surrogate for hazardous drugs in terms of vapor pressure, Henry’s volatility constant, chemical structure, and physicochemical behavior. Furthermore, 2-POE can be efficiently cleansed from the testing chamber following a high-level release using a validated cleaning protocol, enabling CSTD testing to continue without delays. In contrast, potential challenge agents that are much more persistent and difficult to remove from the test chamber following a high release are impractical to use. Recent published work by Wilkinson  testing . In addiThe study data obtained using our modified, universal protocol showed that CSTDs with both air filtration and physical barrier technology can prevent the release of hazardous drugs as vapor, liquid, and liquid-based aerosols during compounding and administration tasks performed in the clinic. Our data also demonstrated that Tevadaptor (OnGuard), which employs air filtration technology, produced containment efficacies equivalent to the two physical barrier CSTDs tested, including the reference predicate product PhaSeal .There are additional challenges that NIOSH may address in relation to CSTD assessments. As no acceptance criteria have been suggested by NIOSH, it is currently impossible for hospital pharmacists to make an informed purchasing decision based on the release data from the protocol. We recommend that acceptance criteria should be established based on either providing a protection factor (PF) over that of using an open system of needle and syringe, or on the experimental LOQ value of the CSTD when challenged. In this study we report efficacies of CSTDs based on the release values being above or below LOQ using the assay described, which has sub-1 ppb sensitivity. A CSTD that has containment performance yielding below-LOQ values represents the highest efficacy that can be obtained. Other air sampling approaches could be adopted as an alternative to ATD-GC-MS; for example, diffusive sampling using solid phase microextraction or sorbent pen, and active sampling using a needle trap. However, investigations performed by BSTL and HSL using alternative sampling approaches did not show potential for use under the NIOSH protocol due to lack of sensitivity in detection for 2-POE. As such, the ATD-GC-MS was shown to be the most sensitive air sampling detection strategy.2-POE has proved to be an ideal surrogate for testing of CSTDs using the NIOSH protocol. Equashield, Tevadaptor (OnGuard), and PhaSeal all produced release values that were below the LOQ for the assay, whereas the ChemoClave Vial Shield with Spinning Spiros system released higher amounts than the open system of needle and syringe, showing that physical barrier as well as air filtration technology can provide hazardous drug containment.We propose that acceptance criteria be established for scoring the containment performance of CSTDs either based on the experimental LOQ or a PF approach where: 1.) a PF must be significantly higher than the proportion between the values obtained for the open system divided by the experimental LOQ of that system and 2.) the experimental LOQ must not be greater than 1 ppb.In our system, Equashield, Tevadaptor (OnGuard), and PhaSeal provided a PF greater than 5.8 for task 1 (4.1 ppb/0.71 ppb) and a PF greater than 7.2 for task 2 (5.13/0.71) and therefore these systems meet the acceptance criteria for significantly reducing operator exposure and are qualified for handling hazardous drugs.CSTDs that do not provide any additional protection to the operator over an open system when handling hazardous drugs or even release higher amounts of hazardous drugs, cannot be considered a form of protective equipment, and these devices are therefore unsuitable for use with hazardous drugs.S1 Text(DOCX)Click here for additional data file.