Gata4 gene as a driver. 80% of G2-Gata4Cre;Wt1fl/fl embryos developed typical Bochdalek-type CDH. We show that the posthepatic mesenchymal plate coelomic epithelium gives rise to a mesenchyme that populates the pleuroperitoneal folds isolating the pleural cavities before the migration of the somitic myoblasts. This process fails when Wt1 is deleted from this area. Mutant embryos show Raldh2 downregulation in the lateral mesoderm, but not in the intermediate mesoderm. The mutant phenotype was partially rescued by retinoic acid treatment of the pregnant females. Replacement of intermediate by lateral mesoderm recapitulates the evolutionary origin of the diaphragm in mammals. CDH might thus be viewed as an evolutionary atavism.Congenital diaphragmatic hernia (CDH) is a severe birth defect. Wt1-null mouse embryos develop CDH but the mechanisms regulated by WT1 are unknown. We have generated a murine model with conditional deletion of WT1 in the lateral plate mesoderm, using the G2 enhancer of the DOI:http://dx.doi.org/10.7554/eLife.16009.001 Congenital diaphragmatic hernia (CDH) is a severe birth defect, characterized by incomplete formation or muscularization of the diaphragm and, as a consequence, herniation of the stomach, intestines, liver or spleen into the pulmonary cavities, leading to pulmonary hypoplasia. CDH occurs approximately in 1 out of 3000 births, accounting for about 8% of all severe congenital anomalies. 80–90% of all the cases are posterolateral hernias, also known as Bochdalek-type CDH, characterized by a defect in the postero  lateral area of the diaphragm. In most cases (>85%), this defect is located at the left side . The claCrePrrx1 driver leads to the development of amuscular, weak areas in the diaphragm and CDH  signaling pathway has also been involved in diaphragmatic development. A classical animal model for CDH consists in the treatment of pregnant rats with nitrofen, a substance that inhibits the synthesis of RA. It has been shown that nitrofen treatment leads to reduced size of the PPFs/PHMP, especially in the left side, decreasing cell proliferation in this area  and alsoWt1 in mice leads to defective diaphragms . The t-test for one sample compared with an expected value = 1  gave a result of 5,21 . Thus, the right PHMP contains about 50% more cells from the G2-Gata4 lineage than the left one.A clear asymmetry was observed in the distribution of PHMP mesenchyme in embryos of E10.5-E12.5 . MesenchThis is a basically descriptive study of the phenotype of mouse embryos with conditional deletion of Wt1 in lateral mesoderm. We have included in the study 36 out 104 mutant embryos detected by genotyping. The rest of them had been previously used for a study of the cardiac phenotype .Wt1 gene using the described G2-CreGata4 line. Deletion of WT1 in the PHMP/PPFs continuum, i.e. in the G2+ domain, causes a phenotype characterized by defects in the inflow tract of the heart, coronary vessels  of the epicardium by repression of E-cadherin and activation of Snail1 , we checing PHMP . Howevering PHMP .10.7554/Cre;Wt1fl/flGata4 CDH phenotype with that displayed by the systemic WT1 knockout mouse embryos, which do not survive beyond E13.5. Despite the early embryonic death of these models, we have observed a more severe defect, with a complete lack of PHMP and displaying, in some cases, persistent thoracic nephric ridges in E12.5 and E13.5 embryos  signaling pathway has been involved in the formation of diaphragm. To test whether RA signaling might be disturbed in our CDH model, we checked the expression of RALDH2 by immunofluorescence in G2- embryos . The PPFthe PHMP . This prthe PHMP .Cre; Wt1fl/flGata4 mutant embryos. Five of them  displayed a complete septation of the pleural cavity by E15.5, i.e. about three times the proportion of normal phenotypes found in the non-treated mutants . Control littermates were not affected by the RA-treatment.RA treatment partially rescues the pulmonary phenotype and promotes alveologenesis in the nitrofen model of induced CDH in rats . In ordeThen, we designed a second experiment to assess the effect of the RA dietary supplement on the development of the diaphragmatic defect, comparing E13.5 mutant embryos with control embryos of the same age obtained from pregnant females fed with the same diet without the retinoic acid supplement . Seven m+) and the intermediate (G2−) mesodermal domains. The lateral mesoderm domain would be characterized by the expression of Gata4 driven by the enhancer G2 while the intermediate mesoderm domain would lack Gata4 expression. The limit between G2+ and G2− domains is established as early as E10.5 in the coelomic epithelium lateral to the mesentery  through the so called PHMP . This coesentery , betweenWe think that another important factor in order to understand the development of the CDH is the asymmetrical distribution of the mesenchyme in the PHMP/PPFs continuum. Asymmetry has already been described in the gut mesenchymal compartment, where cells are more densely packed on the left than on the right . HoweverOur results suggest that CDH could be caused by a defective population of the PHMP/PPF continuum by mesenchymal cells. A process of migration of liver mesenchymal cells into the PPFs relevant for the closure of the pleuroperitoneal canal had been suggested in human embryos . Since t+ domain (PHMP and anterior part of the PPFs) for the formation of the pleuroperitoneal septa. The immediate consequence of its deletion in this territory is a lesser number of mesenchymal cells in the PHMP. Furthermore, WT1-null embryos completely lack of PHMP. WT1 is critically involved for EMT of the epicardium  administration or plasmid-mediated overexpression, resulted in ectopic kidney gene expression  in its primitive anterior position.The presence of lateral plate mesoderm into the PPFs coincides with the backward movement of the intermediate mesoderm from the thoracic levels. Significantly, pression . This isCre;Gata4 fl/flPrrx1 model of Merrell et al.  and our WT1 model, much more similar to the human Bochdalek hernia . Incidentally, we think that the claim made by WT1 has a dual function concerning the epithelial-mesenchymal transition and the reverse process, mesenchymal-epithelial transition (MET). The first process is promoted by WT1 in heart and liver (G2+ domain), but MET is induced by WT1 in the kidneys (G2- domain)  that lacIn summary, two types of CDH would originate in different moments of development. CDH due to defective generation of the PHMP mesenchyme appears in models of RA deficiency and also in models with loss of function of WT1. In later stages CDH can develop by defective muscularization of the pleuroperitoneal septa. This type of CDH occurs in models of GATA4 conditional deletion  and alsoThe dietary supplement of RA to pregnant females that rescues pulmonary hypoplasia in the nitrofen model of CDH  was alsoThe continuity between septum transversum, PPFs and nephric ridges suggests an evolutionary scenario about the origin of the diaphragm in mammals. The diaphragm probably derives from the cephalic end of the nephric ridges, which run throughout the peritoneal cavity in fish, amphibians and reptiles. We suggest that the thoracic nephric ridges were invaded in the ancestors of mammals by lateral plate mesodermal mesenchyme, giving rise to the pleuroperitoneal septa. In fact, the idea that PPFs derive from nephric ridges is not new. It was already proposed by In summary, Bochdalek's hernia could be regarded as an atavistic condition related with the failure in the ontogenetic/evolutionary process which replaced the thoracic intermediate mesoderm by lateral plate mesoderm expressing GATA4 under control of the G2 enhancer. WT1 was probably crucial in the process of generating the cellular substrate which made this replacement possible. A right/left asymmetry in this process is probably behind the left prevalence of this disease.The animals used in our research program were handled in compliance with the institutional and European Union guidelines for animal care and welfare. The procedures used in this study were approved by the Committee on the Ethics of Animal Experiments of the University of Malaga (procedure code 2015–0028). All embryos were staged from the time point of vaginal plug observation, which was designated as E0.5. Embryos were excised and washed in PBS before further processing.LacGata4Z and G2-CreGata4 transgenic lines have been previously described  mouse has been used in previous studies to trace or delete specific genes in WT1-expressing cells (Gata4Cre+/- mice and homozygote Wt1Cre+/+ were crossed with Rosa26R (B6.129S4-Gt(ROSA)26Sortm1Sor/J, RRID:IMSR_JAX:003474) and EYFPRosa26 (B6.129X1-Gt(ROSA)26Sortm1(EYFP) Cos/J, RRID:IMSR_JAX:006148) mice to generate permanent reporter expression in G2-Gata4 and Wt1-expressing cells. Generation of loxP-flanked Wt1 mice were performed as described , and frozen in liquid N2-cooled isopentane. Samples were sectioned on a cryostat (10 μm) and cryosections were stored at −20°C until use. Other samples for routine histology were dehydrated and paraffin-embedded.For in situ hybridization the embryos were isolated in ice-cold PBS and were fixed overnight in 4% paraformaldehyde in PBS. In situ hybridization was performed as previously described . The PaxAB_476701) was used at a 1:100 dilution for immunofluorescence. Polyclonal rabbit anti-RALDH2  was used at a 1:5000 dilution. Polyclonal goat anti-GATA4  was used at 1:50 dilution for immunofluorescence. Polyclonal chicken anti-GFP  was used at 1:200 dilution. Monoclonal anti E-cadherin  and monoclonal anti-mouse WT1  were used at 1:100 dilution. For WT1, E-cadherin and GATA4 immunostaining, citrate buffer antigen retrieval was required. Negative controls were performed by incubation with non-immune mouse serum or rabbit IgG instead of the primary antibody.The monoclonal mouse anti-smooth muscle cell α-actin antibody . Between 6 and 9 Images of consecutive 10 µm thick serial sections covering all the PHMP were processed by splitting the channels, transforming the green (YFP+) channel into a 8-bit format, and measuring the total area occupied by YFP+ cells in the left and right PHMP using Fiji image analysis software (RRID:SCR_002285). The total sum of the areas along all the serial sections was used as an estimation of the total volume occupied the YFP+ cells. We then calculated the R/L volume ratio for each embryo and this value was used for statistical comparisons.Image analysis was performed on digital images obtained from four Cre;Wt1fl/flGata4 embryos was performed by supplying RA in the diet of the pregnant female mice between E8.5 and E10.5, i.e. when the ST and PPFs develop. At 8.5 and 9.5 days of pregnancy, female mice were fed with a paste composed of 5 g of normal chow, 5 mL of distilled water, 1 mL of corn oil and 1 mg of RA (Sigma). Control females received the same diet without RA. The food was placed in a cage protected from light and pregnant female mice consumed the RA-supplemented diet normally. By the beginning of the day 10.5, RA-supplemented diet was replaced by normal chow. We collected the embryos at stages E13.5 and E15.5.Rescue of the phenotype in G2-We used the Z-test for comparison of the R/L PHMP volume ratio and for comparison of the percentages of CDH incidence in RA treated and control embryos. For comparison of the size of the left pleural cavity opening in RA treated and control embryos we used the Student’s test. In both cases, calculations were performed using Excel 2013 software. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Harry Dietz as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Conditional deletion of WT1 in the septum transversum mesenchyme causes congenital diaphragmatic hernia in mice" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This is a thought-provoking study that deals with a topic of much clinical and fundamental interest. Congenital Diaphragmatic Hernia (CDH) is a common birth defect, affecting 1 in 3000 children. The most prevalent form, known as Bochalek-type CDH is characterised by a defect in the postero lateral area of the diaphragm which is asymmetric, the majority of cases affecting the left side.There has been much speculation about the developmental origins of CDH, a problem that had been addressed in several recent papers using mouse models. Carmona and colleagues have investigated the role of the Wilms tumor transcription factor in septum transversum mesenchyme and identified a role in diaphragm development, with implications for both ontogeny and evolution of the diaphragm. They now provide the first animal model that appears to mimic the most common form of CDH. The authors trace the origins of the CDH to a deficiency of the posthepatic mesenchyme plate (PHMP) that arises from the coelomic epithelium. Importantly the authors show for the first time that the right part of the PHMP has more mesenchyme cells than the left at the time of septa development and this may explain the asymmetry. It has been shown previously by the authors that WT1 transcriptionally activates Raldh2 in the developing liver mesothelial derived cells. Raldh2 is essential for synthesis of retinoic acid (RA). In another study it was shown that maternally introduced RA can partially rescue CDH in a chemically induced model. Interestingly this chemical model also mimicked Bochdalek-type CDH, showing left side predominance. Carmona et al. show here that Raldh2 levels are much reduced in the mutant PHMP mesenchyme.Overall, this is a scholarly descriptive study that had many interesting features. While the complex embryology of diaphragm development is carefully documented using a range of molecular and genetic markers, a number of major points need to be addressed to justify the authors' conclusions.Essential points:1) It would help to provide some marker analysis to inform on cell state . In particular, detailed analysis of EMT markers seems necessary to support the authors' arguments.2) When do the embryos die and what other defects are observed? This is referred to very loosely.3) The RA rescue is potentially important but very poorly described at the moment. First, rigorous statistical analysis has to be applied. Second, if not all animals are rescued could it be that some have partial rescue that indicates a qualitative difference between RA exposed and non RA exposed embryos? Has detailed anatomical analysis been carried out on all the embryos? It would help to provide some way of representing these data, perhaps a table with a few pictures of embryos.4) Carmona et al. show here that Raldh2 levels are much reduced in the mutant PHMP mesenchyme. Supplementation of the pregnant females' diets with RA at 8.5 and 9.5 dpc seemed to result in rescue of the phenotype as 65% of animals with the mutant genotype have complete septation of the pleural cavity by E15.5. This would be a very important observation, but needs to be better documented.5) Experimental evidence is required in support of cell migration from the coelomic epithelium to the PHMP or the PHMP to PFF. In the absence of direct evidence from DiI labelling, for example, comparison of Gata4 G2 Cre and conditional YFP expression may be helpful.6) Is it possible to quantify the asymmetry in PHMP mesenchyme? Can the authors provide genetic evidence that this is connected to embryonic left right asymmetry?7) How do the authors explain the variability in the conditional mutant phenotype? Do they consider that this is due to variable Cre expression or another cause? For example, variability in RA synthesis? Might removing one allele of Raldh2 influence the phenotype?8) Please provide information as to the relative timing of the onset of Gata4 and WT1 expression in septum transversum mesenchyme.9) Can the authors provide any data on WT1 expression in different species to support the evolutionary arguments in the eleventh paragraph of the Discussion? 1) It would help to provide some marker analysis to inform on cell state . In particular, detailed analysis of EMT markers seems necessary to support the authors' arguments.We had performed a preliminary study using proliferation markers (PCNA and BrdU). We did not find clear differences, although a systematic study was not attempted. No signs of apoptosis such as pyknotic or fragmented nuclei were observed. The claim of a detailed analysis of EMT markers is very reasonable. Thus, we performed an analysis of E-cadherin expression in the left PHMP of four mutant and four control E11.5 embryos. E-cadherin is a good surrogate marker of EMT, since different factors  can trigger EMT, but E-cadherin is always a target of them. The results are shown in the new 2) When do the embryos die and what other defects are observed? This is referred to very loosely.We have included a new table  detailin3) The RA rescue is potentially important but very poorly described at the moment. First, rigorous statistical analysis has to be applied. Second, if not all animals are rescued could it be that some have partial rescue that indicates a qualitative difference between RA exposed and non RA exposed embryos? Has detailed anatomical analysis been carried out on all the embryos? It would help to provide some way of representing these data, perhaps a table with a few pictures of embryos.4) Carmona et al. show here that Raldh2 levels are much reduced in the mutant PHMP mesenchyme. Supplementation of the pregnant females' diets with RA at 8.5 and 9.5 dpc seemed to result in rescue of the phenotype as 65% of animals with the mutant genotype have complete septation of the pleural cavity by E15.5. This would be a very important observation, but needs to be better documented.Yes, both criticisms are right. Thus, we have performed new experiments in order to study the development of the pleuroperitoneal septa at the stage E13.5, comparing embryos from females fed with the RA-supplemented diet and embryos from females fed with the same diet, without RA. The results are shown in the new 5) Experimental evidence is required in support of cell migration from the coelomic epithelium to the PHMP or the PHMP to PFF. In the absence of direct evidence from DiI labelling, for example, comparison of Gata4 G2 Cre and conditional YFP expression may be helpful.Yes, direct labelling is really difficult to perform. However, we think that 1) the progression of cells from the G2+ domain towards the PPF is clearly supported by our description of the YFP+ cells that become more abundant in the PPF as the pleural cavities become closed and 2) this migration of cells had been described in human embryos by Hayashi et al. (2011). Since proliferation seems not to be a main role in the growth of the pleuroperitoneal septa, migration is the most probable explanation of this growth. Anyway, we have moderated the statements about the migration of cells, for example in the new version of the abstract.6) Is it possible to quantify the asymmetry in PHMP mesenchyme? Can the authors provide genetic evidence that this is connected to embryonic left right asymmetry?Cre;R26RYFP embryos.Yes, we did a quantification of the asymmetry by estimating the volume occupied by the YFP+ cells in the PHMP and calculating the R/L volume ratio. The right PHMP was 45% larger than the left one. The t-test for one sample compared with the expected value of 1  gave a result of 5,21 . Incidentally, we moved this paragraph from the mutant phenotype description to the first section of the results, where we describe the G2We think that there is a close relationship between the PHMP asymmetry and the main mechanisms regulating L/R embryonic asymmetry. We have recently collaborated in a manuscript about this issue that has just been submitted to Nature. It could be quoted in this context if accepted, but we cannot forward information in our manuscript at this moment.7) How do the authors explain the variability in the conditional mutant phenotype? Do they consider that this is due to variable Cre expression or another cause? For example, variability in RA synthesis? Might removing one allele of Raldh2 influence the phenotype?This is relatively frequent when using the Cre;LoxP technology. Sometimes the levels of Cre recombinase are not enough to produce full recombination. Alternatively, it is possible that the genesis of the CDH depends on a critical amount of cells in the PHMP, and small variations in this number of cells can lead to different outcomes. We also think that the levels of RA are critical, and this would explain the partial rescue of the phenotype just increasing the exposure of the embryos to this morphogen. So, yes, it is possible that Raldh2 hemizygous mice would be even more sensible to the conditional deletion of Wt1.8) Please provide information as to the relative timing of the onset of Gata4 and WT1 expression in septum transversum mesenchyme.Done. We have indicated in the text that Wt1 starts its expression in the ST/proepicardium by E9.0 and the enhancer G2 is active in this area about the same stage.9) Can the authors provide any data on WT1 expression in different species to support the evolutionary arguments in the eleventh paragraph of the Discussion?Not really. This is because our proposal of a colonization of nephric ridges by cells coming from the G2+ domain can only be applied to mammals, and we only have data on Wt1 expression and the activity of the G2 enhancer in mouse embryos. A way to support the evolutionary model would be to check if Gata4 expression in driven by a specific mesodermal enhancer in chick embryos, and what would be the anatomical domain where this enhancer is active.


The ICH score is a validated tool for predicting 30-day morbidity and mortality in patients with intracerebral hemorrhage.The aim of this study is to determine if the ICH score calculated 24 hours after admission is a better predictor of mortality than the ICH score calculated on admission.Patients presenting to our center with ICH from 7/08-12/10 were retrospectively identified from our prospective stroke registry. ICH scores were calculated based on initial Glasgow coma scale (GCS) and emergent head computed tomography (CT) on initial presentation and were recalculated after 24 hours.P = 0.018), but ICH score on admission was not .A total of 91 patients out of 121 had complete data for admission and 24-hour ICH score. The ICH score changed in 38% from baseline to 24 hours. After adjusting for age, NIHSS on admission, and glucose, ICH score at 24 hours was a significant, independent predictor of mortality (OR = 2.71, 95% CI 1–19–6.20, and Early determination of the ICH score may incorrectly estimate the severity and expected outcome after ICH. Calculations of the ICH score 24 hours after admission will better predict early outcomes. The intracerebral hemorrhage (ICH) score was developed as a predictive tool for mortality at thirty days after hemorrhagic stroke . The ICHAlmost 40% of patients with brain imaging obtained in the first 3 hours after onset of symptoms of ICH experience hematoma expansion and this is highly associated with neurological deterioration .We hypothesize that, due to the dynamic nature of early ICH, with high risk of hematoma expansion, new IVH, and decreasing level of consciousness, a delayed measure of the ICH score would be more useful in predicting outcomes. In our center, the ICH score is calculated once all five clinical indicators are available, which is typical at the time when the baseline CT is performed, rather than when the patient went to the OR or neurological intensive care unit (ICU). We sought to determine the reliability of the ICH score calculated in the first hour of arrival compared to the reliability of the ICH score calculated 24 hours later.A retrospective chart review of all patients admitted with ICH from July 2008 to December 2010 was performed. Patients included in this study were spontaneous, nontraumatic ICH with no structural underlying defect. Patients without the variables needed to calculate the admission and 24-hour ICH scores were excluded. ICH volume was calculated using the previously published ABC/2 method -8. InitiOf the 119 patients who were eligible, 89 met inclusion criteria. Fourteen patients had mild stroke or had subacute presentations (median baseline ICH score 0) and were either not monitored in the neurological ICU or with routine follow-up CT. Fifteen patients had catastrophic stroke and either died prior to 24 hours or did not have follow-up CT (median baseline ICH score 3) within the specified timeframe. One patient was in the OR for evacuation and did not have GCS available during the specified timeframe. While the initial ICH score ranged from 0 to 3 (median 1), the 24-hour ICH score ranged from 0 to 5 (median 1). On admission, 21 (23.5%) patients had an ICH score of 0,33 (37%) patients had an ICH score of 1,19 (21.3%) patients had an ICH score of 2, and 16 (18%) patients had an ICH score of 3. No patients included in this study had an admission ICH score of 4, 5, or 6.The distribution of ICH scores and frequencies of worsened, improved, or unchanged ICH score at 24 hours are presented in P < 0.0001) and death  based on the 24-hour ICH score were greater than the admission ICH score odds ratios for both poor functional outcome  and death . After adjusting for age, baseline NIHSS, and admission glucose, baseline ICH score was not an independent predictor of poor functional outcome  or death . In the adjusted model, 24-hour ICH score was an independent predictor of poor functional outcome  and death  (n = 14), followed by volume category increase (n = 5) and development of IVH (n = 3). Similarly, among patients with improved ICH score at 24 hours, the GCS improved in the majority of cases (n = 8) and volume reduced after evacuation in the others (n = 4).In the crude model, the odds ratio for poor functional outcome  . It was Our study found that 24-hour ICH score was better predictor of short-term functional outcome and mortality than initial ICH score. More than one-third of patients had a shif in the ICH score at 24 hours with similar proportions of patients with low scores worsening and high scores improving, mostly related to changes in GCS category. Although the ICH score was shown to be predictive of outcome, the exact time the ICH scores were calculated from the time of symptom onset is not known. Given that components of the ICH score are dynamic soon after ICH onset, particularly with hematoma expansion or interventions and procedures actively changing ICH volume, GCS, and IVH, the utility of using a predictive scoring system at 24-hours instead of on admission to predict outcomes increases the predictive value of the scoring system. The 24 hour ICH score is more precise. During this hyperacute phase, the ICH score on admission may underestimate the severity of the stroke since an ICH score can change rapidly due to decline in GCS or hematoma expansion. Likewise, early interventional therapies may improve the score with hematoma evacuation, hemicraniectomy, and appropriate management of intracranial pressure or intraventricular tissue plasminogen activator. Our data supports that early prognostication of outcomes should be avoided when possible, and using an ICH score calculated at a later time during the ICH admission is a better indicator of patient outcomes, after the hematoma has been stabilized and definitive therapies have been provided.These findings further support how crucial the treatment of a patient within the first few hours after development of ICH is, particularly for interventions focused on prevention of hematoma expansion by blood pressure control, consideration of hemostatic agents and evacuation of hematoma or hemicraniectomy when appropriate, and prevention of complications such as aspiration. The ICH score is not a predictor of the outcome of an individual patient but a population-based guide that can aid in predicting the potential outcomes for ICH patients. These results provide continued evidence that treating physicians should remain vigilant with patient care for the ICH patient and provide aggressive measures until the course is declared.Our study was limited by small sample size and our ability to adjust for other factors, such as blood pressure control and interventional procedures, both of which could affect the outcome. Evaluation of patients with more severe hemorrhages  was not possible due to unavailable data including follow-up ICH volume and/or GCS, raising the question of a self-fulfilling prophecy with early withdrawal of aggressive measures. This is, however, a study of early evolution of ICH score among patients with a reasonable chance of survival. It is possible that some patients with baseline ICH scores of 4 or 5 could be converted to lesser ICH scores, but we were unable to assess this in our population due to the information not being available. Further investigation in a larger sample is needed to validate our findings. A prospective study of the impact of time from symptom onset to calculation of the ICH score may be warranted to determine when, after ICH, the ICH score becomes reliable.


A fundamental challenge in soft-tissue surgery is that target tissue moves and deforms, becomes occluded by blood or other tissue, and is difficult to differentiate from surrounding tissue. We developed small biocompatible near-infrared fluorescent (NIRF) markers with a novel fused plenoptic and NIR camera tracking system, enabling 3D tracking of tools and target tissue while overcoming blood and tissue occlusion in the uncontrolled, rapidly changing surgical environment. In this work, we present the tracking system and marker design and compare tracking accuracies to standard optical tracking methods using robotic experiments. At speeds of 1 mm/s, we observe tracking accuracies of 1.61 mm, degrading only to 1.71 mm when the markers are covered in blood and tissue. Accurate and precise tracking of tools, tissues, and dynamic background scenes is increasingly important in computer-assisted surgery, providing intraoperative guidance, motion stabilization, and dynamic constraints for increased safety and better surgical outcome , 2]. In. In2]. IOptical trackers measure the coordinates of one or more special markers in the visual and near-infrared spectrum , 9]. On. On9]. OThere are other visual methods using high-contrast dyes or inks to differentiate tissue. For example, India ink can be tattooed to target tissues for optical tracking , but alsTo overcome the problem of occlusion, electromagnetic (EM) trackers , 13] ge ge13] geGold markers  can be iNear-infrared (NIR) imaging has the potential to overcome occlusion problems because NIR light penetrates deeper than visual light. NIR fluorescent (NIRF) imaging with Indocyanine green (ICG) has been shown to be very effective in detecting subsurface (<5 mm) liver tumors, intraoperative blood vessels and graft patency invisible to human eyes –20]..20].We developed small biocompatible NIRF markers that are easily attached to tissue in an irrigated surgical setting. Together with a novel fused plenoptic and NIR camera tracking system, the proposed NIRF markers enable 3D tracking of tools and target tissue while overcoming blood and tissue occlusion in the uncontrolled surgical environment. We have previously explored the feasibility of these markers in simple tasks such as visualization of markers occluded by tissue , and 2D tracking accuracy against standard optical tracking methods using robotically-controlled reference trajectories. We investigate the performance of our fused NIRF-3D system while tracking at various speeds and occlusion conditions, and during clinically relevant ex-vivo and in-vivo situations. Tested speeds are informed by respiratory motion  and too-10 mm/s ).The imaging system consists of 2 cameras: an NIR-sensitive camera  with CMOSIS CMV2000 2048×1088 pixel CMOS sensor running at 20 Hz, a plenoptic camera  running at 5 Hz, and an NIR LED light source  . The NIRCamera calibration enables our vision system to translate measurements in pixels to real-world units such as millimeters. First, the intrinsic camera parameters such as focal length and principal point must be found, so that we may translate pixel coordinates to world coordinates in each camera coordinate frame. Secondly, extrinsic calibration  is required to find the position and orientation of the camera coordinate frames with respect to a common base frame, in our case a checkerboard pattern.K for the NIR (n) and plenoptic (p) cameras as seen in  relative to the checkerboard XYZ frame, where the pose is a 4×4 matrix combining a rotation and translation into the full homogeneous transformation between reference frames, shown in  framework. A checkerboard  is observed with both cameras, and the corner detection measurements are fed to a pose estimator, which publishes each camera's pose  with 0.5 milliliters Acetone and 0.25 milliliters (265 mg) cyanoacrylate (Permabond 102) for a consistency viscous enough to remain on tissue in an irrigated surgical field, yet fluid enough to permit easy application of the markers. Acetone is used as a solvent for the ICG and cyanoacrylate; it is mildly toxic and flammable. However, Acetone has been studied extensively and is recognized to have low acute and chronic toxicity, currently being used in a variety of consumer products from cosmetics to processed foods . T. T3 of Ied foods , and is Tracking of descriptive 3D features within a surgical field is not an easy task. Organ surfaces are typically smooth and have similar visual appearances, and may also be occluded by blood or tissue. The motivation behind using NIRF markers is to create high contrast areas on the 2D NIR image, which can be reliably tracked and projected onto the 3D point cloud obtained by the plenoptic camera to track the 3D coordinates of the NIRF markers.The gain and exposure parameters of the NIR camera are set to ensure healthy contrast between the tracked blobs and surrounding environment, and to accommodate changing lighting and scene conditions, or occlusion by tissue or blood. Suitable values for gain and exposure are found to be 40 (data number/electrons) and 50,000 μs respectively. These values will vary depending on the type of camera used. In our ex-vivo experiments, the gain and exposure were set while observing markers occluded by tissue, and held constant throughout the ex-vivo tests. This ensures that all ex-vivo results have relevance in dynamically occluding environments.The NIRF tracking is achieved by a custom C++ program written for ROS using the vpDot2 class of the Visual Servoing Platform (ViSP) library . The traTracking parameters include the maximum and minimum grey level intensity thresholds (set to 100 and 255), shape information (ellipsoid) and blob size precision (set to 0.1). The minimum intensity was determined to allow robust tracking while not confusing markers with surrounding tissues, which typically have intensity well under 100 at the proposed gain and exposure settings, while markers often have intensities of 165 or more. While tracking, estimations of marker position are informed by prior marker position, size and velocity. The tracking module computes the center of gravity of the tracked blob from the Freeman chain elements . If the 2D image tracking is fast (∼19 Hz). However, calculating 3D metric coordinates from the plenoptic camera requires searching through the entire point cloud, which is slower than the plenoptic camera capture framerate of 5 Hz . The harx, y]n are the resulting x and y metric coordinates in the NIR camera frame, n is the principal point in the NIR image, n are the x and y focal lengths of the NIR camera, and n are the reported NIR image coordinates of the tracked blob. The zp value is the metric depth in the plenoptic camera frame, transformed into the NIR frame by u, ν ]n are updated at the faster NIR camera framerate. Resulting metric x and y coordinates are calculated at the faster rate while z updates at the slower rate. This hybrid 2D/3D approach allows our system to track dynamic scenes faster and more accurately than either camera in isolation.Where [We tested the ability of our system to track scenes in motion with a mock “tool” , meaningin-vivo experiments detail the use of real blood with our NIR-3D system, which we have used previously under blood occlusion in-vivo [The tracking test was repeated at 1 mm/s with both sets of markers covered in blood to simulate a surgical field. We used artificial blood  as seen in  in-vivo .We repeated the tracking test at 1 mm/s with markers covered in a single layer of porcine small bowel tissue (thickness ∼1.3 mm). This simulated tissue occlusion during surgery. As seen in To investigate the speed of our tracking system with different numbers of trackers, we increased the number of tracked markers from 1 to 10 in the FOV and quantified the change in system speed for the full 3D calculation and the faster hybrid tracking. With each repetition, we added one marker to the scene and tracked with our system. Sufficient spacing between markers was required to ensure they were not confused with each other during tracking. For this purpose we spaced markers at least 5 mm apart. The minimum suggested spacing depends on the camera settings, target speed, and scene lighting, as well as the crispness of the NIRF marker edges.We also tested the brightness of the markers over time. This was done to ensure that they could be reliably tracked over the course of a procedure without a change in brightness and possibly left in the body for long-term tracking applications. The average signal intensity of an NIRF marker, imaged daily using identical lighting conditions, was quantified over 7 days using the ViSP library. The NIRF marker was exposed to natural and artificial lighting in the lab during the seven day test period.We tested our NIR-3D system performance when the markers are adhered to tissue, which is difficult to achieve with other marker types and enables us to perform tissue tracking during surgery. We fixed a layer of excised porcine bowel to the robot and dispensed 4 NIRF markers on top of the tissue, to be tracked by our 3D-NIR system along the same trajectory as the tool tracking test at 1 mm/s. This test was repeated with the occlusion conditions of the tool test, while markers were covered in artificial blood and with another layer of tissue. We also characterized the average single-marker error on tissue, in addition to the error of the averaged path of all four markers. This is so that the reader may compute expected error for various tissue deformation or registration models.Excised tissue may exhibit different optical characteristics than seen during surgery. For this reason we tested our systems ability to handle occlusion in-vivo. We placed 3 markers of each type  on the skin of a sedated, intubated porcine model underneath blood and exposed intestinal mesentery tissue in a study approved by our Institutional Animal Care and Use Committee (IACUC). This tested our systems ability to handle blood and tissue occlusion in a realistic clinical environment.Our system was able to track with no loss of markers, and achieved near-millimeter accuracy in the static case. At 1 mm/s, the error was 1.61 mm, with the 95% one-sided confidence interval at 1.27 mm. For the Polaris, these numbers are 0.29 and 0.18 mm. The spatial error for our system was 0.87 mm in the static case. We attribute the slight degradation in performance using the Polaris in the static case to drift errors, which are exacerbated by longer testing times for the static case.When the scene was covered in blood, our system saw almost no drop in performance as seen in With both the optical and NIRF markers obstructed by a layer of 1.3 mm thick porcine bowel tissue, our system experienced only a slight degradation of tracking accuracy. The error rose to 1.71 mm. The Polaris was unable to track the obstructed markers.The tool tracking test was repeated at speeds of 5 and 10 mm/s. Error increased for both trackers, particularly our NIR-3D system due to the relatively high system lag . HoweverUsing only the NIR-3D system, we observed up to n=10 markers and measured the system framerate. The results showed that with more markers, the update rate of the system decreased due to an increase in computation time. This trend is visualized in Additionally, we monitored the average intensity level of a marker over a seven day period. The average signal intensity, meaning the average intensity of a tracked blob, decreased 26% in the seven days to a value of 120 . HoweverWhile observing markers on tissue, the norm 3D measurement error with our NIR-3D system was 1.83  mm. There was almost no difference between different occlusion conditions while tracking tissue as seen in Three markers of both types  were tracked in-vivo with no loss of markers in the unobstructed case. When splashed with porcine blood, the Polaris markers were unable to be seen. However, even when the NIRF markers were doused in blood and covered by a layer of bowel mesentery , after aOur NIR-3D tracking system provided near-millimeter tracking accuracy at lower speeds, showing promise for the demanding clinical environment. However, the framerate of the full 3D tracking is currently too slow for more demanding tracking applications such as cardiac motion, where speeds can be upwards of 100 mm/s [The general nature of our system permits the user to specify the number and location of markers to be tracked, encouraging tracking of amorphous surfaces typical in surgical environments. More importantly, the biocompatible NIRF marker enabled tracking even during occlusion by blood and tissue, which is not possible with current optical tracking methods.The limiting factor in our tracking system is the framerate of the plenoptic depth calculation, which is currently 1.9 Hz while tracking 4 markers simultaneously. We mitigated this slow depth calculation with faster tracking in the NIR camera XY frame (19.6 Hz for 4 markers). Although the plenoptic camera has advantages compared to stereo cameras like a smaller footprint, the ability to provide a total-focus image, and imaging from multiple vantage points, the plenoptic depth calculation is more computationally expensive than stereo because of the higher number of microlens images, and the fact that every pixel is assigned a depth. However, with faster GPUs and more optimized correspondence searching, we believe these framerates can improve. In the tool tracking tests, we simply averaged the 4 marker trajectories to obtain a single tool trajectory, but we could use more advanced methods such as Kalman filtering  or a modThis work has broader implications for real-time tracking of mobile deformable targets in unstructured surgical environments, even in situations where the line of sight to target of interest is obstructed. We specifically developed a novel biocompatible fluorescent dye-solvent to track target tissue, allowing real-time dynamic visualization. This unique fluorescent dye-solvent enables us to create custom fiducials to accurately follow deformable, mobile, wet tissue targets. This approach obviates the need for cumbersome EM tracking while overcoming the limitations of in-sight optical tracking. Furthermore, the fusion with plenoptic imaging technology permits accurate quantitative registration and knowledge of the target tissue in 3D, providing a real-time quantitative 3D topography of the surgical field, including the tissue of interest and surrounding surgical environment.We presented the design of key elements of our NIR-3D system including the biocompatible marker composition, camera registration and calibration, and software methods used. Our system was able to track markers that adhere to the tissue surface during blood and tissue occlusion. These markers remain luminescent for long periods of time. We saw accuracy errors of 1.42 mm in the best case, and only small changes in accuracy with varying occlusion conditions. Errors were dominated by the Z-component, making our hybrid 2D/3D approach useful to track at faster rates in the XY plane.Future work will include further integration and evaluation of the tracking system in image-guided medical interventions such as robotic surgeries. We also propose to automate gain and camera parameter adjustment to maintain a consistent size of tracked marker through occlusion conditions. We will improve the accuracy and speed of the tracking system through improved metric calibration, automatic adjustments to camera parameters, and optimizations to allow faster runtimes.


Leishmania infantum and is a systemic disease, which can present with variable clinical signs, and clinicopathological abnormalities. Clinical manifestations can range from subclinical infection to very severe systemic disease. Leishmaniosis is categorized as a neglected tropical disease and the complex immune responses associated with Leishmania species makes therapeutic treatments and vaccine development challenging for both dogs and humans. In this review, we summarize innate and adaptive immune responses associated with L. infantum infection in dogs, and we discuss the problems associated with the disease as well as potential solutions and the future direction of required research to help control the parasite.Canine leishmaniosis (CanL) is caused by the parasite  Leishmania infantum is endemic in approximately 50 countries affecting two major regions; South America and the Mediterranean region  are categorized as neglected tropical diseases, and are endemic in the Mediterranean Basin, the Middle East and in sub-tropical and tropical regions of the world Desjeux, . Canine Leishmania has a digenetic lifecycle. Leishmania metacyclic promastigotes are injected under the skin of the vertebrate host by the phlebotomine (sand fly) vector. This form of the parasite is flagellated and they are rapidly engulfed by phagocytic cells such as neutrophils, dendritic cells and macrophages, which are either resident or recruited to the bite site  response. T lymphocytes play a crucial role in immunity to leishmaniosis by influencing cytokine production and subsequently interacting with infected macrophages  and subsequently resulted in the recruitment of immune cells at the inoculation site, which were found to have an adverse effect on Leishmania parasites  found to influence the likelihood and outcome of infection . High IgG levels have been associated with susceptibility  distributed across the genome. Analysis of these SNPs allowed the phenotype to be predicted with an accuracy of ~0·29 in new samples, providing evidence of a significant genetic component to CanL within the breed  is part of the major histocompatibility complex (MHC) in dogs. DLA has been found to be the main genetic component associated with canine diabetes. In prone breeds , the common alleles/haplotypes are DLA-DBR1*009, DQA1*001 and DQB1*008 , control of the infection is mediated by the early induction of an IL-12 driven Th1 immune response along with the production of IFN-γ by CD4+ T cells  production and eventual parasite killing by macrophages  dichotomy was observed in murine leishmaniosis . However, IL-4 and TNF-α were not expressed -17A, inducible NO synthase (iNOS) and IL-10 in the spleen and liver in symptomatic dogs compared with asymptomatic dogs  and anti-inflammatory cytokines (IL-10 and TGF-β) , IL-22 transcription was significantly lower when compared with non-infected healthy control dogs  in both subclinical and sick dogs compared with non-infected controls , have an important role in suppression of host immunity in murine  are manipulated so that the parasites use granulocytes as host cells, permitting a productive infection. This enables L. major to use the PMNs as ‘Trojan horses’ before entering the macrophages , which converts one of the terminal nitrogens of the guanidine group of L-arginine to NO, producing citrulline  production is the last step involved in the destruction of γ and TNF-α, which kill intracellular amastigotes via the L-arginine NO pathway. This has been demonstrated following successful chemotherapy of L. infantum dogs  and subsequently infected with L. infantum promastigotes showed high levels for fluorescence for NOS2 in their cytoplasm, which was not seen in uninfected macrophages  compared with dogs with moderate or severe disease, supporting an association between TLR2 and the pathogenesis of cutaneous lesions in CanL  burden, which is recognized by the host TLR3 to induce pro-inflammatory cytokines and chemokines and exacerbate disease  and innate immunity genes 1, NOD2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7 and TLR9) in canine macrophages that were resistant or susceptible to intracellular survival of L infantum. The study revealed that the decreased intracellular survival of L. infantum in macrophages was associated with increased production of IFN-γ and TNF-α, and decreased production of IL-10 , inducing protection against L. infantum infection in mice . There were previously two, but Leishmune®  is no longer available. Leish-Tec® appeared to partially protect dogs against CanL, but only limited data from field trials have been reported  and is composed of purified excreted-secreted proteins of L. infantum (LiESP) and adjuvanted with QA-21, which is a purified fraction of the Quillaja saponaria saponin  for immunotherapy of CanL (Ferreira et al.γ and low IL-10 concentrations in comparison with control dogs (Ferreira et al.et al.There are several drugs currently available for the treatment of CanL. However, while many of these drugs can result in improved clinical signs, none of them have the ability to eliminate the infection. There is also the issue of toxicity and drug resistance (Noli and Auxilia, L. infantum is complicated. Visceral dissemination of the parasites makes sampling from infected animals difficult. In response experimental in vivo studies using mice have commonly been undertaken, although unfortunately these offer a poor reflection of immune responses associated with L. infantum in dogs. It is essential that as much information as possible be gathered from both naturally and experimentally infected dogs to better our understanding of this disease. Taken together, the findings summarized in this paper illustrate the multi-systemic effect of L. infantum in dogs. Although a great deal of progress has been made towards understanding the immune responses associated with CanL, a vast amount remains to be determined. A more comprehensive understanding of both the local and systemic responses to L. infantum in the dog is needed to be able to develop more effective treatment regimens and/or vaccines. Improving the control of the spread of CanL might also have a positive impact on reducing the number of human cases of the disease.In conclusion, understanding of CanL caused by


In October 2014, during the Ebola outbreak in Liberia healthcare services were limited while malaria transmission continued. Médecins Sans Frontières (MSF) implemented a mass drug administration (MDA) of malaria chemoprevention (CP) in Monrovia to reduce malaria-associated morbidity. In order to inform future interventions, we described the scale of the MDA, evaluated its acceptance and estimated the effectiveness.MSF carried out two rounds of MDA with artesunate/amodiaquine (ASAQ) targeting four neighbourhoods of Monrovia (October to December 2014). We systematically selected households in the distribution area and administered standardized questionnaires. We calculated incidence ratios (IR) of side effects using poisson regression and compared self-reported fever risk differences (RD) pre- and post-MDA using a z-test.In total, 1,259,699 courses of ASAQ-CP were distributed. All households surveyed  attended the MDA in round 1 (r1) and 96% in round 2 (r2) . 52% (643/1233) initiated ASAQ-CP in r1 and 22% (256/1154) in r2. Of those not initiating ASAQ-CP, 29% (172/590) saved it for later in r1, 47% (423/898) in r2. Experiencing side effects in r1 was not associated with ASAQ-CP initiation in r2 . The incidence of self-reported fever decreased from 4.2% (52/1229) in the month prior to r1 to 1.5% (18/1229) after r1 (p<0.001) and decrease was larger among household members completing ASAQ-CP (RD = 4.9%) compared to those not initiating ASAQ-CP (RD = 0.6%) in r1 (p<0.001).The reduction in self-reported fever cases following the intervention suggests that MDAs may be effective in reducing cases of fever during Ebola outbreaks. Despite high coverage, initiation of ASAQ-CP was low. Combining MDAs with longer term interventions to prevent malaria and to improve access to healthcare may reduce both the incidence of malaria and the proportion of respondents saving their treatment for future malaria episodes. In 2014 and 2015 the largest Ebola outbreak in history occurred in West Africa with more than 28,000 confirmed, probable and suspect Ebola cases reported in three countries with intense transmission–Guinea, Sierra Leone and Liberia [As a consequence of the Ebola outbreak existing healthcare structures in Monrovia were rapidly overwhelmed. By October 2014 few health facilities in Monrovia were fully operational and those that were open seemed to offer limited services. Laboratory testing for any medical condition, including rapid diagnostic tests (RDTs) for malaria was largely unavailable outside Ebola treatment units (ETUs) and Liberian National Malaria Control Program (NMCP) recommended presumptive malaria treatment.At the same time, malaria, the main killer of children under five years of age and among the most common causes of outpatient consultations and inpatient deaths in Liberia , 4, was Clinical presentation of Ebola and malaria cases is similar and differentiation without laboratory tests often impossible. In Liberia the case definition used for suspect Ebola cases (fever plus three unspecific symptoms) was highly sensitive in order to capture all Ebola cases. However, it also captured patients with malaria. As a result, an unknown number of malaria patients remained either unattended to due to the collapse of the healthcare system or were admitted to an ETU as a suspect Ebola case. Consequently, malaria patients were put at risk of exposure to Ebola and ETU resources were further strained by the admission of substantial numbers of non-Ebola patients. While the number of confirmed Ebola cases in Monrovia began to decline in October 2014 , malariaThe administration of therapeutic antimalarial regimens to entire populations regardless of clinical symptoms or laboratory tests has been part of malaria control programs for more than a century , 9. AfteMass administrations of antimalarial drugs to reduce the number of fever cases during an Ebola outbreak had never been carried out before. We aimed to describe the scale of the MDA, to evaluate community acceptance of the strategy, adherence to treatment, and estimate the effectiveness of the intervention with regards to the reduction of fever cases in order to inform future public health interventions targeting fever reduction in an Ebola context.Four zones in Monrovia were targeted for the MDA based on their high Ebola incidence, high population density, precarious living conditions and limited access to healthcare. The population of these zones, New Kru Town, Clara Town, Westpoint and Logan Town , was iniTwo rounds of MDA were carried out in a stepwise fashion at 56 fixed points of distribution between October and December 2014 with a one-month interval between rounds. The drug distributed was ASAQ and was specifically procured for the MDA. One course of ASAQ consists of three doses to be taken once a day over three consecutive days in age-specific, fixed-dose-combinations. ASAQ is used as treatment for uncomplicated malaria infections and the post-treatment prophylactic effect is thought to last for at least one month after the full course is taken .The distribution team met with the governor of each zone prior to the distribution .After agreement with the governor, the chairmen/chairwomen and community leaders (CCL) of all areas in the zone were contacted by the distribution team. Together with CCL a local malaria committee comprised of numerous community authorities including representatives of minority groups was established and population numbers were verified. In collaboration with the malaria committee, one community volunteer (CV) was identified per 100 households and trained by the distribution team to provide sensitization messages to the population. These messages included the information i) that preventive malaria treatment will be distributed, ii) age-specific doses will be provided, iii) doses should be taken with food, three days in a row. These messages were adapted regularly during the intervention. CVs went door-to-door (without entering the house) the day before the MDA explaining the purpose and content of the MDA to household members and distributing one voucher to each household. The voucher entitled one woman of the household to receive one pre-prepared medication pack for their household, from the designated distribution point, on the day of the MDA.To avoid crowding, the MDAs were carried out at fixed points in the early morning during curfew for a maximum of two hours. Crowd-management and security were the responsibility of CCL and the malaria committee. Women attending the distribution were asked to stand in lines, CVs were present to re-emphasize sensitization messages and to ensure adherence to the no-touch policy. The set-up of the easy-to-carry and easy-to-disinfect distribution site was organized in a way that avoided crowding and ensured rapid distributions.On the distribution day, women could exchange the voucher they received from the CV for a pre-prepared medication pack of ASAQ containing a full course of ASAQ-chemoprevention (ASAQ-CP) for seven household members (two adults and five children of various age groups). Children below 6 months did not receive ASAQ-CP. The medication packs were pre-prepared to keep logistics light and to facilitate rapid distribution. A catch-up distribution was carried out if less than 85% of the expected households attended the distribution at the respective site. In addition to the MDA, a back-up stock of medication packs remained with designated members of the community after the MDA. Households that did not receive sufficient ASAQ for all household members had the possibility to collect the missing ASAQ-CP doses.The distribution team documented the number of vouchers and medication packages distributed by area in each round and the number of necessary catch-up distributions.We used data collected by the distribution team to assess the target population size, the absolute numbers of vouchers and medications packages distributed during the MDA and the number of catch-up distributions carried out.For further evaluation of the MDA, one in every 200 households that received vouchers in round 1 (r1) of the MDA was randomly selected for telephone follow-up through systematic sampling during voucher distribution.The objectives of the evaluation were to:Describe the scale of the MDA: We described the absolute numbers of vouchers and medication packs distributed and the number of catch-up distributions.Estimate acceptance of the MDA and adherence to ASAQ: We estimated attendance at the MDA and described the attitude towards the distributions on household level. On individual level, we estimated the availability of ASAQ and compliance with initiation of and adherence to ASAQ-CP and identified reasons for non-compliance and non-adherence. Additionally, we estimated the rate of self-reported side effects and examined the association between side effects and ASAQ-CP initiation.Estimate the effectiveness of MDA: We estimated the incidence of self-reported fever episodes before and after the first round of MDA.th voucher was marked by a triangle shaped punch cut into the plastic voucher. The CVs that carried vouchers informed each household that received a marked voucher about the purpose of the evaluation and asked the head of the household  for consent. If they refused, the next household to whom the CV distributed a voucher was asked for consent. For logistical reasons, no information about households was collected by the CVs during telephone number collection. After consenting, the respondents provided their telephone number. One week after both MDA r1 and r2 had taken place in the area, a trained surveyor interviewed the respondents by telephone using a structured questionnaire. The questionnaires collected information on MDA attendance and acceptance. The respondent provided information for every member of his/her household regarding availability of ASAQ-CP for all household members, initiation and completion of the ASAQ-CP course, side effects and fever in the previous month. A question regarding the availability of the appropriate age-specific dose was added during the first round.Every 200To assess the feasibility of the evaluation and the composition of the medication packs, we conducted a pilot evaluation. This included households from the first five distribution days, approximately 20% of all households. Data from these households was excluded from the evaluation.A household was defined as a group of people living under the same roof and sharing the same meal at least three times a week regardless of family ties.Self-reported fever was used as a proxy for malaria infection and defined as history of fever or malaria within the past month reported by the respondent. Reports could not be confirmed by laboratory testing due to the ongoing Ebola outbreak.Reported side effects were defined as any sign or symptom reported as a side effect by the respondent.Compliance with treatment initiation was defined as taking at least one of the three doses of ASAQ-CP as reported by the respondent.Adherence was defined as taking all three doses of ASAQ-CP, thus completing the full course as reported by the respondent.We described absolute population numbers and MDA attendance and number of catch-up distributions as collected by the distribution team.For further evaluation, households that were reached for telephone interviews in both rounds of MDA were included in the analysis. As one individual was reporting for his or her whole household in both rounds, individual household members needed to be re-identified in the second round to allow for individual level analysis of data; re-identification was based on reported age and sex as data collection was anonymous and names were not recorded. First we re-identified individuals with identical ages in both rounds. Next, in order to account for imprecise age estimations by household members, we re-identified individuals ± 1 year (or months for infants below 12 months of age) during the second round followed by individuals ±2 years of age. Household members that were only reported in one of the two rounds were not included in the analysis as measures of associations cannot be calculated for them.On household level, we calculated proportions. On an individual level we calculated proportions with 95% confidence intervals (95% CI) where appropriate, allowing for the potential cluster effect of the household on treatment availability, compliance with treatment initiation and adherence. Differences between household members initiating and adhering to treatment and those that did not were calculated with chi squared tests.We used poisson regression to calculate incidence ratios (IR) as a measure of association between reported side effects and treatment initiation, adjusting for possible confounders and also taking into account the cluster effect of the household.As fever was reported for the past month, the evaluation after r1 captured self-reported fever incidence prior to the start of the MDA and the evaluation after r2 captured the self-reported fever incidence the month after r1. We calculated the difference in reported fever by subtracting the incidence of reported fever episodes after r2 from the incidence of reported fever episodes after r1 and presented risk differences (RD) with 95% CI. We compared the reported pre- and post-r1 incidences using a z-test for difference in proportions. Additionally, we compared the RD among household members that took the full course of ASAQ in r1 with the RD among household members that did not take the full course of ASAQ with a z-test for difference in proportions.Analyses were conducted with STATA version 13 .The data presented here are from an analysis of MSF programmatic data. The procedures conducted during the evaluation were in accordance with the ethical standards of the Helsinki Declaration. The CVs read a consent form to the head of the household and explained that participation was voluntary. Respondents provided verbal consent. No personal identifiers were collected and only aggregated data was reported. All evaluation documents were stored in a locked cabinet or a password protected computer.In the four targeted zones of Monrovia a total population of 551,971 individuals in 102,372 households was verified with CCL and the malaria committee in round 1 and corrected to 558,483 individuals in 103,497 households during r2. 102,372 vouchers for a pre-packaged medication pack were distributed during round 1 and 103,497 during r2. The total number of vouchers exchanged for medication packs was 90,411 (94%) in r1 and 89,546 (93%) in r2. In total, 1,259,699 courses of ASAQ-CP were distributed.At each of the 56 distribution points in both rounds, between 347 and 3,853 women received medications packs, depending on community size.Catch-up distributions needed to be carried out in three (5%) of the 56 sites in r1. In r2, two catch-up distributions were deemed necessary but only one was carried out; in the other community, chairmen were reluctant to organize a new distribution as they were convinced that it was not necessary.th was asked to participate.The communities included in the evaluation  consisted of 426,760 individuals in 72,585 households of which every 200We aimed to contact 365 selected households by phone in rounds 1 and 2. CVs reported that every selected household agreed to participate and had a phone. Of the 365 selected households, 222 (61%) were reached by phone in both rounds. The remaining 39% (143/365) of sampled households either i) did not answer their phone, or ii) had their phone turned off, or iii) the connection was too poor to collect any information during three tries. Of the 143 households that were not included in the evaluation, 47% (67/143) were only reached in one of the rounds of the MDA and 53% (76/143) were not reached in any round. All 222 households that were reached in both rounds had provided informed consent to the CV beforehand and agreed to be interviewed.In total, 1,643 individuals were reported to live in the 222 households reached in both rounds (average household size: 7.4). Using household-code, sex and age, 1,236  individuals could be re-identified in both rounds. The 1,236 individuals were principally female  with a median age of 16 (Interquartile range: 8–28 years). The 407 (25%) individuals who were unable to be re-identified were reported to have lived in the household during only one of the two rounds of MDA.All 222 households attended the distribution during r1 (100%), accounting for 1,236 household members of which 1,233 were eligible for ASAQ-CP (i.e. household members older than six months). During r2, 212 (96%) households attended the MDA, accounting for 1,157 household members of which 1,154 were eligible for ASAQ-CP. Of the eight households that could not attend the MDA in r2, six were not in the distribution area during the distribution of medication packs, one did not receive a voucher and one lost the voucher.Despite the availability of buffer stocks, some household members did not receive ASAQ-CP. In r11, 1,113 (90%) of 1,233 household members received sufficient ASAQ-CP, compared with 1,144 (99%) of 1,154 in r2 .Among those household members that received any dose, the dose received was reported to be age appropriate for ≥98% of household members in both rounds. During r1, 98%  of household members for whom information was available, reported receiving the correct dosage of ASAQ-CP for their age, 21 (2%) did not know, and one (0.1%) reported receiving an incorrect dose. In r2, 100%  household members reported receiving the correct dose.Compliance with ASAQ-CP initiation among those that attended the MDA was 52% (643/1233) in r1 and 22% (256/1154) in r2 (In r1 48% (592/1233) of all household members that received ASAQ-CP adhered to the full course, in r2 21% (253/1154)  of 462 household members not initiating the course of ASAQ-CP reported they did not feel sick, and 172 (37%) that they had saved ASAQ for later. In r2, 423 (48%) of 888 not compliant with ASAQ-CP initiation reported saving it for later, 270 (30%) reported not feeling sick and 183 (21%) that they waited for one month between the two rounds of chemoprevention .Adherence to the three day ASAQ-CP course among those who initiated ASAQ-CP increased from 91% (592/643) in r1 to 99% (253/256) in r2 of MDA; those completing ASAQ-CP and those not completing ASAQ-CP did not differ in age and gender. In r1, 14 household members reported side effects as the reason for interruption, 12 did not understand the importance of completing a full course, eight shared their course with someone else and 17 reported other or unknown reasons. In r2, three household members did not complete the ASAQ-CP course; one reported side effects as the reason and two reported not understanding the importance of continuing the three-day course. Among the 605 household members who initiated ASAQ-CP in r1 and attended the MDA in r2, 406 (67%) decided not to initiate ASAQ-CP in the second round. The most frequently reported reasons were saving ASAQ for later , waiting for the recommended time between two ASAQ courses to pass  and not feeling ill .Reported side effects among household members starting ASAQ-CP in each round included: drowsiness , dizziness , fever , nausea , headache , vomiting  and skin reactions . Overall attack rate of any reported side effect in the first and second round of MDA was 17% (107/643) and 6% (16/256), respectively. Among those household members that started ASAQ-CP, 15 (1.7%) of 899 ASAQ-CP courses during both rounds of MDA were reported to be interrupted for possible ASAQ side effects.In r1, 23 (2%) of 1,113 household members that received sufficient ASAQ reported that they did not initiate ASAQ-CP because they feared side effects. In r2, 11 (1%) of 1,144 household members did not initiate due to ASAQ side effects. Yet, occurrence of side effects during r1 was not associated with ASAQ-CP initiation during r2 when adjusted for age and gender .Among household members that initiated ASAQ-CP in r1 and provided information on side effects in r1 and ASAQ-initiation in r2, the proportion of household members initiating ASAQ-CP in r2 was 32%, both among those that experienced side effects (33/104) and among those that did not experience side effects (158/487).The incidence of self-reported fever episodes decreased significantly after r1 compared to the month prior to r1, from 4.2% to 1.5% (p<0.0001) .Reported fever incidence was initially higher in children than in adults: self-reported fever incidence was 6.9% in children ≤ 5 years of age and 3.8% in older household members and decreased to 1.1% and 1.6% respectively after the first round of the MDA. Incidence of self-reported fever was 4.9% lower after r1 among those household members who took a full course of ASAQ-CP and 0.6% lower among household members who did not start or not complete a full course of ASAQ-CP . While rThe MSF-lead MDA in Monrovia was a novelty and a challenge as MDAs have rarely been carried out on such large scale, never with ASAQ , 11 and ■Verification of population estimates with local leaders prior to the distribution substantially increased the estimated population size but was necessary to ensure appropriate coverage.■Use of fixed points for distribution of pre-packed medications using stringent infection prevention and control procedures was challenging but fast and feasible.■Coordination of messages with relevant actors in the target area is important; adaptation of messages according to monitoring results can improve compliance and adherence.■If directly observed treatment (DOT) is impossible, treatment initiation can possibly be improved by distributing familiar and uniformly packed medication and by provision food and ORS at the same time.■Occurrence of treatment related side effects did not affect adherence.■Adherence to ASAQ-CP was high and no risk of drug resistance development was identified.■No amplification of the Ebola outbreak in the target areas was observed during or after the intervention.■Self-reported fever cases were significantly lower after the MDA, particularly among household members that initiated ASAQ-CP.■Combining MDAs in malaria endemic areas with longer term interventions to prevent malaria and to improve access to healthcare may reduce the proportion of respondents saving their treatment for future malaria episodes.After discussions with CCL and the malaria committee in all 56 distribution sites the initial population estimate used to plan the distribution increased by 184% . While a challenge for planning, overcrowding and multiple families using the same shelter in shifts may explain the initial underestimation. An additional increase of approximately 7,000 people was noted from the first to second round and was due to the initial accidental exclusion of several small minority communities. These minority communities were not deemed part of their community by CCLs during planning for r1 and were only discovered by local MSF staff after the first distribution.Engagement of local staff and close collaboration with both the local CCL and malaria committee ensured one woman per household would attend the distribution. Similar to other distributions where the importance of community engagement was highlighted as critical to ensuring high campaign coverage , 11, theCompliance with ASAQ-CP initiation may have been improved by utilization of directly observed treatment (DOT), a strategy cited as an essential component of successful MDAs in the past . Indeed,Even though almost 100% of recipients reported a positive attitude towards the MDA, the majority did not initiate ASAQ-CP; most reported not feeling sick and saving ASAQ for later. In other MDA interventions with similar experiences, more than one third of respondents reported being reluctant to take the available drugs because they did not feel sick . In MonrSome of the ASAQ doses however, were procured from a manufacturer different to that manufacturer which provided the most commonly available ASAQ drugs in Monrovia prior to the outbreak. This may have led to confusion among recipients as the packaging was different and community members may have been less familiar with the drug name than the packaging itself. Due to issues with supply, the household medication packs were pre-prepared in plastic bags of varying colours. This may have caused some additional confusion among recipients who suspected that the contents of each bag were different based on its colour.Anecdotal reports from local nurses and CVs highlighted the role that the absence of food and oral rehydration solution (ORS) distributions concurrent with the drug distribution may have had on treatment initiation. Indeed, it was recommended to take ASAQ with food and to use ORS in case of vomiting or diarrhoea–both unavailable to some recipients living in the most precarious areas of Monrovia. Finally, ASAQ-CP may have been saved more frequently during the second round because residents realised that MSF would not be carrying out a third MDA and health facilities had yet to reopen. The risk of future infection with malaria and the lack of available treatment in the continuous absence of healthcare facilities or future MSF distributions may have been perceived as more important. Consequently, the best use of ASAQ would be as treatment when sick.Although a concern, no evidence has been found in the past that MDAs of full course therapeutic treatment with combination drug regimens lead to increased drug resistance –11. We wASAQ was selected as CP due to its availability, proven efficacy , safety,The initial self-reported fever incidence in children under five years of age of 6.5% prior to the first round of MDA is low when compared with previously reported estimates of laboratory confirmed malaria prevalence in Monrovia . UnderreThe reduction in fever incidence among those completing the full course of ASAQ in the first round was significantly larger compared to those not initiating or not completing the ASAQ course. This decrease in incidence is coherent with previous knowledge as ASAQ has not been used in MDAs for chemoprevention previously but proved efficient as treatment in Liberia  and prevWith the observed adherence to a full course of ASAQ-CP of 48% in the first round, extrapolation of the overall reduction in incidence of self-reported fever from 4.2% to 1.5% in the sample to the population of the target zone  suggests that 14,821  fever episodes per month may have been averted as a result of the MDA in Monrovia. Mathematical modelling conducted in early 2015 suggested that in the absence of adequate healthcare provision, three rounds of ASAQ-MDA with coverage of 70% in the whole of Liberia from January 2015 would have had averted 300,000 to 700,000 malaria cases . Given tIn addition to the reduction in incidence of self-reported fever, the availability of left over ASAQ likely resulted in a reduction of movements to search for malaria treatment and may have reduced transmission of communicable diseases in health facilities.Potential selection biases may have occurred and affected the findings. First, our sample was based on households selected during voucher distribution, and completeness of voucher distribution could not be concluded from the data available. Therefore, no inference about coverage in the target area can be made. Second, 20% of the target community was included in the sampling frame for the pilot evaluation and could not be included in this evaluation. Third, 39% of sampled households were not reached in both rounds using the phone number provided. We do not know if those individuals that were not reached after three attempts differed systematically from those that were reached in both rounds. Comparing households that were reached in only one round with those reached in both, did not indicate differences in household composition, self-reported side effects or fever incidence. Nevertheless, there is possible sampling bias introduced by those households that were not reached in any of the rounds as they might have a more critical attitude towards the MDA and did not answer the phone for that reason. Additionally, individuals that did not respond may have died or lived in very remote corners of the MDA area without stable phone connections and might differ in other characteristics as well. Unfortunately, it was not possible to train all CVs in basic data collection and the no-touch policy heavily restricted opportunities for direct interaction with the community, so no background information on sampled but not reached households could be obtained. On the other hand, CVs reported that all selected households agreed to participate and provided a phone number. This indicates a) high acceptability of the phone survey and b) a sufficiently good coverage of phone ownership even in disadvantaged areas of Monrovia.Reporting bias may have been introduced as the prevalence of malaria parasitemia could not be established and self-reported episodes of fever were used as a proxy for malaria, leading to possible estimation-bias of malaria incidence, particularly in the first round . Fever mLastly, this analysis does not include fever incidence figures from non-MDA areas. It is therefore difficult to determine whether the observed differences in reported incidence were exclusively attributable to the intervention.The apparent reduction in self-reported fever cases following the MDA of malaria chemoprevention suggests that the intervention may have been effective in reducing the number of fever cases during the Ebola outbreak in Monrovia. Despite high acceptance and coverage of the MDA and the small impact of side effects, initiation of malaria chemoprevention was low, possibly due to health messaging and behaviour in the pre-Ebola outbreak period and the ongoing lack of healthcare services. Stronger community involvement by dedicated local staff prior to the intervention and improved coordination with other actors in the target area may have been helpful in identifying relevant sensitization messages and hidden communities earlier, and could have possibly addressed misconceptions, fears and rumours more effectively. Combining MDAs during Ebola outbreaks with longer term interventions to prevent malaria and to improve access to healthcare might reduce the proportion of respondents saving their treatment for future malaria episodes.


Tuberculosis (TB) still remains the thorn in the flesh of efficient therapeutics affecting one-third of global population annually. There are several factors that enhance the susceptibility to TB infections including malnutrition, smoking, and immunocompromised conditions such as AIDS. In the recent years, growing body of evidence has gained considerable prominence which suggests that Diabetes Mellitus (DM) is individual risk factor leading to complicated TB infections. In this article the authors have attempted to summarize the link of type 2 DM with TB, the mechanistic action of how DM sensitizes for developing the active TB infection from the latent infection, and problems faced during treatment followed by possible preventive measures. We have tried to give account of the alterations that occurred in DM making a person more prone to develop TB. Tuberculosis (TB) continues to be a considerable worldwide health issue, provoking millions of new cases and one-third deaths annually. Around 2 billion of the population of the world are considered to be infected with latent TB and out of which approximate 5–10% grows in to active TB –6. AmongTB is an airborne disease that is transferred from one person to another either through aerosol formation or through contaminated air within the population. However based on the PPD test results survey, usually, only 30% of the population gets successfully infected when coming in contact with MTB. In remaining 70% of the population the boosted level of immunity prevents the onset of MTB infection and prolongs the latent condition. Under this condition most MTB are distorted and the few viable ones are transferred to the latent state because of their hampered metabolism. TB and DM both interact with each other on various levels, one making worse situation for the other . Under dα, IL-1β, and IL-12), lymphocytes, monocytes, natural killer T cells, and B lymphocytes that form granulomas and kill almost all MTB preventing the growth of those that remain viable. However in the diabetic conditions the immunologic responses weaken making the individual immunocompromised  along with several cytokines  [β (TGF-β), a cytokine responsible for lowering the GCLC expression. This suggests that the lower level of GSH in DM type 2 individuals makes MTB more susceptible. In DM individuals, level of cytokines such as IL-6 and IL-17 was also found to be increased leading to the enhanced free radicals formation which confirms the degraded level of GSH in DM type 2 individuals. However, it was observed that the increment in the GSH level advances the action against MTB infections. Moreover, cytokines accountable for MTB infection at cellular level, such as TNF-α, IL-1β, IL-2, IFN-γ, and IL-12, were found to be altered in DM type 2 individuals; interestingly the augmented level of some immunosuppressive cytokines such as IL-10 was also found in the same individual [Although the immunologic responses in TB and DM conditions are extensively studied, the responses against MTB in diabetic condition are still not completely understood. However in recent study it was shown that the intracellular level of cytokines, such as IFN-type 17) . Elevatitype 17) . In sometype 17) . GSH levdividual . Even shdividual . These lControlling the level of glucose probably minimizes the chances of developing TB among DM people; however sufficient research is still to be conducted which might give awareness whether it is minimizing the TB risk or saving people from death. Gliptins, a new agent for the treatment of type 2 DM also known as DPP-4 inhibitors (inhibitors of dipeptidyl peptidase 4), control the glucagon release and stimulate the release of insulin which lowers the blood glucose level . TherefoPatients suffering from DM are more prone to develop TB as it compromises treatment of TB in diabetic patients. Moreover, DM is associated with enhanced risk of multiple outcomes of not only treatment failure but relapse and death during TB treatment. Therefore, extensive studies on TB management with DM are required. Considering the increasing disease burden of DM, particularly in highly prevalent TB areas, TB control programs will need to expand their efforts and focus on treatment of patients with DM and TB.