The aim of this study was to populate the Equitable Impact Sensitive Tool (EQUIST) framework with all necessary data and conduct the first implementation of EQUIST in studying cost–effectiveness of community case management of childhood pneumonia in 5 low– and middle–income countries with relation to equity impact.Wealth quintile–specific data were gathered or modelled for all contributory determinants of the EQUIST framework, namely: under–five mortality rate, cost of intervention, intervention effectiveness, current coverage of intervention and relative disease distribution. These were then combined statistically to calculate the final outcome of the EQUIST model for community case management of childhood pneumonia: US$ per life saved, in several different approaches to scaling–up.The current ‘mainstream’ approach to scaling–up of interventions is never the most cost–effective. Community–case management appears to strongly support an ‘equity–promoting’ approach to scaling–up, displaying the highest levels of cost–effectiveness in interventions targeted at the poorest quintile of each study country, although absolute cost differences vary by context.The relationship between cost–effectiveness and equity impact is complex, with many determinants to consider. One important way to increase intervention cost–effectiveness in poorer quintiles is to improve the efficiency and quality of delivery. More data are needed in all areas to increase the accuracy of EQUIST–based estimates. Three years are left until the 2015 deadline to achieve the Millennium Development Goals (MDGs) and much progress has been achieved. A recent Inter–Agency Group for Child Mortality Estimation (IGME) meeting reported a child mortality decrease of over one third from 1990 to 2010 [To test this hypothesis, a tool is required that can address the many determinants in the multifaceted relationship between cost–effectiveness and equitable impact in child mortality reduction. Although a number of tools have been developed to assist intervention prioritization at local and national levels – such as Marginal Budgeting for Bottlenecks  ,Choice oEQUitable Impact Sensitive Tool [We recently described a conceptual framework that helps understanding the complex interplay between determinants of cost–effectiveness and equitable impact in child mortality reduction (see ive Tool . This st). These were selected because of their large size and relatively adequate information reported by equity strata. It was also decided to focus on a single disease – pneumonia, which is still the leading cause of child deaths globally [To test EQUIST, five exemplar countries representative of larger WHO regions were used: Nigeria (Sub–Saharan Africa), Egypt (Eastern Mediterranean), Bangladesh (South–East Asia), Cambodia (Western Pacific) and Peru (Americas) , a commonly used measure of equity . For alThe second step was to estimate the cost of scaling–up CCM in each quintile from its existing level of coverage. For more accurate estimation, cost was split for CCM into antibiotic costs and non–antibiotic costs. It was assumed that the direct costs of antibiotics  would be constant across countries and quintiles, while the non–antibiotic costs were likely to be different due to factors including geography, infrastructure and human resources .Direct antibiotic costs for CCM were taken as US$ 0.27 for all quintiles in all areas . Non–antThe third step was to determine coverage levels of the chosen intervention in the five countries in 2008. Coverage with CCM was assumed to be the same as the indicator “% under–fives with suspected pneumonia receiving antibiotics” used in UNICEF’s The State of the World’s Children (SOWC) reports.The fourth step was to estimate how CCM’s effectiveness varied according to the quintile in which it was implemented and therefore calculate the quintile–specific potential impact fraction (PIF). Effectiveness was modelled by graphing effectiveness reported in each study used in a review of CCM , againstFinally, it was necessary to populate the model with disease burden estimates for each disease in each quintile. This was initially attempted through systematic literature review; however an attempt  yielded insufficient data therefore it was decided to model them instead. Data on distributions of under–five mortality deaths by cause for all countries (from the CHERG report ) were coOnce the model was fully populated with data necessary to evaluate cost–effectiveness and impact on mortality and equity of community case management for under–five pneumonia mortality, it was decided to compare the cost per number of lives saved for scaling–up the intervention in the next wealthiest 10% of the uncovered population (‘inequity promoting’ approach ), in theFurther detailed information on the methods described above in each section is presented in Online Supplementary DocumentFigure 4 shows estimates of coverage by wealth quintile for community case management (CCM). Although the estimates of coverage by quintile for community case management generally follow expected trends of decreasing coverage with increasing poverty (the greatest differences by quintile being found in Nigeria and Cambodia), Egypt exhibits a slightly unexpected pattern with increased CCM of suspected pneumonia in Q2 and Q3 as compared with Q1. This is thought to be due to the fact that in rich urban communities , medical professionals are trying to avoid over–treating (and therefore promoting antibiotic resistance), but in poorer quintiles this is not the case and more cases are treated aggressively with antibiotics, explaining the higher coverage levels. There is then a dip again in coverage observed in Q4 and Q5 in Egypt, likely to be explained by poor access to health care in the poorest part of the population.Table 2highlights scarcity in CCM effectiveness data, as although these papers were carefully screened in a recent review and found to be high–quality [–quality , none ofBangladesh shows an interesting pattern of birth asphyxia, which takes up highest proportion of mortality in Q2 and Q3, potentially suggesting that in these quintiles although the infectious diseases which are prevalent in poorer quintiles are still well treated, the health care facilities in these quintiles are not as good as in Q1 and so more babies die of birth asphyxia.WHO region–specific disease proportion estimates and models for disease proportion against U5MR and quintile–specific numbers of deaths in 2008 from each of these causes of death in each of the five exemplar countries are presented in the Online Supplementary DocumentStrikingly, the ‘mainstream’ approach for CCM in all countries is not the most cost–effective, instead an equity–promoting approach always delivers the greatest cost–effectiveness in terms of US$ per life saved. The absolute cost differences between this and the next most costly approach differ with context, varying from US$ 59.92 per life saved in Peru to US$ 1.10 in Bangladesh, where an equity–promoting approach is of almost the same cost–effectiveness as an equity–neutral approach of scaling up in the middle uncovered 10%. It is thought this is due to the differences in U5MR from Q3 to Q5 being relatively small in Bangladesh, resulting in the differences in disease burden, coverage, effectiveness and cost also not being large. This can be contrasted with Peru where the greatest difference in cost–effectiveness is between equity–promoting and equity–neutral and the greatest difference in U5MR is between Q5–Q3. This potentially suggests that in more inequitable contexts such as Peru (which has the highest QR of the countries studied), an equity–promoting approach will have a greater impact when compared with more equitable contexts. Egypt is the only modelled country where the next most cost–effective scale–up option is the ‘mainstream’ approach, possibly due to an already relatively equitable coverage of CCM across quintiles (a difference of only 7.6% coverage from Q1 to Q5).Nigeria is an interesting context to study as due to its exceedingly high U5MR in poorer quintiles, the effectiveness modelled for Q5 is 50.4%. It was thought that this might result in an equity–promoting scale–up delivering poor results however what is observed in actuality is that scale–up in Q5 is still the most cost–effective. This highlights that the childhood pneumonia burden in this stratum is so great that even treating 50% will result in a huge improvement, but also that any intervention which could improve effectiveness of CCM could further enhance this and result in extremely significant reductions in Nigeria’s overall childhood pneumonia burden.The results for all aspects of this study are further explained the Online Supplementary DocumentThis study aimed to populate EQUIST  with reaAn extensive review of the literature found only one paper that attempted to model any child health data split by wealth quintile. Amouzou et al used the LiST to model child mortality data for richest and poorest quintiles in Bangladesh and found this to be within a 95% confidence interval of current DHS data . This isIn absence of information, it was necessary to model much of the data needed to populate EQUIST including data for non–antibiotic costs, as although there are several studies estimating overall cost of global scale–up of health systems ,33 and sEstimates of relative disease proportions split by wealth quintile were the most extensive modelling exercise undertaken and are therefore central to consider when assessing this EQUIST implementation’s robustness. The modelling was based on data from the highly–cited CHERG report on child mortality  and the Limitations of the data in this study are further explained in the Online Supplementary DocumentHaemophilus influenzae (Hib) are rolled out across an increasing number of countries through the GAVI Alliance [The results of this first implementation of EQUIST provide important conclusions. Firstly, one of the main findings of this study was the lack of good data in this important area. The need for extensive future research to fill gaps should be emphasized, especially into variables such as effectiveness and cost of interventions across population wealth strata. One potential way of doing this would be to further expand the DHS or MICS (Multiple Indicator Cluster Surveys ) to collAlliance ,42, usinAnother potential facet for future research is the inclusion within EQUIST of other indicators of inequity apart from wealth. Policy makers are likely to find targeting interventions strictly by wealth quintiles difficult, therefore decomposing the components of the EQUIST for other sub–population group measures may be of more use. One potential way to do this is to consider using geographical areas to split populations as significant variances in U5MR are typically seen -16 and gOne of the most important findings in this first implementation of EQUIST is that the current, “mainstream”, approach never showed the highest cost–effectiveness in studied examples. Therefore for CCM scale–up, the current approach is unjustifiable. If countries are already not delivering interventions maximally cost–effectively, and many are increasing inequity, could an equity–focus lead to improvement in both areas? The CCM cost–effectiveness data generated in this paper suggest that indeed the most cost–effective approach is in actuality scale–up in the poorest, as although poorer quintiles display a decrease in effectiveness and an increase in cost of scale–up, the higher burden of disease and case fatality observed in these strata is great enough to offset this. This potentially lends increased weight to policy makers and academics increasingly calling for exactly this kind of equity–focus in scale–up of interventions ,37,43-46This implementation of EQUIST and the conceptual process involved behind thinking about intervention scale–up in this manner also suggests possible means of further enhancing cost–effectiveness, resulting in more lives saved for a given investment. The limiting factor in CCM in poorer quintiles such as Nigeria seems to be the very low effectiveness of the intervention and so it is implied that enhancement of the efficiency or quality of provision will also significantly decrease cost and therefore increase cost–effectiveness . This deChild health information split by wealth strata in LMICs is severely lacking. This first implementation of the EQUIST framework has expanded knowledge and delivered important analyses on cost–effectiveness of different strategies in scaling up of community case management to tackle pneumonia in five LMICs, demonstrating EQUIST’s potential future value. It has highlighted the complexity of interactions between equity, cost–effectiveness and their determinants, also reinforcing important suggestions for future policy such as the significant effect on cost–effectiveness of increasing efficiency and quality of interventions in poorer quintiles.


Primary breast sarcomas are very rare entities, accounting for 0.04% of all malignant neoplasms. Angiosarcoma of breast is infrequent and is an endothelial malignant tumor with bad prognosis because of the frequency of metastasis and recurrence. We present a case of a 30-year-old female who presented with an ulcerated left breast lesion which on further workup revealed to be a primary angiosarcoma of breast with metastasis to right breast. Angiosarcomas are uncommon malignant neoplasms characterized by rapidly proliferating and extensively infiltrating anaplastic cells derived from blood vessels and lining irregular, blood-filled spaces. The term angiosarcoma is applied to a wide range of malignant endothelial vascular neoplasm's that affect a variety of sites. Angiosarcomas are aggressive and tend to recur locally and spread widely and have a high rate of lymph node and systemic metastases.A 30-year-old unmarried lady presented with an ulcerated lump in the left breast. Initially, patient had noticed a peanut sized painless swelling in her left breast six months back which rapidly increased in size and the skin over the swelling spontaneously ulcerated with seropurulent discharge with occasional bleeding from the ulcer. There was no history of breast surgery or breast irradiation. On examination the lump was 8 cms × 8 cms in diameter and was fixed to skin but free from underlying muscle and chest wall. There were multiple enlarged axillary lymph nodes with the largest measuring 3 cms × 3 cms. In the opposite breast, a lump measuring 5 cms in diameter was detected which the patient had failed to notice. Axillary nodes were palpable on the right side as well. A provisional diagnosis of stromal sarcoma of the breast was considered after a Fine Needle Aspiration Cytology (FNAC) from both lumps. The patient underwent bilateral modified radical mastectomy with axillary clearance. The histology revealed multiple irregular vascular spaces of different sizes lined by single layer of endothelial cells which were surrounded by groups, bundles and masses of oval, and spindle shaped and pleomorphic cells having ovoid and pleomorphic hyperchromatic nuclei Figures  and 2. TAngiosarcoma of the breast is a rare and highly lethal neoplasm accounting for less than 0.1% of malignant breast tumors . AdvanceIn most cases, the tumor is larger than 4 cm in diameter. In a series at Mayo Clinic tumor size was a more valuable prognostic factor than tumor grade .Low grade angiosarcomas are readily mistaken for benign hemangioma. Angiolipoma features intermingling vessels and fat lobules which may be mistaken for invasion of fat by an angiosarcoma. Papillary endothelial hyperplasia as described by Branton et al. is the “great imposter for angiosarcoma” . The tre


Objective: To prospectively assess the efficacy of valproate in controlling infantile spasms in West syndrome. Methods: Consecutive patients presenting with West syndrome to the Pediatric Neurology Clinic or general outpatient department (OPD) were enrolled for study. Those who were not on any treatment were given valproate in a dose of 30 mg/kg/day while awaiting investigations. Patients were followed up every 2 weeks. Predefined criteria for definition of West syndrome and response were used. Those showing partial/poor response or relapse on valproate were given hormonal therapy. Results: One hundred children with West syndrome were enrolled. Ninety one children were started on valproate. Of these 36 (39.5%) showed a good response, but seven later relapsed while on same dose of valproate and three were lost to follow up. Later age at onset and typical hypsarrhythmia on EEG were associated with good sustained response to valproate while a history of delayed cry at birth was associated with partial or poor response. Sixty two patients who responded poorly to or relapsed on valproate were put on hormonal treatment in addition. Of these 36 (58.1%) had a good response but 11 later relapsed after stopping treatment and two were lost to follow up. Conclusion: Valproate may have a role in treatment of West syndrome in a selected group of patients.Management of West syndrome is unsatisfactory. In our clinic we observed that a significant proportion of patients respond to usual dose of valproate.  West syndrome is an age-dependent epileptic encephalopathy characterized by the clinico-electrical triad of infantile epileptic spasms, arrest, or regression of psychomotor development and hypsarrhythmia, although the latter element may be missing  and Pediatric Neurology Clinic of the King George Medical University Hospital, Lucknow. Lucknow is the capital of the state of Uttar Pradesh – India’s most populous state and also one of the poorest. The King George Medical University Hospital is a state run teaching hospital which caters to poor or seriously sick patients from Lucknow city and the surrounding districts.Children aged 1 month to 5 years attending either the general pediatric OPD or the Pediatric Neurology Clinic were enrolled for the study if they had infantile spasms and psychomotor retardation or regression with or without hypsarrhythmia on EEG. Acutely sick children, and those whose parents did not consent for the study were excluded. Patients already receiving antiepileptic drugs were enrolled but excluded from the comparison of factors predicting response to treatment. Socioeconomic status was judged by the Kuppuswamy scale Phatak, . A histoEnrolled patients were worked up according to a standardized protocol. Details of history, examination, investigations, and follow up were entered in predesigned data collection forms. A 16 channel EEG and neuroimaging  was obtained. Tests for developmental quotient  showed initial good response (response categories 4 or 5), while 55 patients showed partial or poor response. However of the 36, seven patients relapsed within 3 months on same dose of valproate, three were lost to follow up, and only 26 (29.5%) patients continued to have a sustained good response  and typical hypsarrhythmia on EEG (p = 0.001) were found to be significantly associated with a sustained good response to valproate therapy, while history of delayed cry at birth was associated with a poor response (p = 0.040).Clinical, demographic, and risk factors were compared in those showing good (26 patients) and partial/poor response/relapse (62 patients) by univariate analysis to look for factors associated with good sustained response to valproate  showed good initial response but of these 11 later relapsed after hormonal therapy was discontinued and two patients were lost to follow up. So only 23/62 (37.1%) could be counted as having a sustained response to hormonal treatment  as also seen by other Indian workers (Kalra and Passi, Availability of newer antiepileptic drugs has led to various treatment regimens for West syndrome being tried all over the world, topiramate (Peltzer et al., We tried valproate initially in our patients only while they were undergoing investigations. Patients were recalled every 2 weeks and those who showed partial or poor response to valproate were immediately shifted to the next line of therapy. An 80% response in seizures was taken as meaningful because we feel that with a lesser response, the physician would be prompted to use another drug. Previous workers have taken a 50% or greater response in seizures as a good response (Kalra et al., Almost 2/5th of our patients had a good response to valproate initially within 2 weeks but sustained response to valproate was seen in less than 30%. Later age of onset was found to be associated with good sustained response to treatment. This was found by Riikonen also (Lux et al., Efficacy of valproate has not been adequately studied in West syndrome and no randomized controlled trial data is available (Riikonen, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In Kenya, infant mortality rate increased from 59 deaths per 1000 live births in 1988 to 78 deaths per 1000 live births by 2003. This was an increase of about 32 percent in 15 years. The reasons behind this upturn are poorly understood. This paper investigates the probable factors behind the upsurge in infant mortality in Kenya during the 1988–2003 period. Understanding the causes behind the upsurge is critical in designing high impact public health strategies for the acceleration of national and international public health goals such as the Millennium Development Goals (MDGs). The reversals in early child mortality is also regarded as one of the most important topics in contemporary demography.http://www.measuredhs.com. Stata version 11.0 was used for data analysis. The paper used regression decomposition techniques as the main method for analysing the contribution of the selected covariates on the upsurge in infant mortality.A merged dataset drawn from the Kenya Demographic and Health Surveys of 1993, 1998 and 2003 was used. The merged KDHS included a total of 5265 singletons. Permission to use the KDHS data was obtained from ICF international on the following website: The duration of breastfeeding; maternal education, regional HIV prevalence and malaria endemicity were the factors that appeared to have contributed much to the observed rise in infant mortality in Kenya over the period. If all the live births that occurred in the 1996/03 period had the same mean values of all explanatory variables as those of live births that occurred in the 1988/95 period, then infant mortality would have increased by a massive 14 deaths per 1000 live births. However, if the live births that occurred in the 1988/95 period had the same mean values of all explanatory variables as those that occurred in the 1996/03 period, the upsurge in infant mortality would have been negligible. While the role of HIV in the upturn in infant mortality in Kenya and other sub Saharan African countries is indisputable, this study demonstrates that it is the duration of breastfeeding and Malaria endemicity that played a more significant role in Kenya’s upsurge in infant mortality during the 1988–2003 period.Efforts aimed at controlling and preventing malaria and HIV should be stepped up to avert an upsurge in infant mortality. There is need to step up alternative baby feeding practices among mothers who are HIV positive especially after the first six months of breastfeeding. Owing to the widely known inverse relationship between maternal education and infant mortality, there is need for concerted efforts to promote girl child education. Owing to the important role played by the short preceding birth interval to the upsurge in infant mortality, there is need to promote family planning methods in Kenya. Despite having an average annual decline in infant mortality of about 4 per cent during the 1960–1980 period, ; Kenya eAn understanding of the reasons behind the upsurge in infant mortality in Kenya is critical and urgent in designing child survival programs and strategies. If clearly understood, priority interventions can be put in place for the quick realization of national and international targets such as target four under the Millennium development goals (MDGs). This paper had the following objectives: -a) To examine the levels and trends in infant mortality during the 1988–2003 in Kenyab) To identify the factors associated with the upturn in infant mortality in Kenya during the 1988–2003 period.http://www.measuredhs.com. We used the Mosley and Chen framework [This paper used a merged data of the 1993, 1998 and 2003 Kenya Demographic and Health Surveys (KDHS). In total, 5265 singletons born five years preceding the 1993 and 2003 and three years before the 1998 KDHS were included in the study. The data were freely obtained from MEASURE DHS on the following website: We used live births that occurred between 1988–95 to analyze infant mortality changes during the 1988–95 period and live births that occurred between 1996–2003 to analyze infant mortality changes during the 1996–2003 period. The dependent variable was the probability of dying between ages 0 and 11 months of life. This was a binary variable with 1 representing the occurrence of the event (i.e. infant death) during 0–11 months and 0 for censuring (i.e. for infants who never died). A detailed description of the Cox regression is available in any standard biostatistics textbook.a place of delivery, whether the mother received a tetanus injection during pregnancy, HIV and malaria endemicity. These are some of the variables that were found to have changed the most during the 1988–2003 and hence were likely to be associated with the upsurge in infant mortality [The following explanatory variables were used: the maternal age at child birth, the length of preceding birth interval, size of the baby at birth, duration of breastfeeding, source of water, type of toilet facility, maternal education, paternal education, ethnicity, household wealth index,ortality ,7. In thIn 1970s, Preston demonstrated that when demographic events such as infant mortality change, it is not only factors that determine such events that could be changing over time and space but rather, the events themselves could be changing at the same time as well -16.In order to apply regression decomposition techniques, it is normally advisable that all selected explanatory variables are converted into dichotomous variables ,9. The da) Maternal age at birth (1 = Below 20 years & above 35 years 0 = 20–34 years)b) Length of the preceding birth interval (0 < 24 months 1 = > 24 months)bc) Size of the baby at birthd) Duration of breastfeeding (0 = Less than 10 months 1 > 10 Months)e) Source of water (1 = Piped water 0 = Other)f) Presence of toilet facility (1 = Yes 0 = No)g) Maternal education: Years of schoolingh) Paternal education: Years of schoolingi) Ethnicity (1 = Kikuyu 0 = Other)c(1 = Low 0 = High)j) Household wealth statusk) Place of delivery l) Had tetanus injection (1 = Yes 0 = No)m) Period (0 =1988-95 1 = 1996/03)d(0 = Low 1 = High)n) Malaria endemicitye(0 = Low 1 = High)o) Level of HIV prevalencet-test statistic. In addition, it is usually important to establish the structure of the relationship between each explanatory variable and infant mortality in each period. Stata version 11.0 was used for statistical analysis. For purposes of accounting for censoring, Cox regression model was used to estimate the time of experiencing death between 0–11 months. The Cox regression provides the hazard ratios and the associated standard errors for each explanatory variable in the two periods under investigation [The next step entails computing means of the dichotomized variables for each period while assessing whether the means are statistically different using a tigation ,8,9. In Ceteris paribus, what level of infant mortality would we have, say in the later period (1996/03) if a given explanatory variable in the initial period (1988/95) had the same mean value as in the later period (1996/03)? This question is repeated for each explanatory variable to assess their individual contribution to the observed upsurge in infant mortality [The critical question in using hazard ratios to predict the role of each explanatory variables on infant mortality between periods is: od 1988/9 had the Assume that we would like to know the contribution of any covariate, say, household wealth Index (HWI) on infant mortality (IMR) in 1988/95 period if this variable values were applied to the 1996/03 period. We would have to use the following formula:-Where0 = change in infant mortality rate for the 1988/95 periodΔ in IMR0 = the hazard ratiof of HWI for the 1988/95 periodβ HWI0 = Mean value of the Household wealth Index for the 1988/95 periodHWI1 = the hazard ratio of HWI for the 1996/03 periodβ HWI1 = Mean value of the Household wealth Index for the 1996/03 periodHWIIn the above formula, the equation is multiplied by 1000 to assess the contribution of the variable to infant mortality per 1000 live births.Table The duration of breastfeeding especially during the first six months of an infant has been found to improve infant survival due to the nutritional value of mother’s milk, the immunities against communicable diseases that it provides, and because it is hygienic . Table 1g recorded an upsurge in infant mortality. This is likely due to the incessant inter-ethnic wars among this groups leading to displacement and death especially of women and children [Infants belonging to minority ethnic groups categorized as “other”children ,17. LikeThe results obtained indicate a decline in infant mortality among mothers belonging to the upper socio-economic class (22 percent) while those in the middle and lower tertiles experienced an upsurge in infant mortality during the 1988–95 and 1996–2003 period. Infant mortality rate among mothers who delivered at home rose by 21 percent. While being born in a government or a private health facility was associated with a lower risk of dying as compared to being born at home, generally, deliveries at a health facility recorded the highest upsurge in infant mortality rate (58 percent and 62 percent respectively). It is likely that there was an increase of high risk births delivered in health facilities.Generally, infant mortality rate decreased among babies of mothers irrespective of whether such mothers had received tetanus injection or not. Among the infants born of mothers who had not received the tetanus injection, infant mortality decreased by 39 percent.While trend analysis is insightful in bringing out the changes in infant mortality over the study period by selected characteristics, it fails to show whether the observed changes are statistically significant. We address this gap in the next section.Results from Table Below, we assess the role of the explanatory variables in relation to the rising levels of infant mortality between 1988–95 and 1996–2003 periods.Table In Model V when the regional HIV prevalence and Malaria variables were added to Model 1, the risk of dying in the 1996–2003 period was 28 percent higher than that of the 1988–95 period . Finally, when the full model was considered, the risk of dying in 1996–2003 was 38 percent higher as compared to the 1988–95 period and this association was statistically significant (p < 0.05).As shown in Table vis a vis explanatory variables in the observed upsurge in infant mortality. This section and the subsequent sections address this issue by first assuming a fixed structure analysis and later, a changing structure analysis. Table As discussed earlier, infant mortality is bound to have changed independent of the explanatory variables considered in this study . It is tThe effect of the baby size at birth was much greater during the 1996–03 period than in 1988–95 period. The effect of duration of breastfeeding was more pronounced during the 1988–95 than in the 1996–03 period. Duration of breastfeeding and regional HIV status had each a positive significant effect on infant mortality. The effect of duration of breastfeeding on infant mortality was however greater during the 1996/03 period than during the 1988–95 period. On the other hand, the effect of HIV seemed to have been more pronounced during the 1988–95 period as compared to the 1996/03 period. The effect of HIV regional prevalence and maternal education was positive and significant while that of paternal education, source of water and type of toilet facility was significant but negative.The preceding section presumed a fixed structure analysis . This section introduces a changing structural analysis . Table In general, the results shown in Table hHad infants belonging to the 1996–03 period experienced the same mean values of duration of breastfeeding as those belonging to the 1988–95 period, infant mortality could have increased by a massive 19 deaths per 1000 live births. In contrast, had infants belonging to mothers in 1988–95 period experienced the same mean values of duration of breast feeding as those of children belonging to mothers in the 1996–03 period, infant mortality would have reduced by 5 deaths per 1000 live birthsIn particular, duration of breast feeding, preceding birth interval, the regional level of HIV prevalence and regional Malaria prevalence appear to account for the largest share of the differences in mortality between the two periods.Regarding malaria endemicity, had infants belonging to period 1996–03 experienced the same mean values of the disease as those belonging to the 1988–95 period, then infant mortality would have reduced by 7 deaths per 1000 live births. In the case of HIV/AIDS, the reduction would have been 3 deaths per 1000 live births. However, had infants belonging to the 1988–95 period experienced the same mean values of malaria endemicity and HIV/AIDS as those of belonging to the 1996–03 period, infant mortality would have increased by 5 deaths and 4 deaths per 1000 live births, respectively. The results also seem to indicate that if the rest of the explanatory variables for the 1996–03 period had values as those born in the 1988–95 period, then infant mortality rate in the 1996/03 would have remained largely unaltered and vice versa.This paper sought to explain the upturn in infant mortality in Kenya during the 1988–2003 period using regression decomposition techniques. The findings revealed that apart from maternal age at child birth, tetanus toxoid, ethnicity and household wealth index, the effect of the rest of the explanatory variables were significantly associated with explaining the differences in infant mortality over the study period. In particular, the duration of breastfeeding, maternal education, regional HIV and malaria prevalence contributed much to the observed rise in infant mortality. However, there could be other important factors behind the upsurge that were not captured by KDHS. For instance, KDHS does not collect information on injury related factors yet an upsurge in these factors could have influenced the upturn in infant mortality during the study period.The above findings have several implications. First, there is need for the urgent promotion of family planning services in order to reduce the adverse effect of short birth intervals as well as prevent high risk births which tend to be associated with a heightened risk of infant deaths ,11. SecoHousehold wealth index- a proxy measure of the socio-economic status of the economy played an increasing role in infant mortality upturn. The downturns in Kenya’s economy during the study period with some years recording a negative growth in the GDP must have depressed the household level spending on health. This was further worsened by the introduction of the structural adjustment programs (SAPs) in the late 1980s which among other things included cost sharing in health and education and was also accompanied by job cuts in the public sector ,19. TherFindings show that malaria prevalence played an important role in the observed upturn in infant mortality. This was expected owing to the fact that malaria resistance peaked in Kenya in 1997 (when resistance to chloroquine ranged from 66–87 percent). This forced the government to change the first line of treatment for malaria from chloroquine to artemisinin combination therapy (ACT) for management treatment of uncomplicated Malaria cases ,20. TherThe deterioration in gross enrolment rates in education especially after the introduction of SAPs in the late 1980s appears to have locked out many young girls in the education system. There is need for renewed efforts to promote the girl child education owing to the widely known role of maternal education in reducing infant mortality ,16.Unlike past studies ,3 this sa We computed the household wealth index using the principle component analysis (PCA). The first principle component was used to compute household tertiles i.e. low, middle and high income households.b Conventionally, infants born with a birth weight of less than 2500grams are categorized as low birth weight (LBW) and vice versa. While our study did not use this variable due to large number of missing cases (55%) arising from low health facility deliveries in Kenya (only 42%), we used baby size as a proxy measure of low birth weight.c Household wealth index was computed using the principle component analysis (PCA) based on the household possessions of each sampled household. Those with low and medium income were categorized as “low” while those with high income were categorized as “high.” In our preliminary analysis, we found that low and medium households experienced an upsurge in mortality while the high income households experienced a decline in mortality.d The categorization of whether a region was low or high malaria endemicity was based on the categorization of Kenya Medical and Research Institute which was used in one of the author’s earlier study [er study . Accordie This paper categorized regions with higher than national HIV prevalence (6.7%) as high HIV prevalence regions and vice versa [ce versa . Using tf For ease of interpretation, the hazard ratios were exponentiated to give regression coefficients (i.e. exp(HR) = β) used as shown in Tables g Ethnic groups categorized as “other” included all tribes apart from the Kikuyu. These include the Kalenjin, Luhya, Luo, Kisii, Kamba and other small tribes such as the Somali, Rendile, Pokomo, TaitaTaveta, Borana, etc.h Although our decomposition analysis focuses on the relative importance of maternal, socio-economic, and health seeking factors on the change in infant mortality over this time period, our study does not examine the important interrelationships occurring between these groups of variables. For instance, maternal education is widely known to be related with age at birth as well as maternal health seeking behavior. It is beyond the scope of this paper to disentangle whether changes in socio-economic factors are causing changes in maternal and health seeking factors or the other way round or if their simultaneous changes are caused by another factor.The authors affirm they have no competing interests.SWW wrote the draft paper which is based on a chapter of his PhD dissertation. LI and BK reviewed the paper. All the three authors approved the manuscript prior to submission.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/441/prepub


AC Biosusceptometry (ACB) was previously employed towards recording gastrointestinal motility. Our data show a reliable and successful evaluation of gastrointestinal transit of liquid and solid meals in rats, considering the methods scarcity and number of experiments needed to endorsement of drugs and medicinal plants. ACB permits real time and simultaneous experiments using the same animal, preserving the physiological conditions employing both meals with simplicity and accuracy. Gastrointestinal (GI) motor activity consist of an intricate group of functions that are essential for life , and disA number of techniques have been employed to evaluate GI motility and transit, but few techniques are able to evaluate more than one GI motility parameter simultaneously .GI transit can be quantified in rats, by measuring the movement of charcoal, dye, radiopaque markers or other non-absorbable materials ,6. This Scintigraphy is the gold standard method for gastric emptying in humans ,8. Such Alternating Current Biosusceptometry (ACB) is an inexpensive, radiation-free and noninvasive method that was previously employed as a reliable technique to record GI transit and contractility in humans, dogs and rats. ACB data showed accuracy and close agreement with standard techniques in humans and dogs -15. ReceThe aim of this study was to monitor in real time GI transit of liquid and solid magnetic meals by using ACB technique in order to establish this biomagnetic method as a reliable technique for multiple records of the GI motility in the same rat.A single ACB sensor works as a double magnetic flux transformer without any nucleus and has two coaxial pairs of coils separated by a baseline of 15 cm ,17. Each2MnO4 – microparticles 50 ≤ ϕ ≤ 100 μm) remained completely inert in all pH solutions and cannot be absorbed by GI tract due to its inter-molecular ligations and size. The ferrofluid  employed in liquid meals (nanoparticles with diameter of 200 nm) consists of iron oxide magnetic particles that were coated with chitosan by spray-drying method. Based on earlier studies, it is reasonable to assume that these particles are not absorbed by mucosa in the GI superior tract [Ferrite powder  utilized in solid meals  were used in the study. All procedures were performed in accordance with the Guide of the Care and Use of Laboratory Animals  and were approved by the local Animal Ethics Committee. Animals were fasted 20 h before experiments, with free access to water. For comparison tests, six groups with three animals each were killed at pre-determined time intervals (10 min) after ingestion of solid meal similarly as it is performed in activated charcoal test. In vivo tests, the studies  were performed in the same animal in a randomized order and were separated by an interval of 1 wk.Magnetic monitoring was achieved by measuring the intensity values recorded by the single-sensor ACB  placed on abdominal surface. The animals were handled gently by the neck and the sensor was positioned on their gastric and cecum projection after ingestion of either solid or liquid magnetic meals Figure .Liquid meal: Ferrofluid (1.0 ml – 50 mg/mL) diluted in 1.5 ml of distilled water was administered by gavage to rats and five minutes later the abdominal surface was scanned by ACB sensor. The point of maximum magnetic signal intensity was identified as corresponding to the stomach and the magnetic value was registered. After that, the ACB sensor was placed in the cecum projection  and the magnetic signal intensity was also recorded. Subsequent measurements were made in awake rats at these two points at regular 10-min intervals for at least 7 hours .Solid meal: Pellet (2 g) made of powder ferrite (0.5 g) and laboratory chow (1.5 g) was quickly ingested by the animals, 10 min before starting the experiments. The abdominal surface was scanned by ACB sensor following the same protocol described above for liquid meal.All raw signals were analyzed in MatLab  by visual inspection and the statistical moment was calculated. The statistical moment was obtained through the temporal average pondered by magnetic intensity curves, normalized by area under curve . By usinp < 0.05.All the results are expressed as mean ± standard deviation (SD). Values of MGET, MCAT, and MSITT obtained after ingestion of liquid or solid meal were correlated. By using paired Student’s t-test statistically significant difference was considered at A magnetic pellet (ferrite) was quickly ingested by animals (n = 18) and 10 minutes later the experiments starts. The animals have gastric magnetic intensity recorded and immediately after that, at pre-determined time intervals , they were killed. The small intestine was carefully removed and the distance traveled by ferrite  up to the last portion that contained at least a continuous 1 cm trace of this marker was determined. The results were expressed as intensity (mV) and distance (cm) traveled by ferrite.Figure p < 0.04).The GI transit times parameters for liquid and solid meals are summarized in Table p < 0.04). The mean values of MSITT on both liquid and solid meals were 103.28 ± 18.73 min and 103.22 ± 37.26 min, respectively. There is no statistically significant difference between the liquid and solid values for MSITT.MCAT values for liquid and solid meals were 202.86 ± 16.31 min and 243.74 ± 62.12 min, respectively. MCAT also presented a significant increase for solid meals (Gastric emptying (MGET), cecum arrival (MCAT) and small intestinal transit (MSITT) profiles obtained by ACB after ingestion of liquid (gray cicles) and solid meals (black squares) are illustrated in the Figure A variability profile was obtained using the same animal (n = 3) in three ACB recordings with one week of interval among them. The biological variability in these measurements was determined in around 6 % for MGET and 11 % for MCAT.Our data show that ACB is useful for monitoring gastric emptying, cecum arrival and small intestinal transit time using both liquid and solid meals. The ACB is a flexible method combining reliability assessment of GI transit and does not require anesthesia or death for studies using laboratory animals.It is important to emphasize that there are specific techniques for measurements of emptying, contractility, accommodation and sensation , howeverAs previously demonstrated, gastric emptying profile can be obtained by ACB because when magnetic material moves into duodenum the intensity of magnetic signal decreases in a point representative of the stomach. Likewise, signal intensity increases when material arrives to the cecum and it is also possible to evaluate the orocaecal transit time .In this study, ACB was employed for the first time to evaluate magnetic liquid meal since this was greatest challenge for this biomagnetic technique for several years. Hereafter, the nanotechnology provides an evaluation of liquid transit that can be compared with solid allowing to establish a complete profile for GI transit times.50) is adopted and consists of the signal decay by half-intensity. This procedure has been severely criticized because is associated with a pure exponential model, which is not realistic for most of the processes of emptying [The gastric emptying of noncaloric liquids meal has usually been exhibited as exponential pattern, while after solid meal the emptying have been characterized by a significant delay ,23. Typiemptying . Meanwhiemptying .p = 0.04). As reported by others, our data showed that small intestinal transit time (MSITT) of both meals exhibited essentially the same transit rates [As expected, gastric emptying for liquid meal has a shorter time than solid; the arrival process of food to the cecum is not concurrent with the emptying, as occurs for solid meal. Even before of the complete gastric emptying, magnetic material have already been detected on caecum region. This profile can be explained by the time it takes for meal leaving the stomach and travel through the bowel of the animal. Still, it is important to note that the average time of arrival of meal in the cecum (MCAT) for liquid meal is considerably less than the same coefficient for solid meal (it rates .Several physiological conditions can be responsible for an altered GI transit . A slow The potential weaknesses of the current study are the multiple handling of the animal that is known to increase plasma cortisol and catecholamines which potentially may confound stress effects with the chosen experimental stimuli . HoweverAnimals have been used as experimental models for centuries and their use has enabled researchers to make significant advances in many areas of human health and disease . NowadayOur magnetic technique allows the in vivo experiments, have a high correlation with standard technique for GI transit in rats and preserving the physiological conditions. Also, both liquid and solid magnetically marked meals may be used paralleling a normal diet and this protocol can be applied in drugs and medicinal plant tests without unnecessary animal death.The authors declare that they have no competing interests.AMF, ORB and MJRA designed the research; QCC, AM and CMFF performed the research; QCC, AM and ORB contributed analytic tools; QCC, CMFF and MJRA analyzed the data; AMF and CLA wrote the paper. All authors have read and approved the final manuscript.


To the Editor: Wang et al. detected severe acute respiratory syndrome–associated coronavirus (SARS-CoV) from throat wash and saliva specimens and suggested that these specimens have advantages over other specimens, including ease of procurement and safety for medical personnel (0R to the transmission rate β indicates that efforts in finding intervention strategies that manage to systematically lower the contact rate of persons of all age groups promise an effective means for lowering 0R” (An important component of a comprehensive strategy to lower the contact rate is improving the safety measures recommended for clinical specimen collection by healthcare workers. Recognizing the importance of obtaining multiple specimens and the difficulties associated with obtaining samples from the 3 usual portals of entry, we devised and tested a novel method of specimen collection, conjunctiva–upper respiratory tract irrigation (


The trends to investigate larger complexes and more transient phenomena pose some challenges to software developers. This is an introduction to four papers based on presentations given at a workshop entitled Integrated Software for Integrative Structural Biology. The use of hybrid techniques, and other trends in structural research, pose new challenges to software developers. A structural biology work bench that meets these needs would provide seamless data transfer between processing steps, and accumulate archival data and metadata without intruding into the scientist’s work process. Acta Crystallographica Section D are based on presentations given at a workshop entitled Integrated Software for Integrative Structural Biology organised by the Computational Centre for Integrative Structural Biology (CCISB) on 21–23 May 2012.Four papers in this issue of et al., 2011This workshop discussed some new challenges which structural biologists are accepting. They are addressing larger macromolecular machines rather than single gene products . ‘Google widgets’ provide similar functionality. The work bench should similarly make it easy to develop and share new functional web pages, to provide novel structural and bioinformatic functionality. Important steps have already been taken in this direction, for example by weNMR, Developing infrastructure software that supports the daily work of a broad community needs a different approach to developing new algorithms. Experience shows that success depends on an effort to understand the context of use and then more detailed requirements, to take into account the user experience, and then design a software architecture (Morris & Segal, 2012


This special issue focused on the intersection of epigenetics with nursing research and practice. The first paper in this series addresses the role of epigenetic modifications in pain and analgesia response, highlighting the need for future research on epigenomic modification in the development of chronic pain, and summarizes the therapeutic potential to alter epigenetic processes to improve health outcomes. The second studies the epigenetic alterations and an increased frequency of micronuclei in women with fibromyalgia, highlighting a difference in an epigenetic biomarker in participants versus controls. The third paper explored the role of epigenetics in critical illness and the need for clinicians to understand and navigate the novel therapies of the future based on advances in epigenetic science. The fourth paper described the complexity of recruiting participants for epigenetic research and highlighted strategies, including scripts for facilitating the informed consent process. The fifth paper was an insightful review of the epigenetic mechanisms that may contribute to the biological response to trauma and risk for posttraumatic stress disorder. The sixth paper discussed the state of the science in understanding measures of cellular aging in depression.Debra  E.  LyonDebra  E.  LyonSusan  G.  DorseySusan  G.  DorseyLeorey  SaliganLeorey  Saligan


While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates analyzed. Therefore, our results suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely.In excess of  Many human cancers are caused by infections with tumor viruses and identification of these pathogens is considered a critical contribution to cancer prevention. Deep sequencing enables us to systematically investigate viral nucleotide signatures in order to either verify or exclude the existence of viruses in idiopathic human cancers. We have developed Virana, a novel computational approach for identifying tumor viruses in human cancers that is applicable to a wide variety of tumors and viruses. Virana firstly addresses several important biological confounding factors that may hinder successful detection of these pathogens. We applied our approach in the first systematic search for cancer-causing viruses in metastatic neuroblastoma, the most common form of cancer in infancy. Although the heterogeneous clinical progression of this disease as well as epidemiological and virological findings are suggestive of a pathogenic cofactor, the viral etiology of neuroblastoma is currently contested. We conducted an analysis of experimental controls, comparisons with related approaches, as well as statistical analyses in order to validate our method. In spite of the high sensitivity of our approach, analyses of neuroblastoma transcriptomes did not provide evidence for the existence of any known or unknown human viruses. Our results therefore suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely. To date, pathogenic agents are known to be causally related to 20% of human cancer cases in utero. Metastatic neuroblastoma has two biologically divergent subtypes. Stage 4S is characterized by an age of presentation between in utero and Neuroblastoma is a heterogeneous embryonal tumor The variation of clinical outcomes between neuroblastoma subtypes indicates distinct genetic and environmental factors affecting the development of this malignancy. Interestingly, the early onset of the disease overlaps with periods of high susceptibility to viral infections and is reminiscent of acute lymphoblastic leukemia – another pediatric tumor with uncertain etiology for which an infective cofactor has long been suspected In general, the search for suspected viral cofactors of idiopathic diseases requires systematic screening of human tissues for viral biomarkers such as virus-derived nucleotide sequences. Unfortunately, viruses are of polyphyletic origin and thus lack common universal marker genes as they are frequently exploited in metagenomics studies targeting cellular microorganisms. Consequently, it is not currently possible to specifically PCR-amplify viral nucleotide sequences within a given tissue without prior information about the infective agent being sought digital transcript subtraction that discards human sequence homologs from the sequence data and considers the remaining transcripts as potential viral signatures de-novo sequence assembly that aims to reconstruct whole viral genomes from overlapping reads Deep sequencing technologies have enabled detection of both known and novel viruses with unprecedented sensitivity Identification of tumor viruses in particular poses several important challenges to existing computational pipelines. Confounding factors such as loss of viral genetic material from progressed tumors as well as limited replication competence or latent replication strategies often result in low or selective transcription of tumor viruses multimaps’). By using this combined and sensitive mapping strategy, our approach is especially well suited for detecting human-viral chimeric transcripts and viruses diverging from known references. In contrast to existing subtractive approaches for viral transcript discovery, our method abstains from discarding reads homologous to the human genome from further analysis. Instead, Virana exploits multimaps to assign sequence reads to a homologous context comprising human reference transcripts and viral reference genomes. These homologous regions retain the full, unfiltered information contained in the raw sequence data while also being amenable to further analyses by multiple sequence alignments, human-viral phylogenies, and orthogonal taxonomic annotations, thus greatly aiding in the interpretation of the results.We have developed Virana, a novel computational approach specifically tailored to detecting low-abundance transcripts that diverge from known viral reference sequences or share significant sequence homology with human factors. In particular, our method maps sequence reads to a combined reference database comprising the human genome and all known viral reference sequences. The approach is configured to allow for high mismatch rates and mappings to multiple reference sequences  patients  was received from the Pediatric Oncology and Hematology Department of the Hannover Medical School. Deep-sequencing reads obtained from full transcriptome libraries of two HPV18-positive HeLa samples (HeLa) and a HPV16-positive primary cervical squamous cell carcinoma (ceSCC) were downloaded from the Short Read Archive (SRA) and preprocessed as specified in the original publication mRNA libraries of the EBV-positive B-cell lymphoma and gag region of HERVK22I  as well as corresponding refGene splice-site annotations were obtained from UCSC. Splice variant annotations and cDNA sequences for the human genome were downloaded from Ensembl Paired-end reads from the neuroblastoma panels and positive control panels were quality-controlled with an in-house sequence analysis framework in order to identify sample contamination, adapter contamination, and batch effects. After quality control, the sequence data consisted of vely see .seedSearchStartLmax and winAnchorMultimapNmax parameters to chimSegmentMin) was reduced to All data were mapped against a combined human-viral reference database with the splicing-aware and gapped read mapper STAR de-novo transcriptome assemblers Trinity In order to identify putative new viral transcripts, read pairs with at least one unmapped read end were extracted from BAM files by the Samtools suite Virana supports detection of human-viral chimeric transcripts in two different manners. First, the read mapper employed in our study is able to partially align reads that contain a human-viral chimeric breakpoint to multiple reference sequences. Consequently, these partially aligned reads can be detected by Virana within the generic analysis of homologous regions (see below). The second, more sensitive approach to detecting chimeric transcripts is based on paired-end read information. Since the STAR mapper assigns reads to a combined reference database comprising both human and viral reference sequences, ends of paired-end reads whose inserts span the breakpoint of a chimeric transcript will be aligned to different reference sequences. These discordant read pairs are reported by STAR during read mapping (see above) and can further be filtered by mismatch score or sequence complexity in order to yield a high-confidence list of chimeric transcripts.A distinguishing feature of Virana is its ability to automatically reconstruct the homologous context of reads that map to both viral and human reference sequences. This homologous context is constructed in four steps:First, reads that map to at least one viral reference are extracted from the mapping together with their primary  and secondary  mapping positions see . Since vhomologous group (HOG) of that family. The same HIT may, in principle, be assigned to several HOGs.Second, pooled HITs originating from the same analysis panel are assigned to viral taxonomic families based on the viral genomic references they refer to  within the HOG using a highly sensitive BLASTN 3bEach HIT is assigned to a singleton cluster. Clusters containing reads that map to the same reference are merged if their reference mapping locations (as determined by BLASTN) are less or equal than 3cFor each HOR, parts of reference sequences that are neither covered by a read mapping location nor by an Last, due to the high mutual similarity of sequences within trimmed HORs, sequences within each HOR are now amenable to sequence alignment against the longest reference sequence within that HOR using LASTZ, the successor of BLASTZ Algorithm 1. Construction of homologous groups.Data: Reads Result: Homologous groups  Initialise: homologous_groups;forread in mappingdo   obtain all mapped references of read;ifmapped against viral referencethen     Initialize: read_hit;   add read to read_hit;forviral reference the read maps todo       add alignment positions of viral reference to     read_hit;end   forhuman cDNA reference the read maps todo       add alignment positions of cDNA to read_hit;end   forviral reference of a viral taxonomic family thus      addeddo       add read_hit to the homologous group for that     family;end   end  end Algorithm 2. Construction of homologous regions.Data:Homologous groupsResult: Homologous regionsInitialise: homologous_regions;forhomologous_group in homologous_groupsdo Initialize: homologous_regions;forread_hit in homologous_groupdo forhomologous_region in homologous_regionsdo  ifread_hit and homologous_region share a reference     then   ifalignment positions of shared reference are      within l basepairsthen         merge read_hit into homologous_region;     extend all reference aligment positions within      homologous_region;end    end   end  end ifread_hit not mergedthen   Initialize: homologous_region;  merge read_hit into homologous_region;  add homologous_region to homologous_regions;end endwhilemergeable_pair_of_homologous_regions existsdoforpair_of_homologous_regions in homologous_regions   do ifpair_of_homologous_regions shares a reference    then  ifalignment positions of shared reference are within l     basepairsthen       merge pair_of_homologous_regions;    extend all reference aligment positions within     new homologous_region;end   end  end endhttp://hiv.lanl.gov, GTR model of nucleotide substitution, transition/transversion ratio: 4, gamma shape parameter: 1, number of substation rate categories: 4, approximate Likelihood Ratio Test (aLRT) using SH-like supports where applicable). We note that the topology of the phylogenetic trees constructed in this manner is stable with regard to the model choice; while more complex model parameters may yield better likelihoods in some instances, these differences do not influence interpretation of our results.Consensus sequences can be further processed by phylogenetic analyses. For generating phylogenies, Virana employs the software PhyML de-novo assembled sequence contigs to nucleotide (NCBI NT) and protein (NCBI NR) reference archives in order to assign transcripts to a taxonomic origin. To this end, we employ several BLAST In this study, we additionally compare consensus sequences of aligned HOGs as well as We quantify the ability of our novel method Virana and the related methods RINS All five data sets  are analyzed by Virana, RINS, and CaPSID using identical reference sequences as described in Section “Reference genomes”. Sensitivity  and specificity  was conducted by configuring CaPSID, RINS, and Virana analogous to the previous section. For the validation of fusion transcript detection, the number of true positives is set to the number of all reads originating from the human-viral fusion transcript. Since all detection methods in this validation are configured to only report reads mapping to the viral part of the fusion transcript, sensitivity estimates are scaled down equally for all methods in this particular validation. Analysis of discordant read ends in order to detect the origins of chimeric transcripts was performed as described before (see Section “Detection of chimeric transcripts”).Expanding on related work in silico simulation of paired-end reads that are assigned randomly independently and uniformly to transcripts of different lengths and at varying coverages. This simulation process addresses the aforementioned confounding factors by considering transcript boundaries and sampling insert sizes from a normal distribution parametrized according to neuroblastoma sequence data employed in this study (see Section “Library preparation and sequencing”). An mean estimator for Although the probability All sequence data generated in this study are publicly available in the European Nucleotide Archive (ENA) at study accession number PRJEB4441. Software implementations of our method and all validation procedures are available at http://mpi-inf.mpg.de/∼sven/virana.de-novo assembly of unmapped sequence reads followed by taxonomic annotation in order to discover proviral integration events and novel viruses, respectively.This study presents a novel approach to detecting viral transcripts in human tumor transcriptomes. In contrast to related approaches such as RINS and CaPSID that rely on subtracting reads homologous to human transcripts from the analysis, our novel method Virana assigns sequence reads to a combined human-viral reference database without discarding homology information see . By emplRetroviridae are detected in validation data at all levels of sequence divergence. Since the detected HERV reads originate from the human rather than from the viral part of the validation data, these reads classified as false positive (FP) hits for the purpose of this validation. As a result of this artifact, Virana exhibits a slightly lowered specificity compared to subtractive approaches . However, we note that HERV reads are correctly classified by Virana during homologous region construction and by optional BLAST-based taxonomic annotation. These reads can therefore be safely and automatically ignored in subsequent analyses if HERV expression is of no interest to the researcher.In order to compare Virana and the two subtractive approaches CaPSID and RINS in a controlled environment we rely on a previously published simulated data set consisting of a negative control data set free of viral reads, here denoted as background set. The background set is used to construct four additional validation data sets spiked with viral reads at increasing rates of sequence divergence 22I, a class of human endogenous retroviruses associated with some forms of breast cancer Having established Virana's ability to detect reads sampled at comparatively high coverage from viral genomes with low or no human-viral sequence similarity, we next test the sensitivity of the viral detection methods in a more challenging scenario involving gene regions of animal viruses that have close human homologs and are sampled at low sequencing coverages. Three such human-viral homologs are used in the analysis: V-ABL of the acutely transforming retrovirus A-MuLV, Bo17 of herpesvirus BoHV-4 . Remarkably, the Coliphage phi-X174 genome of the family Microvirodae could be fully assembled by Virana's homologous region construction, yielding a single fragment of 99% sequence identity and 100% coverage compared to the phi-x174 reference genome. In addition, several retroviral and flaviviral hits at low abundances of Retroviridae and Flaviviridae, respectively. The taxonomic ambiguity of these regions is automatically identified during Virana's homologous region construction and confirmed by optional BLAST-based annotation compared to NCBI nt and nr databases  or from human proto-oncogenes that have close homologs in pestiviruses and acutely transforming retroviruses. HORs associated with these viral families were automatically assigned human or ambiguous taxonomic origin by Virana, as indicated by narrower bars in Reads assigned to viral families No significant differences in viral expression signatures between neuroblastoma 4 and 4S stages could be detected except for HERV-K endogenous retroviruses which display tion see . While sde-novo transcriptome assemblies of all unmapped reads. We applied the two de Bruijn graph based assembly methods OasesIn order to identify transcripts of novel viruses that do not map to known references, we generated vely see . Assemblases see . Overallpolyomaviridae and adenoviridae are indeed suspected to acquire rare transforming properties as a consequence of viral latency or defective replication Neuroblastoma is a pediatric tumor of the sympathetic nervous system that represents the most common form of cancer in infancy. It is characterized by a striking diversity in biology and clinical behaviour of its subtypes. This heterogeneity as well as supporting epidemiological findings are highly suggestive of infectious cofactors involved in genesis and maintenance of the disease Several high-throughput methods for detecting viral sequence reads among human RNA-Seq data have been developed. Among these methods, PathSeq, CaPSID and RINS are most prominent due to their design as reusable computational pipelines. In this study we selected CaPSID and RINS due to their high performance and public availability and compared their detection performance with that of our novel method Virana. Both CaPSID and RINS follow a subtractive approach, e.g. they separately map input data to viral and human reference sequences and subtract viral read mappings that are similar to the human genome from the analysis. While CaPSID is conceptualised as a generalised framework that supports the subtraction process by means of a database and a web server, RINS features an integrated pipeline that splits input reads into shorter fragments in order to increase mapping sensitivity, followed by transcriptome assembly of putative viral reads into full length transcripts.Both RNA and DNA viruses may share considerable sequence homology to human factors due to reasons such as lateral gene transfer, oncogene capture, ancestral endogenization, or insertional mutagenesis leading to chimeric transcripts We quantitatively validated Virana's approach both in settings involving simulated reads as well as in real-world scenarios involving experimental positive and negative controls. In these validations, Virana displays significantly higher detection sensitivities than competing approaches especially at high rates of viral sequence divergence exceeding Interestingly, viral reads analyzed in the sequence divergence validation originate from a broad array of viral species, only two of which infect mammalian hosts and none of which display significant human-viral sequence homology. As a consequence, this validation favors subtractive approaches by reducing the danger of erroneous subtraction of viral reads that are similar to the human genome. In addition, the sequence divergence validation contained reads sampled at high coverage. However, transcripts of tumor viruses are often expressed at only low cellular abundances and are thus expected to have low sequence coverage. We therefore next validated the ability of viral detection approaches to detect viral transcripts homologous to human factors at varying levels of sequence coverage. Virana, by virtue of not relying on digital subtraction, demonstrated superior sensitivity at this validation both in settings of natural sequence homology as well as in cases of human-viral chimeric transcription. Specifically, Virana was the only method able to detect evidence for all viruses even at low twofold coverages. We observed that both RINS and CaPSID discarded a substantial amount of human-viral homologous transcripts due to their high similarity to the human reference genome, a fact that explains the lower performance of these methods in this validation scenario.Flaviviridae and Retroviridae) that display high sequence similarity to human factors. These hits are additionally annotated with an ambiguous taxonomic origin by Virana. In addition, Virana provides extensive support for investigating such ambiguous viral hits by analyzing the homologous context of putative viral reads in a context of multiple sequence alignments and phylogenies.Analysis of positive and negative experimental controls further reveals that Virana is able to detect viral transcripts associated with four types of cancer at a high dynamic range of relative abundances. While Virana displays a slightly reduced specificity in simulated and experimental evaluations, these false positive hits are limited to only two viral families (In principle, several biological confounding factors may hinder detection of viral transcripts by any sequence-based method. Low concentration and extratumoral location of viral producer cells Our approach aims to counteract these confounding factors by two strategies: first by sequencing neuroblastoma transcriptomes at comparatively high depth in order to detect rare transcripts and second by using several biological replicates at different tumor stages, thus reducing the probability of total loss of viral material from all analyzed samples. Based on statistical estimations concerning Virana's homologous region construction process and the sequencing depth of our experimental data, we can conclude that our approach requires minimal abundances of only two average-length viral transcripts per cell even under adverse conditions such as high viral divergence or extensive human-viral sequence homology. While representing a theoretical sensitivity that may be altered by sequencing biases After applying Virana to several positive control panels of human cancers with known viral cofactors and accurately reconstructing large fragments of viruses that are causally related to the respective tumors, we analyzed neuroblastoma transcriptomes at high sequencing depth and using two different sequencing platforms. Analyses of neuroblastoma transcriptomes resulted in the detection of putative viral transcripts with high local sequence similarity to several viral families. However, automatic taxonomic annotation as well as detailed manual inspection of homologous regions pertaining to these families revealed the human or bacteriophage origin of all transcripts. While we could find differences in the abundance of HERV-K transcripts between neuroblastoma stages 4 and 4S, the causative role of HERV transcription with regard to oncogenesis is currently unclear In conclusion, our observations provide negative evidence regarding the contested question of putative viral cofactors of metastatic neuroblastoma by suggesting that viruses are unlikely to be frequent cofactors in the maintenance of metastatic neuroblastoma.


The goal of this meeting was to mentor future Kenyan scientists and prioritize research topics that would lead to improved cancer care and survival for the citizens of Kenya.This is a summary of the 1 Medical Scientists drawn from the Academic Model Providing Access to Healthcare (AMPATH) Consortium collaborated with the Kenya Medical Research Institute (KEMRI) in Kisumu to host the first United States, National Cancer Institue (NCI) sponsored oncology-focused grant writing workshop. The goal of this workshop was to mentor health care researchers and to prioritize research topics that could lead to improved cancer care and survival for the citizens of Kenya. The spectrum of cancer research included prevention, early detection and screening as well as palliative care. Twenty mentees out of 34 applicants were selected through a competitive process in which submitted applications were reviewed based on research interests, how those interests could improve cancer care and outcomes, and how this training would facilitate their careers in medical research. Ideas for research proposals were stimulated by a series of Provocative Questions given to applicants prior to the meeting (Appendix 1).Kenya, like much of the developing world, is rapidly undergoing an ‘epidemiologic transition’ from a health scene dominated by infectious diseases to one in which the chronic diseases such as cancer are becoming major causes of death and disability. Under these circumstances, applying science to the management and control of cancer has become as relevant to Kenya as it is in the United States and other countries. Cancer research in Kenya, whose population is the most genetically diverse in the world , will cahttp://www.ampathkenya.org/). AMPATH opened its first clinics in 2001, at a time in which the AIDS epidemic dominated the health landscape of sub-Saharan Africa. The AMPATH model is a partnership between Moi University School of Medicine (MUSOM), Moi Teaching and Referral Hospital , and a consortium of U.S. and Canadian medical schools led by Indiana University School of Medicine. North American AMPATH consortium partners also include the Alpert Medical School of Brown University, Hubert-Yeargan Center for Global Health at Duke University Medical Center, Lehigh Valley Health Network of Pennsylvania, Eck Institute for Global Health at Notre Dame University, Providence Portland Medical Center, Purdue University, University of Massachusetts Medical School, University of Utah School of Medicine and University of Toronto Faculty of Medicine. Institutions participating in the AMPATH Consortium engage in student and faculty exchange, clinical care, training, and research, all essential for successfully addressing the short- and long-term challenges of global health. While training and research are critically important to the program, AMPATH has always been determined to “lead with care.” Care is not only the most pressing obligation when faced with the needs of an under-served population, it is also the foundation upon which training and research is conducted.AMPATH stands for the Academic Model for Providing Access to Healthcare . The goal of AOI is to be the premier cancer program in Sub-Saharan Africa, noted for excellence in cancer research, prevention, treatment and palliative care. And in order to meet this need, specialized training in oncology and oncology-related research is imperative.th to 18th, 2013 in Kisumu, Kenya which included doctors, nurses and other health-allied scientists affiliated with the Hemato-Oncology Department at the MTRH and AMPATH Oncology, located in Eldoret. Faculty mentors from the United States and Kenya dedicated three days to teaching participants about writing grant proposals for conducting research in oncology.A three-day symposium was conducted from January 16The most prevalent cancers seen at AOI are listed in Table The workshop was chaired by Drs. Ann Moormann and Jodi Skiles. Presenters at the workshop included Prof. Thomas Inui , who provided an overview of grant writing and how important it is to learn how to convey your research ideas, objectives and desired outcomes. Clearly explaining how research can translate into better care and survival for patients is an important aspect of successfully funded grant proposals.Dr. Stephen Munga  followed with a talk on “Conducting Health Services Research in Low Income Countries.” He stressed the importance of understanding local and regional determinants of health, knowledge of ongoing health research efforts – including how social factors such as family, community, and the central government play a role in contributing to health and health behaviors. He further emphasized that limited resources, inadequate infrastructure, the effects of “brain drain” and limited communication between researchers and policy makers pose great challenges to researchers in developing countries.Prof. Patrick Loehrer (Director of the IUSCC and the AOI), covered the basic tenets of writing a clinical trial. Another important aspect of entering into the research arena was presented by Prof. Anthony Mega  who delivered a lecture titled: “Mentorship: the art of choosing, using and becoming a mentor.” Mega said that a mentor should be a role model, an advisor, an encourager, a resource person, a counselor, sponsor and a friend. They ought to inspire, support and invest in their mentees. Research is often a team endeavor whereby scientists with different strengths bring their expertise to the table.Another key topic of interest “Integrating Translational Research into Clinical Research” was introduced by Prof. Alan Rosmarin . He opened the discussion by posing a question to participants about what barriers exist to implementing translational research in Kenya. The participants cited limited availability of laboratory technology available, academic silos within the government, and a lack of access to care for all citizens.While delivering a talk on Manuscript Writing, Prof. Ann Moormann  emphasized that upon getting results, the researcher must strive to tell their target audience what those results mean by interpreting the data and explaining how those results add to what is already known about the topic. Writing the manuscript should take high priority because it is the end product of research efforts and adds to the body of knowledge within the medical community. Another salient point highlighted was that publication costs for scientists in resource-limited settings may be waived or greatly reduced by many journals. She also mentioned the value of publishing in open-access journals which are available to your colleagues without a subscription fee.Other presenters included Prof. Edwin Were , who delivered a lecture on ethical considerations for conducting research in resource-constrained settings and Dr. Christine Wasunna,  who gave a talk on cultural considerations in community-based cancer research in Kenya. David Mulama, , presented a brief tutorial on data management and analysis, mentioning statistical programs that are often available as open source software. Prof. Greg Gramelspacher  shared his experience on palliative care research in low and middle-income countries. Providing comfort and relieving suffering for those diagnosed with cancer is a critically important aspect of cancer care.The morning presentations were followed by afternoon small group sessions where four to five mentees worked directly with two to three mentors. These sessions allowed the mentees to ask more specific questions about their research proposals and seek feedback from their peers. The evenings were free time for the mentees to work on their project presentations.The workshop format included short (10–15 minute) individual research proposal presentations from each of the mentees. After each participant presented their work, the proposals underwent a peer-review process. The three best proposals were selected by the other participants and given special recognition. This process was to simulate peer-review for grant applications whereby only the best proposals get selected for funding. This also allowed the mentees to understand how they could improve their research proposal by integrating constructive suggestions into their revisions.The concluding presentation was an inspiring talk by Dr. Kirtika Patel  who gave a brief overview of her research journey covering the key elements of making the transition from mentee to mentor. She urged the future researchers to exercise patience when writing their proposals, bearing in mind that persistence is a critical key to success.The long-term goal of this workshop is to continue to strengthen research partnerships between collaborators from various institutions that can be leveraged to apply for grant applications. This is being accomplished by continued mentor-mentee communication via the internet and weekly conference calls, helping to identify requests for grant applications that would be amenable to the situation in Kenya, and ongoing grant writing mentorship.Provocative Research QuestionsBreast Cancer● Is breast cancer in Kenya different from North American patient populations?● Why do younger women get breast cancer in Sub-Saharan Africa?● What obstacles to early detection of breast cancer can be overcome in Kenya?● How effective is standard chemotherapy or hormonal therapy in breast cancer in Kenyan women?● Are there associated risk factors for breast cancer in women of Kenya?Cervical Cancer● What are the Human Papillomavirus (HPV) serotypes for cervical cancer in Kenya?● What is the impact of antiretrovirals in patients diagnosed with and treated for cervical cancer?● Is single agent chemotherapy effective in advanced cervical cancer?● Are there regional differences in incidence and outcome of cervical cancer?Lymphoma● What are the most common histologies of non-Hodgkin lymphoma (NHL) in western Kenya?● How accurate is histologic identification of NHL in Kenya?● Will improved subclassification of NHL improve outcomes of lymphoma therapy in Kenya?● What is the safest and most effective treatment for NHL in resource constrained setting?Palliative % Supportive Care● What is the scope of pain management in Kenya and what opportunities exist to enhance it?● Do vitamin  supplements improve outcome when combined with chemotherapy?● What are public perception and beliefs of cancer?● What are obstacles to vaccine therapy in adolescent girls and boys in Kenya?● Can a culturally appropriate model to end-of-life communication be developed and implemented in western Kenya?Kaposi Sarcoma● Why is Kaposi sarcoma (KS) more prevalent in Kenya compared to US?● Why is severity and extent of KS worse in Kenya?● Can optimization of dermatologic care improve palliation of KS patients?● Is there a drug-drug interaction with antiretroviral therapy and chemotherapy in patients with KS?Pediatric Oncology● Is acute lymphoblastic leukemia (ALL) different based on cell phenotype or cytogenetics in Kenya patients compared to children in North America (NA)?● Is Wilm’s tumor in Kenya different compared to NA by protein profiling?● What obstacles to early detection of leukemia/lymphoma can be overcome in Kenya?● How effective is standard chemotherapy in ALL?● Is it feasible to utilize flow cytometry for the diagnosis of leukemia and lymphoma in Kenya?● What is the impact of antiretroviral therapy in patients diagnosed with NHL/Burkitt’s?● Are there regional/ethnic differences in incidence and outcome of leukemia?● What are the most common histologies of pediatric NHL in western Kenya?● How accurate is histologic identification of pediatric NHL in Kenya?● What is the safest and most effective treatment for pediatric NHL and ALL in a resource-limited setting?● What are the factors that contribute to abandonment of care in pediatric oncology patients in Kenya – and are they different than those seen in adults?● How can pediatric cancers  be identified and referred earlier for care?● Can a cost-effective sustainable model for cancer care be established in Kenya?● Why is there such a discrepancy in survival between pediatric ALL patients in North America and Kenya?● What are the main causes of mortality in the context of therapy for pediatric malignancies?Health behavior, health education topics● Why do many patients with potentially preventable conditions fail to present for cancer screening services within the AMPATH system of care.● Corollary question: Why do some AOI patients in care for malignancies drop out of care?Other topics● What are the Human Papilloma Virus (HPV) serotypes in head and neck cancer in Kenya?● What is prevalence of smoking and exposure to smoke (homes) in Kenya and association with cancers?● Can a cost-effective sustainable model for cancer care be established in Kenya?● What is the impact of economic barriers  in seeking and sustaining cancer care?● What financing model  is appropriate in Kenya?The authors state no conflict of interests.AMM and JLS drafted manuscript, organized meeting and mentored attendees. EK drafted manuscript and attended meeting. FCA and NB drafted manuscript and helped organized meeting. PL drafted manuscript, organized meeting and mentored attendees. All authors approved the final manuscript.


Imaging has played a leading role, including such techniques as contrast echocardiography,2 x‐ray angiography with measurement of myocardial perfusion grade,3 and contrast‐enhanced cardiac magnetic resonance (CMR) imaging.5fter acute reperfused myocardial infarction (AMI), there is a variable degree of microvascular damage due to reperfusion injury. This injury is multifactorial, and potential culprits include cellular inflammation (leukocytes and macrophages), reactive oxygen species, shifts in metabolic substrate, and alterations in mitochondrial and cytosolic calcium ion homeostasis.6 for the left ventricle (LV) over time because of adverse remodeling,7 and for the patient in terms of risk of morbidity and mortality.8 In addition, myocardial hemorrhage has been recognized as another marker of severe injury9 and can be imaged on T2‐weighted MRI as areas of low signal intensity. Yet another marker of prognosis by CMR is myocardial salvage index. Myocardial salvage index is calculated as the difference between the area at risk, identified by T2‐weighted (W) imaging as regions of bright signal consistent with myocardial edema,10 and infarct size, which is measured by late gadolinium enhancement.11 In a recent study of patients followed up for 6 months after AMI, myocardial salvage index was a powerful predictor of outcome, inasmuch as nearly all of the cardiac events occurred in patients with myocardial salvage index less than the median.12Markers of reperfusion injury on CMR include microvascular obstruction, which appears as regions of hypointense signal in the first few minutes after gadolinium contrast administration. CMR studies have demonstrated that microvascular obstruction is associated with poor outcome for the particular segment of myocardium in which it occurs,13 IMR is measured with a pressure sensor / thermistor–tipped guidewire and is calculated as mean distal coronary pressure × mean transit time of a 3‐cc saline bolus measured during adenosine hyperemia. Fearon et al13 demonstrated in 29 patients after primary percutaneous coronary intervention for AMI that IMR correlated significantly with 3‐month post–myocardial infarction echocardiographic wall motion score and was its strongest predictor on multivariate analysis. IMR also predicted recovery of regional LV function.In the past few years, a catheter‐based technique has been developed for assessment of microvascular injury in the setting of AMI—namely, the index of microcirculatory resistance (IMR).Journal of the American Heart Association (JAHA), Payne et al14 studied the use of this technique in a larger cohort (n=108) of post‐AMI patients and compared the results with CMR studies at 2 days after AMI and 3 months after AMI. The authors are to be congratulated on the completion of this tour‐de‐force, which involved a large number of patients and 2 follow‐up imaging studies, for which they had an 89% completion rate. They found that the measurement of IMR was extremely reproducible. IMR was a multivariate predictor of early myocardial salvage and predicted the presence and extent of microvascular obstruction and myocardial hemorrhage. In anterior myocardial infarction, IMR predicted late myocardial salvage. Thus, a catheter‐based measurement in AMI can accurately assess the extent of microvascular injury while percutaneous coronary intervention is being performed, which is a potential way to obviate the need for further imaging of microvascular injury. Still, the correlations with 3‐month ejection fraction and end‐systolic volumes were modest and, in fact, fell out in multivariate models because IMR correlated much better with baseline measures of acute microvascular injury on CMR. This could have been, at least in part, because the infarct sizes were modest and ejection fractions fairly preserved, and thus the subsequent LV remodeling, which is dependent on initial infarct size, was likewise modest.In this issue of the yes, given that measurement of Thrombolysis In Myocardial Infarction (TIMI) grade is an inadequate way of assessing microvascular function. Next, does IMR obviate performance of further imaging, such as CMR to assess the status of the microvasculature? This is a more difficult question to answer. Certainly, IMR adds to the understanding of myocardial physiology after reperfused myocardial infarction. One could ask, even though IMR correlated with the presence and extent of microvascular obstruction and myocardial hemorrhage, “Why not just perform the CMR and get a complete and definitive evaluation?” Certainly, cost and availability are issues with CMR. Not every hospital has CMR capability, and the additive cost of a study is not inconsequential. Payne et al14 contend in the introduction to their article that CMR is “usually performed up to 7 days after hospital admission in medically stabilized patients.” Perhaps that is true in Scotland. In the United States, hospital stays are generally on the order of 2 to 3 days after reperfused myocardial infarction, and thus CMR, if it is to be performed, must be obtained quite early on in the admission. It is safe to perform CMR even in the first 24 hours. Yet another question is, “Why was the correlation with subsequent LV remodeling less strong than the correlation with measures on the CMR at day 2?” What one would really like to have at the time of index evaluation is the ability to predict late outcomes over and above assessment of the current state of the myocardium.Several questions are raised by this interesting study. First, does the IMR measure add to what we already know at the time of intervention? The answer is a decided 15 but further studies have not been performed since that publication. A multitude of other pharmacological agents have fallen by the wayside. Does it make sense to examine the microvasculature after AMI if we have no proven therapy to improve microvascular function and thus change subsequent LV remodeling and outcome? As it stands now, all post‐AMI patients leave the hospital on 5 drugs, including aspirin, a statin, a β‐blocker, an angiotensin‐converting enzyme inhibitor, and clopidogrel—the latter if they received a stent as part of the percutaneous coronary intervention. No adjustments are made on the basis of the status of the microvasculature because these therapies are considered standard‐of‐care, and hospitals are monitored for adherence to this post‐AMI medical regimen. Until such time as we have specific therapies for microvascular injury, it might not make sense to look for it, even though we have accurate ways to assess it with CMR and now IMR. Although the physiology is certainly of great interest, no method of assessing microvascular function after reperfused AMI has reached the current high bar of practicality and clinical utility.The larger question is, “In which patients should microvascular function be assessed after reperfused AMI?” The issue here is that despite exhaustive studies over the past 3 decades, little progress has been made in mitigating reperfusion injury. Intravenous adenosine had some promise in that regard in the Acute Myocardial Infarction study of Adenosine (AMISTAD‐II), in which infarct size was reduced with the higher dose and trends toward benefit were seen in larger anterior infarcts,