Discovery of insect and human dengue virus host factors.

Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world's population at risk of infection. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat. Given their compact genomes, dengue viruses (DENV-1-4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and alpha-glucosidases), most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host.

Using Rainfall and Temperature Data in the Evaluation of National Malaria Control Programs in Africa.

Since 2010, the Roll Back Malaria (RBM) Partnership, including National Malaria Control Programs, donor agencies (e.g., President's Malaria Initiative and Global Fund), and other stakeholders have been evaluating the impact of scaling up malaria control interventions on all-cause under-five mortality in several countries in sub-Saharan Africa. The evaluation framework assesses whether the deployed interventions have had an impact on malaria morbidity and mortality and requires consideration of potential nonintervention influencers of transmission, such as drought/floods or higher temperatures. Herein, we assess the likely effect of climate on the assessment of the impact malaria interventions in 10 priority countries/regions in eastern, western, and southern Africa for the President's Malaria Initiative. We used newly available quality controlled Enhanced National Climate Services rainfall and temperature products as well as global climate products to investigate likely impacts of climate on malaria evaluations and test the assumption that changing the baseline period can significantly impact on the influence of climate in the assessment of interventions. Based on current baseline periods used in national malaria impact assessments, we identify three countries/regions where current evaluations may overestimate the impact of interventions (Tanzania, Zanzibar, Uganda) and three countries where current malaria evaluations may underestimate the impact of interventions (Mali, Senegal and Ethiopia). In four countries (Rwanda, Malawi, Mozambique, and Angola) there was no strong difference in climate suitability for malaria in the pre- and post-intervention period. In part, this may be due to data quality and analysis issues.

Impact of Insecticide-Treated Net Ownership on All-Cause Child Mortality in Malawi, 2006-2010.

Insecticide-treated nets (ITNs) have been shown to be highly effective at reducing malaria morbidity and mortality in children. However, there are limited studies that assess the association between increasing ITN coverage and child mortality over time, at the national level, and under programmatic conditions. Two analytic approaches were used to examine this association: a retrospective cohort analysis of individual children and a district-level ecologic analysis. To evaluate the association between household ITN ownership and all-cause child mortality (ACCM) at the individual level, data from the 2010 Demographic and Health Survey (DHS) were modeled in a Cox proportional hazards framework while controlling for numerous environmental, household, and individual confounders through the use of exact matching. To evaluate population-level association between ITN ownership and ACCM between 2006 and 2010, program ITN distribution data and mortality data from the 2006 Multiple Indicator Cluster Survey and the 2010 DHS were aggregated at the district level and modeled using negative binomial regression. In the Cox model controlling for household, child and maternal health factors, children between 1 and 59 months in households owning an ITN had significantly lower mortality compared with those without an ITN (hazard ratio = 0.75, 95% confidence interval [CI] = 0.62-90). In the district-level model, higher ITN ownership was significantly associated with lower ACCM (incidence rate ratio = 0.77; 95% CI = 0.60-0.98). These findings suggest that increasing ITN ownership may have contributed to the decline in ACCM during 2006-2010 in Malawi and represent a novel use of district-level data from nationally representative surveys.

Malaria Control Interventions Contributed to Declines in Malaria Parasitemia, Severe Anemia, and All-Cause Mortality in Children Less Than 5 Years of Age in Malawi, 2000-2010.

Malaria control intervention coverage increased nationwide in Malawi during 2000-2010. Trends in intervention coverage were assessed against trends in malaria parasite prevalence, severe anemia (hemoglobin < 8 g/dL), and all-cause mortality in children under 5 years of age (ACCM) using nationally representative household surveys. Associations between insecticide-treated net (ITN) ownership, malaria morbidity, and ACCM were also assessed. Household ITN ownership increased from 27.4% (95% confidence interval [CI] = 25.9-29.0) in 2004 to 56.8% (95% CI = 55.6-58.1) in 2010. Similarly intermittent preventive treatment during pregnancy coverage increased from 28.2% (95% CI = 26.7-29.8) in 2000 to 55.0% (95% CI = 53.4-56.6) in 2010. Malaria parasite prevalence decreased significantly from 60.5% (95% CI = 53.0-68.0) in 2001 to 20.4% (95% CI = 15.7-25.1) in 2009 in children aged 6-35 months. Severe anemia prevalence decreased from 20.4% (95% CI: 17.3-24.0) in 2004 to 13.1% (95% CI = 11.0-15.4) in 2010 in children aged 6-23 months. ACCM decreased 41%, from 188.6 deaths per 1,000 live births (95% CI = 179.1-198.0) during 1996-2000, to 112.1 deaths per 1,000 live births (95% CI = 105.8-118.5) during 2006-2010. When controlling for other covariates in random effects logistic regression models, household ITN ownership was protective against malaria parasitemia in children (odds ratio [OR] = 0.81, 95% CI = 0.72-0.92) and severe anemia (OR = 0.82, 95% CI = 0.72-0.94). After considering the magnitude of changes in malaria intervention coverage and nonmalaria factors, and given the contribution of malaria to all-cause mortality in malaria-endemic countries, the substantial increase in malaria control interventions likely improved child survival in Malawi during 2000-2010.

Framework for Evaluating the Health Impact of the Scale-Up of Malaria Control Interventions on All-Cause Child Mortality in Sub-Saharan Africa.

Concerted efforts from national and international partners have scaled up malaria control interventions, including insecticide-treated nets, indoor residual spraying, diagnostics, prompt and effective treatment of malaria cases, and intermittent preventive treatment during pregnancy in sub-Saharan Africa (SSA). This scale-up warrants an assessment of its health impact to guide future efforts and investments; however, measuring malaria-specific mortality and the overall impact of malaria control interventions remains challenging. In 2007, Roll Back Malaria's Monitoring and Evaluation Reference Group proposed a theoretical framework for evaluating the impact of full-coverage malaria control interventions on morbidity and mortality in high-burden SSA countries. Recently, several evaluations have contributed new ideas and lessons to strengthen this plausibility design. This paper harnesses that new evaluation experience to expand the framework, with additional features, such as stratification, to examine subgroups most likely to experience improvement if control programs are working; the use of a national platform framework; and analysis of complete birth histories from national household surveys. The refined framework has shown that, despite persisting data challenges, combining multiple sources of data, considering potential contributions from both fundamental and proximate contextual factors, and conducting subnational analyses allows identification of the plausible contributions of malaria control interventions on malaria morbidity and mortality.

Implementing Impact Evaluations of Malaria Control Interventions: Process, Lessons Learned, and Recommendations.

As funding for malaria control increased considerably over the past 10 years resulting in the expanded coverage of malaria control interventions, so did the need to measure the impact of these investments on malaria morbidity and mortality. Members of the Roll Back Malaria (RBM) Partnership undertook impact evaluations of malaria control programs at a time when there was little guidance in terms of the process for conducting an impact evaluation of a national-level malaria control program. The President's Malaria Initiative (PMI), as a member of the RBM Partnership, has provided financial and technical support for impact evaluations in 13 countries to date. On the basis of these experiences, PMI and its partners have developed a streamlined process for conducting the evaluations with a set of lessons learned and recommendations. Chief among these are: to ensure country ownership and involvement in the evaluations; to engage stakeholders throughout the process; to coordinate evaluations among interested partners to avoid duplication of efforts; to tailor the evaluation to the particular country context; to develop a standard methodology for the evaluations and a streamlined process for completion within a reasonable time; and to develop tailored dissemination products on the evaluation for a broad range of stakeholders. These key lessons learned and resulting recommendations will guide future impact evaluations of malaria control programs and other health programs.

Transcriptional regulation of the melanoma prognostic marker melastatin (TRPM1) by MITF in melanocytes and melanoma.

Determining the metastatic potential of intermediate thickness lesions remains a major challenge in the management of melanoma. Clinical studies have demonstrated that expression of melastatin/TRPM1 strongly predicts nonmetastatic propensity and correlates with improved outcome, leading to a national cooperative prospective study, which is ongoing currently. Similarly, the melanocytic markers MLANA/MART1 and MITF also have been shown to lose relative expression during melanoma progression. Recent studies have revealed that MITF, an essential transcription factor for melanocyte development, directly regulates expression of MLANA. This prompted examination of whether MITF also might transcriptionally regulate TRPM1 expression. The TRPM1 promoter contains multiple MITF consensus binding elements that were seen by chromatin immunoprecipitation to be occupied by endogenous MITF within melanoma cells. Endogenous TRPM1 expression responded strongly to MITF up- or down-regulation, as did TRPM1 promoter-driven reporters. In addition, MITF and TRPM1 mRNA levels were correlated tightly across a series of human melanoma cell lines. Mice homozygously mutated in MITF showed a dramatic decrease in TRPM1 expression. Finally, the slope of TRPM1 induction by MITF was particularly steep compared with other MITF target genes, suggesting it is a sensitive indicator of MITF expression and correspondingly of melanocytic differentiation. These studies identify MITF as a major transcriptional regulator of TRPM1 and suggest that its prognostic value may be linked to MITF-mediated regulation of cellular differentiation.

Genomic analysis of the Microphthalmia locus and identification of the MITF-J/Mitf-J isoform.

The deafness-pigmentary disorder Waardenburg Syndrome Type 2 is caused by mutations in the human Microphthalmia-associated transcription factor (MITF) gene. Multiple related deafness-pigmentary disorders result from mutations in genes that regulate MITF expression or its activity. Similarly in mouse, homozygous mutations in the Mitf gene disrupt the development of melanocytes as well as retinal pigment epithelial (RPE) cells, osteoclasts, mast cells, and NK cells. Because abnormalities in Mitf/MITF function are associated with numerous inherited disorders of mouse and man, a detailed understanding of its gene structure is important for both diagnostic and structure/function analyses. While at least eight distinct isoforms of MITF/Mitf have been identified to date, each differing in their promoter and initial exon usage, the positions of these exons and their order within the locus have yet to be fully defined. In this study, we provide a detailed description of the MITF/Mitf locus, identify corresponding human and mouse isoforms, and utilize an informatics-based approach to identify a novel ninth MITF/Mitf isoform, MITF-J/Mitf-J, which we show is expressed in multiple cell types. The MITF/Mitf locus is over 200 kb in length, with strong but imperfect exon conservation between human and mouse. MITF/Mitf tissue expression data are presented from multiple datasets, including EST expression patterns and isoform-specific RT-PCR. The majority of isoforms were found to be broadly expressed, with the M- and Mc-isoforms being tissue-restricted to melanocytes and mast cells, respectively. Consequently, a detailed characterization of this complex locus may help to identify additional unknown deafness-pigmentary syndrome mutations in human kindred and permit a better understanding of tissue-regulated expression that likely underlies divergent biological functions of this factor across multiple cell types.

Impact of Malaria Control on Mortality and Anemia among Tanzanian Children Less than Five Years of Age, 1999-2010.

BACKGROUND: Mainland Tanzania scaled up multiple malaria control interventions between 1999 and 2010. We evaluated whether, and to what extent, reductions in all-cause under-five child mortality (U5CM) tracked with malaria control intensification during this period. METHODS: Four nationally representative household surveys permitted trend analysis for malaria intervention coverage, severe anemia (hemoglobin <8 g/dL) prevalence (SAP) among children 6-59 months, and U5CM rates stratified by background characteristics, age, and malaria endemicity. Prevalence of contextual factors (e.g., vaccination, nutrition) likely to influence U5CM were also assessed. Population attributable risk percentage (PAR%) estimates for malaria interventions and contextual factors that changed over time were used to estimate magnitude of impact on U5CM. RESULTS: Household ownership of insecticide-treated nets (ITNs) rose from near zero in 1999 to 64% (95% CI, 61.7-65.2) in 2010. Intermittent preventive treatment of malaria in pregnancy reached 26% (95% CI, 23.6-28.0) by 2010. Sulfadoxine-pyrimethamine replaced chloroquine in 2002 and artemisinin-based combination therapy was introduced in 2007. SAP among children 6-59 months declined 50% between 2005 (11.1%; 95% CI, 10.0-12.3%) and 2010 (5.5%; 95% CI, 4.7-6.4%) and U5CM declined by 45% between baseline (1995-9) and endpoint (2005-9), from 148 to 81 deaths/1000 live births, respectively. Mortality declined 55% among children 1-23 months of age in higher malaria endemicity areas. A large reduction in U5CM was attributable to ITNs (PAR% = 11) with other malaria interventions adding further gains. Multiple contextual factors also contributed to survival gains. CONCLUSION: Marked declines in U5CM occurred in Tanzania between 1999 and 2010 with high impact from ITNs and ACTs. High-risk children (1-24 months of age in high malaria endemicity) experienced the greatest declines in mortality and SAP. Malaria control should remain a policy priority to sustain and further accelerate progress in child survival.

Mitf and Tfe3: members of a b-HLH-ZIP transcription factor family essential for osteoclast development and function.

The Microphthalmia-associated transcription factor (Mitf) is required for the proper development of several cell lineages including osteoclasts, melanocytes, retinal pigment epithelial cells, mast cells and natural killer cells. Mutations in Mitf in multiple organisms result in osteopetrosis due to defective osteoclast development. Mitf is a member of the basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) transcription factor subfamily named MiT, which also includes Tfe3. Genetic evidence indicates that Mitf and Tfe3 carry out essential functions in osteoclast development. Mitf has been shown to reside downstream of the macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) signaling pathways that are critical for osteoclast proliferation, differentiation and function. Mitf and Tfe3 have been shown to regulate the expression of several target genes necessary for bone degradation by mature osteoclasts. Here, we review the bone and osteoclast phenotypes of animals with mutations in Mitf and Tfe3, Mitf's interaction partners and signaling pathways, and known target genes which, together with others yet to be identified, likely represent key effectors of bone resorption.

Incidence and risk factors for Malaria, pneumonia and diarrhea in children under 5 in UNHCR refugee camps: A retrospective study.

BACKGROUND: United Nations High Commissioner for Refugees (UNHCR) refugee camps are located predominantly in rural areas of Africa and Asia in protracted or post-emergency contexts. Recognizing the importance of malaria, pneumonia and diarrheal diseases as major causes of child morbidity and mortality in refugee camps, we analyzed data from the UNHCR Health Information System (HIS) to estimate incidence and risk factors for these diseases in refugee children younger than five years of age. METHODS: Data from 90 UNHCR camps in 16 countries, including morbidity, mortality, health services and refugee health status, were obtained from the UNHCR HIS for the period January 2006 to February 2010. Monthly camp-level data were aggregated to yearly estimates for analysis and stratified by location in Africa (including Yemen) or Asia. Poisson regression models with random effects were constructed to identify factors associated with malaria, pneumonia and diarrheal diseases. Spatial patterns in the incidence of malaria, pneumonia and diarrheal diseases were mapped to identify regional heterogeneities. RESULTS: Malaria and pneumonia were the two most common causes of mortality, with confirmed malaria and pneumonia each accounting for 20% of child deaths. Suspected and confirmed malaria accounted for 23% of child morbidity and pneumonia accounted for 17% of child morbidity. Diarrheal diseases were the cause of 7% of deaths and 10% of morbidity in children under five. Mean under-five incidence rates across all refugee camps by region were: malaria [Africa 84.7 cases/1000 U5 population/month (95% CI 67.5-102.0), Asia 2.2/1000/month (95% CI 1.4-3.0)]; pneumonia [Africa 59.2/1000/month (95% CI 49.8-68.7), Asia 254.5/1000/month (95% CI 207.1-301.8)]; and diarrheal disease [Africa 35.5/1000/month (95% CI 28.7-42.4), Asia 69.2/1000/month (95% CI 61.0-77.5)]. Measles was infrequent and accounted for a small proportion of child morbidity (503 cases, < 1%) and mortality (6 deaths, < 1%). CONCLUSIONS: As in stable settings, pneumonia and diarrhea are important causes of mortality among refugee children. Malaria remains a significant cause of child mortality in refugee camps in Africa and will need to be addressed as part of regional malaria control and elimination efforts. Little is known of neonatal morbidity and mortality in refugee settings, and neonatal deaths are likely to be under-reported. Global measles control efforts have reduced the incidence of measles among refugee children.