Scaling-up co-trimoxazole prophylaxis in HIV-exposed and HIV-infected children in high HIV-prevalence countries.

Co-trimoxazole (trimethoprim-sulfamethoxazole) is a widely available antibiotic that substantially reduces HIV-related morbidity and mortality in both adults and children. Prophylaxis with co-trimoxazole is a recommended intervention of proven benefit that could serve not only as an initial step towards improving paediatric care in young children with limited access to antiretroviral treatment, but also as an important complement to antiretroviral therapy in resource-limited settings. Despite co-trimoxazole's known clinical benefits, the potential operational benefits, and favourable recommendations by WHO, UNAIDS, and UNICEF, its routine use in developing countries--particularly sub-Saharan Africa--has remained limited. Out of an estimated 4 million children in need of co-trimoxazole prophylaxis (HIV-exposed and HIV-infected), only 4% are currently receiving this intervention. We discuss some of the major barriers preventing the scale-up of co-trimoxazole prophylaxis for children in countries with a high prevalence of HIV and propose specific actions required to tackle these challenges

Treatment outcomes of new tuberculosis patients hospitalized in Kampala, Uganda: a prospective cohort study.

BACKGROUND: In most resource limited settings, new tuberculosis (TB) patients are usually treated as outpatients. We sought to investigate the reasons for hospitalisation and the predictors of poor treatment outcomes and mortality in a cohort of hospitalized new TB patients in Kampala, Uganda. METHODS AND FINDINGS: Ninety-six new TB patients hospitalised between 2003 and 2006 were enrolled and followed for two years. Thirty two were HIV-uninfected and 64 were HIV-infected. Among the HIV-uninfected, the commonest reasons for hospitalization were low Karnofsky score (47%) and need for diagnostic evaluation (25%). HIV-infected patients were commonly hospitalized due to low Karnofsky score (72%), concurrent illness (16%) and diagnostic evaluation (14%). Eleven HIV uninfected patients died (mortality rate 19.7 per 100 person-years) while 41 deaths occurred among the HIV-infected patients (mortality rate 46.9 per 100 person years). In all patients an unsuccessful treatment outcome (treatment failure, death during the treatment period or an unknown outcome) was associated with duration of TB symptoms, with the odds of an unsuccessful outcome decreasing with increasing duration. Among HIV-infected patients, an unsuccessful treatment outcome was also associated with male sex (P = 0.004) and age (P = 0.034). Low Karnofsky score (aHR = 8.93, 95% CI 1.88 - 42.40, P = 0.001) was the only factor significantly associated with mortality among the HIV-uninfected. Mortality among the HIV-infected was associated with the composite variable of CD4 and ART use, with patients with baseline CD4 below 200 cells/µL who were not on ART at a greater risk of death than those who were on ART, and low Karnofsky score (aHR = 2.02, 95% CI 1.02 - 4.01, P = 0.045). CONCLUSION: Poor health status is a common cause of hospitalisation for new TB patients. Mortality in this study was very high and associated with advanced HIV Disease and no use of ART

Operational research in Malawi: making a difference with cotrimoxazole preventive therapy in patients with tuberculosis and HIV.

BACKGROUND: In Malawi, high case fatality rates in patients with tuberculosis, who were also co-infected with HIV, and high early death rates in people living with HIV during the initiation of antiretroviral treatment (ART) adversely impacted on treatment outcomes for the national tuberculosis and ART programmes respectively. This article i) discusses the operational research that was conducted in the country on cotrimoxazole preventive therapy, ii) outlines the steps that were taken to translate these findings into national policy and practice, iii) shows how the implementation of cotrimoxazole preventive therapy for both TB patients and HIV-infected patients starting ART was associated with reduced death rates, and iv) highlights lessons that can be learnt for other settings and interventions. DISCUSSION: District and facility-based operational research was undertaken between 1999 and 2005 to assess the effectiveness of cotrimoxazole preventive therapy in reducing death rates in TB patients and subsequently in patients starting ART under routine programme conditions. Studies demonstrated significant reductions in case fatality in HIV-infected TB patients receiving cotrimoxazole and in HIV-infected patients about to start ART. Following the completion of research, the findings were rapidly disseminated nationally at stakeholder meetings convened by the Ministry of Health and internationally through conferences and peer-reviewed scientific publications. The Ministry of Health made policy changes based on the available evidence, following which there was countrywide distribution of the updated policy and guidelines. Policy was rapidly moved to practice with the development of monitoring tools, drug procurement and training packages. National programme performance improved which showed a significant decrease in case fatality rates in TB patients as well as a reduction in early death in people with HIV starting ART. SUMMARY: Key lessons for moving this research endeavour through to policy and practice were the importance of placing operational research within the programme, defining relevant questions, obtaining "buy-in" from national programme staff at the beginning of projects and having key actors or "policy entrepreneurs" to push forward the policy-making process. Ultimately, any change in policy and practice has to benefit patients, and the ultimate judge of success is whether treatment outcomes improve or not

Assessment of Minimal Residual Disease in Standard-Risk AML

BACKGROUND: Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission. METHODS: We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction. RESULTS: Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status. CONCLUSIONS: The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.)

Outcomes of allogeneic stem cell transplantation among patients with acute myeloid leukemia presenting active disease: Experience of a single European Comprehensive Cancer Center

Introduction: Allogeneic hematopoietic stem cell transplantation (ASCT) represents a potentially curative approach for patients with relapsed or refractory acute myeloid leukemia (AML). We report the outcome of relapsed/refractory AML patients treated with ASCT.Method: A retrospective cohort from 1994 to 2013 that included 61 patients with diagnosis of relapsed/refractory AML. Outcomes of interest were transplant-related mortality (TRM), incidence of acute and chronic graft-versus-host disease (GVHD), relapse incidence, progression-free survival (PFS) and overall survival (OS). Statistical significance was set at p<0.05.Results: The median age was 61 years (range 1 to 65). The cumulative incidence of 90 days, 1 year, and 3 years TRM were 60%, 26.7%, and 13.3%, respectively (p< 0.001). The incidence of relapse was 21.7% at 1 year, 13% at 3 years, and 8.7% at 5 years. Median OS was estimated to be 8 months (95CI 3.266-12.734) and median PFS, 3 months (95CI 1.835-4.165).Conclusion: In our cohort, TRM in first years after ASCT remains considerable, but ASCT in this setting seems to be a good choice for AML patients with active disease. However, novel approaches are needed to reduce TRM and relapse in this set of patients

Autocrine Activation of the MET Receptor Tyrosine Kinase in Acute Myeloid Leukemia

Although the treatment of acute myeloid leukemia (AML) has improved significantly, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified aberrant expression of the hepatocyte growth factor (HGF) as a critical factor in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance due to compensatory upregulation of HGF expression, leading to restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked compensatory HGF upregulation, resulting in sustained logarithmic cell kill both in vitro and in xenograft models in vivo. Our results demonstrate widespread dependence of AML cells on autocrine activation of MET, as well as the importance of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers

Identification and functional characterisation of a locus for target site integration in Fusarium graminearum

Background Fusarium Head Blight (FHB) is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusarium graminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists of identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration. Results Here, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities of Fg native promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations, cassette truncations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1. Conclusion The TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, protein secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work together with PCR amplification of the insert could be an alternative to Southern blotting, the gold standard technique used to identify ectopic integrations, cassette truncations and tandem insertions in fungal transformation

Bone marrow transplantation in AML, and socioeconomic class: a UK population-based cohort study

<p>Abstract</p> <p>Background</p> <p>We have previously shown that in the UK mortality in people with Acute Myeloid Leukaemia (AML) was nearly 50% greater among the most socio-economically deprived. The aim of this study was to determine whether AML patients from lower socioeconomic classes had a lower chance of receiving a bone marrow transplant.</p> <p>Methods</p> <p>Using Hospital Episode Statistics (HES) data, we identified all incident cases of AML admitted to UK hospitals between 1998 and 2007. We calculated the number of bone marrow transplantations undertaken in AML patients, stratifying our results by gender, age at diagnosis, year of diagnosis, degree of socioeconomic deprivation and co-morbidity. We used logistic regression to calculate odds ratios for bone marrow transplantation, adjusting for gender, age at diagnosis, year of diagnosis, degree of socioeconomic deprivation and co-morbidity score.</p> <p>Results</p> <p>We identified a total of 23 910 incident cases of AML over this 10-year time period, of whom 1 140 (4.8%) underwent BMT. Bone marrow transplantation declined with increasing socioeconomic deprivation (p for trend < 0.001) such that people in the most deprived socioeconomic quintile were 40% less likely to have a transplant than those in the most advantaged group (Odds Ratio 0.60, 95% confidence interval 0.49, 0.73), even after adjusting for gender, age at diagnosis, year of diagnosis and co-morbidity.</p> <p>Conclusion</p> <p>This large cohort study demonstrates that AML patients from lower socioeconomic classes are less likely to undergo bone marrow transplantation than their better off counter-parts.</p

Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia

In acute myeloid leukemia (AML) quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC) with treatment-resistance. LSC in CD34+ and more mature CD34- AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC) but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS) profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS). In control BM (n = 24), ROS levels were highest in granulocyte-macrophage progenitors (GMP) and CD34- myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12). Erythroid CD34- precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93) and MDS-with-excess-blasts (MDS-RAEB) (n = 14), immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34- SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs) but also in CD34- AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.</p

Molecular Pathogenesis of Secondary Acute Promyelocytic Leukemia

Balanced chromosomal translocations that generate chimeric oncoproteins are considered to be initiating lesions in the pathogenesis of acute myeloid leukemia. The most frequent is the t(15;17)(q22;q21), which fuses the PML and RARA genes, giving rise to acute promyelocytic leukemia (APL). An increasing proportion of APL cases are therapy-related (t-APL), which develop following exposure to radiotherapy and/or chemotherapeutic agents that target DNA topoisomerase II (topoII), particularly mitoxantrone and epirubicin. To gain insights into molecular mechanisms underlying the formation of the t(15;17) we mapped the translocation breakpoints in a series of t-APLs, which revealed significant clustering according to the nature of the drug exposure. Remarkably, in approximately half of t-APL cases arising following mitoxantrone treatment for breast cancer or multiple sclerosis, the chromosome 15 breakpoint fell within an 8-bp “hotspot” region in PML intron 6, which was confirmed to be a preferential site of topoII-mediated DNA cleavage induced by mitoxantrone. Chromosome 15 breakpoints falling outside the “hotspot”, and the corresponding RARA breakpoints were also shown to be functional topoII cleavage sites. The observation that particular regions of the PML and RARA loci are susceptible to topoII-mediated DNA damage induced by epirubicin and mitoxantrone may underlie the propensity of these agents to cause APL