Quantification of Bt δ-endotoxins in leaf tissues of tropical Bt maize populations

In Kenya, stem borers destroy an estimated 13.5% of farmers' annual maize harvest. Maize transformed using Bacillus thuringiensis (Bt) derived genes controls stem borers without negative effects to humans, livestock or the environment. The effectiveness and sustainability of Bt transgenic technology in the control of stem borers depends on the levels of concentration of the Bt δ-endotoxins in plant tissues. Kenya introduced Bt maize events to test the efficacy of Bt maize in controlling stem borers, and to develop high-yielding and locally adapted Bt maize germplasm for farmers. The objective of this study was to assess under greenhouse conditions the concentration levels of Bt δ-endotoxins in the leaf tissues of the parents, the F1, and the F2:3 populations of tropical maize, as a measure of stability and sustainability. Kenya introduced Bt maize events to test the efficacy of Bt maize in controlling stem borers, and to develop high-yielding and locally adapted Bt maize germplasm for farmers. The objective of this study was to assess under greenhouse conditions the concentration levels of Bt δ-endotoxins in the leaf tissues of the parents, the F1, and the F2:3 populations of tropical maize, as a measure of stability and sustainability. Two public Bt maize lines (Event 216 and Event 223) containing the cry1Ab::ubi gene were crossed with two non-Bt maize inbred lines, CML144 and CML159, to assess how the concentrations of Bt δ-endotoxins are transmitted from parents to F1 and to F2 generations. The mean concentration of Bt δ-endotoxins (μg/g) was 4.93 and 4.63 in Events 216 and 223 respectively. As expected, F1 generations of all the crosses had similar concentrations of Bt δ-endotoxins. However, the F2 generations showed a spread of concentrations. These findings may imply that genotypes with a higher mean concentration of Bt δ-endotoxins also have a lower level of plant damage traits expressed. In addition, these observations indicate that the cry1Ab gene was dominant and was inherited following the Mendelian segregation and that Events 216 and 223 could be utilized as reliable sources of resistance to stem borers in maize breeding programmes.Keywords: Bt maize, stem borers, Bt δ-endotoxins, enzyme-linked immunosorbent assay (ELISA), dot blot analysis, cry1A

Testing public Bt maize events for control of stem borers in the first confined field trials in Kenya

Transgenic maize (Zea mays L), developed using modified genes from the bacterium Bacillus thuringiensis (Bt), controls stem borers without observable negative effects to humans, livestock or the environment, and is now sown on 134 million hectares globally. Bt maize could contribute to increasing maize production in Kenya. Nine public Bt maize events of cry1Ab and cry1Ba genes were tested in confined field trials site (CFTs) to assess the control of four major Kenyan stem borer species. Leaf damage rating, number of exit holes and tunnel length were scored in the field evaluations. Leaf area consumed and mortality rates among stem borers were scored in the leaf bioassays in a Biosafety Level II laboratory, located at the Kenya Agricultural Research Institute (KARI), National Agricultural Research Laboratories (NARL). Field evaluations showed that Bt maize controlled Chilo partellus with mean damage scores of 1.2 against 2.7 for the non-Bt CML216 control. Laboratory bioassays showed high control for Eldana saccharina and Sesamia calamistis, with mean larval mortality of 64 and 92%, respectively. However, substantial control was not observed for Busseola fusca. These results showed that Bt maize could control three of the four major stem borers in Kenya with mortality records of 52.7% for B. fusca, 62.3% for E. saccharina and 85.8% for S. calamistis. Additional Bt genes need to be sought and tested for effective stem borer control in all maize growing ecologies in Kenya.Key words: Maize, Bt, stem borers, confined field trials

Post-harvest evaluation of selected hybrids to maize weevil Sitophilus zeamais resistance

Sitophilus zeamais has been identified as one of the most destructive pests of maize stored in tropical regions. While most maize hybrids are being developed, it is necessary to evaluate their resistance to this pest. This study determined the resistance of selected maize hybrids to infestation by S. zeamais. Twenty two hybrids with varying resistance to weevil infestation and two checks DUMA 41-suceptible and MTP0701-resistant were used in a randomized complete block design experiment. Assessment was done at 10, 60 and 120 days of maize storage. Data was collected on percent weevil damage, grain weight loss and number of live and dead weevils. Heritability and correlation of factors were also estimated. Analysis of variance showed significant differences (P ≤ 0.05) on weight loss. The selection of the resistant genotypes was based on percent weight loss after 60 days. KH631Q and PH4 were selected as the most resistant and moderately resistant hybrids, respectively. The resistant check MTPO701 was also found to maintain resistance to weevil attack. There was a strong positive correlation between weight loss, number of live weevils and percent damage. Moderate heritability estimates of hybrids at 60 days of storage indicated the possibility of their parents to transfer the desirable traits to subsequent generations. Therefore, parents of the resistant hybrids could be utilized in breeding programs for maize weevil resistance and be deployed to farmers for use, respectively

Testing public Bt maize events for control of stem borers in the first confined field trials in Kenya

Transgenic maize (Zea mays L), developed using modified genes from the bacterium Bacillus thuringiensis (Bt), controls stem borers without observable negative effects to humans, livestock or the environment, and is now sown on 134 million hectares globally. Bt maize could contribute to increasing maize production in Kenya. Nine public Bt maize events of cry1Ab and cry1Ba genes were tested in confined field trials site (CFTs) to assess the control of four major Kenyan stem borer species. Leaf damage rating, number of exit holes and tunnel length were scored in the field evaluations. Leaf area consumed and mortality rates among stem borers were scored in the leaf bioassays in a Biosafety Level II laboratory, located at the Kenya Agricultural Research Institute (KARI), National Agricultural Research Laboratories (NARL). Field evaluations showed that Bt maize controlled Chilo partellus with mean damage scores of 1.2 against 2.7 for the non-Bt CML216 control. Laboratory bioassays showed high control for Eldana saccharina and Sesamia calamistis, with mean larval mortality of 64 and 92%, respectively. However, substantial control was not observed for Busseola fusca. These results showed that Bt maize could control three of the four major stem borers in Kenya with mortality records of 52.7% for B. fusca, 62.3% for E. saccharina and 85.8% for S. calamistis. Additional Bt genes need to be sought and tested for effective stem borer control in all maize growing ecologies in Kenya

The cost‐effectiveness of prophylaxis strategies for individuals with advanced HIV starting treatment in Africa

Introduction Many HIV‐positive individuals in Africa have advanced disease when initiating antiretroviral therapy (ART) so have high risks of opportunistic infections and death. The REALITY trial found that an enhanced‐prophylaxis package including fluconazole reduced mortality by 27% in individuals starting ART with CD4 <100 cells/mm3. We investigated the cost‐effectiveness of this enhanced‐prophylaxis package versus other strategies, including using cryptococcal antigen (CrAg) testing, in individuals with CD4 <200 cells/mm3 or <100 cells/mm3 at ART initiation and all individuals regardless of CD4 count. Methods The REALITY trial enrolled from June 2013 to April 2015. A decision‐analytic model was developed to estimate the cost‐effectiveness of six management strategies in individuals initiating ART in the REALITY trial countries. Strategies included standard‐prophylaxis, enhanced‐prophylaxis, standard‐prophylaxis with fluconazole; and three CrAg testing strategies, the first stratifying individuals to enhanced‐prophylaxis (CrAg‐positive) or standard‐prophylaxis (CrAg‐negative), the second to enhanced‐prophylaxis (CrAg‐positive) or enhanced‐prophylaxis without fluconazole (CrAg‐negative) and the third to standard‐prophylaxis with fluconazole (CrAg‐positive) or without fluconazole (CrAg‐negative). The model estimated costs, life‐years and quality‐adjusted life‐years (QALY) over 48 weeks using three competing mortality risks: cryptococcal meningitis; tuberculosis, serious bacterial infection or other known cause; and unknown cause. Results Enhanced‐prophylaxis was cost‐effective at cost‐effectiveness thresholds of US$300 and US$500 per QALY with an incremental cost‐effectiveness ratio (ICER) of US$157 per QALY in the CD4 <200 cells/mm3 population providing enhanced‐prophylaxis components are sourced at lowest available prices. The ICER reduced in more severely immunosuppressed individuals (US$113 per QALY in the CD4 <100 cells/mm3 population) and increased in all individuals regardless of CD4 count (US$722 per QALY). Results were sensitive to prices of the enhanced‐prophylaxis components. Enhanced‐prophylaxis was more effective and less costly than all CrAg testing strategies as enhanced‐prophylaxis still conveyed health gains in CrAg‐negative patients and savings from targeting prophylaxis based on CrAg status did not compensate for costs of CrAg testing. CrAg testing strategies did not become cost‐effective unless the price of CrAg testing fell below US$2.30. Conclusions The REALITY enhanced‐prophylaxis package in individuals with advanced HIV starting ART reduces morbidity and mortality, is practical to administer and is cost‐effective. Efforts should continue to ensure that components are accessed at lowest available prices

Late Presentation With HIV in Africa: Phenotypes, Risk, and Risk Stratification in the REALITY Trial.

This article has been accepted for publication in Clinical Infectious Diseases Published by Oxford University PressBackground: Severely immunocompromised human immunodeficiency virus (HIV)-infected individuals have high mortality shortly after starting antiretroviral therapy (ART). We investigated predictors of early mortality and "late presenter" phenotypes. Methods: The Reduction of EArly MortaLITY (REALITY) trial enrolled ART-naive adults and children ≥5 years of age with CD4 counts .1). Results: Among 1711 included participants, 203 (12%) died. Mortality was independently higher with older age; lower CD4 count, albumin, hemoglobin, and grip strength; presence of World Health Organization stage 3/4 weight loss, fever, or vomiting; and problems with mobility or self-care at baseline (all P < .04). Receiving enhanced antimicrobial prophylaxis independently reduced mortality (P = .02). Of five late-presenter phenotypes, Group 1 (n = 355) had highest mortality (25%; median CD4 count, 28 cells/µL), with high symptom burden, weight loss, poor mobility, and low albumin and hemoglobin. Group 2 (n = 394; 11% mortality; 43 cells/µL) also had weight loss, with high white cell, platelet, and neutrophil counts suggesting underlying inflammation/infection. Group 3 (n = 218; 10% mortality) had low CD4 counts (27 cells/µL), but low symptom burden and maintained fat mass. The remaining groups had 4%-6% mortality. Conclusions: Clinical and laboratory features identified groups with highest mortality following ART initiation. A screening tool could identify patients with low CD4 counts for prioritizing same-day ART initiation, enhanced prophylaxis, and intensive follow-up. Clinical Trials Registration: ISRCTN43622374.REALITY was funded by the Joint Global Health Trials Scheme (JGHTS) of the UK Department for International Development, the Wellcome Trust, and Medical Research Council (MRC) (grant number G1100693). Additional funding support was provided by the PENTA Foundation and core support to the MRC Clinical Trials Unit at University College London (grant numbers MC_UU_12023/23 and MC_UU_12023/26). Cipla Ltd, Gilead Sciences, ViiV Healthcare/GlaxoSmithKline, and Merck Sharp & Dohme donated drugs for REALITY, and ready-to-use supplementary food was purchased from Valid International. A. J. P. is funded by the Wellcome Trust (grant number 108065/Z/15/Z). J. A. B. is funded by the JGHTS (grant number MR/M007367/1). The Malawi-Liverpool–Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine (grant number 101113/Z/13/Z) and the Kenya Medical Research Institute (KEMRI)/Wellcome Trust Research Programme, Kilifi (grant number 203077/Z/16/Z) are supported by strategic awards from the Wellcome Trust, United Kingdom. Permission to publish was granted by the Director of KEMRI. This supplement was supported by funds from the Bill & Melinda Gates Foundation

Raltegravir-intensified initial antiretroviral therapy in advanced HIV disease in Africa: A randomised controlled trial

Background In sub-Saharan Africa, individuals infected with HIV who are severely immunocompromised have high mortality (about 10%) shortly after starting antiretroviral therapy (ART). This group also has the greatest risk of morbidity and mortality associated with immune reconstitution inflammatory syndrome (IRIS), a paradoxical response to successful ART. Integrase inhibitors lead to significantly more rapid declines in HIV viral load (VL) than all other ART classes. We hypothesised that intensifying standard triple-drug ART with the integrase inhibitor, raltegravir, would reduce HIV VL faster and hence reduce early mortality, although this strategy could also risk more IRIS events. Methods and findings In a 2×2×2 factorial open-label parallel-group trial, treatment-naive adults, adolescents, and children >5 years old infected with HIV, with cluster of differentiation 4 (CD4) 0.7) and despite significantly greater VL suppression with raltegravir-intensified ART at 4 weeks (343/836 [41.0%] versus 113/841 [13.4%] with standard ART, p < 0.001) and 12 weeks (567/789 [71.9%] versus 415/803 [51.7%] with standard ART, p < 0.001). Through 48 weeks, there was no evidence of differences in mortality (aHR = 0.98 [95% CI 0.76–1.28], p = 0.91); in serious (aHR = 0.99 [0.81–1.21], p = 0.88), grade-4 (aHR = 0.88 [0.71–1.09], p = 0.29), or ART-modifying (aHR = 0.90 [0.63–1.27], p = 0.54) adverse events (the latter occurring in 59 [6.5%] participants with raltegravir-intensified ART versus 66 [7.3%] with standard ART); in events judged compatible with IRIS (occurring in 89 [9.9%] participants with raltegravir-intensified ART versus 86 [9.5%] with standard ART, p = 0.79) or in hospitalisations (aHR = 0.94 [95% CI 0.76–1.17], p = 0.59). At 12 weeks, one and two raltegravir-intensified participants had predicted intermediate-level and high-level raltegravir resistance, respectively. At 48 weeks, the nucleoside reverse transcriptase inhibitor (NRTI) mutation K219E/Q (p = 0.004) and the non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E/P (p = 0.03) and P225H (p = 0.007) were less common in virus from participants with raltegravir-intensified ART, with weak evidence of less intermediate- or high-level resistance to tenofovir (p = 0.06), abacavir (p = 0.08), and rilpivirine (p = 0.07). Limitations of the study include limited clinical, radiological, and/or microbiological information for some participants, reflecting available services at the centres, and lack of baseline genotypes. Conclusions Although 12 weeks of raltegravir intensification was well tolerated and reduced HIV viraemia significantly faster than standard triple-drug ART during the time of greatest risk for early death, this strategy did not reduce mortality or clinical events in this group and is not warranted. There was no excess of IRIS-compatible events, suggesting that integrase inhibitors can be used safely as part of standard triple-drug first-line therapy in severely immunocompromised individuals. Trial registration ClinicalTrials.gov NCT01825031