HIV treatment support services in Tanzania: a cost and efficiency analysis at facility and community levels.

Differentiated service delivery models provide an opportunity to reduce the cost of HIV treatment while increasing the focus on advanced patients. Also, stable patients who need less attention can visit facilities less frequently, saving time and money and reducing overcrowding. Tanzania needs treatment support services that can improve the quality of life, ensure adherence and result in viral suppression. Treatment support services can be provided through a variety of models, including at the community level, through the facility or through some combination of the two. Understanding the cost of each model is essential for policy-makers who must allocate resources. Data from neighbouring countries suggests that community-level support services can be beneficial to patients, especially in reducing loss to follow-up and death. Though community-based HIV services are available in Tanzania, uncertainty about the costs of these models remain. This study assessed the costs of treatment support services at 27 sites in Tanzania. The cost analysis found that the average unit cost of treatment support services is US$39 per patient per year. The analysis found that community-based models have clear advantages with regard to the number of patients that can be reached with support services. Costing data indicated that community-based models are also less expensive. The lack of data on retention limited any conclusions about whether community-based models are the most effective within the Tanzanian context.

Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppression of salutaridinol 7-O-acetyltransferase in opium poppy.

We demonstrate that both over-expression and suppression of the gene encoding the morphinan pathway enzyme salutaridinol 7-O-acetyltransferase (SalAT) in opium poppy affects the alkaloid products that accumulate. Over-expression of the gene in most of the transgenic events resulted in an increase in capsule morphine, codeine and thebaine on a dry-weight basis. The transgenic line with the highest alkaloid content had 41%, 37% and 42% greater total alkaloids than the control in three independent trials over 3 years. DNA-encoded hairpin RNA-mediated suppression of SalAT resulted in the novel accumulation of the alkaloid salutaridine at up to 23% of total alkaloid; this alkaloid is not detectable in the parental genotype. Salutaridine is not the substrate of SalAT but the substrate of the previous enzyme in the pathway, salutaridine reductase. RNA transcript analysis of 16 primary T0 transformants and their segregating T1 progeny revealed an average reduction in SalAT transcript to about 12% of the control. Reduction in SalAT transcript was evident in both leaves and latex. Reverse transcriptase PCR and high-performance liquid chromatography analyses confirmed cosegregation of the expressed transgene with the salutaridine accumulating phenotype.

Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum.

Only plants of the Papaver genus (poppies) are able to synthesize morphinan alkaloids, and cultivation of P. somniferum, opium poppy, remains critical for the production and supply of morphine, codeine and various semi-synthetic analgesics. Opium poppy was transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1), the penultimate step in morphine synthesis. Most transgenic lines showed significant increases in capsule alkaloid content in replicated glasshouse and field trials over 4 years. The morphinan alkaloid contents on a dry weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control non-transgenic segregants. Transgenic leaves had approximately 10-fold greater levels of Cor transcript compared with non-transgenic controls. Two cycles of crossing of the best transgenic line into an elite high-morphine genotype resulted in significant increases in morphine and total alkaloids relative to the elite recurrent parent. No significant changes in alkaloid profiles or quantities were observed in leaf, roots, pollen and seed.

RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy.

We report on the silencing of codeinone reductase (COR) in the opium poppy, Papaver somniferum, using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi). After gene silencing, the precursor alkaloid (S)-reticuline-seven enzymatic steps upstream of codeinone-accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. Analysis verified loss of Cor gene transcript, appearance of 22-mer degradation products and reduction of enzyme activity. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine-specific branch. However transcript levels for seven other enzymes in the pathway, both before and after (S)-reticuline, were unaffected. This is the first report of gene silencing in transgenic opium poppy and of metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline.

Opium Poppy (Papaver somniferum).

The genetic transformation of opium poppy, Papaver somniferum, offers the opportunity to study the mechanisms involved in the regulation of benzylisoquinoline and morphinan alkaloid biosynthesis. The development of an efficient transformation protocol for opium poppy has allowed us to transform a range of genotypes from all around the world, including previously recalcitrant high-yielding commercial Australian cultivars. The method involves Agrobacterium tumefaciens infection of hypocotyl explants, followed by the production of antibiotic or herbicide resistant embryogenic callus, the subsequent induction of somatic embryos and development into normal plants. The use of different selective agents, binary vectors, and poppy genotypes has demonstrated the robustness and reliability of this protocol in the production of many hundreds of confirmed transgenic poppies.

Genetic transformation in commercial Tasmanian cultivars of opium poppy, Papaver somniferum, and movement of transgenicpollen in the field.

We report a new transformation protocol for the pharmaceutically important opium poppy, Papaver somniferum L.; the protocol allows transformation for the first time of high yielding commercial cultivars. The method involves Agrobacterium tumefaciens infection of hypocotyl explants, followed by the production of antibiotic- or herbicide-resistant embryogenic callus and the subsequent induction of somatic embryos and plants. Key elements of the improvement are the use of buffering agents to stabilise medium pH and bottom-cooling of the cultures. Transformation was verified by PCR and Southern blot hybridisation. Transcription of transgenes was confirmed by RT-PCR and product sequencing. Expression of transgenes was detected by histochemical GUS staining, phosphinothricin acetyltransferase (PAT) enzyme assays for bar and pat genes, and western analysis of transgenic sunflower seed albumin protein. Expression of various transgenes was detected in stem, leaf, seed, capsule and latex. The pat gene was demonstrated to be stably inherited to the T2 generation and to confer phosphinothricin (PPT) herbicide resistance. Most T0 plants showed normal morphology, were self-fertile and the transgenes displayed the expected Mendelian segregation. The percentage of explants producing somatic embryos that developed into plantlets able to be transplanted to soil, ranged from 6-11% in two Tasmanian cultivars.A field trial using pat transgenic plants was designed to estimate the frequency of hybridisation at various distances into buffer rows of non-transgenic poppies and to related weed species, P. somniferum spp. setigerum and P. dubium. The frequency of hybridisation to completely compatible poppy fell sharply with distance, being 2.6% at 20 cm, 2.13% at 0.5 m and falling to 0% at 2.5 and 5 m. No hybridisation could be detected to two weed species under open pollination conditions, including the compatible P. somniferum spp. setigerum, when grown as close as 20 cm, despite flowering at the same time as the transgenic plants in the presence of foraging bees.

Transformation of opium poppy (Papaver somniferum L.) with antisense berberine bridge enzyme gene (anti-bbe) via somatic embryogenesis results in an altered ratio of alkaloids in latex but not in roots.

The berberine bridge enzyme cDNA bbe from Papaver somniferum L. was transformed in antisense orientation into seedling explants of the industrial elite line C048-6-14-64. In this way, 84 phenotypically normal To plants derived from embryogenic callus cultures were produced. The selfed progeny of these 84 plants yielded several T1 plants with an altered alkaloid profile. One of these plants T1-47, and its siblings T2-1.2 and T2-1.5 are the subject of the present work. The transformation of these plants was evaluated by PCR, and northern and Southern hybridisation. The transgenic plants contained one additional copy of the transgene. The alkaloid content in latex and roots was determined with HPLC and LC-MS. We observed an increased concentration of several pathway intermediates from all biosynthetic branches, e.g., reticuline, laudanine, laudanosine, dehydroreticuline, salutaridine and (S)-scoulerine. The transformation altered the ratio of morphinan and tetrahydrobenzylisoquinoline alkaloids in latex but not the benzophenanthridine alkaloids in roots. The altered alkaloid profile is heritable at least to the T2 generation. These results are the first example of metabolic engineering of the alkaloid pathways in opium poppy and, to our knowledge, the first time that an alkaloid biosynthetic gene has been transformed into the native species, followed by regeneration into a mature plant to enable analyses of the effect of the transgene on metabolism over several generations.