Multi-centred mixed-methods PEPFAR HIV care & support public health evaluation: study protocol

BACKGROUND: A public health response is essential to meet the multidimensional needs of patients and families affected by HIV disease in sub-Saharan Africa. In order to appraise current provision of HIV care and support in East Africa, and to provide evidence-based direction to future care programming, and Public Health Evaluation was commissioned by the PEPFAR programme of the US Government. METHODS/DESIGN: This paper described the 2-Phase international mixed methods study protocol utilising longitudinal outcome measurement, surveys, patient and family qualitative interviews and focus groups, staff qualitative interviews, health economics and document analysis. Aim 1) To describe the nature and scope of HIV care and support in two African countries, including the types of facilities available, clients seen, and availability of specific components of care [Study Phase 1]. Aim 2) To determine patient health outcomes over time and principle cost drivers [Study Phase 2]. The study objectives are as follows. 1) To undertake a cross-sectional survey of service configuration and activity by sampling 10% of the facilities being funded by PEPFAR to provide HIV care and support in Kenya and Uganda (Phase 1) in order to describe care currently provided, including pharmacy drug reviews to determine availability and supply of essential drugs in HIV management. 2) To conduct patient focus group discussions at each of these (Phase 1) to determine care received. 3) To undertake a longitudinal prospective study of 1200 patients who are newly diagnosed with HIV or patients with HIV who present with a new problem attending PEPFAR care and support services. Data collection includes self-reported quality of life, core palliative outcomes and components of care received (Phase 2). 4) To conduct qualitative interviews with staff, patients and carers in order to explore and understand service issues and care provision in more depth (Phase 2). 5) To undertake document analysis to appraise the clinical care procedures at each facility (Phase 2). 6) To determine principle cost drivers including staff, overhead and laboratory costs (Phase 2). DISCUSSION: This novel mixed methods protocol will permit transparent presentation of subsequent dataset results publication, and offers a substantive model of protocol design to measure and integrate key activities and outcomes that underpin a public health approach to disease management in a low-income setting

Proteomic Analysis of Rta2p-Dependent Raft-Association of Detergent-Resistant Membranes in Candida albicans

In Candida albicans, lipid rafts (also called detergent-resistant membranes, DRMs) are involved in many cellular processes and contain many important proteins. In our previous study, we demonstrated that Rta2p was required for calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Here, we found that Rta2p was co-localized with raft-constituted ergosterol on the plasma membrane of C. albicans. Furthermore, this membrane expression pattern was totally disturbed by inhibitors of either ergosterol or sphingolipid synthesis. Biochemical fractionation of DRMs together with immunoblot uncovered that Rta2p, along with well-known DRM-associated proteins (Pma1p and Gas1p homologue), was associated with DRMs and their associations were blocked by inhibitors of either ergosterol or sphingolipid synthesis. Finally, we used the proteomic analysis together with immunoblot and identified that Rta2p was required for the association of 10 proteins with DRMs. These 5 proteins (Pma1p, Gas1p homologue, Erg11p, Pmt2p and Ali1p) have been reported to be DRM-associated and also that Erg11p is a well-known target of azoles in C. albicans. In conclusion, our results showed that Rta2p was predominantly localized in lipid rafts and was required for the association of certain membrane proteins with lipid rafts in C. albicans

ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in Saccharomyces cerevisiae

The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily

Characterization of vertebral strength using digital radiographic analysis of bone structure.

Bone mineral densitometry (BMD) is useful in predicting fracture risk, but, unfortunately, there is a significant degree of overlap in BMD measurements of patients who have a high risk of fracture and patients with a low risk of fracture. In this study, a method of characterizing trabecular bone structure in digitized radiographs of vertebrae is proposed and assessed. A significant correlation between bone "structure" and the compressive strength of vertebral bodies was found. The utility of the parameter for distinguishing between "weak" and "strong" bone samples was assessed using receiver operating characteristic (ROC) analysis. Using this analysis, the structural parameter produced an area under the ROC of 0.88 +/- 0.05, while a bone density measure produced an area of 0.79 +/- 0.07. The results suggest that the addition of a measure of bone structure to the conventional measures of bone density may prove useful in predicting the quality of bone when considering surgical or medical intervention for osteoporotic conditions

Characterization of vertebral strength using digital radiographic analysis of bone structure.

Bone mineral densitometry (BMD) is useful in predicting fracture risk, but, unfortunately, there is a significant degree of overlap in BMD measurements of patients who have a high risk of fracture and patients with a low risk of fracture. In this study, a method of characterizing trabecular bone structure in digitized radiographs of vertebrae is proposed and assessed. A significant correlation between bone "structure" and the compressive strength of vertebral bodies was found. The utility of the parameter for distinguishing between "weak" and "strong" bone samples was assessed using receiver operating characteristic (ROC) analysis. Using this analysis, the structural parameter produced an area under the ROC of 0.88 +/- 0.05, while a bone density measure produced an area of 0.79 +/- 0.07. The results suggest that the addition of a measure of bone structure to the conventional measures of bone density may prove useful in predicting the quality of bone when considering surgical or medical intervention for osteoporotic conditions

A study of micromotion and appositional bone growth to a canine madreporic-surfaced femoral component.

A canine total hip arthroplasty model was used to examine micromotion and bone apposition to a proximally two-thirds madreporic-surfaced femoral prosthesis. Micromotion was also measured following initial press-fit implantation into canine cadaveric femora. After initial press-fit fixation and either 6 or 24 months of biologic fixation, micromotion was less than 23 microns in the proximal and midstem regions, a magnitude consistent with bone apposition. Bone apposition was greatest near the junction of the madreporic and smooth surfaces and was not significantly different between 6 and 24 months (51% at 6 months and 47% at 24 months). The quality of the interface tissue appears to be conductive to long-term fixation