Will an Unsupervised Self-Testing Strategy for HIV Work in Health Care Workers of South Africa? A Cross Sectional Pilot Feasibility Study

Background

In South Africa, stigma, discrimination, social visibility and fear of loss of confidentiality impede health facility-based HIV testing. With 50% of adults having ever tested for HIV in their lifetime, private, alternative testing options are urgently needed. Non-invasive, oral self-tests offer a potential for a confidential, unsupervised HIV self-testing option, but global data are limited.

Methods

A pilot cross-sectional study was conducted from January to June 2012 in health care workers based at the University of Cape Town, South Africa. An innovative, unsupervised, self-testing strategy was evaluated for feasibility; defined as completion of self-testing process (i.e., self test conduct, interpretation and linkage). An oral point-of-care HIV test, an Internet and paper-based self-test HIV applications, and mobile phones were synergized to create an unsupervised strategy. Self-tests were additionally confirmed with rapid tests on site and laboratory tests. Of 270 health care workers (18 years and above, of unknown HIV status approached), 251 consented for participation.

Findings

Overall, about 91% participants rated a positive experience with the strategy. Of 251 participants, 126 evaluated the Internet and 125 the paper-based application successfully; completion rate of 99.2%. All sero-positives were linked to treatment (completion rate:100% (95% CI, 66.0–100). About half of sero-negatives were offered counselling on mobile phones; completion rate: 44.6% (95% CI, 38.0–51.0). A majority of participants (78.1%) were females, aged 18–24 years (61.4%). Nine participants were found sero-positive after confirmatory tests (prevalence 3.6% 95% CI, 1.8–6.9). Six of nine positive self-tests were accurately interpreted; sensitivity: 66.7% (95% CI, 30.9–91.0); specificity:100% (95% CI, 98.1–100).

Interpretation

Our unsupervised self-testing strategy was feasible to operationalize in health care workers in South Africa. Linkages were successfully operationalized with mobile phones in all sero-positives and about half of the sero-negatives sought post-test counselling. Controlled trials and implementation research studies are needed before a scale-up is considered.     

Molecular Cloning, Bacterial Overexpression and Characterization of L-myo- inositol 1- Phosphate Synthase from a Monocotyledonous Resurrection Plant, Xerophyta viscosa Baker

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS), an evolutionarily conserved enzyme-protein, catalyses the first and rate limiting step of inositol biosynthesis. Inositol and its derivatives play important roles in biological kingdom like growth regulation, membrane biogenesis, signal transduction and also acts as an osmolyte or osmoprotectant in abiotic stress tolerance. Here we report the cloning, sequencing and the characterization of the INO1 gene from Xerophyta viscosa (XINO1), a monocotyledonous resurrection plant. Nucleotide sequences of XINO1 show striking homology (70–99%) with a number of INO1 genes from plant sources particularly with the monocots. The gene is functionally identified through genetic complementation using a yeast inositol auxotrophic strain FY250. The gene is expressed in E. coli BL21, recombinant protein purified to homogeneity, biochemically characterized and compared with Oryza INO1 (RINO1) gene product. The XINO1 gene product is catalytically active in a broader range of lower temperature (between 10–40 °C) than the RINO1 gene- product. This is the first report of MIPS gene from any resurrection plant.     

Are Treponema pallidum Specific Rapid and Point-of-Care Tests for Syphilis Accurate Enough for Screening in Resource Limited Settings? Evidence from a Meta-Analysis

Background

Rapid and point-of-care (POC) tests for syphilis are an invaluable screening tool, yet inadequate evaluation of their diagnostic accuracy against best reference standards limits their widespread global uptake. To fill this gap, a systematic review and meta-analysis was conducted to evaluate the sensitivity and specificity of rapid and POC tests in blood and serum samples against Treponema pallidum (TP) specific reference standards.

Methods

Five electronic databases (1980–2012) were searched, data was extracted from 33 articles, and Bayesian hierarchical models were fit.

Results

In serum samples, against a TP specific reference standard point estimates with 95% credible intervals (CrI) for the sensitivities of popular tests were: i) Determine, 90.04% (80.45, 95.21), ii) SD Bioline, 87.06% (75.67, 94.50), iii) VisiTect, 85.13% (72.83, 92.57), and iv) Syphicheck, 74.48% (56.85, 88.44), while specificities were: i) Syphicheck, 99.14% (96.37, 100), ii) Visitect, 96.45% (91.92, 99.29), iii) SD Bioline, 95.85% (89.89, 99.53), and iv) Determine, 94.15% (89.26, 97.66). In whole blood samples, sensitivities were: i) Determine, 86.32% (77.26, 91.70), ii) SD Bioline, 84.50% (78.81, 92.61), iii) Syphicheck, 74.47% (63.94, 82.13), and iv) VisiTect, 74.26% (53.62, 83.68), while specificities were: i) Syphicheck, 99.58% (98.91, 99.96), ii) VisiTect, 99.43% (98.22, 99.98), iii) SD Bioline, 97.95%(92.54, 99.33), and iv) Determine, 95.85% (92.42, 97.74).

Conclusions

Rapid and POC treponemal tests reported sensitivity and specificity estimates comparable to laboratory-based treponemal tests. In resource limited settings, where access to screening is limited and where risk of patients lost to follow up is high, the introduction of these tests has already been shown to improve access to screening and treatment to prevent stillbirths and neonatal mortality due to congenital syphilis. Based on the evidence, it is concluded that rapid and POC tests are useful in resource limited settings with poor access to laboratories or screening for syphilis.     

Salt-induced abnormalities on root tip mitotic cells of Allium cepa: prevention by inositol pretreatment

Salt-induced growth reduction of plants is a well-known phenomenon which poses major problem in crop productivity in places where vast majority of land plants are affected by salt. In this report, studies were carried out to reveal the effect of salt injury on the cell division pattern in roots and the role of myo-inositol in preventing the salt-induced ion disequilibrium on the chromosome and DNA degradation in roots. Present study revealed induction of various chromosomal abnormalities on the root tip mitotic cells of Allium cepa by treatment with different concentrations of NaCl (0–500 mM) for 24 h as also the amelioration of such effect by prior treatment of the roots with different concentration of myo-inositol (0–300 mM). Results showed that a narrow albeit definite range of extracellular myo-inositol (100–150 mM) is effective in preventing internucleosomal fragmentation which is the early response in roots under salt stress. Transgenic tobacco plants overexpressing Oryza (OsINO1) as well as Porteresia (PcINO1) cytosolic l-myo-inositol-1-phosphate synthase coding genes can withstand and retain their chromosomal and DNA integrity in 100 mM NaCl solution and can subsequently prevent DNA fragmentation, caused by intracellular endonuclease activity at this salt concentration.     

Disruptin,a cell-penetrating peptide degrader of EGFR: Cell-Penetrating Peptide in Cancer Therapy

Disruptin is a cell-permeable decoy peptide designed to destabilize activated EGFR, both by inhibiting Hsp90 chaperoning and dissociating the active asymmetric EGFR dimer, which leads to an increase in engagement of activated EGFR with the proteolytic degradation machinery and subsequent loss from the cells. Disruptin is an N-terminally biotinylated nonadecapeptide, with 8 amino acids from the αC-helix-β4 sheet loop of EGFR (S⁷⁶⁷-C⁷⁷⁴) fused to a TAT undecapeptide. The S⁷⁶⁷-R⁷⁷⁵ loop is at the interface with juxtamembrane domains in the active EGFR dimers and is a binding site for Hsp90. Cellular studies in EGFR-activated tumor cells demonstrated that Disruptin causes the disappearance of EGFR protein from cells over a few hours, a growth inhibitory effect, similar but more effective than the EGFR kinase inhibition. Interestingly, cells without activated EGFR remained unaffected. In vivo studies showed that Disruptin slowed the growth of small tumors. Larger tumors responded to intratumoral injections but did not respond to systemic administration at tolerated doses. Investigation of these results revealed that systemic administration of Disruptin has acute toxicities, mainly related to its TAT peptide moiety. Therefore, we conclude that although the efficacy of both in vitro and in vivo intratumoral injection of Disruptin supports the therapeutic strategy of blocking activated EGFR dimerization, Disruptin is not suitable for further development. These studies also highlight the importance of the chosen models and drug-delivery methods for such investigations.     

Analysis of the substrate specificity loop of the HAD superfamily cap domain

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.     

Glucose deprivation induced upregulation of phosphoenolpyruvate carboxykinase modulates virulence in Leishmania donovani

Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP⁺ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.