Treatment outcome of HIV-1 infected children on antiretroviral therapy in the Limpopo Province of South Africa

Magister Public Health - MPHBackground:HIV is a worldwide pandemic with an estimated 2.5 million children under the age of 15 living with HIV in the world in 2009. Children account for approximately 14% of all HIV-related deaths around the world. Several studies have shown that the use of antiretroviral drugs greatly improve the lives of HIV-1 infected individuals, however, most of these studies report on outcomes of ART programmes in developed world and for adult patients. Very few settings have published outcomes of paediatric ART programmes.Objectives This research was aimed at describing the long term (at least one year) treatment outcome of HIV-1 infected children in the HIV/AIDS Prevention Group (HAPG) program in Bela-Bela in the Limpopo province of South Africa.Study design and methods: A quantitative approach involving a retrospective cohort design was used for the study. The study included all children under the age of 15 that were enrolled in the HATG treatment programme in Bela-Bela between February 2004 and December2009.Immunological, virological, clinical outcomes and loss to follow-up were determined for this cohort. Mortality and survival was also determined. Results: The median age of children in this study was 5 years (IQR: 2-7) with 14% (10/71) of them being less than 18 months. Median CD4 count at commencement of ART, viral load and weight were 358 cells/mm3 (IQR 203.5-, 125673 RNA copies/μL (IQR 58094-328424.5) and 14.5Kg (IQR: 11.0-18.35) respectively. CD4 counts and weight showed increase within the study period, and there was also a decline in viral load. Loss to follow-up was 7.04% while mortality was 19% with 21.43% of mortality cases being children who were ≤18months. Mortality occurred within the first year of ART initiation and occurred in cases that had advanced disease.Conclusion: This study shows that the ART program in Bela-Bela has a positive outcome on HIV positive children.The high mortality rate was due to children starting ART at an advanced disease stage. Despite the good outcome, it is recommended that a system be put into place that will aid in identifying children at an early stage of the disease and treatment initiated promptly

Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion

The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp 3.49(125) and Arg 6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr 2.56(82) , in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr 2.56(82) mutants were fully stabilized in active conformations. The Thr 2.56(82) Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr 2.56(82) Lys mutation with an Arg 6.32(225) Gln mutation partially reversed the decrease in expression. Mutants with Thr 2.56(82) Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr 2.65(82) Pro substitution exhibited full co-receptor function. Our results suggest that the Thr 2.65(82) Lys and Thr 2.65(82) Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion

Functional consequences of South African mutations of the HIV-1 co-receptor, CCR5

Includes bibliographical references (p. 100-119).Four mutations of the CCR5 receptor have been identified in the South African population, but the effects of these mutations on CCR5 function and HIV infection are unknown. We have used in vitro methods to assess the ffect of the mutations, Asp2Val, Leu107Phe, Arg225Gln and Arg225stop, on CCR5 interactions with chemokine ligands and HIV

Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009–2013)

Abstract Background Bangladesh is a malaria hypo-endemic country sharing borders with India and Myanmar. Artemisinin combination therapy (ACT) remains successful in Bangladesh. An increase of artemisinin-resistant malaria parasites on the Thai-Cambodia and Thai-Myanmar borders is worrisome. K13 propeller gene (PF3D7_1343700 or PF13_0238) mutations have been linked to both in vitro artemisinin resistance and in vivo slow parasite clearance rates. This group undertook to evaluate if mutations seen in Cambodia have emerged in Bangladesh where ACT use is now standard for a decade. Methods Samples were obtained from Plasmodium falciparum-infected malaria patients from Upazila health complexes (UHC) between 2009 and 2013 in seven endemic districts of Bangladesh. These districts included Khagrachari (Matiranga UHC), Rangamati (Rajasthali UHC), Cox’s Bazar (Ramu and Ukhia UHC), Bandarban (Lama UHC), Mymensingh (Haluaghat UHC), Netrokona (Durgapur and Kalmakanda UHC), and Moulvibazar (Sreemangal and Kamalganj UHC). Results Out of 296 microscopically positive P. falciparum samples, 271 (91.6%) were confirmed as mono-infections by both real-time PCR and nested PCR. The K13 propeller gene from 253 (93.4%) samples was sequenced bi-directionally. One non-synonymous mutation (A578S) was found in Bangladeshi clinical isolates. The A578S mutation was confirmed and lies adjacent to the C580Y mutation, the major mutation causing delayed parasite clearance in Cambodia. Based on computational modeling A578S should have a significant effect on tertiary structure of the protein. Conclusion The data suggest that P. falciparum in Bangladesh remains free of the C580Y mutation linked to delayed parasite clearance. However, the mutation A578S is present and based on structural analysis could affect K13 gene function. Further in vivo clinical studies are required to validate the effect of this mutation

Interaction of PfHsp90 with PfCRT is demonstrated using a combination of co-immunoprecipitation, LC-MS/MS and, and PfHsp90 inhibition studies.

<p>The Coomassie stained gels are shown to indicate loading. (A) Under non-denaturing conditions, immunoprecipitation with anti-PfHsp90 (upper panel) pulled down both itself and the PfCRT as demonstrated by western blot. The converse experiment (with anti-PfCRT immunoprecipitation (lower panel) achieved the reciprocal result. The mock control used was beads alone plus extracts. (B) Anti-PfCRT (top) and anti-PfHsp90 (middle) Western blots of immunoprecipitates under denaturing conditions with associated controls. These western blots are displayed as composites because they were run on two separate gels. The exposure levels were matched. The lowermost panel displays western blots of both PfHsp90 and PfCRT from <i>P. falciparum</i> culture protein extracts. (C) Anti-PfCRT Western blot of proteins which bound to histidine-tagged full length (FL) PfHsp90 coupled to Ni-NTA beads. (D) Extracts from <i>P. falciparum</i> strain W2 treated and untreated for 24 hours with 111 nM PU-H71 and immunoblotted with anti-PfCRT and anti-PfHsp90 antibody to determine the level of PfCRT and PfHsp90 available following PfHsp90 inhibition. The upper right panel demonstrates equal loading of protein in drug treated and untreated fractions. (E) Unweighted protein-protein interaction network of LC-MS/MS analyzed immunoprecipitates using anti-PfHsp90 and anti-PfCRT. The complete list of constituents in the interactome is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446.s001" target="_blank">Table S1</a>. The image was generated using Cytoscape v2.8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Shannon1" target="_blank">[43]</a>. Lines connecting proteins suggest a direct interaction.</p

A Purine Analog Synergizes with Chloroquine (CQ) by Targeting <i>Plasmodium falciparum</i> Hsp90 (PfHsp90)

<div><p>Background</p><p>Drug resistance, absence of an effective vaccine, and inadequate public health measures are major impediments to controlling <i>Plasmodium falciparum</i> malaria worldwide. The development of antimalarials to which resistance is less likely is paramount. To this end, we have exploited the chaperone function of <i>P. falciparum</i> Hsp90 (PfHsp90) that serves to facilitate the expression of resistance determinants.</p><p>Methods</p><p>The affinity and activity of a purine analogue Hsp90 inhibitor (PU-H71) on PfHsp90 was determined using surface plasmon resonance (SPR) studies and an ATPase activity assay, respectively. In vitro, antimalarial activity was quantified using flow cytometry. Interactors of PfHsp90 were determined by LC-MS/MS. <i>In vivo</i> studies were conducted using the <i>Plasmodium berghei</i> infection mouse model.</p><p>Results</p><p>PU-H71 exhibited antimalarial activity in the nanomolar range, displayed synergistic activity with chloroquine <i>in vitro</i>. Affinity studies reveal that the PfHsp90 interacts either directly or indirectly with the <i>P. falciparum</i> chloroquine resistance transporter (PfCRT) responsible for chloroquine resistance. PU-H71 synergized with chloroquine in the <i>P.berghei</i> mouse model of malaria to reduce parasitemia and improve survival.</p><p>Conclusions</p><p>We propose that the interaction of PfHsp90 with PfCRT may account for the observed antimalarial synergy and that PU-H71 is an effective adjunct for combination therapy.</p></div

Biochemical evaluation of the affinity of PU-H71 for PfHsp90.

<p>(A) Illustration of geldanamycin (GA, left) and PU-H71 (right) docked within the ATP-binding site of PfHsp90. The models were generated using the PfHsp90 crystal structure (PDB ID: 3K60) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Corbett1" target="_blank">[45]</a>. By convention, the electrostatic potential surface in the background denotes acidic residues in red and basic residues in blue. (B) Surface plasmon resonance (SPR) measurements for PU-H71 binding to the ATP-binding domain of PfHsp90. The colors on the sensorgrams represent varying concentrations of the respective drug (5–1000 µM) injected over the surface with the immobilized PfHsp90. The steady state responses were fitted using non-linear regression to a single binding site model (as shown on the right) to obtain the K_d value indicated. The number in parentheses represents standard error on the K_d obtained from fits. The sensorgrams shown have been double referenced as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Zhao1" target="_blank">[34]</a>. (C) 17-AAG was used as a positive control drug. (D) SPR measurements for PU-H71 binding of the R98K mutant ATP-binding domain of PfHsp90. (E) Full-length PfHsp90 was expressed and ATPase activity tested in the presence of PU-H71. Results are shown as a percentage of total ATPase activity in the absence of drug and IC₅₀ indicated (511 nM) for a single experiment. Positive control drug treatments included radicicol (144 nM) and 17-AAG (146 nM). The inset shows the logarithmic curve fitting of ATPase activity with increasing concentration of PU-H71.</p

Summary of response modification indexes (RMI) for various combinations of PU-H71 and chloroquine <i>in vitro</i> based on the checkerboard assay.

*<p>A response modification index (RMI) of ≈1 correspond with no effect of one drug on another when used in combination; RMI <<1 potentiation of antimalarial activity (ie. synergistic activity); RMI>>1 antagonistic activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Oduola1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Pereira1" target="_blank">[39]</a>. Standard error of the mean is indicated.</p