Johannesburg, South Africa
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Romani B.,Stellenbosch University | Glashoff R.H.,Stellenbosch University | Engelbrecht S.,Stellenbosch University | Engelbrecht S.,National Health Laboratory Services NHLS
Virus Research | Year: 2010

HIV-1 Vpr, an accessory protein with multiple functions, is involved in the induction of apoptosis, cell cycle G2 arrest, and modulation of gene expression. Many functions of this protein have been documented for the wild-type subtype B Vpr, however the functionality of other subtypes has not sufficiently been addressed. In this study, the functionality of Subtype B Vpr, 6 subtype C mutant Vpr proteins and the consensus sequence of subtype C Vpr were compared with each other. All the subtype B and C Vpr proteins localized to the nucleus of human 293T cells. Subtype C Vpr proteins induced cell cycle G2 arrest in a lower proportion of human 293T cells compared to subtype B Vpr. Subtype B and the naturally mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.33% to 98.64%. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability in induction of apoptosis. The study of mRNA profile of the transfected cells indicated that all Vpr proteins modulated the apoptotic genes triggering the intrinsic pathway of apoptosis. Our results indicate that subtype C Vpr is able to exert the same functions previously reported for subtype B Vpr. Most natural mutations in Vpr not only do not disturb the functions of the protein but also potentiate the protein for an increased functionality. The natural mutations of Vpr may thus not always be regarded as defective mutations. The study suggests the adaptive role of the natural mutations commonly found in subtype C Vpr. © 2010 Elsevier B.V.


Moyo S.,Stellenbosch University | Moyo S.,Botswana Harvard AIDS Institute Partnership | Vandormael A.,University of KwaZulu - Natal | Wilkinson E.,University of KwaZulu - Natal | And 14 more authors.
PLoS ONE | Year: 2016

Background: Cross-sectional, biomarker methods to determine HIV infection recency present a promising and cost-effective alternative to the repeated testing of uninfected individuals. We evaluate a viral-based assay that uses a measure of pairwise distances (PwD) to identify HIV infection recency, and compare its performance with two serologic incidence assays, BED and LAg. In addition, we assess whether combination BED plus PwD or LAg plus PwD screening can improve predictive accuracy by reducing the likelihood of a false-recent result. Methods: The data comes from 854 time-points and 42 participants enrolled in a primary HIV-1C infection study in Botswana. Time points after treatment initiation or with evidence of multiplicity of infection were excluded from the final analysis. PwD was calculated from quasispecies generated using single genome amplification and sequencing.We evaluated the ability of PwD to correctly classify HIV infection recency within <130, <180 and <360 days postseroconversion using Receiver Operator Characteristics (ROC) methods. Following a secondary PwD screening, we quantified the reduction in the relative false-recency rate (rFRR) of the BED and LAg assays while maintaining a sensitivity of either 75, 80, 85 or 90%. Results: The final analytic sample consisted of 758 time-points from 40 participants. The PwD assay was more accurate in classifying infection recency for the 130 and 180-day cut-offs when compared with the recommended LAg and BED thresholds. A higher AUC statistic confirmed the superior predictive performance of the PwD assay for the three cut-offs. When used for combination screening, the PwD assay reduced the rFRR of the LAg assay by 52% and the BED assay by 57.8% while maintaining a 90% sensitivity for the 130 and 180-day cut-offs respectively. Conclusion: PwD can accurately determine HIV infection recency. A secondary PwD screening reduces misclassification and increases the accuracy of serologic-based assays. © 2016 Moyo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Moyo S.,Stellenbosch University | Moyo S.,Botswana Harvard AIDS Institute Partnership | Wilkinson E.,University of KwaZulu - Natal | Novitsky V.,Botswana Harvard AIDS Institute Partnership | And 10 more authors.
Viruses | Year: 2015

In this paper, we review serological and molecular based methods to identify HIV infection recency. The accurate identification of recent HIV infection continues to be an important research area and has implications for HIV prevention and treatment interventions. Longitudinal cohorts that follow HIV negative individuals over time are the current gold standard approach, but they are logistically challenging, time consuming and an expensive enterprise. Methods that utilize cross-sectional testing and biomarker information have become an affordable alternative to the longitudinal approach. These methods use well-characterized biological makers to differentiate between recent and established HIV infections. However, recent results have identified a number of limitations in serological based assays that are sensitive to the variability in immune responses modulated by HIV subtypes, viral load and antiretroviral therapy. Molecular methods that explore the dynamics between the timing of infection and viral evolution are now emerging as a promising approach. The combination of serological and molecular methods may provide a good solution to identify recent HIV infection in cross-sectional data. As part of this review, we present the advantages and limitations of serological and molecular based methods and their potential complementary role for the identification of HIV infection recency. © 2015 by the authors; licensee MDPI, Basel, Switzerland.


Jacobs G.B.,Stellenbosch University | Wilkinson E.,Stellenbosch University | Wilkinson E.,University of KwaZulu - Natal | Isaacs S.,Stellenbosch University | And 5 more authors.
PLoS ONE | Year: 2014

South Africa has the largest worldwide HIV/AIDS population with 5.6 million people infected and at least 2 million people on antiretroviral therapy. The majority of these infections are caused by HIV-1 subtype C. Using genotyping methods we characterized HIV-1 subtypes of the gag p24 and pol PR and RT fragments, from a cohort of female participants in the Western Cape Province, South Africa. These participants were recruited as part of a study to assess the combined brain and behavioural effects of HIV and early childhood trauma. The partial HIV-1 gag and pol fragments of 84 participants were amplified by PCR and sequenced. Different online tools and manual phylogenetic analysis were used for HIV-1 subtyping. Online tools included: REGA HIV Subtyping tool version 3; Recombinant Identification Program (RIP); Context-based Modeling for Expeditious Typing (COMET); jumping profile Hidden Markov Models (jpHMM) webserver; and subtype classification using evolutionary algorithms (SCUEAL). HIV-1 subtype C predominates within the cohort with a prevalence of 93.8%. We also show, for the first time, the presence of circulating BC strains in at least 4.6% of our study cohort. In addition, we detected transmitted resistance associated mutations in 4.6% of analysed sequences. With tourism and migration rates to South Africa currently very high, we are detecting more and more HIV-1 URFs within our study populations. It is stil unclear what role these unique strains will play in terms of long term antiretroviral treatment and what challenges they will pose to vaccine development. Nevertheless, it remains vitally important to monitor the HIV-1 diversity in South Africa and worldwide as the face of the epidemic is continually changing. © 2014 Jacobs et al.


Wallis C.L.,University of Witwatersrand | Mellors J.W.,University of Pittsburgh | Venter W.D.F.,University of Witwatersrand | Sanne I.,University of Witwatersrand | And 2 more authors.
AIDS Research and Treatment | Year: 2011

Limited data exist on HIV-1 drug resistance patterns in South Africa following second-line protease-inhibitor containing regimen failure. This study examined drug resistance patterns emerging in 75 HIV-1 infected adults experiencing virologic failure on a second-line regimen containing 2 NRTI and lopinavir/ritonavir. Ninety six percent of patients (n=72) were infected with HIV-1 subtype C, two patients were infected with HIV-1 subtype D and one with HIV-1 subtype A1. Thirty nine percent (n=29) of patients had no resistance mutations in protease or reverse transcriptase suggesting that medication non-adherence was a major factor contributing to failure. Major lopinavir resistance mutations were infrequent (5 of 75; 7%), indicating that drug resistance is not the main barrier to future viral suppression. © 2011 Carole L. Wallis et al.


Wallis C.L.,University of Witwatersrand | Mellors J.W.,University of Pittsburgh | Venter W.D.F.,University of Witwatersrand | Sanne I.,University of Witwatersrand | And 2 more authors.
Journal of Acquired Immune Deficiency Syndromes | Year: 2010

BACKGROUND: The South African national antiretroviral therapy roll-out program is entering its sixth year, with over 570,000 adults accessing treatment. HIV-1 drug resistance is a potential consequence of therapy. This study determined the pattern of HIV-1 drug resistance mutations after failure of first-line treatment regimens in South Africa. METHODS: Two hundred and twenty-six patients virologically failing first-line regimens were studied to determine resistance patterns. RESULTS: The most common reverse transcriptase mutation was M184V/I (72%; n = 163); 11% of patients (n = 25) had only nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations and 17% (n = 38) had no known resistance mutations. The K65R mutation was detected in 4%. The frequency of thymidine analog mutations was significantly higher with azidothymidine-containing (31 of 57) than stavudine-containing regimens (39 of 169; P < 0.001). The Y181C mutation was more frequent with failure of nevirapine (NVP)-containing (26%) than efavirenz (EFV)-containing therapy (3%; P < 0.001). The V106M mutation was more frequent with EFV (30%) than NVP (4%; P = 0.012). CONCLUSIONS: HIV-1 drug resistance patterns varied broadly after failure of first-line therapy, ranging from no known resistance mutations (17%) to multinucleoside reverse transcriptase inhibitor and NNRTI resistance (23%). NNRTI mutation profiles differed for patients on EFV-compared with NVP-containing regimens. Overall, these findings suggest that HIV-1 drug resistance testing would be useful in identifying most appropriate second-line regimens. Copyright © 2010 by Lippincott Williams & Wilkins.


McCormack V.A.,International Agency for Research on Cancer | Joffe M.,Chris Hani Baragwanath Academic Hospital Breast Clinic | van den Berg E.,National Health Laboratory Services NHLS | Broeze N.,Chris Hani Baragwanath Academic Hospital Breast Clinic | And 6 more authors.
Breast Cancer Research | Year: 2013

Introduction: Estimates of the proportion of estrogen receptor negative (ERN) and triple-negative (TRN) breast cancer from sub-Saharan Africa are variable and include high values. Large studies of receptor status conducted on non-archival tissue are lacking from this region.Methods: We identified 1218 consecutive women (91% black) diagnosed with invasive breast cancer from 2006-2012 at a public hospital in Soweto, South Africa. Immunohistochemistry based ER, progesterone receptor (PR) and human epidermal factor 2 (HER2) receptors were assessed at diagnosis on pre-treatment biopsy specimens. Mutually adjusted associations of receptor status with stage, age, and race were examined using risk ratios (RRs). ER status was compared with age-stratified US Surveillance Epidemiology and End Results program (SEER) data.Results: 35% (95% confidence interval (CI): 32-38) of tumors were ERN, 47% (45-52) PRN, 26% (23-29) HER2P and 21% (18-23) TRN. Later stage tumors were more likely to be ERN and PRN (RRs 1.9 (1.1-2.9) and 2.0 (1.3-3.1) for stage III vs. I) but were not strongly associated with HER2 status. Age was not strongly associated with ER or PR status, but older women were less likely to have HER2P tumors (RR, 0.95 (0.92-0.99) per 5 years). During the study, stage III + IV tumors decreased from 66% to 46%. In black women the percentage of ERN (37% (34-40)) and PRN tumors (48% (45-52)) was higher than in non-black patients (22% (14-31) and 34% (25-44), respectively, P = 0.004 and P = 0.02), which remained after age and stage adjustment. Age-specific ERN proportions in black South African women were similar to those of US black women, especially for women diagnosed over age 50.Conclusion: Although a greater proportion of black than non-black South African women had ER-negative or TRN breast cancer, in all racial groups in this study breast cancer was predominantly ER-positive and was being diagnosed at earlier stages over time. These observations provide initial indications that late-stage aggressive breast cancers may not be an inherent feature of the breast cancer burden across Africa. © 2013 McCormack et al.; licensee BioMed Central Ltd.


Huang K.-H.G.,University of Oxford | Goedhals D.,University of the Free State | Goedhals D.,National Health Laboratory Services NHLS | Carlson J.M.,Microsoft | And 26 more authors.
PLoS ONE | Year: 2011

We lack the understanding of why HIV-infected individuals in South Africa progress to AIDS. We hypothesised that in end-stage disease there is a shifting dynamic between T cell imposed immunity and viral immune escape, which, through both compensatory and reverting viral mutations, results in increased viral fitness, elevated plasma viral loads and disease progression. We explored how T cell responses, viral adaptation and viral fitness inter-relate in South African cohorts recruited from Bloemfontein, the Free State (n = 278) and Durban, KwaZulu-Natal (n = 775). Immune responses were measured by γ-interferon ELISPOT assays. HLA-associated viral polymorphisms were determined using phylogenetically corrected techniques, and viral replication capacity (VRC) was measured by comparing the growth rate of gag-protease recombinant viruses against recombinant NL4-3 viruses. We report that in advanced disease (CD4 counts <100 cells/μl), T cell responses narrow, with a relative decline in Gag-directed responses (p<0.0001). This is associated with preserved selection pressure at specific viral amino acids (e.g., the T242N polymorphism within the HLA-B*57/5801 restricted TW10 epitope), but with reversion at other sites (e.g., the T186S polymorphism within the HLA-B*8101 restricted TL9 epitope), most notably in Gag and suggestive of "immune relaxation". The median VRC from patients with CD4 counts <100 cells/μl was higher than from patients with CD4 counts ≥500 cells/μl (91.15% versus 85.19%, p = 0.0004), potentially explaining the rise in viral load associated with disease progression. Mutations at HIV Gag T186S and T242N reduced VRC, however, in advanced disease only the T242N mutants demonstrated increasing VRC, and were associated with compensatory mutations (p = 0.013). These data provide novel insights into the mechanisms of HIV disease progression in South Africa. Restoration of fitness correlates with loss of viral control in late disease, with evidence for both preserved and relaxed selection pressure across the HIV genome. Interventions that maintain viral fitness costs could potentially slow progression. © 2011 Huang et al.


Motswaledi H.M.,University of Limpopo | Monyemangene F.M.,University of Limpopo | Maloba B.R.,National Health Laboratory Services NHLS | Nemutavhanani D.L.,National Health Laboratory Services NHLS
International Journal of Dermatology | Year: 2012

Background Blastomycosis is a chronic granulomatous and suppurative mycosis caused by the fungus Blastomyces dermatitidis. This is a dimorphic fungus, which exists as a non-pathogenic mold in mycelial form in nature and converts to pathogenic yeast at body temperature. Infection is acquired by either inhalation or inoculation. We report a case of blastomycosis with severe involvement of the scalp, face, and neck, with no evidence of systemic involvement. Methods Biopsy specimen was stained with hematoxylin and eosin, periodic acid-Schiff (PAS), PAS with diastase digestion, and Grocott. Culture was performed on a Sabouraud's dextrose agar plate using an aseptic technique as per standard operating procedure for processing mycology specimens at our institution. A lactophenol cotton blue preparation from the cultured material was performed. Results Histopathologic examination showed pseudoepitheliomatous hyperplasia and a granulomatous inflammation with round to oval organisms, with refractile cell walls in the cytoplasm of giant cells. PAS, PAS with diastase digestion, and Grocott stains enhanced the organisms. Cultured material showed growth after 10days, and the lactophenol cotton blue preparation from the cultured material showed the organism to be Blastomyces dermatitidis. Sensitivity studies favored treatment with itraconazole. Radiological examination of the patient showed no evidence of systemic involvement. Conclusions Our case may represent the rare primary cutaneous inoculation blastomycosis as lesions started on an area of previous trauma. Treatment with itraconazole was successful. © 2012 The International Society of Dermatology.


Cordier W.,University of Pretoria | Cromarty A.D.,University of Pretoria | Botha E.,National Health Laboratory Services NHLS | Steenkamp V.,University of Pretoria
Human and Experimental Toxicology | Year: 2012

The use of herbal preparations for staunching blood flow and reducing the risk of vascular thrombosis is common worldwide. In this study, aqueous and methanolic extracts of plants used to treat blood-associated complaints were investigated to determine their effects on red blood cell haemolysis and coagulation. The extent of haemolysis was determined spectrophotometrically. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) as indicators of coagulation rate were determined using a coagulatometer. All of the plant extracts tested had a significant effect on coagulation time, prolonging the aPTT. Cassia petersiana had the greatest prolonging effect on PT compared to the control, phosphate buffered saline (PBS). As all of the herbal extracts tested had a delaying effect on coagulation, patients using herbal/plant therapies should be cautioned to stop their medication before surgery. © SAGE Publications 2012.

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