Optical micro-manipulation in HIV-1 infected cells for improved HIV-1 treatment and diagnosis

Laser application in the field of biological and medical sciences has significantly grown, thereby strengthening the field of Biophotonics. Research conducted in Biophotonics focuses on the concept of using light especially in the visible and near infrared regions of the electromagnetic radiation for the evaluation of living systems. In this thesis new discoveries are presented about low level laser therapy, optical trapping, transmission spectroscopy, luminescence spectroscopy and structured illumination microscopy (SIM), displaying the impact each technique has on HIV infected cells. The results showed that the irradiation of HIV-1 infected TZM-bl cells with low power red laser reduces HIV-1 infection. The outcomes of this study further proved that when irradiation is used in conjunction with efavirenz, an antiretroviral drug, HIV-1 infection could be reduced to undetectable levels in TZM-bl cells. Through the coupling of transmission spectroscopy with optical trapping, and separately, use of luminescence spectroscopy, label free diagnosis of HIV in infected cell samples was achieved. This finding affirms that HIV-1 infection can be detected in a label free manner when using laser based techniques. Furthermore, the photoluminescence spectrometer system was employed to generate a decay curve, which was necessary so as to have some understanding on lifetime of the luminescent signal in infected TZM-bl cells. Finally, in order to confirm that indeed TZM-bl cells were infected, an established super-resolution microscopy system SIM was used to detect HIV-1 infection in TZM-bl cells. Indeed in the infected cells viral molecules p24 and gp41 were detected through SIM, while they were not detected in uninfected cells. In future studies, super resolution microscopy would be coupled to an optical trapping system in order to confirm that each trapped cells is whether infected or uninfected so as to improve HIV diagnosis.College of Science, Engineering and TechnologyPh. D. (Science, Engineering and Technology

A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR + Ki67 + on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67 + HLA-DR + Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67 + HLA-DR + T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ + IL2 + TNFα + CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67 + HLA-DR + ) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment

Using sodium bisulphite treatment and PCR to construct mammalian anti-HIV-1 long hairpin RNA expression cassettes

M.Tech.RNA interference (RNAi) is a gene silencing mechanism that uses short RNA duplexes to block gene expression. This mechanism has been widely explored to determine functions of genes. Furthermore, this phenomenon has been used to silence unwanted genes such as viral genes. RNAi has been successfully employed in non-mammalian organisms such as plants, where long dsRNAs (more than 30 bp) have been used without inducing non-specific effects. However, in mammalian cells, cytoplasmic dsRNAs of more than 30 bp trigger non-specific induction of many genes, which may result from the activation of dsRNA-dependent protein kinase (PKR) and 2’,5’-oligoadenylate synthetase (2’,5’-OAS), via the interferon response pathway. In this study, we describe a novel and simple strategy to overcome nonspecific effects induced by longer RNA duplexes. This strategy uses sodium bisulphite which is a mutagen that deaminates cytosine residue to uracil residues in order to introduce mutations in the sense strand of the duplex. Introduction of these mutations results in the formation of G:U pairings between the sense and antisense strands of the long hairpin RNA. RNA duplexes with mismatches have been shown to be able to prevent interferon induction in mammalian cells. According to the obtained results, long hairpins RNA with and without mismatches were unable to inhibit the expression of the target region, which was the U5 region of the HIV-1 subtype C LTR. The U5 region of the LTR is actively involved in the reverse transcription of HIV-1. Therefore silencing of this region would have led to the inhibition or reverse transcription blockage. Furthermore, data showed that the interferon response was induced when using these long hairpin RNA duplexes. Due to the sensitivity of mammalian cells, the action of sodium bisulphite could have stimulated certain genes of the interferon pathway. Even though hairpins constructed in this study were unable to prevent the induction of the interferon response pathway and also could not silence the target, this strategy of using sodium bisulphite has a great potential as shown by its ability to induce changes in cytosine residues and leaving other nucleotides unchanged

Point of Care Diagnostics for HIV in Resource Limited Settings: An Overview

Human immunodeficiency virus (HIV) is a global health problem. Early diagnosis, rapid antiretroviral therapy (ART) initiation and monitoring of viral load are the key strategies for effective HIV management. Many people in resource limited settings where timely access to medical care is a challenge and healthcare infrastructure is poor have no access to laboratory facilities and diagnosis is dependent on the presence of point of care (POC) devices. POC instruments have shown to be easy to operate, maintain and transport and can easily be operated by less skilled health workers. Additionally, POC tests do not require laboratory technicians to operate. POC devices have resulted in a growing number of people testing for HIV and thereby receiving treatment early. In recent years, there has been great improvement in the development of POC technologies for early HIV diagnosis, HIV viral load and cluster of differentiation 4 (CD4) measurement. This review discusses POC technologies that are currently available and in the pipeline for diagnosing and monitoring HIV. We also give an overview of the technical and commercialization challenges in POC diagnostics for HIV

Nanotechnology-Based Diagnostics for Diseases Prevalent in Developing Countries: Current Advances in Point-of-Care Tests

The introduction of point-of-care testing (POCT) has revolutionized medical testing by allowing for simple tests to be conducted near the patient’s care point, rather than being confined to a medical laboratory. This has been especially beneficial for developing countries with limited infrastructure, where testing often involves sending specimens off-site and waiting for hours or days for results. However, the development of POCT devices has been challenging, with simplicity, accuracy, and cost-effectiveness being key factors in making these tests feasible. Nanotechnology has played a crucial role in achieving this goal, by not only making the tests possible but also masking their complexity. In this article, recent developments in POCT devices that benefit from nanotechnology are discussed. Microfluidics and lab-on-a-chip technologies are highlighted as major drivers of point-of-care testing, particularly in infectious disease diagnosis. These technologies enable various bioassays to be used at the point of care. The article also addresses the challenges faced by these technological advances and interesting future trends. The benefits of point-of-care testing are significant, especially in developing countries where medical care is shifting towards prevention, early detection, and managing chronic conditions. Infectious disease tests at the point of care in low-income countries can lead to prompt treatment, preventing infections from spreading

A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion.

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR+ Ki67+ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67+ HLA-DR+ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67+ HLA-DR+ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ+ IL2+ TNFα+ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67+ HLA-DR+) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment

Quest Volume 19 Number 3

Generation SPACE: South Africa’s contribution to a new generation of space exploration; SA’s first National Space Conference: Celebrating South Africa’s ‘brilliant’ space future; A celestial journey: From stargazing in Ancient Africa to the James Webb Space Telescope (JWST) era; The #AfricaLookup campaign: Protecting our dark skies; Science in (Karoo) silence: Breaking ground on the future of ground-breaking space science; Turning stars into stories: The power of the naked eye to observe celestial objects; Careers focus: Exploring careers in astronomy and space science: Schools competition!; Win a trip to visit the SKA-Mid site in the Karoo; The power of solid-state lasers: A new way of exploring nature; Africa’s big dream: Making our own vaccines: Breaking barriers, blooming strength: Towards women’s health empowerment; NSTF Brilliants Programme winner profile: Matimba Manganyi is ready to join Generation SPACE; Chandrayaan-3: India puts lunar rover on the moon’s south pole - a historic first; Moon base incoming: South Africa joins China’s International Lunar Research Station; Home grown satellites: Dragonfly Aerospace ensures EOS SAT-1 sustainability with Neuraspace partnership.Academy of Science of South Africa: Department of Science and Innovatio

ROC curve analysis of mycobacteria-specific Ki-67⁺HLA-DR⁺ CD4 T cells with time to sputum culture conversion.

<p>Receiver Operating Characteristic curve for the proportion of mycobacteria-specific Ki-67⁺HLA-DR⁺ CD4 T cells at baseline with the time to sputum clearance. The area under the curve (AUC), p-value and 95% confidence interval (c.i.) are shown on the graph. The dotted line represents an AUC of 0.5, which would depict a random test.</p

Comparison of the frequencies of Mtb, PPD and mitogen responsive CD4 T cells at baseline between patients who were sputum culture (SC) negative or positive.

<p>(A) The magnitude of antigen-specific CD4 T cells expressing any of the 3 cytokines measured (i.e. IFNγ, IL2 and TNFα) and expressed as a % of total CD4 population. Open symbols represent SC negative patients (n = 6) and solid symbols represent SC positive patients (n = 12). The horizontal line shows the median. Non-Parametric Mann-Whitney t-test was used for statistical comparisons. (B) Representative flow cytometry dot plots showing the level of expression of IFNγ, IL2 and TNFα after no stimulation (NS), Mtb peptides, PPD and mitogen (MITO) in the whole blood ICS assay. The numbers in the quadrant represent the frequency of cells expressing each cytokine. The gating strategy is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102178#pone.0102178.s001" target="_blank">Figure S1</a>.</p

Changes in the proportion of antigen-specific CD4 T cells co-expressing HLA-DR and Ki67 over time of chemotherapy in individuals with positive SC at baseline.

<p>(A) Representative flow cytometry dot-plots showing the level of Ki-67 and HLA-DR co-expression within PPD-specific CD4 T cells at baseline (BL), 2, 4 and 6 months (M) of treatment. The numbers in the quadrants represent the proportion of antigen-specific CD4 T cells co-expressing Ki-67 and HLA-DR. (B) Proportion of activated (Ki-67⁺HLA-DR⁺) cells within Mtb, PPD and mitogen responsive CD4 T cells over time (months). The statistical differences were assessed using Wilcoxon matched pairs test.</p