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Computational search for nature-derived dual-action inhibitors of HIV-1 reverse transcriptase and integrase: a potential strategy to mitigate drug resistance progression

  • Authors: Mwiinga, Luyando
  • Date: 2024-10-11
  • Subjects: HIV (Viruses) , Reverse transcriptase , Antiretroviral agents , RDKit , Drug resistance , Docking
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/463930 , vital:76458
  • Description: Human immunodeficiency virus Type 1 (HIV-1) is a devastating viral infection affecting millions worldwide and presents significant challenges in treatment and management. In 2022, approximately 39 million people were living with HIV with Sub-Saharan Africa having two thirds of these infections. Devastatingly, there were approximately 300 000 HIV/AIDS related deaths in Sub-Saharan Africa alone in 2022 alone. Antiretroviral therapy (ART) which is fundamental for HIV treatment, comprises of a combination of drugs such as nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTs), protease inhibitors (PIs) and integrase strand transfer inhibitors (INSTIs). However, although 28.7 million people out of the estimated 38.4 million people living with HIV in 2021 were receiving ART, the emergence of drug-resistant strains further complicates treatment efforts, highlighting the need for novel therapeutic approaches. This study aimed to address the challenges raised by drug resistance and significant side effects by identifying potential dual inhibitors against HIV-1 Reverse Transcriptase (RT) and Integrase (IN) using in silico techniques. RT RNase H and IN were chosen as targets for their shared dependency on Mg2+ ions within their active sites, which are crucial for catalytic activity. The selection of dual inhibitors was motivated by the fact that the virus would need to replicate at two points simultaneously to develop resistance, making it less likely. The objectives of this study included the creation of a natural derivative compound library using RDKit with the aid of SciFinder, utilizing (-)-epigallocatechin-3-O-gallate (EGCG), because of its dual inhibitory effects against RT and IN, as indicated by a study conducted by Sanna et al. 2019. The natural derivatives were chosen to take advantage of their chemical diversity and to explore potential novel therapeutic options for combating HIV drug resistance. The compound library created comprised of 125 203 compounds. Then docking studies were conducted to assess proteinligand binding. After the correlation of the RT and IN docking studies, 288 compounds were filtered to have potential dual inhibitory activity. Then quantitative estimation of druggability (QED) analysis identified three compounds with superior properties compared to EGCG and FDAapproved drug raltegravir (RAL). Molecular docking simulations revealed interactions between the inhibitors and the key active site residues of RT and IN, along with the chelation of at least one 3 Mg2+, suggesting the potential for enzymatic disruption. Furthermore, molecular dynamic (MD) simulations were then conducted to assess protein-ligand system behavior, through RMSD and RMSF analysis. The RMSD analysis uncovered instability in the IN-Sci30703 complex, leading to its exclusion as a potential dual action inhibitor. RMSF analysis for IN showed that all the inhibitors had the ability to limit the flexibility of the catalytic loop which is essential for catalytic activity. Therefore, further in vitro studies are required to evaluate the effectiveness of the remaining two EGCG derivatives (Sci33211 and Sci48919) in inhibiting RT and IN through the chelation of at least one Mg2+ ion to determine if they have superior dual inhibitory effects compared to EGCG. This study adds to the ongoing efforts to develop effective strategies against HIV-1 drug resistance and emphasizes the importance of continued research in this field. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
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  • Date Issued: 2024-10-11

Quinolone-Pyrazinamide Derivatives: synthesis, characterisation, in silico ADME analysis and in vitro biological evaluation against Mycobacterium tuberculosis

  • Authors: Rukweza, Kudakwashe Gerald
  • Date: 2023-10-13
  • Subjects: Quinolone antibacterial agents , Mycobacterium tuberculosis , Antitubercular agents , Tuberculosis Chemotherapy , Drug resistance , Moxifloxacin , Isoniazid
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/390901 , vital:68596
  • Description: Tuberculosis is one of the leading causes of death worldwide caused by an infectious species, Mycobacterium tuberculosis (Mtb). Some of the factors that contribute to the prevalence of this disease include the complexity of diagnosis, prolonged period of therapy, side effects associated with current TB drugs, the prevalence of resistance against the current treatment options and a high incidence of co-infection with HIV/AIDS. Thus, there is a need for new alternative drugs to provide safer and shorter treatment therapy options that are not susceptible to the development of drug resistance. In this project, we focus our attention on the quinolone pharmacophore. Quinolones are currently used as alternative options in the treatment of resistant strains of Mtb. Previous work pertaining to quinolone-isoniazid hybrid compounds showed promising in vitro activity against the H37Rv strain of Mtb and served as the inspiration to pursue this project. The journey commenced with the synthesis of quinolone-pyrazinamide hybrid compounds (Figure 3.1). These compounds were synthesised, through the attachment of the quinolone and the pyrazinamide entity through a hydrazine linker. The synthesised compounds were purified, and their structural identity confirmed using common spectroscopic techniques including 1H and 13C NMR, infra-red (IR) and mass spectrometry. In vitro biological assays were performed by testing for the activity against the H37RvMA strain of Mtb. The bioassays were performed in triplicates to ensure the accuracy of the results. Moxifloxacin and isoniazid were tested as control compounds. Finally, the resultant compounds were profiled in silico for physicochemical and ADMET properties using open access software SwissADME. All the synthesised compounds 3.8a-f showed no activity against H37RvMA. In most cases, the resulting compounds showed minimal to no activity (MICs ≥ 57.3 μM) in all three media. During the in vitro studies, the compounds showed significant precipitation in the media over time suggesting poor aqueous solubility. The SwissADME analysis of these compounds indicated poor solubility in aqueous media, which is likely linked to their molecular size and complexity. Despite poor aqueous solubility, compounds 3.8a-f showed acceptable physicochemical properties and ADME parameters. No PAINs (Pan-assay interference compounds) were observed. Minimal to no interaction with CYP enzymes were predicted. Most of the compounds were compatible with the Lipinski’s rules of five. , Thesis (MSc) -- Faculty of Science, Pharmacy, 2023
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  • Date Issued: 2023-10-13

The development of a plate-based assay to detect the activation status of ARF1 GTPase in Plasmodium falciparum parasites

  • Authors: Du Toit, Skye Carol
  • Date: 2023-10-13
  • Subjects: ARF1 , GTPase , Plasmodium falciparum , Malaria , Drug resistance , Drug targeting , Enzyme-linked immunosorbent assay , Proteins
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/424654 , vital:72172
  • Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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  • Date Issued: 2023-10-13

Identification of novel compounds against Plasmodium falciparum Cytochrome bc1 Complex inhibiting the trans-membrane electron transfer pathway: an In Silico study

  • Authors: Chebon, Lorna Jemosop
  • Date: 2022-10-14
  • Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
  • Language: English
  • Type: Academic theses , Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
  • Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
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  • Date Issued: 2022-10-14

The characterization of GTP Cyclohydrolase I and 6-Pyruvoyl Tetrahydropterin Synthase enzymes as potential anti-malarial drug targets

  • Authors: Khairallah, Afrah Yousif Huseein
  • Date: 2022-04-08
  • Subjects: Antimalarials , Plasmodium falciparum , Malaria Chemotherapy , Malaria Africa , Drug resistance , Drug development , Molecular dynamics
  • Language: English
  • Type: Doctoral thesis , text
  • Identifier: http://hdl.handle.net/10962/233784 , vital:50127 , DOI 10.21504/10962/233784
  • Description: Malaria remains a public health problem and a high burden of disease, especially in developing countries. The unicellular protozoan malaria parasite of the genus Plasmodium infects about a quarter of a billion people annually, with an estimated 409 000 death cases. The majority of malaria cases occurred in Africa; hence, the region is regarded as endemic for malaria. Global efforts to eradicate the disease led to a decrease in morbidity and mortality rates. However, an enormous burden of malaria infection remains, and it cannot go unnoticed. Countries with limited resources are more affected by the disease, mainly on its public health and socio-economic development, due to many factors besides malaria itself, such as lack of access to adequate, affordable treatments and preventative regimes. Furthermore, the current antimalarial drugs are losing their efficacy because of parasite drug resistance. The emerged drug resistance has reduced the drug efficacy in clearing the parasite from the host system, causing prolonged illness and a higher risk of death. Therefore, the emerged antimalarial drug resistance has hindered the global efforts for malaria control and elimination and established an urgent need for new treatment strategies. When the resistance against classical antimalarial drugs emerged, the class of antifolate antimalarial medicines became the most common alternative. The antifolate antimalarial drugs target the malaria parasite de novo folate biosynthesis pathway by limiting folate derivates, which are essential for the parasite cell growth and survival. Yet again, the malaria parasite developed resistance against the available antifolate drugs, rendering the drugs ineffective in many cases. Given the previous success in targeting the malaria parasite de novo folate biosynthesis pathway, alternative enzymes within this pathway stand as good targets and can be explored to develop new antifolate drugs with novel mechanisms of action. The primary focus of this thesis is to contribute to the existing and growing knowledge of antimalarial drug discovery. The study aims to characterise the malaria parasite de novo folate synthesis pathway enzymes guanosine-5'-triphosphate (GTP) cyclohydrolase I (GCH1) and 6-pyruvoyl tetrahydropterin synthase (PTPS) as alternative drug targets for malaria treatment by using computational approaches. Further, discover new allosteric drug targeting sites within the two enzymes' 3D structures for future drug design and discovery. Sequence and structural analysis were carried out to characterise and pinpoint the two enzymes' unique sequence and structure-based features. From the analyses, key sequence and structure differences were identified between the malaria parasite enzymes relative to their human homolog; the identified sites can aid significantly in designing and developing new antimalarial antifolate drugs with good selectivity toward the parasites’ enzymes. GCH1 and PTPS contain a catalytically essential metal ion in their active site; therefore, force field parameters were needed to study their active sites accurately during all-atom molecular dynamic simulations (MD). The force field parameters were derived through quantum mechanics potential energy surface scans of the metals bonded terms and evaluated via all-atom MD simulations. Proteins structural dynamics is imperative for many biological processes; thus, it is essential to consider the structural dynamics of proteins whilst understanding their function. In this regard, the normal mode analysis (NMA) approach based on the elastic network model (ENM) was employed to study the intrinsic dynamics and conformations changes of GCH1 and PTPS enzymes. The NMA disclosed essential structural information about the protein’s intrinsic dynamics and mechanism of allosteric modulation of their binding properties, further highlighting regions that govern their conformational changes. The analysis also disclosed hotspot residues that are crucial for the proteins' fold stability and function. The NMA was further combined with sequence motif results and showed that conserved residues of GCH1 and PTPS were located within the identified key structural sites modulating the proteins' conformational rearrangement. The characterized structural features and hotspot residues were regarded as potential allosteric sites of important value for the design and development of allosteric drugs. Both GCH1 and PTPS enzymes have never been targeted before and can provide an excellent opportunity to overcome the antimalarial antifolate drug resistance problem. The data presented in this thesis contribute to the understanding of the sequence, structure, and global dynamics of both GCH1 and PTPS, further disclose potential allosteric drug targeting sites and unique structural features of both enzymes that can establish a solid starting point for drug design and development of new antimalarial drugs of a novel mechanism of actions. Lastly, the reported force field parameters will be of value for MD simulations for future in-silico drug discovery studies involving the two enzymes and other enzymes with the same Zn2+ binding motifs and coordination environments. The impact of this research can facilitate the discovery of new effective antimalarial medicines with novel mechanisms of action. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
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  • Date Issued: 2022-04-08

A retrospective study of antimicrobial prescribing practices in paediatric patients at the Mahalapye District Hospital, Central Botswana

  • Authors: Nyawera, Angella
  • Date: 2022-04-06
  • Subjects: Anti-infective agents Botswana Mahalapye , Drug resistance , Pediatrics Botswana Mahalapye , Pediatrics Formulae, receipts, prescriptions , Drugs Prescribing Moral and ethical aspects
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/290682 , vital:56774
  • Description: Background: The development of antimicrobial resistance (AMR) has been linked to the increased and irrational use of antimicrobial medicines. The aim of this study was to investigate the antimicrobial prescribing practices in the paediatric medical ward at Mahalapye District Hospital (MDH) in Botswana and to determine whether antimicrobial stewardship (AMS) measures were being implemented at the hospital. Methods A cross-sectional, descriptive, mixed methods, observational approach was taken in this study. The study site was the paediatric medical ward (PMW) at MDH. Information about the antimicrobials prescribed for paediatric patients from January 2018 to December 2018 was collected from patients’ information files and compared to national antimicrobial prescribing guidelines to determine prescribers’ adherence. Qualitative semi-structured interviews were conducted with members of staff at MDH to determine whether antimicrobial stewardship (AMS) measures were adopted at the hospital. Results A total of 278 patients were included in this study, 12 of these were admitted twice during the study period. In total 290 admissions were analysed, with 659 antimicrobial medicines prescribed. The most common diagnoses were pneumonia (36.9%), acute gastroenteritis (20.7%), upper respiratory tract infections (3.4%), and bronchiolitis (3.1%). The most prescribed antimicrobials were ampicillin (21.4%), gentamicin (21.2%), and cefotaxime (8.3%). Adherence to guidelines was relatively good, with 82.7% of antimicrobials prescribed for the patients in the study having been prescribed in compliance with the national prescribing guidelines. The semi-structured interviews highlighted the fact that staff knew about AMS and AMR in general, however awareness of an AMS committee at MDH varied. The AMS committee was a multidisciplinary committee, which was a subcommittee of the Drugs and Therapeutics Committee (DTC). Discussion and Conclusion The results suggest that adherence to prescribing guidelines was relatively high compared to other paediatric antimicrobial utilisation studies in African countries. Prescribing of antimicrobial medicines was consistent with other African countries. The long period of time that it takes for microbiological test results to become available means that most prescribers rely on empirical prescribing. The antimicrobial committee is a multidisciplinary committee with defined roles for its members, consistent with international guidelines for implementing an AMS committee at a hospital. , Thesis (MPharm) -- Faculty of Pharmacy, Pharmacy, 2022
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  • Date Issued: 2022-04-06

Creating digital materials for Antimicrobial Resistance One Health awareness and behaviour change for Rhodes University peer educators

  • Authors: Patnala, Shraddha
  • Date: 2021-10-29
  • Subjects: Anti-infective agents South Africa , Drug resistance , Antibiotics , Drug resistance in microorganisms , Health education South Africa , Health risk communication South Africa , Digital media South Africa , Peer counseling South Africa , One Health (Initiative) , Social Behaviour Change Communication (SBCC) , Rhodes University
  • Language: English
  • Type: Master's theses , text
  • Identifier: http://hdl.handle.net/10962/191001 , vital:45048
  • Description: Antimicrobial resistance (AMR) is an urgent, global health problem that stems from the inappropriate use of and poor adherence to antibiotics that treat diseases in human beings. It is further exacerbated by the proliferation of antibiotics into the food chain, particularly from the overuse and misuse of antibiotics in agricultural, meat, and dairy production. The recently developed World Health Organisation (WHO) One Health (OH) approach encompasses and acknowledges the various interconnected pathways that drive AMR between the human, animal, and environmental spheres. Until recently, AMR health challenges have been viewed primarily through a biomedical lens, but this study draws on the more holistic perspective that the One Health approach offers. AMR from food sources (AMR-OH) is an underrepresented topic of research. Creating digital health communication for low-literate end-users on this topic using the One Health approach is an emerging field of research. AMR-OH has not been extensively covered in health communication campaigns and requires developing context-specific digital educational materials, such as the ones this study presents. This study draws on Social Behaviour Change Communication (SBCC) theory elements to create a suggested approach to disseminate AMR-OH information. This intervention was aimed at low-health-literate end-users to accomplish two objectives. First, create awareness and improve knowledge about AMR-OH via a video. Second, offer feasible, easily implementable behaviour change actions in the form of an infographic comprising four food safety steps (Clean, Separate, Cook, and Chill). The study was conducted in three phases. First, recruit participants and conduct a literature review to identify the effective SBCC elements of health communication intervention design. Second, conduct a needs assessment to gauge the volunteering participants’ familiarity with digital media and their current health literacy on AMR-OH. Third, conceptualise and design the two AMR-OH digital educational materials (a video and accompanying infographic). The materials were first evaluated by the researcher using the Clear Communication Index (CCI) test, and then shared with the participants via WhatsApp to be evaluated by them, using two end-user tests: the Patient Education Material Assessment Tool (PEMAT) and the Suitability Assessment of Materials (SAM) test. These two tests assessed the materials’ readability, understandability, and actionability. A post-evaluation, semi-structured interview (SSI) was then conducted with the participants. Deductive thematic analysis was conducted on the SSI data and analysed using the five design benchmarks as themes: Ease of Use of Technology, Clarity of Content, Appropriate Format, Target Audience Resonance (Appropriate for target audience), and Clear calls to Action (Actionable). The rapid onset of COVID-19 restrictions forced the project to scale down and shift entirely online. The study could be conducted due to the active and enthusiastic virtual participation of two Rhodes University Peer Educators (PEs) whose contribution was vital to developing and evaluating the materials. The needs assessment showed that the PEs were comfortable using WhatsApp, had reliable internet connection when on campus, and used this social media platform for professional and personal communication. This assessment also showed that they had prior knowledge of AMR but only from the human health perspective. The video and infographic scored high on the Clear Communication Index, 93.3% and 94.4%, respectively. The PEs’ evaluation of the materials was also high on the PEMAT and SAM assessments: video narration (100%, 80% respectively), video (100%, 99% respectively), and infographic (86%, 90% respectively). This study produced an easy-to-use, accessible and appropriate online repository of AMR-OH information in a novel format with actionable steps. The post-evaluation SSI revealed that the materials and the channel of delivery were welcomed. The PEs expressed their confidence in receiving, using, and sharing this novel presentation of evidence and solutions-based information about AMR-OH. They further highlighted that this is the first time they have received and evaluated context-specific digital multimedia about AMR-OH and that this information equipped them to adopt the food safety behaviours – namely, the four food safety steps. This study demonstrates that the theory-informed creation of engaging digital media for AMR-OH is feasible and viable. Furthermore, it affirms that engaging digital media for AMR-OH can be created to enhance the knowledge of end-users about this health issue. The scaled-down approach created a blueprint to implement a more extensive intervention in the future, informed by this intervention’s methods and tools. Lastly, this blueprint for a particular conceptualisation of an AMR-OH digital media intervention provides effective and empowering tools with which the PEs can disseminate this information to the university's support staff. , Thesis (MA) -- Faculty of Humanities, School of Journalism and Media Studies, 2021
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  • Date Issued: 2021-10-29

Application of machine learning, molecular modelling and structural data mining against antiretroviral drug resistance in HIV-1

  • Authors: Sheik Amamuddy, Olivier Serge André
  • Date: 2020
  • Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
  • Language: English
  • Type: text , Thesis , Doctoral , PhD
  • Identifier: http://hdl.handle.net/10962/115964 , vital:34282
  • Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
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  • Date Issued: 2020

Understanding the underlying resistance mechanism of Mycobacterium tuberculosis against Rifampicin by analyzing mutant DNA - directed RNA polymerase proteins via bioinformatics approaches

  • Authors: Monama, Mokgerwa Zacharia
  • Date: 2020
  • Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
  • Language: English
  • Type: text , Thesis , Masters , MSc
  • Identifier: http://hdl.handle.net/10962/167508 , vital:41487
  • Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
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  • Date Issued: 2020

Investigating assay formats for screening malaria Hsp90-Hop interaction inhibitors

  • Authors: Derry, Leigh-Anne Tracy Kim
  • Date: 2019
  • Subjects: Antimalarials , Heat shock proteins , Drug interactions , Drug resistance , Plasmodium falciparum , High throughput screening (Drug development) , Bioluminescence resonance energy transfer (BRET) , Fluorescence resonance energy transfer (FRET)
  • Language: English
  • Type: text , Thesis , Masters , MSc
  • Identifier: http://hdl.handle.net/10962/63345 , vital:28395
  • Description: Although significant gains have been made in the combat against malaria in the last decade, the persistent threat of drug and insecticide resistance continues to motivate the search for new classes of antimalarial drug compounds and targets. Due to their predominance in cellular reactions, protein-protein interactions (P-PIs) are emerging as a promising general target class for therapeutic development. The P-PI which is the focus of this project is the interaction between the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Hsp70/Hsp90 organising protein (Hop). Hop binds to Hsp70 and Hsp90 and facilitates the transfer of client proteins (proteins undergoing folding) from the former to the latter and also regulates nucleotide exchange on Hsp90. Due to its role in correcting protein misfolding during cell stress, Hsp90 is being pursued as a cancer drug target and compounds that inhibit its ATPase activity have entered clinical trials. However, it has been proposed that inhibiting the interaction between Hsp90 and Hop may be alternative approach for inhibiting Hsp90 function for cancer therapy. The malaria parasite Plasmodium falciparum experiences temperature fluctuations during vector-host transitions and febrile episodes and cell stress due to rapid growth and immune responses. Hence, it also depends on chaperones, including PfHsp90, to maintain protein functionality and pathogenesis, demonstrated inter alia by the sensitivity of parasites to Hsp90 inhibitors. In addition, PfHsp90 exists as a complex with the malarial Hop homologue, PfHop, in parasite lysates. Consequently, the purpose of this study was to explore P-PI assay formats that can confirm the interaction of PfHsp90 and PfHop and can be used to identify inhibitors of the interaction, preferably in a medium- to high-throughput screening mode. As a first approach, cell-based bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays were performed in HeLa cells. To facilitate this, expression plasmid constructs containing coding sequences of P. falciparum and mammalian Hsp90 and Hop and their interacting domains (Hsp90 C-domain and Hop TPR2A domain) fused to the BRET and FRET reporter proteins – yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and Renilla luciferase (Rluc) - were prepared and used for HeLa cell transient transfections. The FRET assay produced positive interaction signals for the full-length P. falciparum and mammalian Hsp90-Hop interactions. However, C-domain-TPR2A domain interactions were not detected, no interactions could be demonstrated with the BRET assay and western blotting experiments failed to detect expression of all the interaction partners in transiently transfected HeLa cells. Consequently, an alternative in vitro FRET assay format using recombinant proteins was investigated. Expression constructs for the P. falciparum and mammalian C-domains and TPR2A domains fused respectively to YFP and CFP were prepared and the corresponding fusion proteins expressed and purified from E. coli. No interaction was found with the mammalian interaction partners, but interaction of the P. falciparum C-domain and TPR2A domain was consistently detected with a robust Z’ factor value of 0.54. A peptide corresponding to the PfTPR2A domain sequence primarily responsible for Hsp90 binding (based on a human TPR2A peptide described by Horibe et al., 2011) was designed and showed dose-dependent inhibition of the interaction, with 53.7% inhibition at 100 μM. The components of the assay are limited to the purified recombinant proteins, requires minimal liquid steps and may thus be a useful primary screening format for identifying inhibitors of P. falciparum Hsp90-Hop interaction.
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  • Date Issued: 2019

Synthesis, characterisation and evaluation of benzoxaborole-based hybrids as antiplasmodial agents

  • Authors: Gumbo, Maureen
  • Date: 2017
  • Subjects: Malaria Chemotherapy , Antimalarials , Boron compounds , Drug resistance , Plasmodium falciparum , Drug development
  • Language: English
  • Type: Master's theses , text
  • Identifier: http://hdl.handle.net/10962/59193 , vital:27456
  • Description: Malaria is a mosquito-borne disease, which continues to pose a threat to the entire humanity. About 40% of the world population is estimated to be at risk of infections by malaria. Despite efforts undertaken by scientific community, government entities and international organizations, malaria is still rampant. The major problem is drug resistance, where the Plasmodium spp have over the past decades developed drug resistance against available drugs. In order to counter this problem, novel antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Benzoxaborole derivatives have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported on the compounds such as 6-(2- (alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles, which showed good antimalarial activity against both W7 and 3D7 strains without significant toxicity. On the other hand, chloroquine (CQ) and cinnamic acids have a wide variety of biological activity including antimalarial activity. Herein, a hybridisation strategy was employed to synthesise new CQ-benzoxaborole and cinnamoyl-benzoxaborole hybrids. CQ-Benzoxaborole 2.12a-c and cinnamoylbenzoxaborole 2.11a-g hydrid molecules were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (1H and 13C NMR, and mass spectrometry). CQ-benzoxaborole compounds, however, showed instability, and only 2.12b was used for in vitro biological assay and showed activity comparable to CQ. Furthermore, in vitro biological assay revealed that compounds 2.11a-g poorly inhibited the growth of P. falciparum parasites. Interestingly, these compounds, however, exhibited satisfactory activity against Trypanosoma brucei with IC50 = 0.052 μM for compound 2.11g. The cell cytotoxicity assay of all final compounds confirmed that all CQ-benzoxaborole 2.12b and cinnamoyl-benzoxaborole 2.11a-g hybrids were non-toxic against HeLa cell lines. However, efforts to further expand the structure-activity relationship (SAR) of CQbenzoxaborole by increasing the length of the linker with one extra carbon (Scheme 2.10) were not possible as an important precursor 6-formylbenzoxaborole 2.29 could not be synthesized in sufficient yields. , Thesis (MSc) -- Faculty of Faculty of Science, Chemistry, 2017
  • Full Text:
  • Date Issued: 2017

An in-silico investigation of Morita-Baylis-Hillman accessible heterocyclic analogues for applications as novel HIV-1 C protease inhibitors

  • Authors: Sigauke, Lester Takunda
  • Date: 2015
  • Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , HIV infections , Drug resistance , Cheminformatics
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:4152 , http://hdl.handle.net/10962/d1017913
  • Description: Cheminformatic approaches have been employed to optimize the bis-coumarin scaffold identified by Onywera et al. (2012) as a potential hit against the protease HIV-1 protein. The Open Babel library of commands was used to access functions that were incorporated into a markov chain recursive program that generated 17750 analogues of the bis-coumarin scaffold. The Morita-Baylis-Hillman accessible heterocycles were used to introduce structural diversity within the virtual library. In silico high through-put virtual screening using AutoDock Vina was used to rapidly screen the virtual library ligand set against 61 protease models built by Onywera et al. (2012). CheS-Mapper computed a principle component analysis of the compounds based on 13 selected chemical descriptors. The compounds were plotted against the principle component analysis within a 3 dimensional chemical space in order to inspect the diversity of the virtual library. The physicochemical properties and binding affinities were used to identify the top 3 performing ligands. ACPYPE was used to inspect the constitutional properties and eliminated virtual compounds that possessed open valences. Chromene based ligand 805 and ligand 6610 were selected as the lead candidates from the high-throughput virtual screening procedure we employed. Molecular dynamic simulations of the lead candidates performed for 5 ns allowed the stability of the ligand protein complexes with protease model 305152. The free energy of binding of the leads with protease model 305152 was computed over the first 50 ps of simulation using the molecular mechanics Poisson-Boltzmann method. Analysis structural features and energy profiles from molecular dynamic simulations of the protein–ligand complexes indicated that although ligand 805 had a weaker binding affinity in terms of docking, it outperformed ligand 6610 in terms of complex stability and free energy of binding. Medicinal chemistry approaches will be used to optimize the lead candidates before their analogues will be synthesized and assayed for in vivo protease activity.
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  • Date Issued: 2015

Comparative study of the effect of silver nanoparticles on the hexokinase activity from human and Trypanosoma brucei

Studies towards the development of novel antimalarial agents

  • Authors: Adeyemi, Christiana Modupe
  • Date: 2015
  • Subjects: Antimalarials , Malaria , Drug resistance , Drug development , Enzyme inhibitors , Plasmodium
  • Language: English
  • Type: text , Thesis , Masters , MSc
  • Identifier: http://hdl.handle.net/10962/54645 , vital:26596
  • Description: Considerable efforts have been made in the modification of existing antimalarial drugs, and the support of incentive programmes have led to a drastic decrease in malaria cases reported by WHO during the past 6 years. However, the development of drug resistance threatens the eradication of this deadly disease and has prompted research on the synthesis of novel antimalarial drugs. Our research has involved the design and synthesis of novel benzylated phosphonate esters as potential 1-deoxy-D-xylose-5-phosphate reductoisomerase (DXR) inhibitors. A series of amidoalkylphosphonate esters were obtained by reacting various 3-subsituted anilines and heterocyclic amines with chloroalkanoyl chlorides and reacting the resulting chloroalkanamides with triethyl phosphite using Michaelis-Arbuzov methodology. Benzylation of the phosphonate esters afforded a series of novel N-benzylated derivatives in good yields and these compounds were fully characterised by NMR and HRMS methods. Several approaches to the introduction of a benzyl group at the C-2 position of the phosphonate ester derivatives have been explored, leading unexpectedly to the isolation of unprecedented tetrahydrofuranyl derivatives. Studies towards the preparation of potential bi-functional PfDXR / HIV-1 RT inhibitors have also been initiated. Preliminary in silico docking studies of selected non-benzylated and benzylated phosphonated derivatives into the Pf-DXR active-site has provided useful insight into the binding potential of these ligands. Bioassays have revealed a very low toxicity for all the synthesised phosphonated compounds and a number of these ligands also exhibit a promising inhibitory activity against the Plasmodium parasite.
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  • Date Issued: 2015

Enterococcus pathotypes as reservoirs of antibiotic resistance determinants in the Kat River and Fort Beaufort abstraction waters

  • Authors: Ntloko, Phindiwe
  • Date: 2014
  • Subjects: Enterococcus , Drug resistance
  • Language: English
  • Type: Thesis , Masters , MSc (Microbiology)
  • Identifier: vital:11290 , http://hdl.handle.net/10353/d1019821 , Enterococcus , Drug resistance
  • Description: In this study, 400 presumptive Enterococcus isolates previously recovered from Kat River and Fort Beaufort Abstraction water dam were subjected to molecular confirmation and pathotyping. Two hundred and seventy-four (68%) of these isolates were confirmed to be enterococci species. Confirmations studies were polymerase chain reaction (PCR) based, using enterococci specific primers targeting the tuf gene. The confirmed enterococci isolates were further differentiated into their pathotypes, the targets of which were: E. faecalis, E. avium, E. hirae, E. casseliflavarus and E. gallinarum using well documented species specific primer sequences. E. faecalis accounted for 20% of the isolates, followed by E. avium (16%), E. hirae (13%), E. casseliflavarus (5%) and E. gallinarum (3%). Furthermore, all the confirmed isolates were analysed for antibiotic susceptibilities using a panel of nine different antibiotics, namely vancomycin, linezolid, ciprofloxacin, ampicillin, gentamicin, chloramphenicol, tetracycline, erythromycin, penicillin, and those that were resistant were assayed for the presence of relevant antibiotic resistance genes. All the 274 isolates were found to harbour vanA resistance gene confirming their phenotypic resistance to the vancomycin. Similarly, 60% (109/180) of the isolates showed phenotypic resistance to erythromycin which was further confirmed by the presence of ermA genes in these isolates. The presence of antibiotic resistant bacteria in surface waters poses a risk to public health.
  • Full Text:
  • Date Issued: 2014

Major spoligotype families of Mycobacterium tuberculosis strains isolated from tuberculosis patients in Port Elizabeth, Eastern Cape, South Africa

  • Authors: Nqini, Babalwa J
  • Date: 2012
  • Subjects: Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Tuberculosis -- Patients -- South Africa -- Eastern Cape , Drug resistance , Multidrug-resistant tuberculosis -- South Africa -- Eastern Cape , HIV infections -- South Africa -- Eastern Cape , AIDS (Disease) -- South Africa -- Eastern Cape
  • Language: English
  • Type: Thesis , Masters , MSc (Microbiology)
  • Identifier: vital:11271 , http://hdl.handle.net/10353/d1006877 , Mycobacterium tuberculosis -- South Africa -- Eastern Cape , Tuberculosis -- Patients -- South Africa -- Eastern Cape , Drug resistance , Multidrug-resistant tuberculosis -- South Africa -- Eastern Cape , HIV infections -- South Africa -- Eastern Cape , AIDS (Disease) -- South Africa -- Eastern Cape
  • Description: South Africa is burdened with tuberculosis (TB) which is aggravated by the concurrent epidemic of HIV as well as the emergence of drug resistance. In most developed countries molecular techniques have been used to look at the dynamics of the TB epidemic however, despite the prevalence that is high in sub-Saharan Africa, there is little data on strain types that are available in Port Elizabeth. This study aims to find the major clades of M. tuberculosis that are circulating in Port Elizabeth. Two hundred MDR-TB DNA samples were obtained from the National Health Laboratory Services TB laboratory in Port Elizabeth. Spoligotyping and MIRU-VNTR were used to genotype the strains. Two hundred strains were sent to the University of Stellenbosch for spoligotyping and 179 of those were typed. Spoligotype defined families were further typed by MIRU-VNTR typing, so as to further differentiate and assess clonal diversity within the spoligotype families. The Beijing family was the dominant family and the MANU family being the least dominant, with percentages of 71 percent and 0.5 percent respectively. A comparison of spoligotyping results with the international spoligotyping database (SITVIT2) showed a total of 15 shared international types. Forty four percent (44 percent) of the isolates that were typed by MIRU-VNTR showed similarities, suggesting epidemiological relatedness. Thirty eight percent of isolates from spoligotyping were from the same family, the Beijing family, with the same shared international type STI1, but when typed by 12 MIRU-VNTR they showed no epidemiological relatedness and 18 percent of the isolates showed no relatedness when typed by 12 MIRU-VNTR but spoligotyping showed that they were from the LAM family. Results from our study illustrate the effectiveness of MIRU-VNTR typing together with spoligotyping in epidemiological studies in the region of Port Elizabeth.
  • Full Text:
  • Date Issued: 2012
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