Repository logo

Browsing by Author "Pym, Alexander S."

Filter results by typing the first few letters
Now showing 1 - 11 of 11
  • Thumbnail Image
    Item
  • Thumbnail Image
    Item
    Development of novel reagents for tuberculosis detection.
    (2013) Ngubane, Nqobile Angel Cebile.; Pym, Alexander S.; Khati, Makobetsa.; Rubin, Eric.
    Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and causes high morbidity and mortality, despite the widespread availability of effective antibiotics against most strains of Mycobacterium tuberculosis, which is the causative agent of TB. One of the primary reasons that hinder TB control is that many cases of active disease go undetected or are discovered late. This is, in large part, due to the relative insensitivity and limited specificity, amongst other limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis infection can be asymptomatic and latent, or cause active disease. Therefore, an ideal or effective TB diagnostic needs to distinguish between these two states. The aim of this study was to develop novel diagnostic reagents for M. tuberculosis using phage displayed peptides and nucleic acid aptamers with a view to discerning latent from active TB. Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of selection (biopanning) were performed. Ten phage displayed peptides that bind to the mycobacteria surface were selected. These phage clones were identified using both random clone picking and high throughput (HTP) sequencing. A phage clone displaying the CPLHARLPC peptide was identified by HTP sequencing as the most enriched, representing 82.49% of the selected CX7C phage population. Further characterization showed that it bound better to different mycobacteria species, including M. tuberculosis, than the unselected phage library. Moreover, using surface plasmon resonance (SPR) technology, the chemically synthesised CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate and not non-mycobacteria lysates. In addition, using the systematic evolution of ligands by exponential enrichment (SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five aptamers were identified after five rounds of selection. Two of these aptamers, GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the EsxA homologue. Taken together, these findings suggest that a combination of phage display, SELEX and HTP sequencing can be a useful tool for the identification of specific detection reagents that can bind to mycobacteria and its associated targets. These reagents could be exploited to develop alternative molecular probes for TB diagnostics.
  • Thumbnail Image
    Item
    Evaluation of a synthetic peptide for the detection of anti-Mycobacterium tuberculosis curli pili IgG antibodies in patients with pulmonary tuberculosis.
    (Elsevier., 2018) Naidoo, Natasha.; Pillay, Balakrishna.; Bubb, Martin Owen.; Pym, Alexander S.; Chiliza, Thamsanqa Emmanuel.; Naidoo, Kogieleum.; Ndung'u, Peter Thumbi.; Kasprowicz, Victoria.; Pillay, Manormoney.
    Abstract available in pdf.
  • Thumbnail Image
    Item
    Evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of Mycobacterium tuberculosis isolates from KwaZulu-Natal.
    (Public Library of Science., 2015) Cohen, Keira A.; Abeel, Thomas.; McGuire, Abigail Manson.; Desjardins, Christopher A.; Munsamy, Vanisha.; Shea, Terrance P.; Walker, Bruce J.; Bantubani, Nonkqubela.; Almeida, Deepak V.; Alvarado, Lucia.; Chapman, Sinéad B.; Mvelase, Nomonde R.; Duffy, Eamon Y.; Fitzgerald, Michael G.; Govender, Pamla.; Gujja, Sharvari.; Hamilton, Susanna.; Howarth, Clinton.; Larimer, Jeffrey D.; Maharaj, Kashmeel.; Pearson, Matthew D.; Priest, Margaret E.; Zeng, Qiandong.; Padayatchi, Nesri.; Grosset, Jacques H.; Young, Sarah K.; Wortman, Jennifer.; Mlisana, Koleka Patience.; O'Donnell, Max Roe.; Birren, Bruce W.; Bishai, William R.; Pym, Alexander S.; Earl, Ashlee M.
    Abstract available in pdf.
  • Thumbnail Image
    Item
    Genetic mechanisms of D-cycloserine resistance.
    (2016) Munsamy, Vanisha.; Pym, Alexander S.
    Abstract available in PDF file.
  • Thumbnail Image
    Item
    Genomic and functional analyses of mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance.
    (Nature Publishing Group., 2016) Desjardins, Christopher A.; Cohen, Keira A.; Munsamy, Vanisha.; Abeel, Thomas.; Maharaj, Kashmeel.; Walker, Bruce J.; Shea, Terrance P.; Almeida, Deepak V.; Manson, Abigail L.; Salazar, Alex.; Padayatchi, Nesri.; O’Donnell, Max Roe.; Mlisana, Koleka Patience.; Wortman, Jennifer.; Birren, Bruce W.; Grosset, Jacques H.; Earl, Ashlee M.; Pym, Alexander S.
    Abstract available in PDF file.
  • Thumbnail Image
    Item
    Host induced microevolution of ESX secretion systems of M. Tuberculosis.
    (2013) Sukkhu, Melisha.; Pym, Alexander S.
    The ESX family of genes (esxA-W) in Mycobacterium tuberculosis (Mtb) encodes 23 effector molecules influencing immunogenicity and pathogenicity. This study was aimed at identifying and evaluating variations in ESX sequence and protein expression profiles in clinical isolates and examining how diversity might influence immune responses. 23 ESX genes from 55 clinical isolates (20 Beijing, 25 KZN and 10 Other) and 3 Laboratory strains (H37Rv, H37Ra and BCG) were sequenced. 482 single nucleotide polymorphisms (SNPs) were identified in 12 ESX genes relative to H37Rv. Majority of the identified 363 nsSNPs occured in Beijing isolates. No mutations were observed in esxA, B, C, E, G, H, J, R, S and T. Six unique nsSNPs were identified in the Beijing isolates: esxI (Q20L), esxO (E52G), 2 in esxP (T3S; N83D), esxU (P63S) and esxW (T2A). Three unique nsSNPs were identified in the KZN isolates: esxK (A58T), esxL (R33S). The esxL polymorphism resulted from a dinucleotide change. ESX gene transcription levels were evaluated using RT-qPCR. Varying expression levels were observed for esxA, B, C, F, M and Q across all clinical isolates with lowest levels seen amongst the Beijing isolates. This correlated with immunoblots with confirmed decreased esxAB protein expression relative to the other strains. The Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) spectral protein profiles were quantitatively compared within and between Mtb clinical and laboratory isolates. Protein spectral profiles within the mass range of the CFP-10 protein with variations in peak intensities were observed across all isolates. QILSS and Mtb9.9 peptides were tested individually for immune responses in TB infected patients. Healthy patients displayed no responses to QILSS and Mtb9.9, strong but variable immune responses were detected for specific regions of QILSS and Mtb9.9 in TB infected patients. These findings demonstrate that differences in sequence, transcriptional profiles and protein expression patterns in ESX secreted proteins exist between clinical isolates, and may translate into differences in human immune responses. Further research is needed to correlate human host immune responses to the phenotype and genotype of the infecting strain of Mtb to determine the consequences of specific variations of the other ESX members. These studies are important for the development of improved immune diagnostics and vaccines.
  • Thumbnail Image
    Item
    The isolation of antibiotic survivors in mycobacterium tuberculosis using fluorescence activated cell sorting (FACS).
    (2016) Shumba, Patience.; Pym, Alexander S.
    Abstract available in PDF file.
  • Thumbnail Image
    Item
    A novel reporter phage to detect tuberculosis and rifampin resistance in a high-HIV-burden population.
    (American Society for Microbiology., 2015) O’Donnell, Max Roe.; Pym, Alexander S.; Jain, Paras.; Munsamy, Vanisha.; Wolf, Allison.; Karim, Farina.; Jacobs, William R.; Larsen, Michelle H.
    Abstract available in pdf.
  • Thumbnail Image
    Item
    A pharmacokinetic study of rifabutin and its interaction with antiretrovirals in African patients with TB-HIV co-infection.
    (2012) Naiker, Suhashni.; Pym, Alexander S.; McIlleron, Helen.
    The management of HIV-associated tuberculosis (TB) is complicated by the pharmacokinetic interactions between rifampicin (RMP) and co-administered protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors. Rifabutin (RBT) is an alternative rifamycin, preferred in patients requiring PIs. Recent studies suggest the current recommended dose of RBT in combination with boosted lopinavir (LPV/r) is suboptimal and there are insufficient pharmacokinetic data evaluating the interaction between RBT coadministered with efavirenz (EFV) and nevirapine (NVP). Pharmacogenomic studies have shown that RMP concentrations are lower in patients from sub-Saharan Africa with polymorphisms of the SLCO1B1gene but there is currently no data on the pharmacogenetic determinants of RBT exposure. The pharmacokinetics of RBT were evaluated at two different doses in HIV co-infected patients before and after the introduction of LPV/r, EFV and NVPbased antiretroviral therapy (ART). After six weeks of standard TB therapy, RBT 300 mg daily was started for four weeks. Thereafter patients were randomized to receive either RBT 150 mg daily or RBT 150 mg three times a week (TPW) with LPV/r, RBT 300mg or 450mg with NVP or RBT- 450mg or 600mg with efavirenz. After four weeks on the first RBT dose, patients switched to the alternate dose and continued until the end of TB treatment. Serial RBT and 25-O-desacetylrifabutin (dRBT) concentrations were measured during a dose interval before patients switched RBT doses. The median AUC0-24 and Cmax, of RBT in patients taking 150mg RBT TPW was significantly reduced when compared to the other treatment arms. 86% of patients whilst on this intermittent RBT arm had an AUC0-24 < 4.5 μg.h/mL, level that has been associated with acquired rifamycin resistance. Rifabutin exposure was maintained within the range of AUCs that have been shown to prevent acquired rifamycin resistance (ARR) with 150mg daily dosing in combination with LPV/r. In addition, the combination of RBT with NVP 300mg resulted in significantly increased exposure of RBT, with significantly higher exposure observed with 600mg RBT. However, the combination of RBT 450mg with EFV resulted in RBT exposure lower than 300mg RBT given alone in the same patients, whereas RBT 600mg plus NVP results in bioavailability of RBT equivalent to 300mg given alone. Rifabutin was well tolerated at all doses. Only three grade 4 laboratory toxicities, elevated transaminases, neutropenia, and uveitis, possibly related to RBT were reported in patients taking NVP. SLCO1B1 rs4149032 C>T polymorphism occurs frequently in African patients in Durban and may be associated with low RBT bioavailability. These findings support recommendations for the higher dose of RBT in combination with LPV and EFV but not with NVP.
  • Thumbnail Image
    Item
    Transmission of drug-resistant tuberculosis in HIV-endemic settings.
    (Elsevier, 2019) Khan, Palwasha Y.; Yates, Tom A.; Osman, Muhammad.; Warren, Robin M.; van der Heijden, Yuri.; Padayatchi, Nesri.; Nardell, Edward A.; Moore, David B.; Mathema, Barun.; Gandhi, Neel R.; Eldholm, Vegard.; Dheda, Keertan.; Hesseling, Anneke C.; Mizrahi, Valerie.; Rustomjee, Roxana.; Pym, Alexander S.
    Abstract available in PDF.