ETD Collection
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Item Antibiotic resistance in viridans streptococci(1994) Potgieter, ElsaViridans streptococci are aetiological agents of endocarditis, bacteraemia, dental caries and abscesses of the liver, brain and joints. Viridans streptococci had until recently generally been considered to be uniformly susceptible to antimicrobial agents active against Gram-positive cocci. The susceptibility of 211 viridans streptococci isolated from blood in South Africa between 1988-1991 to eight antimicrobial agents was determined. Thirty-eight percent of the isolates were found to be resistant to penicillin (MICs >0.25 /Mg/ml). Five Streptococcus mitis isolates exhibited increased (MIC 64 and 128 ^g/ml) or high-level (MIC >500 /Mg/ml) resistance to gentamicin. In vitro synergy between penicillin and gentamicin was not demonstrated with these isolates.Item The ethics of antibiotics use in animal farming(2017) Ncayiyana, Philisiwe PreciousThe unnecessary use of antimicrobials poses a global threat to human health by contributing to the Antimicrobial Resistance (AMR) development. This report evaluates the ethical and scientific implications of non-therapeutic use of antibiotics in animal farming. The report also critiques O'Neill's (2016) Final Report on AMR. The report provides a normative assessment and analysis of scientific evidence and ethical issues involved in farming with antibiotics making use of Mepham's Ethical Matrix. The report makes the case that non-therapeutic use of antibiotics in animal farming contributes to AMR development and that it is not ethically justifiable for farmers to carry on farming with antibiotics non-therapeutically. The study also argues that intensive factory farming poses the greatest risk in the preservation of all classes of antibiotics because it is customary to use antibiotics where a large number of animals are kept in close proximity for example, in poultry farming. An immediate ban of antibiotics deemed medically important for humans in animal farming is necessary in order to prevent the spread of antibiotic resistance. This however, must go hand in hand with preparation for abandonment of intensive farming systems in order for a ban to be successful. The study also recommends the adoption of O'Neill's (2016) recommendations on tackling AMR. In addition, a national public awareness campaign is justified by the threat posed by AMR. Governments and other relevant stakeholders involved should formulate policies or frameworks to deal with the problem with the urgency it requiresItem Antibiotic usage in an intensive care unit of a tertiary level public hospital(2018) Ejike, Antonietta ChidimmaIntroduction: Antibiotic resistance presents a great challenge as the World Health Organization declared antibiotic resistance a global threat. Considering the high disease burden, prescribers are pressured to treat empirically rather than definitively especially in the intensive care units (ICU) where critically ill patients need rapid treatment. Aim: The aim of this study was to document the utilization of antibiotics in a tertiary level hospital intensive care unit. Method: This was a retrospective record review and data was collected for a two-month period in 2016 and 2017. Information was transcribed from the ICU charts. Variables included antibiotic chosen, number of antibiotics per patient, duration and frequency of treatment as well as information on the microorganisms involved. Data was analysed quantitatively using mean, median and frequency. Result: The majority (67% in 2016 and 75% in 2017) of patients admitted to the Helen Joseph Hospital (HJH) ICU during the study period were on antibiotics and the majority were treated empirically. The most frequently used antibiotics were amoxicillin/clavulanic acid followed by piperacillin/tazobactam. The majority of antibiotics stocked in the ICU were started on day zero of admission compared to the restricted antibiotics. The average antibiotic per patient was one and a maximum of three antibiotics was used concurrently. The average length of stay in HJH ICU was two days. Klebsiella pneumoniae (17%), Enterobacter cloacae (15%), Staphylococcus aureus (11%), Escherichia coli (9%) and Pseudomonas aeruginosa (6%) were the frequently most isolated pathogens. Conclusion: The study concluded that restriction of antibiotics does improve antibiotic utilization. Also the length of stay in the HJH ICU is short. Concurrent use of antibiotics was low. Furthermore, there were some antibiotics utilization patterns seen which are not supportive for a successful antibiotic stewardship. If there are no interventions informed by utilization studies, same patterns will continue.Item In vitro HIV-1 drug resistance phenotyping, genotyping and novel virological failure detection tools for clinical patient management.(2014-03-28) Bronze, Michelle SaltaoOf the 22.5 million individuals infected with the human immunodeficiency virus (HIV) in sub-Saharan Africa, 62% of patients requiring treatment had access to highly active antiretroviral therapy (HAART) in 2011. The delivery of HAART and the appropriate laboratory monitoring of HIV positive individuals in sub-Saharan African countries has become a public health priority, an intervention which has and will continue to dramatically reduce HIV-related morbidity and mortality. Routine laboratory monitoring of HIV infected individuals should ideally include CD4+ T cell testing to assess when to start ART, viral load monitoring to assess virological failure on ART and when indicated, HIVDR genotyping.However, this is often not implemented in resource limited settings due to challenges such as inadequate infrastructure and laboratory capacity, amongst others. Thus the Affordable Resistance Testing for Africa (ART-A) initiative was established to develop an affordable HIV drug resistance testing (HIVDR) algorithm applicable to Africa. The objective of this study was to evaluate the role of in vitro HIVDR phenotyping in the context of HIV-1 subtype C (the most prevalent circulating subtype in sub-Saharan Africa), genotyping and genotypic interpretation tools using existing algorithms, as well as novel virological failure detection tools for clinical patient management. Current gold standard HIVDR phenotyping technologies use an HIV-1 subtype B backbone to create recombinant viruses with patient-derived polymerase (protease and partial reverse transcriptase). This backbone could impact on the in vitro phenotyping results of non-B subtypes, and therefore it was deemed necessary to establish the applicability of HIVDR phenotypic testing of subtype C polymerase when a commercially available subtype B backbone is used. One hundred and fourteen HIV-1 subtype C samples were HIVDR phenotyped against 17 antiretroviral drugs using both subtype B and C backbones and showed a high level of concordance between the two backbone phenotypic resistance profiles (95.8%; 1590 of 1660 fold change comparisons). Natural assay variability was largely responsible for discordant results. Results confirmed that HIV-1 phenotypic reverse transcriptase inhibitor drug resistance test interpretation is independent of the virus backbone subtype. No conclusions could be made for protease inhibitor resistance since limited samples from 2nd line failure were available. Subsequently, the HIVDR genotypic and phenotypic results of the 114 patient samples were compared to determine whether genotyping is a viable alternative to phenotyping. Results showed a 92.3% concordance between genotyping and phenotyping of individual drug comparisons for a number of HIVDR profiles. Discrepancies were attributed to phenotypic assay variability in addition to the role of mutation mixtures, which impacted genotypic interpretations. Overall, HIVDR genotyping is a reliable tool to detect and interpret antiretroviral drug resistance in HIV-1 subtype C infected patients, and can thus be used for clinical patient management. Once the accuracy of HIVDR genotyping was established, the development, validation and evaluation of a potential virological failure assay (ARTA-VFA) and a simplified HIVDR (ARTA-HIVDRultralight) assay was undertaken. A simplified and conceptually novel approach using a qualitative viral load assay with a pre-determined cut-off that gives a threshold above which virological failure (VF) could be confirmed and below which treatment success was likely, was tested. A real-time PCR (ARTA-VFA) assay was developed which involved the amplification of a short sequence of the HIV-1 LTR region from RNA extracted either from plasma and/or dried blood spots (DBS). The ARTA-VFA was tested on 409 patient samples,and successfully amplified samples from all major HIV-1 group M subtypes with equal specificity. The VF was qualitatively classified as a viral load >1000 RNA copies/ml in plasma samples, and >5000 RNA copies/ml in DBS samples. Comparative testing yielded accurate VF determination for therapy-switching in approximately 93% of clinical cases tested, compared to current gold standard quantitative viral load assays. A simplified HIVDR genotyping assay (ARTA-HIVDRultralight) targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line regimen failures was developed and assessed. The ARTAHIVDRultralight assay was designed to be practical, faster, and more affordable, show flexibility with respect to equipment (open platform), use DBS or plasma as starting material and amplify and sequence a smaller amplicon (RT). The assay performed well when compared to the in-house assay used in the laboratory at the time for both 212 plasma and 25 DBS samples, yielding identical mutations and subsequent resistant profiles. Furthermore, a theoretical in silico exercise to investigate the consequences of using 125,329 shortened RT genotype (ARTA-HIVDRultralight) as compared to full-length RT sequences showed >95% and >90% concordance when using the Stanford HIVdb algorithm and the virco®TYPE tool, respectively. Differences noted were minor and unlikely to have any impact on clinical decision-making. Overall, this study illustrated that the short RT sequences can be reliably used to generate HIVDR genotypes using the Stanford HIVdb and virco®TYPE algorithms and reduce sequencing costs substantially. A field evaluation using the ARTA-VFA and ARTA-HIVDRultralight on 288 clinical samples was conducted, showing that the accuracy and precision of both assays (using 248 plasma or 40 DBS sampling methods) compared well to the reference methodology, thereby extending access of testing to more remote settings.These assays were designed to either be used as a testing strategy of initially assessing VF,and once confirmed performing an HIVDR assay, or alternatively to be used separately as stand-alone, or within different laboratory tiers in resource limited settings. It is envisaged that the ARTA-VFA could be used in the middle laboratory tier, and if confirmatory, patient samples can be referred to a reference laboratory with the available infrastructure for HIVDR testing using the ARTA-HIVDRultralight. Lastly, an automated sequence analysis and editing software for use in correct base calling of nucleotide/mutation mixtures in HIVDR genotyping was validated on 1624 sequences. Compared to reference software, where interpretation is often operator dependent, this software performed extremely well, with minor discrepancies noted. The automated software can be used to reduce subjectivity, time taken for analysis which is often the rate-limiting step and thus improving the turn-around time and clinical relevance of HIVDR genotyping. Overall, the results obtained describe the validation of using HIVDR genotyping as an alternative tool to phenotyping, and the subsequent development and validation of simple, affordable, "open-platform" alternatives to currently used methods for virological failure monitoring, and accommodate a centralized approach to HIVDR with DBS testing in resource limited settings.Item The fitness costs of drug resistance mutations in Mycobacteria(2012-01-17) Koch, Anastasia SiderisThe increasing emergence of drug-resistant pathogens poses a major threat to public health. Although influenced by multiple factors, resistance is often associated with mutations in drug target-encoding or associated genes. The potential fitness cost of such resistance mutations is, in turn, a key determinant of the spread of drug-resistant strains. Rifampicin (RIF) is a frontline anti-tuberculosis agent that targets the rpoB-encoded β-subunit of the DNA-dependent RNA polymerase (RNAP). RIF resistance (RIFR) maps primarily to mutations in rpoB that might be expected to affect transcription and so the ability of the organism to cause disease. Accordingly, numerous studies have assessed the impact of RIFR on key fitness indicators in pathogens including Mycobacterium tuberculosis (MTB). In contrast, the specific consequences of RIFR for bacterial physiology remain poorly understood. Notably, previous studies of the effects of RIFR-associated rpoB mutations on mycobacterial physiology have been conducted using strains generated by RIF exposure, without accounting for the potential impact of second-site mutations that may compensate for fitness costs or contribute to drug resistance. In this study, site-directed mutagenesis and allelic exchange were employed to generate a panel of M. smegmatis (MSM) strains containing clinically-relevant RIFR-associated point mutations. Importantly, this methodology enables the introduction of rpoB mutations into defined strain backgrounds in the complete absence of RIF. Using this approach, we constructed “RIF naive” MSM rpoB mutant strains carrying either an S531L or H526Y mutation. The resulting mutants were 100-fold less susceptible to RIF than the isogenic, parental strain. Notably, the inclusion of selected efflux inhibitors in susceptibility assays had little impact on mutant susceptibility to RIF. In contrast, restoration of the wild-type allele returned the observed susceptibility to parental levels, thereby providing strong evidence of the sufficiency of a single rpoB mutation for clinical RIFR in mycobacteria. Competitive growth assays utilizing the S531L mutant and the parental strain exposed a growth defect for the S531L mutant. However, discriminating between wild-type and mutant rpoB strains proved a significant technical challenge, again highlighting the difficulties associated with inferring in vivo fitness from in vitro assays conducted under a limited number of different conditions. In summary, our results suggest the benefit of a deeper exploration of the physiological and fitness implications of RIFR-associated mutations. In addition, in coupling a system which enables an evaluation of the physiological consequences of drug resistance-associated mutations with evolutionary analyses, we provide preliminary evidence of the benefits of a multipronged approach to elucidating the physiological implications of drug resistance in MTB.