Doctoral Degrees (Medicine)

Permanent URI for this collectionhttps://hdl.handle.net/10413/8104

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Identifying mechanisms associated with metronidazole resistance in Trichomonas vaginalis and investigating newer therapeutics against this pathogen.
(2023) Govender, Rowen.; Abbai, Nathlee Samantha.
Introduction Trichomonas vaginalis, the etiological agent of trichomoniasis, a prevalent non-viral sexually transmitted infection, presents a growing public health concern due to the emergence of metronidazole resistance, which compromises the effectiveness of this frontline treatment. To address this challenge, this PhD thesis comprises four research manuscripts aimed at understanding the mechanisms associated with metronidazole resistance in T. vaginalis and investigating innovative therapeutic approaches against this pathogen. T. vaginalis, a flagellated protozoan parasite, has long been susceptible to metronidazole. However, the alarming increase in drug-resistant strains necessitates a comprehensive examination of this resistance. This study explored the interaction of T. vaginalis with endosymbionts, particularly T. vaginalis viruses (TVVs) and Mycoplasma hominis, delving into their association with drug resistance. Additionally, alternative treatments such as plant-based nanoemulsions and nanotechnology-mediated approaches were investigated. This thesis serves as a beacon of knowledge and innovation in the ongoing battle against metronidazole resistant T. vaginalis isolates, offering new insights and promising strategies for the future of trichomoniasis management. Methods This research encompassed a multifaceted methodological approach, systematically examining metronidazole resistance in T. vaginalis and exploring alternative therapeutic avenues. The initial set of investigations utilized a combination of in vitro studies and molecular assays to analyse clinical isolates of T. vaginalis. This entailed scrutinizing the presence of T. vaginalis viruses and intracellular M. hominis within these isolates and delving into the intricate associations between these endosymbionts and metronidazole resistance. Further studies conducted in vitro susceptibility assays, gene expression analyses, and investigated the influence of iron supplementation on metronidazole resistance in T. vaginalis isolates. This multifaceted approach aimed to uncover the role of iron in regulating genes associated with metronidazole resistance and to assess how alterations in iron levels might affect the susceptibility of T. vaginalis isolates to the drug. In separate investigations, plant based nanoemulsions were meticulously prepared from select medicinal plants and rigorously tested for efficacy against metronidazole-resistant T. vaginalis isolates, exploring their potential as alternative therapeutic agents. Additionally, our research ventured into the realm of nanotechnology, focusing on the synthesis of iron nanoparticles, modified with chitosan and small interfering RNA (siRNA). This novel approach aimed to explore the potential of these nanocomplexes for gene silencing and targeted therapy in the battle against T. vaginalis infections. Results The research findings have unveiled valuable insights into the mechanisms associated with metronidazole resistance in T. vaginalis. Twenty-one clinical isolates of T. vaginalis were included in the endosymbiosis analysis. The prevalence of TVV and M. hominis were 76% (16/21) and 86% (18/21), respectively. The presence of any TVV was significantly associated with metronidazole susceptibility patterns (p=0.012). No significant associations were noted between the coinfection of both endosymbionts and metronidazole resistance. This study went on to show that iron concentrations of 30 μM and 60 μM had led to a loss of resistance in certain isolates of T. vaginalis. Furthermore, gene expression analysis indicated that iron played a role in modulating the expression of resistance-associated genes. Additionally, the antimicrobial properties of iron on T. vaginalis growth was also evident, which potentially influenced the significance of these gene expression alterations. The efficacy of plant-based nanoemulsions from medicinal plants against metronidazole-resistant T. vaginalis isolates was highlighted. The nanoemulsions shifted resistant isolates towards susceptibility in a concentration-dependent manner, with minimum inhibitory concentrations (MICs) decreasing from 4 μg/ml to 1 μg/ml, promising the restoration of plant-based treatments' effectiveness. Finally, the synthesis of iron nanoparticles modified with chitosan and small interfering RNA (siRNA) for potential gene silencing and targeted therapy against T. vaginalis was presented. These nanoparticles were successfully synthesized and efficiently bound with siRNA, offering a potential for innovative therapeutic interventions. Concentration-dependent effects on cell viability were observed, highlighting the need for precise optimization in therapeutic strategies. These comprehensive results collectively signify alternative strategies for addressing metronidazole resistance and mark a significant step in advancing precision medicine and targeted therapies for T. vaginalis infections. Conclusion The collective findings from this research provides compelling evidence for alternative approaches to address metronidazole resistance in T. vaginalis. The multifaceted nature of this thesis illustrates the complexity of metronidazole resistance and the potential for innovative therapies. These discoveries pave the way for a more comprehensive understanding of T. vaginalis infections and resistance mechanisms. They hold the promise of precision medicine, drug delivery, gene silencing, and targeted therapies. However, for these research outcomes to translate into effective clinical therapies, further exploration, in vivo validation, and rigorous clinical studies are essential. These steps are crucial for addressing the growing challenges posed by T. vaginalis infections and metronidazole resistance while ensuring the continued efficacy of treatment options in the field of public health.
Investigating the host-binding properties of Neisseria gonorrhoeae and newer therapeutics against this pathogen.
(2023) Naicker, Deshanta.; Abbai, Nathlee Samantha.
Background With 80 million annual cases, gonorrhoea is a common sexually transmitted disease caused by Neisseria gonorrhoeae (N. gonorrhoeae). The current study investigated a reverse vaccinology approach (determining the CEACAM binding patterns of N. gonorrhoeae) and alternative therapeutics (nanoemulsion and plant extracts) to combat this disease as well as evaluated cost-effective assays that target virulence proteins such as the opa gene (in-house opa-based real-time PCR assay). Methodology This was a retrospective laboratory-based study using stored bacterial isolates and primary genital swabs collected from larger studies. For the binding assays, we investigated the host-pathogen associations by determining the CEACAM binding patterns of N. gonorrhoeae isolated from symptomatic (G180) versus asymptomatic (G136) pregnant women. To identify the complete repertoire of G136 and G180 Opa proteins, DNA was isolated and a PCR with primers targeting the conserved regions of opa genes was performed. Thereafter, opa amplicons were ligated into pCR Blunt II TOPO, and single clones from this opa amplicon library were sequenced to identify the respective unique opa genes. The Opa proteins identified in this study were subjected to test expression and Western blotting was performed to verify these Opa proteins with a monoclonal antibody against neisserial Opa proteins. Thereafter, binding assays were performed to analyse the host-binding properties of N. gonorrhoeae. For the objective which involved the development and evaluation of the performance of an in-house opa-based real-time PCR assay, three primer sets targeting the opa gene of N. gonorrhoeae were designed and evaluated against published opa gene primers (comparator assay). The in-house and published primers were tested against laboratory and clinical isolates of N. gonorrhoeae as well as non-gonococcal Neisseria control isolates. The antimicrobial properties of Ocimum tenuiflorum (“holy basil”), Moringa oleifera, and Azadirachta indica plants against N. gonorrhoeae were then explored. The plants were collected from the Botanical Gardens, Durban, South Africa. Upon collection and transport to the laboratory, the plants were left to dry naturally from sunlight for about 4 to 5 days and then used in the preparation of the aqueous extracts. The nanoemulsions were produced according to published methods. Different concentrations of the extracts (1000 μM, 100 μM, 10 μM, and 1 μM) were tested against N. gonorrhoeae isolates using the disk diffusion method. The extracts were also tested for their toxicity against human erythrocytes. Results In this study, were able to identify nine distinct Opa proteins from G136 and ten distinct Opa proteins from G180. For isolate G136 (asymptomatic patient), 66.7% of the Opa proteins bound to CEACAM3, 55.6% bound to CEACAM1, and 88.9% bound to CEACAM5. For isolate G180 (symptomatic patient), 30% of the Opa proteins bound to CEACAM3, 80% bound to CEACAM1, and 70% bound to CEACAM5. In this study, it was shown that the N. gonorrhoeae Opa proteins from a symptomatic patient bound at a higher frequency to CEACAM1 and 5 which causes the pathogen to invade the host cell and cause infections. However, N. gonorrhoeae Opa proteins from the asymptomatic patient bound at a higher frequency to CEACAM3. According to the real-time PCR evaluation assays, the opa 1 primer performed better than opa 2, opa 3, and the comparator opa primer sets. The opa 1 assay produced positive amplification for the five WHO and the six N. gonorrhoeae clinical isolates, whereas the comparator assay amplified 90.9% of the samples. For the endocervical DNA samples, 82.8% were amplified with the opa 1 assay, while the comparator assay had only amplified 27.6% of the samples. For the vaginal DNA samples, the opa 1 assay amplified 95.0% of the samples, whereas the comparator assay amplified 25.0% of the samples. All eleven (100%) urine DNA samples were amplified with the opa 1 assay, in contrast to 36.4% with the comparator assay. The opa 1 assay showed no cross-reactivity with non-gonococcal isolates, whilst cross-reactivity was observed with the comparator assay. The opa 1 assay also had a higher limit of detection when compared to the other assays. In this study, all six gonococcal isolates exhibited clear zones of inhibition when exposed to the 1000μM concentration of all three of the nanoemulsion-based plant extracts investigated. Conversely, no zones of inhibition were detected at extract concentrations of 100μM, 10μM, and 1μM for five of the isolates. Isolate G176, however, displayed zones of inhibition at both 1000μM and 100μM concentrations when exposed to the nanoemulsion plant-based extracts derived from Ocimum tenuiflorum. Additionally, the WHO control strains included in the assay exhibited zones of inhibition at the 1000μM concentration. Notably, the WHO Y strain displayed zones of inhibition at both 1000μM and 100μM concentrations when exposed to nanoemulsion-based extracts from Ocimum tenuiflorum and Azadirachta indica. Importantly, the analysis revealed 0% haemolytic activity against human erythrocytes, indicating the non-toxic nature of the extracts. Conclusion In this study, it was demonstrated that N. gonorrhoeae Opa proteins obtained from symptomatic patients exhibited a higher affinity for CEACAM1 and CEACAM5, facilitating the pathogen's invasion of host cells and subsequent infection. Conversely, Opa proteins from asymptomatic patients displayed a higher binding frequency to CEACAM3. These Opa-CEACAM interactions are believed to potentially restrict the dissemination of gonococci by promoting granulocyte-mediated opsonin-independent phagocytosis. Investigating interactions represents a crucial step forward in the quest for vaccine development against this pathogen. This study contributes valuable insights to the expanding body of knowledge concerning the host-receptor binding profiles of this pathogen. This study demonstrated that the opa 1 primer was the superior primer when compared to opa 2, opa 3, and the comparator opa primers from Verma et al., (2012) study. Therefore, this in-house opa 1 assay can be potentially used and further evaluated for its use as a diagnostic assay. This study also demonstrated that the combination of nanoemulsions with plant extracts (specifically, Ocimum tenuiflorum, Moringa oleifera, and Azadirachta indica) presents a promising alternative to traditional antibiotics for combatting N.gonorrhoeae infections. This strategy capitalizes on the antimicrobial attributes of naturalcompounds and utilizes nanoemulsion technology to optimize their delivery and efficacy. Nevertheless, continuous research and development efforts must be undertaken to validate and enhance the viability of this approach for clinical application.
Differential effects of early life stress and schizophrenia on the development of impulse control disorder = Imiphumela ehlukene yengcindezi yempilo engasekuqaleni kanye nokusangana ekuthuthukiseni ukulawula isifo esivumbuka kungahlelekile.
(2024) Oginga, Fredrick.; Mpofana, Thabisile.
Background: Early life stress (ELS) and parental psychopathology, such as schizophrenia, have profound effect on neurobiological and behavioral outcomes in later life. While previous studies in human have explored the individual effects of ELS and parental schizophrenia (PSZ), this study investigates their interactive effects. Objectives: This study aimed to comprehensively examine the impact of ELS and schizophrenia like symptoms on locomotion, anxiety and depressive like behavior, spatial memory, social interaction and neuro-inflammation in Sprague-Dawley rats. Methods: Male and female Sprague-Dawley pups were randomly assigned to eight groups: control, ELS, Ketamine to induce schizophrenia like symptoms (KSZ), and ELS + KSZ. ELS was induced through prenatal stress and maternal separation (MS), while schizophrenia-like behaviour was induced by ketamine administration (KSZ). Ketamine was administered intraperitoneal to the dams, while subcutaneous to the pups as per previously published studies. Behavioral assay, including open field, Morris water maze, social interaction behaviour, and sucrose preference test, was conducted. Neuro-inflammation was through quantification of glial fibrillary acidic protein astrocytes density and inflammatory biomarkers. Results: ELS and KSZ on dams exhibited enduring effects on particularly psychomotor retardation (p < 0.05). Anxiety and depressive like behavior was elevated in the ELS (p = 0.023) and KSZ on dams (p =0.017) groups compared to controls. The combined ELS and KSZ groups showed the highest anxiety and depressive like outcomes (p = 0.006). Additionally, spatial memory and cognitive impairment in pups were observed due to the combined impact of ELS and KSZ, which was associated with a decrease in astrocyte density and dysregulation of neuro-inflammatory markers (p < 0.05). Conclusion: This study highlights the interactive effects of ELS and KSZ on behavior, neurodevelopment, and neuro-inflammation in rats. Both ELS and KSZ in parents were linked to anhedonia, subsequently anxiety-like behavior, and ultimately psychomotor, spatial memory, and cognitive decline in rats. Positive parenting was associated with astrocyte regeneration (p < 0.05) and cognitive improvement. Understanding these complex interactions provides insights into the challenges associated with these stressors and offers potential therapeutic avenues. Iqoqa. Isendlalelo: ingcindezi ekuqaleni kwempilo kanye nobuzali ekuziphatheni kwesifo sengqondo, njengokusangana, kuba nomphumela omkhulu ohlelwenimizwa kanye nemiphumela yokuziphatha ngokuhamba kwesikhathi empilweni. Izifundo ezedlule ngomuntu ziyiphenyile imiphumela ngomuntu mayelana nengcindezi ekuqaleni kwempilo kanye nobuzali ekuphazamisekeni kwengqondo (PSZ). Lolu cwaningo luphenya imiphumela ethelelanayo. Izinhloso: lolu cwaningo luhlose ngokubanzi ukucwaninga imiphumela yengcindezi ekuqaleni kwempilo kanye nokusangana njengezimpawu zokudlathuzela, ixhala kanye nokuziphatha okunobukhwantalala, ukugcinwa kolwazi engqondweni, ukuphilisana ngokwenhlalo kanye nokuvuvukala kwemizwa yengqondo ngokukaSprague-Dawley emagundaneni. Izindlelakwenza: imidlwane yenduna neyensikazi kaSprague-Dawley yakhethwa ngokungahlelekile yanikezwa amaqoqa ayisishiyagalombili: ukulawulwa, i-ELS, Ketamine ukulandela ukusangana njengezimpawu i-KSZ, kanye ne-ELS+KSZ. I-ELS yalandelwa ngokwengcindezi engaphambi kokuzalwa ngesikhathi sokumitha nokuhlukaniswa nonina (MS), ngesikhathi ukuziphatha okusakusangana kwalandelwa ukusetshenziswa kweketamine (KSZ). Iketamine yafakwa ngaphakathi ontwentwesini lwenxasisu yemidlwane esemadamini, ngesikhathi futhi ifakwe ngaphansi kwesikhumba semidlwane njengokusho kocwaningo olushicilelwe ngokwedlule. Ukuziphatha ngokuhlola ingqondo, kufaka insimu evulekile, amanzi amazombe ngokukaMorris, ukuziphatha ngokwenhlalo, kanye nesivivinyo esikhethekile sikashukela, kwenziwa. Ukuvuvukala kwaba ngenxa yequantification yeglial fibrillary yensiza zakhamzimba nokugqishelana kwe-astrocytes kanye nokuvuvukala kwebiomarkers. Imiphumela: Ixhala kanye nengcindezi njengokuziphatha kwaphakanyiswa kuyo i-ELS (p=0.023) and KSZ emadamini (p=0.017) yamaqoqo yaqhathaniswa nabaqashelwe. Ukuhlanganiswa kwe-ELS kanye neKSZ kwatshengisa ixhala elikhulu kanye nengcindezi njengemiphumela (p=0.006). Ngokunezezela, isikhala ngokukhumbula kanye nokulimaza umqondo kwafakelwa izibuko ngenxa yokuhlangana kwesisindo se-ELS kanye neKSZ, okumataniswa nekwehla kwesisindo se-astrocyte kanye nokungalawulwa kwezinkomba zokuvuvukala ngokomqondo (p<0,05). Isiphetho: lolu cwaningo lugqamisa imiphumela ethelelanayo ye-ELS kanye ne-KSZ ekuziphatheni, ukuthuthuka ngokwasengqondo, nokuvuvukala kwengqondo emagundaneni. Kokubili i-ELS kanye ne-KSZ kubazali kwaxhunyaniswa ne-anhedonia, kwalandela ukuziphatha okusaxhala, kwasekuthi ekugcineni ubudlelwanokusebenza kwengqondo nomzimba, ukugcinwa kolwazi engqondweni, nokufadalala kwenqubokucabanga emagundaneni. Ubuzali obuhle kwamataniswa nokuvuselelwa kwe-astrocyte (p<0,005) kanye nokubangcono komqondo. Ukuqonda ukuthelelana okudidayo kokuphilisana kwanikeza ulwazi mayelana nezinselelo ezimataniswa nezimbangangcindezi kwanikeza namathuba okwelapha okungase kwenziwe.
Evaluation of laboratory tests for COVID-19 in South Africa = Ukuhlaziya izivivinyo zaseLabhorethri ze-COVID-19 eNingizimu Afrika.
(2023) Samsunder, Natasha.; Kharsany, Ayesha Bibi Mahomed.; Sivro, Aida.
The emergence of SARS-CoV-2 prompted urgent needs for accurate diagnosis, management, and containment strategies. This study evaluated diagnostic tests, including point-of-care (POC) tests, to aid in rapid diagnosis across different stages of COVID-19 in South Africa. A scoping review highlighted the variability in test performance, with no single assay achieving optimal sensitivity and specificity simultaneously. Sensitivity was influenced by the timing of sample collection, emphasizing the importance of early sampling. Rapid antigen tests were evaluated against RT-PCR, revealing reasonable sensitivity, especially in samples with lower Ct values and within the first week of symptom onset. However, performance varied across SARS-CoV-2 variants. Notably, PanbioTM and SD Biosensor tests maintained high sensitivity and specificity across different variants, including Omicron sub-lineages. Additionally, the study explored alternative sample types, such as saliva, finding comparable results to nasopharyngeal swabs. Serological tests were also assessed, with the Orient Gene Rapid test showing comparable performance to standard assays, while the MILLIPLEX® MAP Kit demonstrated higher detectability. Overall, despite extensive testing efforts, the sensitivity of diagnostic tests remained limited, underscoring the need for improved performance to effectively diagnose and manage SARS-CoV-2 infections and limit transmission. These findings provide valuable insights for enhancing testing strategies in South Africa and globally amidst evolving pandemic challenges. Iqoqa. Ukuqubuka kwe-SARS-CoV-2 kwaphusha izidingo eziphuthumayo zamasu okuhlonza isifo okuyikho, ukulawula nokunqanda. Lolu cwaningo lwahlola izivivinyo, okufaka nezivivinyo ezaziwa ngelepoint-of-care (POC), ukusiza ukuhlonza isifo ngokushesha ezigabeni ezehlukene ze-COVID-19 eNingizimu Afrika. Ukubuyekeza umumo kwagqamisa ukwehlukahlukana ekuhloleni ukusebenza, kungekho neyodwa i-asayi efikisa ekuzweleni okukhulu nasekuqondeni kanyekanye. Ukuzwela kwakudalwa yisikhathi sokuqoqwa kwesampula, kugcizelelwa ukubaluleka kokuqoqwa kwamasampula kwasekuqaleni. Izivivinyo eziningi zedalasihlungu zahlaziywa ziqhathaniswa ne-RT-PCR, okuveza ukuzwela okuzwakalayo, ikakhulukazi emasampuleni ane-Ct ephansi esontweni lokuqala lokubonakala kwezimpawu. Kodwa, ukusebenza kwehlukana ngokwezinhlobo ze-SARS-CoV-2. Okuqaphelekayo, yi-v Notably, PanbioTM nezivivinyo ze-SD Biosensor ezasimama ngokuzwela okukhulu namavariyenti ehlukene, okufaka nama-Omicron sub-lineages. Ukwengeza, ucwaningo lubheke ezinye izinhlobo zamasampula, njengamathe, ukuthola imiphumela eqhathaniseka nemisubelo yenasopharyngeal. Izivivinyo zeseroloji nazo zahlolwa, kanye nesivivinyo se-Orient Gene Rapid okukhombisa ukusebenza okuqhathanisekayo nama-asayi asezingeni, nakuba i-MILLIPLEX® MAP Kit yakhombisa ukutholakala ngezinga eliphezulu. Ngaphezu kwalokho, nakuba kunemizamo yokuhlola okunzulu, ubuthaka bezivivinyo eziyinhlonzasifo zazilokhu zincane, ukuthola okungaphansi isidingo sokusebenza okuphuculiwe ukuze kuhlonzwe ngendlela futhi kulawulwe ukutheleleka nge-SARS-CoV-2 bese kunqanda ukwedluliseka. Lokhu okutholakele kuhlinzeka imibono enesisindo ukuphucula amasu okuhlola eNingizimu Afrika nasemhlabeni jikelele ezinselelweni zobhubhane eziguquguqukayo.
Immune biomarkers of pulmonary tuberculosis treatment response and disease severity among HIV-infected and uninfected individuals from Kwazulu-Natal, South Africa.
(2023) Rambaran, Santhuri.; Sivro, Aida.; Naidoo, Kogieleum.
Background: Tuberculosis is one of the major causes of morbidity and mortality worldwide. The COVID -19 pandemic has had a devastating impact on TB, contributing to increased incidence of both TB and drug-resistant TB. Identification of host immune biomarkers of TB risk, treatment outcome and disease severity are key to the development of more efficient diagnostics and treatment modalities. There is an urgent need for accurate and easily detectable non-sputum-based biomarkers that can correlate with the activity or burden of Mycobacterium tuberculosis. Here, we characterised soluble and cellular phenotypes during active TB and TB/HIV co-infection and assessed their associations with time to negative culture conversion and disease severity. Methods: The study was performed utilizing stored plasma and peripheral blood mononuclear cells from the Improving Retreatment Success (IMPRESS) trial. Multiplex immunoassays and ELISAs were used to evaluate 24 cytokine and chemokine expression during active TB (n=132). Flow cytometry was used to evaluate phenotypic profiles of monocytes, dendritic cells (n=90) and CD4+ T cells (n=75). A Cox proportional hazards and logistic regression models were used to assess the associations between the measured cytokines and chemokines, phenotypic profiles of monocytes, dendritic cells and CD4+ T cells and time to negative culture conversion and lung cavitation in individuals with TB and TB/HIV co-infection. Results: We identified soluble inflammatory signatures of treatment response and disease severity. IP-10 expression during active TB was associated with increased odds of sputum culture conversion by 8-weeks in the total cohort and among the HIV-infected individuals. Increased MCP-3 expression was associated with a shorter time to culture conversion in the total cohort. While among the HIV-infected individuals, higher expression of IL-1RA, IP-10 and IL-1α associated with a shorter time to culture conversion. Higher expression of IL-6 was significantly associated with shorter time to culture conversion and increased risk of lung cavitation in the overall cohort and among TB/HIV co-infected individuals. Additionally, higher IL-1RA expression was associated with the presence of lung cavitation in the total cohort and in HIV-infected individuals. We observed distinct monocyte and dendritic cell profiles in TB/HIV co-infection. Individuals with TB/HIV co-infection had a significantly higher percentage of total monocytes and dendritic cells compared to healthy controls. Increase in CCR2, CD11b and CD40 was associated with active TB while decrease in CX3CR1 and increase in CD163 was associated with HIV infection. Expression of CX3CR1 on non-classical monocytes was associated with longer time to culture conversion while expression of CD86 on intermediate monocytes was associated with presence of lung cavitation. With respect to CD4+ T cells HIV positive individuals with active TB had significantly lower percentage of CD4+ T cells and significantly higher proportion of activated CD4+ T cells compared to TB and healthy control groups. Percentage of CD4+ T cells was significantly associated with increased risk, while the percentage of activated CD4+ T cells was associated with decreased risk of lung cavitation. Integrin α4β7 expressing CD4+ T cells were increased in TB/HIV compared to TB group and was associated with longer time to TB culture conversion in co-infected individuals. Conclusion: The data from this study provides valuable insight into the role that plasma immune biomarkers, monocytes, dendritic and CD4+ T cells play in TB treatment response and disease severity in active TB and TB/HIV co-infection. Iqiqa. Isendlalelo: Isifo sofuba, ituberculosis (TB) singenye yezimbangela zokugula nokufa emhlabeni wonke. Ukutholwa kwezimpawu ezikhombisa ukuba sengcupheni yesifo sofuba, Imiphumela yokwelashwa kanye nezinga lokugxila kwesifo kungasiza kakhulu ekwakhiweni kwezinsiza zokuhlola ngempumelelo kanye nezindlela zokwelapha. Kunesidingo esiphuthumayo sezinkomba ezinembayo nezibonakala kalula ezingahlangene nezikhwehlela ezihambisana nokwenziwa yigciwane noma ezingakhombisa umthwalo wegciwane lesifo sofuba. Kulolu cwaningo kwabhekwa izimpawu ezincibilikayo namacellular phenotypes kulabo abane-TB noma inhlanganisela ye-TB ne-HIV kwase kuhlolwa ukuhambisana kwako ngokuhamba kwesikhathi kuze kufike lapho igciwane lofuba lingasaveli kanye nezinga lokujula kwesifo. Izindlela zokuqhuba ucwaningo: Lolu cwaningo lwenziwa ngokuba kusetshenziswe okusegazini okwaziwa ngeplasma kanye namaperipheral blood mononuclear cells ayetholakale ekuvivinyweni okwaziwa nge-Improving Retreatment Success (IMPRESS). Kwasetshenziswa neMultiplex immunoassays kanye nama-ELISA ukuhlola icytokine nechemokine kulabo asebengenwe yi-TB. Kwasetshenziswa neflow cytometry ukuhlola isimo nobunjalo bamamonocytes, amadendritic cells kanye nama-CD4+ T cells. Izindlela ezaziwa ngamacox proportional hazards kanye nelogistic regression zasetshenziswa ukuhlola ukuhlobana phakathi kwamacytokines, amachemokines, amaphenotypic profiles amamonocytes, amadendritic cells kanye namaseli e-CD4+ T nesikhathi sokushabalala kofuba kanye nokuhlaseleka kwamaphaphu kulabo abane-TB noma inhlanganisela ye-TB ne-HIV. Imiphumela: Ucwaningo lwahlaziya imiphumela yenhlanganisela ye-TB ne-HIV kusetshenziswa okuyizimpendulo ezikaliwe nokwaholela ekutholakaleni kwezindlela ezintsha lapho ushintsho oludalwe yi-HIV luvimbela ukulawulwa kwe-Mtb. Kwatholakala ukuthi i-IP-10 kanye ne-IL-6 yizona zinkomba zokuba khona kwe-Mtb emzimbeni kanye nezinye izifo ezivela ngoba umzimba uzama ukuzivikela kubantu abanesandulela ngculazi nalabo abangenaso. Kwabonakala ukwahluka kwezinga eliphezulu mayelana namamonocyte, amadendritic cell subsets kanye namaphenotypes ngesikhathi sokuhlasela kwe-TB kanye ne-TB/HIV okwaba nomphumela wokugudluka kwamaseli, ukunyakaziseka kwezicutshana, kanye nokusebenza kwezindlela zokuzivikela ezihambisana nezimo nokwaletha inguquko ekulweni namagciwane esifo sofuba nezinye izifo. Ekugcineni, kwatholakala iqhaza elisha elibanjwa amaseli e-integrin α4β7 CD4+ T ekwelapheni isifo sofuba: Le integrin α4β7 yanyusa ama-CD4+ T cells kulabo abanenhlanganisela ye-TB ne-HIV uma beqhathaniswa nalabo abane-TB kanti lokhu kwamanyaniswa nokuhlolwa Isikhathi eside kwalabo abasulelekile. Isiphetho: Imininingo etholakale kulolu cwaningo inikeza ukuqonda kabanzi okubalulekile ekusebenzeni kwamaplasma immune biomarkers, amamonocytes, amadendritic kanye namaseli e-CD4+ T ekutheni imithi yokwelapha i-TB izwela kanjani kanye nobunzima besifo kulabo abane-TB nabanenhlanganisela ye-TB ne-HIV.
Effect of HIV-1 subtype C Transactivator of transcription (Tat) A21P variant on TAR binding ability, nuclear levels of active positive transcription elongation factor b (P-TEFb) and viral latency = Umthelela we-HIV-1 subtype C Transactivator of transcription (Tat) A21P okuhlukile ekhonweni lokubopha i-TAR, amazinga enyukliya we-active transcription elongation factor b (P-TEFb) kanye neviral latency.
(2023) Mkhize, Zakithi Zinhle.; Madlala, Paradise Zamokuhle.
The HIV-1 Transactivator of transcription (Tat) enhances the ability of the viral promoter 5’ long terminal repeat (LTR) to drive viral gene transcription and is important for HIV-1 pathogenesis. Tat binds to the transactivator RNA (TAR) element of the 5’LTR and subsequently recruits the host positive transcription elongation factor b (P-TEFb) for efficient viral gene transcription. Inter- and intra-subtype Tat genetic variation that translates to functional differences has been reported. Specifically, HIV-1 subtype C (HIV-1C) exhibiting Alanine at position 21 of the Tat protein (TatA21) was reported to be associated with reduced LTR transcriptional activity compared to Tat exhibiting Proline at position 21 mutation (TatP21). However, the effect of Tat variation on its ability to recruit P-TEFb is unknown. Therefore, this study seek to determine the effect of HIV-1 subtype C TatA21 mutant on the ability of Tat to recruit P-TEFb to 5’ LTR to enhance viral gene transcription. To this effect, site-directed mutagenesis (SDM) was performed on the Plasmid pcDNA3.1(+) HIV-1C BL43/02 TatA21 to introduce TatP21 alone or together with other mutations using designed primers and the Q5 DNA polymerase kit. The effect of Tat mutations was measured using Tat transactivation assay where the luciferase activity was the measured output in TZM-bl cell lines and the impact of TatA21 was further assessed on ability of the LTR to drive GFP and Gag expression in Jurkat and A72 cells respectively. Next, protein modelling was performed using Hdock software, followed by RNA immunoprecipitation (RNA IP) was performed using stably expressing TatA21 and TatP21 in Jurkat cells. Lastly, co-immunoprecipitation of TatA21 and associated with significantly reduced LTR transcription activity compared to TatP21 (p = 0.0004). TatA21 resulted in had significantly lower GFP expression Jurkat cells (p = 0.0439) and lower Gag expression in A72 cells compared to TatP21. Although TatA21 reduced the LTR transcription activity compared to TatP21, protein modelling using Hdock software revealed that TatA21 and TatP21 protein structures were the same. Consistently, molecular docking showed that TatA21 had a lower binding affinity than TatP21. The RNA IP showed that TatA21 had significantly reduced affinity to bind to TAR compared to TatP21 (p = 0.0151). Moreover, TatA21 and TatP21 formed a complex with cycT1 and CDK9. Taken together, our data shows that HIV-1C TatA21 significantly reduced its transactivation activity but does not affect its ability to recruit P-TEFb. Interestingly, TatP21 is able to bind TAR more efficiently than TatA21 thus revealing a possible mechanism but which the reduced functionality of SDMs and patient derived TatA21 variants was observed. The effect of TatA21 and TatP21 on the propensity of HIV-1 latency development or reversal. To this effect, a recombinant viral vector exhibiting either TatA21 (C731CTatA21C) or TatP21 (C731CTatP21C) were generated. The C731CTatA21C or C731CTatP21C were separately co-transfected together with VSV-G and R8.91 into Jurkat cells for virus production. This virus was then used to infect Jurkat cells for 3 days. Followed by cell sorting of GFP- cells, which represented either truly negative or latently infected cells was then performed. We were able to successfully generate C731CTatA21C virus and characterized it to a 1.2% reactivation. However, the generation of C731CTatP21C recombinant viral vector was unsuccessful and thus could not be used for comparison. Future studies should involve the characterization of TatP21 in the propensity of latency development and/ or reactivation. Iqoqa. Iphrotheni eyaziwa ngeTransactivator of transcription (Tat) le-HIV-1 inamandla okuthuthukisa amandla egciwane 5’ le-LTR ukulawula ukuhlonzwa kofuzo lwegciwane futhi ibalulekile ekwelashweni kokukhula kwe-HIV-1. I-Tat ihlanganisa izinhlasiyana ze-RNA (TAR) ye-5'LTR futhi ikwazi ukudonsa i-P-TEFb ukuze ihlonze ngempumelelo isakhi sofuzo. Ukuhlukahluka kofuzo kwangaphakathi nokwangaphandle ekuhlonzweni komehluko nakho kuveziwe. Ngokukhethekile, uhlobo C lwe-HIV-1 (HIV-1C) ebonisa i-alanine endaweni engu-21 yephrotheni i-Tat (TatA21) kubikwe ukuthi ihlotshaniswa nomsebenzi wokuhlonza oncishisiwe we-LTR uma kuqhathaniswa ne-Tat ebonisa iproline ekuguqulweni kwe-21ye-Tat (TatP21). Nokho, umthelela wokuhluka kwe-Tat ekuphumeleni kwayo ukudonsa i-P-TEFb awaziwa. Ngakho-ke, lolu cwaningo beluhlose ukuhlonza umthelela we-HIV-1 lohlobo C lwe-TatA21 eguquguqukayo emandleni e-Tat okuhlonza i-P-TEFb kuya ku-5’ LTR ukuze kuthuthukiswe ukuhlonzwa kofuzo lwegciwane. Kulokhu, isite-directed mutagenesis (SDM) yenziwa kwiPlasmid pcDNA3.1(+) HIV-1C BL43/02 TatA21 ukwethula i-TatP21 iyodwa noma kanye nezinye izinguquko kusetshenziswa amathuluzi aklanyelwe kanye neDNA ye-Q5. Umthelela wokuguqulwa kwe-Tat ukalwe kusetshenziswa i-Tat lapho umsebenzi weluciferase wawungumphumela olinganiselwe emigqeni yenhlasiya ye-TZM-bl futhi umthelela we-TatA21 wabuye wahlolwa mayelana nokuphumelela kwe-LTR uhlonza i-GFP ne-Gag ezinhlasiyeni zeJurkat nama-A72 ngokulandelanayo. Okulandelayo, ukubheka amaprotheni kwenziwa kusetshenziswa isofthiwe ye-Hdock, kwalandelwa yi-RNA immunoprecipitation (RNA IP) kwenziwa kusetshenziswa okuveza ngokuzinzile i-TatA21 ne-TatP21 ezinhlasiyeni zeJurkat. Okokugcina, ico-immunoprecipitation ye-TatA21 ne-TatP21 yenziwe nge-cycT1 ne-CDK9. Imiphumela yethu ibonisa i-TatA21 eguquguqukayo iyodwa ihlotshaniswe nomsebenzi wokuhlonzwa kwe-LTR owehliswe kakhulu uma kuqhathaniswa ne-TatP21 (p = 0.0004). I-TatA21 iholele ekutheni ibe nezinhlasiya ze-GFP yeJurkat ephansi kakhulu (p = 0.0439) kanye ne-Gag ephansi ezinhlasiyeni ze-A72 uma kuqhathaniswa ne-TatP21. Nakuba i-TatA21 yehlise umsebenzi wokuhlonzwa kwe-LTR uma kuqhathaniswa ne-TatP21, ukubhekwa kwamaprotheni kusetshenziswa isofthiwe i-Hdock kuveze ukuthi izakhiwo ze-TatA21 ne-TatP21 zazifana. Ngokuvumelanayo, ukuhlonzwa kwezinhlasiya kubonise ukuthi i-TatA21 inobudlelwane obubophezelayo obuphansi kune-TatP21. I-RNA IP ibonise ukuthi i-TatA21 inciphise kakhulu ukuhambisana ukuze izibophezele kwi-TAR uma kuqhathaniswa ne-TatP21 (p = 0.0151). Ngaphezu kwalokho, i-TatA21 ne-TatP21 bakhe inkimbinkimbi ene-cycT1 ne-CDK9. Sekuhlangene, imiphumela yethu ibonisa ukuthi i-HIV-1C TatA21 iwunciphise kakhulu umsebenzi wayo wokwenza izinto kodwa ayithinti impumelelo yayo yokuhlonza i-P-TEFb. Kuyajabulisa ukuthi i-TatP21 iyakwazi ukuhlanganisa i-TAR kahle kakhulu kune-TatA21, ngaleyo ndlela iveze indlela engase ibe khona kodwa okuye kwabonwa ukusebenza okuncishisiwe kwama-SDM kanye nokuhluka okutholwe esigulini se-TatA21. Umthelela we-TatA21 kanye ne-TatP21 ekuthembekeni kokuthuthukiswa kokubambezeleka kwe-HIV-1 noma ukuguqulwa. Kulokhu, inhlanganisela yegciwane ekhombisa i-TatA21 (C731CTatA21C) noma i-TatP21 (C731CTatP21C) yenziwe. I-C731CTatA21C noma i-C731CTatP21C yadluliselwa ngokuhlukana ndawonye ne-VSV-G kanye ne-R8.91 kuzinhlasiya zeJurkat ukuze kukhiqizwe igciwane. Leli gciwane labe selisetshenziselwa ukuthelela izinhlasiya zeJurkat izinsuku ezi-3. Kulandelwa ukuhlungwa kwezinhlasiya ze-GFP, okwakumele izinhlasiya ezingezinhle ngempela noma ezisanda kungenwa amagciwane kwase kwenziwa. Sikwazile ukukhiqiza ngempumelelo igciwane le-C731CTatA21C futhi silibeke ku-1.2%. Nokho, ukukhiqizwa kwe-C731CTatP21C akuphumelelanga, ngakho-ke akukwazanga ukusetshenziselwa ukuqhathanisa. Ucwaningo lwangomuso kufanele luhlonze ubunjalo be-TatP21 ekuthambekeni kokuthuthukiswa kokubambezeleka nokusebenza kohlelo lokwelapha.
Inflammation and cellular immune phenotypes in TB/HIV co-infection = Ukuvuvukala nezinswebu zamasosha omzimba ezinhlayiya ku-TB/HIV.
(2023) Maseko, Thando Glory.; Sivro, Aida.; Archary, Derseree.
South Africa has the highest burdens of TB and HIV. HIV induced inflammatory and immune changes are known to increase the risk of TB recurrence and lead to poor disease outcome in co-infected patients. Here we characterised soluble inflammatory, NK and CD4+ T cell profiles in TB and TB/HIV disease. We utilized peripheral blood specimens from the CAPRISA 011 IMPRESS study to characterize NK and memory CD4+ T helper cell phenotypes during active TB and post TB treatment in individuals with or without HIV co infection. We also characterized the effects of these phenotypes on mycobacterial clearance and TB disease severity measured by the presence of lung cavitation. We additionally characterised plasma cytokine/chemokine markers of cavitary disease in drug-resistant TB patients from the CAPRISA 020 InDEX study. TB/HIV co infection led to the expansion of functionally impaired CD56neg NK cell subset. TB treatment completion resulted in restoration of total NK cells, NK cell subset redistribution and downregulation of several NK cell activating and inhibitory receptors. Higher percentage of peripheral CD56bright cells was associated with longer time to culture conversion, while higher expression of NKp46 on CD56dim NK cells was associated with lower odds of lung cavitation in the overall cohort and the TB/HIV co infected participants. With regards to memory CD4+ T cell responses, TB/HIV co infection led to higher percentage of Th2 cells, α4β1 and α4β7 integrin expressing memory CD4+ T cells, and lower percentage of Th9 cells. Increased IL-6 expression during MDR/XDR-TB was associated with higher risk of lung cavitation in CAPRISA 020 participants. Additionally smoking and previous history of TB were associated with increased risk of cavitary disease while HIV and higher BMI were associated with reduced risk of cavitation during MDR/XDR TB. We identified distinct changes in systemic inflammatory and NK cell and memory CD4+ T cell populations with respect to active disease, treatment completion, bacterial clearance and disease severity in TB and TB-HIV co-infected individuals. These results highlight biologically plausible and novel mechanisms by which concurrent HIV infection impairs the host immune control of Mtb infection. Iqoqa. INingizimu Afrikha inomthwalo omkhulu we-TB ne-HIV. I-HIV ifike nokuvuvukala nezinguquko kumasosha omzimba okwaziwa njengokukhulisa ubungozi bokubuya kwe-TB okuholela emiphumeleni emibi yesifo ezigulini eziphethwe nangezinye izifo. Lapha sibona ukuvuvukala okuncibikalayo, i-NK ne-CD4+ ubunjalo bezinhlayiya zika-T ezifweni ze-TB ne-HIV. Sasebenzisa izimelabunjalo zegazi ezingasekugcineni ocwaningweni lwe-CAPRISA 011 IMPRESS ukuze kubonakale i-NK nememori ye-CD4+ yenswebu yenhlayiya engumsizi ka-T ngesikhathi i-TB isenamandla nangemuva kokwelashelwa i-TB kulowo osuke enayo noma engenayo i-HIV nezinye izifo. Sichaza nomthelela wezinswebu zokucaciswa kwemycobacterial nokwenzeka ngamandla kwesifo se-TB okulinganiswa ngobukhona bezimbobo emaphashini. Sibuye sichaze ngabakhombisi besifo sezimbombo zesifo seplasma cytokine/chemokine ezigulini ezimelana nekhambi le-TB ocwaningweni lwe-CAPRISA 020 InDEX. Izifo ezingosomathuba ze-TB/HIV ziholela ekukhuleni kokuphazamiseka kokusebenza kwesethi encane yenhlayiya ye-CD56neg NK. Ukuqedelwa ukwelashelwa i-TB kuholela ekwenziweni kabusha kwezinhlayiya ze-NK, ukusabalaliswa kabusha kwesethi encane ye-NK kanye nokulawulwa maphansi kwezinhlayiya eziningi ze-NK ezenza izamukeli zisebenze noma ziphazamiseke. Iphesenti eliphezulu lezinhlayiya ze-CD56bright zazihlobaniswa nesikhathi eside sokubonakala kwenguquko, ngenkathi izinga eliphezulu lokuziveza kwezinhlayiya ze-NKp46 ku-CD56dim NK kwakuhlobaniswa nezinga eliphansi lokubhoboka kwamaphaphu eqoqweni lonke labantu beminyaka elinganayo kanye nababambiqhaza abane-TB/HIV kodwa bebenezinye izifo ezibaphethe. Ngokwezimpendulo zenhlayiya yememori ye-CD4+ T, izifo mixhantela ye-TB/HIV kwaholela ephesentini eliphezulu lezinhlayiya ze-Th2, i-α4β1 ne-α4β7 i-integrin ikhombisa izinhlayiya zememori ye-CD4+ T nephesenti eliphansi lezinhlayiya ze-Th9. Ukukhula kokuziveza kwe- IL-6 ngesikhathi i-MDR/XDR-TB ihlobaniswa nobungozi bezinga eliphezulu bokubhoboka kwamaphaphu kubabambiqhaza be-CAPRISA 020. Ngaphezu kwalokho ukubhema nomlando owedlule we-TB wahlobaniswa nokukhula kobungcuphe kwesifo sezimbobo emaphashini ngenkathi i-HIV ne-BMI ephezulu kwahlobaniswa nokwehla kobungcuphe bezimbobo emaphashini ngesikhathi se- MDR/XDR TB. Sathola izinguquko ezibonakalayo zabasengcupheni ohlelweni lokuvuvukala, izinhlayiya ze-NK kanye nezinhlayiya ze-CD4+ T ngokwesifo esimandla, ukuqedelwa kokwelashwa, ukuqedwa kwegciwane nokuba mandla kwesifo se-TB ne-HIV kulowo onezinye izifo ezimphethe. Imiphumela yagqamisa iqiniso elikholekayo nendlela yokwelapha okuyiyona okubuye kube nokutheleleka nge-HIV ngesikhathi esisodwa okuyikhona okuphazamisa umgcinikulawulwa kwamasosha omzimba esifo seMtb.
Genotypic and phenotypic analysis of Neisseria gonorrhoeae for the identification of resistance to various antibiotics in pregnant women attending antenatal care at the King Edward VIII Hospital in Durban, KwaZulu-Natal, South Africa.
(2021) Oree, Glynis.; Abbai, Nathlee Samantha.; Ramsuran, Veron.
Introduction: Worldwide antimicrobial resistance (AMR) is making the clinical management of sexually transmitted infections (STIs) increasingly challenging with a particular emphasis on the emergence of antibiotic resistant strains of Neisseria gonorrhoeae (N. gonorrhoeae). In the current study, the detection and emerging patterns of drug resistant clinical isolates of N. gonorrhoeae to previous and current antibiotics to treat cervicitis pathogens as per syndromic management guidelines was investigated. Methodology: This cross-sectional study was conducted at King Edward VIII Hospital and included 307 antenatal attendees. Two endocervical swabs were collected from each enrolled woman. Each enrolled women also provided data on socio-demographic, behavioural and clinical factors. The first swab was placed in Amies Charcoal media immediately after collection. This swab was used to confirm the identification of N. gonorrhoeae clinical isolates using culture based assays. Culture confirmed isolates were grown in Mueller Hinton Broth and were each adjusted to have a bacterial suspension turbidity of a 0.5 McFarland Standard. These isolates were then subjected to antibiotic susceptibility testing to ceftriaxone, tetracycline, spectinomycin, azithromycin, ciprofloxacin, penicillin G and cefixime using the Etest™ method. The second swab was processed for molecular based assays. Extracted DNA from the second swab was subjected to the TaqMan quantitative Polymerase Chain Reaction (qPCR) assay, an in-house 16S ribosomal RNA (rRNA) PCR and PCR detection of the opacity (opa) gene. DNA extracted from the endocervical swabs and cultured isolates were used for the detection of specific targets (genes/plasmids/mutations) associated with resistance to penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, azithromycin and ceftriaxone. All statistical analysis performed in this study was conducted in RS Studio. Results: The prevalence of N. gonorrhoeae was 7.8% (24/307) when detected by the TaqMan qPCR assay and 1.9% (6/307) by culture. When compared to culture, PCR for the opa gene and PCR for the 16S rRNA, the TaqMan qPCR assay was a more superior assay demonstrating a diagnostic accuracy of 94.5%. Susceptibility testing of the six isolates obtained after culturing showed resistant phenotypes for penicillin G (12 - 64 mg/L), tetracycline (1.9 - 32 mg/L) and ciprofloxacin (1.16 - 3 mg/L). Isolates displayed either dual or triple resistance to these 3 antibiotics. However, all isolates showed susceptibility to spectinomycin (>64mg/L), azithromycin (1mg/L), ceftriaxone (>0.125 mg/L) and cefixime (>0.125 mg/L). This study also detected the resistance determinants associated with penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, ceftriaxone and azithromycin from the molecular level using the primary endocervical swab sample. Gene mutations and plasmids associated with resistance to tetracycline (tetM gene carried on a plasmid), penicillin G (penicillinase producing plasmid) and ciprofloxacin (Ser-91 mutation) were detected confirming the results obtained with the susceptibility assays. Resistance mutations associated with the remaining antibiotics were not detected. There was a 100% correlation of cultured isolates and endocervical swabs for detecting the specific AMR determinants conferring resistance to tetracycline, penicillin G, and ciprofloxacin. Conclusion: The TaqMan qPCR assay has the ability to serve as a future diagnostic assay for the detection of N. gonorrhoeae. Despite the lack of resistance to spectinomycin, cephalosporins and azithromycin in our study population, continuous surveillance for emerging patterns of resistance to these antibiotics is still required since they form part of the current South African treatment guidelines. The detection of resistance determinants from the molecular level without the need for culture may prove to be more feasible for future epidemiological investigations focused on tracking antimicrobial susceptibility or resistance patterns in N. gonorrhoeae.
Trauma care in Sub-Saharan Africa: challenges and opportunities in Botswana and Tanzania for implementing Afrocentric systems.
(2021) Mwandri, Michael Bartholomew.; Hardcastle, Timothy Craig.
Summary available in PDF.
An investigation into the mode of action of pyrazinamide on mycobacterium tuberculosis.
(1999) Dwarka, Rahana Mohan.; Sturm, Adriaan Willem.
Abstract available in PDF.
The Clinico-pathological manifestations of schistosomiasis in the African and the Indian in Durban.
(1964) Bhagwandeen, Surridhine Brumdutt.; Elsdon-Dew, R.
Abstract available in PDF.
Aspects of gastrointestinal tuberculosis at King Edward VIII Hospital.
(1987) Pettengell, Keith Edward.; Mayet, Fatima G. H.
Abstract available in PDF.
Increase in live infected cell number with drug and generation of a quasispecies are consequences of multiply HIV infected cells.
(2018) Jackson, Laurelle.; Sigal, Alexander.
HIV may form reservoirs in anatomical compartments and evolve a quasispecies in order to survive under selective pressures such as antiretroviral drugs. Lymph nodes and lymphoid tissue - critical sites for reservoir formation - are environments conducive to cell-to-cell spread, an efficient mode of HIV transmission. Cell-to-cell spread can lead to multiple infections per cell which in turn profoundly changes how the virus responds to selective pressure. In this thesis, my goal was to understand the consequences of multiple infections per cell on how the infection responds to and evolves in the face of inhibitors. The specific aims were to: (1) model and experimentally examine the effect of attenuating cell-to-cell spread by using antiretrovirals (ARVs) on infected cell viability; (2) test whether a stable quasispecies can be formed and maintained by complementation– a process where virions derived from different HIV genotypes infecting the same cell share components; (3) test the feasibility of new single-cell RNA-Seq methodology that can be applied to quantify the frequency of multiply infected cells in vivo. These studies showed that: (1) partially attenuating infection involving multiple virions per cell with drug resulted in an increase in the number of live infected cells in both cell line and lymph nodes at suboptimal drug strengths. The increase in live infected cells was a result of fewer HIV DNA copies per cell, relative to no drug; (2) under the selective pressure of efavirenz (EFV), when drug-resistant and drug sensitive HIV co-infect the same cell during drug resistant evolution, complementation takes place, driving the formation and maintenance of a quasispecies; (3) Novel single-cell RNA-Seq approaches are feasible to quantify the number of cells that are multiply infected in vivo. Inhibiting mechanisms such as cell-to-cell spread may therefore reduce infection in the face of ARVs and limit viral diversity and hence the ability of HIV to evolve resistance.
The epidemiology and impact of pretreatment HIV drug resistance in adults in South Africa.
(2018) Chimukangara, Benjamin.; De Oliveira, Tulio De Paiva Nazareth Andrade.; Naidoo, Kogieleum.; Samuel, Reshmi.
HIV drug resistance (HIVDR) present prior to initiating or re-initiating antiretroviral therapy (ART), is known as pretreatment drug resistance (PDR). Conventionally, PDR is detected by Sanger sequencing. Drug resistant minority variants (DRMVs) that are not reliably detected by Sanger sequencing can be detected by next generation sequencing. The aims of this research were to assess levels of PDR in HIV hyper-endemic areas (with high HIV incidence and prevalence) in KwaZulu-Natal (KZN) province, trends of PDR in South Africa, and the impact of DRMVs on ART. To assess PDR in adults from KZN hyper-endemic areas, 1845 sequences were analyzed from two population-based HIV surveillance studies; a longitudinal HIV surveillance programme in northern KZN (2013-2014), and the HIV Incidence Provincial Surveillance System (HIPSS) in central KZN (2014-2015). Overall, 182/1845 (10.0%) had NNRTI-PDR mutations, and when analyzed by study year, NNRTI-PDR was 10.2% (CI:7.5-12.9) for the HIPSS study in 2014. To assess PDR trends in South Africa, 6880 HIV-1 sequences were collated from 38 datasets of ART-naïve adults (2000-2016). Increasing levels of PDR were observed, most marked from 2010. Crude pooled prevalence of NNRTI-PDR reached 10% in 2014, with a 1.18-fold (CI:1.13- 1.23) annual increase (p<0.001), consistent with findings from the HIPSS data. This provided the first evidence of high-level NNRTI-PDR in KZN and South Africa, supporting the transition to dolutegravir in standard first-line ART, as recommended by the World Health Organization when NNRTI-PDR reaches ≥10%. A case-control (2:1) study in HIV/TB co-infected adult patients was done to assess the impact of DRMVs at different thresholds. Cases were patients that initiated ART and had viral loads ≥1000 copies/mL after ≥6 months on ART, and controls were those that initiated ART and achieved virologic suppression through 24 months. Pre-ART NNRTI-resistance was associated with ART failure. NGS improved detection of HIVDR at lower thresholds, but reduced the specificity of identifying patients at risk of virologic failure, with the specificity reducing from 97% (CI:92-99) at 20% threshold, to 79% (CI:71-86) at 2% threshold. In all, the findings presented in this thesis provide a broad message about the need to improve quality in HIV prevention and treatment services.
A standardised approach to the treatment and management of significant acinetobacter species infection at academic complex hospitals in KwaZulu-Natal.
(2017) Swe Swe-Han, Khine.; Mlisana, Koleka Patience.; Baba, Kamaldeen.; Pillay, Manormoney.
Introduction: Carbapenem-resistant Acinetobacter species (Acinetobacter spp.) are increasingly recognised as important pathogens, whose resistance patterns present a high-risk global challenge. However, there is limited scientific data and a lack of a standardised approach to help the clinician select optimal therapy in local setting. This study aimed to provide a standardised approach for the management of significant Acinetobacter spp. infection based on phenotypic and genotypic characterisation of local isolates, as well as clinical characteristics and outcomes of patients at academic complex hospitals in KwaZulu-Natal. Objectives: The significance of Acinetobacter spp. infections and the most effective drug combinations for optimal therapy were determined. Acinetobacter spp. isolates were phenotypically and genotypically characterised. This was followed by the development of a standard management guideline for local use, based on the data obtained in the different objectives. Methods: The research consisted of a retrospective and prospective observational and experimental laboratory component. The laboratory component included synergy testing of colistin, susceptibility to antimicrobial agents in use at local hospitals, polymerase chain reaction and sequencing for analysis of the resistant genes related to carbapenem, colistin and amikacin. Phenotypic, genotypic, and clinical characterisation were utilised to develop a standardised management approach of significant Acinetobacter spp. infection. Results: Acinetobacter spp. was identified as a significant cause of sepsis and mortality among patients in a surgical intensive care unit (ICU). Cases of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Acinetobacter spp. increased over seven years, together with the emergence of pandrug-resistant (PDR) isolates. The results of synergy testing of colistin combinations with amikacin, carbapemens (imipemen, meropenem), ciprofloxacin, tazocin, linezolid, rifampicin and vancomycin against Acinetobacter spp. was highly diverse and speciesdependent. Characterisation of Acinetobacter spp. isolates showed that oxacillinase β-lactamase (OXA-23)-producing MDR isolates correlated with their antibiogram. Pulsed field gel electrophoresis (PFGE) showed horizontal transfer between seven clusters, each containing two patients each, totalling 14 patients. However, the PFGE typing revealed a diverse collection of MDR Acinetobacter spp. clones, and that isolates from not more than two patients were related. This suggests, therefore, that no outbreak had occurred based on the PFGE typing interpretation. Further genetic investigation revealed that the aphA6 gene were associated with amikacin resistance and IpxA gene may be associated with colistin resistance in our local setting. Conclusion: The results highlighted the importance of antibiotic stewardship in the treatment of Acinetobacter spp. infection. Individual-specific antibiograms are recommended as the best 2 approach for treatment in KwaZulu-Natal (KZN) and synergy testing should be performed for individualised direct therapy. The clinical and microbiological indicators of significant infection are crucial when establishing the decision to treat. The study provided a valuable standardised approach, including a flow chart of criteria for sepsis and colonisation; a standardised algorithm for the management; and synergy test at academic complex hospitals, Medical Microbiology laboratory, National Health Laboratory Service (NHLS) in KZN.
Culturally competent patient-provider communication with Zulu patients diagnosed with osteosarcoma.
(2016) Brown, Ottilia.; Aldous, Colleen Michelle.
Background: Communicating the diagnosis and prognosis of cancer is widely documented as a challenging task. Furthermore, ensuring that patients understand their treatment options is considered good practice; however literature in this regard tends to be limited. Performing these tasks in cross-cultural clinical settings complicates patient-provider communication. This study focused on Zulu patients diagnosed with osteosarcoma and was conducted at a tertiary (training) hospital in the province of KwaZulu-Natal (KZN), South Africa. The primary motivation for undertaking this research stemmed from observations in clinical practice that Zulu cultural beliefs and practices play a significant role in the management of osteosarcoma and hence culturally competent communication was an essential requirement at this site. In addition, patients typically present at the study site with locally advanced or metastatic disease. The late presentation of patients and further delays stemming from patients’ preferences to fulfil cultural practices results in treatment limitations and very poor prognosis. Healthcare providers in this setting are therefore expected to simultaneously inform patients of the diagnosis of osteosarcoma, the significant limitations with regard to treatment options, and prognostic considerations in a culturally sensitive manner that engenders cooperation in the patient while allowing them the opportunity to fulfil their cultural obligations. Aim and Objectives: This study aimed to develop an evidence-based practice guideline with recommendations for engaging in culturally competent communication with adult Zulu patients regarding the diagnosis, treatment and prognosis of osteosarcoma. Four objectives were devised in order to meet the aim of the study. Objective 1: Conduct an integrative literature review to gather evidence from previous research. Objective 2: Gather evidence from healthcare providers about the approach taken when they discuss osteosarcoma, its treatment and prognosis with Zulu patients as well as the cultural aspects considered during these discussions. Objective 3: Gather evidence from Zulu patients by exploring their understanding of the osteosarcoma diagnosis, its treatment and prognosis, and their experience of patient-provider communication throughout the illness experience was conducted. Patients’ cultural descriptions related to the management of osteosarcoma were also elicited. Objective 4: Develop an evidence-based practice guideline for culturally competent patient-provider communication with osteosarcoma patients based on the evidence collected in Objectives 1, 2 and 3. Methods: Objective 1: Whittemore and Knafl’s approach to conducting an integrative literature review was used. A number of databases were systematically searched and a manual search was also conducted. Specific inclusion and exclusion criteria were set and documents were critically appraised independently by two reviewers. Thirty-five documents were included following these processes. Data extraction and synthesis followed and were also independently verified. Objective 2: We used an exploratory descriptive contextual study design and conducted focus group interviews with professional nurses, allied health professionals, and orthopaedic physicians. Three focus groups with a total of twenty-three participants were conducted. Focus group interviews were audiotaped and transcribed verbatim. We thematically analysed the interview transcripts using Guba’s Model of Trustworthiness to ensure rigour. Objective 3: We used a qualitative case study approach with in-depth interviews that were conducted in isiZulu, audiotaped and transcribed verbatim. The transcripts were translated into English and back translated. Transcripts were then analysed thematically. Data were verified using Guba’s model of trustworthiness. Objective 4: The AGREE II (Appraisal of Guidelines, Research and Evaluation) appraisal instrument was used as a guide for developing the evidence-based practice guideline. The AGREE II is a 23 item tool comprising six domains, five of which were considered in developing the guideline. Results: The integrative literature review provided directives on how to deliver culturally competent communication to cancer patients. The review also highlighted the grave need for scientifically rigorous research in the field of culturally competent communication in the management of cancer. Our research with the healthcare providers produced a number of strategies for communicating with Zulu patients about the diagnosis, treatment and prognosis of osteosarcoma. These strategies also addressed cultural considerations and provided detailed information on the cultural factors that have to be taken into account when managing Zulu patients diagnosed with osteosarcoma. Challenges encountered with regard to discussing diagnosis, treatment and prognosis also emerged. In addition to revealing strategies and challenges that are confirmed in the literature, this study also unearthed unique strategies and challenges peculiar to this cross-cultural clinical setting. Despite the uniqueness of some of these strategies, they could be useful in other cross-cultural clinical settings where patients belong to collectivistic cultures, and observe traditions and other practices that are significantly different to Western medical approaches. Our findings also emphasised the importance of training healthcare providers on communication of sensitive information in cross-cultural clinical settings. Our research with Zulu patients diagnosed with osteosarcoma revealed that these patients had extensive understanding of the diagnosis of osteosarcoma, diagnostic procedures, the treatment options applicable to treating osteosarcoma and the side-effects of chemotherapy. These findings also revealed patients’ varied perceptions of and emotional responses to diagnosis and treatment and exposed difference in healthcare provider and patient perceptions of amputation. A significant contribution of the patient study is embedded in Zulu patients’ descriptions of their cultural and health beliefs and practices. Specific rituals that are performed to ensure successful outcome of medical procedures, to cleanse patients from bad luck and to address the issue of witchcraft were outlined. Consultation with a reputable traditional healer was flagged as an important cultural practice. However, patients varied in their adherence to traditional belief systems, participation in rituals and the extent to which they deferred decision-making to the familyreinforcing the importance of not stereotyping based on pre-existing knowledge of a cultural group. The evidence-based practice guideline was developed based on the findings from the integrative literature review and the studies conducted with the healthcare providers and the Zulu patients. These three sources of evidence facilitated the development of a guideline that presents generic requirements and recommendations for culturally competent communication, and denotes specific strategies for communicating diagnosis, treatment, and prognosis to Zulu patients diagnosed with osteosarcoma. The evidence-based practice guideline also explicates areas that require further research and refinement. Conclusions: The obvious contribution of this research is represented in the evidence-based practice guideline. However, each of the objectives makes a significant contribution to knowledge and practice. This study breaks ground and alerts to the magnitude of research that is required in cross-cultural clinical settings, especially in the South African context as literature in this context with regard to culturally competent communication is very limited. The need for training our healthcare providers in communication of sensitive information in cross-cultural clinical settings strongly emerged from the data. Policy directives that support culturally competent patient-provider communication at a healthcare systems level could significantly contribute to addressing resource constraints and creating clinical environments that are conducive to culturally competent communication.
An investigation into the renewed need for the care and prevention of congenital disorders in South Africa.
(2017) Malherbe, Helen.; Aldous, Colleen Michelle.
Abstract available in PDF file.
The prevalence of bacterial vaginosis in KwaZulu-Natal and its association with the vaginal immune response and shedding of HIV and HSV-2.
(2017) Naido, Kavitha.; Sturm, Adriaan Willem.
Introduction: South Africa has a high burden of sexually transmitted infections (STIs) and HIV. The role of Gardnerella vaginalis in the development of BV has been disputed after the recovery of G. vaginalis from healthy patients and the discovery of new bacteria using molecular identification. Infection with HSV has been associated with increased vaginal HIV RNA copies and bacterial vaginosis has been implicated as a risk factor for the transmission of HIV. Methodology: Consenting patients of > 18 years were recruited from two different primary health clinics. Microscopy was used to diagnose BV and serology for HIV, HSV-2 and syphilis testing. Chlamydia trachomatis and Neisseria gonorrhoeae were detected by BD Probetec, and conventional PCR was used for the diagnosis of Trichomonas vaginalis and recognised ulcer pathogens. Quantitative bacteriology and HIV viral loads were done using the Applied biosystems ABI 7500 Real Time instrument. Immune cells from vaginal tampon specimens were analysed using flow cytometry. Results: In both clinics, of the discharge pathogens T. vaginalis had the highest prevalence. The prevalence of both T. vaginalis and N. gonorrhoeae was significantly higher in Boomstreet clinic (p<0.05). The Umlazi D clinic had significantly more patients with BV (p<0.0001) and HSV-2 (p<0.05). Of the patients with ulcers, HSV-2 was detected in a one third of the specimens in each of the clinics. One patient was diagnosed with lymphogranuloma venereum (LGV). The Nugent score group 0-3 was dominated by Lactobacillus spp. while the Nugent score group 7-10 was dominated by Gardnerella vaginalis. The group with Nugent score 7-10 was shown to have significantly higher levels of immune cells that are proposed HIV targets. Lactobacillus spp. was associated with the group that was HIV antibody negative and Prevotella spp. with the HIV antibody positive group (p < 0.05). Prevotella spp. was not associated with shedding of HIV. The number of bacterial copies of G. vaginalis was significantly higher in patients shedding HIV (p < 0.05). In those shedding HSV-2 the number of copies of G. vaginalis was also higher but this did not reach statistical significance. Conclusion: The trend in STI prevalence was similar to that described previously. We report circulating LGV and there is a possible increase in gonorrhoeae which needs to be confirmed. The potential pathogenic role of G. vaginalis in BV as well as the increased risk of HIV transmission is emphasized.
“Sowing the seeds” the use of feedback in postgraduate medical education : a key factor in developing and enhancing clinical competence.
(2016) Bagwandeen, Chauntelle Ingrid.; Singaram, Veena S.
Background: The importance of feedback in enhancing clinical competency in the postgraduate medical education arena is well documented. Many definitions of, and models and frameworks for delivering feedback exist. Trainee specialists must learn how to use the feedback that they receive to hone their knowledge, skills and professional performance. Clinical supervisors must be equally effective in delivering the best feedback possible in all spheres of the training platform so as to impact positively on performance. However, while many studies have explored how feedback is given and received in postgraduate medical education, these studies have been conducted in homogenous settings. Aim: This study set out to examine how contextual and demographic factors affect the provision of feedback in a clinical training environment with heterogeneous demographics. This study aimed to investigate the perceptions of the registrars, consultants and Clinical Training Heads regarding the quality and factors that influence the process of giving and receiving feedback, so as to make recommendations for improvement and to develop policy guidelines for the enhancement of postgraduate clinical speciality training in diverse clinical training environments. Methods: A mixed methods approach was adopted for this study. Qualitative and quantitative analysis was done regarding the perceptions of the quality of the current delivery of feedback across six disciplines at a teaching hospital. Consultants and registrars consented to complete a questionnaire consisting of open- and close-ended questions to determine the quality, quantity, type and timing of feedback. Responses were coded on a five-point Likert Scale and combined to give an overall positive or negative response. The relationship between demographic factors such as age, race, gender, home language and discipline of study were also evaluated, with responses to open-ended questions used to extend and enrich the quantitative data. Descriptive statistics were used to analyse the data. Differences between groups were calculated using Pearson’s Chi Square test for independent variables, with a p–value of < 0.05 regarded as being statistically significant. Semi-structured interviews were conducted with the Clinical Training Heads to explore their feedback regarding the feedback received about feedback from the consultants and registrars. The Walt and Gilson (1994) triangular framework for policy analysis was used to explore the perceptions of current practice of the Clinical Training Heads of six major disciplines. A thematic analysis was conducted of their perceptions of how feedback was currently given and received by consultants and registers, with a view to developing policy guidelines to improve the practise of giving and receiving feedback. Results: The results revealed a disparity in the perceptions of consultants and registrars regarding current practise. Although consultants believed that they provided adequate feedback, registrars disagreed, citing an overall dissatisfaction with the process. Registrars believed that consultants lacked training in how to give feedback , and that important elements such as prior provision of the standards to be obtained, as well as feedback being based on directly observed performance were missing. Consultants concurred that they lacked capacity in how to give adequate feedback, but felt that heavy workloads, fear of negative reactions and the apathy of registrars as well as their failure to act on feedback when given, hampered the process. Male consultants and registrars both reported better experiences of giving and receiving feedback overall. Registrars who were English second language speakers had statistically significantly more favourable outcomes with feedback compared to English first language speakers. The Clinical Training Heads reported that lack of appropriate institutional support and an overall guiding framework, combined with multiple administrative bodies of registrars as well as language barriers, were challenges to be overcome. They identified areas for future improvement, including standardisation of the process, more effective use of logbooks and better monitoring and evaluation. Conclusion: Registrars and consultants agreed that feedback was essential to ensuring that clinical competencies were achieved. However, ongoing in-service education and training of consultants and registrars was necessary to ensure that consultants were fully capacitated to provide constant, high quality feedback and that registrars were able to recognise feedback when it was given. Feedback needs to be an integral part of the culture of the university teaching and learning ethos. To this end, policy guidelines incorporating elements of identified ‘Best Practices’ on how to give feedback were developed and recommended for implementation under the auspices of an overarching Postgraduate Committee for Teaching and Learning.
Metabolic complications of antiretroviral therapy (ART) in a South African black population..
(2014) Magula, Nombulelo Princess.; Lalloo, Umesh Gangaram.; Motala, Ayesha Ahmed.
Aims To determine the prevalence and incidence of lipodystrophy (fat distribution [lipoatrophy and lipohypertrophy] and metabolic complications [insulin resistance-dysglycaemia and dyslipidemia]) in HIV-1 infected adult subjects of second generation Zulu descent at baseline and during 24 months of follow-up on antiretroviral therapy (ART). Methods The total study group included three groups: HIV infected ART naive patients eligible for ART (HIV-ART, n=150), age, gender and ethnically matched HIV infected not eligible for ART (HIV-no ART, n=88) and HIV negative (control, n=88) subjects. All participants had demographic, anthropometric, biochemical and radiological assessments at baseline; in addition, the HIV-ART group had follow-up assessments for 24 months on ART (tenofovir, lamivudine and nevirapine or efavirenz). Fat distribution was assessed using FRAM questionnaires, computerized tomography (CT) scans and dual energy absorptiometry X-ray (DXA). Disorders of glycaemia (diabetes mellitus (DM), impaired glucose tolerance and impaired fasting glucose) were defined using WHO criteria. Total, LDL, HDL cholesterol and triglycerides were measured for each group; CD4 cell count and HIV RNA for group 2 and 3, at baseline, 3, 6, 12, 18 and 24 months. Poisson approximations estimated incidence of disorders of glycaemia. Results At baseline, when compared with the control group, the mean BMI (kg/m2) was significantly lower in the HIV-ART and HIV-no ART subjects (26.4 vs. 28.6 vs. 29.1; p =0.01). Prevalence of lipoatrophy as measured by participant and physician examination questionnaires was similar in the three groups. Visceral and subcutaneous fat area by CT scan were similar between the groups but limb and trunk fat mass by DXA scan was significantly lower in the HIV-ART compared to control subjects. In the HIV-ART group, at the 24 month follow-up, there was a significant mean reduction in HIV RNA (p<0.0001) and increase in CD4 cell count (p<0.0001). The mean BMI increased to 29.4 kg/m2 and no lipoatrophy developed; DXA scan showed a 33.6% increase in trunk fat mass (mean difference 4.2 kg, p <0.0001) and 30.8% increase in total fat mass (mean difference 9.4 kg, p < 0.0001); visceral (p 0.005) and subcutaneous (p 0.0002) fat area also increased. At baseline, the prevalence of DM was 0% in HIV-ART and HIV-no ART and 4.9% in control subjects (p 0.005); the prevalence of “any dysglycaemia” was 3.7% in HIV-ART and HIV-no ART compared to 8.6% in control subjects. When compared with group 1, mean values in group 3 were lower for the following serum lipids: total cholesterol (p<0.0001), LDL (p=0.0007) and HDL (p<0.0001). There was no difference in mean total triglycerides in the three groups (p=0.3). During follow-up, in the HIV-ART group, using glucose-based WHO criteria, the incidence of diabetes mellitus was 2.3 per 100 person year follow-up (PYFU) and of “any dysglycaemia” 7.6 per 100 PYFU. The only independent predictor of DM was visceral: subcutaneous fat ratio measured by CT scan (HR 2.95 [95% CI 1.25-6.98], p 0.01). Significant predictors for development of “any dysglycaemia” included systolic blood pressure (HR 1.04 [95%CI 1.02-1.07], p=0.0006), serum albumin (HR 0.85 [95% CI 0.76-0.94], p=0.002), CD4 cell count (HR 0.988 [95%CI 0.978-0.997], p=0.01) and efavirenz (HR 6.27 [95%CI 1.65-23.80], p=0.01) Serum total (p<0.0001), LDL (p<0.0001) and HDL-cholesterol (p<0.0001) increased significantly during follow-up. Conclusion: In this cohort of South Africans with HIV-1 infection, at baseline (prior to ART) there was no significant fat redistribution or lipoatrophy and an absent to low prevalence of dysglycaemia. In the follow-up study, ART use was not associated with lipoatrophy although there was significant increase in BMI and in limb and trunk fat mass by DXA scan. ART was associated with increased incidence of dysglycaemia. These findings underscore the importance of clinical monitoring on ART. The association of efavirenz with dysglycaemia warrants further evaluation.

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