School of Pathology (ETDs)
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Item Investigation of Contamination of Community Groundwater Sources with Antibiotics in Informal Settlements of Kisumu, Kenya(University of the Witwatersrand, Johannesburg, 2023-09) Karimi, Kellen Joyce; Ahmad, Aijaz; Duse, Adriano; Mwanthi, MutukuAntibiotics have been used to cure diseases, but there are growing concerns about the risk to human health caused by inadvertent exposure to low levels of antibiotics in the environment. Despite extensive reporting from the developed world on antibiotic pollution of groundwater, relatively little study has been conducted on antibiotic contamination of groundwater in the developing countries, particularly informal settlements. Antibiotic usage and misuse have long been seen as clinical events, with little understanding of the role of disposal in the development of environmentally induced resistance. Exposure pathways that contribute to groundwater contamination in informal settlements put residents at odds because they already face inequalities, such as a high disease burden exacerbated by antibiotic resistance; thus, proper antibiotic disposal is critical in protecting human and environmental health. The purpose of this cross-sectional study was to establish the prevalence of groundwater contamination with the common antibiotics’ such as sulfamethoxazole, trimethoprim, and metronidazole, and the related antibiotic resistance and the human health risk of exposure. Ethical clearance to conduct research was obtained from three institutions as follows: - the Health Research Ethic Committee of the university of the Witwatersrand (HREC. Protocol Number M190412); the Kenyatta National Hospital and University of Nairobi Ethics and Research Committee (KNH/UoN-ERC. Ref No. P71910/2018); and the National Commission for Science, Technology, and Innovation (Ref No. NACOSTI/P/19/3232/28732). Each respondent gave informed consent to participate in the study. Anonymity was maintained at all levels of the study to protect the study participants from identification. Antibiotic use, which is connected to antibiotic disposal, was evaluated in a random sample of 447 families. From the 188 mapped groundwater sources, a random sample of 49 groundwater sources was chosen, and water samples were taken for antibiotic concentration analysis utilising a solid-phase extraction and liquid chromatography coupled to magnetic sector high resolution mass spectrometry (SPE-LC-MS/MS). The Kirky-Bauber diffusion method was used to test antibiotic resistance in Escherichia coli. The community's potential groundwater contamination routes were assessed by determining antibiotic use and disposal among households as well as assessing the environmental risk of exposure. In the households visited, 75% (n=337) were female and 25% (n=110) were male. The prevalence of antibiotic use in informal settlements was 43% (n=193), with 70% (n=137) users reporting that they obtained the antibiotics through a prescription from a health practitioner. A significant relationship was observed between having HIV/AIDS and acquiring antibiotics through a prescription; p=0.001. An association was also observed among the informal settlements, where a lower number of MNY B dwellers did not receive a prescription for the antibiotics acquired. There was no statistically significant difference in antibiotic use between males and females; odds ratio=1.33; whereas there was a difference in HIV/AIDS status; odds ratio=0.313; and among informal settlements where the odds of using antibiotics were reduced in NY B; odds ratio=0.42. Respondents who used antibiotics either kept the unused antibiotics for future use 87.1% (n=27) or disposed them. Among the disposals 51.6% (n=16) disposed in pit latrines, 16.1% (n=5) dispose in compost pits, and 6.5% (n=2) dispose the remaining antibiotics by burning. Females completed their antibiotic doses at a higher rate (36.3%; n=117) than males (32.5%; n=39). Significant difference was observed in completion rate among the HIV/AIDS positive and negative respondents as well as among informal settlements; p<0.000 and p=0.001 respectively. On the other hand, groundwater use in these communities is widespread. Respondents used it for a variety of purposes, including drinking (9%; n=39), though they declined to report. Awareness of the health consequences of drinking antibiotic-contaminated water was also low (35%; n=158), especially among households that reported antibiotic use; p=0.003. Only Sulfamethoxazole was detected in 7 out of 49 groundwater samples at a detection frequency of 14.3%; with concentrations ranging from nd to 258 ng/L. Escherichia coli and Cryptosporidium parvum were isolated from all the 49 water samples and E. coli isolates from 3 (6%) water samples were resistant to sulfamethoxazole with Inhibition Zone Diameters of 0.8 mm, 10.5 mm, and 11.5 mm. The 3 water samples were however not among samples where sulfamethoxazole was detected. The Hazard Quotient was 0 (zero), and therefore no risk of exposure to sulfamethoxazole in the environment, but the level of antibiotics that trigger antibiotic resistance is not known. Because of the rising problem of antibiotic resistance due to overuse and incorrect disposal, teaching on safe antibiotic prescription should be incorporated into medical training for all cadres. In addition to educating patients on proper use and disposal, the ministries of health should ensure the antimicrobial stewardship standards are adhered to both locally and worldwide. Follow-up research of antibiotic resistance discovered in three groundwater sources must be done to eliminate the possible sources and prevent further spread. This study is instrumental in informing the inclusion of antibiotics on the list of frequently monitored contaminants during water treatment, as well as serving as a starting point for antibiotic surveillance in Kenya.Item HIV infection, antiretroviral therapy and the haemostasis of pregnancy(University of the Witwatersrand, Johannesburg, 2023-07) Schapkaitz, Elise; Libhaber, Elena; Jacobson, Barry; Büller, HarryThe human immunodeficiency virus (HIV) epidemic affects an estimated 30% of pregnant women living in South Africa. Increasing evidence suggests that women living with HIV are at a heightened risk for venous thrombo-embolism (VTE), which is a significant contributor to maternal mortality. In addition to a higher prevalence of obstetric and venous risk factors, this increased risk of VTE has been attributed to the effects of HIV and/or its treatment. HIV is characterized by immune activation and inflammation, which promote endothelial dysfunction and activation of coagulation. This is more pronounced with untreated HIV, yet this pro-inflammatory and pro-thrombotic balance may persist with long-term suppressive antiretroviral therapy (ART). However, the extent to which ongoing inflammation disrupts maternal haemostasis and predisposes pregnant women living with HIV to its prothrombotic consequences, is currently unknown. The aims of the work presented in this thesis in women living with HIV with access to ART were firstly to identify antepartum and postpartum risk factors for VTE; secondly to assess procoagulant changes in maternal haemostasis; and thirdly to determine risks of thrombosis and bleeding associated with thromboprophylaxis for VTE prevention. An epidemiological case-control study was performed in 128 cases with pregnancy related VTE and 640 matched controls. This study found at least a two-fold increased risk for VTE among pregnant and postpartum women living with HIV. In addition, antepartum risk factors, that may explain the disproportion of VTE risk in HIV, included medical co-morbidities and chronic hypertension, while postpartum risk factors included a personal history of VTE, medical co-morbidities, systemic infection, prolonged hospital admission and postpartum haemorrhage. Opportunistic infections, ART and the degree of immunosuppression were not associated with VTE risk. A sub-study followed and investigated antiphospholipid antibodies (aPL) in 215 women with thrombosis and/or obstetric complications. In this study, 15 (13.2%) of the women with HIV were positive at baseline for one of the five criteria aPL. The prevalence of aPL was not significantly increased among women with HIV, as compared to HIV negative women. Furthermore, the aPL profiles were not significantly different between the two groups. Lupus anticoagulant (LAC) positivity, on a single occasion, was associated with thrombosis (p < 0.003). Subsequently two prospective cross-sectional studies were conducted which assessed endothelial activation as well as fibrinolysis, coagulation and platelet activation in pregnant women with HIV, in each trimester. The studies included three groups: HIV negative, HIV with virological suppression (< 50 copies/mL) and HIV with viral load (VL) of >50 copies/mL. Endothelial activation was evaluated by measuring von Willebrand factor (VWF) antigen, VWF propeptide, multimer patterns and ADAMTS-13 antigen, activity, and antibody levels. The results showed an increase in the ratio of VWF propeptide to VWF antigen in the first, second and third trimester, in the HIV virologically suppressed group (1.7 ± 0.7, 1.7 ± 0.4, 1.6 ± 0.5) and the HIV group with VL > 50 copies/mL (1.9 ± 0.9, 1.7 ± 0.9, 1.6 ± 1.1) compared to the HIV negative group (1.4 ± 0.6, 1.3 ± 0.4, 1.2 ± 0.3, p < 0.05). Virological suppression was not associated with a significant reduction in this ratio, in each trimester. In addition, increased high molecular weight multimers were observed in the HIV groups, despite only a mild reduction in ADAMTS-13 activity compared to the HIV negative group (p < 0.001). Thereafter, fibrinolytic activity was evaluated by measuring d-dimer and plasminogen activator inhibitor-1 (PAI-1). Coagulation activity was determined by measuring thrombin-antithrombin (TAT) complex concentrations, and platelet factor-4 and platelet indices, namely mean platelet volume (MPV) and platelet distribution width as a measure of platelet activation. The results showed increased log d-dimer levels in the first, second and third trimester, in the HIV virologically suppressed group (-1.2 ± 0.5, -0.9 ± 0.4, -0.5 ± 0.3) and the HIV group with VL > 50 copies/mL (- 1.1 ± 0.4, -0.7 ± 0.4, -0.5 ± 0.5) compared to the HIV negative group (-1.4 ± 0.2, -1.1 ± 0.3, -0.8 ± 0.3, p < 0.05). Additionally, log PAI-1 levels were increased in the first, second, and third trimester, in the HIV virologically suppressed group (1.0 ± 0.4, 1.3 ± 0.4, 1.5 ± 0.4) and the HIV with VL > 50 copies/mL (0.8 ± 0.5, 1.2 ± 0.4, 1.5 ± 0.3) compared to the HIV negative group (0.4 ± 0.5, 0.8 ± 0.3, 1.3 ± 0.3, p < 0.05). Virological suppression was not associated with a significant reduction in first and third trimester d-dimer and PAI-1 levels. Thrombin-antithrombin complex levels were not increased, in the HIV virologically suppressed group as compared to the HIV negative group, beyond the first trimester. With regard to platelet parameters, only log MPV measured in the third trimester was decreased in in the HIV virologically suppressed group (2.3 ± 0.1) and the HIV group with VL > 50 copies/mL (2.3 ± 0.1) compared to the HIV negative group (2.5 ± 0.2) (p < 0.001). The last study was a longitudinal study of 129 pregnant women at intermediate or high risk of VTE, who received thromboprophylaxis. Venous thrombo-embolism occurred antepartum in 1.4%, 95% confidence interval (CI) 0.04-7.7 of intermediate and 3.4%, 95% CI 0.4-11.7 of high risk pregnancies. Major, clinically relevant non-major and minor bleeding events occurred in 7.1%, 95% CI 2.4-15.9 of intermediate and 8.5%, 95% CI 2.8-18.7 of high risk pregnancies. Owing to the small number of events, this study could not assess for HIV as a predictor of thrombosis and bleeding. Thus, in conclusion, the findings described in the studies in this thesis contribute to our knowledge in pregnant women living with HIV in the following ways. Firstly, HIV emerged as a significant antepartum and postpartum risk factor for VTE. Traditional obstetric and venous risk factors were also linked to the risk of thrombosis and could be useful for identifying women with HIV, who may benefit from postpartum and/or antepartum thromboprophylaxis. Secondly, this thesis identified heightened markers of endothelial activation and impaired fibrinolysis. Markers such as the ratio of VWF propeptide to VWF antigen, d-dimer and PAI-1 may provide a biological mechanism for the increased risk of pregnancy-related VTE in in HIV. Finally, this thesis provided rates of thrombosis and bleeding in women who received thromboprophylaxis in pregnancy and the postpartum period which can be used to advise women with HIV of the associated risks.Item Life History Trade-offs associated with Evolution of Cancer(University of the Witwatersrand, Johannesburg, 2023-07) Worsley, Catherine Mary; Durand, Pierre; Mayne, Elizabeth; Veale, RobThe evolution of multicellularity requires cooperation between single cells to form new multicellular individuals. Changes in levels of selection occur during this process, with selection at the multicellular level overriding that at the single cell level. For a multicellular individual to function, somatic mutations and selection must be under tight regulation. Nevertheless, mutations and selective environmental pressures can select for cells with fitness advantages relative to normal cells, resulting in cancer. Therapeutic drugs and radiation are forms of artificial selection that can drive the development and selection of cell populations that are resistant to treatment. Cancer occurs because of the failure of multicellular systems to suppress somatic evolution. This somatic evolution results in tumour cells with a wide range of phenotypes with either fast (proliferating) or slow (quiescent) life history strategies. Evolutionary theory provides a framework for understanding what drives the formation of these phenotypes and the ecological niche that supports them, and helps in predicting tumour progression and response to therapy. The key hypothesis of this study was that selective pressures in the tumour microenvironment drive trade-offs between tumour cell survival, proliferation, and apoptosis. An extensive literature review was conducted to identify key selective pressures affecting tumour progression. Low extracellular pH was identified as a component of the tumour microenvironment that affects life history trade-offs, and particularly drives escape from immune-mediated destruction. A protocol was then developed to expose cancer cells to low pH in cell culture. Breast carcinoma and oesophageal squamous cell carcinoma cell lines were selected for these experiments based on the prevalence of these cancers and because of their different anatomical locations. Exposure to low pH induced different levels of apoptosis in each cell line. This also affected cell cycle progression and the secretion of growth factors and immunomodulatory cytokines. The oesophageal cell line, WHCO6, adapted to moderate acidity levels with some cells undergoing apoptosis. Factors released by these cells supported the growth and survival of related cells. In contrast, in the breast carcinoma MCF-7 cell line, low pH induced high rates of apoptosis, and factors released by dying cells stimulated death in related cells. This study highlights that different life history strategies are employed by different cancer types. It also shows the importance of the tumour microenvironment, and acidity in particular, in driving tumour cell adaptation and survival. This study also identifies apoptosis as a pro-tumorigenic driver of cancer progression which has important therapeutic implications.Item Biomarkers to predict Tuberculosis treatment response(University of the Witwatersrand, Johannesburg, 2023-06) Boshielo, Itumeleng Tania; Tiemessen, Caroline; Kana, BaveshTuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis (Mtb). Despite the implementation of multifaceted TB prevention and control efforts, a significant number of people still dee from TB. Consistent with this, an uptick in TB-related mortality was recently noted, which has been ascribed to the negative effects of Coronavirus disease-2019 (COVID 19) on TB programs. The complex life cycle of Mtb is largely due to the use of immune evasion mechanisms to establish initial infection, remain dormant in the host, and reactivate pathogenicity under favourable circumstances. The prolonged TB treatment regimen is necessitated by the slow response of bacterial populations to standard TB chemotherapy, a phenomenon that may be caused by persistent, drug-tolerant bacteria. Scientific literature has provided evidence for these types of bacterial populations in the form of Differentially Culturable Tubercle Bacilli (DCTB). It has been demonstrated that DCTB represent drug tolerant bacteria that appear to be cleared at slower rate than organisms detected by routine culture methods. However, it remains unclear if DCTB populations elicit different immune responses when compared to their conventionally culturable counterparts. Herein, we address this question by optimizing a laboratory model for the generation of DCTB in vitro and test the capacity of clinical isolates of Mtb from Lineage 2 (Beijing) and Lineage 4 (LAM) to adopt the DCTB state. Using the Most probable number (MPN) assay, in the presence of culture filtrate (CF) as a source of growth factors to resuscitate DCTB, and colony forming units, the amount of DCTB in our model was quantified. As demonstrated by the limited growth on agar plates and increased growth in liquid media supplemented with CF from an axenic culture of Mtb, our findings demonstrated that carbon starvation was able to generate DCTB from clinical Mtb strains. After generating these populations, we stimulated whole blood with DCTB and conventionally culturable populations and report on the stimulation of a select set of cytokines (IFN-γ, IL-4, IL-5, IL-6, IL-12p70 and TNF-α) using a Bead Array Multiplex Immunoassay. In comparison to H37Rv-DCTB and LAM-DCTB, Beijing-DCTB induced significantly reduced levels of IL-5 and TNF-α. When comparing cytokine production between culturable and DCTB populations, within a single strain, we noted that LAM-DCTB was delayed in the production of IFN-γ whilst Beijing-DCTB was not able to induce production of this cytokine when compared to conventionally culturable counterparts. These data suggest that shifting to a non-replicating DCTB state does indeed affect the ability of clinical isolates to induce immune responses. Based on these observations, we next set out to determine if DCTB affects immune responses during treatment of Mtb infected individuals. In prior work, using a prospective observational cohort, we demonstrated a substantive heterogeneity in clearance of DCTB in individuals with drug susceptible TB. We were able to classify these response patterns into three broad groups including (I) participants who were able to clear DCTB within the first two weeks of treatment (treatment-responsive); (II) those with delayed ability to clear these organisms (delayed-responsive) and (III) a group of individuals where DCTB did not change substantively during treatment (non-responders). Given these stark differences in treatment response patterns, we hypothesized that the immune responses associated with these patterns would be substantively different. In the second component of this work, we set out identify immune biomarkers that predict an effective response of DCTB to TB treatment. To quantify cytokines, chemokines and growth factors in plasma from these groups, we used a 65-plex Luminex assay, with a broad selection of targets. Statistically significant differences between these groups were analysed using the Kruskal-Wallis test with Dunn’s multiple comparisons, with p<0.05 was considered as statistically significant. When compared to patients who had TB and HIV co-infection, the number of cytokines that may possibly be used to report on the effectiveness of TB treatment was significantly higher in Mtb-only infected patients. This suggests that HIV infection significantly reduces the number of cytokines that can be used to report on TB treatment response. The ROC analysis of I-TAC, G-CSF and VEGF-A showed that these cytokines have a significant discriminatory power to distinguish treatment-responsive and non-responsive patients from HCs using DCTB as the measure of treatment response. No unifying cytokine signature that predicted DCTB response in all groups was identified. Together, our results indicate that some inflammatory markers are elevated in individuals with TB that rapidly clear bacteria during treatment. Given that these responses are based on DCTB, which represent drug tolerant populations, these select cytokines may be useful in evaluating the effectiveness of novel shorter TB treatment regimens.Item Exploring the Interplay of Chemokine Receptors CCRS and CXCR6 in Mechanisms of Natural Control in HIV-1-lnfected Black South Africans(University of the Witwatersrand, Johannesburg, 2023-06) Koor, Gemma Whitney; Tiemessen, Caroline T.; Paximadis, Maria; Shalekoff, SharonIn sub-Saharan Africa, HIV-1 is a significant cause of morbidity and mortality. However, research remains primarily focused on North American and European population groups, who have remarkably different genetic backgrounds to individuals from sub-Saharan Africa. HIV-1 controllers represent a model of HIV-1 functional cure, with some individuals able to control viral replication, and some able to sustain immune function in the presence of high viral loads, both in the absence of antiretroviral therapy (ART). The chemokine receptors CCR5 and CXCR4 are the major coreceptors HIV-1 utilises to enter cells. The use of alternative coreceptors, such as the CXCR6 coreceptor, is thought to contribute to the lower pathogenicity exhibited by the HIV-2 and SIVsmm strains. Building on previous work conducted in our research unit on these two coreceptors in South African populations, this thesis firstly describes CCR5 genetic variants that associate with HIV-1 control or risk of progressive infection in black South Africans, and then explores constitutive expression levels of CCR5 and CXCR6 on various peripheral blood immune cell subsets in the absence of HIV-1 infection in ethnically divergent population groups. The effect of sex, age, and select CCR5 and CXCR6 single nucleotide polymorphisms (SNPs) on expression levels of these two receptors was also investigated. The CCR5 5’UTR and 3’UTR regions were PCR-amplified and sequenced from genomic DNA extracted from 145 ART-naive black South African individuals living with HIV-1 (71 HIV-1 controllers – 23 elite controllers, 37 viraemic controllers, 11 high viral load long-term non-progressors and 74 progressors). Findings confirmed results from other studies in showing that the CCR5 HHE haplotype is deleterious for HIV-1 disease progression, and the HHA haplotype and HHA/HHC genotype associated with protection from HIV-1 disease progression. Novel haplotypes were characterised, both in the 3’UTR and spanning the CCR5 5’UTR and 3’UTR. Overall, findings suggest that two CCR5 promoter SNPs (-2459 G>A and -2135 T>C) and one CCR5 3’UTR SNP (+2919 T>G) may be key functional variants with regards to HIV-1 control in black South Africans. To gain further insight into the constitutive expression of CCR5 and CXCR6 on peripheral blood immune cells and explore the relationship between select genetic variants and expression, immunophenotyping by flow cytometry was conducted using whole blood from age- and sex-matched ethnically distinct South African HIV-uninfected individuals (17 black, 21 white). Expression levels of CCR5 and CXCR6 were assessed on CD4+ and CD8+ T cells, B cells, monocytes and NK cells, and their respective subsets. The effects of age and sex on expression levels of these two receptors was also investigated. Population-specific differences with regards to CCR5 expression on all cell types, except for B cells, were evident. Generally, black South Africans exhibited a lower expression level of CCR5 compared to white South Africans. CXCR6 expression only differed with regards to percentage of CXCR6-expressing cells, not CXCR6 density (numbers of cell surface receptors). Black individuals had a lower percentage of CXCR6-expressing CD8+ T cell subsets (naïve and effector memory) and a higher percentage of CXCR6-expressing CD14+CD16+ monocytes compared to white individuals. Overall, we found significant population-specific differences in expression levels of both CCR5 and CXCR6, multiple associations with cell activation (as measured by HLA-DR expression) and CCR5 and CXCR6 expression, and CCR5 and CXCR6 expression was positively significantly correlated on multiple cell subsets. Furthermore, both sex and age influenced CCR5 and CXCR6 expression, however results varied widely across the two population groups studied. Sex differences were only evident in white individuals; predominantly CXCR6 expression was increased in males compared to females. Age associations with CCR5 and CXCR6 expression were also primarily found in white individuals. Four CCR5-related SNPs that are associated with HIV-1 control in this or other studies (rs553615728 -4223 C>T SNP, rs1799987 −2459 G>A SNP, rs746492 +2919 T>G SNP and rs1015164 G>A SNP) were assessed for their potential association with CCR5 expression levels. The +2919 TG genotype significantly associated with a higher percentage of CCR5-expressing total CD8+ T cells, transitional memory and terminally differentiated CD8+ T cells compared to the GG genotype. The +2919 GG genotype associated with a lower percentage of CCR5-expressing B cells compared to the TT and TG+TT genotypes, however, only in white South Africans. The +2919 TG and TG+TT genotypes associated with significantly higher CCR5 density on all CD8+ T cell subsets, except for naïve CD8+ T cells, when compared to the GG genotype. When evaluating two CXCR6 genetic variants previously associated with HIV-1 viraemic control (rs2234355 G>A and rs2234358 G>T) in relation to CXCR6 expression, possession of the rs2234355 SNP GA genotype associated with lower CXCR6 expression on select CD4+ and CD8+ T cell subsets as well as on B cells, while possession of the rs2234358 SNP TT genotype associated with higher CXCR6 expression on multiple cell types, primarily in white South Africans. Possession of the -358TT/+355GA genotype combination associated with lower CXCR6 expression on select subsets of CD4+ T cells and monocytes. In summary, this study provides information on genetic variation in the CCR5 gene in a South African context, describes genetic variants associating with HIV-1 control in black South Africans, adds novel insight into constitutive CCR5 and CXCR6 expression levels on CD4+ and CD8+ T cells, B cells, monocytes and NK cells in HIV-1-uninfected black and white South Africans, and describes the potential associations of select genetic variants and expression. Black and white individuals differed in their baseline expression levels of CCR5 or CXCR6, which was partly driven by host genetic factors that were explored. This work highlights the importance of considering effects of ethnicity, age, and sex in any studies addressing any immune molecules in relation to differential HIV-1 outcomes of infection susceptibility/protection, disease progression, or HIV-1 virological control on antiretroviral therapy. Although conducted on small numbers of individuals, these variables clearly influenced constitutive expression of CCR5 and CXCR6, and further population-specific studies are warranted to gain further insights. Findings from this study have implications for risk of acquisition of HIV-1 infection and for disease progression in people living with HIV-1. Understanding the role of these molecules is important for informing strategies for both HIV-1 prevention and HIV cure.Item The role of the 20-hydroxyecdysone (20E) signaling pathway in modulating Anopheles arabiensis reproduction, gut microbiome and anti-bacterial immunity(University of the Witwatersrand, Johannesburg, 2023-05) Ekoka, Elodie; Dahan-Moss, Yael; Koekemoer, LizetteThe 20-hydroxyecdysone (20E) signaling pathway, which is activated when 20E binds to its ecdysone receptor, EcR, is a promising target to reduce Anopheles mosquitoes’ ability to transmit malaria. The function of this pathway is typically assessed by altering the pathway and assessing how this manipulation affects a phenotype of interest. Two ways to alter this pathway include injecting mosquitoes with 20E or reducing EcR transcript levels with RNA interference (RNAi). Whether the 20E signaling pathway regulates An. arabiensis fecundity, fertility, gut bacteria, and immunity has never been investigated. These questions were addressed in this study by using a South African An. arabiensis strain. First, RNAi was used to investigate whether EcR silencing affects An. arabiensis reproductive output. While EcR depletion did not affect the mosquito fecundity, both vitellogenesis and egg fertility were impaired, as indicated by adecrease in the expression of some yolk genes and the number of eggs that hatched into larvae. Next, a link between the gut bacteria and EcR expression was established, by showing that antibiotic-fed (i.e., with less gut bacteria) mosquitoes displayed fewer EcR transcripts. To investigate whether the relationship between An. arabiensis gut microbiome and EcR expression was mediated by the mosquito innate immune defenses, the expression of ten selected anti-bacterial immune genes was measured in the gut and the whole mosquito after disturbing the 20E signaling pathway. This experiment uncovered that the 20E signaling pathway down-regulates the mosquito anti-bacterial immune defenses, which may favour bacterial proliferation post feeding. Finally, the effect of Gram-negative and Gram-positive bacteria on EcR expression was assessed by injecting mosquitoes with each type of bacteria and quantifying EcR transcripts. The results suggested that only Gram-negative bacteria influenced EcR expression. Altogether, these results demonstrated that An. arabiensis reproduction, gut microbiome, and antimicrobial peptides are regulated by 20E.Item Synthetic cytology image generation to augment teaching and quality assurance in pathology(University of the Witwatersrand, Johannesburg, 2023-05) McAlpine, Ewen David; Michelow, Pamela; Celik, TurgayINTRODUCTION: Urine cytology offers rapid and relatively inexpensive screening for the detection of high-grade urothelial neoplasia in patients with haematuria. In our setting of a public sector laboratory in South Africa, however, there is a paucity of such specimens with which to train cytotechnologists and cytopathologists. Advancements in Generative Adversarial Networks present a potential solution to this problem by allowing for the generation of synthetic urine cytology images to supplement teaching and training. We illustrate an end-to-end machine learning model – from dataset creation to testing synthetic images in pathology personnel – to assess this technology in a real-world setting. METHODS: Two hundred and fourteen urine cytology slides were digitised and processed to construct a morphologically balanced dataset containing examples of benign, atypical and malignant urine cytology images. This dataset was used to train a StyleGAN3 model to generate synthetic urine cytology images. These synthetically generated images were then tested in a group of pathology personnel – both pathologists and trainees – to assess whether a difference between real and synthetic urine cytology images exists. Diagnostic error rate and subject image assessment were tested. RESULTS: StyleGAN3 was able to generate a wide morphological diversity of realistically appearing benign, atypical and malignant urine cytology images. When testing how these synthetic images were perceived by pathology personnel, there was no significant difference in diagnostic error rate, subjective image quality or inclusion of synthetic images in a cytology teaching set. DISCUSSION: This work presents a proof-of-concept illustration of the feasibility of the use of synthetic cytology images to supplement pathology teaching when real examples may be difficult to obtain. Furthermore, this work presents important insights into the dynamics of pathology dataset creation and discusses the use of synthetic data in health education and the ethical and legal issues that arise with the use of synthetic patient data. CONCLUSION: Our work demonstrates that realistic, morphologically diverse urine cytology images can be generated using existing GANs technology and that human observers find such synthetic data visually acceptable. Additionally, our data indicate that there is no significant difference in synthetic data in terms of subjective image quality or diagnostic classification as determined by pathology personnel.Item Comparison of second and third generation parathyroid hormone (PTH) assays performed at Charlotte Maxeke Johannesburg Academic Hospital – Are they fit for purpose?(University of the Witwatersrand, Johannesburg, 2023-11) Nhlapo, Nokuthula; Jacob, Doreen; Maphayi, MphoBackground: Parathyroid hormone (PTH) measurement is crucial in the investigation of calcium and phosphate disorders and in the management of chronic kidney disease (CKD). Available PTH assays include second (intact PTH) and third generation (PTH 1-84) assays. Intact PTH assays are widely available and used for clinical guidelines but overestimate PTH in CKD. PTH 1-84 assays are more specific, but lack of standardisation has complicated clinical interpretation. The study aimed to compare the second and third generation assays to determine the difference in analytical performance and the effect on clinical interpretation. Methods: A method comparison was done on 481 patient samples with PTH requested at Charlotte Maxeke Johannesburg Academic Hospital. PTH was measured in each sample using both intact PTH and PTH 1-84 assays. Passing Bablok regression and Bland Altman plots were performed to determine method agreement and bias. Analytical performance was assessed using the European Federation of Clinical Chemistry and Laboratory Medicine biological variation specifications. Clinical performance was compared in the diagnosis of hypo- and hyperparathyroidism, and in predialysis and dialysis CKD based on current Kidney Disease Improving Global Outcomes guidelines. Results: Intact PTH had a higher median concentration than PTH 1-84 (9.93 vs 8.60 pmol/L, p<0.0001) but showed good correlation (r = 0.994 and p<0.0001). Regression analysis revealed significant systematic and proportional differences, with increased deviations at higher concentrations. The average bias was above allowable bias of 7.1%. Clinical interpretation of hypo- and hyperparathyroidism and predialysis and dialysis groups was unchanged. Conclusions: There was significant bias observed between the two PTH assays thus, they should not be used interchangeably. However, no significant changes in clinical interpretation were found when one assay was used over the other. The decision to use third over second generation PTH assay should consider the impact on clinical interpretation in the population.Item Epidemiology of laboratory-confirmed SARS-CoV-2 hospitalized cases in a tertiary hospital, Gauteng Province, South Africa, 1 April 2020 to 31 March 2021(University of the Witwatersrand, Johannesburg, 2021-12) Sikhosana, Mpho Lerato; Makatini, ZinhleGauteng Province (GP) was the most affected province in South Africa during the first year of the COVID-19 pandemic. We aimed to describe the epidemiology of COVID-19 cases admitted in one of the largest quaternary hospitals in the province during the two pandemic waves. We used data from the national hospital surveillance system, DATCOV, that recorded COVID-19 admissions at Charlotte Maxeke Johannesburg Academic Hospital in (GP) from 5 March 2020 to 27 March 2021. We used multivariable logistic regression to determine a) factors associated with hospitalization in the second compared to the first pandemic wave, and b) factors associated with in-hospital mortality. There were 1861 cases admitted during the study period. The mean age of the cases was 50 (IQR 37-61), 51.80% were females, and 58.68% were black. Of the total number of admissions, 2.10% were healthcare worker, 53.85% of whom were nurses. On admission, 91.99% of cases were admitted at a general ward while 5.86% were admitted at an intensive care unit. Overall, 10.59% of the cases required intensive care during their hospital stay. The case fatality ratio was the highest (28.54%) during wave 2 and lowest during pre-wave (11.49%). Compared to the first wave, factors associated with hospitalization during the second wave included age >80 years (adjusted odds ratio [aOR] 3.43, 95% CI 1.07-10.98) compared to ages 0-19 years, as well as being of other race (aOR 5.63, 95%CI 1.84-17.20) compared with White race. Regarding in-hospital mortality, associated factors included age groups 60-79 (aOR 4.53, 95%CI 1.03-19.86) and >80 (aOR 9.63, 95%CI 1.93-48.01) compared to ages 0-19 years; male sex (aOR 1.55, 95%CI 1.16-2.08); presence of an underlying comorbidity (aOR 1.99, 95%CI 1.45-2.71) 106 as well as being admitted during the second wave (aOR 1.54, 95%CI 1.12-2.10). Our study found that there was a higher risk of mortality during the second compared to the first wave, and other factors associated with mortality included older age, being male as well as having an existing comorbidity. These findings will help inform prevention strategies required to prevent high mortality rates during future waves of infection.Item Comparison of measured LDL cholesterol with calculated LDL-cholesterol using the Friedewald and Martin-Hopkins formulae in diabetic adults at Charlotte Maxeke Johannesburg Academic Hospital/NHLS Laboratory(University of the Witwatersrand, Johannesburg, 2023-01) Dintshi, Mogomotsi Portia; Kone, Ngalulawa; Khoza, SiyabongaBackground: National Cholesterol Education Programme Adult Treatment Panel III (NCEP ATP III) and the European Society of Cardiology recommends using low-density lipoprotein cholesterol (LDL-C) as a treatment target for cholesterol lowering therapy. The Friedewald formula underestimate LDL-C in non-fasted and hypertriglyceridemia patients. This study aimed to compare measured LDL-C to calculated LDL-C in diabetic patients using the Friedewald and Martin-Hopkins formulae. Methods: The data of 1 247 adult diabetes patients were retrospectively evaluated, and included triglycerides (TG), LDL-C, total cholesterol, and high-density lipoprotein cholesterol that were measured on the Roche Cobas® c702. Passing-Bablok regression analysis was used to determine the degree of agreement between measured LDL-C and calculated LDL-C using both formulae. The Bland-Altman plots were used to assess the bias at medical decision limits based on the 2021 European Society of Cardiology (ESC) guidelines on cardiovascular disease prevention in clinical practice. Results: Both formulae showed a good linear relationship against measured LDL-C. However, the Martin-Hopkins formula outperformed the Friedewald formula at LDL-C treatment target <1.4mmol/L. The Friedewald formula and the Martin-Hopkins formula had 14.9% and 10.9% mean positive bias, respectively. At TG-C ≥1.7 mmol/L, the Martin-Hopkins formula had a lower mean positive bias of 4.2 % (95 % CI 3.0-5.5) compared to the Friedewald formula, which had a mean positive bias of 21.8 % (95 % CI 19.9-23), which was higher than the NCEP ATP III recommended total allowable limit of 12%. Conclusion: The Martin-Hopkins formula performed better than the Friedewald formula at LDL-C of 1.4 mmol/L and showed the least positive bias in patients with hypertriglyceridemia.
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