*Electronic Theses and Dissertations (Masters)

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Extended characterization of multi-drug resistant organisms colonising neonates at a tertiary hospital in Johannesburg, South Africa.
(2024) Mntla, Nonkululeko Marcia
Neonatal deaths remain high globally, particularly in sub-Saharan Africa. A third of deaths are due to infections, often secondary to multi-drug resistant (MDR) organisms. The purpose of this study was to investigate the prevalence of MDR ESKAPE+ C. auris colonisation amongst hospitalised neonates, to determine risk factors associated with MDR colonisation, and perform antimicrobial resistance characterization of these isolates. Two hundred and fifty-eight swabs were collected from 86 hospitalised neonates at a tertiary South African hospital between November and December 2020. A total of 135 ESKAPE+ C. auris isolates were identified; 68% were MDR. Majority of neonates (65%) were colonised with extended spectrum betalactamase (ESBL) producing Klebsiella pneumoniae, followed by extensivelydrug resistant (XDR) Acinetobacter baumannii. New Delhi metallo-beta-lactamase (NDM) producing A. baumannii were more prevalent than carbapenemase producing Enterobacterales (CPE). A prolonged hospital stay, median=14 days (p<.001) was identified as a risk factor for MDR organism colonisation. The high prevalence of MDR ESKAPE+ C. auris colonisation supports use of non-invasive samples to determine colonisation prevalence. More data are needed to develop improved surveillance systems which should incorporate colonisation swabs and clinical biomarkers in neonates with independently established risk factors.
Colonisation with ESKAPE organisms and Candida auris among primary caregivers and healthcare workers in a neonatal unit at a public sector tertiary South African hospital
(2024) Rees, Nicki
Background: Nosocomial neonatal infection remains a significant cause of mortality and morbidity, particularly in the high care and intensive care settings. Among implicated pathogens ESKAPE- C organisms are considered particularly worrisome due to their virulence, ability to gain resistance and propensity to affect multiple sites. Transmission to neonates is postulated to occur through contact with colonised adults. Objective: This study aims to describe the prevalence of colonisation of both primary caregivers and healthcare workers in contact with admitted neonates. As a secondary objective this study aims to identify the most common resistance patterns in ESKAPE-C organisms isolated from primary caregivers and healthcare workers in a neonatal unit. The overall aim of the study is to provide insight into how best to prevent hospital acquired infections in this group. Methods: This cross-sectional prevalence study describes colonisation of healthcare workers (HCW) and primary caregivers in a neonatal unit in a tertiary South African hospital. Over one week in August 2021, twenty-five primary caregivers and twenty-nine healthcare workers submitted specimens which were processed for the identification of ESKAPE-C organisms. Susceptibility was performed on identified organisms. Results: Of the healthcare worker participants 13,8% (4/29) were shown to be colonised with one or more ESKAPE-C pathogen, while 52% (13/25) of primary caregivers were shown to be colonised with one or more ESKAPE-C pathogens. Of the S. aureus organisms isolated 28,6% were MRSA, of the A. baumannii organisms isolated 66.7% were XDR and of the Enterobacteriaceae isolated 60% were ESBL producing. No CRE or VRE organisms were isolated in this study. Conclusion: This study demonstrates that the prevalence of colonisation of healthcare workers and primary caregivers is significant and reinforces the need for stringent infection prevention and control strategies to prevent transmission to vulnerable neonates.
Machine Learning on biochemical data for the prediction of mutation presence in suspected Familial Hypercholesterolaemia
(2024) Hesse, Reinhardt
Background Familial hypercholesterolemia (FH) is a common monogenic disorder and, if not diagnosed and treated early, results in premature atherosclerotic cardiovascular disease. Most individuals with FH are undiagnosed due to limitations in current screening and diagnostic approaches, but the advent of machine learning (ML) offers a new prospect to identify these individuals. Our objective was to create a ML model from basic lipid profile data with better screening performance than low-density lipoprotein cholesterol (LDL-C) cut-off levels and diagnostic performance comparable to the Dutch Lipid Clinic Network (DLCN) criteria. Methods The ML model was developed using a combination of logistic regression, deep learning and random forest classification and was trained on a 70% split of an internal dataset consisting of 555 individuals clinically suspected of having FH. The performance of the model, as well as that of the LDL-C cut-off and DLCN criteria, were assessed on both the internal 30% testing dataset and a high prevalence external dataset by comparing the area under the receiver operator characteristic (AUROC) curves. All three methodologies were measured against the gold standard of FH diagnosis by mutation identification. Furthermore, the ML model was also tested on two lower prevalence datasets derived from the same external dataset. Results The ML model achieved an AUROC curve of 0.711 on the high prevalence external dataset (n=1376; FH prevalence=64%), which was superior to that of the LDL-C cut off alone (AUROC=0.642) and comparable to that of the DLCN criteria (AUROC=0.705). The model performed even better when tested on the medium prevalence (n=2655; FH prevalence=20%) and low prevalence (n=1616; FH prevalence=1%) datasets, with AUROC curve values of 0.801 and 0.856 respectively. Conclusions Despite the absence of clinical information, the ML model was better at correctly identifying genetically confirmed FH in a cohort of individuals suspected of having FH than the LDL-C cut-off tool and comparable to the DLCN criteria. The same ML model performed even better when tested on two cohorts with lower FH prevalence. The application of ML is therefore a promising tool in both the screening for, and diagnosis of, individuals with FH.
Comparison between lupus nephritis in HIV positive patients and HIV associated immune complex glomerulonephritis with “lupus–like” features: a clinicopathologic study
(2024) Mathaba, Margaret Masala
Background: Systemic lupus erythematosus (SLE) is an autoimmune disease seen commonly in black females of childbearing age. More than half of the patients present with renal disease or lupus nephritis complications. Coinfection with HIV in patients with lupus nephritis is rare. Despite Africa having the highest rate of HIV infection in the world, and there is no available data on the coexistence of HIV and Lupus nephritis. HIV is associated with a wide spectrum of renal diseases, including “lupus-like” HIV-Associated Immune Complex Kidney Disease (HIVICK). The most prevalent renal lesions in “lupus-like” HIVICK is diffuse proliferative lupus nephritis Objectives: This study aimed to compare and correlate the demographics, epidemiology, pathological and clinical findings of HIV positive patients with lupus nephritis and those with “lupus-like” HIVICK. Methods: This retrospective chart review study was conducted at the Charlotte Maxeke Johannesburg Academic Hospital in 5 years (2014-2018). We reviewed case reports that met our criteria for cases with lupus nephritis and cases with “lupus-like” HIVICK and allocated a lupus class according to the report findings. Results: Out of 2174 renal reports, 25(1.14%) patients were diagnosed with lupus nephritis and nine (0.41%) with “lupus-like” HIVICK. There were significant differences in age, serology (urea and creatinine), clinical presentation and lupus class. Conclusion: The occurrence of both HIV associated lupus nephritis and ‘lupus like’ HIVICK is rare. In our setting, the former is more common than the latter. We observed clinical and pathological differences which may be used to diagnose these disease entities.
Delineating the ontogeny of an N332-directed anti-HIV-1 broadly neutralising antibody lineage
(2024) Naidoo, Thamara
Broadly neutralising antibodies (bNAbs) develop in chronically HIV-1 infected individuals and can neutralise a variety of HIV-1 Env strains, making them an important element contributing to the design of HIV-1 vaccines. By understanding and delineating the long affinity maturation pathways that bNAb lineages follow against evolving viruses, it is hoped that this process will be able to be replicated in uninfected people with a series of vaccine immunogens. Investigating the ontogeny of a N332-directed bNAb lineage will provide insights into the key somatic hypermutations necessary for the development of neutralisation breadth. CAP255.G3 is an N332-directed bNAb isolated from CAP255, a participant in the CAPRISA 002 cohort, at 149 weeks post-infection (wpi). Six key antibody intermediates were selected from the CAP255.G3 lineage arm between 17 and 47 wpi, based on their position on a phylogenetic tree and because they contained mutations that were present in broad members of the lineage. We hypothesized that these intermediates would exhibit breadth similar to CAP255.G3. The intermediates were tested against a panel of eight autologous and six heterologous pseudoviruses using a TZM-bl neutralisation assay. The sequence identity of the first heavy chain complementarity-determining region (CDRH1) of CAP255.G3 differed from the closest intermediate and therefore, a CDRH1 chimera was constructed to determine if the CDRH1 from CAP255.G3 affected neutralisation activity. Later intermediates from 39 and 47 wpi, had moderate neutralisation activity against the autologous pseudoviruses, but only theintermediate closest to CAP255.G3 had limited neutralising activity against the heterologous pseudoviruses. Although the CDRH1 chimera had increased neutralisation activity against the heterologous viruses relative to the intermediates, it was less broad and potent than CAP255.G3. This indicates that additional affinity maturation between 47 and 149 wpi, at sites outside of the CDRH1, was required for the development of neutralisation breadth in the CAP255.G3 lineage arm..
Off-label evaluation of alternative specimen types: Cobas® plasma separation card for HIV viral load and dried blood spots for COVID-19 serology testing
(2024) Mampa, Thabiso Mmammitsi
Plasma is the preferred specimen for HIV viral load (VL) monitoring and COVID-19 serology testing but poses a challenge in resource-limited settings due to the need for venous blood, skilled phlebotomy, and cold storage for specimen integrity. In this study dried blood spots and novel plasma separation devices (PSC, HSSE, and VLPlasma) versus plasma were investigated as alternative specimen types. The plasma separation devices (PSD) were compared to DBS to determine if eliminating cellassociated nucleic acids could improve HIV VL performance. Paired PSD (n=72), DBS (n=72) and plasma (n=72) were prepared from HIV positive residual whole blood. Similarly, paired PSC, DBS (n=91) and plasma (n=91) were prepared from HIV positive prospective whole blood to assess PSC as an alternative specimen for use on the Abbott m2000. The eluates were processed on the GeneXpert (residual blood), Abbott m2000 (residual and prospective blood) and Roche cobas® 68/8800 (prospective blood). Using plasma as reference, residual blood: DBS outperformed PSC, HSSE and VLPlasma in terms of accuracy 91.8%, compared to 87.8%, 79.1% and 75%. Prospective blood: PSC had improved performance over DBS in terms of sensitivity (92.2% and 87.1%), specificity (65% and 61.9%), and accuracy (86.9% and 80.7%). Additionally, the performance of DBS was evaluated for COVID-19 serology testing in 45 PCR-confirmed, COVID-19 positive individuals by preparing laboratory paired DBS-plasma samples. DBS were eluted using two diluents followed by manual ELISA and results compared to reference plasma testing. DBS-PBS and DBS-manufacturer’s diluent showed the same accuracy (93.6%). Kappa values (0.817 and 0.845) and sensitivity (100% and 91.4%) were similar, but DBS-PBS showed low specificity (75%) compared to DBS-diluent (100%). Off-Label use of the cobas® PSC for HIV VL and DBS for COVID-19 serology testing provides expanded options for testing in resource-limited settings. Further evaluation on capillary blood and automated laboratory workflow optimisation would still be required prior to scaled implementation.
Exploring the interaction of host genetics and the gut microbiome in obesity in an African population
(2024) Schnell, Samantha Susan
Obesity is a highly prevalent health concern that is on the rise in Sub-Saharan Africa. Even though genetic variation and gut microbiota have been implicated in the development of obesity independently, the interactions between these factors have not been previously explored in a South African cohort. This study aimed to identify possible associations between host genomes, body mass index as a measure of obesity, and gut microbiota composition (in the form of V3-V4 16S rRNA sequencing) in a female African cohort. Polygenic risk scores predictive of body mass index in this cohort were generated to categorise the participants into high- and low-risk groups. Subsequently, several statistical analyses were performed comparing gut microbiota between these groups. High-risk participants with high body mass index had associations identified with increased abundances of Prevotella_9 and VadinBE97. In contrast, the low polygenic risk and low body mass index sub-group was associated with greater Bacteroides levels. This study acknowledges the plausible interactions between these factors in an African cohort.
The use of insecticide treated eave ribbons as a protection tool against pyrethroid-resistant populations of mosquitoes that transmit malaria and dengue fever
(2024) Shirima, Ruth Severin
Vector control methods such as insecticide treated nets (ITNs) and indoor residual spraying (IRS) have been successful in preventing mosquito-borne diseases like malaria and dengue. However, these methods face challenges including insecticide resistance, high costs, logistical difficulties, low adoption rates, and limited durability. Therefore, there is a need for simpler and more affordable interventions that can be used on a large scale in disease-endemic communities to supplement current approaches. This study evaluated the effectiveness of using insecticide-treated eave ribbons as a potential tool for complementing the current vector control methods. Eave ribbons are pieces of hessian fabric that can be placed around the eave spaces of poorly constructed houses to kill or repel mosquitoes. Laboratory cone bioassays were conducted to assess the efficacy of eave ribbons treated with the organophosphate, pirimiphos-methyl, for killing the malaria vectors, Anopheles funestus and Anopheles arabiensis, and the dengue vector, Aedes aegypti, under varying exposure durations and insecticide doses. In addition, a semi-field experiment was done to assess the efficacy of eave ribbons treated with pirimiphosmethyl against the malaria vectors. Indoor and outdoor biting was assessed by the number of mosquitoes captured indoors in window exit traps and outdoors by human landing catches, respectively. Mortality of recaptured mosquitoes was recorded after 24, 48, and 72 hours. The findings revealed that treated eave ribbons resulted in higher mosquito mortality than the untreated ribbons, but the impact increased with increased exposure duration or dose. The semi-field study indicated moderate levels of bite prevention and mortality of the mosquitoes. At the doses of 1 g a.i./m2 and 2 g a.i./m2 pirimiphos-methyl, there was no significant protection against An. arabiensis, but at the dose of 4 g a.i./m2 pirimiphos-methyl, there was only significant protection against outdoor biting An. arabiensis, but not An. funestus. In conclusion, while insecticide-treated eave ribbons may have potential for controlling malaria and dengue vectors, further research is needed to validate their efficacy in field settings and to identify suitable insecticides or insecticide combinations that are safe and effective.
Characterisation of variation in the CYP2C19 gene in African populations
(2024) Booyse, Ross Peter
Background: CYP2C19 pharmacogenetic testing is important clinically to optimise patient response to clopidogrel and anti-depressants. This study aimed to characterise the distribution of CYP2C19 star alleles (haplotypes) across diverse African populations compared with global populations, with a view towards informing future pharmacogenetic implementations. Methods: CYP2C19 star alleles and diplotypes were called from 604 high coverage genomes from continental African populations using the StellarPGx pipeline. Results: From our analysis, CYP2C19*1 (51%), *2 (17%), and *17 (22%) were the most common star alleles across African populations in this study. We also identified 3% of African participants that had potentially novel CYP2C19 haplotypes. Over 70% of the SSA participants had either poor, intermediate, rapid, and ultrarapid metabolizer status, and would likely benefit from dosage and/or treatment alterations, especially for clopidogrel Conclusion: This study supports the necessity for CYP2C19 pharmacogenetic testing in African clinical settings and the importance of comprehensive star allele characterisation in the African context.
Delineating antibody responses elicited by four SARSCoV-2 variants against the Mu variant
(2024) Kgagudi, Prudence
SARS-CoV-2 is the causative agent of the acute respiratory syndrome known as coronavirus disease 2019 (COVID-19). The SARS-CoV-2 spike protein plays an essential role in virus attachment, fusion and entry and is the main target of neutralizing antibodies. Continued evolution of the spike has led to the emergence of variants of concern or interest (VOC/VOIs) which pose a greater risk to the population due to augmented transmission risk, increased disease severity, antigenic escape and/or decreased effectiveness of vaccines. The Mu variant was first identified in Columbia in January 2021, however the immune escape potential of the variant remains poorly characterised. It is typified by T95I, Y144S and Y145N mutations in the N-terminal domain; R346K, E484K, and N501Y mutations in the receptor-binding domain and D614G, P681H, and D950N mutations in the S2 region. This study aimed to assess targeting of the Mu variant by neutralizing and non-neutralizing antibodies elicited by four variants during four COVID-19 waves in South Africa namely D614G, Beta, Delta and Omicron (BA.1). This was achieved by measuring antibody binding, neutralization and antibody dependent cellular cytotoxicity (ADCC) activity against Mu, and comparing these responses across each of the four variants tested. All variants elicited cross-reactive binding antibodies to Mu although the magnitude of binding to Mu was reduced. This cross-reactivity was a result of multiple binding antibody epitopes on the spike protein. We observed variable neutralization escape of Mu from antibodies triggered by infection with WT D614G and Delta. Antibodies induced by the Beta variant exhibited increased breadth towards the Mu variant when compared to those elicited by other variants. Neutralization escape of Mu from antibodies elicited by WT D614G and Delta may be caused by the 95I, 484K and 346K mutations in Mu, which are known to confer resistance in other variants. Compared to neutralization, ADCC was mostly preserved against Mu. However, the infecting variant impacted the potency of ADCC responses. WT D614G triggered significantly higher ADCC activity against Mu compared to Beta, Delta and Omicron. These data suggest that different variants trigger qualitatively different neutralizing and Fc effector responses, providing a rationale to study humoral resistance following infection by different variants. As Mu shares mutations with other VOCs, this study may aid in predicting the impact of mutations that may be common to emerging VOCs
Characterisation of RSV fusion proteins from South African patients with RSV disease, 2019 to 2020
(2024) Mabilo, Prince
Respiratory syncytial virus (RSV) is classified into subtypes A (RSV-A) and B (RSV-B), which are classified into different genotypes based on genetic variability of the G surface glycoprotein gene. The F surface protein gene is more conserved however variability in signal peptide, transmembrane domain, and antigenic sites have been reported. The study was conducted in the Virology laboratory, Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), South Africa. Study participants included patients of all ages from whom respiratory samples were submitted for respiratory viruses diagnoses from 2019 to 2020. The complete RSV F genes were amplified, library prep was preformed using the Nextera DNA prep kits and sequenced using amplicon-based next generation sequencing on the Illumina MiSeq sequencing platform. The Genome Detective Virus tool v2.27 was used to assemble sequencing reads and MEGA X was used for phylogenetic analysis. N-linked glycosylation and amino acid sequence variation was assessed. The overall prevalence of RSV was 5.8% (101/1 734). Seventy (69.3%; 70/101) RSV-positive samples were available for genetic characterisation of the F protein gene and thirty-one (30.7%; 31/101) were excluded due to insufficient sample volume. Only RSV-A strains were identified (91.2%; 31/34). Twenty three of thirty-one (74.2%) of the RSV F gene sequences from 2019 to 2020 clustered together with bootstrap values ranging from 64% to 99% and were NA1-like. A N-glycosylation site at position 120 gained by South African strains from 2018 is retained in strains from this study. This N-glycosylation site is present in approximately 25.8% of RSV strains from this study. The diversity of RSV-A F proteins was low, with amino acid variations observed at 30/571 (5.3%) sites. Ten mutations were detected in 4/6 antigenic sites (I, II, IV and V), with frequencies ranging from 0.3 to 100%. Antigenic changes seen exclusively among South African strains are: Y33H (0.3%) and V384T (7.3%) at site I and S275F (0.3%) at site II. Seven mutations associated with escape of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocyte (CTL) were predicted in seven epitopes. Overall amino acid mutation frequency for 2019 to 2020 RSV-A F genes is similar to that reported for South African strains from 2018 (3.3% to 6.7%). For the first time in South Africa we detected the S275F mutation which causes palivizumab resistance.
Natural killer cell phenotypic, functional, and nutrient transporter profiles during spontaneous control of HIV-1 infection in black South Africans
(2024) Batohi, Nikayla
Despite the devastation caused by the human immunodeficiency virus (HIV) for over four decades, a subset of individuals termed HIV-1 elite controllers (ECs) can control the virus in the absence of antiretroviral therapy (ART) and may provide a model for a functional cure. This study aimed to understand the role of natural killer (NK) cells as important innate effector cells in the control of HIV-1 infection in African populations. We measured the phenotypic, functional, and nutrient transporter profiles of NK cells on cryopreserved peripheral mononuclear cells from HIV-1 ECs (n=15), viraemic progressors (VPs) (n=19), antiretroviral therapy-treated individuals (ART-treated) (n=20), and HIV-1 uninfected donors (HCs) (n=21) from Johannesburg, South Africa using multicolour flow cytometry. Functional and metabolic profiles were assessed after stimulation with a major histocompatibility complex-devoid cell line. The frequency of NK cells and their subsets in ECs were similar to HCs and ART-treated and altered compared to VPs. Total NK cells in ECs had similar expression of NKG2A (inhibition), NKG2C (activation), PD-1 (exhaustion), and CD57 (maturation) markers compared to ART-treated and increased expression of CD38 and CD69 (activation markers) compared to ART-treated individuals. CD107a (cytotoxicity) was reduced in total NK cells in all people living with HIV-1 compared to HCs, whereas IFN-γ (cytokine production) was comparable across the ECs, HCs, and ART-treated groups and significantly lower in VPs compared to the other study groups. Metabolic profiles (glucose transporter 1 (Glut1), CD98, and CD71) were similar in ECs compared to ART-treated and significantly increased in VPs compared to other study groups. Together these findings show that NK cells from ECs have an inhibitory, mature profile with low levels of immune exhaustion and reduced metabolic phenotype suggesting functional competence. These new insights could be employed in novel immunotherapeutic strategies for the treatment of HIV-1 in an African population.
Evaluating the clinical utility of a SNP-based microarray platform: a comparative study
(2024) Mayisela, Minenhle Peniel
Developmental disorders make up a majority of cases seen in genetic clinics at the National Health Laboratory Service (NHLS). Chromosomal microarray analysis (CMA) is the first line testing for individuals with developmental disorders and congenital anomalies. This study compared the clinical utility of single nucleotide polymorphism (SNP) - based array (CytoScan® Optima array) and a comparative genomic hybridization (CGH) array (SurePrint G3 Unrestricted CGH ISCA v2, 8x60 kb microarray) in diagnosis of developmental disorders. This was done by looking at the differences in copy number variant calls, segment size differences and gene content between the two microarrays. The cost to run each type of microarray test for diagnosis was also compared. The copy number variant calls made by the two platforms were comparable. The SNP-based array did provide additional information that would be useful in molecular diagnosis. The final recommendation was for the CGH array and the SNP-based array to be used interchangeably based on clinical requests at the Division of Human Genetics
Combination interaction of fluconazole with efflux pump inhibitors against antifungal resistant Candida parapsilosis
(2024) Alati, Marwah Omar Ali
Introduction C. parapsilosis is the most common fungal pathogen recovered in neonatal intensive care units and is associated with a higher mortality rate due to its multidrug resistance. This study investigated the virulence factors and reversal of efflux activity by using a well-known antifungal drug (fluconazole) in combination with two efflux pump inhibitors (pantoprazole and omeprazole). In addition the effect of most active combination on pathogenicity markers of C. parapsilosis has studied. Materials and methods The microbroth dilution method was used to obtain antifungal susceptibility tests of conventional antifungal drugs (fluconazole and amphotericin B) against twelve clinical isolates of C. parapsilosis, as well as antifungal susceptibility tests of two efflux pump inhibitors (EPI). In addition, the combination of efflux pump inhibitors (pantoprazole and omeprazole) with fluconazole, an antifungal drug, was determined by calculating the fractional inhibitory concentration index (FICI) based on zero-interaction theory of Loewe additivity. a time-kill kinetics assay was used to study the antimicrobial activity of fluconazole and efflux pump inhibitors against C. parapsilosis. The virulence factors in C. parapsilosis isolates were determined using in vitro virulence assays. The effects of the efflux pump inhibitors on virulence factors were also studied, and comparison of percent reduction of virulence factors was determined in both fluconazole-resistant and susceptible C. parapsilosis. The effects of Pantoprazole and Omeprazole on efflux pump were also investigated using two assays (Rhodamine 6G accumulation and Hoechst 33342 accumulation). Results The efflux pump inhibitors showed enhanced antifungal activity in combination with antifungal agents against C. parapsilosis. When pantoprazole was combined with fluconazole, the median Minimum inhibitory concentration for fluconazole and pantoprazole decreased from 125 to 31.25 µg/ml and from 620 to 310 µg/ml respectively. In the time-kill kinetics assay, fungistatic activity of Fluconazole was changed to fungicidal by addition of efflux pump inhibitors (pantoprazole and omeprazole) at their ½MIC values. On the other hand, this study demonstrated that C. parapsilosis had a variety of virulence characteristics, including the ability to adhere to epithelial cells and secretion of proteinase hydrolytic enzyme. Pantoprazole and omeprazole reduced the ability of C. parapsilosis cells to adhere to epithelial cells. Adhesion was reduced in a concentration-dependent manner. The reduction was significant (p < 0.01 However, the results also showed that pantoprazole and omeprazole and their combinations was significantly reduced efflux pump activity. Conclusion Candida resistance has increased, particularly to azoles, and advent of C. parapsilosis as most prevalent non albicans Candida species poses a significant threat to the future. Combination therapy is shown to be therapeutically efficacious, and MIC values of fluconazole decreased when it was combined with omeprazole and pantoprazole. Further study is needed to understand the epidemiology, microbiology, genetics, and antibiotic susceptibility of this pathogen. The ability to express different virulence factors, such as adherence, proteinase, and phospholipase production, is strain-dependent and the widespread use of antifungal agents leads to drug resistance in C. parapsilosis.
Combination interaction of proton pump inhibitors with fluconazole against multidrug resistant Candida auris
(2024) Galane, Kamogelo
A significant challenge with controlling C. auris infections is its resistance to multiple antifungal agents, including azoles. Alternative strategies are required to combat the problem of antifungal resistance. Some of the suggested approaches include combination therapy and anti-virulence treatment strategies. There is growing interest in combining proton pump inhibitors (PPIs) with available antifungal drugs for better treatment outcomes. In this study, we investigated the antifungal activity of PPIs omeprazole and pantoprazole alone and in combination with fluconazole against resistant C. auris isolates. The anti-virulence activity of the synergistic combination against resistant C. auris isolates was determined. Lastly, the effect of the synergistic combination on C. auris energy-dependent efflux-activity was established. The CLSI broth micro-dilution method was used to determine the antifungal susceptibility of 25 C. auris isolates against antifungal agents (fluconazole and amphotericin B) and PPIs (pantoprazole and omeprazole). The combination interaction of fluconazole and PPIs was established by determining the fractional inhibitory concentration index (FICI) of each combination. Time-kill curves were used to determine the antifungal activity of the synergistic combination over time and to confirm the combination interaction. All 25 C. auris isolates were screened for their ability to adhere to epithelial cells and proteinase secretion using microscopy and culture technique respectively. The effect of the synergistic combination on isolates with positive pathogenicity markers was determined. Most of the C. auris isolates were resistant to fluconazole (92%) and all were sensitive to amphotericin B (100%). Omeprazole and pantoprazole had antifungal activity against C. auris at MIC values of 3125 µg/ml and 15625 µg/ml, respectively. The combination of fluconazole and pantoprazole reduced fluconazole MIC values by 4-fold, from 125 µg/ml to 31.2 µg/ml. The combination had synergistic effect against most C. auris isolates (56%), additive effect in 36% and indifference in 8%. There was no synergism in the fluconazole and omeprazole combination. However, the combination had antagonist effect against most C. auris isolates (56%). All C. auris isolates displayed some degree of adherence to epithelial cells and 96% produced proteinases. Fluconazole and pantoprazole combination significantly reduced adherence (p values < 0.05) and proteinase secretion (p values< 0.001) at inhibitory and subinhibitory concentrations. Pantoprazole inhibited fluconazole efflux at inhibitory and subinhibitory concentrations. The study showed that C. auris isolates express virulence factors such as adherence and proteinase production. The combination of pantoprazole and fluconazole has antifungal and anti-virulence activity against resistant C. auris isolates. In addition, it was shown that pantoprazole can attenuate fluconazole resistance by inhibiting the activity of energydependent efflux-pumps and it has synergistic effect with fluconazole. Therefore, pantoprazole has the potential to improve therapy outcomes when azoles are used.
Maternal vaccination in South Africa: timing and completeness
(2024) Bourne, Julia
Maternal immunisation is an invaluable public health measure that protects not only the mother, but also the foetus and new-born infant against a host of diseases; and is recommended by both the World Health Organisation (WHO) and South African national health authorities. Pregnancy induces a heightened state of immune system vulnerability, leaving women more susceptible to severe influenza outcomes, whilst neonatal tetanus has a fatality rate of between 80-100% in the absence of medical intervention. Maternal immunisation against influenza and tetanus has been successfully utilised as a public health strategy across the globe to uphold maternal and neonatal health. Maintaining coverage is imperative for both diseases as influenza strains change seasonally and tetanus cannot be eliminated, highlighting the importance of continued maternal immunisation. This study aimed to describe the uptake of both influenza and tetanus vaccinations during pregnancy, the completion of the tetanus vaccination schedule and the timing of both influenza and tetanus immunisation within South African antenatal care facilities. In addition, this study described clinical and demographic factors affecting maternal immunisation uptake. Clinical and demographic data were collected in a parent study and were retrospectively analysed in this study using the statistical software Stata. Influenza vaccination uptake within the sampled population was found to be 16.62% (806/4851). The odds of influenza vaccination were significantly higher in women aged 21-30 years, and women with six or more ANC visits. Metro East Cape Town site in the Western Cape outperformed Gauteng sites, with significantly increased odds of influenza vaccination amongst women frequenting that site. Appropriate influenza immunisation: defined as immunisation occurring during the period of either 01/04/2017-31/07/2017 or 01/04/2018-30/06/2018, occurred in 74.86% (530/708) of the cohort. Women who were alcohol users were significantly more likely to receive an influenza vaccine – yet this may be explained by the Metro East site which had the higher influenza coverage having the highest prevalence of alcohol use during pregnancy. Of 7105 women, 7031 (98.96%) received at least one dose of tetanus toxoid vaccine (TTV). Of these women, 39.24% (2759) received one dose; 51.06% (3590) received two doses and 9.70% (682) received the recommended three doses of TTV in their index pregnancy. Tetanus schedule completion was significantly more likely in women ≤20 years, and those who presented for their booking antenatal care (ANC) visit in the first trimester. In addition, women with more than three visits had an increased likelihood of TTV schedule completion. The odds of TTV schedule completion were decreased by negatively parity and gravidity, values over one and less than six, and greater than one respectively. Women with hypertension were significantly less likely to receive three TTV doses compared to women without hypertension. Julia Bourne MSc(Vaccinology) Research Report: Version 2.0 v Tetanus immunisation schedule adherence prevalence was 0.60% (4/670) in women with three doses and 90.34% (3209/3552) in women with two. Improvements may be made in South African maternal immunisation coverage, with this study supporting the idea of targeted educational campaigns and a revision of the maternal immunisation schedule to include the tetanus, diphtheria & acellular pertussis vaccine instead of the tetanus toxoid vaccine.
Molecular epidemiology of M and E protein coding genes from South African SARS-CoV-2 strains, 2020 to 2021
(2024) Marsden, Fabian
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the current pathogen causing the COVID-19 pandemic across the world. While vaccines that elicit anti-SARS-CoV2 antibodies have been developed and licenced, there is a reduced protection against variants of concern (VOCs) such as Beta, Delta and Omicron. This is due to the mutations within the spike (S) protein which is the antigen targeted by most vaccines. Other potential vaccine targets include the structural proteins namely the membrane (M) and envelope (E) proteins of SARSCoV-2 which are more conserved. In this study we aimed to determine the extent of genetic diversity in the M and E protein genes from South African SARS-CoV-2 strains and its impact on predicted B and T cell epitopes. M and E gene sequences were extracted from South African SARS-CoV-2 genomes obtained from the Global Initiative on Sharing All Influenza Data (GISAID) database for the period 01March 2020 to 31 December 2021. Maximum-likelihood phylogenetic tree analysis shows that among South African E gene sequences only the Omicron VOC sequences form a distinct cluster. Similarly, the Omicron M gene sequences also form a distinct cluster compared to the Wuhan reference strain, Beta and Delta sequences. The predicted T cell and B cell epitopes of M and E proteins were identified with specific regions that have shown to have identical regions in both the variants and the reference strain, this shows the conserved nature of the M and E genes. SARS-CoV-2 are shown to have varying antigenic probabilities for M and E proteins from each of the variants considered as probable antigens. The allergenicity and toxicity of the M and E proteins was assessed in the context of potential vaccine development with certain peptides of each shown to have toxic properties. The predicted B and T cell epitopes show that despite the presence of mutations in the VOCs’ derived protein sequences, there is a common epitopic region that is shared between the reference and the variants. There is a strong 9-mer coverage by the natural sequences despite some non-coverage due to non-silent mutations. The results from the epitope predication and HLA typing shows the conserved nature of the M and E proteins which highlights the potential use for the development of vaccines.
Impact of donor CYP3A5 genotype on pharmacokinetics of tacrolimus in South African paediatric liver transplant patients
(2024) Wheeler, Caitlin
Tacrolimus is characterised by a narrow therapeutic target range and wide interpatient variability. Pharmacogenetic research attributes CYP3A5 single nucleotide polymorphisms to the variable inter-patient tacrolimus concentration dose ratios (CDR). In this study, we compared the mean tacrolimus CDR in paediatric liver transplant recipients and their living liver donors’ CYP3A5 rs776746 T>C genotypes(*1/*1, *1/*3 and *3/*3), accounting for donor and recipient characteristics. The graft-to-recipient weight ratio and the CYP3A5 donor genotypes were found to be independent factors significantly impacting the mean tacrolimus CDR. Donor CYP3A5 expressors (*1/*1 and *1/*3) were shown to have significantly lower recipient tacrolimus CDRs, therefore, higher dosages would be required in comparison to CYP3A5 non-expressors to reach the same therapeutic target range. The potential implementation of a stratified medicine dosage algorithm, combining living liver donor CYP3A5 genotyping with the calculation of the graft-to-recipient weight ratio, may predict the optimal tacrolimus dosage schedules for liver transplant recipients.
Molecular characterization of invasive Streptococcus agalactiae in South Africa, 2019 – 2020
(2024) Ntozini, Buhle
Background: Group B streptococcus (GBS) is a leading cause of neonatal meningitis and sepsis. Capsular polysaccharide and protein-based GBS vaccines are currently in development. Therefore, it is important to understand the serotype and antigen distribution of GBS to assess potential vaccine coverage. We aimed to use whole genome sequencing (WGS) and phenotypic methods to assess the serotype and antigen distribution, antimicrobial susceptibility patterns, and the phylogeny of invasive GBS isolates in South Africa (SA). Methods: As part of national laboratory-based surveillance, 661 invasive GBS isolates cultured from normally sterile-site specimens (e.g. blood and cerebrospinal fluid) from patients of all ages, were collected from 2019-2020. Phenotypic identification was based on colony morphology and β-hemolysis. Serotyping was performed using latex agglutination. Antimicrobial susceptibility testing was based on disc diffusion and broth microdilution methods. Whole genome sequencing (WGS) was performed using the NextSeq 550 System and the 2 x 100-bp paired-end mode. WGS based prediction of serotypes, surface protein genes, and resistance gene detection was performed using a validated GBS bioinformatics pipeline developed by the US Centers for Disease Control and Prevention (CDC). Results: A total of 658/661 isolates yielded good quality sequences from WGS and three isolates poor quality sequences. The isolates belonged to 6 major clonal complexes (CCs): CC1 (37/658, 5.6%), CC8/10 (69/658, 10.5%), CC17 (272/658, 41.3%), CC19 (45/658, 6.8%), CC23 (188/658, 28.6%) and CC24 (37/658, 5.6%). Five singletons (10/658, 1.5%) were identified. Excluding phenotypically and genotypically non-typeable isolates, concordance for serotyping using latex agglutination and WGS was 99.7% (647/649). Overall, 6 serotypes were detected: III (281/656, 42.8%), Ia (183/656, 27.9%), V (78/656, 11.9%), II (955/656, 8.4%), Ib (44/656, 6.7%), and IV (15/656, 2.3%). Three percent (20/658) of the isolates were non-susceptible to penicillin. Erythromycin and clindamycin resistance was detected in 16.1% (106/658) and 3.8% (25/657) of the isolates respectively. Majority (91.5%, 625/657) of the isolates were resistant to tetracycline. Fifty-five percent (11/20) of penicillin non-susceptible isolates had mutations in the PBP2x gene known to confer resistance, while no PBP2x resistance mutation were detected in the remaining resistant isolates. ermTR (34.9%, 37/106) and mefA/E (29.2%, 31/106) resistance genes were the most common determinants of erythromycin resistance. Clindamycin resistance was mediated by the erm (ermB, T, and TR) genes. Tetracycline resistance was driven by the presence of the tet genes (tetM, tetO, and tetL), with tetM being the most common (95.8%, 599/625). Nearly all the isolates carried at least one of the 3 main pilus gene clusters (657/658, 99.8%), 1 of the 4 homologous alpha/Rib family determinants (656/658, 99.7%) and 1 of the serine-rich repeat (Srr) protein genes (626/658, 95.1%). The hvgA virulence gene was found exclusively in CC17 isolates. Conclusion: Our results show that the majority of isolates circulating in SA belong to the 5 major GBS CCs (CC1, CC8/10, CC17, CC19, and CC23). This study suggests that vaccines currently under development should provide good coverage in our setting. We show that the GBS6 vaccine that targets serotypes Ia, Ib, II, III, IV, and V has the potential to cover 100% of invasive GBS disease in SA. A pilus protein-based vaccine has a potential coverage of 99.8% and the GBS-NN and the latch peptide vaccines have a potential coverage of 68.5% and 95.3% respectively. Βeta-lactams remain appropriate for treatment and intrapartum antibiotic prophylaxis (IAP). However, detecting the emergence of non-susceptibility requires ongoing surveillance.
Evaluation of the genetic and metabolic determinants of postprandial glucose variability in Black South Africans
(2024) Masango, Bontle
This study aimed to determine the metabolic and genetic factors that account for the variation in postprandial glucose (PPG) in Black South Africans (SA). The study included 794 participants from the middle-aged Sowetan Cohort (MASC). PPG was calculated using the integrated area under the curve (iAUC) in response to an oral glucose tolerance test. Principal component analysis was applied to 31 metabolic factors to generate clusters represented by principal component variables. Polygenic risk scores (PRS) were computed for each participant using variants and weights from a validated African type 2 diabetes PRS. Linear regression models were used to evaluate the variance. The PRS did not contribute to the PPG variability in men or women. Central fat, serum lipids, and liver enzymes explained 10.8% of PPG variability in women. In men, peripheral fat, serum lipids, liver enzymes, and steroid hormones explained 10.6% of PPG variability. Our work has identified metabolic factors that predict PPG variability in Black SA.

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