School of Pathology (ETDs)

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A laboratory based retrospective study of plasma cell myeloma in the public sector of South Africa from 2017 to 2019
(University of the Witwatersrand, Johannesburg, 2024) Wilding, Bradley Thomas; George, Jaya
Background Plasma cell myeloma is a haematological malignancy characterized by clonal proliferation of plasma cells. This malignancy is frequently associated with the production of a monoclonal protein in either serum and / or urine, referred to as an M protein, which is used as a screening test for patients. Patients are then further investigated to assess if they meet the International Myeloma Working Group (IMWG) diagnostic criteria for plasma cell myeloma. There is limited literature describing plasma cell myeloma in South Africa, particularly in people living with HIV. Objective The primary objective of this study was to describe plasma cell myeloma in patients diagnosed in the public sector of South Africa over a three-year period. The secondary objective was to compare demographic features (age, sex) and diagnostic criteria, between the myeloma patients living with HIV and the HIV negative myeloma patients. Methods A retrospective analysis was performed on data from 4518 patients who had a positive immunofixation on serum and / or urine from public sector hospitals, between 2017 and 2019. A total of 718 of the 4518 patients met the laboratory criteria for plasma cell myeloma and were included in the analysis. Demographics (age, sex) and laboratory investigations used in the diagnostic criteria for plasma cell myeloma were analysed and statistically compared across the different HIV status of patients. Results Plasma cell myeloma patients presented at a mean age of 59.46 years with a female to male ratio of 1.2:1. In the patients that met the diagnostic criteria the most common end-organ damage present was anaemia in 77.16% patients and the most common biomarker of malignancy was a bone marrow trephine biopsy plasma cell percentage t60% in 55.71% patients. IgG isotype was the most common paraprotein detected on serum immunofixation in 58.5% of the patients. Kappa was the most common Bence-Jones protein detected in 27.16% of patients which was 1.76 times more common than lambda Bence-Jones protein. People living with HIV were younger 55.11 (±9.79) as compared to their HIV negative counterparts (p value 0.010). No other statistically significant difference was noted when comparing HIV status groups Conclusion In conclusion, this study described the demographics, laboratory investigations and diagnostic features of plasma cell myeloma patients diagnosed in the South African public sector from 2017 to 2019. We found that people living with HIV were diagnosed at younger age when compared to their HIV negative counterparts
A multicentre study to evaluate an in-house multiparameter immunophenotypic panel to identify precursor B-cells in the determination of measurable residual disease in paediatric B-cell acute lymphoblastic leukaemia
(University of the Witwatersrand, Johannesburg, 2024) Nell, Zanre; Glencross, Deborah; Geel, Jennifer
Background: Periodic assessment of measurable residual disease (MRD) is an important prognostic factor in the management of paediatric B-cell acute lymphoblastic leukaemia (ALL). Conventional polymerase chain reaction (cPCR) and multiparameter flow cytometry (MFC) are well-established in MRD determination, the latter with no current optimal immunophenotypic panel by international consensus. Objective: To determine whether an in-house immunophenotypic panel containing the discriminatory CD58-FITC (cluster of differentiation; fluorescein isothiocyanate) marker compares with cPCR in the detection of paediatric B-cell ALL MRD. Methods: This prospective descriptive validation study was performed on diagnostic and follow-up bone marrow aspirate samples, comparing an in-house immunophenotypic panel against the standardised commercial ClearLLab 10CTM B-cell/myeloid cell-2 (M2) panels in MRD assessment. These findings were then compared to cPCR to determine individual panel performance and predictive power. Results: Both immunophenotypic panels demonstrated 100% concordance in the identification of the leukaemia-associated immunophenotype (LAIP) on all diagnostic samples. The in-house immunophenotypic panel showed a higher sensitivity and specificity, and greater association with cPCR in MRD assessment in follow-up samples. In combination with shared backbone markers of the ClearLLab 10CTM B-cell/M2 panels, inclusion of CD58-FITC and CD81-APC-H7 (allophycocyanin- cyanine dye) proved most informative in accurate distinction between regenerating B-cell precursors and residual leukaemic cells. Conclusion: This work confirms the findings of previous studies, where discriminatory marker CD58- FITC in combination with backbone informative markers demonstrates both superior diagnostic and monitoring utility in paediatric B-cell ALL. The in-house immunophenotypic panel offers an attractive, comparable alternative in MRD determination in this patient population whilst awaiting cPCR results, raising the possibility of earlier clinical decision-making with potential improvement of morbidity and mortality outcomes
A review of genetic testing for females referred to genetic counselling for a personal or family history of gynaecological cancer
(University of the Witwatersrand, Johannesburg, 2023) Barnard, Sebastian
Hereditary cancer syndromes, caused by pathogenic variants in specific genes, substantially increase an individual’s risk for cancer and are estimated to cause 10% of all uterine cancers and 20% of all ovarian cancers. However, these data are primarily based on high-income countries and to date there is no published data on the known variants and testing of cancer predisposition genes associated with gynaecological cancers in South Africa. In this study, patient records for those with either a confirmed diagnosis or family history of gynaecological cancer that were seen by the Division of Human Genetics (NHLS/Wits) were retrospectively analysed (n = 104). Associations between patient characteristics, genetic testing availability and the detection of pathogenic variants as well as the utility of risk assessment tools were investigated using statistical analysis. The majority of patients underwent diagnostic genetic testing (78/104, 75.0%), 25 (32.1%) were positive, 41 (52.6%) were negative, and 12 (15.4%) returned a variant of unknown significance. Test results were significantly different between European and non-European patients (p << 0.05) with non-European patients being 30% less likely to have a pathogenic variant detected (OR 0.7, 95% CI 0 0.22, 2.21). Patients who met genetic testing criteria according to online risk assessments were more likely to have a positive genetic test result than those who did not (p < 0.05). A disparity exists not only in genetic testing availability but also clinic attendance between public and private healthcare which is likely limiting the ability to diagnose hereditary cancer syndromes associated with gynaecological cancers in public healthcare hospitals
An Audit of Adult Vaccination in HIV-Positive Patients Attending a Tertiary Centre HIV Clinic
(University of the Witwatersrand, Johannesburg, 2024) Muchichwa, Mitchell Tanaka; Feldman, Charles
Background: People living with HIV (PLWH) have a greater risk of acquiring vaccine preventable diseases (VPDs) and of developing severe disease due to impaired immune responses. The risk of acquiring VPDs among PLWH is greater in adults aged 15-49 years as a result of common infections found in occupational, social and travel settings. At the same time, studies have demonstrated low vaccination coverage among this population. For this reason, international and local guidelines for the vaccination of HIV-infected adults have been drawn up to address challenges in immunization in the HIV setting. However, there is little evidence on the compliance to these guidelines and on the knowledge and attitudes of PLWH to vaccination for VPDs. Aim: The aim of the study was to investigate vaccination uptake, knowledge, and acceptance of vaccination among PLWH. Methodology: In-depth interviews and retrospective file reviews among PLWH attending the Charlotte Maxeke Johannesburg Academic Hospital HIV Clinic over a 1-month period. Results: Among the 107 PLWH who were recruited and interviewed, it was found that 24.3% had received at least one vaccine following their HIV diagnosis. The majority of participants (76.6%) had a history of tuberculosis (TB), which was the most common vaccine-preventable disease among the sample. Influenza vaccination had the highest uptake rate at 76.9%. Overall, 65% of participants believed that vaccines could prevent diseases, while 41.1% thought vaccines were harmful. Interestingly, 56% did not believe that vaccines worsened their HIV condition. Furthermore, 67.2% of participants expressed their willingness to accept a vaccine if it was recommended by their healthcare provider. The most significant factors associated with vaccine uptake were awareness of the influenza vaccine and the willingness to accept a vaccine after a recommendation. Conclusion: Our study revealed poor adherence to the vaccination guidelines for PLWH in South Africa. Only four vaccines against influenza, hepatitis B, shingles and pneumococcal diseases had been taken and commonly known among this population.
Analysis of Whole Exome Sequence Data from African Patients with HD-Like Features and No Known HDPhenocopy Syndrome
(University of the Witwatersrand, Johannesburg, 2023) Naicker, Racilya; Krause, Amanda; Baine-Savanhu, Fiona
Huntington disease (HD) is a rare progressive neurodegenerative disorder that results from a CAG repeat expansion within the huntingtin gene (HTT). Several syndromes present with HD- like features in the absence of the HTT expansion and are termed HD phenocopies. Huntington disease-like 2 (HDL2), a known phenocopy, is most commonly observed in individuals with African, or probable African, ancestry. Therefore, previous diagnostic testing in the Division of Human Genetics, National Health Laboratory Service (Johannesburg, South Africa) screened for both HD and HDL2 in patients with HD-like phenotypes and an indication of African ancestry. Patients who tested negative for both syndromes remain undiagnosed, highlighting the need for further testing strategies. This study aimed to identify variants implicated in the HD-like phenotype of these patients. In a group of nine undiagnosed patients with Black African ancestry, a virtual gene panel was analysed through a whole exome sequencing (WES) approach. The data was filtered, and candidate variants were prioritised according to the frequency, type, and location of the variants as well as in-silico pathogenicity prediction scores. A total of 20 candidate variants in 15 genes were shortlisted and classified according to ACMG/AMP guidelines. Notably, variants in the mitochondrial DNA polymerase subunit gamma (POLG; c.2246T>C; p.Phe749Ser) and the glutaryl-CoA dehydrogenase (GCDH; c.877G>A; p.Ala293Thr) genes were classified as likely pathogenic and pathogenic, respectively. Genotype-phenotype correlation indicated a potential diagnosis of autosomal dominant progressive external ophthalmoplegia in the patient carrying the POLG variant, whereas the GCDH variant was considered an incidental finding due to a lack of correlation with the characteristics of glutaric aciduria type 1. These findings highlight the diagnostic challenges faced in the African context for patients with HD-like clinical features and call for further validation studies and re-analysis of the WES data using alternative gene panels for the patients for whom no putative pathogenic variants were identified
Assessing the propensity of drug resistant tuberculosis to enter and exit the differentially culturable state
(2024) Nonkula, Bomikazi
Tuberculosis (TB), one of the oldest and most contagious infectious diseases, continues to be a global health concern. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC) which comprises of several species. These species are further subdivided into strains based on subtle genetic differences. The success of M. tuberculosis as a pathogen can be attributed to its ability to survive various stresses by adopting different growth states. Previous studies have shown that sputum from TB infected patients harbours a large proportion of drug-tolerant bacteria that are unable to form colonies on agar plates but can grow in liquid media. This population of organisms, termed differentially culturable tubercle bacilli (DCTB), could be resuscitated to grow by supplementing liquid media with cell free culture filtrates from axenic cultures of wild type M. tuberculosis H37Rv or mutant H37Rv lacking all five resuscitation promoting factors (Rpfs). Laboratory models that induce this differentially culturable state are critical for studying the physiology and metabolism of these bacteria in order to develop new TB diagnostic tests. In this study, five Beijing and five LAM drug resistant strains of M. tuberculosis were selected and used to robustly generate DCTB through an in vitro stress model using carbon starvation. The most probable number (MPN) assay and colony forming units were used to determine the amount of DCTB. Furthermore, the phenotype of these cells was studied using microscopy as well as metabolic probes that target the peptidoglycan (PG) component of the bacterial cell wall. Our findings demonstrated that applying the carbon starvation model to clinical M. tuberculosis strains (Beijing and LAM) resulted in robust levels of DCTB, as evidenced by limited growth on agar plates and enhanced growth in liquid media supplemented with culture filtrate from LAM and Beijing strains. Comparison of cell length between carbon starved cells to those grown in routine laboratory media suggested that DCTB appeared to be non-replicating and significantly shorter. The metabolic activity of the starved cultures was restored when they were supplemented with H37Rv, LAM and Beijing culture filtrate. Our results also demonstrated that Beijing strains had a higher propensity to produce DCTB compared to LAM strains and that the supplementation with Beijing culture filtrate resuscitated more DCTB. Collectively, our findings allow for the advancement of experimental systems that enable further investigation of DCTB and the properties of the Beijing strain that facilitate better adoption of the differentially culturable state.
Assessment of the in vitro phenotypic drug susceptibility of HIV-1 subtype C drug-resistant variants to Doravirine (DOR)
(University of the Witwatersrand, Johannesburg, 2024) Reddy, Nikita; Basson, Adriaan
The high prevalence of antiretroviral drug resistance warrants approval of novel antiretroviral drugs that are effective against drug resistant-variants. Doravirine (DOR) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) with an improved safety profile, requires only a single daily dose, and has shown activity against prevalent NNRTI drug resistance mutations. To understand the impact of NNRTI drug resistance mutations on DOR in South Africa, in which HIV-1 subtype C is prevalent, this study assessed the in vitro phenotypic drug susceptibility of DOR against prevalent HIV-1 subtype C drug-resistant variants. Replication-defective HIV-like pseudoviruses (PSV) containing the most prevalent NNRTI drug resistance mutation combinations (n=20), as well as the single mutations (n=15) they contain, were generated and screened in a single-cycle in vitro phenotypic assay for susceptibility to DOR. An initial screen of DOR against wild-type PSVs showed no significant differences in Islatravir (ISL) potency among subtypes B and C. Furthermore, the combination of DOR with ISL showed a significant synergistic inhibition of a wild-type HIV-1 subtype C PSV. High-level DOR resistance was observed with the V106M and Y188L single mutants, while the remaining single mutants remained susceptible or showed a low level of resistance to DOR. Combinations of mutations that included V106M, Y188L, and P225H showed a high level of resistance to DOR. Given the lower prevalence of the latter mutations in treatment-naïve and -experienced patients in South Africa, DOR would likely be an effective treatment option. However, genotypic drug resistance testing may be advised prior to initiation of DOR-containing regimens for NNRTI-experienced individuals.
Association of catestatin with insulin resistance disparity in normoglycaemic Black and White South African women
(University of the Witwatersrand, Johannesburg, 2024) Ntuli, Philisiwe; Toman , Marketa
Background: Unexplained ethnic disparities exist in insulin resistance (IR) worldwide, affecting negatively more women of African ancestry than their white female counterparts. Insulin resistance is a pathological condition preceding the development of Type 2 diabetes (T2D), which in 2016 was the second leading cause of death in South Africa. The studies evaluating IR disparities in South Africa are very sparse, focussing mainly on obesity and fat distribution. However, IR has multifactorial aetiology and disparities have been reported irrespective of Body Mass Index (BMI) or age. Additional studies are therefore needed. From rodent-based and in-vitro studies it becomes clear that glycoprotein Chromogranin A (CgA) and its cleavage products catestatin (CST; found in pancreatic β-cells, having insulin- sensitising effects) and pancreastatin (PST; derived from pancreatic α-cells, having anti-insulin effects) are involved in the regulation of glucose metabolism. We hypothesise that women of African ancestry may have low CST levels due to dysregulated CgA cleavage, yielding in an increased proportion of deregulatory PST. Therefore, we aim to determine whether circulatory levels of CST (or CgA; CgA/CST ratio) differ between healthy normoglycaemic black and white South African women independently of obesity and whether they are significantly involved in the regulation of IR. Methods: “Apparently healthy” black (n=67) and white (n=54) women volunteers below 50 years of age from Gauteng province were enrolled in the study, following the enrolment criteria. Anthropometric measurements including weight, height, waist (WC) and hip (HC) circumferences and blood pressure (BP) were recorded. All participants underwent shortened oral glucose tolerance test (OGTT) to evaluate their glycaemic status except for externally enrolled white women (n=47). Samples were collected for measurements of glucose (fasting and 30-minutes), insulin (fasting and 30-minutes) and C-peptide (fasting) which were analysed by an automated Roche Cobas analyser. Other samples collected at fasting for CgA, CST, Interleukin 6 (IL-6), leptin, monocyte chemotactic protein-1 (MCP-1) and adiponectin were analysed by individual enzyme-linked immunosorbent assay (ELISA) kits. Surrogate indices for IR (HOMA-IR), early insulin secretion (Insulinogenic index; IGI), -cell function (Disposition index; DIo) and hepatic insulin clearance (HIC) were calculated. vi Indices of adiposity (BMI, waist to hip ratio (WHR), waist to height ratio (WHtR) and a body shape index (ABSI)) were calculated from obtained measurements. Demographic information and socio-economic status (SES) were evaluated by questionnaires. Results: Black women were younger 26 (21; 32) than white women 30 (24; 35) and had higher BMI than white women (26.9 (22.4; 31.4) kg/m2 vs 23.5 (20.8; 25.9) kg/m2 respectively). Although normoglycaemic, white women had higher basal glucose levels (4.8 ± 0.3 mmol/L vs. 4.7 ± 0.3 mmol/L). No ethnic differences were found for fasting insulin or HOMA-IR. The only difference was attributable to adiposity in obese (OB) vs non-obese (NOB) black women. Similarly, no ethnic differences were observed for DIo and IGI. However, NOB black women had reduced (p<0.001) HIC in comparison with NOB white women. Black NOB women had reduced CST and elevated CgA (and CgA/CST ratio) than white NOB women, all with p<0.001. No associations of CST, CgA or CgA/CST were observed in white women. Catestatin correlated inversely with HIC (p=0.045) and positively with leptin (p=0.010) in black women. Correction for confounders removed this significance. CgA and the CgA/CST ratio correlated negatively with HOMA-IR and fasting insulin and positively with HIC. Only after removal of the strongest determinants, positive associations for CgA (p<0.001) and the ratio (p=0.009) remained in the model for HIC. Conclusion: In the study population of normoglycaemic, normotensive and apparently healthy black and white women, we have not found elevated insulin levels or higher IR in the black ethnic group. However, we demonstrated for the first time the ethnic differences in CST with reduced levels and elevated levels of CgA and the CgA/CST ratio in black women vs white women. This difference is independent of obesity; we have not identified its reason but a genetic origin cannot be excluded. There were no direct regulatory effects of CST (CgA or CgA/CST) on IR. However, the study results indicate that HIC is the most closely associated variable of glucose- insulin dynamics with CST (CgA and CgA/CST ratio) in the black women in our study. The effects of CST on IR and glucose regulation may be indirect. The study found a positive correlation with leptin and CST-leptin interaction has been described previously in the literature. The binding of CST to insulin receptor results in reduction of obesity and this, in turn, can reduce IR and improve insulin and glucose levels.
Audit of Lysosomal Storage Diseases Testing at the National Health Laboratory Service in Johannesburg from 2011-2020
(2023) Novellie, Michael
Lysosomal Storage Diseases (LSDs) are a group of Inborn Errors of Metabolism (IEM), due to the lack of a lysosomal enzyme. This results in toxic accumulation of metabolic waste products in various organs leading to neurodevelopmental regression, organ failure and premature death in the absence of treatment. Treatments for LSDs are limited. This study audited LSD diagnostic test requests received by the Division of Human Genetics, National Health Laboratory Service (NHLS) in Johannesburg from 2011 to 2020 with the aim of understanding the demand, appropriateness, and patient management of suspected LSD cases. A quantitative survey of all samples (1861 tests) referred to NHLS Johannesburg during the study period was performed. A total of 198 (13.3%) samples were rejected for testing mainly because of faulty sample collection. Of the 1663 that were accepted for testing 1457 (87.6%) tested negative, 73 (4.4%) were inconclusive and 133 (8.0%) tested positive. Fifty-five (3.1%) patients with LSD test requests, all of which were positive, were known to a Clinical Genetics unit. The most frequently requested test was for Fabry disease: 620 (33.3% of all requests), even though this disease is not the most prevalent LSD. Of the 603 accepted test requests for Fabry disease, only 6 (1.0%) tested positive. This suggests that some referring clinicians had unrealistic expectations of encountering this disease. It should be noted, however, that testing for Fabry disease is part of a broad diagnostic workup that may be applied even if the indication for testing is not specific. Access to LSD testing was unequal: private facilities were proportionally over-represented compared to public facilities; certain provinces with large referral centres (in KZN and Gauteng) were over-represented compared to smaller centres. Feedback and education of referring clinicians regarding indications for testing and importance of patient follow up, especially by clinical genetics services, are recommended. Follow up of positive MPS screening tests with specific diagnostic tests is essential. A system should be implemented where a medical geneticist phones the referring clinician and discusses further sample requirements (blood for enzyme analysis) and referral to a genetics clinic for all positive LSD screening tests. Future consideration should be given to designing a more systematic testing process, with the introduction of molecular testing to supplement biochemical testing.
Biomarkers to predict Tuberculosis treatment response
(2024) Boshielo, Itumeleng
Tuberculosis (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 die 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 LAMDCTB 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 Mtbonly 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 VEGFA showed that these cytokines have a significant discriminatory power to distinguish treatmentresponsive 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.
Biomarkers to predict Tuberculosis treatment response
(University of the Witwatersrand, Johannesburg, 2023-06) Boshielo, Itumeleng Tania; Tiemessen, Caroline; Kana, Bavesh
Tuberculosis (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.
Ceftazidime-avibactam and Aztreonam susceptibility of Carbapenem Resistant Enterobacterales and Pseudomonas aeruginosa isolates at Chris Hani Baragwanath Academic Hospital
(University of the Witwatersrand, Johannesburg, 2024) Phooko, Bontle Bessie; Mothibi, Lesego
Background: The rising resistance of multidrug resistant organisms (MDROs) to currently available last resort antibiotics accompanied by increased mortality, has led to organisms such as Carbapenem Resistant Enterobacterales (CRE) and drug resistant P. aeruginosa becoming a global threat. New antibiotics such as Ceftazidime-Avibactam (CZA) and its use in combination with Aztreonam (ATM), has presented a glimmer of hope to this threat. There is a lack of susceptibility data of these MDROs to CZA and ATM in low to middle income countries such as South Africa. Finding out such information, may assist in motivating for acquisition and usage of these lifesaving antibiotics. Objectives: The aim of the study was to determine the in vitro susceptibility data of CZA and ATM against CRE, Drug resistant P. aeruginosa at Chris Hani Baragwaneth Academic Hospital (CHBAH). Secondarily, we aimed to investigate the invitro synergism of combined CZA and ATM to the CRE and P. aeruginosa isolates that were both CZA and ATM resistant. Methods: This was a prospective laboratory-based study conducted at the National Health Laboratory Services (NHLS), Department of Microbiology, CHBAH. We tested 101 CRE isolates and 32 drug-resistant P. aeruginosa isolates, including multidrug-resistant (MDR) and extremely drug resistant (XDR) strains, against CZA and ATM. Those that were resistant to both CZA and ATM were then tested for synergy using the combination of CZA and ATM. Results: Majority of the CRE isolates were Klebsiella species (84%) with OXA-48 & its variants being the predominant carbapenemase gene detected (67%). The P. aeruginosa isolates were predominantly carbapenem-resistant (88%), with 53% harbouring metallo-β- lactamases (VIM and NDM), and 78% classified as XDR. The overall CZA in vitro susceptibility amongst the serine active (OXA-48 positive) CRE in the study was 100%. The ATM susceptibility was 6% for all the CRE isolates tested. Synergism was seen in 96% of the CRE isolates that were resistant to both CZA and ATM. The activity of CZA and ATM against P. aeruginosa was 34% and 44% respective. Furthermore, the synergistic effect of combined CZA and ATM was seen in only 64 % of the P. aeruginosa isolates qualifying for testing. Conclusion: Local evaluation of CZA and CZA-ATM synergy is important in this era of antimicrobial resistance where there is great need of new treatment options. The high number v of CZA susceptible organisms amongst the OXA-48 isolates is encouraging. Synergistic activity of CZA and ATM presents as a good alternative for the MBL producers. More rigorous studies are needed to determine the clinical impact of usage of these antimicrobials.
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.
Characterisation of the dynamic gut microbiota of members of the Anopheles gambiae complex
(University of the Witwatersrand, Johannesburg, 2024) Singh, Ashmika; Allam, Mushal; Oliver, Shüné V.
The gut microbiota of mosquitoes plays a pivotal role in their life history. This includes insecticide resistance and immune responses, which makes it a potential target for vector control interventions. Although a growing body of research characterises the gut microbiota of various mosquito species, there is limited information on most members of the Anopheles gambiae complex. This study characterised the gut microbiota of four laboratory reared strains, namely SENN (Anopheles arabiensis- an insecticide susceptible major vector), SENN DDT (Anopheles arabiensis- an insecticide resistant major vector), MAFUS (Anopheles merus- minor vector) and SANGWE (Anopheles quadriannulatus- non-vector). The gut microbiota of fourth instar larvae, three-day old, fifteen-day old non-blood fed and fifteen-day old blood fed females was characterised and compared, bacterial identification was performed using Next-Generation Sequencing (NGS) of the bacterial 16S ribosomal RNA (rRNA) gene. The larval environment plays a role in acquiring gut microbes. This study characterised the gut microbiota of laboratory reared and wild F1 population An. arabiensis adult females and how changes in the larval environment affect the dynamics of the adult gut microbiota. Additionally, the effects of heavy metals on the gut microbiota of laboratory reared and F1 population of An. arabiensis adult females post-exposure to heavy metals in the larval breeding site were assessed. Furthermore, the effect of changes in the salt concentration in the larval environment on the gut microbiota of An. merus larvae and adults were examined. All bacterial identification of the gut microbiota was performed using NGS of the bacterial 16S rRNA. The dynamic gut mycobiota of SENN, SENN DDT and fourth instar larvae, three-day old, fifteen-day old non-blood fed and fifteen-day old blood fed laboratory reared and wild F1 population An. arabiensis females were characterised. Fungal identification was performed using NGS of the fungal internal (ITS) transcribed spacer of the rRNA cistron. Distinct differences were observed between the diversity and abundance of the gut microbiota of major malaria vectors and the minor and non-vectors, with the minor and non-vector harbouring more Plasmodium-protective bacterial genera. Pesticide-degrading bacteria were found in insecticide susceptible and insecticide resistant strains of An. arabiensis, suggesting that the gut microbiota does not contribute to insecticide resistance. However, differences between these strains indicate that the diversity of the gut microbiota is affected by the insecticide resistant phenotype. The F1 population and wild population of An. arabiensis had a greater gut microbiota diversity than laboratory reared An. arabiensis strains. Exposure to heavy metals as larvae significantly changed the abundance of gut bacteria in adults. The F1 population and SENN DDT adults had more heavy metal degrading bacterial genera present viii than SENN. Changes in the salinity of the larval environment of An. merus significantly affected larval development time, adult longevity, fecundity and deltamethrin tolerance. Additionally, An. merus adults exposed to the highest salt concentration at the larval stage had more Plasmodium-protective bacterial genera than the lower concentrations. The insecticide resistant phenotype of laboratory reared An. arabiensis affected the diversity and the differential abundance of fungi observed in the gut mycobiota. Furthermore, the gut mycobiota of the F1 population of An. arabiensis was similar to that of the laboratory reared insecticide resistant strain. In conclusion, these findings have implications for using gut bacteria as a vector control intervention in South Africa. The gut microbiota of the An. gambiae complex is a dynamic network consisting of what seems to be a core microbiota crucial to its function. The gut microbiota of the selected members of the An. gambiae complex corresponds to their capacity to transmit Plasmodium. Furthermore, the larval environment strongly influences the dynamics of the gut microbiota of members from the An. gambiae complex. Therefore, the impact of the larval environment on the adult microbiota must be considered for the development and implementation of future microbial-based strategies.
Characterisation of the genetic variation in pharmacogenes involved in anti-tuberculosis drug metabolism across African populations
(University of the Witwatersrand, Johannesburg, 2024) Malinga, Thandeka Vuyiswa Bongiwe; Twesigomwe, David; Othman, Houcemeddine
Tuberculosis (TB) is a major health burden in Africa. Although TB is treatable, anti-TB drugs are associated with adverse drug reactions (ADRs) which are partly attributed to pharmacogenetic variation. The distribution of star alleles (haplotypes) influencing anti-TB drug metabolism, is unknown in many African populations. This presents challenges in implementing genotype-guided therapy in Africa to decrease the occurrence of ADRs and enhance the efficacy of anti-TB drugs. Therefore, this study aimed to characterise the distribution of star alleles in genes that are involved in anti-TB drug metabolism (mainly isoniazid), namely CYP2E1, NAT1, NAT2, GSTM1 and GSTT1, across diverse African populations. We used 794 high-depth whole genome sequence datasets representative of eight Sub-Saharan African (SSA) population groups. Data sources included the 1000 Genomes Project and H3Africa AWi-Gen. CYP2E1, NAT1, NAT2, GSTM1 and GSTT1 star alleles were called from the WGS data using StellarPGx. Subsequently, novel star alleles were analysed, and their allele defining variants were annotated using the Ensembl Variant Effect Predictor. We present the distribution of both common and rare star alleles influencing anti-TB drug metabolism across various SSA populations, in comparison to other global populations. Various key star alleles were identified in the SSA study populations at relatively high frequencies including NAT1*10, GSTT1*0 (>50%), GSTM1*0 (49%), and NAT2*5B (21%). Additionally, we predicted varying phenotypic proportions for NAT1 and NAT2 (acetylation) and the GST enzymes (detoxification activity) between SSA and other global populations. Fifty potentially novel haplotypes were identified computationally across the five genes. This study provides insight into the distribution of star alleles in genes relevant to isoniazid metabolism across various African populations. The high number of potentially novel star alleles exemplifies the need for pharmacogenomics studies in the African context. Overall, our analysis provides a foundation for implementing pharmacogenetic testing in Africa to reduce the risk of ADRs related to TB treatment.
Characterisation of the murine immune response to self-amplifying mRNA sequences encoding Hepatitis B virus surface proteins
(University of the Witwatersrand, Johannesburg, 2024) Samudh, Nazia; Bloom, Kristie
Vaccination against Hepatitis B virus (HBV) remains the most effective means of preventing infection. However, approximately 10% of vaccinated individuals fail to develop neutralising antibodies necessitating the development of improved vaccines which target the more immunogenic large HBV surface antigen (L-HBsAg) and can elicit cell-mediated immunity. Although Africa bears a high burden of HBV infections, placing many individuals at risk of contracting the disease, we rely on imported vaccines for prophylactic vaccination programmes. The COVID-19 pandemic was a stark reminder of Africa’s vaccine dependence and since then great interest has been generated in establishing vaccine manufacturing capabilities on the African continent. Herein, we explored the Alphavirus-based self-amplifying RNA (saRNA) vaccine platform to produce dose-sparing HBV vaccines which could contribute to vaccine independence. saRNAs encoding reporter proteins, small HBV surface antigen (S-HBsAg) or L-HBsAg were synthesised by optimised in vitro transcription. Expression of reporter proteins from saRNAs was achieved even at low concentrations and was observed for extended periods of time in vitro. saRNAs encoding S-HBsAg were able to trigger the interferon response in a dose-response manner in vitro, however, this did not hamper antigen expression. Expression of L-HBsAg was achieved but restricted to the intracellular space and will require sequence modification to facilitate secretion. In vivo delivery of saRNAs by electroporation or commercially available cationic liposomes was found to be unsuccessful, and further optimisation of in vivo saRNA delivery is required before determining the prophylactic potential of candidate vaccines. This preliminary study has produced promising results demonstrating the dose-sparing properties and self-adjuvanting nature of the saRNA vaccine platform
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.
Characterising the combined eects of cytochrome P450 missense variants within the star allele nomenclature
(University of the Witwatersrand, Johannesburg, 2024) Khoza, Nhlamulo; Othman, Houcemeddine; Hazelhurst, Scott
Genetic variations in Cytochrome P450 (CYP) enzymes shape drug responses. However, many CYP haplotypes (star alleles) lack functional annotations, posing a barrier to under- standing drug metabolism comprehensively. To address this, our study investigates combined missense variant eects on CYP enzyme structures, analyzing 261 variants across 91 CYP haplotypes. We utilized Normal Mode Analysis (NMA; FoldX and ENCoM) to explore variant impact on protein stability. Subsequently, we conducted Molecular Dynamics (MD) simulations on key alleles, CYP2D6*2 and CYP2D6*17, to reveal star allele impact on protein dynamics. Integrating NMA and MD, we uncover the interactions that collectively shape the conformation and attributes of CYP enzymes. Notably, our investigation highlights significant deviations between wild-type and CYP2D6*17 -encoded proteins in the F/G loop region, pivotal for CYP functionality. Additionally, we compare NMA results with CYP2C9 and CYP2C19 Deep Mutational Scanning (DMS) results, identifying 65% concordance. Furthermore, our NMA predictions show 80% concordance with commonly used Variant Eect Predictor tools. This alignment underscores our approach’s reliability in predicting variant eects. Our study illuminates missense variants’ nuanced impact on CYP protein structures, significant for personalized medicine and drug response prediction, as accurate drug response predictions hinge on a comprehensive understanding of CYP missense variants. Moreover, our study highlights multi-scalemodelling potential for interpreting CYP missense variants, especially in star alleles. The synergy of NMA, MD simulation, and assays like DMS is invaluable, each with distinct strengths and limitations. This research deepens our understanding of the complexity of CYP metabolism profiles, providing insights into the functional assessment of CYP star alleles and missense variants with unknown functional classifications.
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.
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.