School of Pathology (ETDs)
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Item Comparison of second and third generation parathyroid hormone (PTH) assays performed at Charlotte Maxeke Johannesburg Academic Hospital – Are they fit for purpose?(University of the Witwatersrand, Johannesburg, 2023-11) Nhlapo, Nokuthula; Jacob, Doreen; Maphayi, MphoBackground: Parathyroid hormone (PTH) measurement is crucial in the investigation of calcium and phosphate disorders and in the management of chronic kidney disease (CKD). Available PTH assays include second (intact PTH) and third generation (PTH 1-84) assays. Intact PTH assays are widely available and used for clinical guidelines but overestimate PTH in CKD. PTH 1-84 assays are more specific, but lack of standardisation has complicated clinical interpretation. The study aimed to compare the second and third generation assays to determine the difference in analytical performance and the effect on clinical interpretation. Methods: A method comparison was done on 481 patient samples with PTH requested at Charlotte Maxeke Johannesburg Academic Hospital. PTH was measured in each sample using both intact PTH and PTH 1-84 assays. Passing Bablok regression and Bland Altman plots were performed to determine method agreement and bias. Analytical performance was assessed using the European Federation of Clinical Chemistry and Laboratory Medicine biological variation specifications. Clinical performance was compared in the diagnosis of hypo- and hyperparathyroidism, and in predialysis and dialysis CKD based on current Kidney Disease Improving Global Outcomes guidelines. Results: Intact PTH had a higher median concentration than PTH 1-84 (9.93 vs 8.60 pmol/L, p<0.0001) but showed good correlation (r = 0.994 and p<0.0001). Regression analysis revealed significant systematic and proportional differences, with increased deviations at higher concentrations. The average bias was above allowable bias of 7.1%. Clinical interpretation of hypo- and hyperparathyroidism and predialysis and dialysis groups was unchanged. Conclusions: There was significant bias observed between the two PTH assays thus, they should not be used interchangeably. However, no significant changes in clinical interpretation were found when one assay was used over the other. The decision to use third over second generation PTH assay should consider the impact on clinical interpretation in the population.Item Comparison of measured LDL cholesterol with calculated LDL-cholesterol using the Friedewald and Martin-Hopkins formulae in diabetic adults at Charlotte Maxeke Johannesburg Academic Hospital/NHLS Laboratory(University of the Witwatersrand, Johannesburg, 2023-01) Dintshi, Mogomotsi Portia; Kone, Ngalulawa; Khoza, SiyabongaBackground: National Cholesterol Education Programme Adult Treatment Panel III (NCEP ATP III) and the European Society of Cardiology recommends using low-density lipoprotein cholesterol (LDL-C) as a treatment target for cholesterol lowering therapy. The Friedewald formula underestimate LDL-C in non-fasted and hypertriglyceridemia patients. This study aimed to compare measured LDL-C to calculated LDL-C in diabetic patients using the Friedewald and Martin-Hopkins formulae. Methods: The data of 1 247 adult diabetes patients were retrospectively evaluated, and included triglycerides (TG), LDL-C, total cholesterol, and high-density lipoprotein cholesterol that were measured on the Roche Cobas® c702. Passing-Bablok regression analysis was used to determine the degree of agreement between measured LDL-C and calculated LDL-C using both formulae. The Bland-Altman plots were used to assess the bias at medical decision limits based on the 2021 European Society of Cardiology (ESC) guidelines on cardiovascular disease prevention in clinical practice. Results: Both formulae showed a good linear relationship against measured LDL-C. However, the Martin-Hopkins formula outperformed the Friedewald formula at LDL-C treatment target <1.4mmol/L. The Friedewald formula and the Martin-Hopkins formula had 14.9% and 10.9% mean positive bias, respectively. At TG-C ≥1.7 mmol/L, the Martin-Hopkins formula had a lower mean positive bias of 4.2 % (95 % CI 3.0-5.5) compared to the Friedewald formula, which had a mean positive bias of 21.8 % (95 % CI 19.9-23), which was higher than the NCEP ATP III recommended total allowable limit of 12%. Conclusion: The Martin-Hopkins formula performed better than the Friedewald formula at LDL-C of 1.4 mmol/L and showed the least positive bias in patients with hypertriglyceridemia.Item Validation of Roche immunoassay for severe acute respiratory virus 2/SARS-COV-2 in South Africa(University of the Witwatersrand, Johannesburg, 2023-01) Grove, Jurette Simone; George, Jaya; Mayne, ElizabethBackground: Serology testing is an important ancillary diagnostic to the reverse transcriptase polymerase chain reaction (RT-PCR) test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We aimed to evaluate the performance of the Roche Elecsys™ chemiluminescent immunoassay (Rotkreuz, Switzerland), that detects antibodies against the SARS-CoV-2 nucleocapsid antigen, at an academic laboratory in South Africa. Methods: Serum samples were collected from 312 donors with confirmed positive SARS CoV-2 RT-PCR tests, with approval from a large university’s human research ethics committee. Negative controls included samples stored prior to December 2019 and from patients who tested negative for SARS-CoV-2 on RT-PCR and were confirmed negative using multiple serology methods (n = 124). Samples were stored at –80 °C and analysed on a Roche cobas™ 602 autoanalyser. Results: Compared with RT-PCR, our evaluation revealed a specificity of 100% and overall sensitivity of 65.1%. The sensitivity in individuals > 14 days’ post-diagnosis was 72.6%, with the highest sensitivity 31–50 days’ post-diagnosis at 88.6%. Results were also compared with in-house serology tests that showed high agreement in majority of categories. Conclusions: The sensitivity at all-time points post-diagnosis was lower than reported in other studies, but sensitivity in appropriate cohorts approached 90% with a high specificity. The lower sensitivity at earlier time points or in individuals without symptomatology may indicate failure to produce antibodies, which was further supported by the comparison against in-house serology tests.Item The stability of C-peptide and insulin in plasma and serum samples under different storage conditions(University of the Witwatersrand, Johannesburg, 2023-10) Nkuna, Delhia Xikombiso; Maphayi, MphoObjectives: C-peptide and insulin are peptide hormones and their stability is affected by a number of pre-analytical factors. The study aimed to investigate the impact of sample type, storage temperature and time delays before centri-fugation and analysis on the stability of C-peptide and insulin. Methods: Ten healthy non-diabetic adults in fasting and non-fasting state were enrolled. 40 mL of blood was collected from each participant into SST and dipotassium EDTA tubes. Samples were centrifuged immediately or at timed intervals (8, 12, 48 and 72 h). After baseline measurements on the Roche Cobas e602 analyzer using electrochemiluminescence immunoassays, aliquots were stored at room temperature (RT), 2–8 and −20 °C for 4 h to 30 days. The percentage deviation (PD) from baseline was calculated and a change greater than desirable biological variation total error was considered clinically significant. Results: C-peptide was more stable in separated serum than plasma (PD of −5 vs. −13 %) samples stored at 2–8 °C for 7 days and was most unstable at RT when centrifugation was delayed (PD −46 % in plasma and −74 % in serum after 48 h). Insulin was more stable in plasma than in serum under the different storage conditions with a minimum PD of −1% when stored at −20 °C for 30 days. When samples were kept unspun at RT for 72 h, PD was −23 and −80 % in plasma and serum, respectively. Conclusions: C-peptide was more stable in serum provided the sample was centrifuged immediately and stored in the fridge or freezer while insulin was found to be more stable in EDTA plasma.Item The usefulness of monocyte fluorescence as a biomarker of Tuberculosis infection at Chris Hani Baragwanath Academic Hospital(University of the Witwatersrand, Johannesburg, 2023-11) Moosa, Aamina Yunus; Vaughan, Jenifer; Hodkinson, KatherineIntroduction: South Africa has the 5th highest burden of Tuberculosis (TB) as well as coinfection with Human immunodeficiency virus (HIV) worldwide. Routine laboratory methods have varying sensitivity and specificity. The Xpert MTB/RIF (GXPU) (Cepheid, Sunnyvale, CA), has lower sensitivity in sputum smear negative cases and poor quality sputum samples. A robust, non-sputum based, inexpensive biomarker of TB would be of value in such cases. Monocytes are the major leucocyte involved in the immune response to TB. The Sysmex haematology analysers (Sysmex, Kobe, Japan) measure monocyte activation via monocyte fluorescence (MO-Y). This study aimed to evaluate the MO-Y and other Sysmex extended differential parameters (EDPs) as biomarkers of TB infection in the local setting. Methods: The MO-Y and EDPs were retrieved from the analyser for 121 adult cases (56 with TB, 65 controls). Further information was obtained from the laboratory information system, including patient demographics and other laboratory results; TB culture, SARS-CoV-2 results, C-reactive protein level, HIV status, bone marrow biopsies and the cycle threshold (CT) values on positive GXPU analysis. The MO-Y, EDPs and full blood count (FBC) values were compared among patients with and without TB (HIV positive and negative). Statistical significance was assessed (P-value of <0.05). Results: The MO-Y did not show utility in identifying patients with TB. A sub-population of patients living with HIV (PLWH) with a CD4 <100 cells/ul showed significantly higher MO-Y levels, due to other opportunistic infections affecting monocytes. Neutrophil surface fluorescence (a marker of neutrophil activation), was significantly higher in PLWH and with concomitant TB infection, possibly due to immune activation, worse illness, or increased bacterial infection. Among the PLWH, those with TB had significantly lower CD4 counts, absolute lymphocyte counts and mean cell volume (MCV) values. The MCV (cut-off value 87 fL) showed the strongest diagnostic utility for discriminating PLWH with and without TB (AUC 0.79). Conclusion: The MO-Y is not a useful biomarker of TB, but is significantly elevated in PLWH with low CD4 counts. The MCV showed adequate discriminatory power for differentiating patients with and without TB, at a cut-off level of 87fL.Item The contribution of common genetic variants to breast cancer risk in South African black populations(University of the Witwatersrand, Johannesburg, 2023-08) Hayat, Mahtaab; Brandenburg, Jean-Tristan; Ramsay, Michèle; Mathew, ChristopherBreast cancer is the second most common cancer in South African black women. The contribution of common genetic variants to breast cancer risk is well studied in non-African populations, but little is known about their role in resident African populations, and there are no published genome-wide association studies (GWAS) on breast cancer in Africa. This PhD thesis aimed to determine the contribution of common genetic variants to breast cancer in a South African black population. A GWAS was carried out in 2,573 black female breast cancer patients from the Johannesburg Cancer Study and 744 population-matched, female controls from the AWI-Gen study. All participants were from Soweto, Johannesburg, South Africa. Samples were genotyped on the H3Africa SNP array. Replication testing was done of existing loci from European and African American (AA) populations in the resident African data, and loci from the resident African data in European and AA populations. A meta-analysis was carried out with an AA population. Finally, existing polygenic risk scores (PRSs) were tested in the resident African dataset. Three variants at two loci were strongly associated with breast cancer in this study. Two variants (rs77422433, p-value=2.89x10-08, odds ratio (OR):0.46, 95% confidence interval (95%CI): 0.40-0.52 and rs112410019, p-value=3.01x10-08, OR: 0.47, 95%CI: 0.41-0.53) were located within the DNA repair gene XRCC5. These variants were not previously associated with breast cancer, suggesting that it may be an African specific risk locus. The second locus is on chromosome 16 in CES5A (rs3859109, p-value=4.54x10-08, OR=0.70, 95%CI: 0.68-0.73), and had not previously been associated with breast cancer. None of these SNPs were replicated in European and AA populations. The meta-analysis with AA data revealed strong association of an intergenic SNP with breast cancer (rs139299680, pmeta=7.25x10-08) on chromosome 3. A polygenic risk score (PRS) developed in European populations demonstrated poor transferability to this African dataset. This GWAS is the first to be conducted in a resident black African population. This study suggests that there may be African-specific genetic risk factors for African breast cancer, and that large genome-wide studies in African populations are essential to develop a comprehensive understanding of the genetics of breast cancer in Africa.Item Development of an Anopheles arabiensis sex separation strain and optimisation of mosquito handling, packaging and transport conditions for the South African Mosquito Sterile Insect Technique programme(University of the Witwatersrand, Johannesburg, 2023) Mashatola, Thabo; Munhenga, Givemore; Koekemoer, LizetteSouth Africa is taking significant strides towards eliminating malaria transmission within its borders. However, existing vector control strategies that focus on indoor mosquito management face challenges with Anopheles arabiensis, the primary malaria vector. To bolster these efforts, the sterile insect technique (SIT) is being considered as an additional vector control strategy. SIT involves mass-rearing and sterilising specific pest insects, which are then released to mate with wild insects, effectively reducing the pest population. The South African SIT project faces a crucial challenge of efficiently separating female mosquitoes from the production line. This is because female mosquitoes are capable of transmitting the malaria parasite, making their elimination vital. Current methods like manual separation and resistance-based sorting have operational limitations and require further optimisation for field trials. To address this challenge, this thesis conducted optimisation and acclimatisation experiments on adult Anopheles arabiensis females, aiming to transition them to an artificial membrane feeding technique. Comparative assessments demonstrated that artificial blood-feeding methods utilising a Hemotek® membrane feeding system and hog casing could effectively replace conventional methods without significant detriment to reproductive fitness. Subsequently, the study explored the use of ivermectin, a toxicant, to spike blood during artificial feeding to target and eliminate females. An optimal concentration of ivermectin (7.5 ppm) was identified, showing potential for segregating females from males during laboratory rearing. However, complete female elimination within the desired timeframe was not achieved, indicating that this method serves as a secondary phase for female elimination. The study also investigated the use of genetic sexing strains (GSSs) to selectively eliminate females. Although efforts to induce temperature-sensitive lethal mutations temperature sensitive lethality mutations and random morphological variations were unsuccessful, further cross mating studies and insights from previous studies on thermosensitive strains from Cameroon informed future research aimed at developing GSSs tailored to the South African genetic background Another challenge addressed was the optimal temperature and compaction conditions for chilling and immobilising sterile males during handling and transport. This is crucial for maintaining the quality of sterile males. Optimal knockdown temperature ranges (4°C – 8°C for 20 minutes) and packaging conditions (1000 sterile, marked adult males per 27000 cm³ Bugdorm-1® cage) were identified for laboratory handling and transport, facilitating recent small-scale pilot trials with successful packaging and transportation of sterile males to field sites. These advancements signify significant strides in South Africa's malaria elimination endeavours, while also playing a pivotal role in shaping the development of efficient operational protocols for SIT. Furthermore, as the country remains steadfast in its commitment to eliminating malaria, these innovative approaches offer a promising trajectory forward and establish a robust framework for orchestrating the SIT operational phase.Item Development of a multiplex HIV/TB point-of-care diagnostic assay based on the microarray(University of the Witwatersrand, Johannesburg, 2023) Malatji, Kanyane BridgettHIV/AIDS mortality is caused by opportunistic illnesses/infections that take advantage ofthe weakened immune system in infected individuals. In Africa, the most common of these opportunistic illnesses include infection by Mycobacterium tuberculosis (M.tb) responsible for tuberculosis (TB). HIV co-infection with M.tb has negative implications for disease management given that each pathogen accelerates the morbidity caused by the other. The effective management of patients infected with both pathogens is restricted by the fact that their diagnosis is done separately. The situation is more difficult in remote areas where patients must wait for much longer to obtain their TB diagnostic results. In addition, the current diagnostic tests for the detection of TB such as chest X-ray and bacterial culture have a long turnaround time, are expensive to perform, and require sophisticated equipment and trained personnel. It is in this context that this project sought to develop an HIV and TB multiplex microarray-based assay for the detection of the two diseases using one test. The project used a 2.5 x 7.6 cm epoxy-coated glass slide as well as high- binding 96 well plates to which HIV-1 p24 and M.tb CFP10, ESAT6 and pstS1 antigens, known to be markers of active TB, were immobilized. The immobilized antigens were then incubated with anti-p24, anti-CFP10, anti-ESAT6 and anti-pstS1 primary antibodies diluted in human serum to mimic physiological conditions where the antibodies would exist in the presence of other proteins. Detection of binding between the antigens and primary antibodies was achieved by means of secondary antibodies conjugated to either a fluorescence dye or horseradish peroxidase (HRP). In chapter two of the study, the immobilization of the HIV and TB antigens on the epoxy-coated glass slides as capture molecules of the HIV and TB antibodies diluted in human serum was performed. The antigen-antibody reactions detection were achieved by means of fluorescence dye conjugated secondary antibodies. This chapter also covered the sensitivity and specificity of the technology where the epoxy-coated glass slides were compared to the gold standard 96 well high-binding plates. Data showed that the HIV and TB antigen-antibody reactions were specific, and the slides were more sensitive relative to the 96 well high-binding plates with limits of detection many folds lower. To be specific, the limit of detection from the slides averaged 0.954 ng/ml compared to 4474.6 ng/ml for the plates. The detection limit concentrations of the slides were lower than the reported physiological concentrations of HIV and TB antibodies in infected individuals. Chapter two also focused on the evaluation of the antigens’ stability on the epoxy-coated glass slides by determining the optimal experimental pH buffer, temperature, storage condition (dry or wet), as well as the shelf-life. Data showed that the optimal pH and temperature for the HIV and TB antigens immobilized on the slides were pH 7.4 and 25 ˚C. Moreover, the antigens could be stored dry for at least 90 days without losing their function. Overall, this chapter showed that the epoxy-coated microarray slides performed better than the gold standard 96 well high-binding plates in terms of sensitivity; and that the immobilized antigens could remain stable for a long period, and do not require specialized storage conditions; thus, making the microarray technology a potential diagnostic tool for the multiplex detection of HIV and TB in the case of co-infection. Chapter three of the study focused on the proof-of-concept of the technology using human serum samples infected with HIV. The chapter showed that the technology could detect p24 antibodies in six out of seven samples infected with HIV, i.e., it detected p24 antibodies in 85.7% of samples known to be HIV positive. Furthermore, HIV negative samples also proved to be negative with this technology, thus no false positives were observed. Moreover, the technology was specific for HIV detection as no binding was observed on TB antigens. Therefore, these data support what was observed in the previous chapter when the HIV antibodies were spiked in normal human serum. Chapter four explored the application of the diagnostic technology for the point-of-care (POC) detection of HIV and TB antigen- antibody reaction, using HRP conjugated secondary antibodies, as well as the 2,2′-azino- bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and 3,3',5,5'-tetramethyl Benzidine (TMB) substrates for colour change based endpoint. This chapter also covered the sensitivity and specificity of the immunoassay in the high-binding 96 well plates and on epoxy-coated glass slides. Similar to what was observed in the previous chapter, the HIV and TB antigen-antibody interactions were specific, and the epoxy-coated microarray slides were more sensitive than the 96 well high-binding plates with limits of detection averaging 815-folds lower than the plates. Nevertheless, both platforms were found to be sensitive enough to be used for the POC detection of HIV and TB co infection using visual inspection. Furthermore, the stability of the antigens in the 96 well high-binding plates using colour change detection was also evaluated. The antigens were found to be stable in the high-binding plates at different pH and temperature conditions; however, pH 7.4 and 25 ˚C were optimal. In addition, the antigens were stable when stored dry in the plates for a period of three months. In addition, between the two HRP substrates used, TMB was faster and more sensitive to the HIV and TB antigen-antibody reactions than the ABTS substrate, and the difference was statistically significant (p<0.05). The importance of this chapter is that it eliminated the need for sophisticated equipment to detect the presence of HIV and TB antibodies, as the detection could be achieved by visual inspection. Overall, data in this chapter supported further development of the microarray technology for the POC HIV and TB co-infection diagnosis. Chapter five attempted to produce the CFP10, ESAT6, and pstS1 TB antigens in plants to reduce the cost associated with the current commercially available bacteria-produced antigens.Item Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker(University of the Witwatersrand, Johannesburg, 2023) Tsolo, Semakaleng Theresia; Ranchod, HeenaThe World Health Organization has prioritized the development of non-sputum-based assays that are capable of detecting active Tuberculosis (TB). Tryptophan (tryp) is converted to kynurenine (kyn) by the rate-limiting enzyme indoleamine 2, 3- dioxygenase (IDO). IDO activity may serve as a biomarker for active TB. Dried blood spots (DBS) can be collected outside of medical institutions and are simple to transport. We wanted to explore the use of DBS as an alternative sample type to measure the kyn/tryp ratio and IDO mRNA gene expression in healthy people. METHODS We optimised methods for elution of dried blood spots, exploring various elution buffers. Following method optimisation, we enrolled 40 healthy participants, and collected whole blood and DBS samples. Kyn and tryp concentrations were measured using ELISA (ImmuSmol, France). IDO mRNA gene expression was determined by real-time PCR using two housekeeping genes GAPDH and BACT. Statistical analysis was performed to determine the correlation and agreement between peripheral blood samples and DBS. RESULTS For IDO activity, tryp showed good agreement between plasma and DBS with a median percentage similarity of 91.1%. In contrast, no agreement was observed for kyn with a median percentage similarity of 56.6%. The kyn/tryp ratio performed poorly due to poor detection of kyn in DBS. Percentage similarity between whole blood and DBS IDO mRNA expression using GAPDH 87.1%, while using BACT was 84.6%. Using either traditional sample types or DBS, there was no correlation between IDO gene expression and kyn/tryp ratio. CONCLUSION We showed that tryp was measurable in DBS. Tryp in DBS was 91.1% similar to values in plasma. Despite method optimization, there was poor agreement between DBS and plasma for kyn. Although IDO mRNA gene expression was detectable in DBS, method agreement with whole blood was unsatisfactory. Alternative methods for the stabilization of kyn in DBS should be explored in future studies. IDO mRNA expression should be measured from whole blood in future studiesItem 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, SebastianHereditary 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