School of Pathology (ETDs)

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The role of regulatory T cells in adults in South Africa with active tuberculosis
(2010-01-28T10:11:24Z) Mayne, Elizabeth Sarah
Introduction: Regulatory T cells (Tregs) are increasingly being recognized as key immunological players in immunosuppression and have been seen to be permissive for certain infections. Aim: This study aimed to elucidate the role that Tregs play in symptomatic infection with Mycobacterium tuberculosis (TB), both with and without co-infection with human immunodeficiency virus type 1 (HIV 1) by quantification of these cells at ex vivo. It was then attempted to characterise the behaviour of FoxP3 positive cells in culture with stimulation. Methods: Peripheral blood mononuclear cells were purified from uninfected controls, patients with active TB, patients with HIV infection and patients with HIV infection and active TB. The frequencies of Tregs were assessed by flow cytometry at ex vivo and again after four days of culture with stimulation with anti-CD3, Purified protein derivative, tetanus toxoid and HIV peptide superpools (gag and nef). These frequencies were compared between the four groups of patients. The ability of Tregs and effector T cells to proliferate was also assessed. Interferon-γ secretion was used as a measure of effector T cell response to stimulation. vi Results: Frequencies of Tregs were significantly reduced in patients with active TB as compared with HIV infected patients and uninfected controls. Co-infected individuals showed a broad range of frequencies which were not significantly different from controls. These frequencies remained stable in culture with the exception of those individuals infected with HIV who showed a decline in the frequency of those cells expressing FoxP3 over the period. Cells expressing FoxP3 were not anergic and responded to stimulation. HIV specific proteins, in addition, resulted in specific effects on the Tregs with a positive interferon response to gag correlating with increased Treg frequencies and FoxP3 expression in CD4+ T cells correlated with the proliferative response of CD4+ T cells to Nef in HIV infected individuals. Conclusions: This study shows significant differences of frequencies of FoxP3 positive producing cells in the peripheral blood at ex vivo in patients with active TB. The function of these cells in this population is uncertain and further functional data and long-term clinical follow-up is required. In addition, the frequencies of these cells remained constant over time and showed proliferative response to stimuli (most notably CD3) suggesting that these cells may be generated in the periphery.
Unmasking serial murder: a comparison of a South African murder series with characteristics from the Federal Bureau of Investigation Serial Murder Database
(2015) Holland, Shakeera
The term ‘serial killer’ brings to mind notorious criminals whose crimes are so heinous as to test the limits of the most vivid imagination and make us question their humanity. What is the reality of serial murder? In 2005, the Federal Bureau of Investigation (FBI) hosted a symposium on serial murder, which brought together international experts in the field of serial murder with the aim of clarifying and understanding this multifarious crime. On the 12th of March 2008, Gcinumzi Richman Makhwenkwe, ‘The Moffat Park Serial Murderer’ was convicted of 5 counts of murder, 3 counts of rape and 3 counts of robbery with aggravating circumstances. The Department of Forensic Medicine and Pathology of the University of the Witwatersrand, based at the Johannesburg Forensic Pathology Service (FPS) Medicolegal Mortuary Facility performed the medicolegal investigations of death in all the victims. This research report explores the characteristics of serial murder and serial murderers as documented in the literature; documents the features and characteristics of the Moffat Park murder series; compares the features of this South African murder series to those from the findings of the FBI serial murder symposium; explores the role of the forensic medical practitioner in the investigation of the Moffat Park series and serves to educate and inform forensic medical practitioners of the features of serial murder as awareness may potentially lead to earlier identification of a murder series. This could ultimately lead to earlier implementation of specialist investigative methods, earlier apprehension of the serial murderer and most importantly fewer victims.
Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker
(University of the Witwatersrand, Johannesburg, 2023) Tsolo, Semakaleng Theresia; Ranchod, Heena
The 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 studies
Exome Sequencing of individuals with vitiligo and their relatives with and without autoimmune disorders
(University of the Witwatersrand, Johannesburg, 2023) Rabinda, Kentie Rofhiwa
Autoimmune disorders result from the immune system attacking and damaging its healthy cells because of an acquired immune system malfunction. Vitiligo is an example of an autoimmune disorders. It is a common depigmentation skin disorder with an estimated prevalence of 0.5–2% of the population worldwide. It is shown by selective loss of melanocytes causing non- scaly, chalky-white macules. Individuals with vitiligo often have other autoimmune disorders or first-degree relatives with at least one other autoimmune disorder. This study aimed to identify genetic variants in selected genes in individuals with vitiligo and their unaffected close relatives who have other autoimmune disorders. Upon DNA extraction, whole exome sequencing was performed, and five non- major histocompatibility complex (MHC) genes were analysed. Identified variants were annotated on Ensembl Variant Effect Predictor (VEP) and compared between individuals with vitiligo and their close relatives with and without autoimmune disorders in a family-specific manner. Eight variants were identified in two families, however, the three missense variants in the NLRP1 gene (rs12150220, rs11651270, and rs2301582) were observed between and across two families in individuals with autoimmune disorders. SMOC2 rs13208776, was identified as a risk locus for vitiligo in individuals from two Romanian isolate villages with vitiligo and other autoimmune disorders. None of the genotyped individuals were homozygous for the minor allele (A) and 2/12 individuals were heterozygous therefore it is unlikely that rs13208776 has any role in the development of vitiligo or any of the autoimmune disorders present in the two families. This study showed that NLRP1 gene variants segregated with vitiligo and autoimmune disorders
Mutation Profiling of Paediatric Solid Tumours in a Cohort of South African Patients
(University of the Witwatersrand, Johannesburg, 2023) Manolas, Erin; Krause, Amanda; Lamola, Lindie
Childhood cancers are an emerging global health burden, with the highest increase in incidence and mortality rates occurring in low-middle income countries, such as South Africa (SA). Adding to this burden is the contribution of cancer-predisposing genes (CPGs), whose germline variants increase the risk of cancer development in childhood. These genes are largely associated with a set of disorders, known as cancer-predisposing syndromes (CPSs), which are characterised by an increased likelihood of cancer development and/or additional phenotypic malignant/non- malignant features. Next-Generation Sequencing (NGS) technologies have been key in determining the occurrence and contribution of such germline variants to paediatric cancer development across international research and diagnostic settings. However, these technologies have not been applied to paediatric cohorts in SA and thus there is a paucity of data regarding the contribution of germline variants to childhood cancer development in this setting. Through the design and evaluation of an NGS targeted-CPG panel, this study aimed to generate germline variant profiles of SA paediatric cancer patients, thereby gaining insight into their potential role in the pathogenesis of childhood cancers. NGS was performed on the genomic DNA from 32 solid-tumour paediatric cancer patients using an Ion Ampliseq 50 CPG panel design and the Ion Torrent S5 sequencing instruments. Germline variants were called using the Ion Torrent Suite™ software (v.5.12.0) and annotated using the Ensembl Variant Effect Predictor. Variants were filtered using a bioinformatics pipeline assessing variant data from population and public databases, computational data, functional studies data, genetic pedigrees indicating family history and phenotypic data. Variant evidence was further interpreted for variant prioritisation and classification according to the ACMG-AMP guidelines. All putative pathogenic/likely-pathogenic (P/LP) variants identified were validated via Sanger Sequencing. Seven pathogenic and/or likely pathogenic germline variants were identified and validated in seven patients. Three of these variants, identified in the NF1, RET, and TP53 genes, were detected in patients who presented with phenotypes consistent with their genetic findings and are associated with well-known CPSs (diagnostic yield - 3/32, ~9.4%). The remaining four variants, identified in the BRCA1, ERCC3, FAH, and RB1 genes, have not been previously associated with the patient’s cancer phenotype and therefore require further investigation. At the time of this project’s data generation, this is the first global report of the novel heterozygous, likely pathogenic FAH p.R162H variant. Additionally, although reported elsewhere, the majority of the variants identified in this study (6/7, ~86.7%) have been reported for the first time within the SA paediatric population. To our knowledge, this is the first study to utilise NGS technologies in the germline variant profiling of paediatric solid-tumour patients in SA and therefore has greatly added to filling the current knowledge gap. In addition, these findings have contributed towards the foundation for the development of a CPG sequencing panel suitable for implementation in a SA diagnostic setting
Laboratory Evaluation of Aspergillus Galactomannan Lateral Flow Assays
(University of the Witwatersrand, Johannesburg, 2023) Ubbink, Anja; Chibabhai, Vindana
Invasive aspergillosis diagnosis is based on a combination of clinical, radiological, and mycological factors, including the detection of Aspergillus galactomannan antigen in serum and bronchoalveolar lavage fluid (BALF). Lateral flow assays (LFA) introduced for rapid detection of galactomannan in serum and BALF include the IMMY sōna Aspergillus LFA (IMMY LFA) and the Dynamiker QuicGMTM Aspergillus Galactomannan Ag LFA (QuicGM LFA). Objective To evaluate the performance of the IMMY LFA and QuicGM LFA in South Africa. Methods Serum and BALF samples previously tested by Platelia BioRad Aspergillus GM-EIA were analysed using the two different LFAs. Percentage agreement and precision was assessed. Results Forty-six serum- and 13 BALF samples were tested using the IMMY GM LFA and 48 serum- and 6 BALF samples were tested using the QuicGM LFA. Using an optical density ≥0.5 as positive, results were compared to the BioRad Aspergillus GM-EIA. For the IMMY LFA, serum samples had a positive percent agreement (PPA) of 0% (0/1); negative percent agreement (NPA) of 91% (41/45) and overall percent agreement (OPA) of 89% (41/46). BALF samples had a PPA of 75% (3/4), NPA 50% (5/10) and OPA of 57% (8/14). For the QuicGM LFA, serum samples had a PPA 0% (0/3), NPA of 96% (43/45) and OPA of 90% (43/48). BALF samples had a PPA of 100% (1/1), NPA of 100% (5/5) and OPA of 100% (6/6). For the IMMY LFA, between-day reproducibility for 72% (13/18) and 63% (5/8) for serum and BALF samples, respectively. Between-batch reproducibility was 89% (16/18) and 50% (4/8), respectively for serum and BALF samples. For the QuicGM LFA, between-day reproducibility was 75% (9/12) and 75% (3/4) for the serum and BALF samples, respectively. The between-batch reproducibility was 100% (8/8) for serum and 100% (3/3) for BALF. Conclusion A follow-up evaluation with a larger sample size utilizing clinical, radiological, and laboratory data is warranted to determine the assays’ clinical utility. What this study adds Invasive aspergillosis is a life-threatening disease, where a prompt diagnosis improves outcome. Currently there is no Aspergillus galactomannan assay available in the South African state sector. This study evaluates two lateral flow assays for the detection of Galactomannan in South Africa
Sequencing for high-risk type 1 diabetes genotypes in the South African black population using AmpliSeq Nanopore next generation sequencing
(University of the Witwatersrand, Johannesburg, 2023) Mathabela, Nomfundo Mathabela; Ali, Stuart
Introduction: Type 1 diabetes (T1D) is a chronic autoimmune disorder characterized by the destruction of the -cells of the pancreas, resulting in the inability to produce/maintain insulin. This results in an inability to maintain the blood glucose at homeostasis. Prolonged hyperglycaemia leads to micro- and macro-vascular complications. Thus, it is vital to diagnose and treat patients in a timely manner. It is important to identify individuals at increased risk of developing T1D allowing for appropriate follow ups. Numerous mutations/variants in specific genes confer an increased risk of T1D with the HLA gene accounting for approximately 40-50 % of the risk. Therefore, it is possible that by looking at genetic variation in T1D associated genes we can develop a tool that can determine the likelihood of an individual developing T1D. Study aims and objectives: The development, implementation, and validation of a Nanopore NGS method to sequence and genotype polymorphisms associated with T1D in a cohort of black South Africans which could be used to develop a genetic risk score (GRS) for T1D in this population. In addition, we aimed to compare sequencing results to two HLA genotypes obtained using PCR-RFLP. Method: Participants with T1D (n=19) and control participants (n=5) were genotyped for 12 T1D associated polymorphisms through Ampliseq Nanopore sequencing. In addition, Ion Torrent was used to confirm the Ampliseq Nanopore results. A bioinformatics pipeline that involves reference sequence generation using an in-house script, alignment of sequencing data with the reference sequence, filtering and variant calling was developed. A genetic risk score (GRS) was calculated for the participants. Participants (n=73) were genotyped by PCR-RFLP for the HLA rs2040410 and rs7454108 polymorphisms. Results: Sequencing samples individually was found to have a slightly higher Qscore than samples sequenced in multiplex (9.73 vs. 9.5). Samples sequenced individually had higher average reads (187.60 vs. 151.14 Mb), passed reads (41.47 vs. 25.99 Mb), and estimated bases (54.72 vs. 49.57 Mb) than those sequenced in multiplex. In addition, samples sequenced in a multiplex had higher average failed reads (475.58 Mb) in comparison to those sequenced individually (13.58 Mb). The average percentage difference in sequencing data generated using Ion Torrent compared to Nanopore was 5.67%. Variant calling produced average Phred-scale quality scores of 73.89 for standards (The Coriell Trio) and 89.77 for participants. The GRS calculator was not able to accurately predict which participants had T1D. Discussion and Conclusion: A next generation sequencing method and bioinformatics pipeline for the screening of participants for 645 T1D associated polymorphisms was investigated. The method combined two sequencing techniques i.e., Ion AmpliSeq and Nanopore sequencing to achieve this. The data can then be processed by in-house variant callers. With a larger sample group, this method will be useful for the investigation of genetic variants linked to T1D.
Germline Cancer Predisposition Variants in Paediatric Rhabdomyosarcoma
(University of the Witwatersrand, Johannesburg, 2023) Pillhofer, Gabriella Peta; Lamola, Lindie; Mnika, Khuthala
Rhabdomyosarcoma (RMS) is cancer that originates from undifferentiated skeletal muscle cells. RMS is the most common tissue sarcoma in children and adolescents and has over 50% of cases occurring in individuals under the age of 10 and thus there is reason to suspect that RMS may have a hereditary predisposition. However, much of the existing research of germline predisposition in paediatric RMS is focused on ethnically European populations, and currently very little data is available from African populations. In this study, we aimed to identify RMS predisposition variants by performing whole exome sequencing (WES) on germline DNA from eight paediatric patients diagnosed with RMS. Following WES, variant annotation and filtering was performed to identify variants in genes of interest that were potentially germline causes of malignancy. Filtered variants were then classified according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. This study identified four variants of uncertain significance (VUSs) in four patients. Two of these were in genes that have previously been associated with an RMS phenotype (SDHB and BRCA1), and two were in genes that have been associated with a hereditary cancer syndrome that is not linked to RMS (CBL and CREBBP). While the highlighted variants are not of clinical significance, this study emphasises the importance of cataloguing and reporting VUSs in research in Africa. By expanding the genomic database on African patients, the analysis of variants on the continent may be made more accurate and efficient. It is the goal that the knowledge gained from this study will contribute to the information base of hereditary paediatric cancers in Africa, and that it may encourage similar research so that the field may continue to expand.
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, Lizette
South 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.
Development of a multiplex HIV/TB point-of-care diagnostic assay based on the microarray
(University of the Witwatersrand, Johannesburg, 2023) Malatji, Kanyane Bridgett
HIV/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.
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.
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
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
Rationalising laboratory workflow to improve the efficiency of diagnostic service delivery: A critical review of haematological malignancies by flow cytometric immunophenotyping at the Charlotte Maxeke Johannesburg Academic Hospital
(University of the Witwatersrand, Johannesburg, 2023) Naidoo, Maynolia; Glencross, Debbie
The 2006 Bethesda medical indications guideline provides concise indications for flow cytometric immunophenotyping (FCI), to enable rationalising a decision for sample processing. The local practice of processing every sample received for FCI places an enormous burden on the resources of the laboratory, and leads to unnecessary expenditure for state health. A ‘triage’ process based on the current Bethesda medical indications guideline may be beneficial in developing countries. The aim of this study was to determine how the implementation of a triage process would impact on the diagnostic service delivery, and the ability to detect or miss disease. A retrospective review of 500 bone marrow aspirate (BMA) samples submitted for FCI analysis was performed from October to December 2019. The sensitivity, specificity, and predictive values of the BMA cytomorphology against the FCI outcomes (‘test-all’) were determined. Thereafter, the Bethesda medical indications guideline was retrospectively applied to the same data set (‘triage’), to compare the decision to process or not to process samples, against objective evidence of disease in various BMA investigations. After exclusion of inadequate quality samples that preclude comparison, 429 ‘test-all’ and 455 triage cases were evaluated. The ‘test-all’ analysis revealed a 97.1% sensitivity and 89.8% specificity, with a 64.1% positive predictive value (PPV) but striking 99.4% negative predictive value (NPV). The triage was largely effective in identifying cases with disease, revealing a 100% sensitivity and 83.3% specificity, with a PPV of 32.5% and very high NPV of 93.8%. Without impacting clinical outcomes, the implementation of a triage process can reduce the burden of FCI testing by 18%. Preliminary cytomorphological review of the accompanying BMA is strongly recommended as an additional step to improve the overall PPV of the triage, while safely reducing unnecessary FCI sample processing in a further 56% of cases. The implementation of a triage process with modifications for local use in flow cytometry laboratories, would enable the appropriate rationalisation of resources, improve the cost- effectiveness, and overall diagnostic service delivery in developing countries like Sub-Saharan Africa
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, Elizabeth
Background: 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.
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, Christopher
Breast 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.
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, Mpho
Objectives: 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.
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, Katherine
Introduction: 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.
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
Synthetic cytology image generation to augment teaching and quality assurance in pathology
(2024) McAlpine, Ewen David
INTRODUCTION- Urine cytology offers rapid and relatively inexpensive screening for the detection of high-grade urothelial neoplasia in patients with haematuria. In our setting of a public sector laboratory in South Africa, however, there is a paucity of such specimens with which to train cytotechnologists and cytopathologists. Advancements in Generative Adversarial Networks present a potential solution to this problem by allowing for the generation of synthetic urine cytology images to supplement teaching and training. We illustrate an end-to-end machine learning model – from dataset creation to testing synthetic images in pathology personnel – to assess this technology in a real-world setting. METHODS- Two hundred and fourteen urine cytology slides were digitised and processed to construct a morphologically balanced dataset containing examples of benign, atypical and malignant urine cytology images. This dataset was used to train a StyleGAN3 model to generate synthetic urine cytology images. These synthetically generated images were then tested in a group of pathology personnel – both pathologists and trainees – to assess whether a difference between real and synthetic urine cytology images exists. Diagnostic error rate and subject image assessment were tested. RESULTS- StyleGAN3 was able to generate a wide morphological diversity of realistically appearing benign, atypical and malignant urine cytology images. When testing how these synthetic images were perceived by pathology personnel, there was no significant difference in diagnostic error rate, subjective image quality or inclusion of synthetic images in a cytology teaching set. DISCUSSION This work presents a proof-of-concept illustration of the feasibility of the use of synthetic cytology images to supplement pathology teaching when real examples may be difficult to obtain. Furthermore, this work presents important insights into the dynamics of pathology dataset creation and discusses the use of synthetic data in health education and the ethical and legal issues that arise with the use of synthetic patient data. CONCLUSION- Our work demonstrates that realistic, morphologically diverse urine cytology images can be generated using existing GANs technology and that human observers find such synthetic data visually acceptable. Additionally, our data indicate that there is no significant difference in synthetic data in terms of subjective image quality or diagnostic classification as determined by pathology personnel.