Department of Pathology

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Analysis of hereditary haemochromatosis and clinical correlations in the elderly
(Stellenbosch : Stellenbosch University, 2000-12) Bouwens, C. S. H.; Kotze, Maritha J.; Maritz, F. J.; De Villiers, J. N. P.; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Hereditary haemochromatosis (HH) is an autosomal recessive iron storage disease where the accumulation of iron in parenchymal organs may lead to diabetes, heart failure, liver cirrhosis, arthropathy, weakness and a variety of other ailments if preventive measures are not taken. HH is often not considered as a cause of these conditions, particularly not in the elderly where the background frequencies of type II diabetes, osteoarthritis and heart failure are generally high. Heterozygosity for C282Y, the HFE-mutation causing HH in approximately 80% of affected individuals worldwide, has been linked to a raised incidence of malignancies of the colon and rectum, stomach and the haematological system. One of the highest carrier-frequencies (116) in the world for this mutation has been reported in the South-African Afrikaner population, resulting in C282Y-homozygosity in approximately 1 in every 115 people in this group. A sample of 197 elderly Afrikaner volunteers was recruited for genotype/phenotype association studies. Their clinical presentation was denoted, biochemical iron-status determined and HFE genotyping performed. Either an increase or decrease in survival, or both, were proposed, depending on possible gender effects. HH has been positively associated with various cancer types, but may also protect against iron-deficiency anaemia which is by far the most frequent cause of anaemia in the older person. This study has led to the following findings: 1. The carrier frequency of mutation C282Y was found to be 1/8 in the elderly population (similar in males and females), which is slightly lower than the 1/6 reported in younger adults from the same population. Only one C282Y homozygote and two C282YIH63D compound heterozygotes were detected, all of them female. 2. The prevalence of diabetes, heart disease, arthropathy or a combination of these conditions did not differ significantly in C282Y heterozygotes and the mutationnegative group. 3. Among 24 C282Y heterozygotes only one individual with rectal carcmoma was detected compared with two cases with rectal- and seven with colonic malignancies in 153 mutation-negative individuals. The single female C282Y homozygote identified suffered from both rectal and colon carcinoma and died approximately 6 months ago as a consequence of her colon malignancy. 4. Serum ferritin appears to be a highly unreliable parameter of iron status, particularly in the elderly where a variety of factors that may influence the levels are often present in elderly individuals. This may be due to ageing alone or as a result of multiple comorbidities. 5. Serum ferritin levels were lower than expected in elderly subjects with mutation C282Y and compound heterozygotes with both C282Y and H63D, which may be related to a variable penetrance of the HFE gene mutations. It is possible that variation in other genes exist that confer protection against iron-loading by gene-gene interaction. The probability that environmental factors (e.g. a low iron diet) are more important in this respect cannot be excluded, although this is considered less likely in the light of the fact that the same trend was observed in all mutation-positive elderly individuals. It is therefore highly likely that C282Y -positive subjects with significant iron loading have died before reaching their seventies, particularly since none of the males included in this study were homozygous or compound heterozygous for the mutations analysed. In conclusion, possession of a mutant HFE gene does not appear to confer a survival advantage in old age, neither does it seem that mutation carriers with significant ironloading are overlooked by the medical fraternity. Further investigations are warranted to shed more light on the contributions of gene-gene and gene-environment interaction in the clinical manifestation of Hll, and how these processes can be manipulated to prevent the symptoms of this largely underdiagnosed disease.
Analysis of HIV-1 diversity and inflammatory markers in HIV-associated neurocognitive disorders (HAND).
(Stellenbosch : Stellenbosch University, 2022-04) Ruhanya, Vurayai; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH SUMMARY: HIV-associated neurocognitive disorders (HAND), which involve impairment or disruption of neurocognitive functioning have become one of the most frequent complications in adult HIV-1 infections with global estimates ranging from 42% to 45%. The screening and diagnosis for HAND relies on multiple clinical and neuro-psychometric methods. However, these methods have a low reliability because they are not precise as most of possess inadequate psychometric properties and diagnostic accuracy. Therefore, this study aimed to describe and characterise viral and immunological determinants of HAND and evaluated their relationship with specific clinical, neuromedical and neuropsychological data to identify putative easy-to-measure biological markers for diagnosis of the condition. This study demonstrated that higher peripheral blood lymphocyte-derived HIV-1 proviral DNA is a predictor of global and domain-specific neurocognitive impairment among individuals infected with HIV-1 subtype C. The study also determined proviral load cut-off /threshold value for neurocognitive impairment and associated diagnostic accuracy. It also identified IP-10 and RANTES as a plasma chemokine bio-signature for HIV-associated neurocognitive impairment with diagnostic accuracy comparable to standard psychometric tests used to screen and describe severity of HAND. In addition, the study identified 3 viral genetic signatures for cognitive impairment, namely Lysine at codon 24, (24K) and Arginine at codon 29 (29R) on Tat protein and Tyrosine (Y) at position 45 (45Y) of Vpr. These three signature amino acids were related to classical markers for monitoring HIV infection. Finally, we identified 4 conserved fragment sequences, PEDQGPQREPYNEWTLE (5 to 21), LGQYIY (42 to 47), TYGDTW (49 to 54), PEDQGPQREPYNEW (5 to 18) on viral protein R, that were associated with higher plasma viral load and proviral load. The study has identified novel cytokine/chemokine and viral biomarker signatures for HIV associated neurocognitive impairment with low to moderate diagnostic accuracy. The findings demonstrated a need for interdisciplinary approach to elucidate possible molecular interactions between the peripheral blood immune markers, viral signatures and the CNS that are linked to observed clinical outcomes of neurodegradation in HIV infection. The identified biomarkers can be further investigated for use as screening tools and treatment endpoints for HAND.
Analysis of single nucleotide polymorphisms with opposite effects on serum iron parameters in South African patients with multiple sclerosis
(Stellenbosch : Stellenbosch University, 2014-04) Moremi, Kelebogile Elizabeth; Van Rensburg, S. J.; Kotze, Maritha J.; Geiger, D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Chemical Pathology.
ENGLISH ABSTRACT: There is growing interest in how genetic and environmental risk factors interact to confer risk for dysregulated iron homeostasis, which is considered a possible pathogenic mechanism in multiple sclerosis (MS). While iron deficiency has been associated with greater disability and disease progression, cerebral accumulation and overload of insoluble iron has also been reported in MS patients. Variation in the matriptase-2 (TMPRSS6) gene has recently been described that may lead to reduced iron levels, which raised the question of whether it may be involved in dysfunctional iron regulation as a pathogenic mechanism in MS. The aims of the study were as follows: 1)) comparison of the allele frequencies and genotype distribution for TMPRSS6 A736V (rs855791, c.2207C>T) and HFE C282Y (rs1800562, c.845G>A) between patients diagnosed with MS and unaffected controls; 2) determination of the effects of clinical characteristics, relevant lifestyle factors and genotype on serum iron parameters in MS patients compared to population matched controls; and 3) determination of clinical outcome in relation to age of onset and degree of disability in MS patients. The study population included 121 Caucasian MS patients and 286 population-matched controls. Serum iron, transferrin, ferritin and transferrin saturation levels were available from previous studies and lifestyle factors were subsequently documented in a subgroup of 68 MS patients and 143 controls using the study questionnaire. Genotyping of TMPRSS6 A736V and HFE C282Y were performed using allele-specific TaqMan technology. The genotype distribution and allele frequencies of TMPRSS6 A736V and HFE C282Y did not differ between MS patients and controls. MS patients homozygous for the iron-lowering minor T-allele of TMPRSS6 A736V had significantly lower serum iron levels (p=0.03) and transferrin saturation levels (p=0.03) compared to CC homozygotes. In MS patients the iron-loading minor A-allele of HFE C282Y was also associated with a paradoxical decrease in serum ferritin (p<0.01) compared to GG homozygotes. When considering the combined effect of the minor alleles of TMPRSS6 A736V and HFE C282Y with opposite effects on iron levels, we found a significant reduction in serum ferritin levels (p<0.05), independent of age, sex, body mass index (BMI) or dietary red meat intake in MS patients. A similar effect was not observed in the population- and age-matched controls. Higher dietary red meat intake correlated significantly with increased ferritin only in controls (p=0.01 vs. 0.21 for MS patients). In the presence of the minor allele of HFE C282Y, the TMPRSS6 A736V CT and TT genotypes were associated with a significantly earlier age of onset of MS when the post hoc test was applied (p=0.04). All the study aims were successfully accomplished. Our results support the possibility of an epistatic effect between TMPRSS6 A736V and HFE C282Y associated with reduced ferritin levels in MS patients. Pathology-supported genetic testing (PSGT) applied in this study as a new concept for analysis of complex diseases with a genetic component, is well placed to optimise clinical management in patients with MS.
Analysis of the clinical utility of gene expression profiling in relation to conventional prognostic markers in South African patients with breast carcinoma
(Stellenbosch : Stellenbosch University, 2014-12) Grant, Kathleen Ann; Kotze, Maritha J.; Wright, Colleen A.; Apffelstaedt, Justus P.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Anatomical Pathology.
ENGLISH ABSTRACT: Breast cancer is a heterogeneous disease characterised by marked inter-individual variability in presentation, prognosis and clinical outcome. The recognition that morphological assessment has limited utility in stratifying patients into prognostic subgroups led to clinico-pathological classification of tumour biology, based on receptor expression using immunohistochemical (IHC) techniques. This standard is currently complemented by the development of gene expression profiling methodology that led to the identification of intrinsic molecular subtypes, reflecting tumour genetics as the true driver of biological activity in breast cancer. The study was based on the hypothesis that molecular classification of breast carcinomas integrated with established clinico-pathological risk factors will improve current diagnostic and risk management algorithms used in clinical decision-making. A pathology-supported genetic testing strategy was used to evaluate microarray-based gene profiling against diagnostic pathology techniques as the current standard. Clinico-pathological factors including age, number of positive axillary nodes, tumour size, grade, proliferation index and hormone receptor status was documented for 141 breast cancer patients (143 tumours) referred for microarray-based gene expression profiling between 2007 and 2014. Subsets of patients were selected from the database based on the inclusion criteria defined for three phases in which the study was performed, in order to determine 1) the percentage of patients stratified as having a low as opposed to high risk of distant recurrence using the 70-gene MammaPrint profile within the inclusion criteria, 2) correlation of HER2 status as determined by IHC and fluorescence in situ hybridisation (FISH) with microarray-based mRNA readout (TargetPrint), and 3) the relationship between hormone receptor determination as reported by standard IHC and molecular subtyping using the 80-gene BluePrint profile. Similar distribution patterns for MammaPrint low- and high-risk profiles were obtained irrespective of whether fresh tumour biopsies or formalin-fixed paraffin embedded (FFPE) tissue was used. During the first phase of the study, 60% of the 106 tumour specimens analysed with MammaPrint were classified as low-risk and 40% as high-risk using a newly-developed MammaPrint pre-screen algorithm (MPA) aimed at cost-saving. In the second phase of the study, performed in 102 breast tumours, discordant or equivocal HER2 results were found in four cases. Reflex testing confirmed the TargetPrint results in discordant cases, achieving 100% concordance regardless of whether fresh tumour or FFPE tissue was used for microarray analysis. For the third phase of the study 74 HER2-negative tumour samples were selected for comparative analysis. Statistically significant positive correlations were found between protein expression (IHC score) and mRNA (TargetPrint) levels for estrogen receptor (ER) (R=0.53, p<0.0001) as well as progesterone receptor (PR) (R=0.62, p<0.0001), while combined ER/PR tumour status was reported concordantly in 82.4% of these tumours. BluePrint was essential for interpretation of these results used in treatment decision-making. The MPA developed in South Africa in 2009 was validated in this study as an appropriate strategy to prevent chemotherapy overtreatment in patients with early-stage breast cancer. The use of microarray-based analysis proved to be a reliable ancillary method of assessing HER2 status in breast cancer patients. Risk reclassification based on the TargetPrint results helped to avoid unnecessary high treatment costs in false-positive cases, in addition to providing potentially life-saving treatment to those for whom it was indicated. While neither IHC nor TargetPrint estimation of intrinsic subtype correlated independently with the molecular subtype as indicated by BluePrint profiling, the ability to distinguish between basal-like and luminal tumours was enhanced when the combined protein and mRNA values was considered. Genomic profiling provided information over and above that obtained from routine clinico-pathological assessments. This finding supports the relevance of a pathology-supported genetic testing approach to breast cancer management, whereby advanced genomic testing is combined with existing clinico-pathological risk stratification methods for improved patient management.
An anti-SARS-CoV-2 quantitative study to determine the effect of time on the antibody titre, post-vaccination
(Stellenbosch : Stellenbosch University, 2024-01) Meyer, Burnet Adriaan; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: In 2019, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) outbreak emerged in Wuhan, China, rapidly evolving to a pandemic. Vaccines such as messenger ribonucleic acid (mRNA)- based and Adenovirus-based, have been developed to counter severe Coronavirus Disease 2019 (COVID-19). However, many factors, for example, immunosuppression and prior infection can affect the antibody titre. This study evaluated the effectiveness in Anti-SARS-CoV-2 immunoglobulin (Ig) G production and decline over time, indicating the potential requirement for additional boosters. Furthermore, this study focused on how Adenovirus-based vaccine boosters and breakthrough infections affected the antibody titre. A total of 184 samples were collected from two cohorts which consisted of 67 participants. Samples were collected at baseline, week 3, and week 6 post-booster for Cohort A (laboratory workers at the Western Cape Blood Service). Cohort B (staff and students at the Faculty of Medicine and Health Sciences, Stellenbosch University) had samples collected at baseline, week 2, week 4 and week 8 post-booster. The samples were tested for the presence of the SARS-CoV-2 Anti-nucleocapsid (N) as well as Anti-spike (S) antibodies. The Anti-N antibodies indicated that a participant had a natural infection to SARS-CoV-2 while the Anti-S antibodies were utilised to examine the influence of age, gender and time on vaccine-induced antibodies. This findings of this study demonstrated that vaccine-induced antibodies were still detectable six months following the initial vaccination. A significant increase was observed for both cohorts, when comparing the baseline titre with the different timepoints, post-booster. Multiple samples were found to be Anti-N positive, suggesting that these participants were potential asymptomatic cases. Variations were found in the Anti-S antibody titre based on different age groups, although these variations were not statistically significant. Moreover, the results have indicated that gender does not affect the Anti-S antibody titre. This research enhances the understanding of antibody titre dynamics, contributing to a better grasp of immune longevity. Further studies are recommended to determine the impact of combining different boosters on the antibody titre, and assess the difference in antibody titre between monovalent boosters with bivalent boosters.
The apoptotic potential of different HIV-1 subtype C Tat mutations in cell culture
(Stellenbosch : Stellenbosch University, 2013-03) Isaacs, Shahieda; Engelbrecht, Susan; Glashoff, Richard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
The efficiency in which HIV-1 can infect, spread and evade the attack of therapeutic agents can be attributed to a high mutation rate and frequent recombination events. These factors have collectively contributed to the diversity observed in HIV-1 and resulted in a multitude of subtypes, sub-subtypes, circulating recombinant forms (CRF’s) and unique recombinant forms (URF’s). The aim of this study was to investigate HIV-1 diversity in Cape Town using a small cohort of treatment naive patients being investigated for HIV Associated Neurocognitive Disorders (HAND). Four different genomic domains: gag, pol, accessory and gp41 genes were sequenced to subtype the virus. HIV-1 tat was further investigated because the dicysteine motif has been reported to play a role in HAND. Viral RNA and proviral DNA was extracted from 64 patients and used for the amplification and sequencing of the genes. Rega and jpHMM online tools were used to identify HIV-1 subtypes and recombinants while Neighbor-joining phylogenetic trees were constructed for phylogenetic analysis. The pol gene was further investigated using SCUEAL to detect possible intra-subtype recombination and was also screened for the presence of transmitted drug resistance. In addition tat sequence datasets retrieved from the Los Alamos sequence database were investigated and compared with the newly generated sequences for the detection of point mutations and amino acid signature patterns. Sequencing identified most of the samples as subtype C; however six inter-subtype recombinants (AE, A1G, A1CU and two BC) and 9 intra-subtype C recombinants were identified. In addition 13% of pol sequences were identified with resistance mutations. Signature pattern analysis identified a high level of variability in the tat sequences: 68% were identified with C30S31; 29% with the C30C31 mutation and a single sequence with a novel mutation C30A31. Functional analysis of these mutations indicated that all mutations investigated were capable of inducing apoptosis in cell culture. The C30C31 mutation generated the highest level of apoptosis, closely followed by the C30A31 mutation. However no statistical significance could be detected between tat mutations and the observed levels of apoptosis.
The application of wastewater-based epidemiology to Hepatitis E virus in South Africa
(Stellenbosch : Stellenbosch University, 2024-02) Roberts, Bronwyn; Maponga, Tongai; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Background Hepatitis E virus (HEV) has a global presence, but the highest burden of disease is found in Africa and Asia. Unfortunately, in Sub-Saharan Africa, there is a gap in knowledge on the molecular epidemiology of the disease, as often serological methods only are applied. In recent years a high seroprevalence of the virus has been detected in populations in the Western Cape province. Evidence of viraemia has been published on a few South African patients from the Western Cape. Viral ribonucleic acid (RNA) was also detected in swine samples from an abattoir and in porcine meat products in Cape Town. Based on the existing literature, HEV genotype 3 has been detected in patients and swine samples. In this study, the aim was to determine whether HEV was circulating in communities in the Western Cape by testing wastewater extracts for the presence of HEV RNA. The study also sought to determine the HEV genotype in wastewater sample extracts. Methods One hundred and forty-three wastewater extracts were tested for the presence of HEV RNA using real-time polymerase chain reaction (PCR). Samples were sourced from four locations: two wastewater treatment works and sewer manholes from two Stellenbosch University residences. Nested reverse-transcriptase PCR (RTPCR) targeting three regions of the HEV genome was applied to samples which tested positive for HEV RNA. The sequenced regions were a portion of open reading frame (ORF) 2 covering 347 base pairs (bp), a 286 bp region of ORF1 and a 126 bp region of the overlapping ORF2/3. The generated PCR products were then sequenced with Sanger sequencing. The generated consensus sequences were placed in a phylogenetic tree with reference sequences for HEV genotypes 1 to 8, to determine which genotype is likely circulating in the selected communities. Results Out of the 143 wastewater samples, 130 valid results were generated. Out of 130, 21 (16.2%%) were positive and 109 (83.9%) were negative. Of the 21 positive samples, 5 (23.8%) were collected from Athlone wastewater treatment works (WWTW), 9 (42.9%) from Zandvliet WWTW, 1 (4.8%) from Meerhoff residence in Tygerberg and 6 (28.6%) from Metanoia residence in Stellenbosch. The positive samples had a median cycle threshold value of 37.9 (interquartile range: 36.7-39.7). Out of the 21 positive samples, 4 (19.1%) sequences were obtained from the ORF2/3 region. The sequences clustered most closely with a sequence from a South African patient and HEV genotype 3 reference sequences. Conclusions Based on the real-time PCR results, it appears that HEV is circulating among communities in the Western Cape province. Based on the detection of HEV genotype 3 in the wastewater samples, it suggests that zoonotic transmission may be the mostly likely route of infections. Further investigation to identify porcine and other food products which may be contaminated with HEV are necessary to break chains of transmission.
Aspects of the pharmacokinetics of hypoxoside in rodents
(Stellenbosch : Stellenbosch University, 1993) Bester, Christoffel Carolus; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Microbiology.
ENGLISH ABSTRACT: Certain aspects of the pharmacokinetics of a novel diglucoside, hypoxoside, were investigated in mice and rats. After intragastric dosage of the mouse, hypoxoside was hydrolysed to its aglycone, rooperol. Most enzyme activity occurred in the mouse caecum with intestinal bacteria playing a major role in producing the hydrolytic enzymes. Caecalase extracts hydrolysed hypoxoside in vitro but this activity ceased after the mice were treated with antibiotics, suggesting bacteria as the enzyme source. However, in vivo tests with similar antibiotic-treated mice seemed to demonstrate a minor second, possibly mucosal source of hypoxoside hydrolysing B-glucosidase. An attempt to isolate a single strain of faecal bacteria capable of this activity was unsuccessful although hypoxoside was hydrolysed by pure cultures of some non-intestinal clinical isolates
The association between vitamin D, vitamin D Binding proteins and VDR polymorphisms in diabetic and non-diabetic patients
(Stellenbosch : Stellenbosch University, 2019-03) Maepa, Setjie Welcome; Matsha, Tandi E.; Erasmus, Rajiv T.; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Pathology. Chemical Pathology.
ENGLISH ABSTRACT: Introduction: Type 2 diabetes mellitus (T2DM) is by far the most prevalent form of diabetes manifesting with insulin resistance (IR), abnormal pancreatic β-cell function and hyperglycaemia. Evidence from epidemiological and observational studies have shown that vitamin D deficiency is associated with increased risk for T2DM although the findings are inconsistent and inconclusive. In the circulation vitamin D is transported bound to vitamin D binding protein (VDBP), evidence showed that vitamin D levels are positively associated with VDBP levels. Several genes such as vitamin D receptor gene (VDR), involved in the metabolic pathway of T2DM have been considered good candidate for susceptibility to T2DM. The present study aimed to investigate the association between vitamin D, vitamin D binding proteins (VDBP) and vitamin D receptor (VDR) polymorphisms in T2DM and non-diabetic patients in the mixed ancestry population. Materials and methods: The current study comprised of 1603 participants (387 males and 1216 females). Vitamin D levels were measured using the paramagnetic particle chemiluminescence test on a Beckman DXI. Vitamin D binding protein (VDBP) in serum samples was measured using the Human Vitamin D BP Quantikine ELISA kit. Fok1 (rs2228570), Apa1 (rs7975232) and Taq1 (rs731236) single nucleotide polymorphisms (SNPs) of the VDR gene were genotyped from a genomic DNA using the TaqMan SNP Genotyping Assays and were confirmed by direct sequencing. Results: Vitamin D deficiency (44%) and insufficiency (42.6%) were highly prevalent and optimal 25(OH)D levels were very low with only 13% having optimal levels. The overall vitamin D status of the whole population group was insufficient (22.0±7.6 ng/mL). 25(OH)D levels and serum VDBP varied according to gender with males having higher 25(OH)D levels (23.6±7 vs 21.5±7.5ng/mL, P=0.0006) and females with significantly higher serum VDBP levels (299.1±71.2 vs 315.9±76.1 µg/mL, P<0.0001). 25(OH)D levels were generally significantly decreased in the hyper-glycemic subgroups. Screen-detected DM males had low 25(OH)D levels compared to normoglycaemic group (17.0±6.1vs 24.2±8.2, P=0.0214). A similar trend was observed in the female groups (21.1±6.0 vs 22.4±7.9, P=0007). Anthropometric measurements including the BMI (kg/m2), Waist C (cm) and Hip C (cm) were significantly higher in hyper-glycaemic group than in normo-glycaemic males and females (All, P<0.0001). In contrast, there were no significant differences in serum VDBP (µg/mL) between the glycaemic sub-groups in either male (P=0.5614) or females (P= 0.4813). The glycaemic parameters, as expected, were significantly increased in the hyperglycaemic sub-groups in both genders, including FBG (mmol/L), 2 hr BG (mmol/L), HbA1c (%), FBI (mIU/L), 2 hr BI (mIU/L) and HOMA-IR (All, both males and females P<0.0001). In general, the lipids, including the triglycerides (mmol/L), LDL-C (mmol/L) and Cholesterol (mmol/L) were also significantly increased in both genders in the hyper-glycaemic sub-groups (All, males P≤0.0300, females P<0.0001), while HDL-C (mmol/L) was significantly decreased in both males and females in the hyperglycaemic sub-groups (All, P≤0.0308). The variant genotype GG of the Fok1, AA of Apa1 and GG of the Taq1 SNPs were not significantly different in hyper-glycaemic patients compared to normo-glycaemic group (58.5% vs 55.1%, P-value, 40.1% vs 38.0%, P-value and 6.9% vs 8.5%, Pvalue,) respectively. Similarly, there was no significant difference in the alleles frequency distribution of these SNPs between the groups. Results also demonstrated no significance difference in the genotype or allele frequency distribution of Fok1 (rs2228570), Apa1 (rs7975232) and Taq1 (rs731236) SNPs between subjects with optimal Vitamin D (25(OH)D ng/mL) levels and those with insufficient/deficient levels (P≥0.2036 and P≥0.6347 respectively). These trends were also observed when serum VDBP levels were evaluated against Fok1, Apa1 and Taq1 genotypes. Multiple linear regression showed that low 25(OH)D was associated with increased LDL-C and PTH in both male and females irrespective of T2DM, but serum VDBP was associated with low 25(OH)D in hyper-glycaemic females only. In normo-glycaemic males 19.5% of the variation in 25(OH)D was attributed to increased LDL-C and in the hyper-glycaemic group 15.5% it was attributed to PTH and CRP. In normo-glycaemic females 12.8% variation in 25(OH)D was attributed to LDL-C, serum creatinine and PTH, whereas in hyper-glycaemic group 16.1% was attributed to increased age, serum VDBP, triglycerides, LDL-C, creatinine and PTH. Conclusion: This study showed prevalence of vitamin D deficiency/insufficiency in the mixed ancestry population group. There was no association between vitamin D (25(OH)D), vitamin D binding proteins (serum VDBP) and VDR polymorphisms in T2DM patients. Serum VDBP levels were associated with low vitamin D levels in hyper-glycaemic females only. Increased LDL-C, PTH and CRP were predictors of low vitamin D levels.
Automated sputum screening using the BD FocalPointTM Slide Profiler : correlation with transbronchial and transthoracic needle aspirates in a high risk population
(Stellenbosch : Stellenbosch University, 2014-04) Neethling, Greta Sophie; Schubert, Pawel T.; Koegelenberg, C. F. N.; Diacon, A. H.; Wright, C. A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Division of Anatomical Pathology.
ENGLISH ABSTRACT: Background: Sputum is a non-invasive, economic investigation whereby bronchogenic carcinoma can be identified. Manual cytological screening is labour intensive, time-consuming and requires a continuous high level of alertness. Automation has recently been successfully introduced in gynaecological cytology. Since sputum samples are similar to cervical smears, the question arises as to whether they are also suitable for automated screening. Objective: This study presented with various objectives: 1) To test automated sputum screening using the BD FocalPoint™ Slide Profiler (FP) and compare with manual sputum screening. 2) To determine the sensitivity and specificity of sputum in identification of bronchogenic carcinoma. 3) To ascertain if any clinical, radiological or bronchoscopy findings would be predictors for bronchogenic carcinoma. 4) To determine the significance of adequacy. Method: Sputum samples were collected prospectively from patients attending the Division of Pulmonology at Tygerberg hospital for a transbronchial fine needle aspiration biopsy (TBNA) or a transthoracic fine needle aspiration biopsy (TTNA) for the period from 2010 to 2012. A pre-bronchoscopy sputum was collected and submitted for processing. Stained slides were put through the FP for automated screening. After slides were qualified, sputum slides were put back in the routine screening pool. Correlation was done using the TBNA/TTNA result as the standard to evaluate the sputum results. Results: 108 sputum samples were included in this study. Of the 84.3% malignant (n=91) and 15.7% benign (n=17) cases confirmed with a diagnostic procedure, sputum cytology had a sensitivity of 38.5% (35/91 malignant cases), and a specificity of 100% (17/17 benign cases). Automated screening had a better sensitivity of 94.3% (33/35 positive sputum cases), while manual screening showed a sensitivity of 74.3% (26/35 positive sputum cases) when compared to the final sputum result. Individual parameters with a significant association with positive sputum included the presence of an endobronchial tumour, partial airway obstruction / stenosis, round mass, spiculated mass (negative association), loss of weight (negative association) and squamous cell carcinoma as the histological subtype. Adequacy was not as significant as hypothesised since 85.3% of true positive sputum, but also 65.5% of false negative sputum, had large numbers of alveolar macrophages present. Conclusion: Sputum cytology remains an important part of the screening programme for bronchogenic carcinoma in the public health sector of South Africa. Results confirm that sputum cytology is very specific, and automated screening improves sensitivity. Automated screening proved to be more time efficient, resulting in 83.1% reduction (p<0.0001) in the screening time spent per case by a cytotechnologist. Results confirm that the quantity of alveolar macrophages is not directly proprtional to pathology representation. Positive sputum results did however improve with sputum adequacy, but had no significant association. Recommendations from this study include adopting automated sputum screening.
B lymphocyte activation and exhaustion in chronic HIV : novel surrogate markers of generalised immune activation and selective modulation of aberrant B cell responses using vasoactive intestinal peptide (VIP)
(Stellenbosch : Stellenbosch University, 2015-04) Reid, Timothy Dawson; Glashoff, Richard H.; Ipp, Hayley; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Introduction: Chronic HIV-1 infection is characterized by immune activation and dysregulation of immune homeostasis, which impacts on multiple immune cell types. The B-cell compartment, which plays an important role in the producing neutralizing antibodies, is also dysregulated in HIV- 1 infection. In this study we investigated peripheral blood B-cell subset distribution, and changes in expression of cellular activation, inhibition, and apoptosis signaling markers in both untreated chronic HIV-1 infected individuals and healthy uninfected controls. The neuropeptide immune modulator, vasoactive intestinal peptide (VIP) is known to selectively down-regulate activation of CD4+ T-cells in various disease settings including HIV-1, however to our knowledge, no studies have investigated the effect of VIP inhibition on B-cell activation. Materials & Methods: A total of 21 HIV+ve (CD4 count >250 cells/µl), and 19 HIV-ve individuals were recruited from the Emavundleni voluntary testing and counseling clinic in Crossroads, Western Province, South Africa. Whole blood was stained to distinguish B-cell subsets (activated memory (AM: CD21-CD27+), resting memory (RM: CD21+CD27+), mature naïve (MN: CD21+CD27-), or tissue-like memory (TLM: CD21loCD27lo). In addition expression of markers of B-cell activation (CD126, CD86, CD38, CD284, CD287), inhibition (CD72, CD85j, CD300a, CD305, CD307d), and apoptosis signaling (CD95), was assessed ex vivo by flow cytometry (BD FACSCanto II). For determination of functional responsiveness isolated B-cells (RosetteSep, Stemcell Technologies) were cultured for 18h (37°C, 5%CO2) without stimulation or stimulated with TLR ligands (LPS or R848). Stimulation experiments were also performed in the presence or absence of VIP. Results: Chronic HIV-1 infection affected B-cell subset distribution. The percentage (%) of TLM was increased by 59.24%, and %RM was decreased by 22.73% (both p<0.01). Total expression of the VIP receptor VPAC2 was decreased by 47.35% (p=0.0296). Subsets had a mixed phenotype ex vivo; HIV infection upregulated CD38 (by 59.56%, p=0.0004), CD72 (by 60.70%, p=0.0396), CD307d (by 68.63%, p=0.0015) on AM, while RM B-cells had increased expression of TLR4 (by 107.04%, p=0.0057) and TLR7 (by 208.14%, p=0.0199). TLM B cells (i.e. exhausted phenotype) displayed upregulated TLR7 (by 550%, p=0.0128) and CD307d (by 72.40% p=0.045) expression. MN B-cells had increased CD72 expression (by 70.98%, p=0.0026). R848 upregulated CD86 expression by 42.20% on AM (p<0.01), and by 56.06% on RM B-cells (p<0.01), which was significantly downregulated with VIP inhibition (both p<0.05). Similarly, CD95 expression on RM, TLM, and MN B-cells increased by 31.10% (p<0.001), 21.46% (p<0.01), and 39.92% (p<0.01) with R848 stimulation respectively, which was also significantly downregulated with VIP inhibition. Conclusion: These data indicate that B-cells in untreated HIV infection display increased levels of activation, and also the potential for increased susceptibility to apoptosis as evidenced by increased FAS (CD95) expression. VIP significantly down-regulated markers of activation, inhibition, and apoptosis signaling. Dysregulation of B-cells is thus apparent in asymptomatic stable chronic HIV-1 infection, which may impact on both inefficient neutralizing antibody production and hypergammaglobulinemia. The ability of VIP to prevent stimulationassociated marker upregulation may indicate that VIP is a potential therapeutic agent. Its immuno-modulatory properties were demonstrated to limit B-cell hyperactivation, and selectively down-regulate apoptosis and mark it out for further investigation.
Beta-lactam resistance mechanisms in Enterobacter species isolates from Tygerberg Hospital.
(Stellenbosch : Stellenbosch University, 2018-10-31) Okyere, Dora; Newton-Foot, Mae; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
Background Resistance to carbapenem antibiotics in Gram-negative bacteria is a major public health problem due to limited treatment options. In Enterobacter species, carbapenemase production and reduced membrane permeability, resulting from reduced expression of outer membrane proteins OmpF and OmpC, in combination with extended-spectrum β-lactamase (ESBL) production, hyper-production of chromosomal blaAmpC β-lactamases and / or over-expression of the AcrAB/TolC efflux pump have been speculated to promote carbapenem resistance. This study investigated the mechanisms that promote ertapenem resistance in clinical Enterobacter cloacae isolates from Tygerberg Hospital. Materials and methods Twenty ertapenem non-susceptible clinical E. cloacae isolates, four ertapenem susceptible controls and five wild-type controls were selected based on the VITEK2®AES. Ertapenem MICs were determined using broth microdilution (BMD) and gradient diffusion. Resistance mechanisms were characterized using the phenotypic assays VITEK2®AES, Mastdiscs D68C, D69C and D63C combination sets, Rapidec® Carba NP kit and a synergy assay using disc diffusion in the presence of an efflux pump inhibitor (EPI). Molecular assays included multiplex PCR to identify carbapenemases, multiplex PCR and sequencing to identify ESBLs, SDS-PAGE to characterize OmpC and OmpF abundance and RT-qPCR to quantify expression of blaAmpC, ompC, ompF and acrB. Results and Discussion Seventeen (85%) ertapenem non-susceptible isolates were confirmed non-susceptible by BMD and six (30%) by gradient diffusion, suggesting possible undercalling of ertapenem resistance by gradient diffusion and overcalling by VITEK2®AES. Seven ertapenem non-susceptible isolates were predicted to be carbapenemase producers by the VITEK2®AES and one by the Rapidec® Carba NP kit, however no carbapenemases were detected by PCR. Nineteen ESBL producers were identified by the VITEK2®AES, eight by the D68C combination set and eleven by the D63C combination set. ESBLs were detected in 12 (60%) isolates by PCR and sequencing; of which eight were blaCTX-M, three were blaSHV-12 and one isolate contained both genes. Twelve (60%) ertapenem non-susceptible isolates were predicted to be derepressed blaAmpC producers by the VITEK2®AES, thirteen (65%) by the D68C combination set and six (30%) by the blaAmpC RT-qPCR.
Blood dendritic cells in chronically HIV-1 infected individuals in South Africa: Phenotype, function and immune modulation
(Stellenbosch : Stellenbosch University, 2016-12) De Swardt, Dalene; Glashoff, Richard H.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology
ENGLISH ABSTRACT :HIV-1 infection detrimentally affects CD4 T lymphocytes as well as the blood plasmacytoid (pDC) and myeloid dendritic cell (mDC) compartment. DCs act as innate sensors and as initiators and directors of antigen-specific immune responses. Whereas, mDCs have the unique ability to prime naïve T-lymphocytes and activate adaptive immune responses, pDCs are primary producers of type 1 interferons (IFNs), playing a pivotal role in anti-viral immunity. In the current study both pDCs and mDCs from chronically HIV-1 infected South African individuals (on or naïve for ARV therapy) as well as with and without concurrent TB disease, were compared to matched uninfected controls. Similar to CD4 T lymphocytes, bothpDCs and mDCs, were significantly depleted during HIV-1 infection, (reduction of pDC, mDC and CD4 T lymphocyte was 63% (p 0.001), 80% (p 0.001) and 31% (p 0.01), respectively). In parallel, significantly higher levels of generalised immune activation and exhaustion (CD38+CD8+, PD-1+CD8+ and CD38+PD-1+CD8+ T lymphocytes) were observed. ARV treatment ( 1 year) did not result in DC number recovery despite a significant increase of CD4 T lymphocytes numbers (CD4 T lymphocyte number gain of 89% (p 0.01), it fell short of full recovery).TB co-infection did not exacerbate number loss. Phenotypic characterisation of DC populations in circulation during HIV-1 infection may indicate the underlying reasons for the loss from circulation. Phenotypic profiling by multiparameter flow cytometry included: markers of activation (CD86, CD80 and CD62L), maturation (CD83), apoptosis (TNF-R2, FAS, FASL and TRAIL R1-R4) and chemotaxis (CCR5, CCR7, CCR9 and CXCR6). HIV-infection was associated with a significantly higher percentage of CD86+mDCs which may be indicative of early maturation or transition to secondary lymphoid tissue. The frequency of the CD86+mDCs subset normalised upon ARV therapy. Also, HIV-1 infection influenced the distribution of TNF-R2+pDCs and TNF-R2+mDCs. Increased TNF-R2 expression in both subsets, may attest to enhanced survival function. Functionally, DCs of HIV-1 infected individuals were reactive to TLR-L stimulation and in some cases showed enhanced responses compared to uninfected individuals. A significantly higher frequency of TNF-R2+pDCs, IFN- +pDCs, and TNF +mDCs was observed in whole blood TLR cultures of HIV-1 infected individuals (TNF-R2+pDCs: LPS (p = 0.002) and R848 (p = 0.01), IFN- +pDCs: R848 (p = 0.04), TNF +mDCs: LPS (p = 0.003))s.In addition, whole blood TLR cultures of the ARV treated study group generally showed normalisation of the responses, however; in certain cases ARV therapy reduced responsiveness to levels significantly lower than the control study group (i.e.TNF-R2+pDCs and TNF-R2+mDCs in CpG ODN stimulation). In contrast, a significantly lower frequency of IL12p40+mDCs was observed during HIV-1 infection (p = 0.02). TLR-L cultures of the ARV treated study groups showed normalisation of IL12p40+mDCsfrequency. Notably, treatment with the immunomodulating peptide VIP induced a decline in IL12p40+mDCs to levels lower than the control study group.The frequency of TNF +pDCs in TLR-L whole blood cultures was similar between the healthy, untreated and treated HIV-1 infected study groups, however, significantly reduced frequencies were observed in these study groups upon VIP treatment. These data indicate unique phenotypic and functional changes in DC subsets in chronic HIV- 1 infection which may provide potential targets for immunotherapeutic intervention.
The carriage of antibiotic resistant Gram-negative organisms in children in the Cape Town community and the impact of antibiotic exposure on the development of resistance (a TB-CHAMP sub-study)
(Stellenbosch : Stellenbosch University, 2019-12) Ocloo, Remous; Whitelaw, Andrew; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
Introduction Antibiotic resistance has become a major issue across the globe and the situation is worsening in low- and middle-income countries. In sub-Saharan Africa and the world at large, antibiotic resistance research is localized and focused on hospitalized individuals. There is, therefore, little or no data on antibiotic resistance in the community; especially in children. This study described the carriage of resistant isolates in children in Cape Town and investigated the effects of antibiotic exposure on the development of resistance in stool using an in-vitro model. Materials and Methods Stool samples from fifty participants of the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP) were cultured onto McConkey agar (MCC) with the addition of ertapenem and cefpodoxime discs to select for carbapenem and cephalosporin-resistant and susceptible E. coli and Klebsiella isolates. Antibiotic susceptibility testing was performed using Kirby Bauer disk diffusion. Carbapenem-, quinolone- and cephalosporin-resistance genes were detected by PCR and resistance-conferring mutations were detected using Sanger sequencing. Ten stool samples were exposed to two sub-clinical concentrations of amoxicillin, ciprofloxacin and colistin for 48 hours, whereafter they were plated onto MCC with the addition of various antibiotic discs (amoxicillin, ertapenem, ciprofloxacin, colistin, cefotaxime and nalidixic acid). The impact of antibiotic exposure on the development of resistance was assessed by enumeration of presumptive resistant E. coli and Klebsiella colonies within the zones of inhibition around the antibiotic discs. Results Twenty-one (42%) of the participants were colonized by quinolone-resistant isolates and 18 (36%) by cephalosporin-resistant isolates (predicted ESBL-producing organisms). Of the 21 quinolone-resistant E. coli isolates, 5 (24%) harbored qnrS while of the 6 quinolone-resistant Klebsiella isolates, 4 (67%) had qnrB. The most common quinolone resistance mutations were S83L in gyrA and S80I, A141V and S129A in parC. blaCTX-M was the only ESBL gene detected. All of the blaSHV and blaTEM genes detected were β-lactamases without extended spectrum activity. One of the participants was colonized by a carbapenem resistant Klebsiella isolate, which carried the blaNDM carbapenemase gene. Exposure of the stool samples to ciprofloxacin selected for resistant bacteria, however exposure to amoxicillin and colistin did not. Conclusion Children in Cape Town are frequently colonized by resistant bacteria and are at risk of becoming infected by these resistant organisms. The presence of plasmid-mediated resistance genes is concerning because they can be transferred between bacteria of the same and different species. There is also a need to further investigate what might be driving the high prevalence of quinolone resistance in the community. This study is the first to report the carriage of carbapenemase resistant bacteria in healthy children in South Africa. Although the in-vitro antibiotic exposure model was crude, the approach provides some evidence for the development of resistance during exposure to sub-clinical concentrations of antibiotics (especially ciprofloxacin); and notably, to agents other than those to which the sample had been exposed. This highlights the need for further investigations into the impact of sublethal antibiotic concentrations on the selection of resistance.
The carriage of antimicrobial resistance genes in children from Cape Town communities.
(Stellenbosch : Stellenbosch University, 2022-12) Venter, Amanda-Jo; Newton-Foot, Mae; Whitelaw, Andrew; Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology.
ENGLISH ABSTRACT: Introduction: The human microbiota is a rich source of microbial diversity and an accessible reservoir of antibiotic resistance genes (ARGs). The collection of ARGs present in the microbiota has been termed the resistome. Antibiotic use is a major driver of antibiotic resistance (ABR) and selection of ARGs. There is limited surveillance data on ABR in undercharacterized populations, particularly in children, in the community and in developing countries. A collection of stool specimens collected from healthy children as part of the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial, which evaluates the efficacy of levofloxacin as prophylaxis in multidrug-resistant tuberculosis exposed children, provides an opportunity to describe the carriage of ARGs in a population of healthy children in the community. Methods: Fifty baseline stool samples from children in Cape Town communities that are part of the TB CHAMP trail, collected from November 2017 to July 2018, were used in this substudy for resistome analysis. Both targeted and functional metagenomics (FM) approaches were used to investigate the resistome. The blaCTX-M-1, -9 and -B (2, -8 and -25) genes were detected with real-time PCR in fifty stool samples. Conventional PCR was used to identify blaSHV and blaTEM genes. A FM approach was developed to detect ARGs from stool samples. DNA was extracted from five stool samples, fragmented and end repaired. Fragmented DNA between 1500 - 8000 bp was selected and ligated into the Eco53KI site in plasmid pHSG298, a plasmid vector with kanamycin as selectable marker. The ligation mix was transformed into chemically competent DH5α Escherichia coli cells, blue-white colony screening was performed, and all white colonies were stored, creating a metagenomic library from each stool sample. The FM libraries were plated on selected antibiotics and Sanger sequencing was used to identify the inserts from resistant colonies. Results: blaCTX-M-1 (72%) was present in more samples than blaCTX-M-9 (8%). SHV and TEM were present in 64% and 100% of the samples, respectively. The FM libraries created ranged from 1.7 x 106 to 2.5 x 107 bp in size. Resistant colonies were detected on media containing ampicillin, amikacin and cefotaxime. Sequencing results identified known ARGs, including an aminoglycoside adenylyl transferase gene selected on the amikacin-containing agar plates and a MATE family efflux pump from the ampicillin-containing agar plates. Various resistance associated and potential resistance proteins were also detected. Conclusion: blaCTX-M-1 was the commonest ESBL, in line with global trends. When compared to a culture-based study on bacterial isolates from the same samples; beta-lactamase genes were detected more commonly using targeted PCR than by culture The high prevalence of ESBL genes in samples from the community is concerning and supports the concept of microbiota acting as a reservoir of resistance genes. We have also developed a FM approach to detect ARGs from stool samples, which is able to identify known and potentially novel resistance mechanisms. This study has contributed to our knowledge of ARGs in under characterized populations in Cape Town.
The carriage of antimicrobial resistance in community children – a TB-CHAMP sub-study.
(Stellenbosch : Stellenbosch University., 2019-12) Brand, Chante; Whitelaw, Andrew Christopher; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Microbiology.
Introduction: The world-wide rise in antimicrobial resistance (AMR) is threatening the effectivity of antibiotics and the control of infectious diseases. The challenge of AMR is considerably exacerbated by the presence of mobile genetic elements harbouring various antibiotic resistance genes, which can easily spread to other species by horizontal gene transfer. This poses serious risks for clinical infections, however, reports on the carriage of these plasmid-mediated resistance genes are still rare in South Africa. This study aimed to describe the rates of carriage and mechanisms of antimicrobial resistance at baseline, as well as the effect of levofloxacin exposure on these rates in children in communities in Cape Town. Methods: Stool samples were collected from 100 children enrolled in the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP) at baseline and at 16- and 24-week follow up visits between November 2017 and November 2019. The stool samples were cultured onto MacConkey agar plates with cefpodoxime and ertapenem disks and in some cases, nalidixic acid and ciprofloxacin disks, in order to select for cephalosporin, carbapenem and quinolone resistant and susceptible E. coli and Klebsiella isolates. Kirby Bauer disk diffusion was used to determine the susceptibility profiles of the organisms and PCR and Sanger sequencing were used for subsequent detection of cephalosporin and quinolone resistance mechanisms. DNA was extracted directly from the stools and targeted molecular detection and quantification of plasmidmediated quinolone resistance (PMQR) genes using real-time PCR. Results: High levels of antibiotic resistance were detected at baseline, with 81% of participants carrying an organism resistant to at least one antibiotic and 49% and 33% carrying quinolone and cephalosporin resistant organisms respectively. These rates increased over time, with significant increases in quinolone resistance after 16 weeks (69.8%). The presence of the extended spectrum -lactamase gene, blaCTX-M, in cephalosporin resistant E. coli and Klebsiella spp. remained relatively constant over time, ranging between 19.7 – 26.8% and 33.3 – 37.5% respectively over 24 weeks. However, this gene was observed in higher proportions in Klebsiella spp. compared to E. coli. We saw high rates of carriage of qnrB (53.3%) and aac(6’)-Ib-cr (66.7%) in Klebsiella spp. at baseline and significant increases in qnrS and aac(6’)-Ib-cr, as well as mutations in gyrA and parC after 16 and, in some cases, 24 weeks. The presence of aac(6’)-Ib-cr also increased significantly in E. coli from baseline (3.8%) to 16 weeks (21.3%). qnrS was detected in 86% of stools in the targeted molecular analysis, while qnrB was only detected in 14%, although it was more abundant than qnrS in the stool samples. Conclusions: We report high rates of resistance to various antibiotics, as well as the presence of -lactamase and PMQR genes, in commensal gut bacteria in children in Cape Town communities before and over 24 weeks of levofloxacin treatment. The high rate of quinolone resistance at baseline is especially worrying as roughly half of these children started levofloxacin treatment after the baseline stool samples were collected. The increase in rates of resistance and presence of PMQR genes is interesting, however, the TB-CHAMP trial is still ongoing and we do not yet know which participants are receiving levofloxacin or placebo. Once the TB-CHAMP study has been completed and the blind has been broken, the results can be stratified according to treatment group to determine the impact of levofloxacin on resistance carriage.
The characterisation and expression of HIV-1 subtype C gag
(Stellenbosch : Stellenbosch University, 2002) Sampson, Candice Corene; Van Rensburg, E.J.; Engelbrecht, S.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.
ENGLISH ABSTRACT: The gag gene of HIV-1 encodes for one of the major structural proteins, which contains several conserved cytotoxic T cell (CTL) epitopes. Gag specific CTL responses are important in controlling viral load during acute infection and asymptomatic stages of the infection. Currently, only one complete South African HIV-1 subtype C gag sequence has been published. The first aim of this study was to characterise the complete gag gene of 15 HIV-1 subtype C isolates, to be used as a set of reference sequences in the design of a South African HIV-1 subtype C vaccine. Fifteen HIV-1 subtype C isolates selected for this study, were isolated during 1998 and 1999 from the HIV-1 positive patients attending the Infectious Disease Clinic at Tygerberg Hospital. The gag gene of these isolates was amplified by PCR, cloned into mammalian expression vectors and sequenced. Restriction digest analyses as well as phylogenetic analyses were performed on the sequencing data. Previously published mutational analyses and CTL epitopes were compared to the predicted amino acid sequences of the gag clones. Sequences of 23 complete gag genes representing the 15 HIV-1 subtype C isolates as well as one complete sequence of an HIV-1 subtype B isolate were compiled. Subtyping by restriction fragment length polymorphism (RFLP) would have correctly identified 14 of the 15 subtype C isolates as subtype C and one as unidentifiable. The subtype B isolate would have also been correctly identified. Phylogenetic analyses showed that our subtype C isolates clustered with reference subtype C strains from various countries, including Botswana, India, Israel, Tanzania and Zambia. Strains from Ethiopia and Brazil formed a separate subtype C cluster. The diversity between our isolates was comparable to the diversity seen between all the HIV-1 subtype C strains. Comparisons of previously published mutational analyses and CTL epitopes to the predicted amino acid sequences of the gag clones, showed conservation in most of the clones throughout the sequence. A second aim was to establish transfection and Western Blot techniques in our laboratory for use in future studies. An in vitro transcription! translation assay was performed on the gag clones and the protein producing clones were used to transfect mammalian cells using electroporation. A Western blot was then used to screen for Gag protein expression in the transfected cell Iysates. The in vitro transcription! translation assay showed that seven of the 23 clones could produce a protein of -55 kDa in size. Four out of the seven of these clones gave a weak expression of a-55 kDa protein after transfection in a mammalian cell line. Since the completion of the experimental work of this study, other cloned HIV-1 genes have successfully been transfected into mammalian cells using the electroporation technique and the proteins produced were screened for by Western blot. To conclude with; the native form of the gag gene does not elicit strong expression of the protein, but studies have shown that expression can be improved by sequence-modification of the gag nucleotide sequence. Due to the conservation of gag, the sequence of any subtype C strain can be used for the development of a Southern African vaccine.
The Characterisation of Antibiotic Resistance Plasmids in Escherichia coli and Klebsiella pneumoniae Isolates from Hospital and Community Settings
(Stellenbosch : Stellenbosch University, 2021-03) Stein, Lisa; Newton-Foot, Mae; Whitelaw, Andrew; Pienaar, Colette; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Antimicrobial resistance has become one of the biggest challenges and threats to public health systems worldwide. Widespread distribution of resistance is commonly due to horizontal gene transfer, which includes mobile genetic elements (MGE) such as plasmids, insertion sequences, transposons, and integrons. This study aimed to characterise plasmids conferring antibiotic resistance in extended-spectrum β-lactamase (ESBL) positive Escherichia coli and Klebsiella pneumoniae isolates from bloodstream infections to determine whether ESBL and plasmid-mediated quinolone resistance (PMQR) genes were mobilised on the same plasmids and whether the same plasmids are disseminated in healthcare and community settings. Methods Illumina MiSeq whole-genome sequencing (WGS) was previously performed on 112 E. coli and 66 K. pneumoniae isolates from blood cultures submitted to the National Health Laboratory Service Microbiology Laboratory at Tygerberg Hospital during 2017 and 2018. Assembled genomes were interrogated for the presence of ESBL and PMQR genes and plasmid replicon types. Based on the results, eight E. coli and nine K. pneumoniae isolates were selected for plasmid sequencing on the Oxford Nanopore Technologies MinION platform. Unicycler assembler was used for hybrid assembly of Illumina short-reads and Nanopore plasmid long-reads. In silico analyses were performed using ResFinder, PlasmidFinder and ISsaga to identify ESBL and PMQR genes, plasmid replicon types, and MGEs. Results Based on Illumina WGS, the ESBL and PMQR containing isolates contained multiple resistance genes and IncF plasmid replicons, individually or in combination with additional plasmid types. The IncF replicons and resistance genes were on separate contigs, therefore associations between different IncF replicons and with resistance genes could not be confirmed. Nanopore sequencing resolved plasmids from several E. coli and K. pneumoniae isolates; however, chromosomal genes could not be visualised and misassembly resulted in fragmented plasmids. Hybrid assembly fully resolved plasmids and chromosomal genes in several E. coli and K. pneumoniae isolates. Amongst the E. coli isolates, three F-type multireplicon plasmids and two single replicon plasmids IncI1-γ and IncB/O/K/Z, which contained resistance genes, were described. Novel multireplicon plasmid FII(FIC)-FIB-X was detected and harboured ESBL blaTEM-135 and PMQR qnrS1. The blaCTX-M genes were confirmed to be chromosomally located in three E. coli isolates and plasmid-mediated on F-type plasmids in two E. coli isolates. K. pneumoniae isolates harboured single replicon F-type plasmids, multireplicon FIB-HI1B fusion plasmids, and a single replicon IncC plasmid. The FIB-HI1B plasmids were associated with blaCTX-M-15, aac(6’)-Ib-cr, and qnrS1. The blaCTX-M-15 was plasmid-mediated in all K. pneumoniae isolates. Conclusion Amongst the E. coli isolates, ESBL and PMQR genes were present both on the same plasmid and on separate plasmids. In K. pneumoniae, ESBLs and PMQRs were found collectively on the same plasmids, and the F-type plasmids harbouring ESBL and PMQR genes differed from those in E. coli. As only two community-acquired K. pneumoniae isolates were selected for Nanopore plasmid sequencing, conclusions regarding the dissemination of K. pneumoniae plasmids in healthcare and community settings could not be made. Plasmids of the same FAB-types were detected amongst E. coli isolates of various sequence types and from both hospital- and community-settings, which is indicative of spread between these settings.
Characterisation of follicular helper T (Tfh) cells in early treated HIV-infected children: relationship to immune activation and inflammation.
(Stellenbosch : Stellenbosch University, 2022-11-24) Olifant, Paulina; Glashoff, Richard Helmuth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.
ENGLISH ABSTRACT: Background: South Africa has a high burden of Human Immunodefiency Virus (HIV) infection. Babies of HIV-positive pregnant women can become HIV-infected or exposed through vertical transmission. Follicular helper T (Tfh) cells have been of particular interest in HIV infection due to their preferential expansion, contribution to the HIV reservoir and dysregulation within HIV-infected individuals. The aim of this study was to investigate the Tfh cell population within children from the Children with HIV Early Antiretroviral Therapy (CHER) trial who started treatment within the first six months of life to determine whether the numbers of these cells is altered as compared to uninfected children and also whether persistent immune activation and inflammation in these children is associated with Tfh cell dysregulation. Methodology: This retrospective cross-sectional observational study consisted of three groups, i.e., early antiretroviral-treated HIT (HIV Infected Treated), HEU (HIV Exposed Uninfected) and HUU (HIV Uninfected Unexposed), of children. Cryopreserved peripheral mononuclear blood cells (PBMCs) were stained with an 11-colour antibody panel designed and optimised for phenotypic identification and quantification of T cell populations using flow cytometry. Tfh cells populations were characterised as CD4+CXCR5+PD-1+ with/without ICOS+. CD4, CD8 and Treg cells were defined as follicular/ follicular-homing based on CXCR5+ expression and activated based on CD38+ and/or CD69+ expression. Secondary data of clinical parameters and inflammatory cytokines for each group were evaluated. Statistical comparisons between groups were made using the Mann-Whitney test to identify significant differences. Significant correlations between Tfh cells and clinical parameters, other T cell populations and inflammatory cytokines were identified using Spearman’s rank order test. Results: Phenotypic results generally indicated significantly increased proportions of CD38+ subsets in HIT group and CD69+ subsets in HEU group. Although there was no significant difference in median CD4+CXCR5+PD-1+ Tfh cell percentage between groups, the ICOS-expressing subset namely CD4+CXCR5+PD-1+ICOS+ Tfh cells was significantly higher in the HIT (33.6% vs 19.2%; p = 0.016) and HEU group (31.6% vs 19.2%; p = 0.006) compared to the HUU group. In the HIT group, CD4+CXCR5+PD-1+ Tfh cells shared significant negative correlations with a majority of activated T cell subsets. A significant positive correlation between CD4+CXCR5+PD-1+ Tfh and CD8+PD-1+ Tc cells, general indicator of immune exhaustion, was demonstrated. Lastly, the HIT group showed the highest level of INF-α and hsCRP inflammatory cytokines and levels of IL-1β and hsCRP significantly correlated with CD4+CXCR5+PD-1+ICOS+ Tfh cells within this group. Conclusions: Overall levels of immune activation were significantly higher in HIT and HEU groups. Several activated T cell subsets inversely correlated with CD4+CXCR5+PD-1+ Tfh cells, suggesting high levels of immune activation can lead to decreased proportions of circulating Tfh cells. Even though no significant difference in the proportion of CD4+CXCR5+PD-1+ Tfh cells was found between groups, the ICOS+ subset was significantly expanded in HIT and HEU children in comparison to HUU children. The significant positive correlation between IL-1β and ICOS-expressing Tfh cells, within the entire study population and HIT group, suggested that increased inflammation resulted in an Tfh cell increase.
Characterisation of fosfomycin resistance in urinary pathogens from the Western Cape, South Africa.
(Stellenbosch : Stellenbosch University, 2021-03) Mosime, Lesedi Bridget; Nel, Pieter; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Introduction: Urinary tract infections (UTI) are the most commonly acquired bacterial infections worldwide. The South African Department of Health advised that fosfomycin, nitrofurantoin and gentamicin be used for the treatment of uncomplicated UTI due to other antibiotics showing adverse side effects. Fosfomycin has effectively been utilised in the management of UTI, however resistance has been detected in urinary pathogens at the Tygerberg Hospital National Health Laboratory Service (NHLS) Medical Microbiology diagnostic laboratory. This study aimed to determine the prevalence of fosfomycin resistance among community-acquired urinary pathogens in the Western Cape and to characterise fosfomycin resistance mechanisms in fosfomycin resistant Escherichia coli and Klebsiella pneumoniae isolates. Methods and Materials: Two-hundred urinary isolates (Enterobacterales and Enterococcus spp.) from antenatal clinics in the Western Cape were collected from the Tygerberg Hospital NHLS Medical Microbiology laboratory during 2019 and 2020 and used to determine the prevalence of fosfomycin resistance. Fosfomycin susceptibility was determined using disc diffusion and Etest®. Fosfomycin resistant E. coli and K. pneumoniae isolates from the prevalence study and another set of fosfomycin resistant isolates (5 E. coli and 19 K. pneumoniae) collected from urine samples submitted to the NHLS at Tygerberg Hospital in 2017 (Ethics #: U17/05/026) were used to characterise fosfomycin mechanisms. FosA mediated resistance was determined using a phenotypic assay and fosA genes were detected by PCR. Mutations in the fosfomycin target gene murA and transporter genes, glpT and uhpT, were characterised by polymerase chain reaction (PCR) and Sanger sequencing. Results: Fosfomycin resistance was detected in 3.5% of community-acquired urinary pathogens. Fosfomycin resistance rates were 2.2% in E. coli (3/139) and 12.9% in other Enterobacterales. All Enterococcus spp. isolates were susceptible to fosfomycin. In the combined sample set of 31 fosfomycin resistant isolates, the phenotypic assay detected FosA in only 7 isolates, while fosA genes were detected by PCR in 25. Chromosomal mutations were identified in 6 isolates, of which three isolates (1 K. pneumoniae and 2 E. coli) had deletions in the uhpT gene, which has previously been reported to confer fosfomycin resistance. The role of other mutations found in the glpT gene of E. coli and the murA and glpT of K. pneumoniae isolates has not been determined. Conclusion: The fosfomycin resistance rate in community-acquired UTI was low, which supports the careful ongoing use of fosfomycin for the treatment of uncomplicated community-acquired UTI. FosA mediated resistance was the most common mechanism of fosfomycin resistance identified in this population.

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