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Introduction: Differentiating follicular adenoma (FA), follicular thyroid carcinoma (FTC) and follicular variant of papillary thyroid carcinoma (FVPTC) remain a diagnostic challenge in some cases. This is as a result of the assessment of nuclear features being highly subjective, and the threshold for confirming cytomorphological features for papillary thyroid carcinoma varying greatly among pathologists which results in poor interobserver agreement. Diagnostic challenges may be encountered with some encapsulated follicular-patterned neoplasms of the thyroid due to uncertainty about the presence of capsular or vascular invasion. Immunohistochemistry may provide a better alternative to distinguishing these entities. Aims and objectives: To study the expression of biomarkers HBME-1, CD15, CK-19 and BRAF V600E in follicular adenoma, follicular carcinoma and follicular variant of papillary thyroid carcinoma. To ascertain the usefulness of these markers in differentiating these follicular-patterned thyroid neoplasms. Materials and methods: This is a ten-year retrospective study in which seventy-nine cases consisting of follicular adenoma (n=26), follicular thyroid carcinoma (n=25) and follicular variant of papillary thyroid carcinoma (n=28) were retrieved and reviewed. Four immunohistochemical stains (CD15, CK-19, HBME-1 and BRAF V600E) were performed and scored in tumour tissue. Data were analysed to determine if there was any correlation between the expression of the immunomarkers and the three follicular patterned thyroid neoplasms. Results: The patients' ages ranged from 13 to 74 years. There was a female bias with a female-to-male ratio of 4:1. HBME-1 expression showing varying intensity and proportion was seen in 7 (28%) FA, 15 (65%) FTC and 22 (79%) FVPTC. CK-19 expression showing varying intensity and proportion was seen in 5 (19%) FA, 3 (13%) FTC and 18 (72%) FVPTC. BRAF V600E expression showing varying intensities and proportions was seen in 3 (11%) of FVPTC. FA and FTC cases were all negative for BRAF V600E. CD15 expression showing varying intensity and proportion was seen in 2 (8%) FA, 6 (24%) FTC and 9 (33%) FVPTC. Statistical analysis detected a significant association between group and HBME-1 and CK-19 (H-score, proportion and intensity). The post-hoc comparison revealed that follicular adenoma was significantly more likely to have negative HBME-1 staining and a lower H-score when compared to follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. The follicular variant of papillary thyroid carcinoma was significantly more likely to have an HBME-1 intensity of three and a higher H-score compared to follicular adenoma. Post-hoc comparison revealed that follicular adenoma was significantly more likely to have a negative CK-19 and a lower H-score when compared to follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. Follicular variant of papillary thyroid carcinoma was significantly more likely to have a CK-19 intensity of three and a higher H-score compared to follicular thyroid carcinoma. HBME-1 had an overall specificity and sensitivity of 72% and 74% for distinguishing FA from FTC and FVPTC (benign from malignant). CK-19 had an overall specificity and sensitivity of 80.8% and 43.8% for distinguishing FA from FTC and FVPTC (benign from malignant). CK-19 had specificity of 87% and sensitivity of 72% for distinguishing FTC from FVPTC. Combining HBME-1 and CK-19 did not significantly increase the sensitivity and specificity of these markers. There was statistically no significant association between group and BRAF V600E and CD15 biomarkers. Conclusion: The study, within the small sample size power limitations, has shown that CK-19 may have a role in distinguishing follicular thyroid carcinoma from follicular variant of papillary thyroid carcinoma. In addition, HBME-1 and CK-19 may be used in differentiating benign follicular-patterned thyroid lesions from malignant follicular patterned thyroid lesions.

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation and inactivation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. A total of 195 samples were tested as part of the validation. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

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Introduction: South Africa has the highest global burden of human immunodeficiency virus (HIV). The HIV seropositive population is at increased risk of developing non-Hodgkin lymphoma, particularly high grade aggressive subtypes such as Burkitt- and Burkittlike lymphoma (also known as B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma). Methods: Ten year retrospective review of clinico-pathological features and survival of adults with newly diagnosed Burkitt- and Burkitt-like lymphoma at a tertiary hospital in South Africa. Results: Burkitt lymphoma (BL) (n=109) was more frequent than Burkitt-like lymphoma (BLL) (n=41) and at presentation there were no significant differences in HIV prevalence (86% vs 78%); median CD4 count (213 vs 207 cells/μL); bone marrow involvement (49% vs 34%), leukaemic dissemination (37% vs 27%) and most frequent site of diagnosis (abdomen/pelvis; 26% vs 29%), respectively. There were significant differences in median age (34 vs 41 years, p=0.0319), median lactate dehydrogenase levels (2052 vs 869 U/l, p=0.0011) and cerebrospinal fluid involvement (12% vs 0%, p=0.046). 43% of patients with an available HIV viral load result showed virological supression defined as lower than detectable limit (LDL) with the median value also being LDL. The patients that received high dose chemotherapy including high dose methotrexate (112/150; 75%) showed a one-year survival of 62% with no significant difference between Burkitt and Burkitt-like lymphoma (66 months versus 51 months, respectively; p=0.267). Patients with leukaemic presentation showed a significantly lower mean survival of 24 months compared to those without (72 months; p< 0.001). The 2 year survival for the whole group, regardless of type of treatment received, was 40% (95% CI, 32-48%) with a median survival of 7.5 months. Conclusion: This is the largest cohort of Burkitt- and Burkitt-like lymphoma patients described in South Africa. There was a high HIV prevalence. The majority received intensive chemotherapy and survival was comparable to certain well-resourced countries. Leukaemic presentation was frequent and associated with less favourable survival.

TCV isolate, TCV-M, supports a family of satellite (sat-) RNAs. The virulent sat-RNA C intensifies TCV symptoms on turnip cultivar Just Right. In this thesis, I report that sat-RNA C (356 b) exacerbates symptoms on all hosts where TCV produces visible symptoms including cultivars of Brassica rapa and Arabidopsis thaliana. This finding has led to studies of TCV-resistance using A. thaliana, a small plant with a well characterized genome, as a host. After screening over 6,000 M2 A. thaliana plants from EMS treated Columbia cultivar seeds and 22 ecotypes of A. thaliana, ecotype Dijon was found to be resistant to TCV. A second isolate, TCV-B, supports an RNA species with a size similar to that of sat-RNA C. Northern hybridization and cDNA cloning and sequencing demonstrate that this RNA is actually a defective interfering (DI) RNA, denoted DI RNA G. Infection of turnip with virus derived from cloned transcripts of TCV-B resulted in de novo generation of a DI RNA, DI1 RNA. Unlike DI RNAs associated with other plant viruses (or animal viruses), TCV DI RNAs intensify TCV symptoms. To understand sequences required for DI RNA infectivity, a series of mutation have been generated in a full length cDNA copy of DI RNA G. Stepwise deletions at base 98 of DI RNA G, at which an Apa I linker had been inserted, has shown that DI RNA GA (DI RNA G with a 8-base insertion at base 98) harboring deletions of bases 74-98 or less are infectious. However, deletion of bases 73-98 or more abolishes RNA infectivity. DI RNA GA with a deletion of bases 107-124 is infectious. However, DI RNA GA harboring a deletion of bases 107-138 is not infectious. I have found that infectivity of RNA harboring 31- or 32-base deletions can be restored by inserting foreign sequences into deletion sites. This implies that at least 71 bases (including the 8-base insertion) in DI RNA GA near the 5$\sp\prime$ end are not specifically required for RNA infectivity. By combining deletions of infectious clones to generate larger deletions (40 or 43 bases deleted), I demonstrate that the infectivity of the RNA is abolished. This result suggests that size of the DI RNA is important in maintaining RNA infectivity.

Captive black rhinoceroses in the United States are threatened by three major clinical disorders. Among these, haemolytic anaemia ranks as the number one cause of death. The object of this study therefore was to determine the metabolic lesion responsible for the haemolytic anaemia. Since black rhinoceros red blood cells were shown to be susceptible to oxidative stress, the first stage of the work focused on the characterisation of the hexose monophosphate shunt in these cells. Results were compared to human red blood cells. To determine how these cells responded to oxidative stress and to stimulation of this pathway, rhinoceros red blood cells were incubated with the known shunt stimulants ascorbate and methylene blue. Red blood cells were incubated with 14C-Iabelled glucose and then acid extracted. The rate of flux through the shunt was measured by counting 14C trapped on a filter placed inside the incubation vessel and saturated with NaOH. Red cell extracts were used for lactate and reduced glutathione determinations. These experiments showed that black rhinoceros red blood cells were capable of increasing flux through the shunt in response to oxidative stress although the magnitude of this response was significantly lower than that observed in human red blood cells. To determine whether the cells were capable of recycling metabolites through the shunt in response to prolonged oxidative stress, the red cells were incubated with 2-14C-Iabelled glucose. Recycling through the shunt was observed to function efficiently in these cells. Since no particular metabolic lesion could be defined with respect to the functioning of the lIMP, we shifted our attention to an unusual peak with cytidine-like absorbance properties which dominated the rhinoceros HPLC profile. A range of cytidine nucleotides and other nucleotides were analysed by reverse phase HPLC in an attempt to match the elution position of the unknown peak. This search yielded a surprising candidate, the amino acid tyrosine. Co-elution of this peak with standard tyrosine strengthened this identification. Diode array analysis of the HPLC peak yielded an identical wavelength maximum to standard tyrosine. Amino acid analysis of rhinoceros red blood cell extracts showed that rhinoceros red blood cells did indeed have elevated levels of tyrosine relative to human red blood cells. These levels were in the range of 20 to 50 times that measured in human red blood cells. The peak was then fractionated, concentrated by freeze drying and analysed by mass spectroscopy, which showed that it had the molecular weight expected for the amino acid tyrosine. Red blood cells of wild black rhinoceroses were found to contain 0.78 ± O.llmM (n=8) tyrosine. The finding of such a high concentration of a free amino acid in red blood cells appears to be unprecedented. In an attempt to elucidate the function of tyrosine in rhinoceros red blood cells we drew on the analogy of taurine which is present at very high concentrations in heart epithelial cells and neutrophils. Taurine protects these cells against the respiratory burst oxidants during periods of acute inflammation. Exposure of rhinoceros red blood cells to hydrogen peroxide resulted in the accumulation of dityrosine, a highly fluorescent tyrosine dimer. The formation of dityrosine has previously been shown to occur between protein tyrosyl residues. In our system we describe the crosslinking of free tyrosine in response to hydrogen peroxide in rhinoceros red blood cells. The formation of dityrosine was followed by fluorimetry. Human red blood cells showed no significant production of dityrosine under the same conditions. The accumulation of dityrosine in rhinoceros red blood cells was found to be reciprocally related to GSH concentration. A series of cell-free experiments showed that dityrosine only accumulated in the absence of sufficient GSH and that its production was catalysed by haemoglobin. This study therefore describes the identification of tyrosine present in rhinoceros red blood cells at levels approaching 1 mM. Haemoglobin catalyses the production of dityrosine in response to hydrogen peroxide in a manner that is inversely proportional to the GSH concentration. It is our hypothesis that this tyrosine response to hydrogen peroxide may form a backup antioxidant mechanism for the glutathione peroxidase / reductase system in these cells which are relatively deficient in catalase, a major enzyme for the protection from hydrogen peroxide in other mammals. Analysis of red blood cell extracts of 30 captive black rhinoceroses was found to have significantly lower tyrosine levels (0.37 ± 0.14mM) than the wild rhinoceroses (0.78 ± O.llmM) analysed. These low levels of tyrosine combined with an iron overload syndrome recently described by D. Paglia at UCLA, suggests that black rhinoceroses in captivity may face a higher level of oxidative challenge. The unusual mechanisms in the rhinoceros red cell described here for handling oxidative stress, in which catalase has been replaced by reliance on the glutathione peroxidase system together with a tyrosine buffer, seem to be inadequate under the environmental and dietary circumstances of the captive state. These findings however may give new insight into therapeutic or preventative measures based on dietary modification which will address iron absorption and antioxidants.

Background Tuberculosis (TB), especially extrapulmonary TB, is a difficult diagnosis to make in children due to the paucibacillary nature of paediatric disease and difficulty in obtaining sputum and tissue samples for microbiology confirmation. Lymphadenopathy in children with suspected cervicofacial TB are amenable to FNA or surgery for further cytological and histological assessment. It is therefore important to understand how well the morphologic features (from cytology and histology) correlate with defined reference standards (TB culture and molecular evidence of MTb) for the diagnosis of TB and the reliability of these features. Aim The aim of the study is to determine how well the cytology and histology-made TB diagnoses in children with suspected cervicofacial EPTB correlates with TB culture and MTb PCR results. Materials and methods This is a descriptive retrospective study that involved a re-appraisal of all patients with suspected cervicofacial EPTB who had histology and cytology performed at Red Cross Children's Hospital identified from the National Health Laboratory Service (NHLS) Trakcare system over a 5 year period (2012-2017). Following identification of histopathology accession numbers, histopathology reports and slides were retrieved from the archive of the Division of Anatomical Pathology/ National Health Laboratory Service, Red Cross Children's Hospital, Cape Town for evaluation. In addition, results for Genexpert testing and TB culture were identified using the National Health Laboratory Service (NHLS) Trakcare system. In patients that did not have either of the above, MTb PCR testing was performed. Results Data from the reports of 76 children with suspected cervicofacial TB were included in this study. More biopsies were submitted for histology (48) than for cytology (22). Six children had biopsies for both cytology and histology done. Most children had suspected and confirmed TB involvement of the cervical lymph nodes. On histology, the feature that correlated the best with proven TB was necrotising granulomatous inflammation (79.5% of cases had confirmed TB). On cytology, necrotising inflammation, necrotising granulomatous and non-necrotising granulomatous inflammation correlated well with proven TB. The sensitivity of cytology was 77.3% against TB culture and 81.8% against GXP for TB diagnosis. Whilst for histology the sensitivity was 82.5% against TB culture and 90.3% against GXP as reference standards for TB diagnosis. Conclusion FNA for cytology is a safer procedure with less complications than biopsy for histology. Also, the use of cytology together with a GXP renders a rapid and accurate diagnosis of TB and our findings are supportive for the combined use of these modalities as first line investigations. However, every attempt should still be made to obtain a sample for TB culture (as the WHO recommended gold standard for TB confirmation).

Helminth infections induce systemic changes to host immunity and can impact unrelated infections, even those occurring at anatomical sites not normally colonised by the helminths. A few studies have shown that helminths can increase the risk of infection and pathology resulting from sexually transmitted viral infections in the female reproductive tract, however the evidence is limited and the scope of helminth infection on immunity and infection in the female reproductive tract has not been fully elucidated. In this thesis the impact of hookworm infection on immunity in the female reproductive tract and risk of Human Papillomavirus infection in humans was investigated. The influence of helminth infection on B and T cell responses in the female reproductive tract and how this impacts vaccine mediated responses to another viral infection of the female reproductive tract, Herpes Simplex Virus, Type 2 was also assessed in a mouse model. To determine the risk of Human Papillomavirus among hookworm infected participants, we compared the prevalence of Human Papillomavirus infection among hookworm infected and uninfected women. Hookworm infected women were two times more likely to be Human Papillomavirus positive than women with no hookworm infection. Furthermore, hookworm infection was positively associated with the intensity of Human Papillomavirus infection. To determine whether hookworm infection induced changes in vaginal immunity we employed multiplex assays to measure chemokine, cytokine and antibody levels in the vaginal flushes of our study participants. Hookworm infected women displayed an elevated mixed Type 1 (TNF-a, IL-2 and IL-12) and Type 2 (IL-4, IL-5, IL-13, eotaxin and elevated IgG4/ IgE ratio) immune response in the female reproductive tract in xvi comparison to uninfected women. Type 2 immunity was pronounced in hookworm and Human Papillomavirus co-infected women who maintained an elevated Type 2 signature (IL-4, IL-5, IL-13, eotaxin and elevated IgG4/ IgE ratio) and an increased Th2/Th1 ratio in comparison to uninfected women. We then investigated the impact of primary helminth infection on B and T cell immunity in the female reproductive tract using the mouse model of hookworm infection, Nippostrongylus brasiliensis. Nippostrongylus brasiliensis infection of wild type BALB/c mice resulted in increased B cells, IgG1+ B cells and IgG1+ follicular B cells as well as increased effector memory T cells and T follicular helper cells in iliac lymph nodes, which drain the female reproductive tract. We then infected wild type BALB/c mice with Nippostrongylus brasiliensis and immunised them with formalin inactivated Herpes Simplex Virus, Type 2 then challenged them intravaginally with lethal dose Herpes Simplex Virus, Type 2. Nippostrongylus brasiliensis infection did not significantly impact B cell responses to vaccination and subsequent challenge though there was a trend towards lower B cell responses in mice that received Nippostrongylus brasiliensis treatment prior to vaccination. Mice that had prior Nippostrongylus brasiliensis infection, however, had significantly lower effector memory CD4+ T cells than mice that did not have helminth infection before vaccination. In summary, this thesis demonstrates that helminth infection induces Type 2 associated immune changes in the female reproductive tract in humans and alters B and T cell populations in lymph nodes draining the female reproductive tract of mice. Furthermore, in humans, an increased risk of Human Papillomavirus infection and increased intensity of Human Papillomavirus infection was associated with hookworm infection. In mice, a dampening of Herpes Simplex Virus, Type 2 vaccine mediated xvii effector CD4 T cells responses and increased pathology following viral challenge was observed in mice previously infected with Nippostrongylus brasiliensis. The findings in this thesis highlight helminth infection as a significant risk factor for sexually transmitted viral infections and have implications for control of these infections among women living in helminth endemic areas.

No description available.