Background Human parainfluenza 4 (HPIV 4) is a recognised cause of acute respiratory infection (ARI). However, there is no published data on the epidemiology of this virus in South Africa. This thesis describes the molecular epidemiology of HPIV 4 over a 4-year period (2014-2017). Respiratory samples from infants, children and adults presenting with respiratory illness in the Western Cape, South Africa were studied. Method A retrospective 4-year study using routine diagnostic samples from patients with ARI was conducted in Western Cape, South Africa. A database search of positive HPIV 4 samples detected by the Seegene Anyplex RV 16 diagnostic assay was extracted. Epidemiological information was recorded to determine age, gender, hospital ward (used as a proxy for disease severity), specimen type (upper or lower respiratory tract) and collection date (to indicate seasonality). To determine genetic evolution, novel primers targeting the haemagglutinin-neuraminidase (HN) in both HPIV 4 subtypes were designed to amplify a 733 bp and 738 bp sequence for HPIV 4A and HPIV 4B respectively. This product was then sequenced and aligned with known reference sequences from GenBank, using BioEdit. These aligned sequences were analysed using the phylogenetic analysis tool, MEGA 6, and Highlighter plots to determine sequence divergence events and evolution. A real-time PCR assay, targeting the phosphoprotein, was developed to rapidly distinguish subtype A and B viruses. Results HPIVs were the 6th most common respiratory viruses detected in diagnostic samples. In all, there were 312/7456 (4.2 %) HPIV 4 positive samples in patients with a median age of 12 months. Males had a higher infection rate. HPIV 4 was the most prevalent of the HPIVs accounting for 47% of all HPIVs. Respiratory infections due to HPIV 4 were seasonal, peaking in autumn and mid-winter (March to August). The overall prevalence of HPIV 4 increased over the study period. Of the HPIV 4-positive samples that were subtyped, 59 were subtype A and 26 subtype B. Both subtypes co-circulated during each season. 71 % of patients who were positive for HPIV 4 were co-infected with one or more additional respiratory virus with Adenovirus (27 %), Human Rhinovirus (23 %) and Bocavirus (19 %) as the most common. HPIV 1 and HPIV 3 were both able to co-infect patients with HPIV 4, but no co-infections with HPIV 2 were detected. Phylogenetic trees constructed using neighbour joining (NJ) method showed that most of the South African HPIV 4 subtypes did not group with the closest significant reference sequences from GenBank. The phylogenetic tree for HPIV 4A revealed 4 genetic groupings. There were many nucleotide changes increasing with time as well as a non-synonymous change in HPIV 4A, at location N161D. HPIV 4B had an amino acid change in location G198R in the HN protein sequenced. Conclusion HPIV 4 with an overall prevalence of 4 % over the study period was identified as a significant cause of ARI in the Western Cape, South Africa. Mono-infection with HPIV4 was associated with severe disease. In hospitalized infants who were HPIV 4 positive, between ¼ to 1/3 were from patients in ICU. Of these almost half (46 %) had HPIV 4 as a single infection. Further studies are needed to fully understand the molecular epidemiology of this infection.

Persistent genital infection with oncogenic or high-risk human papillomavirus (HPV) is causally associated with cervical cancer in women and some penile cancers in men. The role of the complex genital microbiota in HPV infection has not been extensively addressed. This study characterised the genital microbiotas of heterosexually-active Black South African women and men, predominantly of the Xhosa ethnicity, recruited from a community in Cape Town, South Africa. The association of the genital microbiotas with prevalent HPV, HIV, demographic, behavioural, and clinical characteristics of the participants was examined. In Chapter 2 the bacterial communities in cervicovaginal samples from 62 HIVseronegative South African women were profiled by Ion Torrent PGM sequencing of the V4 hypervariable region of the bacterial 16S rRNA gene (IT-V4 method). The cervicovaginal microbiotas (CVMs) were found to cluster into three distinct community state types (CSTs): Lactobacillus iners-dominated CVMs (CST I (38.7%, 24/62)), unclassified Lactobacillusdominated CVMs (CST II (4.8%, 3/62)), and diverse CVMs (CST III (56.5%, 35/62)) with an array of heterogeneous bacteria, predominantly the bacterial vaginosis (BV)-associated Gardnerella, Prevotella, Sneathia, and Shuttleworthia. The majority of the women had nonLactobacillus-dominated CVMs. Lactobacilli are recognised as protective against sexually transmitted infections. Among the Lactobacillus species detected in the women, L. iners was the most prevalent and abundant. This species is recognised as the least protective amongst the vaginal lactobacilli. Women in CST I were more likely to be on hormonal contraception compared to women in CST III (relative risk (RR): 2.6 [95% CI 1.3-5.3]; p=0.005). Further research is required to confirm this association and to determine the biological mechanism. Microbiome research methodologies are constantly improving and in Chapter 3 the performance of two bacterial 16S rRNA gene amplicon-based methodologies were compared. The CVMs of 19 women were characterised using the IT-V4 method (Chapter 2) and using the Illumina 16S rRNA metagenomics method (IM-V3/V4 method). The latter method involves sequencing the V3 and V4 hypervariable regions of the 16S rRNA gene on the Illumina MiSeq platform. The two methods showed a high degree of correlation (r=0.89, p< 0.0001) in the average relative abundance of shared bacterial taxa. Procrustes analyses of the weighted UniFrac distances further showed a statistically consistent clustering between the two methods (M 2 =0.3, p< 0.0001). The IM-V3/V4 method proved to have a greater throughput, longer read-length, and lower error rates than the IT-V4 method and was therefore used in the subsequent chapters (4 and 5). In Chapter 4, the CVMs of 87 HIV-seronegative women from the same cohort were examined using the IM-V3/V4 method. The CVMs clustered into eight CSTs. Only 23 women (26.4%) had CVMs dominated by a single Lactobacillus species, this included two women (2.3%) with L. crispatus (CST-1), two (2.3%) with L. jensenii (CST-2), and 19 (21.8%) with L. iners (CST-3). The majority of the women (64.4% (56/87)), however, had diverse and heterogeneous CVMs (CST-8) that were associated with BV (p2, p<0.05). In the final experimental chapter, the penile microbiotas of 238 Black South African men were characterised. This is the first large-scale study of the penile microbiota of South African men. Corynebacteriaceae (47.2%) and Prevotellaceae (6.6%) were found to be the most abundant bacterial families. The penile bacterial communities clustered into six CSTs (designated 1-6). A majority of the men (53.4% (127/238)) had Corynebacterium-dominated microbiotas (CST-1). The remaining CSTs (2-6) had lower relative abundances of Corynebacterium than CST-1 and were colonised with several vaginal bacteria. The prevalences of these CSTs (2-6) in men together with their respective most abundant genera (besides Corynebacterium) were as follows: CST-2 (9.2%; unclassified Clostridiales and Porphyromonas), CST-3 (8.8%; Gardnerella), CST-4 (7.6%; Chryseobacterium and Acinetobacter), CST-5 (18.5%; unclassified Clostridiales and Porphyromonas), and CST-6 (2.5%; Lactobacillus). One hundred and thirty (54.6%) and 102 (42.9%) of the men were positive for HPV and high-risk HPV, respectively, as detected by the Roche Linear Array HPV Genotyping assay. Of the 130 HPV-positive men, 37 (28.5%) and 93 (71.5%) had single and multiple HPV types, respectively. Men in CST-1 were less likely to have high-risk HPV and multiple HPV infections relative to men in CSTs 2-6 (RR: high-risk HPV: 0.8 [95% CI 0.6-1.0]; p=0.027 and multiple HPV: 0.8 [95% CI 0.6-1.0]; p=0.042). LefSe revealed that prevalent HPV infection was strongly associated with higher relative abundances of Sneathia, Porphyromonas, Prevotella, Dialister, and Campylobacter (LDA score >3, p3, p< 0.05). The relative abundances of the latter three bacteria together with Peptoniphilus were strongly associated with high-risk HPV infection (LDA score >3, p <0.05). In our cohort, 88 men (37.0%) were positive for HIV. Of these, 71.6% and 60.2% were positive for HPV and highrisk HPV infection, respectively. Among the HIV-negative men (n=150), 44.7% and 32.7% were positive for HPV and high-risk HPV infection, respectively. Although HIV status did not impact the overall composition of the penile microbiotas, HIV-infected men had higher relative abundances of Staphylococcus, Faecalibacterium, Strenotrophominas, Jonquetella, Ruminococcus, Roseburia, and Lamia (LDA score >2, p<0.05) Men with BV-negative female sexual partners (66.5% (157/236)) had higher relative abundances of Lactobacillus in their penile microbiotas than men with BV-positive female partners (p=0.007). Atopobium, Sneathia, and Saccharofermentans were significantly more prevalent in men with BV-positive female partners than men with BV-negative partners (p<0.020). The main limitations of our study include relatively small sample size of women, insufficient participant information such as host genetics, other STIs (e.g., herpes simplex virus) and abnormal vaginal flora (e.g., aerobic vaginitis), using a less sensitive method to diagnose BV in women, and inherent biases evident in any retrospective study. Moreover, we did not adjust for confounding factors in our analysis due to the small sample size. Despite the underscored limitations, our findings provide insight into the baseline genital microbiotas of the Black South African women and men. The associations identified in this cross-sectional study between specific microbiota members and HPV infection, particularly the association between Sneathia and HPV/high-risk HPV infection, identified in both women and men, are hypothesis-generating and warrant further investigation. The study forms a critical starting point for future longitudinal confirmatory association studies and studies examining these bacteria as potential biomarkers or risk factors for HPV infection.

Alzheimer’s disease (AD) is the most common type of dementia, affecting millions of people around the world. It is a devastating disease characterized by memory issues, cognitive impairment, and personality changes. It is incurable and the only available approved medications alleviate symptoms but have not been shown to substantially modify the disease. Scientists in recent years have looked to non-pharmaceutical interventions and aerobic exercise is one intervention that has come to the forefront. There is mechanistic support for aerobic exercise in human and mouse studies demonstrating that aerobic exercise can benefit brain pathology. It can improve brain glucose metabolism, lower the amount of beta amyloid, and increase brain-derived neurotrophic factor (BDNF). Therefore, an exercise intervention may have the potential to delay and prevent AD pathology and symptoms. In our study, we will attempt to demonstrate this in an expansive single-blind trial. The subjects will be 1246 adults, age 60-70 years, cognitively normal, but at a risk for AD. Participants will be randomized 1:1 to either an active control group that wears an activity smart watch and receives coaching regarding a standard stretching routine or the intervention group which has special engaging features on their smart watch and coaching to encourage individualized aerobic exercise routines. The primary outcome will be the neurological test battery composite score, demonstrating global cognitive function in these patients. We hope to clearly demonstrate that exercise interventions can be utilized to help prevent cognitive decline and delay AD.

Introduction: Maternal cardiovascular changes that occur in pregnant women are usually well tolerated by most women experiencing an uncomplicated pregnancy and are reversable postpartum. However, pregnancy can induce adverse cardiac events in previously healthy women without any known cardiovascular disease. Understanding of the maternal cardiovascular adaptation during healthy pregnancy is important to identify deviations from regular patterns caused by pathological conditions. The main objective of this study was to explore the functional, structural and molecular cardiovascular changes that are involved in healthy pregnancy with the goal to delineate possible mechanisms involved in the lack of reverse cardiovascular remodeling observed in peripartum cardiomyopathy (PPCM). Methods: Cardiovascular functional, morphological and molecular changes during pregnancy and postpartum were assessed in pregnant wild type mice (C57/BL6) and healthy women. An invitro model of cardiac hypertrophy was then used to explore the involvement of pregnancy hormones in the regulation of cardiac hypertrophy. Finally, we assessed the circulatory level of growth differentiation 15 (GDF-15) in PPCM patients and matched healthy controls. Results: Cardiac structural, functional and morphological changes were observed in mice and all the parameters were resolved postpartum. Strikingly, volume load, cardiac hypertrophy and fibrosis were sustained for a longer period postpartum than previously reported. Proteomics profiling confirmed the prolongation of cardiac hypertrophy in the postpartum and the involvement of the ubiquitin proteasome system (UPS) in the reverse remodeling of cardiac changes that occur during pregnancy. We also identified a set of transcription factors that regulates the protein expression in the postpartum. Left ventricular systolic function was significantly reduced in late pregnancy in humans. Finally, the serum level of GDF-15 was significantly lower in PPCM patients compared to healthy controls Conclusion: We conclude that pregnancy induces cardiac stress which is sustained in the postpartum period. The heart remodels and adapts to meet the demand by both the mother and the fetus. Cardiac changes that occur during pregnancy are strictly regulated and reversed postpartum. However, the postpartum period is a period of intense cardiac stress and activity which requires monitoring for any deviation that may lead to pathological conditions.

Background: Pulmonary alveolar proteinosis (PAP) is a rare disorder characterized by excessive accumulation of intra-alveolar surfactant related, lipoproteinaceous material. With the exception of a single pediatric case report of PAP, no data exists in Sub Saharan Africa. The aim of the study is to describe the epidemiological and clinical features of patients with PAP treated at Groote Schuur Hospital since May 2009 and their outcome after the first therapeutic whole lung lavage. Methods: 11 patients with PAP were identified using the Pulmonology whole lung lavage register. A retrospective folder review was undertaken for demographic and clinical data which was captured via a paper data capture sheet and then entered into a REDCap database for ease of statistical analysis. Findings: The median age at diagnosis was 42 years, with a male to female ratio of 1:1.2 . All the patient's tested negative for HIV. A history of smoking was seen in 63.6% (7) with median pack years of 21.5. Common symptoms at presentation included: dyspnoea (100%), dry cough (45.5%), productive cough (45.5%) and weight loss (54.5%). All patients were hypoxic at diagnosis with an average Pa02 on room air of 7.75 kPa (±1.59) and, a mean FEV1/FVC ratio of 87.60% (±7.02) of predicted. Although 36.4 % (4) were unable to perform 6 min walk tests at presentation, the remaining patients had a median distance of 287 m. No mortality was seen at 12 months, despite all patients requiring whole lung lavage during this period. Conclusions: This small, retrospective cohort offers the first insight into the demographic and clinical features of patients in Sub-Saharan Africa with PAP. Interestingly, no patients in this cohort were HIV positive. Within this small cohort very few statistically significant details can be drawn but rather a description of a rare condition. Future plans to continue data collection prospectively and expand to other centres will improve deductions made.

Introduction: Tenofovir disoproxil fumarate (TDF) is vastly used in South Africa (SA) as a first line agent for the treatment of human immunodeficiency virus (HIV). TDF is known to be associated with nephrotoxicity with identified risk factors. This study aimed to describe the demographics, clinico-biochemical features, kidney function and mortality outcomes in TDF exposed patients with acute kidney injury (AKI) in two tertiary centres in SA. Method: This observational cohort study reviewed all HIV infected in-patients presenting with AKI referred to the nephrology units at both Groote Schuur Hospital, Cape Town and Livingstone hospital, Gqeberha. Baseline characteristics, contributory factors to the AKI, associated clinical and biochemical features were recorded. Where a kidney biopsy was indicated, histological features were documented. Kidney and mortality outcomes of the enrolled patients were assessed over a 1-year period. Results: There were 213 patients enrolled from 1 August 2013 to 30 September 2016, 114/213 (51.8%) of the patients were TDF-exposed and 99/213 (45%) were TDF-unexposed. The median age was 37 years (IQR: 31 - 45yrs). The TDF-exposed were significantly older, 40 years versus 34 years (p<0.01) The TDF-unexposed group had a higher prevalence of hypertension: 21/99 (21.2%) versus 11/114 (9.7%), (p=0.02). The median creatinine at referral was 642 µmol/L (IQR: 340 - 1116 µmol/L) and 96/210 (45.7%) required dialysis. HIV/tuberculosis (TB) coinfection was common, 119/199 (59.8%). There was significant exposure to nephrotoxic drugs and drugs associated with idiosyncratic drug reactions in both groups, with anti-tuberculous treatment being the most common. Rifampicin was used by 51/212 (24.1%) [TDF-exposed 31/114 (27.2%) and TDF-unexposed 20/98 (20.4%), p=0.25]. There were no differences in serum and urinary biochemical features between the TDFexposed and unexposed groups. Of the enrolled patients, 57/213 (26.8%) underwent a kidney biopsy. On histology, the incidence of acute tubular necrosis (ATN) was higher in TDFexposed individuals (TDF-exposed: 47% versus TDF-unexposed: 22% p=0.05) whilst in TDF-unexposed, HIV associated nephropathy was most common. In the total cohort, chronic kidney disease (CKD) developed in 22/212 (10.4%) and the mortality was 62/213 (29.1%). There were no significant differences between the TDF-exposed and non-exposed cohorts in terms of CKD or mortality. Conclusion: This study demonstrated that hospitalized people living with HIV in SA have a high rate of tuberculosis co-infection and significant drug exposures. The clinical characteristics, severity of AKI and outcomes were similar in TDF-exposed and -unexposed. TDF exposure was associated with a greater degree of ATN on kidney biopsy. AKI in this HIV infected cohort carried a high mortality, regardless of the aetiology.medicib

Background. The primary immunodeficiency diseases (PIDs) constitute a diverse and everexpanding group of inborn errors affecting a wide range of immune functions. They are not well documented in Sub-Saharan Africa. An important barrier to care is limited awareness of PIDs and their management among health care professionals. This fascinating spectrum of diseases is rapidly expanding worldwide, and not as rare as we think. Genetic characterization and newborn screening for primary Immunodeficiency diseases (PIDs) may be the gold standard in the first world setting but are neither practical nor feasible for our doctors. Yet, other low and middle income countries in the world have also established reasonable services and created registries for children with PIDs, including other African countries. Objective. To describe the spectrum of PIDs at a tertiary paediatric hospital. Methods. A retrospective descriptive study of PIDs diagnosed at Red Cross War Memorial Children's Hospital, Cape Town, South Africa between 1975 and 2017 was undertaken. Results. 252 children with PIDs were identified, spanning 8 of the 9 categories listed in the 2017 classification of the International Union of Immunological Societies. Predominantly antibody deficiencies, combined immunodeficiencies with associated syndromic features, and immunodeficiencies affecting cellular and humoral immunity accounted for 79% of all PIDs. The mean age (standard deviation) at diagnosis was 46 (50) months and the male to female ratio was 1.5:1. A history of parental consanguinity was present in 3 children (1.2%). Recurrent infection was the most prevalent presenting phenotype, manifesting in 70.2% of the patients. Genetic or chromosomal confirmation was obtained in 42/252 (16.7%) of the children. Common interventions used to prevent infection were antimicrobial prophylaxis and immunoglobulin replacement therapy, administered to 37.7% and 36.9% of the patients respectively. Six of seven children who underwent haematopoietic stem cell transplantation (HSCT) had successful outcomes. The 7th patient died 2 months post-HSCT from overwhelming infection. Although we could not account for the children lost to follow up during the study period, 53 (21.0%) deaths were confirmed. Conclusions. Several challenges exist in the recognition and treatment of children with PIDs in our setting. These include limited access to genetic diagnostics and HSCT. Sub-optimal treatment options contribute to the overall mortality of PIDs in South Africa. Greater awareness among clinicians treating children and more laboratory diagnostic capacity are needed to increase the recognition PIDs among children in South Africa. The treatment options that are available in South Africa are unevenly distributed. Hence, treatment capacity should be expanded throughout the country, especially advanced interventions such as HSCT. Ongoing reporting of registries such as ours and increased community awareness should strengthen the lobby for greater investment in rare diseases such as the PIDs.

Includes abstract. / Includes bibliographical references (leaves 100-111). / This study examines the histopathological response to injury following both balloon angioplasty and endovascular stenting in the Zucker rat, a model that allows interpretation of the role of obesity as well as progressive glucose intolerance and hyperinsulimaemia. Lean and obese Zucker fatty rats and Zucker diabetic fatty rat (ZDF) were subjected to balloon injury with or without stenting. The development of IH, along with the histological response to injury was analyzed.

Includes bibliographical references (p. 99-110).

Objective: The main purpose of this study was to evaluate cataract surgical services in Madagascar to help guide the national eye health program. Methods: A retrospective study that used cataract surgical data collected between January 1st and December 31st, 2012 in 8 regional capitals (districts) of Madagascar. 1072 cataract operated eyes from 8 regional capitals supported by Kilimanjaro Centre for Community Ophthalmology (KCCO) satellite centers were analysed. Results: The study findings demonstrated a borderline post-operative visual acuity outcome after 24 hours but showed visual improvements 4 weeks after follow up. Males were more likely to have cataract surgeries compared to females. The cataract surgery rate (CSR) was 1467 in 8 regional capitals of Madagascar. Conclusion: This study demonstrated unequal distribution of cataract surgical services in Madagascar. Therefore, the findings of this study could be used to advocate for equitable provision of cataract surgery across all regions in Madagascar.