Thesis (M.Sc. (Chemical Pathology) --University of Limpopo, 2013 / Cardiovascular diseases (CVDs) are among one of the well documented conditions and pose a significant health burden in the world as they areconsidered to be of adult onset. However, recent studies have shown that in developed countries CVD risk factors are becoming prevalent in young people which isof great concern. Therefore, the aim of this study was to determine if CVD risk factors are present in young subjects aged 15-20 years of age residing in a rural area of a developing country. Methods: Subjects aged between 15-20 years who participated in the “Gene - Environment interaction project” were included in this sub-study. Total cholesterol, triglycerides, HDL-cholesterol, LDLcholesterol, insulin, glucose, creatinine, Lp(a), apoB, apoA-1 and hs-CRPwere determined. Blood pressure, physical activity (number of steps/day), weight, height, waist circumference and hip circumference were obtained from the database. Subjects with CRP levels above 10mg/L and creatinine levels above 130 mmol/L were excluded. Results: The present study showed an overall high prevalence of some CVD risk factors. There was high prevalence of insulin resistance (23.0% in females and 34.7% in males), and high hs-CRP (18.4% in females, 12.9% in males). The prevalence of low HDL-C levels was high (55.2% in females and 16.8 % in males), however, the prevalence of abnormal levels of other lipids such as total cholesterol/HDL-cholesterol ratio was low in both males and females. The prevalence of an increased apoB/apoA ratio was significantly higher in females 26.4% compared to males 7.9%. The prevalence of overweight (12.6%) and obesity (9.2%) was higher in females than in males (overweight 1%, obesity 0 %). The prevalence of hypertension was comparable between the two genders (5.7% in females and 10.9 % in males). Conclusion: The results showed a relatively high prevalence of non-traditional risk factors for cardiovascular diseases in adolescents residing in a rural area, Limpopo Province, while the prevalence of traditional risk factors such as total cholesterol and triglycerides was low.

A large number of studies have reported on the association between the obligate intracellular bacterium, Chlamydia pneumoniae and atherosclerosis. These studies suggest that C. pneumoniae may potentially play a role in the atherosclerotic process, as not all the current atherosclerotic risk factors account for the resulting complications, such as angina, myocardial infarction, heart failure and stroke. The research presented in this thesis analysed whether there are any reliable markers of chronic C. pneumoniae vascular infection, including chlamydial sero-prevalence as defined by two commercial serological tests, detection of C. pneumoniae DNA in the peripheral circulation, the presence or absence of risk factors and symptomatic status. The presence of the bacterium in atherosclerotic carotid specimens was diagnosed directly using a C. pneumoniae-specific polymerase chain reaction (PCR) and a genus-specific immunofluorescent (IF) assay. Eighteen of the 54 (33%) carotid artery diseased (CAD) specimens were positive for the presence of C. pneumoniae DNA by PCR detection, whereas the IF assay detected only six positive samples. PCR analysis found that only two of 43 (5%) patients had C. pneumoniae DNA present within their peripheral blood mononuclear cell (PBMC) fraction. Chlamydial antibodies were detected by Focus microimmunofluorescence and/or Medac recombinant enzyme-linked immunosorbert assay (rELISA) in 56% (24/43) of CAD patients tested. Traditional risk factors, symptomatic status, antigen detection and PCR-based detection of C. pneumoniae in PBMCs, all failed to correlate with the presence of a chlamydial vascular infection. In conclusion, the existing non-invasive diagnostic tests (serology and peripheral blood-based PCR detection) are inefficient for diagnosing a vascular Chlamydia infection, suggesting that a different chlamydial antigen should be tested targeted to identify a chronic C. pneumoniae infection in CAD patients. Given the observation that numerous previously published studies have detected C. pneumoniae in atherosclerotic arterial tissue, yet at widely different detection rates (0% to 100%), it was clear that the location and quantity of clinical specimen could directly affect the detection rate. Previous reports have not used a standard and validated procedure for sampling arterial specimens for C. pneumoniae DNA. The inconsistent detection rates of chlamydial DNA in atherosclerotic plaque are a result of low concentration and irregular distribution of the bacterium, as reported in this study. Our research concluded that a minimum of 15 (30ìm-thick) sections should be analysed by PCR to minimize these sampling variables and obtain a 95% chance of detecting all true C. pneumoniae-positive samples. All previous studies may have under estimated the prevalence of C. pneumoniae, as stringent sampling and repeat testing of the bacterium is required to minimise false-negative results. An interesting finding was that C. pneumoniae DNA was present in all 10 atherosclerotic arteries, although extensive sampling of the carotid was crucial for detection. The third area of research examined the question of possible strain differences between C. pneumoniae isolates infecting human atherosclerotic carotid arteries. Whole genome sequencing as well as specific gene typing suggests that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. We analysed the genotypes of C. pneumoniae present in human atherosclerotic carotid plaque and found several polymorphisms in the variable domain-4 (VD4) region of the outer membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes. Our research identified four different genotypes of C. pneumoniae in human atherosclerotic carotid arteries, including an isolate that appears genetically identical to a strain previously detected in koalas. Two genotypes of C. pneumoniae were present in both human carotid specimens and koala PBMC fractions, suggesting that these genotypes of C. pneumoniae may be capable of crossing the host barrier. The study showed that diversity exists in both the ompAVD4 gene and the ygeD-urk intergenic region enabling fine-detailed differentiation between five different genotypes found in respiratory and/or vascular C. pneumoniae isolates. The importance of the diversity of C. pneumoniae isolates in its role in atherogenesis needs to be further studied.

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