Background Oral candidiasis is the most common infection of the oral mucosa of HIV-seropositive patients, although its frequency is rapidly decreasing with the advent of highly active antiretroviral therapy (HAART). Many questions regarding its complex pathogenesis remain unanswered. The diagnosis is usually established with non-invasive techniques such as mucosal smears. Oral lesions of HIV-associated Kaposi sarcoma (HIV-KS) are routinely biopsied and frequently show secondary infection with Candida albicans or other Candida species. Aims and objectives The aim of this investigation was to determine the frequency and histomorphology of secondary Candidal infection of the surface epithelium of oral HIV-associated KS lesions (HIV-KS), which are routinely biopsied in HIV infected patients. Materials and methods Haematoxylin and eosin (HE), and Periodic Acid-Schiff (PAS) stains of 133 cases of oral Kaposi sarcoma diagnosed between the period 2003 and 2007 within the Division of Oral Pathology were examined histologically for intensity and morphology of Candidal colonisation, depth of invasion, number of organisms, epithelial reactions and associated inflammatory response. The depth of Candidal invasion and severity of infection were correlated with the available CD4 T cell counts of HIV seropositive patients at the time of biopsy. Results Almost forty one percent (40.62%) of all oral HIV-KS cases were secondarily infected with Candida species. The intensity varied from an isolated single pseudohyphus to matted colonies of vegetative yeasts and psuedohyphae. Whilst in most cases the organisms did not invade beyond the parakeratin layer, pseudohyphae were noted extending into the stratum spinosum in 2 cases, and a single case showed a pseudohyphus within the lamina propria. A further 2 cases showed pseudohyphae growing in the pyogenic membrane. Neutrophilic permeation of the epithelium and Munro micro-abscess formation, features commonly associated with Candidal infection, were frequently present even in the absence of Candidal infection. Candidal organisms were often present in the absence of inflammation. Conclusion Oral lesions of HIV-KS are commonly secondarily infected with large numbers of Candidal organisms. The morphological characteristics of secondary Candidal infection within the surface epithelium of HIV-KS lesions suggest an altered pathogenetic pathway. Further studies are necessary in this regard.

Thesis (Ph.D. (Public Health))--University of the Witwatersrand, Faculty of Health Sciences, 2012 / Factors associated with the transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV) are inconclusive. In countries where KS and KSHV are confined to men who have sex with other men (MSM), KSHV is associated with sexual risk factors. In countries where KSHV is endemic, it affects adults and children of all ages and irrespective of sexual orientation, suggesting the existence of non-sexual risk factors for KSHV infection. In this thesis, three distinct cross sectional studies aiming to define the seroprevalence of KSHV in South African populations and to identify plausible risk factors for KSHV infection were undertaken. The studies measured KSHV seropositivity in relation to sociodemographic factors and HIV status. In children, factors associated with horizontal mother to child transmission were also explored. In adults KSHV seropositivity was also measured in relation to sexually transmitted infections and/or measures of sexual behaviour. Calculated risk factors were expressed as odds ratios (95% confidence interval) for KSHV. Methods Mother to Child KSHV seroepidemiology Study: KSHV seroprevalence (reactive to either lytic K8.1 or latent Orf73) was measured in 1287 children and their 1179 biological mothers. Association between KSHV seropositivity in children was measured against KSHV seropositivity and HIV status of their mothers. KSHV seroepidemiology in women attending antenatal clinics: Antibodies to KSHV lytic K8.1 and latent Orf73 antigens were tested in 1740 pregnant women attending antenatal clinics in South Africa in 2001. Information on HIV and syphilis serology, age, education, residential area, gravidity, and parity was anonymously linked to evaluate risk factors for KSHV seropositivity. Clinics were grouped by municipal regions and their proximity to the two main river catchments defined. Carletonville Community KSHV seroepidemiology Study: Sera from 2103 South African individuals (862 miners, 95 sex workers, 731 female and 415 male township residents) were tested for antibodies to KSHV lytic K8.1 and latent Orf73, HIV gonococcus, herpes simplex virus type 2 (HSV-2), syphilis and chlamydia. Information on social, demographic and highrisk sexual behaviour was linked to laboratory data. Results Mother to Child KSHV seroepidemiology Study: KSHV seroprevalence (reactive to either lytic K8.1 or latent Orf73) was 15.9% (204 of 1287 subjects) in children and 29.7% (350 of 1179 subjects) in mothers. The risk of KSHV seropositivity was significantly higher in children of KSHV seropositive mothers compared with those of KSHV-seronegative mothers. The HIV status of mothers was marginally associated with an increased risk of KSHV seropositivity in their children (AOR = 1.6, 95% CI: 1.0 to 2.6; P = 0.07). KSHV seroprevalence was significantly higher in HIV-infected subjects (P = 0.0005), and HIV-infected subjects had significantly higher lytic and latent KSHV antibody levels than HIV-negative subjects. KSHV seroepidemiology in women attending antenatal clinics: KSHV seroprevalence was nearly twice that of HIV (44.6% vs. 23.1%). HIV and syphilis seropositivity was 12.7% and 14.9% respectively in women without KSHV, and 36.1% and 19.9% respectively in those with KSHV. Women who were KSHV seropositive were 4 times more likely to be HIV positive than those who were KSHV seronegative (AOR 4.1 95%CI: 3.4 - 5.7). Although, women with HIV infection were more likely to be syphilis seropositive (AOR 1.8 95%CI: 1.3 - 2.4), no association between KSHV and syphilis seropositivity was observed. Those with higher levels of education had lower levels of KSHV seropositivity compared to those with lower education levels. KSHV seropositivity showed a heterogeneous pattern of prevalence in some localities. Carletonville Community KSHV seroepidemiology Study: Overall KSHV and HIV prevalences were 47.5 and 40%, respectively (P<0.43). The risk of HIV infection was highest in sex workers followed by female residents and miners, compared with male residents (P<0.001). HSV-2 infection was highly prevalent (66%) and lower, but still substantial, prevalence (6–8%) was observed for other sexually transmitted infections (STI). No significant difference in KSHV infection was observed among the residential groups (P>0.05). KSHV was not associated with any of the STI or any measures of sexual behaviour. Conclusion The findings of these three studies contribute substantially to global KSHV seroepidemiology and show that in Southern African settings KSHV is associated with non-sexual mode of transmission. Firstly KSHV is common in very young children up to ten years of age and increases with age until adulthood. The high prevalence of KSHV in the South African populations remained evident in all populations. In children, the risk of acquisition of KSHV was higher among children of KSHV-seropositive mothers than if the mother was KSHV negative. The association between KSHV and HIV was also noted in the study of pregnant women attending antenatal clinics and in the mother to child study. However this association was not evident in the Carletonville population where both KSHV and HIV were highly prevalent. In both the adult studies the lack of association between KSHV and syphilis was evident. KSHV infection was also not associated with other sexually transmitted infections and measures of sexual behaviour. As expected, the pattern of HIV and STI in sex workers suggests high rates of high-risk sexual behaviour in this population; however KSHV seropositivity was the same amongst sexworkers and all the other community groups. This pattern of the lack of association with high-risk sexual behaviour, particularly in sex workers and with any markers of STI strongly suggests that the sexual mode does not play a significant role in KSHV transmission in this South African population. This may also suggest that KSHV transmission may involve geographical and cultural factors other than sexual transmission.

Mechanistic insights on molecular and cellular mechanisms whereby KSHV induces Kaposi?s sarcoma (KS) are key for our understanding of KS tumors and for the development of new therapies. We have previously developed an animal model for KSHV induced KS using murine bone marrow cells transfected with a KSHVBac36. We found that although these cells lacked attributes of transformed cells in vitro, they were able to cause KS-like tumors in vivo. In vivo tumorigenesis correlated with upregulation of both KSHV lytic genes and host angiogenesis suggesting that that cues provided from the microenvironment played a major role in regulating viral and host gene expression related with KSHV-induced tumorigenesis. Our goal thus, was to identify these molecular cues regulating pathogenic gene expression in KSHV infected cells in vivo. An important difference between cells kept in vitro versus in vivo is the lack of environmental extracellular matrix (ECM) signals. Therefore the mECK36 cells were cultured in vitro in matrigel, a basement membrane preparation rich in ECM proteins and its individual components, to discern the effect of host signaling by the ECM on KSHV infected cells. Investigation of gene expression through Real Time RT-PCR identified several viral and host genes associated with tumorigenesis such as KSHV vGPCR and angiogenesis associated VEGF and EGF- receptors were upregulated in response to this environment. Further analysis of the molecular activity of the cell indicated the change in transcription was due to the activation of integrin signaling, as assessed by phosphorylation of the Focal Adhesion Kinase (FAK) protein. Our results show that integrin signaling occurring in vivo through interaction with ECM serves to enhance the pathogenic viral and host gene expression of KSHV infected cells and that EGFR upregulation can be correlated with these conditions. These results points to the integrin signaling pathway or the EGF-Receptor as promising targets for therapy and prevention of KS tumors.