MMed (Paediatrics), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Introduction HIV-Associated Nephropathy (HIVAN) is the commonest cause of proteinuria, especially in black HIV seropositive children. This chronic nephropathy is a late complication of untreated HIV that requires earlier intervention to prevent progression of renal disease. Microalbuminuria is an early marker of the presence of subclinical renal disease in systemic diseases such as diabetes mellitus and hypertension. This study assessed the prevalence and clinical significance of a single screening test for microalbuminuria in a cohort of HIV seropositive children without any symptoms of renal disease at Chris Hani Baragwanath hospital situated in Johannesburg, South Africa. Methods A prospective study was undertaken at Chris Hani Baragwanath hospital (a major tertiary facility that serves the people of Soweto and the surrounding areas of southern Gauteng). HIV seropositive and seronegative patients from both an inpatient and outpatient ambulatory setting were screened for qualitative proteinuria and microalbuminuria. Those on antiretroviral therapy, anti tuberculosis treatment, known chronic kidney disease, hypertension, fever, acute illness and urinary tract infection were excluded from the study. Results 180 patients were enrolled into the study, of which 110 were HIV positive and 70 HIV negative. Majority of the patients were black (98%) with 100 (56%) males and 80 (44%) females. Microalbuminuria was present in 27(25%) of HIV positive patients and 1 (1%) HIV negative patient, p=0.00003. The mean age at presentation of microalbuminuric HIV positive patients was 6 ± 3.2 years. With normal renal function and no proteinuria; microalbuminuria was present in 21 (19%) patients, p=0.03. Microalbuminuric patients were moderately immunosuppressed (mean CD4 % of 16.8 ± 8%, mean viral load 8 ± 18 x 105 RNA copies/ml) and had WHO clinical stage 2 and 3 disease. Absolute CD4 counts appear to correlate better with microalbuminuria than CD4 percentage as the mean CD4 absolute count in HIV positive patients with microalbuminuria (493 ± 330 x 106/l) was significantly lower than those without microalbuminuria (780 ± 702 x 106/l), p=0.03. Conclusion Microalbuminuria screening of HIV positive patients is a more sensitive screening test compared to standard urine dipsticks as it is present in patients with normal renal function who have no proteinuria. This may allow for early identification of subclinical renal disease in patients with some evidence of immunosuppression; thus possibly preventing the deterioration of renal function and severity of HIV disease with early initiation of antiretroviral therapy.

The Human Immunodeficiency Virus (HIV) subtype C accounts for the majority of infections in Southern Africa. The HIV protease is one of the targets in HIV treatment due to its pivotal role in HIV maturation in the host cell. However, because of polymorphisms in the HIV genome, drug resistance becomes a major problem in HIV treatment. Polymorphisms in the HIV protease gene result in altered substrate cavities, and /or flap hinge modifications leading to unfavourable drug interaction with the enzyme. The most common form of drug resistant mutations is single amino acid substitutions. Although, amino acid insertions have been reported, this form of mutation in the HIV protease is rare. L38↑N↑L insertion is a unique form of HIV protease polymorphism that was isolated from a patient failing drug therapy in South Africa. The objective of this research was to assess the impact of the L38↑N↑L insertions, with accompanying background mutations, on the structure and function of this form of polymorphism in HIV-1 South African subtype C protease. The far-UV circular dichroism (CD) spectra of L38↑N↑L protease shows a trough at 203 nm, suggesting alterations in the secondary structure content of this mutant. Whereas the wild type (WTCSA-HIVPR) displays a trough at 215 nm. However, tertiary structure characterisation using fluorescence spectroscopy did not detect changes within the local tryptophan environment of L38↑N↑L protease in comparison with the wild type due to no significant shift in emission wavelength. The specific activity of L38↑N↑L protease and wild type was 28.0±1.3 μmol.min-1.mg-1 and 123.45±6.4 μmol.min-1.mg-1 respectively. The turn-over number for L38↑N↑L protease and wild type was 1.0 × 10-3 ± 6.0 × 10-5 and 7.7 × 10-3 ± 5.6 × 10-4 respectively. As much as the presence of known drug resistance mutations in L38↑N↑L can be attributed to drug resistance, it should also be noted that the insertions may have also caused local structural alterations that may have enhance drug resistance of L38↑N↑L. These changes could have lead to the decreased catalytic activity of the L38↑N↑L protease. Homology modelling studies show that the insertions in L38↑N↑L protease may have resulted in a fold similar to 2HS1 (PDB code), which has a modification on the flap hinge. In addition, the homology modelling studies suggest that L38↑N↑L protease may have a second inhibitor binding site next to one of the flap hinge regions as seen in the 2HS1 model. In conclusion, the L38↑N↑L insertions and accompanying background mutations may have contributed to the local structural modifications that lead to drug resistance in L38↑N↑L protease.

ABSTRACT: Background: Correlation of nutritional status with genital shedding of HIV-1 has not been described. Genital shedding patterns have not been described in Botswana previously. Methods: I conducted a cross-sectional study to describe genital shedding patterns in Botswana and to correlate nutritional status with genital shedding of HIV-1. Between July 2005 and December 2005, samples were collected and analyzed from 50 women participating in an ongoing micronutrient supplementation clinical trial that is examining the effect of supplementation on HIV disease progression. Results: HIV-1 RNA was isolated from both baseline and three months CVL in 24% of the study population, and these were labelled continuous shedders (CS). No HIV-1RNA was isolated from both baseline and three months CVL in 64% of the study population, and these were labelled non-shedders (NS). In 14% of women, HIV-1 RNA was either isolated from baseline CVL only (4/50) or from the three months CVL only (3/50) and these were labelled as Intermittent Shedders (IS). Women who had detectable genital HIV-1 RNA at baseline had lower haemoglobin compared to those who were not shedding (Hb11.7 (95% CI 10.8 ; 12.5) vs. Hb 12.5 (95% CI 12.0 ; 13) P = 0.0877), showing a strong trend, albeit a non significant haemoglobin difference. Women who had detectable genital HIV-1-RNA at baseline had significantly lower CD4 cell percentage compared to those not shedding (22% (95% CI 19 ; 24) vs. 30 % (95% CI 27 ; 34) P < 0.01) and a significantly higher log viral load (4.7 log (95% CI 4.2 ; 5.1) vs. 3.6 log; ((95% CI 3.5 ; 4.0) P < 0.01). Overall there was a non significant higher prevalence of genital infection in women who were shedding HIV-1 at baseline, compared to those who were not (73% vs. 46% P = 0.123). No HIV -1 RNA was isolated in all the19% of the women in the study who were using some form of contraception. Conclusions: The preliminary analysis showed three patterns of HIV shedding in this study population, namely Continuous shedders, Intermittent shedders and Non-shedders. Women with detectable genital HIV-1-RNA at baseline had more advanced disease, and by extension poor nutritional status, than those not shedding, as shown by higher plasma viral load, lower CD4 count, lower haemoglobin level and higher prevalence of genital infections. This study generates hypothesis on the role haemoglobin may play in genital shedding of HIV-1 in females. Recommendation: Due to the small sample size, these results will need to be validated by larger studies of appropriate design. Timely treatment of anaemia in HIV positive women may be important in reducing HIV transmission associated with presence of HIV-1RNA in genital secretions.

ABSTRACT Aim The aim of this study is to compare the degree of severity of chronic periodontitis in HIVseropositive subjects with chronic periodontitis to control subjects with chronic periodontitis, in the Ga-Rankuwa area in South Africa. Methods Two cohorts of subjects with chronic periodontitis were recruited for this study over a period of time: thirty HIV-seropositive subjects; and 30 control subjects presumed to be HIV - seronegative and apparently in good health. Results When all the periodontal indices were compared and evaluated there was no association between HIV-serostatus and the periodontal indices. When periodontal indices were compared between HIV-seropositive subjects using highly active anti-retroviral therapy (HAART), and HAART-naïve subjects, there was no statistical differences regarding gingival recession, plaque index and bleeding index. However, the mean pocket depth in HAARTnaïve seropositive subjects was slightly greater than in HIV-seropositive subjects using HAART. Correlation coefficient of mean pocket depth in relation to log CD4+ T cell count in the HIV-seropositive HAART-naïve group of subjects showed a significant negative correlation (P = -0.947), but there was no correlation between the mean gingival recession values and the log CD4+ T cell counts in the same group (P=0.303). For the HIVseropositive subjects using HAART the correlation coefficient test failed to show significant statistical relationships between log CD4+ T cell count and mean pocket depth (P=0.903) and mean gingival recession (P=0.312) in HIV-seropositive subjects using HAART. Conclusion HIV-seropositive subjects with chronic periodontitis show clinical manifestations of similar degree of their periodontal disease to those of healthy control subjects with chronic periodontitis, with no differences in the mean pocket depth, gingival recession, plaque index and bleeding index.

Introduction Human immunodeficiency virus (HIV) infection is becoming a global epidemic. In HIV-infected individuals, anorectal diseases are common and the commonest indications for surgical intervention. However, it has not been clear whether the cause and management of anorectal conditions differ in patients who are HIV negative and those who are HIV positive. Aim To compare the presentation of perianal diseases in HIV +ve and HIV –ve patients and to determine the best therapy for perianal diseases in HIV +ve patients Methods The study period was from 1999 to 2002. Patients seen at Helen Joseph Hospital during the author’s gastroenterology fellowship period and those from his private practice were recruited. Those who agreed to HIV testing and who were treated by the author were included in the study. The patients were categorized according to HIV status, CDC classification and perianal pathology. Standardized questionnaires were used for every patient. Patients were reviewed every two weeks after the procedure until fit for discharge. Results The sample comprised 241 patients: 100 HIV+ve (63 males and 37 females) and 141 HIV-ve (61 males and 80 females). The mean age of the HIV+ve patients was 34,8y (range 17-62y), and the mean age of the HIV-ve patients was 41y (range 5-82y). Follow-up was from two weeks to two years. The pathology included 62 (25 +ve, 37 -ve) haemorrhoids, 67 (27 +ve, 40 –ve) fistulas, 59 (25 +ve, 34 -ve) abscesses, 46 (11 +ve, 35 -ve) fissures, 24 (22 +ve, 2 -ve) anal ulcers, four (3 +ve, 1 -ve) anal warts, two (both +ve) pilonidal sinuses, three (all -ve) anal cancers and two (1 +ve, 1 -ve) hidradenitis suppurativa. Nine patients with haemorrhoids (4+ve, 5-ve) were treated conservatively, 11 patients with haemorrhoids (6+ve, 5-ve) had rubber band ligation and 42 patients with haemorrhoids (15+ve, 27-ve) had haemorrhoidectomy. Thirteen patients (1+ve, 12-ve; p<0.001) had complex fistulas and 8 patients (7+ve, 1-ve; p=0.006) had multiple fistulas. Ten HIV -ve patients with complex fistulas and seven patients with trans-sphincteric fistulas (3+ve, 4-ve) needed more than six weeks to heal after treatment. Thirty patients with anal fissures had sentinel piles, but there were none in the patients with anal ulcers (p<0.0001). Thirteen HIV +ve patients with anal ulcers had an abnormally weak anal tone, but only one HIV +ve patient with anal fissure had a weak anal tone (p<0.001). In the patients with anal fissure, 37 (8+ve, 29-ve) had a high anal tone, but none of the patients with anal ulcers had increased anal tone (p<0.0001). Of the 59 patients with perianal suppuration, 23 (8+ve, 15-ve) had primary fistulotomy as well as drainage of the abscess. Conclusions Multiple fistulas were more commonly seen in advanced HIV patients, whereas complex fistulas were more commonly seen in HIV negative individuals. Healing after fistula surgery is determined more by the type of fistula than the HIV status or stage. Anal fistulas associated with perianal suppuration can be treated the same way in both HIV negative and positive patients, without increased complications. Careful physical examination is essential to differentiate between anal ulcers and fissures. Both conditions are common, and have similar symptoms in HIV positive patients. They can, however, be readily and safely distinguished on clinical examination, since fissures are associated with high anal tone, and a sentinel pile, while the AIDS ulcer lacks the sentinel pile, and the pressure is low. Anal malignancies, especially squamous cell carcinoma, have been uncommon in our experience. With the addition of anti-retroviral therapy and antibiotics, haemorrhoids may be safely treated according to standard principles (rubber band ligation and haemorrhoidectomy, as appropriate). Our practice has favoured a conservative approach in patients with advanced HIV disease. However, anti-retroviral therapy and antibiotics may improve the safety and outcome after surgical procedures.

MPH, Faculty of Health Sciences, University of the Witwatersrand, 2009. / Introduction Patient retention and loss to follow-up in the antiretroviral programmes in South Africa and indeed the world is important as failures to reduce these lead to higher drug resistances and treatment failures. In the light of the few drugs available to treat HIV and AIDS it is imperative that patients lost to follow-up be traced and brought back into the programme. The objectives of the study were to quantify the number of patients enrolled in the programme between 01st June 2004 and 31st December 2004; determine the demographic profile of enrolled patients with regard to age; sex; education; employment and area of residence; to determine compliance and defaulter rates at every monthly appointment up to 6 months of follow-up and to describe follow-up systems in place for tracking patients on ARVs; identifying those who fail to comply with scheduled appointments; and ensuring complianceand finally to identify challenges faced by the hospitals in tracking patients on ARV therapy. Material and Methods The two hospital chosen were the first public hospitals to rollout antiretroviral treatment in Ekurhuleni in 2004. This was a descriptive study involving review of health facility records and primary data collection through key informant interviews at two district hospitals in Ekurhuleni. The study reviewed mechanisms employed by the two hospitals in tracking those patients who started on the programme during the first six months of the ARV programme (June 2004 to December 2004). Results The two hospitals had after six months of starting with the rollout a combined number of 378 patients on treatment. Far East Rand Hospital (FERH) had registered 208 5 patients and Natalspruit (NSH) had 170 patients on their register. Most of the patients started on treatment were from Townships (82%), and 81% of all patients started on treatment were unemployed. The male(33.7%) to female (62.7) ratio was 1:2. Even though on average 90% of patients at both hospitals kept their first six appointment, defaulter rates at FERH was 23,2% and NSH was sitting at 33,1%. Discussion Our results show tha the two hospitals fall short on achieving the requierements by the Departmentof Health’s HIV plan that states under Priority Area 2, point 6.2, that accredited facilities must have the capacity to increase the retention of children and adults on ART – actively trace people on ART who are more than a month late for clinic/pharmacy appointment. The hospitals do not have proper tracking mechanisms in place, they lack important resources like transport, telephones and get wrong addresses. Based on the evidence we have gathered the hospitals’ defaulter rates and loss to follow-up are a concern but they are also not far off when compared to other places and countries whose defaulter rates are 20% on average. Conclusion and Recommendation Retention of patients in the programmes is an essential health imperative. It is therefore necessary that we make the following improvements to our hospital programmes: Make resources like telephone and transport available to healthcare workers; employ a dedicated team of workers doing only patient tracing and followup; invest in technology that would alert health care workers immediately a patient misses an appointment and finally educate the patients themselves of the importance of adherence to treatment and follow-up.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in the Field of Population-Based Field Epidemiology / Socio-economic status (SES) disparities do not only exist between racial groups in South Africa but also exists within the vulnerable black population with the devastating impacts of the HIV epidemic. Households are important determinants of human welfare. However, little is known about the effect of household socio-economic status on the establishment and break-up of households within a low-resource setting and a severe HIV epidemic. It is in the midst of these challenges in rural South Africa that this study examined the effect of household SES on household formation and dissolution among the black population in rural northern KwaZulu-Natal. METHODS Using longitudinal data from the period 2003-2012 from the Africa Centre for Health and Population Studies, the study used a cross-sectional study design approach to examine the effect of household SES on household formation. It also examined the effect of household SES change (i.e. either positive, negative change or stable SES) between the start and end of observation of a household within the study period. Household formation was defined as when an individual or individuals come from different households to form a new social unit with a new household head. Dissolution occurred when all individuals in a household end their membership to a household due to death, out-migration or by joining other households. Separate regression models for the two outcomes, household formation and dissolution were explored with household SES covariates while adjusting for other household variables. RESULTS Household formation and dissolution trends both decreased over the study period. Out of a total of 18,249 households, newly formed households had a relatively higher percentage of tertiary educated household heads (10.7% versus 2.5%), unemployed household members (41.6% versus 28.5%), grant recipient household members (37.1% versus 8.5 %) and households within the average to richest wealth quintiles (44.1% versus 36.4 %) than pre-existing households. Multivariate analysis showed that tertiary educated household heads (aOR=2.96, 95% (CI) 2.26-3.89) and households within the average to richest wealth quintiles most especially the 4th quintile (aOR=3.29, 95% (CI) 2.69-4.04) were associated with a higher odds of households being newly formed. However, the lesser the employed members (aOR=0.31, 95% (CI) 0.21-0.45) and grant recipients per household size in a household (aOR=0.15, 95% (CI) 0.12-0.18) the lower the odds of formation. Furthermore, small size households (aOR=0.68, 95% (CI) 0.56-0.80) and unmarried household heads (aOR =0.47, 95% (CI) 0.40-0.55) were associated with lower odds of being newly formed. Whereas female headed households (aOR=2.23, 95% (CI) 1.93-2.57) were associated with a higher odds of household formation. With regards to household dissolution, close to a quarter of households had an increase in SES over the study period compared to households with a decreased SES (24.6% versus 8.6 %). Similar to household formation, male headed households dominated the study population with the highest proportion in dissolved households (63.8% and 61.5% at start and end of household observation respectively). Also unmarried household heads were the majority in dissolved households (62.7% and 64.1% at start and end of household observation respectively). Approximately 65.6% of households that never dissolved had an extended family type of composition compared to 36.6% of dissolved households. The area was predominantly rural with about 47.2% households in rural segment of the study area. The study has shown that households had lower odds of dissolving if there is a positive change (i.e. an increase) in household SES compared with households with an unchanged SES over the period. In exact terms, an increment in the number of employed household members over the study period was associated 49% lower odds of a household being a dissolved (aOR=0.51 95% (CI) 0.42-0.61). Also, an increment in the number of household grant recipients over the period of observation was associated with a 69% lower odds to result in the dissolution (aOR=0.31 95% (CI) 0.25-0.39). Households with an improved wealth index over the period of study were associated with 55% lower odds of dissolution (aOR =0.45, 95% (CI) 0.38-0.54). However, households with both male and female death (multiple sex) were more likely to dissolve. Similarly, peri-urban (aOR=0.71; 95% (CI) 0.58-0.86) households were more likely to dissolve compared to urban households. Surprisingly divorced, widowed and separated couples were not significantly associated with household dissolution. CONCLUSION SES is an important determinant of household existence and stability. This study has shown a complex relationship between household SES and household formation. Although education and improved household wealth index were more likely to result in household formation, an increase in the number of employed household members and household grant recipients did not necessary have an effect on household formation. Government cash transfers, education, employment of household members are valuable cushioning mechanisms necessary for household stability. There is need for government and non-governmental organisations to set up interventions to improve the socio-economic conditions of poor households prioritising rural and female headed households. This is especially critical in a high HIV prevalence area where these interventions will also mitigate against the burden of the HIV epidemic on the population. / MT2017

Thesis (Ph.D.)--University of the Witwatersrand, Faculty of Health Sciences, 2012. / The development of an effective HIV-1 vaccine is critical in mitigating the global HIV epidemic. Understanding the interplay between host immune functions, such as cellular memory differentiation, activation, inflammatory cytokine production and the virus, may provide key insight into anti-HIV immunity that can inform vaccine development. This PhD aims at understanding and identifying T cell memory, functional profiles and the effect of immune activation on in vivo HIV-1 control during primary/early infection. Furthermore, this study aims to examine and understand the potential mechanisms related to immune activation during primary HIV-1 infection. Use was made of a unique cohort of individuals recruited during primary HIV-1 infection and using a battery of assays to characterize and identify properties and mechanisms of T cell reactivity and activation. Multiparameter flow cytometry was used to measure memory differentiation (CD27 and CD45RO), activation (CD38, HLA-DR), proliferation (Ki67), and multiple cellular functions (CD107, IFNγ, IL-2, MIP-1β and TNFα) of total and antigen-specific CD4+ and CD8+ T cells from 15 HIV-1 and CMV-coinfected individuals followed over 15 months of HIV-1 infection. Plasma samples were used to measure markers associated with intestinal permeability (LBP, sCD14, I-FABP and IgM EndoCAb) and inflammation (IL-1β, IL-6, IL-7, IL-10, IL-12p70, TNFα and MCP-1). The differentiation profile of HIV-Gag specific memory CD4+ and CD8+ T cells was found to be mainly characterized by an early differentiated (ED) memory phenotype relative to CMV- specific CD4+ and CD8+ T cells. Moreover, the proportion of HIV-specific ED-memory CD4+ T cells inversely associated with viraemia, suggesting that HIV-1 antigen burden could be shaping the differentiation of HIV-specific memory CD4+ T cells during primary infection. Primary HIV-1 infection was also characterized by significantly elevated levels of activated and proliferating total and HIV-specific memory CD4+ and CD8+ T cells, which positively correlated with viraemia. Furthermore, upon sorting of total activated memory CD4+ T cells, these cells harboured more gag provirus DNA than non-activated memory cells, suggesting that activated memory CD4+ T cells support ongoing HIV-1 replication. When examining the relationship between memory differentiation and activation markers, the level of T cell activation was equally expanded across the different memory CD4+ T cell subpopulations, suggesting that memory differentiation of CD4+ T cells was unlikely driven per se by the level of T cell activation. In addition, when teasing out events that may result in T cell activation during primary HIV-1 infection using statistical models, plasma markers of microbial translocation and inflammation were found to correlate with immune activation. The lack of these associations in HIV-uninfected controls suggests that microbial translocation and inflammation were unlikely causative. Analysis of the polyfunctional profile of memory T cells during primary HIV-1 infection showed that HIV-specific CD4+ and CD8+ T cell responses are less polyfunctional relative to CMV-specific memory CD4+ and CD8+ T cell responses. Furthermore, the polyfunctional status of HIV-specific CD4+ T cells significantly correlated with viraemia at 3 months post-infection, indicating that the polyfunctionality of memory CD4+ T cells is likely driven by HIV-1 antigenemia. Overall, these observations suggest that HIV-1 antigenic burden appears to be a central driver of memory differentiation, activation/inflammation and polyfunctionality of T cells. Given the impact of HIV-1 viraemia on immune activation and memory T cell dysfunction (as measured by limited polyfunctional HIV-specific responses), preventing high levels of viral replication, with a vaccine or other early interventions may serve as an important strategy for delaying HIV-1 disease progression.

A Thesis Submitted to the School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, South Africa 2016. / Introduction Human immunodeficiency virus (HIV) has been associated with invasive enteric infections in HIV-infected patients, since it was first described in the 1980s. In South Africa, HIV remains an important health challenge, despite the introduction of antiretroviral therapy (ART) in 2003. In association with this, is an ongoing problem of invasive enteric infections, including those due to Shigella and Salmonella, including Salmonella enterica serovar Typhi (Salmonella Typhi). There are few South African data available as to the incidence of invasive disease due to these pathogens and how these data may contrast with the presentation and outcome in HIV-uninfected patients. The associated risk factors for mortality due to invasive enteric pathogens and whether there has been a response with ART as an intervention also needs further elucidation. Aims This work was undertaken to better describe the burden of invasive enteric infections (Shigella, nontyphoidal Salmonella and Salmonella Typhi) in association with HIV, define risk factors for mortality and establish whether the introduction of ART has impacted on disease burdens due to these pathogens. Methods Laboratory-based surveillance for enteric pathogens was initiated in 2003. Basic demographic details (age and gender) were collected on all patients where possible. In 25 hospital sites in all nine provinces, additional clinical information was collected by trained surveillance officers, including HIV status, data reflecting severity of illness, other immune suppressive conditions, antimicrobial and antiretroviral usage and outcome (survival versus death). Laboratories were requested to transport all isolates to the Centre for Enteric Diseases (CED) at the National Institute for Communicable Diseases of the National Health Laboratory Service (NHLS) in Johannesburg for further characterisation, including serotyping, antimicrobial susceptibility testing and molecular typing where relevant (whether isolates could respectively be classified as Salmonella Typhimurium ST313 and Salmonella Typhi H58). Additional cases were sought through audits of the Central Data Warehouse (CDW) of the NHLS. Annual incidence rates were calculated according to published estimates of population by age group by the Actuarial Society of South Africa for the Department of Statistics of the South African government. Analyses were specifically directed at invasive shigellosis, Salmonella meningitis, typhoid fever in South Africa and nontyphoidal salmonellosis in Gauteng Province, South Africa. Data were recorded in an Access database and analysed using chisquared test to establish differences between HIV-infected and uninfected individuals and univariate and multivariate analysis to compare risk factors for mortality. Data in the number of patients accessing ART were derived through audits of the CDW, by using the numbers of patients on whom viral loads were done annually as a proxy. Results Between 2003 and 2013, a total of 10111 invasive enteric isolates were received by CED. For patients for whom sex was recorded, 3283/6244 (52.6%) of patients presenting with invasive enteric infections were male; invasive disease was predominantly observed in children less than five years of age (1605/6131; 26.2%) and those who were aged between 25 and 54 years (3186/6131; 52.0%), with the exception of typhoid fever where the major burden was in patients aged 5 to 14 years (302/855; 35.3%). KH Keddy 81-11384 PhD iv More HIV-infected adult women were observed with invasive shigellosis (P=0.002) and with typhoid fever compared with adult men (P=0.009). Adults aged ≥ 15 years were more likely to die than children aged < 15 years (invasive shigellosis, odds ratio [OR]=3.2, 95% confidence interval [CI]=1.6 – 6.6, P=0.001; Salmonella meningitis, OR=3.7, 95% CI=1.7 – 8.1, P=0.001; typhoid fever, OR=3.7, 95% CI=1.1 – 14.9, P=0.03; invasive nontyphoidal salmonellosis, OR=2.0, 95% CI=1.6 – 2.5, P<0.001). HIV-infected patients had a significantly higher risk of mortality compared with HIVuninfected patients (invasive shigellosis, OR=4.1, 95% CI=1.5 – 11.8, P=0.008; Salmonella meningitis OR=5.3, 95% CI=1.4-20.0, P=0.013; typhoid fever, OR=11.3, 95% CI=3.0 – 42.4, P<0.001; invasive nontyphoidal salmonellosis OR=2.5, 95% CI=1.7 – 3.5, P<0.001). In all patients, severity of illness was the most significant factor contributing to mortality (invasive shigellosis, OR=22.9, 95% CI=2.7 – 194.2, P=0.004; Salmonella meningitis OR=21.6, 95% CI=3.5 – 133.3, P=0.01; typhoid fever, OR=10.8, 95% CI=2.9 – 39.5, P<0.001; invasive nontyphoidal salmonellosis OR=5.4, 95% CI=3.6 – 8.1, P<0.001). Between 2003 and 2013, ART was significantly associated with decreasing incidence rates of invasive nontyphoidal salmonellosis in adults aged 25 - 49 years (R=-0.92; P<0.001), but not in children (R=-0.50; P=0.14). Conclusion Decreasing incidence rates of invasive nontyphoidal salmonellosis and shigellosis suggest that ART is having an impact on opportunistic enteric disease in HIV. Further work is necessary however, to fully understand the associations between age, sex and invasive enteric pathogens. Specifically, this work would include typhoid fever, Shigella transmission from child to adult carer, development of invasive enteric infections in HIV-exposed children and whether the decreasing incidence rates can be sustained. Moving forward, an understanding of invasive enteric infections in the HIV-uninfected patient may assist in targeting severity of illness as a risk factor for mortality. / MT2017

A Thesis Submitted to the School of Public Health, Faculty of Health Sciences University of the Witwatersrand, Johannesburg, South Africa, in fulfilment of the requirements for the Degree of Doctor of Philosophy 15 June 2017 / Background HIV infection has remained a major global health burden since its discovery in 1983 and Sub-Saharan Africa remains the region hardest hit by the HIV/AIDS pandemic. The HIV pandemic continues to ravage most parts of Southern African countries, current prevalence between 10-20%. Individuals worldwide differ in their degree of susceptibility to HIV infection and genetic polymorphisms play a major role. Mannose Binding Lectin (MBL) is one such immunological factor found in serum/plasma, it is a normal liver-derived protein and is a key component of the innate immune defence system. MBL deficiency, due to mutations in the MBL2 gene and promoter region, leading to decreased plasma/serum MBL concentration, characterised by defective opsonisation activities of the innate immune system and increased susceptibility to infections including HIV-1 and schistosomiasis. Rationale While there is a lot of advancement in HIV prevention and treatment in Southern African countries, there is still need to investigate host genetic molecules in adults and mother-baby pairs that could be playing a role in HIV-1 transmission/acquisition, disease progression and survival. It was imperative to carry out this study because of the need to quantify the burden of MBL deficiency in this Zimbabwean adult and PMTCT study populations. Alsoto contribute to the knowledge gap on the role of MBL deficiency in HIV-1 transmission, disease progression and survival in African populations in adults and children. The available literature shows that the majority of studies on the association of MBL deficiency and HIV-1 infection in adults and children have been done on populations outside the African continent. There is dearth of information on the role of MBL in this era when access to ART has greatly improved even in developing countries like Zimbabwe. This will be the second study that will assess MBL2 genes and promoter typing in mother-infant pairs in HIV vertical transmission/acquisition. This study aimed to identify and explore potential biomarkers for susceptibility to HIV infection and disease progression. We assessed role of MBL deficiency in HIV-1 and schistosoma infections in Zimbabwean adults enrolled in the Mupfure Schistosomiasis and HIV Cohort (MUSH Cohort) (Paper 1).We also assessed the role of MBL deficiency on HIV progression and survival in this African adult population. We hypothesized that MBL deficiency has a role to play in HIV infection by increasing HIV disease progression and decreasing survival (Paper 2). We also determined prevalence of MBL deficiency, as estimated by MBL2 haplotypes among Zimbabwean mothers and their children aged 9-18 months old as well as its association with risk of HIV-1 infection and vertical transmission from their HIV positive mothers (Paper 3). Main Aim The broad objective of this study was to determine the relationship between MBL deficiency and HIV infection in an adult population of males and females and among mother-infant pairs in Zimbabwe. Study Specific Objectives 1. To determine the prevalence of MBL deficiency among the Zimbabwean adult population. 2. To determine the relationship of MBL deficiency with HIV infection among the Zimbabwean adult population. 3. To determine the effect of MBL deficiency on disease progression and survival among the Zimbabwean adult population. 4. To determine prevalence of MBL deficiency among mothers and their infants in a Zimbabwean population. 5. To determine the relationship between MBL deficiency and HIV transmission from mother to child in a Zimbabwean population. Methods DNA and plasma samples for MBL and HIV analysis were collected from the 379 adult males and females from the MUSH cohort and stored dried blood samples from 622 mother infant pairs from a national PMTCT survey. HIV-1, S. haematobium and S. mansoni infections were determined at baseline using HIV commercial kits and parasitologically respectively. Plasma MBL concentration was measured by ELISA and MBL2 genotypes determined by PCR. We calculated and compared the proportions of plasma MBL deficiency, MBL2 structural variant alleles B (codon 54A>G), C (codon 57A>G), and D (codon 52T>C) as well as MBL2 promoter variants -550(H/L), -221(X/Y) and +4(P/Q) between HIV-1 and schistosoma co-infection and control groups using Chi Square test (Paper 1). We also assessed the role of MBL deficiency on HIV disease progression and survival inthe adult (MUSH) cohort.We analysed blood samples for MBL levels, MBL2 genotypes, HIV-1 status, viral load and CD4+ T cell counts (Paper 1). Participants were followed up for 3 years wherein the endpoints were measured at baseline, 6 weeks, 3, 6, 12, 24 and 36 months. Disease progression was measured as the rate of decline in CD4+ T cell counts and the rate of increase in HIV viral load (Paper 2). Generalised Estimating Equations (GEE) models were used to compare rates of change of the CD4+ T cell count and viral load measurements over the three-year follow-up period. The role of plasma MBL deficiency and MBL2 genetic variants on survival over the 3-year period were estimated using the Cox proportional hazard models. Regression analysis was used to test for interaction and confounding between MBL deficiency, MBL2 genetic variance, age and sex. We used the Wald Chi-square statistic to choose between full and nested models. We also assessed MBL2 polymorphisms in Zimbabwean HIV positive mothers and their children enrolled in a national PMTCT survey carried out in 2012. MBL deficiency was defined as presence of A/O and O/O genotypes in the mothers and their children. We extracted DNA from two dried blood spots for 622 mothers and infant pairs using the Gene Extract and Amp kit reagents. MBL2 Exon 1 genotypes and promoter region alleles -221(X/Y) and -550(H/L) SNP were detected by pyrosequencing. Differences in distribution frequency between HIV infected and uninfected children, of the MBL2 genotypes, promoter region variants and MBL2 haplotypes, were determined by the Chi square test or Fisher’s exact tests (Paper 3). Key findings For specific objective number 1, we assessed 379 adults, 80% females, median age (IQR) 30 (17-41) years. HIV-1, S. haematobium and S. mansoni prevalence were 26%, 43% and 18% respectively in the MUSH baseline survey. Median (IQR) plasma MBL concentration was 800μg/L (192-1936μg/L). Prevalence of plasma MBL deficiency was 18% with high frequency of the C (codon 57G>A) mutant allele (20%). For specific objective number 2, we found no significant difference in median plasma MBL levels between HIV negative (912μg/L) and HIV positive (688μg/L), p=0.066. However plasma MBL levels at the assay detection limit of 20μg/L were more frequent among the HIV-1 infected (p=0.007). S. haematobium andS. mansoni infected participants had significantly higher MBL levels than uninfected. All MBL2 variants were not associated with HIV-1 infection but promoter variants LY and LL were significantly associated with S. haematobium infection (Paper 1). For specific objective number 3, we assessed 197 HIV positive adults where 83% (164) were women with a median age of 31 years old. Prevalence of plasma MBL deficiency (less than 100μg/L) and MBL2 deficient genetic variants (A/O and O/O genotypes) was 21% (42 out of 197) and 39% (74 out of 190), respectively. We did not observe a significant role to explain individual variation in mortality, change of CD4+ T cell count and viral load by MBL plasma deficiency or MBL2 genetic variants from baseline to 3 years follow up period in this adult population (Paper 2). For specific objective number 4, from the PMTCT study, the median age (IQR) of the mothers was 30(26 - 34) years and the children mean age (IQR) was 12 (11-15) months old at the time of enrolment. All 622 mothers were HIV-1 infected, 574 babies were HIV negative and 48 were HIV-1 positive babies. MBL2 normal structural allele A and variants B (codon 5A>G), C (codon 57 A>G) and promoter region SNPs -550(H/L) and -221(X/Y) were detected. Prevalence of MBL deficiency was 34% among the mothers and 32% among the children. For specific objective number 5, we found no association between maternal MBL2 deficiency and HIV-1 transmission to their children. We found no difference in the distribution of HIV-1 infected and uninfected children between the MBL2 genotypes of the mothers and those of the children (Paper 3). Conclusions The results from our study indicate high prevalence of MBL deficiency but we found no evidence of association between MBL deficiency and HIV-1 infection. However, lower plasma MBL levels were associated with reduced prevalence of both S. haematobium and S. mansoni infections and MBL2 promoter and variants LY and LL were associated with increased susceptibility to S. haematobium infection (Paper 1). Our findings attest to the large between-population variability in a host of factors that can predispose individuals susceptible to HIV progression and mortality. We therefore cannot recommend at this time the use of plasma MBL levels or MBL2 genetic variants as a prognostic marker in HIV infection, disease progression and survival in this adult population in Africa (Paper 2). MBL deficiency was not associated with HIV-1 infection among the children nor was it associated with HIV-1 vertical transmission in this study population (Paper 3). / MT2017