Vitamin A has well recognised benefits for the reduction in severity of diarrhoeal episodes but the impact of therapeutic doses given during diarrhoea on the biochemical and clinical outcomes is less clear. In this study these potential therapeutic benefits were investigated to establish the optimum time for vitamin A supplementation to improve vitamin A status. Establishing the optimum time for vitamin A supplementation during an infectious stage would improve cost effectiveness and clinical benefit. Young children (174) between the ages of 3 and 60 months with severe diarrhoea were randomised in a double - blinded placebo controlled trial into one of 2 groups. The 1 st group received 60 mg of retinol as retinyl palmitate on admission during the acute diarrhoeal stage. The 2nd group received the same dose of vitamin A once symptoms had resolved, usually between 3 - 7 days. At each of these two time points, children not receiving vitamin A were given an identical placebo dose. Baseline (day 0) and day 3 serum samples were collected for vitamin A, retinol binding protein (RBP) and other biochemical markers. At four and eight weeks after discharge both morbidity and weight gain were recorded. The modified dose response test (MRDR) was conducted at the eight - week follow - up to estimate vitamin A liver stores. Initially, most of the children presented with watery diarrhoea and dehydration and were clinically very ill. At day 3 plasma retinol concentrations improved in both groups viz. from 0.57umol/L to 0.97umol/L in the 1st group and from 0.49umol/L to 0.90umol/L in the 2nd group. Similar improvements were found in retinol binding protein viz. 21.28 mg/L to 31.06 mg/L in the 1st group and 17.05 mg/L to 24.80 mg/L in the 2nd group. At 8 weeks there was also no significant difference between the two groups either for serum retinol (0.69umol/L and 0.73umol/L respectively) nor for MRDR ratios (0.036 and 0.049 respectively). The MRDR results at 8 weeks indicated that these children did not have depleted vitamin A liver stores and that the low serum retinol levels seen at baseline were probably due to the acute phase response during an infectious episode. The results of these analyses showed no significant difference between the two treatment groups thus indicating that there was no benefit to giving vitamin A on recovery from an infectious episode instead of on admission, as is currently practised. / Thesis (M.Med.Sc.)-University of Natal, Durban, 2001.

BACKGROUND Sub-saharan Africa remains to date at the forefront of the HIV/AIDS epidemic. Despite breastfeeding being a significant mode of postnatal HIV transmission it remains the main nutritional source and pillar of child survival for the majority of infants born in Africa. It is therefore, not surprising that considerable research has centred on making breastfeeding safer in terms of HIV transmission. The flash heat treatment method (HTEBM) provides a unique opportunity to safely breastfeed infants but prevent mother-to-child transmission of HIV. Cytotoxic T lymphocyte (CTL) responses have been well documented in HIVinfected adults and children. However, there is a lack of literature on CTL responses in HIV exposed low birth weight infants. This pilot study attempted to examine the association of early neonatal feeding on the clinical outcomes and CTL responses in HIV exposed low birth weight infants. METHODS Seventy-seven patients that fulfilled inclusion and exclusion criteria were enrolled. The clinical outcomes of these patients were evaluated over a 9 month period. Fifty-five of these patients were also investigated for cytotoxic T lymphocyte (CTL) responses by means of the IFNγ ELISpot (megamatrix and confirmation) assays at the 6 weeks, 3, 6,and 9 months follow-up. RESULTS Two HIV-1 infected infants generated a CTL response at a single time point using the ELISPOT matrix screening assay. These responses could not be confirmed and were undetectable at any of the consecutive visits. At the time of detection of responses the infants were fed unheated breastmilk. HIV-1 exposed uninfected infants were unable to elicit a HIV-1-specific CTL response irrespective of feed. With regards to clinical outcomes, infants born o HIV infected mothers with a CD4 count < 500cells/μl were 2x more likely to acquire other infections at birth compared to those infants born to HIV infected mothers with a CD4 count >500cells/μl. Also, infants born to HIV infected mothers with advanced disease (CD4 count 0-200 cells/μl) had a lower birth weight compared to infants born to HIV-1 infected mothers with a CD4 count > 350 cells/μl. We also investigated the feasibility of the flash heat treatment method at birth. While inhospital, 38 HIV-1 infected women fed their infants HTEBM after receiving counseling and support from the nursing staff at the King Edward VIII hospital. The numbers decreased rapidly post hospital discharge, mainly due to mixed feeding. DISCUSSION In conclusion we have shown that it is feasible for HIV infected mothers to heat treat their expressed breastmilk during hospital admission. Furthermore, we were able to demonstrate in this small cohort of patients that the clinical outcomes and growth parameters of infants fed HTEBM were similar to that of infants fed either formula or unheated breastmilk. We were unable to demonstrate HIV-specific responses in the infected infants or the uninfected infants who had been exposed to heat inactivated virus in HTEBM. Our findings indicate that this pilot study was limited in its ability to detect CTL responses in HIV exposed low birth weight infants and further studies are warranted. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

Conventional pertussis vaccine prepared from killed whole cell B. pertussis organisms has been in widespread use since the early 1950's. Despite marked reductions in the incidence of pertussis, the use of the vaccine has caused concern because of questions of significant adverse reactions. Whooping cough is not notifiable in South Africa, and there is consequently a paucity of hard data on efficacy; in addition few cases are proven. Incidence, prevalence, severity and transmission of the disease hence remain a matter of conjecture. In order to provide background information and determine baseline data for undertaking further studies, available clinical and epidemiological data on whooping cough (pertussis) in South Africa was collated. It was intended to compare the pattern of disease seen in this country with that known in other parts of the world. Clinical and epidemiological findings from 1525 whooping cough admissions (diagnosed on the basis of clinical criteria) obtained from 6 major infectious disease hospitals around the country for the period 1980-1987/1988 are reported. The data from Durban were available in some detail. Incidence, morbidity and mortality of the disease in South Africa, as elsewhere, was higher in infancy. Infants and young children were predominantly affected, with 31.3% of cases under 12 months of age and 7.2% less than 2 months. Mortality was disproportionately higher in infancy; 53 .2% of deaths were in those younger than 12 months of age. There was a slight female preponderance, both in respect of prevalence and mortality. Patients were admitted with pertussis throughout the year, although there was a peak during the winter months (May to October). The typical whoop accompanied the cough in 55.6% of patients . A raised white cell count was recorded in 66% of patients. The most commonly detected The average duration of hospital stay was between 1 0-13 days. Of those with vaccination records, 26% were unvaccinated, 44% had 1 or 2 doses, and 27% had been fully vaccinated with a whole-cell pertussis vaccine combined with diphtheria and tetanus toxoids. The picture, incomplete though it is, reveals a pattern of pertussis similar to that described in other developing countries. The study reveals huge gaps in our knowledge of this subject in South Africa. More research needs to be done, particularly with respect to improved diagnosis, prevention and treatment; further pertussis should be made a notifiable disease in South Africa. The whole-cell pertussis vaccine currently used in South Africa has not been adequately evaluated for post-vaccination events and immunogenicity. The development of new purified component pertussis vaccines, which appear to be safer than conventional whole-cell preparations and of equal or almost equal efficacy (although optimal vaccine composition remains to be defined), requires that the concept of early vaccination with this vaccine compared with conventional whole-cell vaccine be examined in order to optimise the immune response to these vaccines in infants; more especially since neonates do not appear to benefit from passive immunity. Acellular pertussis vaccine has not been evaluated previously in neonates. In order to address the problem of high morbidity and mortality from pertussis in early infancy; and the incorporation of the vaccine into routine vaccination schedules, a phase 11 trial of acellular and whole-cell vaccines was undertaken in very young infants. The effect of neonatal vaccination with acellular pertussis vaccine on subsequent immunity; and the immunogenicity and shortterm safety and reactogenicity of routine primary vaccination with acellular vaccine compared with conventional whole-cell preparations was investigated. Three hundred and forty-five healthy, full-term African babies were, enrolled in the study at birth; 58% of whom were successfully followed for 9 months. Infants were assigned to 1 of 3 vaccine groups in sequence at birth and received either acellular or whole-cell pertussis vaccine , combined with 0 and T (A-OTP or W-OTP) at 2, 4 and 6 months of age. Groups I and 11 received A-OTP and Group III W-OTP. In addition, at birth, Group I received an additional dose of A-OTP and Group 11, a saline placebo injection. No unvaccinated controls were studied for ethical reasons. Serologic IgG responses to 3 major protective antigens of B. pertussis, filamentous haemagglutinin (FHA), pertussis toxin (PT) and fimbrial agglutinogens 2,3 (AGG2,3), were measured by ELlSA in sera obtained at birth, and before vaccination at 2, 4 and 6 months and at 9 months of age. The incidence and nature of post-vaccination events were recorded for 14 days after each dose. A-OTP induced serum IgG responses to PT and FHA comparable with those reported in other studies, with peak PT titres occurring at 6 months of age after 2 doses in babies vaccinated according to the routine schedule (Group 11). Surprisingly, response to W-OTP was found merely to restore levels of antibody to PT and FHA to those found in cord blood after 3 doses of vaccine, which questions the immunogenicity of the South African product. Four doses of AOTP (Group I) did not produce a better antibody response than the 3-dose schedule. Incidence of all post-vaccination events to both acellular and whole-cell vaccines was low (85.96/1000 doses, 136.36/1000 doses and 76.30/1000 doses in Groups I, 11 and III respectively). Minor symptoms were more common in recipients of A-OTP, although no significant differences in rates were demonstrated. Neurologic post-vaccination events (without sequelae) occurred more frequently hi recipients of W-OTP. No infant vaccinated with A-OTP from 2 months of age (Group 11) experienced a neurologic symptom. The risks of major post-vaccination events cannot however be fully quantified In a study of this small size and diseases. A-DTP vaccination commencing at birth produced final antibody titres to PT and FHA which were superior to those of South African whole-cell vaccine but were considerably lower than when the vaccine was incorporated into routine schedules commencing at 2 months of age. The study findings suggest that acellular pertussis vaccines, whether given from birth or from the age of 2 months, appeared safer and produced a better serologic response than the South African whole-cell product which may have impaired immunogenicity. During the course of the above study, 11 full-term infants with pertussis infection (10 subclinical) were retrospectively diagnosed on the basis of ser~logic evidence. Of the infants with subclinical disease, all 10 had a ~ 4-fold rise in at least 2 different pertussis IgG antibodies and nine had ~ 4-fold rise in all 3 IgG antibodies measured. Seven infants had raised IgA antibodies to PT and FHA and 4 infants had raised IgA antibodies to AGG2,3. Subclinical infection provoked antibody production to multiple antigens to differing degrees. The role of various factors which may have contributed to asymptomatic infection were analysed, viz - household contacts; type of antibody response (clinical vs. subclinical; vaccinated vs. subclinical); maternally acquired antibody levels; vaccination status (number of vaccine doses received); age and gender; and nutritional status. SpeCial features of the study which require emphasis are: pertussis remained subclinical in unvaccinated babies (most of the subjects were unvaccinated). Subclinical infection followed incomplete primary vaccination with either acellular or whole-cell vaccines commencing at 2 months of age. Subjects were 8 months of age or younger and only 1 had completed primary vaccination. Other infections of infancy were commonly detected; 4 infants had upper Likelihood of subclinical infection was related to significantly lower levels of maternallyacquired pertussis IgG-AGG2,3 antibodies but not associated with infants' nutritional status. Subclinical pertussis is described in very young African babies at an age when the disease is most severe, and therefore has implications for infant morbidity and mortality; it is also relevant to disease surveillance and vaccine-efficacy studies . Some perinatal factors influencing vaccination were also explored . In this context we looked at: i. The acquisition of maternal antibodies to B. pertussis in paired mother-infant sera from both well -nourished and SGA full-term and pre-term infants, and infants who subsequently developed pertussis infection, and effect of these maternally-acquired antibodies on subsequent antibody responses to acellular pertussis vaccine administered soon after birth, and to acellular and whole-cell vaccines administered from the age of 2 months. if. The acquisition of maternal antibodies to diphtheria and tetanus (OT) in paired motherinfant sera from full-term and pre-term infants, and the effect of these maternallyacquired antibodies on serologic responses to neonatal OT vaccination followed by whole-cell OTP vaccination at 2, 4 and 6 months. Maternal antibodies were measured since little is known about materno-fetal transfer of pertussis antibodies, especially in African countries where inhibition of placental transfer might occur for a variety of reasons. Furthermore, the effect of peri-natal malnutrition and prematurity on transplacental transfer of diphtheria, tetanus and pertussis antibodies has not been conclusively established in these areas. sera with levels in the latter frequently being higher, Indicating active transplacental transfer of antibodies. The significant pertussis antibody levels in maternal sera were unlikely to be due to the currently used South African whole-cell vaccine (given the poor antibody response to PT and FHA shown in this study). It is assumed that the presence of these antibodies are the end result of natural infection and therefore that pertussis is widespread in the African community. Maternal and cord IgG-PT and AGG2,3 titres were significantly lower (p <0.05) and maternal IgG-FHA marginally lower (p ... O.05) in SGA infants compared to cohorts, although placental transfer was equally efficient in both groups~ This study has demonstrated that the high titres of maternally derived circulating B. pertussis antibodies do not have an inhibitory effect on the subsequent serologic response to acellular vaccine administered in early infancy (2 months) or with the first dose given soon after birth. Protective levels of diphtheria and tetanus antibodies were detected in 100% and 76% of cord sera in pre-term infants and in 85% and 67% of cord sera in full-term infants. Although the number of infants studied was too small to make a definite comment, there did not appear to be 'tolerance' due to neonatal diphtheria-tetanus vaccination of pre-term infants, or to high levels of maternally-acquired antibody. During analysis of the data from the phase 11 study of acellular pertussis vacclnes, 25 infants with protein-energy-malnutritlon (PEM) were detected on the basis of anthropometric indices. Seventeen infants were smaIJ-for-gestatlonal-age (SGA). of whom 9 developed PEM by the age of 9 months. Eight other infants developed post-natal PEM before the completion of the primary vaccination course. and extent of immunological impairment, if any, on the serologic responses to acellular vaccine and to conventional whole-cell OTP in SGA infants, and in infants who developed PEM before the completion of the primary vaccination course. The following indices were evaluated in malnourished infants; (i) anthropometric indices of nutritional status, (ii) intercurrent illnesses including pertussis infection, (iii) post-vaccination events, (iv) serologic responses to vaccination. Results were compared with those obtained in well-nourished (WN) age- and vaccine-matched cohorts. Overall, peak titres and seroconversion data of all 3 antibodies were not significantly different in malnourished infants though final anti-AGG2,3 titres (at 9 months of age) in Group III were significantly lower (p = 0.035). Although peak and final PT and FHA antibody titres in many SGA and malnourished infants were lower than in WN infants and peak responses were attained at a later age, malnutrition did not significantly affect the response to A-OTP. Peak and final AGG2,3 antibody titres were similar in SGA, malnourished and WN infants. Overall malnourished infants responded no less well to pertussis vaccination than did other vaccinees. Incidence of minor local and systemic post-vaccination symptoms was not significantly different in PEM and WN groups although induration at injection site and irritability were more frequently reported in the latter. No major neurologic post-vaccination symptoms to either vaccine were reported in SGA infants or infants with PEM at the time of vaccination. No significant differences was noted in the incidence of major symptoms in PEM as compared with WN infants. One male infant (Group I) who was malnourished at birth and who had been given 2 doses of AOTP, developed clinical signs of pertussis infection between 2 and 4 months of age. Pertussis antibody levels immediately prior to infection were not significantly different from those of un infected age-matched cohorts. The percentage of infants afflicted with common childhood illnesses were similar in PEM and WN groups (46 vs. 43.2%) although the former group incurred significantly more illnesses at an earlier age' (s6 months) (p=O.05, chi square). These findings, albeit preliminary given the small numbers of subjects studied, suggest that acellular pertussis vaccine may be incorporated into routine vaccination schedules followed in developing countries with the expectation that adequate antibody responses will be provoked in SGA infants and in infants who develop post-natal PEM and that the incidence of vaccinerelated adverse effects will be no higher than in WN infants. Further and more extensive studies are indicated before the use of acellular pertussis vaccines can be recommended for routine primary vaccination of infants in preference to whole-cell preparations in developing countries. / Thesis (Ph.D.)-University of Natal, Durban, 1991.

Introduction: Integrated management of childhood illness (IMCI) is a child survival strategy that has been adopted in South Africa (SA) as the standard of care for managing sick children in the primary health care setting. IMCI includes guidelines for management of paediatric HIV. This study aimed to investigate effectiveness of IMCI as a vehicle to deliver essential child survival interventions, particularly HIV interventions, in routine practise in a high HIV prevalence setting, and to investigate barriers and enabling factors for IMCI implementation. Methods: The study was conducted in Limpopo and KwaZulu-Natal provinces, SA. In the qualitative component, focus group discussions were conducted with IMCI trained health workers and carers of children under 5 years, to explore experiences of IMCI implementation, particularly the HIV component, from the perspective of both target groups. A comparative survey was then conducted. Randomly selected IMCI trained nurses were observed for up to 20 consultations with sick children presenting consecutively to the facility, and their findings compared to those of an IMCI expert who subsequently assessed the child. Observed children were tested for HIV. Results: IMCI trained nurses found IMCI training informative and empowering, and there was agreement among nurses that their skills in managing sick children improved after training. Barriers to IMCI implementation included increased time required for IMCI consultations and lack of support from colleagues. IMCI trained nurses expressed reluctance to implement the HIV component of IMCI, believing it to be unnecessary, unacceptable to mothers and that they lacked the skills to implement HIV care. In total, 77 IMCI trained nurses were observed for a total of 1357 consultations between May 2006 and January 2007; nurses were observed for a mean of 17.7 consultations. Components of the IMCI assessment were frequently omitted; 14/77(18%) nurses asked about all main symptoms in every child. IMCI classifications were often incorrect; 52/112 (46.4%) children with a general danger sign were correctly classified. The HIV component was poorly implemented, 342/1357 (25.2%) children were correctly classified for HIV, although the HIV algorithm performed well when implemented by IMCI experts. Conclusion: IMCI implementation is fragmented and incomplete. Interventions are urgently needed to achieve and maintain high quality health worker performance in implementing IMCI. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

Human immunodeficiency virus type 1 (HIV-1) isolates can be differentiated based on their ability to use particular coreceptors – R5 viruses use CCR5, X4 viruses use CXCR4 and R5X4 (dual tropic) viruses use both CCR5 and CXCR4. It is widely reported that HIV-1 subtype C (HIV-1C) has a unique viral coreceptor evolution pattern in that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for this subtype compared to other subtypes. However, dual tropic HIV-1C isolates have occasionally been described. Furthermore, it has been reported that certain highly active antiretroviral drugs (HAART) may select for X4 viral variants. Therefore, this thesis study was undertaken to better understand the functional and genotypic characteristics of dual tropic HIV-1C isolates, and to characterize drug resistance and coreceptor usage patterns in HAART-naïve versus HAART-failing HIV-1C infected patients. Thirty-five functional HIV-1 env clones derived from seven dual tropic HIV-1C strains were generated and their coreceptor usage characterized in transformed cell lines. All 35 env clones efficiently infected transformed cells expressing CXCR4. Twenty of 35 clones (57%) also utilized the CCR5 receptor. No R5-only clones were detected. Functional coreceptor usage data was correlated to env gene sequence data. The ability of the HIV-1C env clones to facilitate infection of primary lymphocytes and monocyte-derived macrophages was next investigated. The majority of clones characterized as X4 or R5X4 on cell lines used either CXCR4 alone or CXCR4 and CCR5, respectively, in primary cells. A few viruses displayed comparable CCR5 and CXCR4 usage and clones from one virus preferred CCR5 usage in macrophages. Thus in a few cases coreceptor phenotyping in transformed cell lines does not predict usage in primary cells. Genetic determinants for coreceptor usage in primary cells require further investigation. Finally the patterns of drug resistance mutations were studied and coreceptor usage among 45 HAART-naïve and 45 HAART-failing HIV-1C infected patients analyzed. Ninety-five percent of HAART-failing patients had viruses with at least one drug resistance mutation. Thymidine analog resistance mutations (TAMs) were present in 55% of patients. HAART-failing patients had significantly higher prevalence (59%) of X4/R5X4-utilizing viruses compared to HAART-naïve patients (30%) (p<0.02) using the Trofile Co-receptor Tropism Assay while 41% of HAART-failing patients used CCR5 and 70% of HAART-naïve patients used CCR5. Functional results correlated with predictive algorithm methods. This study enhances our understanding of HIV-1 pathogenesis and the results have important implications for the use of coreceptor antagonists for the clinical management of HIV-1C infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2011.

Mortality and morbidity rates associated with tuberculous meningitis (TBM) are substantial. The average duration of the untreated disease from onset to death is about 17 days. The prognosis of TBM is known to correlate with the stage of the disease at the time of diagnosis and commencement of chemotherapy. Early diagnosis improves the chances of recovery without neurological sequelae. Early diagnosis is a problem because the presenting symptoms are non-specific and the onset of the disease is typically insidious. To date no single test is available that is totally reliable and specific for TBM. I have attempted to develop a reliable and easily applicable test for the diagnosis of TBM. In fulfilling this objective, the work undertaken may be divided into three major sections:- 1. Detection of soluble Mycobacterium tuberculosis antigens in the cerebrospinal fluid (CSF) of patients with TBM and in control groups by using Mycobacterium bovis BCG antigens. The technique used was that of inhibition enzyme-linked immunosorbent assay (ELISA). The principle of this technique is illustrated in Fig. 5. 2. Detection of soluble M. tuberculosis antigens in the CSF of tuberculous and control groups of patients by using antibodies raised against M.bovis BCG. The technique used was that of the double antibody sandwich ELISA. An outline of this ELISA is given in Fig. 6. 3. Correlation of chloride levels in the blood and CSF of patients with tuberculous and other forms of meningitis. It has been established that the SERUM/CSF ratio of bromide tends towards unity in patients with TBM because the permeability of the blood-brain barrier is impaired. Since both bromide and chloride are chemically similar (both being halides), it was thought that a similar pattern may exist for BLOOD/CSF chloride ratios; and this was investigated. The method used for the INHIBITION ELISA had to be standardized before the samples could be tested. This involved investigating the acceptability of various microtitre plates; determination of the optimal working dilutions for the coating solution and conjugate; and determination of optimal conditions for the various incubation periods, both in terms of time and temperature. A total of 70 specimens was tested. These consisted of 25 normal CSF controls; 25 pleural and ascitic fluid samples; 10 TBM samples, and 10 bacterial meningitis CSF samples. It was found that a distinction existed between the absorbance values obtained from positive TBM CSF samples (Mean 0,658 + 0,043) and that from normal CSF samples (Mean 1,089 + 0,224). The mean absorbance of the culture-positive bacterial CSF's also differed significantly from the other 2 groups (Tables VII; IX). Some overlap occurred amongst the absorbance values of bacterial culture positive CSF's (Range 0,975-0,879) and normal CSF's (Range 1,486-0,934). The mean absorbance value for bacterial positive CSF samples (0,920 _+ 0,029) differed significantly (p <0,01) from those of normal CSF (1,089 + 0,224) and TBM CSF's (0,658 + 0,043). The difference between the mean values obtained with tuberculous and non-tuberculous groups of pleural and ascitic fluid was also significant (p < 0,01). The method used for the DOUBLE ANTIBODY SANDWICH ELISA was that of Sada et al. (1983). Before the samples could be tested, the method had to be standardized and similar investigations to those for the INHIBITION ELISA were performed. In addition, antibodies raised against M.bovis BCG were conjugated to alkaline phosphatase since no commercial preparation was available. Unfortunately no distinction was recorded between negative and positive test specimens, even on repetition of the entire procedure. Measurement of chloride was done by a fully automated procedure using the BECKMAN ASTRA-8. A total of 149 samples were tested. Of these 10 were tuberculous, 34 were viral, and the remainder were bacterial meningitis. No pattern was established that could differentiate TBM from viral or bacterial meningitis. The results obtained are tabulated in Table III and illustrated in Figures 9, 10, and 11. In summarizing, the use of the INHIBITION ELISA technique for the accurate diagnosis of TBM seems promising. However, its validity in the clinical situation will have to be assessed further and with greater numbers of specimens before it can be adopted as a diagnostic procedure for TBM. OBJECTIVE. To determine 1. The ability and reliability of the INHIBITION ELISA1 technique to detect mycobacterial antigens in pleural, ascitic, and cerebrospinal fluids. 2. The accuracy and reproducibility of the double antibody sandwich ELISA in the detection of mycobacterial antigens in CSF of patients with tuberculous meningitis (TBM). 3. Whether a correlation exists between blood and CSF chloride levels in patients with tuberculous and other forms of meningitis. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1985.

BACKGROUND Sub-Saharan Africa has the highest number of HIV-infected individuals and limited treatment programs. The use of Highly Active Antiretroviral Therapy (HAART) has resulted in a considerable decrease in morbidity and mortality among HIV-infected individuals. Long-term use of HAART has several limitations relating to cost, drug toxicity and adherence. Structured Treatment Interruption (STI) has been proposed as a therapeutic approach which limits the exposure to continuous HAART, but retains the benefits thereof. The role of HIV-specific Tcell responses in the control of viraemia has not been well studied in children and it is not clear when these responses become detectable or whether they are associated with improved viral control. Furthermore, antiretroviral drug resistance is well documented in adults infected with HIV-1 clade B virus but comparable information is lacking for chronic paediatric clade C virus infection. This pilot study focused on a chronic HIV-infected paediatric cohort from Durban, South Africa, to assess the immunologic and virologic responses in perinatal HIVinfected children undergoing STI. METHODS Thirty chronic HIV-infected treatment naïve children were enrolled and randomised into either the treatment interruption or continuous treatment group. Longitudinal measurements of viral loads and CD4 percentages were done at scheduled intervals. Peripheral blood mononuclear cells (PBMCs) were screened for cytotoxic T-lymphocyte (CTL) gamma interferon (IFN-?) enzyme-linked immunospot (ELISpot) assay responses using 410 peptides which spanned the entire HIV-1 clade C proteome. Intracellular cytokine staining (ICS) was done to distinguish between IFN-? Gag-specific T-helper and cytotoxic T cell responses. Pre-HAART drug resistance mutations testing and HLA typing were done for all children. RESULTS There was a significant increase in the median CD4 percentage after HAART was introduced. Six children randomized to the STI arm did not undergo treatment interruption because their viral loads remained detectable at the time of scheduled interruption. Most HIV proteins were targeted in this paediatric cohort. Gag was the most frequently targeted HIV-1 protein (93.1%). In both treatment groups, there were broadening of T-cell responses, however, the magnitude of T-cell responses decreased over time on HAART. Drug-resistant mutations were detectable in 4/29 children before initiation of HAART. CONCLUSION In this pilot study, the HIV-1-specific CD8+ and CD4+ T-cell responses were detected before and during HAART. Although the treatment interruption period was short, there were no adverse outcomes in either the continuous or treatment interruption groups in terms of death or other clinical outcomes. This study suggests that it is important to continue to explore alternative treatment strategies in order to reduce cost and toxicity as well as to enhance adherence. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2010.

Certain HLA-B-restricted CD8+ T cell responses are associated with control of viremia whereas HLA-Cw* restricted responses, including Gag epitopes are associated with high viremia. To better understand the role of HLA-Cw* restricted epitopes in viral control, HLA-Cw* restricted epitopes were optimally defined. Seventy eight study subjects from a cohort of 451 chronically infected participants had HLA-Cw* restricted CD8+ T cells responses as quantified by intracellular cytokine staining assessing IFN-γ secretion. Fine mapping and HLA restriction of the optimally defined HLA-Cw* restricted epitopes were performed using ELISPOT assay. Functional avidity of responses was assessed by peptide dilution in an ELISPOT assay. Two novel HLA-Cw* restricted epitopes Cw*04- TF10 (in reverse transcriptase) and Cw*08-RM9 (in gp120) were optimally defined. A previously described epitope, Cw*07- KY11 (Nef) was the most frequently targeted epitope in this cohort (30/78) and has high functional avidity compared to other HLA-Cw restricted CD8+ T cell responses. The polyfunctionality of HLA-B*57/5801-restricted Gag-specific HIV-1 CD8+ T cell responses and HLA-Cw*07-KY11 restricted CD8+ T cell responses within the same study subject was determined. Polyfunctionality of CD8+ T cell responses to HLAB* 57/5801 and HLA-Cw*07 restricted epitopes were determined in nine study subjects assessing IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a by multicolour flow cytometry. Additionally gag and nef genes were sequenced from plasma. HLA-B*57/5801-restricted IFN-γ-producing CD8+ T cell responses were of lower magnitude than HLA-Cw*07 responses (p=0.0012) for the nine subjects. The majority of responses were monofunctional (75%), irrespective of HLA restriction. HLA-B*57/5801 and HLACw* 07 restricted CD8+ T cells did not differ significantly in polyfunctionality (p=0.84). Possession of ≥3 functions correlated positively with CD4+ T cell counts (r=0.85; p=0.006). The percentages of monofunctional CD8+ T cells inversely correlated with CD4+ T cell counts (r=-0.79; p=0.05). There was no correlation between polyfunctionality and viral load and sequence variation within targeted epitopes did not impact polyfunctionality. These results suggest that polyfunctionality of HIV-1-specific CD8+ T cells is associated with disease progression independent of restricting HLA alleles, and that loss of these polyfunctional cells correlates with increased in the frequency of monofunctional virus-specific CD8+ T cells. In addition, sequence variation does not appear to significantly impact CD8+ T cell polyfunctionality in chronic HIV infection. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2010.

No abstract available.

Background: in 1987, the first child with HIV-1 infection was identified in the paediatric wards at King Edward VIII Hospital in Durban. This made paediatricians aware that the epidemic had spread to the children of KwaZulu/Natal. Although information on transmission and natural history was becoming available from developed countries, little was known about the disease in developing countries. It was important to determine transmission rates and disease patterns in the local population, in order to appropriately counsel women, and for management of infected infants. In addition, with resources for laboratory diagnoses being limited in developing countries, much emphasis had to be placed on clinical findings for identification of infected children. In 1989, a retrospective analysis was made of the HIV-infected children seen over a 2-year period, between 1987 and 1989. Nine such children were identified and their clinical and biochemical features were described. It was concluded that HIV infected children presented with an identifiable pattern of signs, fairly similar to that described for children in industrialised countries. With these findings, a prospective study was undertaken, to determine the vertical transmission rate, the factors affecting this rate, and natural history of vertically transmitted I-IIV-1 infection. ix KwaZulu/Natal, being at the epicentre of the epidemic in South Africa, was a natural site for the study. Patients and Methods: a trained research worker was placed in the antenatal clinic at King Edward VIII Hospital for the specific purpose of educating, counselling, and testing of all women attending the clinic. Women attending the clinic for the first time in the index pregnancy were offered HIV testing if informed consent was obtained. Blood for HIV serology was drawn at the same time as sampling for the obligatory syphilis serology. The acceptance rate for sampling was > 95%. The majority of the women attending the clinic were black, and first attendance was generally late, into the third trimester. The same research worker was responsible for post-test counselling which was offered to all the women, not only those who tested positive. This research worker was also responsible for obtaining maternal consent for entering the newborn infant into the study. All newborn infants were seen within 48 hours of birth. At this time they were examined, growth parameters were recorded, and initial blood samples taken. These infants were then followed-up at 1 month, 2 months, 3 months, then at 3-month intervals up to 18 months, then at 6-month intervals. At each visit, a thorough clinical examination was performed, growth measurements taken, and development assessed. Record was made of any interim illness and visits to health centres, and of hospital admissions. Method of feeding was note& and details on immunisation obtained from the child's immunisation card. The children received all the x routine childhood immunisations according to the national regimen, based on WHO recommendations. Mothers were asked to bring the child to the follow up clinic for any problem, so that episodes of illness would not be missed. The women were reimbursed for transport costs to encourage follow up visits. Calculation of transmission rate and classification of infection status were made according to the recommendations of the Ghent workshop. Children were regarded as infected if they were antibody positive at 18 months or had an HIV related death. They were classified as uninfectd if the antibody test was negative at 9 months of age. Those infants who were lost to follow up before the age of nine months whilst still antibody positive and those whose cause of death could not be determined, were classified as indeterminate. The diagnosis of AIDS was based on the WHO criteria. Blood samples were taken at birth, at age one and three months, then at three month intervals to 18 months; thereafter at six month intervals. Sera were tested for HIV1 antibodies by a commercial enzyme-linked immunosorbent assay,ELISA. Samples that tested positive were confirmed by two tests, a Roche Elisa and by an immunoflourescent assay (IFA). A sample was regarded as being positive if both the second ELISA as well as the IFA or the Western Blot tested positive. xi Results: between October 1990 and March 1993, 234 infants and their 229 mothers were entered into the study. Those who did not attend a single follow up after birth were excluded from the study. The final cohort comprised 181 infants, of whom 48 were classified as infected ( including 17 deaths); 93 not infected, and 40 as indeterminate ( including 8 deaths). Maternal Data: about 60% of the mothers were under 30 years of age and were multiparous; 18% tested positive for syphilis serology; 22.9% were anaemic during pregnancy, and 37% were delivered by caesarean section. Most women lived in urban areas, and 16% chose to bottle-feed exclusively. Vertical Transmission Rate and Factors affecting this Rate: the median vertical transmission rate was 34%, (95% confidence intervals, CI 26%-42%). This figure is similar to that found in most parts of Africa, but much higher than those for Europe and USA. The maternal factors found to be associated with an increased risk of transmission were vaginal deliveries and a low haemoglobin level during pregnancy. Breastfeeding, Transmission, and Outcome: breastfeeding was found to have an increased risk of transmission, by 15 % (CI 1.8-31.8). On assessing growth and morbidity, it was noted that breastfed infants were not protected against such common childhood infections as pneumonia and diarrhoea, and that failure to thrive occurred with equal frequency in both those breastfed as well as those receiving artificial feeds. Newborn Data: when comparing newborn data between those infants who were subsequently found to be infected with those who were uninfected, it was found that there were no major differences between these groups with regard to growth parameters and neonatal complications. However, those infants with rapidly progressive disease (those who died within 24 months), were noted to have lower mean birth weights and lengths, a higher frequency of low birth weights, and tended to have more neonatal problems. Clinical Manifestations: the first differences between the infected and the uninfected infants generally manifested from about 3 months of age. HIV infected children were identifiable by higher frequencies of thrush, lymphadenopathy, skin rash, and hepatosplenomegaly in the early stages, and later on with a higher tendency to neurological and developmental abnormalities, as well as of diarrhoea. Pneumonia was found with equal frequencies in both the infected and uninfected children. The HIV infected child could be distinguished fairly early in life by the combination of the manifestations described above. Progression to AIDS: AIDS was diagnosed in 44% of all the infected children during the study period. Ninety five percent of these children were identified by 12 months of life, showing a rapid progression of the disease Longitudinal Growth: when longitudinal growth parameters were analysed in this cohort, it was found that HIV infected children were stunted from as early as 3 months of age, and remained below the international standards into early childhood. Infected children were also found to be malnourished (i.e. weight for age below international means), from an early age, and this persisted throughout early childhood. Of note, the uninfected childrens' weights, although comparable to international means initially, dropped after the first year of life. However, both groups did not have significant wasting, when compared to international means. Mortality: there were 25 known deaths during the study period. Of these, 17 were classified as HIV-related, and 8 as indeterminate. The mean age at death was 10.1 months, with 83% of all the HIV-related deaths occurring within the first year of life. The commonest diagnoses at the ti me of death were diarrhoea, pneumonia, and failure to thrive; also, thrush was common, as were neurological abnormalities. / Thesis (MD)-University of Natal, Durban, 1999.