Thesis (MScMedSc)--University of Stellenbosch, 2011. / It is estimated that 32.8 million people are living with Human Immunodeficiency Virus (HIV) globally with the number of people receiving antiretroviral therapy in low- and middle- income counties increasing to more than 5 million people in 2009. These successes are threatened by treatment failure and the development of resistance to treatment. With an estimated 3.7% patients failing first line treatment after 2 years and 17.9% after 4 years on treatment there is a need for a practical and cheap in-house drug resistance assay that can be used to provide drug resistance data to clinicians and to use as a research tool to investigate drug resistance. In this study we attempted to optimize and validate an in-house drug resistance assay, adapted from Jacobs et al, 2008, to be used as a diagnostic tool and to study the presence of antiretroviral resistance in patients on the Western Cape Mother-To-Child-Transmission (MTCT) regimen. Quality control samples were received from The National Institute of Communicable Diseases AIDS Virus Research Unit, The Round Robin HIV-1 genotyping assessment system from the University of Würzburg and the QCMD assessment system were used for the optimization and validation of an in-house drug resistance assay. The ViroSeq™ HIV-1 Genotyping System was used for comparison of sample and mutation detection. It was possible to optimise and validate a genotyping assay for diagnostic testing and research use by comparison with the ViroSeq™ HIV-1 Genotyping System and evaluation with external quality assessment systems. This assay could subsequently be used to determine the development of genotypic-antiretroviral resistance in patients treated according to the provincial prevention of mother-to-child-transmission (PMTCT) protocol in the Western Cape (single dose nevirapine (sd-NVP), combined with a short course Zidovudine (AZT)). Patient samples were collected from pregnant women who took part in the Western Cape PMTCT program and visited the Tygerberg Obstetrics Clinic and Delft Community Hospital. EDTA blood was obtained to measure CD4-cell count, viral load, and to do genotyping for viral subtype and the presence of resistance mutations. Information on prior exposure to antiretroviral therapy was also collected. A detected resistance rate of 17.1% in this predominantly HIV-1 subtype C population is lower than previously recorded when sd-NVP was administered to HIV-1 subtype C positive patients in PMTCT programs. This could indicate that a dual PMTCT regimen including AZT and NVP reduces the risk of resistance to NVP relative to a regimen that uses sd-NVP. The genotyping assay uses four primers to amplify the PR and the RT gene separately to obtain PCR products, of 487 and 804 base pairs respectively for sequencing. The two PCR products were sequenced with three and five primers respectively to sequence the complete PR and approximately 250 amino acids of the RT gene. The sequences generated, thus, are analysed and aligned with the Sequencer V4.7 software to obtain a consensus sequence of approximately 1200 base pairs for analysis of resistance mutations in the protease and reverse transcriptase genes. The developed assay was hence further simplified and improved, by combining the PR and RT assay into one, which was optimised and validated for use in the routine diagnostic setting. The final genotyping assay uses 8 primers for sequencing to obtain a 1200 bp sequence for genotyping that contains the protease and the 5’ of the reverse transcriptase genes in which antiretroviral resistance associated mutations are found. The assay was accredited by SANAS in 2008.

Thesis (Ph.D.)--Murdoch University, 2006. / Thesis submitted to the Division of Health Sciences. Bibliography: leaves 138-177.

Thesis (MScMedSc) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The gag gene of HIV-1 encodes for one of the major structural proteins, which contains several conserved cytotoxic T cell (CTL) epitopes. Gag specific CTL responses are important in controlling viral load during acute infection and asymptomatic stages of the infection. Currently, only one complete South African HIV-1 subtype C gag sequence has been published. The first aim of this study was to characterise the complete gag gene of 15 HIV-1 subtype C isolates, to be used as a set of reference sequences in the design of a South African HIV-1 subtype C vaccine. Fifteen HIV-1 subtype C isolates selected for this study, were isolated during 1998 and 1999 from the HIV-1 positive patients attending the Infectious Disease Clinic at Tygerberg Hospital. The gag gene of these isolates was amplified by PCR, cloned into mammalian expression vectors and sequenced. Restriction digest analyses as well as phylogenetic analyses were performed on the sequencing data. Previously published mutational analyses and CTL epitopes were compared to the predicted amino acid sequences of the gag clones. Sequences of 23 complete gag genes representing the 15 HIV-1 subtype C isolates as well as one complete sequence of an HIV-1 subtype B isolate were compiled. Subtyping by restriction fragment length polymorphism (RFLP) would have correctly identified 14 of the 15 subtype C isolates as subtype C and one as unidentifiable. The subtype B isolate would have also been correctly identified. Phylogenetic analyses showed that our subtype C isolates clustered with reference subtype C strains from various countries, including Botswana, India, Israel, Tanzania and Zambia. Strains from Ethiopia and Brazil formed a separate subtype C cluster. The diversity between our isolates was comparable to the diversity seen between all the HIV-1 subtype C strains. Comparisons of previously published mutational analyses and CTL epitopes to the predicted amino acid sequences of the gag clones, showed conservation in most of the clones throughout the sequence. A second aim was to establish transfection and Western Blot techniques in our laboratory for use in future studies. An in vitro transcription! translation assay was performed on the gag clones and the protein producing clones were used to transfect mammalian cells using electroporation. A Western blot was then used to screen for Gag protein expression in the transfected cell Iysates. The in vitro transcription! translation assay showed that seven of the 23 clones could produce a protein of -55 kDa in size. Four out of the seven of these clones gave a weak expression of a-55 kDa protein after transfection in a mammalian cell line. Since the completion of the experimental work of this study, other cloned HIV-1 genes have successfully been transfected into mammalian cells using the electroporation technique and the proteins produced were screened for by Western blot. To conclude with; the native form of the gag gene does not elicit strong expression of the protein, but studies have shown that expression can be improved by sequence-modification of the gag nucleotide sequence. Due to the conservation of gag, the sequence of any subtype C strain can be used for the development of a Southern African vaccine. / AFRIKAANSE OPSOMMING: Die HIV-1 gag geen kodeer vir een van die hoof strukturele proterene en bevat verskeie sitotoksiese T-limfosiet epitope. Gag spesifieke sellulere immuun respons is belangrik vir die beheer van virale lading tydens akute infeksies en tydens asimptomatiese fases van die infeksie. Tans is slegs een volledige Suid Afrikaanse HIV-1 subtipe C nuklerensuur volgorde gepubliseer. Die eerste doel van hierdie studie was om die volledige gag geen van 15 HIV-1 subtipe C isolate te karakteriseer, om gebruik te word as In stel verwysings nukleiensuur volgordes, vir die ontwerp van In Suid Afrikaanse HIV-1 subtipe C entstof. Die 15 HIV-1 subtipe C isolate wat vir hierdie studie geselekteer is, is tydens 1998 en 1999 ge·lsoleer vanaf HIV-1 positiewe pasiente wat die Infeksiesiekte Kliniek, Tygerberg Hospitaal bygewoon het. Die gag geen van hierdie isolate is geamplifiseer deur PKR, gekloneer in soogdier ekspressie vektore en die nukleiensuur volgorde is bepaal. Die nuklerensuur volgorde is gebruik in restriksie ensiem analises asook filogenetiese analises. Reeds gepubliseerde mutasie analises en limfosiet epitope is met die voorspelde aminosuur volgorde van die gag klone vergelyk. Die nukleiensuur volgordes van die 23 volledige gag gene wat die 15 HIV-1 subtipe C isolate verteenwoordig, asook een volledige nukleiensuur volgorde van een HIV-1 subtipe B isolaat, is saamgestel. Subtipering deur middel van restriksie fragment lengte polimorfisme (RFLP) sou 14 uit die 15 subtipe C isolate korrek qerdentifiseer het, maar sou een nie kon identifiseer nie. RFLP sou ook die subtipe B isolaat korrek qerdentifiseer het. Filogenetiese analises het gewys dat ons subtipe C isolate met die verwysings subtipe C stamme van verskeie lande, insluitend Botswana, lndie, Israel, Tanzania en Zambie groepeer. Stamme van Ethiopie en Brasilie het In aparte subtipe C groep gevorm. Die diversiteit tussen ons isolate was vergelykbaar met die diversiteit tussen al die subtipe C stamme. Vergelykings van gepubliseerde mutasie analises en limfosiet epitope met die voorspelde aminosuur volgorde van die gag klone, het konservasie in meeste van die klone, deur die hele nukleiensuur volgorde, getoon. Die tweede doel was om die metodes van transfeksie en Westerse klad in ons laboratorium tot stand te bring. In vitro transkripsie/ translasie toetse is gedoen op die gag klone en die proteten produserende klone is gebruik om soogdierselle te transfekteer deur gebruik te maak van elektroporasie. In Westerse klad is toe gebruik om vir Gag proterenuitdrukkinq in die sellisate te toets. Die in vitro transkripsie/ translasie toets het getoon dat sewe uit 23 klone, In proteren van -55 kDa kon produseer. Vier uit die sewe van hierdie klone het In -55 kDa proteren swak uitgedruk na transfektering van soogdier selle. Sedert die voltooiing van die eksperimentele werk van hierdie stud ie, is ander gekloneerde HIV-1 gene suksesvol in soogdierselle getransfekteer met die gebruik van elektroporasie en die proterene is met In Westerse klad aangetoon. Ten slotte: die natuurlike vorm van die gag geen ontlok nie In sterk ekspressie van die proteren nie, maar ander studies het wei aangetoon dat die ekspressie verbeter kan word met modifikasie van die gag nukleiensuur volgorde. As gevolg van die konservasie van gag, kan die nuklerensuur volgorde van enige subtipe C stam gebruik word vir die ontwikkeling van In Suider Afrikaanse entstof. / The Poliomyelitis Research Foundation / The South African AIDS Vaccine Initiative / Harry Crossley Foundation

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: At the end of 2002, human immunodeficiency virus (HIV) had infected 42 million people worldwide. The morbidity and mortality rate, as well as the epidemic proportions of the disease have led to concentrated scientific efforts to reveal the disease's pathogenesis and develop effective preventative and treatment measures. Advances have been made to inhibit viral replication by suppressing the virus' ability to replicate by developing antiretroviral treatments, although development of a save and effective vaccine is the only way to stem the pandemic. Advances in vaccine design, animal models and clinical research have led to the creation of promising candidate vaccines to counter this rampage, but most of these vaccines entering phase I-III clinical trials are based mainly only subtype B genomes. HIV-1 subtype C is the most commonly transmitted subtype worldwide, and is the predominant subtype in India, China, East and Southern Africa. A subtype C vaccine is critical for the developing nations such as South Africa, where antiretroviral therapies are largely unaffordable. The envelope gene (env) is an attractive target as immunogen to be included in a HIV vaccine. The envelope protein (Env) elicits neutralising antibodies and cytotoxic T-Iymphocyte (CTl) responses. This protein will therefore be useful in creating a humoral and cellular immune response in the host. A shortage in characterised subtype C env gene sequences from South Africa was recognised, and this study focussed on the characterisation of generated sequences, as well as the expression of selected env genes. These immunogens were created for possible use in a prime-boost vaccine modality. The env genes from recent circulating strains in South Africa were amplified by polymerase chain reaction (PCR). The genes were then cloned for sequencing and expression purposes. Phylogenetic relationships were determined by comparing the sequences to reference subtype strains and subtype C strains. Expression of the genes was assessed by Western Blot in 293 cells with HIV- 1 positive patient sera. Sequence analysis showed a more conserved third variable (V3) loop in South African subtype C sequences, with a more variable region downstream from the loop. The crown sequence (GPGQ) and positions of uncharged or negatively charged residues in the V3 loop indicated a non-syncytium-inducing (NSI) phenotype for the isolates. Phylogenetic analysis showed the sequences to all belong to the C subtype, and further that the sequences were not recombinant, which was confirmed by recombination analysis. The intersample diversity observed for strains from South Africa was significantly higher than distances observed to the subtype C consensus sequence. The South African sequences were distributed across several subclusters in a subtype C phylogenetic tree, highlighting the concept that these infections represent a more longstanding epidemic with multiple introductions from different geographic areas. Western Blot with HIV-1 positive patient sera showed the expression of uncleaved gp160 Env proteins, which were Rev dependent. This study has generated much needed subtype C South African env gene sequences that can be used as basis for modification for use as immunogens in a South African vaccine. / AFRIKAANSE OPSOMMING: Teen die einde van 2002 was 42 miljoen mense wêreldwyd geïnfekteer met die menslike immuniteitsgebrekvirus (MIV). Die dode- en sterfte syfers, asook die skaal van die epidemie, het gelei tot 'n wetenskaplike poging om die siekte se patogenese te openbaar en om effektiewe voorkomende en terapeutiese middels te ontwikkel. Vordering is reeds gemaak om die virus se replikasie te hinder deur die ontwerp van antivirale middels, alhoewel die ontwikkeling van 'n doeltreffende en veilige entstof die enigste manier is om die pandemie te stuit. As gevolg van die vordering in entstof ontwerp, diere modelle en kliniese navorsing is belowende kandidaat entstowwe wat die infeksie kan teenwerk ontwikkel, maar die meeste van hierdie enstowwe wat vir fase I-III kliniese proewe gebruik word is gebaseer op subtipe B genome. MIV-subtipe C is wêreldwide die algemeenste subtipe wat oorgedra word en is die oorheersende subtipe in lande soos Indië, China, oostelike en suidelike Afrika. 'n Subtipe C entstof word dringend benodig in ontwikkelende lande soos Suid-Afrika waar antivirale middels onbekostigbaar is. Die membraangeen is 'n aanloklike teiken om as immunogeen in 'n MIV entstof te dien. Die membraanproteïen lok neutraliserende teenliggame en sitotoksiese T-limfosiet reaksies uit. Die proteïen sal dus 'n humorale en sellulêre immuunrespons in die gasheer ontlok. 'n Tekort aan gekarakteriseerde subtipe C membraangeen volgordes van Suid-Afrika is opgemerk, en dus fokus hierdie studie op die karakterisering van gegenereerde volgordes, asook die uitdrukking van geselekteerde membraangene. Die immunogene is geskep om moontlik gebruik te word in 'n stimuleer-versterkingsenstof toedieningstrategie. Die membraangene van onlangs sirkulerende virusstamme in Suid-Afrika was geamplifiseer deur polimerase kettingreaksie (PKR). Die gene is daarna gekloneer vir beide volgordebepalings en uitdrukkingdoeleindes. Filogenetiese verhoudings is uitgewerk deur die volgordes met verwysingsstamme en subtipe C stamme te vergelyk. Uitdrukking van die gene is waargeneem in 293 selle deur die Westerse kladtegniek te gebruik met MIV-1 positiewe pasiëntsera as teenliggaam. Volgorde-analise het aangetoon dat die derde varieerbare (V3) lus meer gekonserveer is, en dat die gedeelte wat op die lus volg meer varieerbaar is. Die kroonvolgorde (GPGQ) asook posisies van ongelaaide of negatief gelaaide aminosure in die V3 lus het aangedui dat die isolate 'n nie-syncytia induserende fenotipe het. Filogenetiese analise het aangedui dat al die volgordes subtipe C is en dat die volgordes nie rekombinant is nie. Dit is ook deur rekombinasie analise bewys. Die inter-monster diversiteit van die Suid-Afrikaanse volgordes was hoër as die waargenome afstand vanaf die subtipe C konsensus volgorde. Die Suid-Afrikaanse volgordes is versprei oor verskeie subgroepe in 'n subtipe C boom, wat die konsep dat hierdie infeksies 'n meer gevestigde epidemie voorstel waar veelvuldige infeksies met verskillende geografiese oorspronge plaasgevind het beklemtoon. Die Westerse klad het ongeprosesseerde gp160 membraanproteïne aangetoon wat Rev afhanklik was. Hierdie studie het hoogs benodigde subtipe C Suid-Afrikaanse volgordes van membraangene geproduseer. Die volgordes kan as basis dien om die gene te modifiseer sodat dit gebruik kan word as immunogene in 'n entstof vir Suid-Afrika.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background: Sudden infant death syndrome (SIDS) is considered the second most frequent cause of infant mortality worldwide. Research specifically pertaining to SIDS is limited in the South African setting. Identifiable causes for sudden infant death remain challenging despite full medico-legal investigations inclusive of autopsy, scene visit and ancillary studies. Viral infections could contribute to some sudden unexpected death in infancy (SUDI) cases, especially since a multitude of respiratory viruses have been detected from autopsy specimens. The specific contribution of viruses in the events preceding death, including the subsequent involvement of the immature immune response in infants, still warrants deciphering. Infancy is characterised by marked vulnerability to infections due to immaturities of their immune systems that may only resolve as infants grow older when these sudden deaths rarely still occur. In South Africa there is a lack of a standard protocol for investigations into the causes of SIDS, including the lack of standard guidelines as to which specimens should be taken, which viruses should be investigated and which laboratory assays should be utilised. Objectives: In this prospective descriptive study we aimed to investigate the prevalence of viruses in SUDI and SIDS cases at Tygerberg Forensic Pathology Service (FPS) Mortuary over a one year period. The primary aim was to explore possible respiratory viral infections in SUDI and SIDS cases and to determine the usefulness of molecular techniques to detect viruses from SUDI cases. To determine the significance of viruses, we assessed signs of infection from lung histology. The secondary objectives included collecting demographic data to investigate possible risk factors for SUDI and to look for possible similarities between viruses confirmed in living hospitalised infants at Tygerberg, during the study period compared to viruses detected from SUDI cases. Methods: Between May 2012 and May 2013 samples were collected from 148 SUDI cases presenting at Tygerberg FPS Mortuary. As part of the mandatory routine investigations into SUDI, shell vial culture (SVC) results were collected from lung and liver tissue specimens and bacterial culture results were collected from left and right lung and heart swabs at autopsy. To investigate the possibility of viruses implicated in some of the infant deaths we used the Seeplex® RV15 Ace detection multiplex polymerase chain reaction (PCR) assay to establish the frequency of 13 ribonucleic acid (RNA) respiratory viruses (influenza A and B, human parainfluenza 1-4, human coronavirus [OC43, 229E/NL63], human rhinovirus A, B and C, respiratory syncytial virus A and B, human enterovirus and human metapneumovirus) from RNA extracted from tracheal and lower left and right lung lobe swabs. Tissue from the lower left and right lung lobes were also assessed for histology signs of infection. Results: During our study we confirmed multiple known demographic risk factors for SIDS, such as the age peak around 1-3 months, the male predominance, bed-sharing, sleeping in the prone position, heavy wrapping in warm blankets, prenatal smoke exposure, and socio-economic factors. With the Seeplex® RV15 Ace detection assay between one and three viruses were detected in 59.5% (88/148) of cases. Of the 88 cases that had viruses detected, 75% (66/88) had one virus and 25% (22/88) had co-detections of two to three viruses. The most common viruses detected were HRV in 77% (68/88) of cases, RSV in 18% (16/88) of cases and HCoV in 14% (12/88) of cases. Many of the viruses we detected from our cases are included in the SVC test that forms part of the medico-legal laboratory investigation for all SUDI cases at Tygerberg FPS Mortuary. SVCs were positive in 9.5% (14/148) of all cases only. We showed that the SVC method is potentially missing most of the 13 respiratory viruses we investigated that could contribute to death in some of the SUDI cases. Conclusion: In some cases that had a Cause of Death Classification - SIDS, the PCR viruses detected cannot be ignored, especially when it is supported by histological evidence of infection. We thus propose that the use of PCR could alter a Cause of Death Classification from SIDS to Infection in some of these cases. Further research is needed to determine the significance of detecting viruses from SUDI cases wherein no significant histological evidence of infection was observed. This questions whether PCR may be too sensitive and is detecting past and latent viral infections that do not play any role in the cause of death. The histological picture also requires further characterisation to determine if it accurately predicts infections or lethal events and can truly support virology findings, especially in young infants whose immune systems are still maturing. Without determining the true prevalence of viruses in SUDI cases and the viral-specific immune response, the contribution of virus-specific infections to this syndrome will remain largely undetermined. / AFRIKAANSE OPSOMMING: Agtergrond: Wiegiedood (“SIDS/SUDI”) word beskou as die tweede mees algemene oorsaak van sterftes in kinders jonger as een jaar wêreldwyd. Toegewyde SIDS-spesifieke navorsing in die Suid-Afrikaanse samelewing is beperk. Dit bly steeds „n uitdaging om oorsake te probeer identifiseer vir hierdie onverwagte sterftes in kinders (SUDI) ten spyte van volledige medies-geregtelike ondersoeke, insluitende die lykskouing, ondersoek van die doodstoneel en aanvullende ondersoeke. Virusinfeksies kan aansienlik bydra tot sommige onverwagte sterftes in kinders, aangesien verskeie respiratoriese virusse alreeds aangetoon is in monsters verkry tydens outopsies. Die spesifieke rol wat virusse speel in die prosesse wat die dood voorafgaan, asook die bydraende rol van „n onder-ontwikkelde immuunrespons in babas, regverdig verdere ondersoek. Die eerste jaar van lewe word gekenmerk deur verhoogde vatbaarheid vir infeksies weens die ontwikkelende immuunstelsels soos wat babas ouer word, en die voorkoms van SUDI neem stelselmatig af met „n toename in ouderdom. In Suid-Afrika bestaan daar tans geen standaard protokol vir die ondersoek van wiegiedood nie en daar is ook nie standaard riglyne oor die tipe monsters wat geneem moet word, watter virusse ondersoek moet word en watter laboratorium toetse uitgevoer moet word nie. Doelstellings: In hierdie prospektiewe beskrywende studie is gepoog om die virusse wat in gevalle van wiegiedood of SUDI voorkom te ondersoek. Die studie is uitgevoer by die Tygerberg Geregtelike Patologie Dienste lykshuis oor 'n tydperk van een jaar. Molekulêre tegnieke om virusse aan te toon in hierdie gevalle is gebruik om spesifieke virusinfeksies te ondersoek. Die resultate is met histologiese tekens van infeksie in longweefsel gekorreleer. Demografiese data is verder versamel om moontlike risikofaktore vir wiegiedood te ondersoek. Dit is verder vergelyk met virusse wat met dieselfde diagnostiese tegnieke in babas geïdentifiseer is wat tydens die studieperiode in Tygerberg Hospitaal opgeneem was met lugweginfeksies. Metodes: Monsters van 148 SUDI gevalle wat by die Tygerberg lykshuis opgeneem is, is versamel tussen Mei 2012 en Mei 2013. As deel van die roetine ondersoeke in SUDI gevalle, was selkultuur resultate verkry van long en lewer weefsel, asook bakteriële kulture van deppers wat van beide longe en hart geneem was tydens die lykskouings. „n Seeplex® RV15 Ace polimerase kettingreaksie (PKR) toets is gebruik om die teenwoordigheid van virusse te ondersoek wat moontlik by die babasterftes betrokke kon wees. Trageale- en longdeppers wat tydens die lykskouings versamel was, was getoets vir 13 ribonukleïensure (RNS) respiratoriese virusse (influenza A and B, human parainfluenza 1-4, human coronavirus [OC43, 229E/NL63], human rhinovirus A, B and C, respiratory syncytial virus A and B, human enterovirus and human metapneumovirus). Resultate: Ons studie het verskeie bekende demografiese risikofaktore vir SUDI bevestig, byvoorbeeld „n ouderdomspiek tussen een en drie maande ouderdom, manlike predominansie, deel van „n bed met ander persone, slaap posisie op die maag, styf toedraai in warm komberse, blootstelling aan sigaretrook voor geboorte en sosio-ekonomiese faktore. Die Seeplex® RV15 Ace toets het tussen een en drie virusse geïdentifiseer in 59.5% (88/148) van die gevalle. Uit die 88 gevalle waarin virusse opgespoor was, was selgs een virus in 75% (66/88) van gevalle gevind en twee en drie virusse in 25% (22/88). Die mees algemene virusse was HRV in 77% (68/88) van gevalle, RSV in 18% (16/88) van gevalle en HCoV in 14% (12/88) van gevalle. Baie van die virusse wat tydens hierdie studie ondersoek was, was ingesluit in die roetine selkultuur toets wat deel vorm van die standaard medies-geregtelike laboratoriumondersoeke in alle SUDI gevalle by die Tygerberg lykshuis, alhoewel die selkulture positief was in slegs 9.5% (14/148) van gevalle. Ons het gevind dat baie respiratoriese virusse potensieel gemisdiagnoseer word wat „n rol kon speel in of bydra tot die dood van sommige SUDI gevalle. Gevolgtrekking: In sommige gevalle waarin SIDS geklassifiseer is as die oorsaak van dood, kan die virusse wat met PKR toetse opgespoor is nie geïgnoreer word nie, veral waar die bevinding ondersteun word deur histologiese bewyse van infeksie. Ons stel dus voor dat die gebruik van PKR toetse die oorsaak van dood klassifikasie kan verander van SIDS na Infeksie in sommige van hierdie gevalle. Verdere navorsing is nodig om die waarde van gelyktydige opsporing van virusse in SUDI gevalle te bepaal wanneer daar geen noemenswaardige histologiese bewyse van infeksie gevind word nie. Dit bevraagteken of die PKR toets dalk te sensitief is en gevolglik vorige en latente virusinfeksies identifiseer wat nie noodwendig 'n rol in die oorsaak van dood speel nie. Die diagnostiese en kliniese waarde van die histologiese beeld in terme van die rol van virusinfeksies as bydraende oorsaak van dood moet verder ondersoek word, veral in jong kinders wie se immuunstelsels nog nie volledig ontwikkel is nie. Indien die werklike voorkoms van virusse in SUDI gevalle en die virus-spesifieke immuunrespons nie bepaal word nie, sal die rol van virus-spesifieke infeksies in hierdie sindroom grootliks onbekend bly. / Harry Crossley Foundation / Poliomyelitis Research Foundation (PRF) / National Health Laboratory Services Research Trust

Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: In 2007, AIDS caused an estimated 2.1 millions deaths worldwide; about 70% in sub-Saharan Africa. HIV preferentially targets activated CD4 T cells, expressing the major HIV receptor CD4, as well as the major chemokine coreceptors CCR5 and CXCR4. These coreceptors play a prominent role during HIV cell entrance phase, HIV transmission and also disease progression. They have been found to be differentially expressed by CD4 T cell subsets. Tuberculosis coinfection may enhance immune activation in vivo thus accelerating HIV disease progression and has become a major challenge in the control of TB in Africa. Introduction of HAART has reduced disease progression to AIDS, as well as risk of further morbidity and mortality. HAART results in a rapid decline of viral load and an initial increase of peripheral CD4 count, however little is known on the effect of HAART in regulation of coreceptor expression, immune activation status and CD4 T cell subset distribution in HIV infection and HIV/TB coinfection. This study is a cross-sectional analysis of coreceptor expression, immune activation status and CD4 T cell subpopulation distribution in South African HIV and HIV/TB coinfected patients before and after ARV. A total of 137 South African individuals were investigated, comprising 15 healthy normal donors (healthy subgroup), 10 patients with active pulmonary tuberculosis (PTB subgroup), 33 HIV-1 positive patients without active PTB (HIV subgroup), 23 positive patients with active PTB (HIV/PTB subgroup), 36 HIV-1 positive patients on ARV (HIV on ARV subgroup) and 20 HIV-1 positive patients with active PTB on ARV (HIV/PTB on ARV subgroup). CD4 absolute count and plasma viral load were determined for all donors. Freshly isolated PBMC were classified by flow cytometry into the following CD4+ T lymphocyte subsets: naïve (CD45+, CD27+), effector memory (CD45-, CD27-), central memory (CD45-, CD27+), and effector (CD45+, CD27-). Coreceptor expression and activation status was assessed by CCR5, CXCR4 and CD38 expression on CD4 T cell subsets. HIV, TB and HIV/TB coinfection was associated with a decrease in percentage CCR5+ T cells as compared to healthy controls, with the HIV/TB group showing the most extensive decrease. In treatment naive patients, CD4 T cells showed elevated surface expression of CCR5 and CD38 as determined by mean fluorescence intensity in HIV/TB co-infection compared to HIV infection alone. The percentage of antigen-experienced cells was higher in the HIV/TB co-infected group compared to the HIV group. The percentage of naïve T cells was decreased in both the HIV infected and the HIV/TB co-infected groups compared to healthy controls. HIV patients with more than 6 months of ARV showed decreased CCR5 and CD38 surface level expression in the HIV and the HIV/ TB co-infected subgroups. An increased percentage of naïve T cells was observed in the HIV infected subgroup, but not in the HIV/TB subgroup, similarly, a decreased percentage of antigen-experienced cells was observed in the HIV subgroup, but not in the HIV/TB co-infected subgroup. A positive correlation was found between CCR5 and CD38 expression, and CXCR4 and CD38 expression (Spearman coefficient of correlation respectively: r=0.59, p<0.001 and r=0.55, p<0.001). Furthermore we found plasma viral load positively associated with CD38 expression (r=0.31, p<0.001) and percentage activated CCR5+ expressing CD4 T cells positively related to viral load (r=0.31, p<0.001). Percentage naïve CD4 T cells was positively associated with CD4 count (r=0.60, p<0.001) and negatively correlated to viral load (r=-0.42, p<0.001). These results indicate that TB coinfection exacerbates certain aspects of dysregulation of CD4 T cell homeostasis and activation caused by HIV infection. In addition, ARV-associated decrease in coreceptor expression, immune activation status and a normalisation of CD4 T cell subset distribution was observed in HIV infected individuals, but not in HIV/TB coinfection. Despite viral suppression after ARV treatment, the decline in the immune activation marker CD38 and coreceptor CCR5 expression, increase in percentage naïve CD4 T cells and decrease of antigen-experienced cells did not reach the levels displayed in the healthy control group. This may indicate that ongoing (albeit reduced) T cell immune activation may occur in the presence of ARV. Further longitudinal studies are needed to closely monitor immune activation during ARV treatment. This study highlighted an association of TB disease with immune activation in HIV infection, the importance of T-cell activation in HIV pathogenesis and its impact on ARV treatment. Further studies are needed to identify causative factors that may lead to a persistent immune activation status during ARV treatment, and how TB coinfection confounds normal responses to ARV. / AFRIKAANSE OPSOMMING: In 2007 was ongeveer 2.1 miljoen sterftes wêreldwyd veroorsaak deur VIGS; ongeveer 70% in Sub-Sahara Afrika. CD4 T selle is die hoof teiken van MIV, aangesien dit die primêre CD4 reseptor, sowel as een of beide van die vernaamste chemokien koreseptore CCR5 en CXCR4 vrystel. Hierdie koreseptore speel ‘n prominente rol wanneer die MIV die sel binnedring, asook tydens MIV oordrag en verloop van die siekte. Dit word ook deur verskillende fraksies van CD4 T selle vrygestel. Gelyktydige TB infeksie mag immuunaktivering in vivo verhoog en dus die siekeproses versnel. MIV het ‘n groot uitdaging geword in die beheer van TB in Afrika. Bekendstelling van HAART het die ontwikkeling van VIGS vertraag, asook die risiko van verdere morbiditeit en mortaliteit. HAART veroorsaak ‘n vinnige afname in virale lading ‘n toename in CD4 telling, hoewel die spesifieke invloed van HAART op die regulering van koreseptor vrystelling, immuunaktivering en verspreiding van CD4 fraksies in MIV en MIV/TB infeksies nog onduidelik is. Hierdie studie het gepoog om koreseptor vrystelling, immuunaktiveringstatus en die verspreiding van CD4 subpopulasies in pasiënte met MIV en MIV/TB voor en na ARV behandeling te ondersoek. ‘n Totaal van 137 Suid-Afrikaanse individue is ondersoek en die studiegroep het bestaan uit 15 normale persone (gesonde subgroep), 10 pasiënte met aktiewe pulmonale TB (PTB subgroup), 33 MIV positiewe pasiënte sonder PTB (MIV subgroep), 23 MIV positiewe pasiënte met aktiewe PTB (MIV/PTB subgroep), 36 MIV positiewe pasiënte op ARV (MIV op ARV subgroep) en 20 MIV positiewe pasiënte met aktiewe PTB op ARV (MIV/PTB op ARV subgroep). Absolute CD4 telling en virale ladings was bepaal vir alle deelnemers. Vars geïsoleerde perifere bloed mononukleêre selle is geklassifiseer deur middel van vloeisitometrie as die volgende CD4 T limfosiet subgroepe: naïewe selle (CD45+, CD27+), effektor geheueselle (CD45-, CD27-), sentrale geheueselle (CD45-, CD27+), en effektor selle (CD45+, CD27-). Koreseptor vrystelling en aktivering was beoordeel volgens CCR5, CXCR4 en CD38 vrystelling op CD4 T sel subgroepe. HIV, TB en MIV/TB ko-infeksie is geassosieer met ‘n afname in die persentasie CCR5+ T selle, vergeleke met gesonde kontroles, waar die MIV/TB subgroep die grootste afname getoon het. In onbehandelde pasiënte het die CD4 T selle verhoogde vrystelling van CCR5 en CD38 op die oppervlakte getoon en dit is bevestig deur die gemiddelde fluoresserende vii intensiteit in die MIV/TB subgroep vergeleke met die subgroep met slegs MIV. Die MIV/TB subgroep het verder ook ‘n verhoogde persentasie totale geheue T selle getoon vergeleke met die MIV subgroep. Die persentasie naïewe T selle was egter verlaag in beide die MIV en MIV/TB subgroepe vergeleke met normale kontroles. MIV pasiënte wat langer as 6 maande op ARV behandeling was in beide die MIV en MIV/TB subgroepe, het ‘n verlaagde vrystelling van CCR5 en CD38 op die oppervlakte van die CD4 selle getoon. ‘n Verhoogde persentasie naïewe T selle het in die MIV subgroep voorgekom, maar nie in die MIV/TB subgroup nie. ‘n Soortgelyke tendens is gevind waar die persentasie totale geheueselle verlaag was in die MIV subgroep, maar nie in die MIV/TB subgroep nie. ‘n Positiewe korrelasie is gevind tussen CCR5 en CD38 vrystelling, asook CXCR4 en CD38 vrystelling (Spearman korrelasie koëffisiënt: r=0.59, p<0.001 en r=0.55, p<0.001 onderskeidelik). Verder het die plasma virale lading ‘n positiewe assosiasie getoon met CD38 vrystelling (r=0.31, p<0.001) en die persentasie geaktiveerde CCR5+ vrystellende CD4 T selle met virale lading (r=0.31, p<0.001). Die persentasie naïewe CD4 T selle het ‘n positiewe assosiasie getoon met CD4 telling (r=0.60, p<0.001) en ‘n negatiewe korrelasie met virale lading (r=-0.42, p<0.001). Volgens hierdie resultate vererger TB ko-infeksie sekere aspekte van die disregulasie van CD4 T selhomeostase en aktivering as gevolg van MIV infeksie. Verder kon ‘n ARVgeassosieerde afname in koreseptor vrystelling, immuunaktivering en normalisering van CD4 T sel fraksies bespeur word in die MIV subgroep, maar nie in die MIV/TB subgroep nie. Ten spyte van virale onderdrukking veroorsaak deur ARV behandeling, het die afname in die immuunmerker CD38 en koreseptor CCR5, toename in die persentasie naïewe CD4 selle en afname in totale geheue CD4 T selle nie die vlakke van die normale kontrolegroep bereik nie. Dit is moontlik dat volgehoue verlaagde T sel immuunaktivering nog steeds mag plaasvind in die teenwoordigheid van ARV. Verdere longitudinale studies is nodig om immuunaktivering tydens ARV behandeling te monitor. Hierdie studie het die belangrikheid van T sel aktivering in MIV patogenese en dit impak daarvan op ARV behandeling beklemtoon. Verdere studies is nodig om moontlike oorsake of bydraende faktore te identifiseer wat tot volgehoue immuunaktivering tydens ARV behandeling kan lei, asook tot mate waartoe TB ko-infeksie kan inmeng met die normale werking van ARV behandeling.

Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Chronic HIV-1 infection is characterized by extensive inflammation/immune activation and also by anaemia. Macrophages and neutrophils produce reactive oxygen species (ROS) which can cause damage to surrounding cells, including erythrocytes. Damaged erythrocytes may die by apoptosis (erythroptosis) or be tagged for clearance by monocytes/ macrophages. In this study we investigated HIV-1-associated anaemia and erythroptosis in asymptomatic, untreated HIV-1 infected individuals and how it relates to oxidative stress and immune activation. / AFRIKAANSE OPSOMMING: Chroniese MIV-1 infeksie word gekenmerk deur uitgebreide inflammasie/immuun aktivering en ook deur anemie. Makrofage en neutrofiele produseer reaktiewe suurstof spesies (ROS), wat kan skade aan omliggende selle, insluitend rooibloedselle veroorsaak. Beskadigde rooibloedselle kan sterf deur apoptose (erythroptosis) of gemerk vir klaring deur monosiete/makrofage. In hierdie studie het ons ondersoek MIV-1-verwante bloedarmoede en erythroptosis in asimptomatiese, onbehandelde MIV-1 besmette individue en hoe dit verband hou met oksidatiewe stres en immuun aktivering. / The Poliomyelitis Research Foundation (PRF

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Despite the decreased rate of HBV horizontal transmission in South Africa (SA) due to the HB vaccine, the risk of perinatal transmission remains of concern, especially in HIV/HBV co-infected women. Loss of HBV immune control, resulting in higher HBV replication and thus increasing the risk of transmission is described in HIV/HBV co-infected women. Chronic hepatitis is a well-recognized risk factor for hepatocellular carcinoma (HCC). The presence of specific HBV mutations has been reported in chronic and HCC patients and is used in algorithms for the prediction of HCC in CHB patients in Asia. While these mutations are extensively described in male patients, little is known regarding the antenatal and paediatric populations. This study aimed to determine the prevalence of HBV infection in HIV-exposed infants and to investigate the presence of HCC-related mutations in pregnant women and HIV-exposed children in SA. Residual samples of infants born to HIV-infected mothers were collected from the P1041 study previously conducted in SA. HBV markers (HBsAg, anti-HBs and anti-HBc) were tested on the Architect (Abbott). HBsAg positive samples were tested for HBV DNA to determine HBV viral loads. HBV strains were characterised by sequencing of the HBsAg gene and genotypes were determined by phylogenetic analysis using HepSEQ (www.hepseq.org.uk). For the HCC-related mutations investigation, samples and data were collected from three HBV-related studies: the NHLS Paediatric Study, an Antenatal Study and the current study. Pre-S, basal core promoter (BCP) and pre-core data was collected from all samples. Multiple alignments were formed and the nucleotide sequences of these extracts were translated into protein sequences. These protein sequences were compared manually to the HBV reference genes to identify HCC-related mutations. Of 850 HIV-exposed infants tested, three infants were positive for both HBsAg and HBV DNA. Two samples show evidence of past, but cleared HBV infection. Sequence analysis showed that the infants were infected with a subgenotype A1. At follow up, only one infant and mother were able to be traced and contacted. The infant was HIV-infected and had been on an ART regimen, including lamivudine for two years. HBV testing showed that the infant was HBsAg positive and had an undetectable viral load. Core sequence analysis showed clustering between mother and infant sequences. Transmission of mutant HBV previously associated with HCC prompted the question of what the prevalence of mutations in the antenatal and paediatric population is. In this investigation of HCC-related mutations study, a higher prevalence of combined pre-S, BCP and pre-core mutations was found in HIV-infected as compared to HIV-uninfected women. This study shows that vertical transmission is occurring in HIV-exposed infants in SA despite HB vaccination. Data described in this study suggests the importance of HB vaccination closer to the time of birth in SA. Moreover, data on the higher prevalence of HCC-related mutations in HIV-infected pregnant women provide a background for further longitudinal studies to confirm these findings and their implications in SA. / AFRIKAANSE OPSOMMING: As gevolg van die beskikbaarheid van die Hepatitis B virus (HBV) entstof , het horisontale transmissie van die virus drasties in Suid-Afrika (SA) verminder. Ten spyte hiervan, is daar steeds ‘n hoë risiko van perinatale transmissie van swanger vroue na hulle babas, dit word veral gesien met MIV/HBV positiewe vroue. Dit is wyd beskryf dat vroue wat mede-besmet is met MIV/HBV gewoonlik beheer verloor oor hulle immuunstelsel, wat lei tot ‘n hoër mate van HBV replikasie en dus ‘n hoër risiko van virus oordrag. Kroniese hepatitis is wel bekend as ‘n hoë risiko faktor vir HCC. Die teenwoordigheid van spesifieke HBV mutasies in kroniese en HCC pasiënte word alreeds in Asië gebruik in sekere algoritmes en formules om infeksie aan te dui en te voorspel. Hierdie mutasies is omvattend beskryf in manlike pasiënte, maar baie min is bekend in voorgeboorte en pediatriese gevalle. In hierdie studie het ons die teenwoordigheid van HCC-verwante mutasies in swanger vroue en MIV-blootgestelde kinders in Suid-Afrika ondersoek. Monsters is verkry van babas gebore van MIV-positiewe moeders van die P1041 studie wat voorheen in SA gedoen is. Die HBV merkers (HbsAg, teen-HBs en teen-HBc) was op die Architect (Abbott) getoets. HBsAg positiewe monsters was getoets vir HBV DNA om die virale lading te bepaal. Die verskeidenheid HBV stamme was gekarakteriseer deur die virus se nukleïensuur volgordes te bepaal. Die verskillende genotipes is bepaal deur filogenetiese analises te doen met behulp van die HepSEQ (www.hepseq.org.uk) program. Vir die HCC-verwante mutasie studie is monsters en data vergelyk met 3 HBV-verwante studies: die NHLS pediatriese studie, ‘n voorgeboorte studie en hierdie spesifieke studie. Voor-S, basale kern promoter en voor-kern data was van alle monsters bekom. ‘n Veelvoudige belyning was gedoen met die nukleïensuur volgordes van die verskeie DNA ekstrakte, wat daarna vertaal is in proteïen volgordes. Hierdie proteïenvolgordes translasie was by hand vergelyk met verwysings gene om die relatiewe HCC mutasies te probeer identifiseer. Van die 850 blootgestelde MIV babas wat getoets is, het 3 positief getoets vir beide HbsAg en HBV DNA. Twee monsters het bewys van verlede , maar vrygestelde HBV infeksie. Data analise bewys dat die babas met subtipe A1 besmet was. Ons kon slegs een moeder en baba paar opvolg en kontak vir verdere toetse. Die baba was MIV-positief en was op antiretrovirale behandeling , insluitend lamivudine, vir ten minste 2 jaar. HBV toetse het gewys dat die baba HbsAg positief is en ‘n onopspoorbare virale lading gehad het. Kern nukleïensuur volgorde analise het groepering getoon tussen die ma en baba se virus monsters . Die transmissie van die mutante HBV wat geassosieer is met HCC het gelei tot die vraag wat die voorkomssyfer is van hierdie spesifieke mutasies in die voorgeboorte en pediatriese populasies in SA. In hierdie studie het ons ‘n hoër gekombineerde voorkomssyfer gevind van die voor-S, basale kern promoter en voor-kern mutasies in MIV-positiewe vroue, in vergelyking met MIV-negatiewe vroue. Hierdie studie bewys dus dat vertikale transmissie van HBV in blootgestelde MIV babas steeds plaasvind, ten spyte van HBV inenting. Die data wat in hierdie studie beskryf was dui daarop dat die belangrikheid van HBV inenting nader aan die tyd van die geboorte in SA gegee moet word.As gevolg van die hoë voorkomssyfer van HCC-verwante mutasies in swanger vroue, is daar verdere longitudinale studies nodig om hierdie bevindinge en hul implikasies in SA te bevestig.

Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: The HIV epidemic in the past couple of decades has spread at an alarming rate throughout Southern Africa. Today the region accounts for roughly one third of all HIV infections, while prevalence rates in other areas of sub-Saharan Africa remain low. In the following study, sampled sequences from Cape Town, spanning over a 21-year period were used to investigate the epidemic history of HIV, which was compared to epidemic trends across Southern Africa. Longitudinal sequence data sets were generated from stored patient samples from Cape Town through standard molecular techniques. Firstly, these sequences were used to estimate the date of origin of the HIV epidemic in Cape Town and to reconstruct a demographic history of the epidemic with advanced Bayesian inference methods. These analyses placed the estimated date of origin of the Cape Town epidemic around the mid 1960‟s with periods of strong epidemic growth observed during the mid 1980‟s and 1990‟s. Secondly, reference strains of HIV from Southern African countries were used to estimate the date of origin of the epidemic in the Southern African region. These analyses placed the date of origin of the epidemic in the Southern African region around the mid 1950‟s roughly ten years before the start of the epidemic in Cape Town/South Africa. These sequences were also used for the reconstruction of the demographic history of the epidemic in the region. A two phased growth in the HIV epidemic in the Southern African region was observed with exponential growth occurring in the mid 1980‟s and 1990‟s. Such findings are also supported by HIV prevalence estimates made by some of the leading HIV research centres and government health departments. Thirdly, a large number of homologous reference strains were used to establish the evolutionary relationship of HIV isolates from Cape Town with those from around the world. A close genetic relationship between Cape Town isolates with other South African and other Southern African isolates was observed in these analyses. Finally, large monophyletic clusters of Cape Town isolates, which was observed during the evolutionary inference, were further investigated. After detailed analyses it appears that these transmission clusters of HIV-1 have been in circulation amongst the infected population of Cape Town for several years or decades. / AFRIKAANSE OPSOMMING: Die MIV-epidemie het in die afgelope paar dekades teen ´n snelspoed deur Suider-Afrika versprei. Een derde van die globale MIV-infeksies kom hiér voor terwyl ander dele van Afrika aansienlik minder infeksies aantoon. Verskeie studies skryf dit toe aan onder andere: manlike besnydenis, seksuele losbandigheid, migrasie en verskeie politike faktore. Die MIV-epidemie in Suider-Afrika word deur ´n enkele subtipe van die virus oorheers (nl. MIV Subtipe C) terwyl ander subtipes sirkuleer deur die res van sub Sahara-Afrika. In die opeenvolgende studie word DNS-monsters uit Kaapstad (wat oor ´n 20 jaar tydperk strek) gebruik om die oorsprong en verloop van die epidemie te bestudeer. Die data van die Kaapstad epidemie word met die geskiedkundige verloop van die epidemie in Suider-Afrika vergelyk. Deur gestoorde bloedmonsters van Kaapstad te gebruik, was DNS-datastelle gegenereer deur middel van standaard molekulêre tegnieke. Die DNS-monsters was eerstens gebruik om die evolusionêre oorsprong en verloop van die epidemie in Kaapstad te bepaal deur Bayesiaanse Markov-ketting Monte Carlo steekproefneming. Volgense die resultate het die epidemie sy oorsprong in die 1960‟s. Klein periodes van epidemiese groei kon waargeneem word gedurende die 1980's en -90's. Die bevindings is toe vergelyk met die geskiedkundige verloop van die epidemie in Suider-Afrika. Die Suider-Afrika epidemie se oorsprong en verloop was afgelei van DNS monsters wat verkry is van publieke databasisse en die gebruik van soortgelyke Bayesiaanse metodes. Die resultate van die ondersoek het bevind dat die epidemie in Suider-Afrika in die 1950‟s ontstaan het. In vergelyking toon dit 'n stadiger liniêre groei met kort periodes van eksponensiële groei. Verder is ´n standard filogenetiese analise onderneem om die evolusionêre verwantskap van die Kaapstad-monsters te bepaal met ander MIV subtipe C isolate. Die filogenetiese steekproef toon dat die Kaapstad-monster baie nou verwant is aan ander isolate van Kaapstad, Suid-Afrika en Suider Afrika. Buiten hierdie bevindings was transmissie-bondels van MIV in Kaapstad ontdek. Na ´n deeglike verdere filogenetiese ondersoek blyk dit of die transmissie bondels al vir ´n paar dekades deur die geïnfekteerde populasie van Kaapstad sirkuleer. / Poliomyelitis Research Foundation (PRF) / Faculty of Medicine and Health of the University of Stellenbosch / National Research Foundation (NRF) of South Africa,

Submiltted in fulfillment of the requirements for the degree of Master of Science in the faculty of Science, University of the Witwatersrand, Johannesburg • Johannesburg 1993. / The roles of the highly conserved aspartic acid residues found at positions 443 and 498 within the RNase H domain of Human Immunodeficiency Virus type-1 reverse transcription were investigated by the defined substitution of these residues using site-directed mutagenesis. [Abbreviated Abstract. Open document to view full version] / MT2016