Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: Reactivation of human cytomegalovirus (HCMV) infection in individuals infected with human immunodeficiency virus (HIV) may lead to life-threatening end-organ diseases (EOD). The EOD becomes clinically apparent when a critical number of cells in the affected organs become damaged as a consequence of HCMV-infection. Treatment of the HCMV-associated disease at this point may not be effective. Therefore, early detection of HCMV reactivation may be useful to guide pre-emptive therapy. Aim: The aim of this study was to determine whether there is a point at which the HCMV-specific cellular immune response breaks down, as determined by the interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay, and HCMV reactivation occurs in HIV-positive, antiretroviral therapy (ART)-naïve individuals in a South African setting. This was done in relation to the CD4+ T cell count and the HCMV viral load as determined by real-time polymerase chain reaction (qPCR). Materials and methods: Fifty-two (52) HIV-infected, ART-naïve subjects were recruited from primary healthcare centres that they attended for the management of their HIV infection. Heparinised blood samples were collected to quantify the HCMV-specific cellular immune response using the IFN-γ-ELISPOT assay and to determine the HCMV IgG serostatus. Ethylenediaminetetraacetic acid (EDTA) blood samples were collected for the determination of the CD4+ T cell counts and the HCMV viral loads. Results: All 52 subjects recruited were confirmed to be HIV-HCMV co-infected based on their HCMV IgG serostatus. The results of 34 subjects with completed data sets were analysed. The CD4+ T cell counts of these subjects ranged from 10 to 784 cells/μl. Twenty-two (22) (65%) subjects had positive HCMV IFN-γ-ELISPOT results with 94% having no detectable HCMV viral loads. All subjects (28) with a CD4+ T cell count above 100 cells/μl had undetectable HCMV viral loads. Two of the six subjects with CD4+ T cell counts <100 cells/μl had detectable HCMV viral loads. There was no statistically significant association between the CD4+ T cell counts and the HCMV IFN-γ-ELISPOT results. Conclusion: No specific point could be determined when there is loss of integrity of the HCMV-specific cellular immune response in HIV-positive individuals. Low CD4+ T cell counts did not correlate with HCMV IFN-γ-ELISPOT results suggesting that the HCMV-specific cellular immunity did not necessarily break down at low CD4+ T cell counts. Nevertheless, a CD4+ T cell count above 100 cells/μl appeared to be protective against viraemia as determined by the HCMV viral load qPCR. The IFN-γ-ELISPOT assay was employed as a tool to determine the integrity of the HCMV-specific cellular immune response in HIV-positive individuals. However, the IFN-γ-ELISPOT assay should be used in conjunction with the CD4+ T cell count and the HCMV viral load qPCR to determine when there is loss of integrity of the HCMV-specific cellular immune response and HCMV reactivation occurs. This may assist clinicians in their choice of management and appropriate pre-emptive treatment in the HIV-HCMV co-infected individual at a risk for HCMV reactivation. / AFRIKAANSE OPSOMMING: Inleiding: Heraktivering van menslike sitomegaalvirus (MSMV) in menslike immuniteitsgebreksvirus (MIV)-MSMV ko-geïnfekteerde individue kan lei tot dodelike end-orgaan siektes (EOS). Die EOS word klinies duidelik wanneer 'n kritieke aantal selle in die organe beskadig raak as gevolg van die MSMV-infeksie. Behandeling van die MSMV-verwante siekte op hierdie punt mag moontlik nie meer effektief wees nie. Daarom kan die vroeë opsporing van MSMV heraktivering nuttig wees in die gebruik van voorkomende terapie. Doel: Die doel van hierdie studie is om die punt te bepaal wanneer die MSMV-spesifieke sellulêre immuun reaksie afgebreek word met behulp van die interferon gamma (IFN-γ) ensiem-gekoppelde immunospot (ELISPOT) toets en MSMV heraktivering voorkom in MIV-positiewe, antiretrovirale terapie (ART)-naïewe individue in' n Suid-Afrikaanse instelling. Dit word gedoen in verhouding met die CD4+ T sel telling en die MSMV virale lading. Materiale en metodes: Twee-en-vyftig (52) MIV-geïnfekteerde, ART-naïewe pasiënte is vanaf primêre gesondheidsentrums, wat hul bywoon vir die behandeling van hul MIV infeksie, genader. Gehepariniseerde bloedmonsters is gebruik om die MSMV-spesifieke sellulêre immuun reaksie met behulp van die IFN-γ-ELISPOT toets en die MSMV IgG serostatus te bepaal. Etileendiamientetra-asynsuur (EDTA) bloed monsters is versamel vir die bepaling van hul CD4+ T sel telling en hul MSMV virale lading met behulp van die ―real-time‖ polimerase kettingreaksie (qPKR) waardes. Resultate: Al 52 pasiënte is bevestigde MIV-MSMV ko-infeksies, gebasseer op hul serologiese status. Die resultate van 34 pasiënte met voltooide data is ontleed. Die CD4+ T sel tellings van hierdie pasiënte het gewissel 10-784 selle/μl. Twee-en-twintig (22) (65%) pasiënte het positiewe MSMV IFN-γ-ELISPOT resultate met 94% wat ‗n negatiewe qPKR resultate. Alle pasiënte (28) met 'n CD4+ T-seltelling bo 100 selle/μl het' n negatiewe qPKR resultate. Twee van die ses pasiënte met 'n CD4+ T-seltelling <100 selle/μl het waarneembare MSMV virale ladings oor die qPKR. Daar was geen statisties beduidende assosiasie tussen die CD4+ T sel tellings en die MSMV IFN-γ-ELISPOT resultate nie. Gevolgtrekking: Geen spesifieke punt wanneer die MSMV-spesifieke sellulêre immuun reaksie afgebreek word kon in MIV-positiewe individue bepaal word nie. Lae CD4+ T sel tellings het nie ooreengestem met die MSMV IFN-γ-ELISPOT resultate nie en dui daarop dat die MSMV-spesifieke sellulêre immuniteit nie noodwendig afgebreek word teen 'n lae CD4+ T sel tellings nie. Tog blyk 'n CD4+ T-seltelling bo 100 selle/μl om beskerming teen viremie te bied. Die meriete van die gebruik van die IFN-γ-ELISPOT toets die integriteit van die MSMV-spesifieke sellulêre immuun response in MIV-positiewe individue te bepaal, is waargeneem in die opgehoopte data. Tog kan die gebruik van die IFN-γ-ELISPOT toets in samewerking met die CD4+ T sel telling en die MSMV virale lading meer voordelig in die bepaling van 'n punt wanneer die MSMV-spesifieke sellulêre immuun reaksie afbreek en herstel plaasvind. Dit kan help om klinici in hul keuse van bestuur en gepaste voorkomende behandeling in die MIV-MSMV mede-geïnfekteerde individu op 'n risiko vir herstel.

Thesis (MMed)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Introduction Acute respiratory tract infections cause significant morbidity and mortality worldwide, and are the main reason for the utilisation of health care services. Identifying the aetiological cause of lower respiratory tract infections (LRTIs) is difficult at the best of times, and more than 20 viruses and bacteria have been associated with LRTIs, which cannot be distinguished with clinical examination alone. Viruses can be detected in respiratory samples by a variety of methods, and without exception molecular methods have proven to be more sensitive than non-molecular-based tests. The increased sensitivity of molecular methods may assist in expanding our knowledge of the pathogenesis of severe respiratory tract infections, and could have a positive influence on patient management, infection control, vaccination strategies and public health. Aims and objectives 1. Determine the viral causes of lower respiratory tract infections requiring admission in using shell vial culture with immunofluorescent staining and two multiplex PCR assays, the Seeplex® RV15 ACE Detection system (Seeplex® RV15 ACE) and the Respiratory Multiplex Real-Time RT-PCR LightMix® Customised Kit (Resp Multiplex RT-PCR). 2. Compare the Seeplex® RV15 ACE and the Resp Multiplex RT-PCR with shell vial culture for the detection of respiratory viruses in routine diagnostic respiratory samples. 3. Examine the demographic and clinical characteristics associated with each respiratory viral pathogen. Materials and Methods One hundred and thirty-eight paediatric patients, admitted to Tygerberg Children’s Hospital from May 2010 to August 2010 with a presumptive diagnosis of an acute respiratory tract infection were included in the study. Nasopharyngeal or tracheal aspirates were collected, and all samples were tested by all three diagnostic methods. Clinical, demographic and laboratory data were collected through a systematic review of medical and laboratory records and subsequently anonymised Results Thirty-seven viruses were detected in 36 samples (26.1%) by shell vial culture with immunofluorescent staining; 169 viruses in 102 samples (73.9%) with the Seeplex® RV15 ACE; and 90 viruses in 73 samples (52.9%) with the Resp Multiplex RT-PCR. Shell vial culture had excellent specificity, but low sensitivity for all of the respiratory viruses. Conversely, the Seeplex® RV15 ACE had excellent sensitivity for all viruses, but slightly lower specificity. This was due to the detection of additional viruses, which may have been true positives due to the increased sensitivity of this assay. The Resp Multiplex RTPCR had excellent sensitivity and specificity. At least one respiratory pathogen could be identified in 80% of the patients. At least one virus was detected in 57% of patients, bacterial micro-organisms in 6%, and both viral and bacterial pathogens in 17%. Viral-bacterial co-infections were associated with increased severity compared to other infections, as these children were more likely to receive steroids and a blood transfusion (p = 0.002), and more likely to require mechanical ventilation (p < 0.001) and admission to the intensive care unit (p = 0.04). Conclusions We confirmed that molecular techniques are significantly more sensitive than shell vial culture for the detection of respiratory viruses in children. Due to their highly specific nature and the genetic variability observed in viruses, an excellent, continuous quality control programme is essential to ensure the continued superiority of these assays. Viral-bacterial co-infection is associated with increased severity of LRTIs in children. Further research is needed to elucidate the precise pathogenic and immunologic mechanism of this interaction. / AFRIKAANSE OPSOMMING: Inleiding Akute lugweg infeksies is verantwoordelik vir beduidende morbiditeit en mortaliteit wêreldwyd en is die hoofrede vir die benutting van gesondheidsdienste. Identifisering van die oorsaak van laer lugweg infeksies is baie moeilik en meer as 20 virusse en bakterieë word hiermee geassosieer. Ongelukkig kan kliniese ondersoek alleen nie onderskei tussen die verskillende organismes nie. Respiratoriese virusse kan deur ‘n wye verskeidenheid van toets metodes aangetoon word. Molekulêre metodes is sonder uitsondering meer sensitief as nie-molekulêre metodes. Hul verhoogde sensitiwiteit mag help om ons kennis oor die patogenese van erge lugweg infeksies te verbreed en kan ’n positiewe invloed op pasiëntbehandeling, infeksiebeheer, immunisasie strategieë en publieke gesondheidsorg hê. Doel van die Ondersoek 1. Bevestig die virale oorsake van laer lugweg infeksies deur gebruik te maak van “shell vial” kultuur met immunofluoressensie en twee veelvoudige molekulêre toetse, die Seeplex® RV15 ACE en die Resp Multiplex RT-PCR. 2. Vergelyk die Seeplex® RV15 ACE en die Resp Multiplex RT-PCR met “shell vial” kultuur vir die aantoning van respiratoriese virusse in roetine diagnostiese monsters. 3. Ondersoek die demografiese en kliniese eienskappe wat met elke respiratoriese patogeen geassosieer word. Metodiek en Materiaal Een honderd agt-en-dertig kinders wat toegelaat is tot Tygerberg Kinderhopitaal vanaf Mei 2010 tot Augustus 2010 met ’n voorlopige diagnose van ’n akute lugweg infeksie is in die studie ingesluit. Nasofaringeale of trageale aspirate is van elke pasiënt gekollekteer en met al drie diagnostiese metodes ondersoek. Kliniese, demografiese en laboratorium data is gekollekteer deur ’n sistematiese ondersoek van mediese en laboratorium rekords en daarna anoniem gemaak. Resultate Sewe-en-dertig virusse is in 36 monsters (26.1%) aangetoon deur “shell vial” kultuur met immunofluoressensie; 169 virusse in 102 monsters (73.9%) deur die Seeplex® RV15 ACE; en 90 virusse in 73 monsters (52.9%) deur die Resp Multiplex RT-PCR. “Shell vial” kultuur het uitstekende spesifisiteit gehad, maar sensitiwiteit was laag vir al die virusse. Teenoorgesteld hiermee het die Seeplex® RV15 ACE hoë sensitiwiteit vir al die viruses gehad, maar effe laer spesifisiteit. Dit was as gevolg van die aantoning van addisionele virusse, wat moontlik ware positiewe resultate kon wees as gevolg van die verhoogde sensitiwiteit van hierdie toets metode. Die Resp Multiplex RT-PCR het uitstekende sensitiwiteit en spesifisiteit gehad. Ten minste een respiratoriese patogeen is in 80% van die pasiënte geidentifiseer. Een of meer virusse was in 57% van die pasiënte aangetoon, bakterieë in 6% en beide virale en bateriële patogene in 17%. Virale-bakteriële ko-infeksies, in vergelyking met ander infeksies, was geassosieer met meer ernstige lugweg infeksies aangesien hierdie kinders meer geneig was om steroïede en ’n bloedtransfusie te ontvang (p = 0.002). Hulle het ook meer waarskynlik meganiese ventilasie (p < 0.001) en toegang tot die intensiewe sorg eenheid benodig (p = 0.04). Gevolgtrekkings Ons het bevesitg dat molekulêre tegnieke aansienlik meer sensitief is as “shell vial” kultuur vir die aantoning van respiratoriese virusse in kinders. As gevolg van hul hoogs spesifieke aard en die genetiese variasie waargeneem in virusse, is ’n uitstekende deurlopende kwaliteitsbeheer program noodsaaklik vir die voortgesette uitneemendheid van hierdie metodes. Virale-bakteriële ko-infeksies word geassosieer met meer ernstige laer lugweg infeksies in kinders. Verdere navorsing is nodig om die presiese patogenetiese en immunologiese meganisme van hierdie interaksie toe te lig.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations. / AFRIKAANSE OPSOMMING: Agtergrond: Die virus protein R (Vpr) van Menslike Immuungebrek Virus tipe 1 (MIV-1) is ‘n bykomstige protein wat met ‘n aantal sellulêre proteine van die gasheer en ander virus proteine in wisselwerking tree. Vpr het 'n invloed op verskeie funksies onder andere die induksie van apoptose, die induksie van selsiklus G2 staking, modulering van geen uitdrukking en onderdrukking van immuun aktivering. Die funksionaliteit van subtipe C Vpr, en veral die van Suid-Afrikaanse stamme, is nie beskryf nie. Die doelwit van die studie was om die diversiteit van Suid Afrikaanse MIV-1 subtipe C vpr gene te beskryf en ook om selektiewe funksies van die Vpr proteine te ondersoek Metodiek: Die MIV-1 vpr streek van 58 stamme is vermeerder, die DNA volgordes is bepaal en die stamme is gesubtipeer deur filogenetiese analise. Fragmente met natuurlike mutasies is in ekspressie vektore gekloon. ‘n Konsensus subtipe C Vpr geen is ontwerp en mutasies in posisies waar geen natuurlike mutasies beskryf is nie, is ontwerp deur mutagenese. Die funksionaliteit van die konstrukte is met die wilde tipe subtype B vergelyk deur 293T sellyn te transfekteer en te ondersoek vir subsellulêre lokalisering, induksie van apoptose, en G2 selsiklus stilstand. Die modulering van geen uitdrukking in die induksie van apoptose is deur TLDA ondersoek. Resultate: Filogenetiese analise het 54 stamme as HIV-1 subtipe C geklassifiseer en 4 stamme as subtype B. Die Vpr aminosuur volgordes was konstant insluitend die FPRPWL en TYGDTW motiewe, maar die C-terminaal was meer variëerbaar. Deur mutagenese is die volgende mutasies ontwerp: P14I, W18C, Y47N, Q65H and Q88S. Subtipe B en al die natuurlike mutante van subtipe C het in die selkern gelokaliseer, maar die W18C mutasie het die lokalisasie versteur. Die G2 selsiklus stilstand van alle mutante en konsensus C was laer as die van subtype B. Al die natuurlike subtipe C mutante het G2 selsiklus tot stilstand gebring in 54.0-66.3% van die selle, terwyl subtype B selsiklus tot stilstand gebring het in 71.5% van die selle. Subtipe B en die natuurlike Vpr mutante het apoptose op ‘n soortgelyke wyse geinduseer, wat wissel tussen 95.3-98.6% van getransfekteerde selle. Die protein met die kunsmatig ontwerpte konsensus C volgorde het egter ‘n verlaagde vermoë gehad om apoptose te induseer. Die konsensus subtipe C het apoptose in 82.0% van getransfekteerde selle geinduseer en die kunsmatige mutante in 88.4 – 96.2% van die selle. Die induksie van die apoptose verwante geen ekspressie deur die mutante was soortgelyk as die van konsensus C en subtipe B Vpr wat ’n aangeduiding is dat apoptose effektief veroorsaak is deur die intrinsieke roete. Gevolgtrekking: Hierdie studie het aangetoon dat kern lokalisering en apoptose op ‘n soortgelyke wyse by beide MIV-1 subtipe B en C Vpr plaasvind. Die induksie van selsiklus G2 stilstand deur MIV-1 subtipe B Vpr is egter meer robuust as baie van die subtipe C Vpr proteïene. Natuurlike mutasies in MIV-1 Vpr kan nie as gebrekkig beskou word nie, aangesien beter funksionaliteit 'n aanduiding is vandie aanpasbare rol. Die verhoogde krag van die gemuteerde Vpr proteïen dui daarop dat Vpr die patogenisiteit van MIV-1 kan verbeter deur die aanpassing van mutasies.

Thesis (MScMedSc))--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Despite the availability of Hepatitis B virus (HBV) vaccination for over three decades, this infection remains a major public health problem. Whilst the WHO recommends giving a birth dose of the vaccine, in South Africa, routine infant HBV vaccination commences at six weeks of age. This schedule is based on data from the pre-HIV era which showed transmission occurred via the horizontal, rather than the vertical route. In the era of HIV however, maternal HIV co-infection may release HBV from immune control, resulting in higher HBV loads and increasing the risk of vertical transmission. The aim of this study was to determine the prevalence and character of HBV infection in HIV-exposed infected and uninfected infants. Residual plasma samples from routine HIV nucleic acid testing of 1000 HIV-exposed infants aged between 0 and 18 months from the Western Cape were tested. Samples were tested for HBsAg by ELISA (Murex HBsAg Version 3) and confirmed by neutralisation. HBV DNA was quantified using an in-house real-time PCR assay. Infants with HBsAg positive samples were followed up and a blood sample was collected from mother and child. Those HBsAg positive samples were tested for HBeAg/antiHBe (Diasorin) and HBsAg negative samples were tested for antiHBs. HBV DNA was quantified. The surface gene was sequenced and the HBV genotype determined by phylogenetic analysis using HepSEQ (www.hepseq.org.uk). Whole genome sequencing was also performed. Of 1000 samples tested, four samples were positive for HBsAg and/or HBV DNA, indicating a prevalence of HBV transmission of 0.4%. At follow-up, two of three infected infants were positive for HBsAg, with HBV viral loads of greater than 108 IU/ml. The third infant was found to have cleared his infection and the fourth child was lost to follow up. These infected infants had all received HBV vaccination. All four mothers were HBeAg positive. Sequencing analysis showed the HBV strains from the two infants and four mothers belonged to subgenotype A1. The two mother-child paired sequences were identical. The data from this study shows that vertical transmission of HBV infection in HIV-exposed infants from the Western Cape is occurring, despite vaccination. Data from the Western Cape, showing an HBV prevalence of 3.4% in HIV-infected pregnant women, and those presented here suggest a vertical transmission rate of HBV of 12%. This is despite the widespread use of tenofovir and lamivudine in HIV-infected women with low CD4 counts. This study provides data supporting calls to bring HBV vaccination closer to the time of birth. Further work is urgently needed to confirm these findings and to determine the rates of transmission in HIV-unexposed infants. / AFRIKAANSE OPSOMMING: Ten spyte van die beskikbaarheid van die Hepatitis B virus (HBV) inenting vir meer as drie dekades, hierdie infeksie bly 'n groot openbare gesondheid probleem. Terwyl die WGO aan beveel dat'n geboorte dosis van die entstof, in Suid-Afrika, roetine baba HBV inenting op die ouderdom van ses weke gegee word. Hierdie skedule is gebaseer op data van die pre-MIV era wat getoon het dat die oordrag plaasgevind het via die horisontale, eerder as die vertikale roete. In die era van MIV egter, moeder MIV ko-infeksie kan HBV vrylaat van immuun beheer, wat lei in hoër HBV vlakke en die verhoging van die risiko van vertikale oordrag. Die doel van hierdie studie was om die voorkoms en karakter van die HBV infeksie in MIV-besmette en onbesmette babas te bepaal. Residuele plasma monsters van roetine-MIV-nukleïensuur toetse van 'n 1000 MIV-blootgestelde babas tussen die ouderdomme van 0 en 18 maande van die Wes-Kaap was getoets. Monsters was getoets vir HBsAg deur ELISA (Murex HBsAg Version 3) en bevestig deur neutralisering. HBV DNA is gekwantifiseer deur gebruik te maak van 'n in-huis real-time PCR assay. Babas met HBsAg positiewe monsters was opgevolg en 'n bloedmonster is versamel van moeder en kind. Die HBsAg positiewe monsters was getoets vir HBeAg/antiHBe (Diasorin) en HBsAg negatiewe monsters was getoets vir antiHBs. HBV DNA was gekwantifiseer. Die oppervlak gene volgorde en genotipes was bepaal deur filogenetiese analise met behulp van HepSEQ (www.hepseq.org.uk). Die hele genoom-volgordebepaling was ook uitgevoer. Van die 1000 monsters wat getoets was, was vier monsters positief vir HBsAg en of HBV DNA, dit dui op 'n voorkoms van HBV oordrag van 0.4%. By op volg, twee van die drie besmette babas was positief vir HBsAg, met HBV virale vlakke van groter as 108 IE/ml. Die derde baba was gevind dat sy infeksie opgeklaar het en die vierde kind was verlore as gevolg van op volg. Hierdie besmette babas het almal HBV inenting ontvang. Al vier moeders was HBeAg positief. Volgordebepaling analise het getoon die HBV stamme van die twee babas en vier moeders behoort aan subgenotype A1. Die twee moeder-kind gepaarde rye was homoloë. Die data van hierdie studie toon dat die vertikale oordrag van HBV infeksie in MIV-blootgestelde babas van die Wes-Kaap vind plaas, ten spyte van inenting. Data van die Wes-Kaap, wat 'n HBV voorkoms van 3.4% in MIV-besmette swanger vroue, en dié wat hier aangebied is dui op 'n vertikale oordrag koers van 12% van die HBV. Dit is ten spyte van die wydverspreide gebruik van tenofovir en lamivudine in MIV-geïnfekteerde vroue met 'n lae CD4-telling. Hierdie studie bied data wat ondersteunende oproepe van HBV inenting nader aan die tyd van die geboorte bring. Verdere werk is dringend nodig om die bevindinge te bevestig en die pryse van die oordrag in MIV-blootgestelde babas te bepaal. / National Health Laboratory Service Research Trust / Poliomyelitis Research Foundation (PRF) / Harry Crossley Foundation / Stellenbosch University

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Hepatitis B virus (HBV) immunisation protocols in much of Africa are based on data from the pre-human immunodeficiency virus (pre-HIV) era that indicated that HBV transmission occurs predominantly horizontally between siblings and play-mates rather than vertically from mother to child. The immunosuppression associated with HIV infection however may release HBV from immune control resulting in higher HBV viral loads, which may increase the risk of perinatal mother to child transmission of HBV. The aim of this study was to determine the prevalence and characteristics of chronic HBV infection in HIV-infected pregnant women compared to HIV-uninfected pregnant women in the Western Cape province of South Africa. Ethical approval was obtained to conduct a retrospective, matched case-control, unlinked anonymous study using residual plasma samples from the 9355 pregnant women included in the Western Cape's 2008 National HIV and Syphilis Antenatal Survey. Samples were tested for HBsAg on the AxSYM (Abbott, Chicago, IL) and confirmed by neutralization. Confirmed HBsAg-positive samples were tested for HBeAg, anti-HBe and anti-HD (Diasorin, Saluggia, Italy) and had HBV viral load and genotyping done. In addition, HBsAg-negative samples were tested for anti-HBc. Samples from 1549 HIV-infected pregnant women were included and matched to the same number of samples from age- and race-matched HIV-uninfected women. Median age of 26 years, parity and education were similar in the two groups. The prevalence of HBsAg was 3.4% for the HIV-infected group and 2.9% for the HIV-uninfected group. HBV DNA loads of greater than 104 IU/ml were detected in 32.1% of HBsAg-positive, HIV/HBV co-infected women, and in 14.3% HBsAg positive, HBV mono-infected women. Among the HIV-infected group 18.9% of HBsAg-positive were HBeAg positive, with a median viral load of 7.93 log10 IU/ml; whilst 15.5% HIV-uninfected women were positive for HBeAg with a median viral load of 6.07 log10 IU/ml. Genotype A was seen in 92.6% of the isolates while 7.4% of the isolates were genotype D. Serum total anti-HBc antibodies that are a marker of past infection were detected in 42.2% of HIV-infected and in 24.1% of HIV-uninfected women that were negative for HBsAg. No positive sample for anti-HD was seen among all HBsAg-positive samples. This data indicates that there is increased exposure to HBV in HIV-infected pregnant women than in HIV-uninfected women and that a greater proportion of HIV-infected pregnant women compared to HBV mono-infected pregnant women may be at increased risk of transmitting HBV to their infants. Further studies are needed to determine the rate of vertical transmission of HBV in the HIV era. / AFRIKAANSE OPSOMMING: Hepatitis B virus (HBV) immunisasie protokolle vir meeste dele van Afrika is gebaseer op data versamel in die era voor MIV. Die data dui aan dat HBV oordrag hoofsaaklik deur horisontale transmissie tussen broers, susters en speelmaats eerder as vertikale transmissie van moeder na kind plaasvind. Die onderdrukking van die immuunstelsel as gevolg van MIV infeksie kan egter lei tot 'n verhoogde risiko van perinatale HBV oordrag van moeder na kind. Die doel van hierdie studie was om die voorkoms en karakter van chroniese HBV infeksie in MIV-positiewe swanger vroue te vergelyk met die van MIV-negatiewe swanger vroue. Etiese goedkeuring is verkry om 'n retrospektiewe, deursnee-, ongekoppelde anonieme studie uit te voer wat gebruik maak van oorblywende plasma monsters van 9355 swanger vroue wat ingesluit is in die Wes-Kaap 2008 Nasionale MIV en Sifilis Voorgeboortelike Opname. Die monsters was getoets vir HBsAg antiliggame (AxSYM, Abbott, Chicago, IL) en bevestig deur neutralisasie toetse. Positiewe monsters was getoets vir HBeAg en anti-HBe (Diasorin, Saluggia, Italië). HBV viruslading en genotipering was ook op HBsAg positiewe monsters gedoen. Die HBsAg negatiewe monsters was getoets vir die teenwoordigheid van anti-HBc. Monsters van 1549 MIV-positiewe swanger vroue was ingesluit in die studie. Dieselfde aantal monsters van MIV-negatiewe vroue, met ooreenstemende ouderdom en etnisiteit, was ingesluit as kontroles. Die gemiddelde ouderdom van albei groepe was 26 jaar. Pariteit en opvoeding was dieselfde in albei groepe. Die voorkomssyfer van HbsAg was 3.4% in die MIV-positiewe groep en 2.8% in die MIV-negatiewe groep. HBV DNS ladings van meer as 104 IU/ml was waargeneem in 32.1% van die MIV-mede-geinfekteerde vroue en in 14.3% van die MIV-negatiewe groep. In die MIV-positiewe groep was 18.9% vroue HBeAg positief, met 'n gemiddelde virale lading van 7.93 log10 IU/ml, terwyl 15.5% MIV-negatiewe vroue positief was vir HBeAg met 'n gemiddelde virale lading van 6.07 log10 IU/ml. In ons studie was 92.6% van die monsters genotipe A en 7.4% genotipe D. Toatale anti-HBc antiliggame, 'n merker van vorige infeksie, was gevind in 42.2% van MIV-mede-geïnfekteerde vroue en 24.1% van MIV-negatiewe vroue wat negatief was vir HBsAg antiliggame. Data van ons studie dui op 'n verhoogde risiko vir vertikale HBV transmissie van MIV-positiewe moeders na hul babas. Verdere studies word benodig om vas te stel of vertikale transmissie van HBV van MIV-positiewe vroue na hul babas plaasvind. / Wellcome Trust / Poliomyelitis Research Foundation