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)--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.