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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Analysis of the clinical utility of gene expression profiling in relation to conventional prognostic markers in South African patients with breast carcinoma

Grant, Kathleen Ann 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Breast cancer is a heterogeneous disease characterised by marked inter-individual variability in presentation, prognosis and clinical outcome. The recognition that morphological assessment has limited utility in stratifying patients into prognostic subgroups led to clinico-pathological classification of tumour biology, based on receptor expression using immunohistochemical (IHC) techniques. This standard is currently complemented by the development of gene expression profiling methodology that led to the identification of intrinsic molecular subtypes, reflecting tumour genetics as the true driver of biological activity in breast cancer. The study was based on the hypothesis that molecular classification of breast carcinomas integrated with established clinico-pathological risk factors will improve current diagnostic and risk management algorithms used in clinical decision-making. A pathology-supported genetic testing strategy was used to evaluate microarray-based gene profiling against diagnostic pathology techniques as the current standard. Clinico-pathological factors including age, number of positive axillary nodes, tumour size, grade, proliferation index and hormone receptor status was documented for 141 breast cancer patients (143 tumours) referred for microarray-based gene expression profiling between 2007 and 2014. Subsets of patients were selected from the database based on the inclusion criteria defined for three phases in which the study was performed, in order to determine 1) the percentage of patients stratified as having a low as opposed to high risk of distant recurrence using the 70-gene MammaPrint profile within the inclusion criteria, 2) correlation of HER2 status as determined by IHC and fluorescence in situ hybridisation (FISH) with microarray-based mRNA readout (TargetPrint), and 3) the relationship between hormone receptor determination as reported by standard IHC and molecular subtyping using the 80-gene BluePrint profile. Similar distribution patterns for MammaPrint low- and high-risk profiles were obtained irrespective of whether fresh tumour biopsies or formalin-fixed paraffin embedded (FFPE) tissue was used. During the first phase of the study, 60% of the 106 tumour specimens analysed with MammaPrint were classified as low-risk and 40% as high-risk using a newly-developed MammaPrint pre-screen algorithm (MPA) aimed at cost-saving. In the second phase of the study, performed in 102 breast tumours, discordant or equivocal HER2 results were found in four cases. Reflex testing confirmed the TargetPrint results in discordant cases, achieving 100% concordance regardless of whether fresh tumour or FFPE tissue was used for microarray analysis. For the third phase of the study 74 HER2-negative tumour samples were selected for comparative analysis. Statistically significant positive correlations were found between protein expression (IHC score) and mRNA (TargetPrint) levels for estrogen receptor (ER) (R=0.53, p<0.0001) as well as progesterone receptor (PR) (R=0.62, p<0.0001), while combined ER/PR tumour status was reported concordantly in 82.4% of these tumours. BluePrint was essential for interpretation of these results used in treatment decision-making. The MPA developed in South Africa in 2009 was validated in this study as an appropriate strategy to prevent chemotherapy overtreatment in patients with early-stage breast cancer. The use of microarray-based analysis proved to be a reliable ancillary method of assessing HER2 status in breast cancer patients. Risk reclassification based on the TargetPrint results helped to avoid unnecessary high treatment costs in false-positive cases, in addition to providing potentially life-saving treatment to those for whom it was indicated. While neither IHC nor TargetPrint estimation of intrinsic subtype correlated independently with the molecular subtype as indicated by BluePrint profiling, the ability to distinguish between basal-like and luminal tumours was enhanced when the combined protein and mRNA values was considered. Genomic profiling provided information over and above that obtained from routine clinico-pathological assessments. This finding supports the relevance of a pathology-supported genetic testing approach to breast cancer management, whereby advanced genomic testing is combined with existing clinico-pathological risk stratification methods for improved patient management. / AFRIKAANSE OPSOMMING: Borskanker is „n heterogene siekte wat gekenmerk word deur merkbare inter-individuele variasie in kliniese beeld, prognose en uitkoms. Die beperkings van morfologiese klassifikasie vir identifikasie van prognostiese subgroepe het gelei tot klinies-patologiese tumor karakterisering op grond van reseptor uitdrukking deur gebruik van immunohistochemiese (IHC) toetse. Hierdie standaard word tans gekomplementeer deur ontwikkeling van geenuitdrukking tegnologie wat gelei het tot die identifikasie van intrinsieke molekulêre subtipes, wat die tumor genetika reflekteer as die ware drywer van biologiese aktiwiteit in borskanker. Die huidige studie is gebaseer op die hipotese dat integrasie van die molekulêre klassifikasie van borskanker met konvensionele risiko klassifikasie skemas huidige diagnostiese en behandelings algoritmes kan verbeter vir kliniese besluitneming. „n Patologie-gesteunde strategie is gebruik om mikroplaat-gebaseerde geen profilering te evalueer teen standaard patologie diagnotiese tegnieke. Kliniese-patologiese faktore insluitend ouderdom, aantal positiewe aksillêre limfnodes, tumor grootte, gradering, proliferasie indeks en hormoon reseptor status is gedokumenteer in 141 borskanker pasiente (143 tumore) wat verwys is vir mikroplaat-gebaseerde geenuitdrukking profilering tussen 2007 en 2014. Pasiënt subgroepe is geselekteer uit die databasis volgens die insluitingskriteria soos gedefiniëer in die drie fases waarvolgens hierdie studie uitgevoer is, om vas te stel 1) watter proporsie pasiënte geklassifiseer word as lae- of hoë-risiko vir latere herhaling van die borskanker deur gebruik van die 70-geen MammaPrint profile binne die insluitingskriteria, 2) hoe korreleer HER2 status soos vasgestel deur IHC en fluoreserende in situ hybridisasie (FISH) toetsing met mikroplaat-gebaseerde RNA lesings (TargetPrint), en 3) wat die verwantskap is tussen hormoon reseptor status soos deur standaard IHC gerapporteer en molekulëre klassifikasie volgens die 80-geen BluePrint profiel. Soortgelyke verdelingspatrone vir MammaPrint lae- teenoor hoe-risiko profiele is waargeneem ongeag of vars tumor biopsies of formalien-gefikseerde paraffin bevattende weefsel gebruik is. Tydens die eerste fase van die studie is 60% van die 106 tumore as lae-risiko en 40% as hoë-risiko geklassifiseer met toepassing van die nuwe MammaPrint Presifting Algoritme (MPA) wat ontwikkel is met die doel op kostebesparing. In die tweede fase van die studie waar 102 tumore ingesluit is, het die resultate van vier gevalle verskil van mekaar of was onbepaald ten opsigte van HER2 status. Refleks herevaluering het die TargetPrint resultate bevestig in alle nie-ooreenstemmende gevalle, en 100% ooreenstemming is bereik ongeag of vars tumor biopsies of formalien-gefikseerde paraffin bevattende weefsel gebruik is vir mikroplaat analise. In die derde fase van die studie is 74 HER2-negative tumore selekteer vir vergelykende analise. Statisties beduidende positiewe korrelasies is waargeneem tussen proteïen uitdrukking (IHC) en mRNA (TargetPrint) vlakke vir die estrogeen reseptor (ER) (R=0.53, p<0.0001) sowel as progesteroon reseptor (PR) (R=0.62, p<0.0001), terwyl gekombineerde ER/PR reseptor status ooreenstemming getoon het in 82.4% tumore. BluePrint was noodsaaklik vir die korrekte interpretasie van die resultate wat gebruik is in kliniese besluitneming vir behandeling van pasiënte. The MPA wat in Suid Africa ontwikkel is in 2009, is gedurende hierdie studie bevestig as n toepaslike strategie om onnodige handeling met chemoterapie te voorkom in pasiënte met vroeë stadium borskanker. Die gebruik van mikroplaat-gebaseerde analise is aangetoon as „n betroubare aanvullende metode om HER2 status te evalueer. Risiko herklassifikasie gebaseer op TargetPrint resultate het onnodige hoë behandelingskoste in vals-positiewe gevalle vermy, sowel as om die verskaffing van potensieël lewensreddende behandeling vir die toepaslike pasiënte te verseker. Genomiese profilering het inligting addisioneel tot dit wat met roetine klinies-patologies metodes verkry kan word verskaf. Hierdie bevinding ondersteun die relevansie van „n patologie-gesteunde genetiese toets benadering tot hantering van borskanker, waardeur genomiese toetsing gekombineer word met bestaande klinies-patologiese risiko stratifisering metodes om pasiënt behandeling te verbeter.
2

Development of selective real-time PCR (SPCR) asays for the detection of K103N resistance mutation in minor HIV-1 populations

Seleka, Mpho Maria 12 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class. Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences. Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants. Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants. Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%. / AFRIKAANSE OPSOMMING: Agtergrond: Konvensionele volgorde bepaling analise is die mees algemeenste metode wat gebruik word vir die opsporing van middel-weerstandige mutasies, maar weens beperkte sensitiwiteit is dit nie moontlik om hierdie mutante op te spoor wanneer dit minder as 20% (minderheids populasie) van die totale viruspopulasie in `n monster uitmaak nie. Nietemin, kwalitatiewe PKR-gebaseerd toetse bied vinnige, sensitiewe, spesifieke en makliker opsporings en kwantifisering van sulke mutante aan. MIV-1 variante wat die K103N mutasie bevat word geassosieer met weerstand teen nevirapine (NVP) and efavirenz (EFV). Volhoudende middel-weerstandige mutasies vergaan stadig na laer vlakke en word daarom na minderheids middel weerstandige mutasies verwys. Gevolglik affekteer dit opvolgende behandeling met die middel van die relevante klas. Doelwitte: Die doel van die studie was om twee TaqMan kwantifiserende PKR gebaseerde selektiewe polymerase ketting reaksies (SPKR), naamlik totale virale kopie SPKR en K103N-SPKR te ontwikkel. Die voormalige toets het die MIV-1 subtipe C omgekeerde transkriptase volgorde bepaal, waar K103N die middel-weerstand variante in hierdie volgorde opspoor. Ontwerp en Metodes: `n Geskikte stel inleiers en peiler was ontwikkel vir die MIV-1 subtipe C omgekeerde transkriptase (OT) vir gebruik in die K103N-spesifieke en die totaal kopie reaksie. Twaalf DNS plasmied standaarde met volgorde diversiteit was saamgestel vir die toets vanaf twee MIV-1 subtipe C monsters wat volgens ons Departement se weerstand databasis geklassifeer is vir die besit van die K103N mutasie (AAC of AAT). Die OT streke was geamplifiseer, gekloneer en geverifieer deur volgorde bepaling. Punt-gerigte mutagenese is gebruik om `n mutasie by die amino suur posisie 103 van sekere klone te induseer om meer standaarde te genereer wat een van die drie kodons (AAA, AAC en AAT) bevat. Die twee toetse is geoptimiseer en gevalideer en `n standard kurwe is genereer vir elk van die toetse deur die gebruik van tienvoud serie verdunnings (107-1 DNS kopie/μL) van `n algemene K103N-mutante plasmied standard. Die geoptimiseerde en gevalideerde SPKR toets was gebruik om vir die minderheids K103N variante in 40 “nested” PKR produkte van voorheen gegenotipeerde pasiënt te soek. Resultate: Twee sensitiewe en herproduseerbare selektiewe kwantitiewe PKR toetse met `n ΔCt afsnypunt van 8.23 en `n deteksie limiet van 0.006% was ontwikkel vir die K103N weerstand variant. Die toets het `n voorkomsyfer van 25.6 % vir die K103N weerstand mutasie in 40 pasiënt monsters bepaal, waar genotipering (populasie volgorde ) 40% van hierdie variante nie opgespoor het nie. Gevolgtrekking: `n Sensitiewe en betroubare selektiewe kwantitatiewe PKR toets vir die opspoor en kwantifisering van die minderheids K103N variante van MIV-1 in PKR produkte was ontwikkel. Hierdie toets het `n laer opsporings limiet van 0.01%. / Poliomyelitis Research Foundation (PRF) / National Research Fund (NRF) / National Health Laboratory Service Research Trust (NHLS RT)
3

Population structure, host cell interactions and pathogenesis of Staphylococcus aureus strains isolated at Tygerberg hospital, South Africa

Oosthuysen, Wilhelm Frederick 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Numerous studies conducted internationally have identified and described several endemic methicillin-susceptible Staphylococcus aureus (MSSA) clones. However, only some of these clones are associated with methicillin resistance (CC5, CC8, CC22, CC30 and CC45). To date, studies reporting on the population structure of S. aureus isolated in South Africa represent limited demographic areas, focus on methicillin-resistant S. aureus (MRSA) only and have displayed little emphasis on virulence. This study was undertaken to elucidate the population structure of S. aureus isolated from specific clinical sources at Tygerberg hospital, and to investigate specific host-pathogen interactions of representative isolates. Consecutive non-repetitive clinical S. aureus isolates were collected over one year (September 2009/2010) with patient demographics and limited clinical information. Strains were typed by PFGE and molecular markers (spa, multi-locus sequence typing (MLST), agr, Staphylococcal Chromosome Cassette mec and Panton-Valentine leukocidin (PVL)). Representative isolates were selected and investigated for the presence of virulence genes, adherence (to immobilised fibronectin [Fn], fibrinogen [Fg], collagens IV [CnIV] and VI [CnVI]), cellular invasion and cell death induction. Statistical association were determined between all in vitro results and methicillin-resistance, clonality, patient HIV status and bacterial PVL status. Fifteen percent of the isolates (n = 367) were MRSA. Forty four present of isolates were PVL+. agr I-IV and SCCmec I-V were identified. The MSSA population was diverse: ST22 (dominant), ST1865 and ST121 were PVL+. ST45, ST1863 and ST15 were PVL-. PVL- MRSA were diverse: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II and ST22-MRSA-IV. The genes fnbA/B (fibronectin-binding protein A/B), clfA/B (clumping factor A/B), eap (extracellular adherence protein), nuc (nuclease), coa (coagulase) and hld (delta toxin) were detected in all representative isolates. The CC8 and CC6 isolates adhered strongly to all ligands (100-700% of control, ligand dependent), while isolates of CC45, CC22 and CC88 adhered strongly only to Fg and Fn. The CC30, CC15, and CC12 isolates adhered extremely strongly to CnIV (>300%) and CC8, CC15, and C6 to CnVI (>200%). Isolates from CC30, CC8, CC15, CC6, CC12, CC97, CC88 and CC45 were highly invasive (>100%). ST121 was non-invasive (>50%). Isolates of CC5, CC30 and CC121 were non-cytotoxic (<50%), while isolates of CC22, CC8, CC15, CC45 and CC88 were very cytotoxic (>70%). No significant difference was observed in adherence or cell death induction of MRSA vs. MSSA clones or between isolates from HIV+ vs. HIV- persons. PVL- isolates displayed higher cellular invasiveness than PVL+ isolates. The presence of ST612-MRSA-IV, ST22-MRSA-V and ST8-MRSA-V points to local SCCmec acquisition, as we found MSSA isolates with the same spa types. Numerous MSSA clones were prevalent, but do not appear to have a major common genetic background with MRSA. PVL was highly prevalent among MSSA, indicating acquisition of PVL genes independently of SCCmec. The abilities to adhere to specific immobilised ligands in vitro were diverse and grouped with the genetic background, while the vast majority of isolates were invasive and induced significant cell death. We can conclude that the population of S. aureus at Tygerberg hospital is composed of a vast number of MSSA and MRSA clones, which display varying patters of adherence to selected ligands and of which, the majority clones are invasive and cytotoxic. / AFRIKAANSE OPSOMMING: Talle internasionale studies het verskeie endemiese metisillien vatbare Staphylococcus aureus (MSSA) klone geïdentifiseer en beskryf. Slegs 'n paar van hierdie klone word geassosieer met metisillien weerstandigheid (Klonale kompleks (KK) 5, KK8, KK22, KK30 en KK45). Studies oor die bevolking struktuur van S. aureus geïsoleer in Suid-Afrika is tot dusver beperk tot demografiese gebiede, fokus slegs op metisillien-weerstande S. aureus (MRSA) en het min klem op virulensie geplaas. Hierdie studie is onderneem om die bevolking struktuur van S. aureus, geïsoleer vanaf spesifieke kliniese bronne, in die pasiëntpopulasie van Tygerberg-hospitaal te ondersoek en om ondersoek in te stel na spesifieke gasheer-patogeen interaksies van verteenwoordigende isolate. Opeenvolgende, nie-herhalende en suiwer kliniese S. aureus isolate is versamel oor ´n periode van een jaar (September 2009/2010), tesame met pasiënt demografiese- en beperkte kliniese inligting. Stamme is deur PFGE en molekulêre merkers (spa, MLST, agr, SCCmec en PVL) beskryf. Verteenwoordigende isolate is gekies en ondersoek vir die teenwoordigheid van virulensie gene, aanhegting ( aan geïmmobiliseerde fibronektien [Fn], fibrinogeen [Fg], kollageen IV [CnIV] en kollageen VI [CnVI]), sellulêre indringing en die induksie van seldood. Statistiese assosiasies is bepaal tussen alle in vitro resultate en methicillin-weerstandigheid, klonaliteit, pasiënt MIV status en bakteriese PVL status. Fyftien persent van die isolate (n = 367) was MRSA. Vier-en-veertig van die isolate was PVL+. agrI-IV en SCCmec I-V is geïdentifiseer. Die MSSA bevolking was divers: ST22 (dominant), ST1865 en ST121 PVL +. ST45, ST1863 en ST15 was PVL+. PVL- MRSA was divers: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II en ST22-MRSA-IV. Die gene fnbA/B (fibronektien A/B), clfA/B (klontings faktor A/B), eap (ekstrasellulêre aanhegtings protein), nuc (nukease), coa (koagulase) en hld (delta toksien) was aangetref in alle verteenwoordigende isolate. Isolate van KK8 en KK6 het sterk aan alle ligande (100-700% van kontrole, ligand-afhanklike) aangeheg, terwyl isolate van KK45, KK22 en KK88 slegs sterk aand fibronektien en fibrinogeen aangeheg het. Isolate van KK30, KK15, en KK12 het baie sterk aan CnIV (> 300%) aangeheg en KK8, KK15, en KK6 and CnVI (> 200%). Isolate van KK30, KK8, KK15, KK6, KK12, KK97, KK88 en KK45 was hoogs indringend (> 100%). ST121 was nie-indringende (> 50%). Isolate van KK5, KK30 en KK121 was nie-sitotoksiese (<50%), terwyl isolate van KK22, KK8, KK15, KK45 en KK88 baie sitotoksies was (> 70%). Geen betekenisvolle verskil is waargeneem in die aanhegting of seldood induksie van MRSA teenoor MSSA klone of tussen isolate van MIV+ teenoor MIV- persone nie. PVL- isolate het hoër sellulêre indringing as PVL+ isolate vertoon. Die teenwoordigheid van ST612-MRSA-IV, ST22-MRSA-V en ST8-MRSA-V verwys na die plaaslike verwerwing van SCCmec, aangesien ons MSSA isolate beskryf het met dieselfde spa-tipes. Talle MSSA klone was algemeen, maar het nie 'n beduidende genetiese agtergrond met MRSA vertoon nie. PVL was baie algemeen onder MSSA isolate en die PVL gene is dalk onafhanklik van SCCmec verkry. Die vermoë om aan spesifieke geïmmobiliseer ligande in vitro aan te heg was divers en groepeer met die genetiese agtergrond, terwyl die meerderheid van die isolate indringend was en kon betekenisvolle sel dood veroorsaak. Ons kan aflei dat die bevolking van S. aureus by die Tygerberg hospitaal saamgestel is uit 'n groot aantal van MSSA en MRSA klone, wat verskillende patrone van aanhegting aan geselekteerde ligande vertoon en waarvan die meeste klone indringende en sitotoksies is. / DFG/NRF International Research and Training Group (IRTG) 1522 “HIV and associated infectious diseases in Southern Africa” / National Research Foundation / Medical Research Council, Medi-Clinic / Harry Crossley Fund (Stellenbosch University) / Stellenbosch University
4

Coreceptor expression and T lymphocyte subset distribution in HIV-infected and TB co-infected South African patients on anti-retroviral therapy

Ngandu, Jean Pierre Kabue 12 1900 (has links)
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.
5

Evaluation of high-throughput methodology for multi-gene screening in patients with Non-Alcoholic Fatty Liver Disease (NAFLD)

Fisher, Leslie Reginald 12 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disease in Western countries and is considered the hepatic manifestation of the Metabolic Syndrome (MetS). Its heterogeneous nature ranges from hepatic steatosis through steatohepatitis to advanced fibrosis and cirrhosis where the ingestion of significant amounts of alcohol has been excluded. The disease profile of NAFLD and its necro-inflammatory subset Nonalcoholic Steatohepatitis (NASH) were described in the parent study, which provided a clinically well-characterised patient cohort for the present investigation. South African patients with NASH had significantly higher mean serum cholesterol and triglyceride levels than those with fatty liver only. The objective of this study was to implement a high-throughput real-time polymerase chain reaction (PCR) method in our laboratory to enable the assessment of cardiovascular genetic risk factors in NAFLD patients. The specific aims were to determine the clinical utility and perform analytical validation of each mutation included in the multi-gene cardiovascular disease (CVD) screening assay. The Pathology Supported Genetic Testing (PSGT) concept developed at our department provides a practical approach to personalized medicine. The CVD multi-gene screen analyses key metabolic pathways relating to atherogenic dyslipidaemia, chronic inflammation, hypercoagulation and iron dysregulation implicated in insulin resistance, which is known to be a universal factor in the pathogenesis of NAFLD. Deleterious low-penetrance mutations in the APOE (APOE2 and E4 alleles), MTHFR (677C>T and 1298A>C), F2 (20210G>A), FV (1691G>A, Leiden) and HFE (C282Y and H63D) genes were included for analysis due to their important role as genetic contributors to these biological processes. A total of 178 patients diagnosed with NAFLD and 75 controls were studied using direct DNA sequencing and a RT-PCR system for mutation detection. In addition, two patients with high ferritin levels were included as case studies. A significant association was found between HFE mutations and elevated Alanine Transaminase (ALT) levels in the NAFLD population (p = 0.04). This discovery is interpreted as the identification of a subset of patients at greater risk of developing progressive liver damage who would benefit most from genetic testing to direct more aggressive therapy at an earlier stage. The necessity of an integrative, systems-based network approach was demonstrated to more accurately distinguish between Hereditary Haemochromatosis (HH) and Insulin Resistance-associated Hepatic Iron Overload (IR-HIO) syndrome in obese patients. The PSGT approach to personalized medicine facilitates diagnosis of CVD subtypes, prevention of cumulative risk and the formulation of gene-based intervention programs tailored to the needs of the patient. These findings support the clinical utility of the CVD multi-gene test to guide chronic disease risk management in patients with NAFLD. The HFE mutation detection component of this test is of particular relevance in directing an effective treatment strategy in patients with a medical history of CVD and/or high iron stores. / AFRIKAANSE OPSOMMING: Nie-Alkoholiese Vettige Lewer Siekte (NAFLD) is die mees algemene kroniese lewer siekte in Westerse lande en word bestempel as die hepatiese manifestasie van die Metaboliese Sindroom (MetS). Die heterogene natuur van NAFLD strek van hepatiese steatose deur steatohepatietis tot gevorderde fibrose en sirrose waar grootskaalse alkohol inname uitgesluit is. Die siekte-profiel van NAFLD en sy nekro-inflammatoriese subtipe Nie-Alkoholiese Steatohepatietis (NASH) is reeds beskryf in die ouer studie, wat ‗n klinies goed-gekarakteriseerde pasiënt groep vir die huidige ondersoek daar gestel het. Suid-Afrikaanse pasiënte met NASH het beduidend hoër gemiddelde serum cholesterol en trigliseried vlakke in vergelyking met slegs vettige lewer. Die doel van hierdie studie was om ‗n hoë deurvoer rieëltyd polimerase kettingreaksie (RT-PCR) metode in ons laboratorium te implimenteer om kardiovaskulêre genetiese risiko faktore in NAFLD pasiënte te ondersoek. Die spesifieke mikpunte was om die kliniese nut en analitiese geldigheid van elke mutasie wat ingesluit is in die multi-geen kardiovaskulêre siekte (KVS) siftings toets vas te stel. Die Patologie Ondersteunde Genetiese Toetsing (PSGT) konsep wat by ons departement ontwikkel is, verskaf ‗n praktiese benadering tot persoonlike medisyne. Die KVS multi-geen toets analiseer belangrike metaboliese weë verwant aan atherogene dyslipidemie, kroniese inflammasie, oormatige bloedstolling en yster disregulering wat betrokke is by insulien weerstand wat bekend is as ‗n universele factor in the patogenese van NAFLD. Nadelige lae-penetrasie mutasies in die APOE (APOE2 en E4 allele), MTHFR (677C>T en 1298A>C) F2 (20210G>A), FV (1691G>A, Leiden) en HFE (C282Y en H63D) gene was ingesluit vir analise as gevolg van hul belangrike rol as genetiese bydraers tot die bogenoemde biologiese prosesse. ‗n Totaal van 178 pasiënte gediagnoseer met NAFLD en 75 kontroles is bestudeer deur gebruik te maak van direkte DNA volgordebepaling en ‗n RT-PCR metode vir mutasie opsporing. Twee pasiënte met verhoogde ferritien vlakke is ook as gevalle studies ingesluit. ‗n Beduidende assosiasie is gevind tussen HFE mutasies en verhoogde Alanien Transaminase (ALT) vlakke in die NAFLD studiepopulasie (p = 0.04) wat aanduidend is van ‗n subgroup van pasiënte wat die meeste baat sal vind uit genetiese toetsing om meer aggressiewe behandeling te rig op' n vroeër stadium. Die noodsaaklikheid van 'n geïntegreerde, stelsels-gebaseerde netwerk benadering is gewys om meer akkuraat te onderskei tussen Oorerflike Hemochromatose (HH) en Insulien Weerstand-geassosieerde Hepatiese Yster Oorlading (IR-HIO) sindroom in vetsugtige pasiënte. Die PSGT benadering tot persoonlike medisyne formuleer geen-gebaseerde intervensie programme aangepas tot die behoeftes van die pasiënt ek maak diagnose van KVS-subtipes en voorkoming van kumulatiewe risiko moontlik. Hierdie bevindinge ondersteun die kliniese nut van die KVS multi-geen toets om riglyne vir die risikobestuur van kroniese siektes soos NAFLD daar te stel. Die HFE mutasie opsporings komponent van hierdie toets is van besondere belang om 'n effektiewe strategie vir die behandeling van pasiënte met 'n mediese geskiedenis van KVS en/of hoë yster vlakke daar te stel.
6

Ontogeny of the innate immune response in healthy South African infants

Adams, Rozanne Charlene McChary 12 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Infection is a major cause of morbidity and mortality in infants within the first few months of life. Susceptibility to infectious disease in this vulnerable population is more prevalent in resource-limited regions, with a higher disease burden. Due to certain deficiencies in their adaptive immune system, neonates rely predominantly on their innate immune system for protection against infection, a vital component in the early host defence against pathogens. Several studies have described differences in neonatal innate toll-like receptor-mediated responses compared to adult counterparts, though very little is known about these receptor responses within resource-limited settings. To address this issue, we assessed the longitudinal development of cytokine-specific responses of TLR4 and TLR7/8 in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells in infants from a resource-limited setting, South Africa, within the first 12 months of life and compared it to adults. Contrary to previously published literature, we observed heightened production of TH-1 cytokines: we showed increased responsiveness to TLR4 and TLR7/8 stimulation in infants at two and six weeks of age, which may be due to vaccination administered at birth. Unexpectedly, the hyper-inflammatory response persisted at six months in response to the LPS (TLR4) stimulus. This increased response at six months may be attributed to decreased passive immunity through infant weaning as well as increased exposure to microbial pathogens in this setting. Maturation of most cytokine responses was reached at twelve months for the TLR4 receptor, and at six months for the TLR7/8 receptor. The first year of life represents a critical period for maturation of the immune response. Data from this study point towards an elevated response within the first six months of life. This heightened response reflects both an ability to mount a sufficient TH-1 response in infancy, but more likely, the increased exposure to microbial stimuli in the environment. Thus, we speculate that these age-specific inflammatory responses may influence the outcome of immune responses to various vaccines administered, which may result in altered responsiveness to immunisation in infancy. / AFRIKAANSE OPSOMMING: Die hoof oorsaak vir morbiditeit en mortaliteit in babas binne die eerste paar maande van hul lewe word toegeskryf aan infeksie. In hulpbron beperkte gebiede, gekenmerk deur `n groter siektelas, is daar `n verhoogde vatbaarheid vir infeksie in hierdie kwesbare populasie. As gevolg van sekere gebreke in die verworwe immuunstelsel, maak pasgebore babas hoofsaaklik staat op hul aangebore immuunstelsel vir beskerming teen infeksie, ’n belangrike komponent vir die vroeë verdediging teen patogene. Verskeie studies het al die verskille in toll-tipe reseptor (TTR) bemiddelde reaksies tussen pasgebore babas en volwassenes vergelyk, maar nie veel is bekend oor hierdie reaksies in areas waar hulpbronne beperk is nie. Om hierdie kwessie aan te spreek is die longitudinale ontwikkeling van sitokien-spesifieke reaksies van die TTR4 en TTR7/8 reseptore van monosiete, miëloïede en plasmasitoïede dendritiese selle van babas in die hulpborn beperkte land Suid-Afrika, oor die eerste 12 maande geëvalueer en dit vergelyk met volwassenes. In teenstelling met vorige literatuur, het hierdie studie ’n polarisasie tot TH-1-sitokien produksie gevind: verhoogde reaktiwiteit van die TTR4 en TTR7/8 is gevind in babas van twee en ses weke oud, wat gedeeltelik as gevolg van die inenting kan wees wat toegedien was na geboorte. Hierdie hiper-inflammatoriese reaksie teen die TTR4 stimulus (Lipopolisakkaried (LPS), het teen verwagting voortgeduur tot op ses maande en kan toegeskryf word aan die vermindering van passiewe immuniteit deur spening, sowel as die toenemende blootstelling aan mikrobiese patogene in die omgewing. Maturasie vir die meerderheid van die sitokiene reaksies, is bereik op 12 maande vir TTR4, en op ses maande vir TTR7/8. Die eerste lewensjaar is ‘n kritiese periode vir die ontwikkeling van die immuunstelsel. Data van hierdie studie dui op ‘n verhoogde reaksie binne die eerste ses maande van ‘n baba se lewe. Hierdie verhoogde reaksie dui op die vermoë om `n voldoende TH-1 reaksie te ontlok, maar meer waarskynlik, verhoogde blootstelling aan mikrobiese stimuli in die omgewing. Dus spekuleer ons dat hierdie ouderdom-spesifieke reaksies dalk die uitkoms van die immuunreaksie teen verskeie entstof toediening kan beïnvloed in babas.
7

Automated sputum screening using the BD FocalPointTM Slide Profiler : correlation with transbronchial and transthoracic needle aspirates in a high risk population

Neethling, Greta Sophie 04 1900 (has links)
Thesis (MMed)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background: Sputum is a non-invasive, economic investigation whereby bronchogenic carcinoma can be identified. Manual cytological screening is labour intensive, time-consuming and requires a continuous high level of alertness. Automation has recently been successfully introduced in gynaecological cytology. Since sputum samples are similar to cervical smears, the question arises as to whether they are also suitable for automated screening. Objective: This study presented with various objectives: 1) To test automated sputum screening using the BD FocalPoint™ Slide Profiler (FP) and compare with manual sputum screening. 2) To determine the sensitivity and specificity of sputum in identification of bronchogenic carcinoma. 3) To ascertain if any clinical, radiological or bronchoscopy findings would be predictors for bronchogenic carcinoma. 4) To determine the significance of adequacy. Method: Sputum samples were collected prospectively from patients attending the Division of Pulmonology at Tygerberg hospital for a transbronchial fine needle aspiration biopsy (TBNA) or a transthoracic fine needle aspiration biopsy (TTNA) for the period from 2010 to 2012. A pre-bronchoscopy sputum was collected and submitted for processing. Stained slides were put through the FP for automated screening. After slides were qualified, sputum slides were put back in the routine screening pool. Correlation was done using the TBNA/TTNA result as the standard to evaluate the sputum results. Results: 108 sputum samples were included in this study. Of the 84.3% malignant (n=91) and 15.7% benign (n=17) cases confirmed with a diagnostic procedure, sputum cytology had a sensitivity of 38.5% (35/91 malignant cases), and a specificity of 100% (17/17 benign cases). Automated screening had a better sensitivity of 94.3% (33/35 positive sputum cases), while manual screening showed a sensitivity of 74.3% (26/35 positive sputum cases) when compared to the final sputum result. Individual parameters with a significant association with positive sputum included the presence of an endobronchial tumour, partial airway obstruction / stenosis, round mass, spiculated mass (negative association), loss of weight (negative association) and squamous cell carcinoma as the histological subtype. Adequacy was not as significant as hypothesised since 85.3% of true positive sputum, but also 65.5% of false negative sputum, had large numbers of alveolar macrophages present. Conclusion: Sputum cytology remains an important part of the screening programme for bronchogenic carcinoma in the public health sector of South Africa. Results confirm that sputum cytology is very specific, and automated screening improves sensitivity. Automated screening proved to be more time efficient, resulting in 83.1% reduction (p<0.0001) in the screening time spent per case by a cytotechnologist. Results confirm that the quantity of alveolar macrophages is not directly proprtional to pathology representation. Positive sputum results did however improve with sputum adequacy, but had no significant association. Recommendations from this study include adopting automated sputum screening. / AFRIKAANSE OPSOMMING: Agtergrond: Die verkryging van ‘n sputummonster is ‘n nie-indringende, ekonomiese ondersoek waardeur bronguskarsinoom identifiseer kan word. Nie-geoutomatiseerde sitologiese ondersoek is arbeidsintensief, tydrowend en vereis ‘n deurlopende hoë vlak van konsentrasie en fokus. Outomatisering is onlangs suksesvol geïmplementeer in ginekologiese sitologie-ondersoeke. Aangesien sputummonsters soortgelyk aan servikale monsters is, het die vraag ontstaan of sputummonsters ook geskik sou wees vir geoutomatiseerde sifting. Doelwit: Hierdie studie het verskeie doelwitte gehad: 1) Om geoutomatiseerde sifting van sputummonsters te toets deur gebruik te maak van BD Focal Point ™ Slide Profiler (FP), en te vergelyk met nie-geoutomatiseerde sputum sifting. 2) Om die sensitiwiteit en spesifisiteit van sputum in die identifikasie van bronguskarsinoom te bepaal. 3) Om vas te stel of enige kliniese, radiologiese of brongoskopiese bevindings bronguskarsinoom sou kon voorspel. 4) Om die belang van ‘n verteenwoordigende monster te bepaal. Metode: ‘n Prospektiewe studie van die pasiënte wat die Divisie van Pulmonologie by Tygerberg Hospitaal vir transbrongiale nodale aspirasie (TBNA) of ‘n transtorakale aspirasie (TTNA) vanaf Julie 2010 tot Mei 2012 bygewoon het, is gedoen. ‘n Prebrongoskopiese sputum is geneem en gestuur vir prosessering. Die gekleurde skuifies is deur die FP gestuur vir geoutomatiseerde ondersoek. Indien die sputumskuifies gekwalifiseer het vir geoutomatiseerde sifting, is hulle in die groep vir ondersoek ingesluit. ‘n Korrelasiestudie, om die sputumresultate te evalueer, is uitgevoer deur die TBNA/TTNA bevindings as standaard te gebruik. Resultate: Vir hierdie studie is 108 sputummonsters ingesluit. Vanuit die 84.3% maligne (n=91) en 15.7% benigne (n=17) gevalle, bevestig deur ‘n diagnostiese prosedure, het sputumsitologie ‘n sensitiwiteit van 38.5% (35/91 maligne gevalle) en ‘n spesifisiteit van 100.0% (17/17 benigne gevalle), getoon. Geoutomatiseerde sifting het ‘n beter sensitiwiteit met 94.3% (33/35 maligne gevalle), terwyl nie-geoutomatiseerde (ondersoek) ‘n sensitiwiteit van 74.3% (26/35 maligne gevalle) wanneer met die finale resultaat vergelyk, gevind. Individuele parameters met ‘n betekenisvolle assosiasie het die teenwoordigheid van ‘n endobrongiale tumor, gedeeltelike lugwegobstruksie / stenose, ronde massa, ‘n spekuleerde massa (negatiewe assosiasie), gewigsverlies (negatiewe assosiasie) en plaveiselkarsinoom as die histologiese subtipe, ingesluit. Geskiktheid van die monster was nie so betekenisvol as wat in die hipotese gestel is nie: aangesien 85.3% van ware positief gediagnoseerde sputummonsters, maar ook 65.5% van die vals negatiewe sputummonsters, groot hoeveelhede alveolêre makrofae ingesluit het. Gevolgtrekking: Sputumsitologie bly steeds ‘n belangrike deel van die siftingsprogram vir bronguskarsinoom in die openbare gesondheidssektor in Suid-Afrika. Resultate van hierdie studie bevestig dat sputumsitologie baie spesifiek is en dat geoutomatiseerde sifting die sensitiwiteit verbeter. Ge-outomatiseerde sifting het bewys dat dit meer tydsbesparend is, met ‘n 83.1% vermindering (p<0.0001) in die siftingstyd wat deur een sitotegnoloog per geval bestee word. Resultate het bevestig dat die hoeveelheid alveolêre makrofae nie direk proporsioneel verwant is tot die patologie nie. Hoe meer verteenwoordigend die sputummonster was, hoe groter was die kanse om ‘n akkurate positiewe diagnose te maak. Die assosiasie van die geskiktheid van die sputummonster en die positiewe resultate het egter nie ‘n statisties betekenisvolle resultaat getoon nie. Aanbevelings vir hierdie studie sluit in die aanwending van geoutomatiseerde sputumondersoeke.
8

Free light chains in patients with HIV: establishing local reference ranges and their association with stage of disease, chronic antigen stimulation and the effect of Haart

Germishuys, Jurie J. 03 1900 (has links)
Thesis (MMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: Serum free light chains (FLC) are associated with imbalances in heavy and light chain production. Abnormal FLC ratios have been associated with risk of progression in certain diseases. Automated assays are available for their determination and they are used in the followup and management of patients with monoclonal gammopathies. Acceptable imprecision, specificity, accuracy and reproducibility between reagent batches is required to prevent under- or overestimation. Method validation is a standard process in every good laboratory to judge the acceptability of a new method. Reference intervals have been established in an older population, but it was considered important to verify these in our population. HIV is associated with B-cell dysfunction. As B-cell abnormalities are associated with disorders leading to monoclonal gammopathies, we postulated that the FLC levels and FLC ratio would be abnormal in HIV infected individuals. Methods and materials: Controls and pooled patient samples were used for the method validation study which included imprecision studies, linearity, recovery and interference studies, and method comparison studies, the latter compared our method to the same method used in another laboratory. For the reference interval study, blood was obtained from 120 healthy subjects. The following blood tests were performed: total protein, IgG, IgA, IgM, creatinine, protein electrophoresis, kappa FLC and lambda FLC. Using the kappa and lambda FLC results, a FLC ratio was determined. Three hundred and sixty-nine HIV positive subjects were then studied. The same tests were performed, as well as CD4+ counts and viral loads on the majority of them. Results: For the method validation study, precision, linearity and recovery was acceptable. Minimal interference was observed with haemolysis, lipaemia, bilirubin and rheumatoid factor. Our method showed comparable performance with the established method. For the reference interval study, all the creatinine values were normal, as were serum protein values. The serum protein electrophoreses were independently reviewed by 3 pathologists. Most were normal, with a few polyclonal increases seen, but no definite monoclonal bands. The 95% reference intervals for FLC’s as well as the FLC ratio were not statistically significantly different to the manufacturer’s recommendations. When examining the HIV positive study population, we found that FLC and FLC ratio were influenced by markers of HIV disease severity, such as CD4+ count, IgG, viral load, use of antiretroviral treatment and abnormal serum protein electrophoreses. Conclusion: The validation study of FLC showed excellent precision, acceptable bias, good linearity, good recovery and minimal interference, allowing routine introduction of the test. The 95% reference intervals obtained for our population were slightly higher than those recommended by the manufacturer. However, as most of the values fell within the manufacturer’s limits, we could accept the manufacturer’s recommended cut-offs. We found that FLC levels were definitely influenced by markers of HIV disease severity in our population and we postulate that they may be of use for follow-up of patients with HIV. / AFRIKAANSE OPSOMMING: Agtergrond: Serum vry ligte kettings (VLK) word geassosieer met ‘n wanbalans van ligte en swaar ketting produksie. Abnormale VLK ratios is geassosieer met ‘n risiko van verloop in sekere siektes. Geoutomatiseerde laboratorium toetse vir VLK is beskikbaar vir hul bepaling en word gebruik om pasiënte met monoklonale gammopatieë op te volg en te behandel. Aanvaarbare impresisie, spesifisiteit, akkuraatheid en herhaalbaarheid tussen reagens besendings is belangrik om onder- of oorbepaling te verhoed. Metode validasie is ’n standaard proses in elke goeie laboratorium om die aanvaarbaarheid van ’n nuwe metode te bepaal. Verwysingswaardes is al bepaal in ’n ouer populasie. Ons het besluit om die verwysingswaardes in ons populasie te bepaal. Mens-immuungebrekvirus (MIV) word geassosieer met B-sel disfunksie. Omdat B-sel abnormaliteite geassosieer word met afwykings wat tot monoklonale gammopatieë lei, het ons gepostuleer dat die VLK vlakke en VLK ratio abnormaal sal wees in MIV geïnfekteerde persone. Metodes en Materiale: Kontroles en pasiënt monsters is gebruik vir die metode validasie studie wat impresisie studies, lineariteit, herwinning, inmenging en metode korrelasie studies ingesluit het. In laasgenoemde geval is ons metode met dieselfde metode van ’n ander laboratorium vergelyk. Vir die verwysingswaardes studie is 120 gesonde persone se bloed gebruik. Die volgende toetse is bepaal: totale proteïen, IgG, IgA, IgM, kreatinien, proteïen elektroferese, kappa en lambda VLK. Die VLK ratio is bepaal deur die kappa en lambda resultate te gebruik. Driehonderd nege en sestig MIV-positiewe pasiente is gebruik vir die studie. Dieselfde toetse was gedoen, asook CD4+ tellings en virale ladings op die meerderheid van pasiente. Resultate: Vir die metode validasie studie, was presisie, lineariteit en herwinning aanvaarbaar. Minimale inmenging van hemolise, lipemie, bilirubien en rumatoïede factor is waargeneem. Ons metode het goed gekorreleer met die bepaalde metode. Die serum kreatinien en serum totale proteïen waardes was normaal tydens die verwysingswaardes studie. Die serum proteïen elektroferese was onafhanklik beoordeel deur 3 patoloë. Die meeste was normaal met enkele poliklonale verhogings, maar geen definitiewe monoklonale bande nie. Die 95% verwysings intervalle vir VLK en VLK ratio het nie statisties betekenisvol verskil van die vervaardiger se aanbevelings nie. In die studie van die MIV-positiewe studie populasie, het ons gevind dat VLK en VLK ratio beïnvloed word deur merkers van ernstige MIV siekte, soos CD4+ telling, IgG, virale lading, die gebruik van antiretrovale medikasie en abnormale serum proteïen elektroferese. Gevolgtrekking: Die validasie studie van VLK het uitstekende presisie, aanvaarbare partydigheid, goeie lineariteit, goeie herwinning en minimale inmenging gewys, wat die roetine instelling van die toets toegelaat het. Die 95% verwysingsintervalle wat vir ons populasie bepaal is, was effens hoër as die vervaardiger se aanbeveling. Die meeste van die waardes het egter binne die vervaardiger se limiete geval, dus kon ons die vervaardiger se afsnypunte aanvaar. Ons het gevind dat VLK vlakke definitief beïnvloed word deur merkers van die ernstigheidsgraad van MIV siekte in ons populasie en ons postuleer dat VLK van waarde kan wees met die opvolg van MIV pasiente. / NHLS / Harry Crossley for funding obtained
9

The occurance of genetic variations in the MYH9 gene and their association with CKD in a mixed South African population

Masconi, Katya 12 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The purpose of this study was to investigate the association of the selected MYH9 single nucleotide polymorphisms (SNPs) with chronic kidney disease (CKD) and its related co-morbidities in the South African mixed ancestry population residing in Bellville South, Cape Town. In 2008, two landmark studies identified SNPs in the MYH9 gene which explained most of the increased risk for non-diabetic CKD in African Americans. These polymorphisms were later found to be weakly associated with diabetic nephropathy. Three SNPs that exhibited independent evidence for association with CKD were selected (rs5756152, rs4821480 and rs12107). These were genotyped using a Taqman genotyping assay on a BioRad MiniOpticon and confirmed by sequencing in 724 subjects from Bellville South, Cape Town, South Africa. Prevalent CKD was defined based on the estimated glomerular filtration rate calculated using the modification of diet in renal disease (MDRD) formula. Chronic kidney disease was present in 214 subjects (29.6%), 96.3% were stage 3 and only 8 subjects were stage 4. In additive allelic models, adjusted for age and gender, rs5756152 demonstrated an association with kidney function whereby each G allele of rs5756152 increased eGFR by 3.67 ml/min/1.73, reduced serum creatinine by 4.5% and increased fasting plasma glucose by 0.51 mmol/L. When an interaction model was used, the effect of rs5756152 on serum creatinine, eGFR and blood glucose levels was retained, and enhanced, but only in diabetic subjects. In addition, rs4821480 T allele increased eGFR while rs12107 A allele decreased glucose levels in diabetic subjects. In contrast to reports that MYH9 SNPs are strongly associated with non-diabetic end stage renal disease, our study demonstrated that rs5756152 and rs4821480 are associated with early kidney function derangements in type 2 diabetes whilst rs12107 is associated with glucose metabolism. Our findings, along with previous reports, suggest that the MYH9 gene may have a broader genetic risk effect on different types of kidney diseases than previously thought. / AFRIKAANSE OPSOMMING: Hierdie studie het ondersoek ingestel na die verband tussen drie gekose MYH9-enkelnukleotied-polimorfismes (SNP’s) en chroniese niersiekte (hierna ‘niersiekte’), wat verwante ko-morbiditeite insluit, onder ’n Suid-Afrikaanse populasie van gemengde afkoms in Bellville-Suid, Kaapstad. Twee rigpuntstudies het in 2008 op SNP’s in die MYH9-geen afgekom wat verklaar het waarom Afro-Amerikaners ’n hoër risiko vir niediabetiese niersiekte toon. Later is bevind dat hierdie polimorfismes ook ’n swak verband met diabetiese nefropatie het. Drie SNP’s wat elk onafhanklik bewys gelewer het van ’n verband met niersiekte is vervolgens gekies (rs5756152, rs4821480 en rs12107). Die SNP’s is daarná met behulp van die Taqman-toets op ’n BioRad MiniOpticon aan genotipering onderwerp, en is toe deur middel van reeksbepaling by 724 proefpersone van Bellville-Suid, Kaapstad, Suid-Afrika, bevestig. Die voorkoms van niersiekte is bepaal op grond van die geraamde glomerulêre filtrasietempo (eGFR), wat aan die hand van die ‘niersiekte-dieetveranderings’- (MDRD-)formule bereken is. Daar is bevind dat 214 proefpersone (29,6%) aan chroniese niersiekte ly – 96,3% was in fase 3 en slegs agt proefpersone in fase 4. In toegevoegde alleliese modelle wat vir ouderdom en geslag aangepas is, het rs5756152 ’n verband met nierfunksie getoon: Elke G-allel van rs5756152 het eGFR met 3,67 ml/min/1,73 verhoog, serumkreatinien met 4,5% verlaag en vastende plasmaglukose met 0,51 mmol/L verhoog. Toe ’n interaksiemodel gebruik is, is die effek van rs5756152 op serumkreatinien, eGFR en bloedglukosevlakke behou en versterk, hoewel slegs by diabetiese proefpersone. Daarbenewens het die T-allel van rs4821480 eGFR verhoog, terwyl die A-allel van rs12107 ook glukosevlakke by diabetiese proefpersone verlaag het. In teenstelling met bewerings dat MYH9-SNP’s ’n sterk verband met niediabetiese eindstadiumniersiekte toon, het hierdie studie bewys dat rs5756152 en rs4821480 met vroeë nierfunksieversteurings by tipe 2-diabetes verband hou, terwyl rs12107 weer met glukosemetabolisme verbind word. Tesame met vorige studies, doen hierdie navorsingsbevindinge dus aan die hand dat die MYH9-geen dalk ’n groter genetiese risiko-effek op verskillende tipes niersiekte het as wat voorheen vermoed is. / Cape Peninsula University of Technology Research Fund / University of Stellenbosch Merit Bursary
10

The investigation of genotypic antiretroviral drug resistance in the context of the South African national antiretroviral roll-out programme

Van Zyl, Gert Uves 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: Since the South African public sector antiretroviral roll-out programme started in 2004, the success of antiretroviral combination therapy (cART) has been experienced in terms of survival, prevention of mother-to-child transmission (PMTCT) and quality of life. However, as the programme matures, viral resistance to the constituent drugs will increase. Monitoring antiretroviral drug resistance (ARVDR) should therefore be a priority in the public health approach to HIV treatment. Methods: A cross-sectional investigation of genotypic antiretroviral drug resistance in: a) HIV-infected mothers who were exposed to a PMTCT regimen of short course azidothymidine (AZT) with single dose nevirapine (NVP) during labour. b) HIV-infected adults and children who were cART-naïve (transmitted or initial resistance). c) HIV-infected adults and children who were failing cART (drug-induced or acquired resistance). In case of adults, this includes patients on a first-line, non-nucleoside reverse transcriptase (NNRTI)-based regimen, or on a second-line, protease inhibitor (PI)-based regimen, and in case of children, this includes patients on a first-line PI-based regimen. Results: In mothers who received a PMTCT-regimen that combined AZT and NVP the prevalence of NNRTI resistance mutations was 17.1% (95% CI: 8.7-25.6%). The prevalence of transmitted ARVDR in adults was low, as was initial ARVDR in young children (mostly PMTCT-exposed), except for NNRTI resistance in children who had received NVP as part of PMTCT. Drug-induced resistance was found in adults failing first-line NNRTI-based cART, with 83% having resistance to ≥1 drug. In contrast, adult patients failing second-line PI-based cART had a low prevalence of PI resistance; the predominant reason for failure was poor drug exposure, as detected by measuring lopinavir concentrations in blood plasma and hair samples. In contrast, PI resistance in children was not rare, largely due to historic exposure to un-boosted PIs. This resulted in extensive resistance to PIs and reverse transcriptase inhibitors (RTI) in some children. Conclusions: A combined regimen of short course AZT with intrapartum NVP for PMTCT may, in addition to reducing the risk of neonatal infection, also reduce the risk of NVP resistance in the mothers compared to a regimen of NVP only. In South Africa, the prevalence of transmitted ARVDR remains low relative to industrialised countries, probably as comparatively little time has elapsed since the scale-up of cART. Adults failing first-line cART are likely to respond to second-line cART, without failure due to resistance. However some children with PI and RTI resistance cannot be adequately treated with drugs currently available through the roll-out programme. This emphasizes the urgent need for a rational and science-based approach to managing cART-experienced children, including access to additional drugs to form a third-line paediatric cART regimen. / AFRIKAANSE OPSOMMING: Inleiding: Sedert die begin van die Suid Afrikaanse publieke sektor antiretrovirale uitrol program in 2004 is die sukses van antiretrovirale kombinasie-behandeling (k-ARB) ervaar in terme van oorlewing, voorkoming van moeder na kind oordrag (VMKO) en lewenskwaliteit. Nietemin, sal weerstandigheid teen die middels wat in die antiretrovirale program gebruik word toeneem soos wat die program gevestig raak. Die monitoring van antiretrovirale middel-weerstandigheid is derhalwe ‘n prioriteit in gemeenskap-gesondheid benadering tot MIV behandeling. Metodes: ‘n Deursnit ondersoek van genotipiese antiretrovirale middel-weerstandigheid in: a) MIV-geïnfekteerde moeders wat blootgestel is aan VMKO regimen bestaande uit ‘n kort kursus AZT met ‘n enkeldosis nevirapien (NVP) tydens kraam. b) MIV-geïnfekteerde volwassenes en kinders wat komibinasieterapie-naïef (oorgedraagde of inisiële weerstandigheid) is. c) MIV-geïnfekteerde volwassenes en kinders wat k-ARB faal (middel-geïnduseerde weerstandigheid). In geval van volwassenes, sluit dit pasiënte op ‘n eerste-linie, non-nucleosied tru-transkriptase inhibitor (NNRTI)-regimen, en tweede-linie protease inhibitor (PI)-gebaseerde regimen, en in geval van kinders, sluit dit pasiënte in op ‘n eerste-linie PI-gebaseerde regimen. Resultate: In moeders wat ‘n gekombineerde AZT en NVP VMKO-regimen ontvang het, was die voorkoms van NNRTI weerstandigheid 17.1% (95%-vertrouensinterval: 8.7-25.6%). Die voorkoms van oorgedraagde ARVMW in MIV-geïnfekteerde volwassenes en kinders wat kombinasieterapie-naïef is, was laag, so ook ARVMW in jong kinders (meestal VMKO-blootgestel), behalwe vir non-nukleosied tru-transkriptase inhibitor (NNRT) weerstandigheid in kinders wat NVP ontvang het deur VMKO. Middel-geïnduseerde weerstandigheid was gevind in volwassenes wat die eerste-linie NNRTI-gebaseerde k-ARB gefaal het, met 83% wat weerstandigheid teen ≥1 middel het. Volwassenes wat ‘n tweede-linie protease inhibitor (PI) –gebaseerde k-ARB gefaal het , het ‘n lae voorkoms van PI weerstandigheid, met die oorwegenede oorsaak, swak middel-bloostelling, soos bepaal deur van lopinavir-konsentrasies in bloed plasma en hare. In teenstelling hiermee was PI weerstandigheid nie skaars in kinders nie, hoofsaaklik weens historiese blootstelling an ongeskraagde PI-behandeling. Dit het tot uitgebreide weerstandigheid tot PIs en tru-transkritptase inhibitors (RTI) in sommige kinders gelei. Gevolgtrekkings: ‘n Gekombineerde regimen van ‘n kort kursus AZT met NVP tydens kraam vir VKMO, mag bykomend tot die vermindering die risiko van pasgebore infeksie, ook die kans vir weerstandigheid teen NVP in die moeders verlaag in vergelyking met ‘n regimen van NVP-alleen. Die voorkoms van oorgedraagde ARVMW is tans laag in vergelyking met geïndustrialiseerde lande, waarskynlik aangesien daar nog betreklik min tyd verloop het sedert k-ART wyd beskikbaar gemaak is. Volwassenes wat eerstelyn kombinasie terapie faal sal waarskynlik goed reageer op tweede-linie terapie, sonder terapie faling weens middelweerstandigheid. Daarenteen kan sommige kinders met protease inhibitor en tru-transkriptase weerstandigheid nie voldoende behandel word met die huidig-beskikbare middels in die uitrol program nie. Dit beklemtoon die dringende noodsaaklikheid van ‘n rasionele en wetenskaplike benadering tot k-ART in kinders, met ‘n lang terapie geskiedenis, wat toegang tot bykomende medikasie behels om `n derde-linie regimen saam te stel.

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