Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / Introduction: Obesity, which is implicated in the development of the metabolic syndrome (MS) is reaching epidemic proportions worldwide. MS significantly increases the risk of developing cardiovascular disease, which includes coronary artery disease. The current absence of animal models of diet induced obesity and the MS makes the investigation of the cardiovascular consequences of MS virtually impossible. As a result the effects of the MS on cardiac function, morphology and susceptibility to ischaemia are not well understood. Aims: We set out to: 1) develop and characterize a rodent model of dietinduced obesity and the MS, 2) investigate the susceptibility of hearts from these animals to ischaemia/reperfusion induced injury and, 3) determine whether angiotensin II (Ang II) and endothelin-1 (ET-1) plays a role in cardiac remodelling and/or the severity of ischaemia and reperfusion injury in this model. Methods: Male Wistar rats were fed a standard rat chow diet or cafeteria diet (CD) for 16 weeks. After the feeding period rats were sacrificed and blood and myocardial tissue samples were collected to document biochemical changes in these animals. Hearts were perfused on the isolated working rat heart perfusion apparatus to assess myocardial mechanical function before and after ischaemia. In a separate series of experiments, hearts underwent coronary artery ligation to determine the incidence and duration of ventricular arrhythmias during ischaemia and reperfusion, using electrocardiography. To assess a possible link between myocardial remodelling and ischaemia/reperfusion injury and myocardial Ang II and ET-1 content, we also measured these peptides under basal conditions and during ischaemia. Two-dimensional targeted Mmode echocardiography was used to assess in vivo myocardial mechanical function in control and obese rats. Results: After 16 weeks on the CD, obese rats satisfied the World Health Organization (WHO) criteria for the MS by having visceral obesity, insulin resistance, dyslipidaemia and an elevated systolic blood pressure, compared to control rats. Circulating Ang II levels, but not ET-1 levels, were elevated in CD fed rats. Obese rats had cardiac hypertrophy and ex vivo basal myocardial mechanical function was depressed in the CD fed rat hearts compared to control rat hearts. CD fed rat hearts had poorer aortic output (AO) recoveries compared to hearts from control rats. These hearts also had a higher incidence and duration of reperfusion arrhythmias. No such functional differences were seen in the in vivo experiments. No differences in basal or ischaemic myocardial Ang II and ET-1 levels were seen in either group. Conclusion: We have developed and characterized a model of diet-induced obesity and the MS. Obesity is associated with cardiac hypertrophy and an increased myocardial susceptibility to ischaemia and reperfusion injury in our model. The hearts from obese rats were also more prone to reperfusion ventricular arrhythmias. As myocardial function was only poorer in the ex vivo obese animal experiments, our data suggests that the obesity associated changes in function observed in the ex vivo studies may be related to the absence of circulating substrates or factors, which are essential for their normal mechanical function.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / Insulin stimulates the production of nitric oxide (NO) in endothelial cells and cardiac myocytes by a signalling pathway that involves the insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase and protein kinase B (PKB/Akt). Physiological concentrations of NO play an important part in maintaining normal vascular function. It has been suggested that nitric oxide synthase (NOS) activity and NO production are chronically impaired in diabetes mellitus by an unknown mechanism. The reninangiotensin system and subsequent production of angiotensin II (Ang II) are elevated in obesity and diabetes while antagonism of the AT1 receptor with Losartan has beneficial effects in patients with insulin resistance and type II diabetes. Aims: We therefore aimed to investigate (i) the effect of Ang II on myocardial insulin signalling with regards to key proteins (IRS-1, PKB/Akt, eNOS and p38 MAPK) in correlation with NO production, (ii) the effect of Losartan on these parameters. Methods: Hyperphagia-induced obese, insulin resistant rats (DIO=diet supplemented with sucrose and condensed milk) were compared to age-matched controls. Half the animals were treated with 10mg/kg Losartan per day for 1 week. Isolated hearts were perfused with or without 0.03 μIU/mL insulin for 15 min. Blood glucose, bodyweight, intraperitoneal fat and plasma insulin and Ang II were recorded. Proteins of interest and their phosphorylation were determined by Western blotting. NO production was flow cytometrically analyzed. ANOVA followed by the Bonferroni correction was used with a p< 0.05 considered significant. Results: DIO animals had significant elevated bodyweight, blood glucose, plasma insulin and Ang II levels. Our data showed that the hearts from the DIO animals are insulin resistant, ultimately reflected by the attenuated activation of the key proteins (IRS-1, PKB/Akt and eNOS) involved in insulin signalling as well as NO production. AT1 receptor antagonism improved NO production in isolated adult ventricular myocytes from DIO animals while concurrently enhancing expression of eNOS, PKB/Akt and p38 MAPK. In contrast, NO production as well as expression of eNOS and PKB/Akt was attenuated in control animals after Losartan treatment. Conclusion: These results suggested that Ang II via AT1 or AT2 receptors, modulates protein expression of both PKB/Akt and eNOS. This encouraged us to investigate the involvement of AT2 receptors in the observed changes. To investigate this we needed to establish a culture of neonatal rat cardiac myocytes treated with raised fatty acids and Ang II. If similar changes were induced as observed in the hearts of DIO animals, the involvement of the AT1 and AT2 receptors could be investigated using specific antagonists against these receptors. Primary cultured ventricular myocytes were isolated from 1-3 day old Wistar rat pups. They were cultured for 48 hours before the addition of palmitate and oleate at a concentration of 0.25 mM each and were treated with or without the fatty acids for a period of 4 days. After 18 hours of serum starvation, cells were stimulated with or without 10 nM insulin for 15 minutes. The effect of fatty acid treatment on cell viability and glucose uptake were assessed by trypan blue and propidium iodide staining and 2-deoxy-D-3[H] glucose uptake respectively. Protein levels and phosphorylation of key proteins (PKB/Akt, PTEN and p38 MAPK) in insulin signalling was determined by Western blotting. 0.25 mM Fatty acids did not result in the loss of cell viability. Contrary to expectation, fatty acid treatment led to enhanced basal glucose uptake but lower Glut 1 protein expression. Basal protein expression of PPARα was, however, upregulated as was the expression of the phosphatase, PTEN. The latter could explain the lower PKB/Akt phosphorylation also documented. From these results we conclude that neonatal cardiac myocytes, cultured in the presence of elevated fatty acids, did not respond in a similar manner as the intact hearts of our animals and further modifications of the system might be needed before it can be utilized as initially planned.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: INTRODUCTION Mycobacterium tuberculosis (MTB) and Cryptococcus neoformans are the most common causes of chronic meningitis in South Africa. Conventional microbiology has limited utility in diagnosing these pathogens due to the paucibacillary nature of cerebrospinal fluid (CSF) and the diagnostic delay associated with culturing methods. This study aimed to evaluate the utility of an in-house polymerase chain reaction (PCR) method for the detection of the etiological agent of chronic meningitis. METHODS CSF samples (where volume exceeded 5ml) were submitted to the Medical Microbiology diagnostic laboratory of the Tygerberg Hospital from patients with suspected tuberculosis meningitis (TBM). Following routine bacteriology, the sample was used to inoculate two mycobacterial growth indicator tubes (MGIT A and B) and subsequently incubated in the BACTEC 960 automated system. MGIT A followed standard operating procedures and the time to culture positivity was noted. Weekly aliquots (up to 6 weeks) were removed from MGIT B. These samples were boiled to inactivate the bacteria and then the DNA was extracted using the Promega Wizard SV Genomic DNA kit. The DNA was then speciated by PCR and high-resolution melting analysis (HRM) by using primers specific to either the RD9 region of MTB complex or primers specific to the partial internal transcribed spacer 1 (ITS1), 5.8S rRNA gene and partial ITS2 sequence of C. neoformans. RESULTS Routine CSF microscopy indicated that 14 of the 78 patients (17.9%) had typical CSF findings of TBM (lymphocytes predominant, increased protein levels and decreased glucose levels). IV Ziehl-Neelsen (ZN) stains were positive for 12 (15.4%) samples, and MTB was cultured from 19 samples (24.4%). Our optimized PCR and HRM method was able to detect M. tuberculosis in 17 of the 19 culture positive specimens with a sensitivity of 89.5% and a specificity of 62.7%. The sensitivity of this method was higher than that of direct microscopy. In all of the PCR positive samples, the time to detection, compared to culture, could be shortened by 1 to 2 weeks. Only one sample was positive for Cryptococcus culture and another sample was positive with a Cryptococcus latex test. PCR for Cryptococcus was positive in 2 cases (n=78), sensitivities and specificities could not be reported due to the low number of positive cases. CONCLUSION We demonstrated that a short culture period and the use of commercial DNA extraction kit on CSF samples increases the sensitivity of molecular tests to diagnose tuberculosis. Furthermore, the molecular techniques could significantly reduce the time to positivity of results, when compared to culture. Due to the low occurrence of Cryptococcus in the samples included in our study, we could not comment on the diagnostic utility of PCR in the diagnosis of Cryptococcal meningitis, when compared to the conventional methods. / AFRIKAANSE OPSOMMING: INLEIDING Mycobacterium tuberculosis (MTB) en Cryptococcus neoformans is die mees algemeenste oorsake van kroniese meningitis in Suid-Afrika. Routine mikroskopie dra beperkte waarde in die diagnose van hierdie patogene as gevolg van die klein hoeveelhede organismes wat in die SSV (serobrospinale vog) voorkom en die lang tyd wat dit benodig om hierdie organisms te kweek. Hierdie studie beoog om die diagnostiese waarde van ‘n polymerase ketting reaksie (PKR) metode wat intern ontwerp is te evalueer vir die identifikasie van patogene verantwoordelik vir kroniese meningitis. METODES SSV monsters (waarvan die volume 5ml oorskry) en waar daar ‘n kliniese vermoede van tuberkulose meningitis (TBM) was, is na die diagnostiese Mediese Mikrobiologie laboratorium van Tygerberg hospitaal gestuur vir roetine bakteriologiese ontleding. Die oorblywende monsters is gebruik om twee mikobakteriële groei-indikasiebuise (MGIT A en B) te innokuleer en hulle is geïnkubeer in ‘n BACTEC 960 geautomatiseerde sisteem. MGIT A is volgens roetine diagnostiese metodes geanaliseer en die tyd tot ‘n positiewe resultaat is aangeteken Weeklikse monsters (tot en met week 6) is uit MGIT B verwyder en die monsters is gekook om sodoende die bakterië te inaktiveer. Die Promega Wizard SV Genomiese DNS ekstraksiemetode is gebruik om die DNS te versuiwer. Spesiëring van die DNS is deur middel van ‘n intern ontwerpte PKR en hoëresolusiesmeltingsmetode (HRS) gedoen met inleiers wat spesifiek is tot die RD9 gedeelte van die MTB kompleks en inleiers spesifiek tot die gedeeltelike interne getranskribeerde spasieerder 1 (ITS1), 5.8S rRNS geen en die gedeeltelike ITS2 DNS volgorde van C. neoformans. VI RESULTATE Roetine SSV mikroskopie het aangedui dat 14 uit 78 (17.9%) pasiënte tipiese SSV bevindings van TBM (oorwegend limfosiete, verhoogde proteïene en verlaagde glukose) gehad het. Ziehl- Neelsen (ZN) kleurings was positief vir 12 (15.4%) monsters, en MTB is gekweek in 19 (24.4%) van hierdie monsters. Ons geoptimaliseerde PKR en HRS metode het daarin geslaag om M. tuberculosis in 17 van die 19 kultuurpositiewe monsters aan te toon met ‘n sensitiviteit van 89.5% en ‘n spesifisitiet van 62.7%. Die sensitiwiteit van die direkte PKR was hoër in vergelyking met mikroskopie. In al die PKR positiewe monsters was die tyd tot aantoning, in vergelyking met kultuur, verkort met 1 tot 2 weke. Slegs een monster het C. neoformans gekweek en ‘n ander monster was positief met die kriptokokkale latekstoets. PKR vir C. neoformans was positief in 2 gevalle (n=78). Die sensitiwiteit en spesifisiteit van die C. neoformans PKR kon nie bepaal word nie weens te min gevalle. GEVOLGTREKKINGS Ons het aangetoon dat ‘n verkorte inkubasieperiode en die gebruik van ‘n kommersiële DNS ekstraksiemetode op SSV monsters die sensitiwiteit van die molekulêre tegniek vir die diagnose van tuberkulose verhoog en dat hierdie metode die tyd na positiwiteit aansienlik verkort in vergelyking met kultuur. Weens die lae getalle van kriptokokkale meningitis in ons studie kon ons nie kommentaar lewer op die akkuraatheid van PKR in die diagnose van kriptokokkale meningitis, in vergelyking met meer konvensionele metodes, nie.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility. / AFRIKAANSE OPSOMMING: Sperm morfologie bly ‘n belangrike parameter in die voorspelling van vrugbaarheid, beide in vivo en in vitro. Tog is daar nogsteeds ‘n aansienklike vlak van kommer rondom die ware potensiaal van hierdie parameter weens die gebrek aan standardisering van verskillende kleuringstegnieke wat gebruik word vir die evaluering van spermmorfologie. Hierdie studie is daarop gemik om ondersoek in te stel na twee algemeen gebruikte kleurings tegnieke naamlik, Rapidiff® (RD) en Papanicolaou (PAP), asook ‘n nuwe kommersiëel beskikbare kleurstof, SpermBlue® (SB), vir die evaluering van spermmorfometrie en morfologie. Resultate dui aan dat beduidende verskille in sperm morfometriese afmetings ontstaan as gevolg van die gebruik van die verskillende kleurstowwe. Bevindinge dui verder daarop dat RD swelling van die sperm se kop versoorsaak, terwyl PAP die spermkop laat krimp. Resultate wat verkry is met behulp van die SB kleuringstegniek dui daarop dat hierdie kleurstof aanleiding gegee het tot afmetings naaste aan die verkry tydens die beoordeling van vars, ongekleurde sperme. Die gebrek aan standardisasie en die uiteenlopende effekte wat verskillende kleurstowwe het op die spermstruktuur en die evaluering van sperm morfologie ingeheel is kommerwekkend, veral tydens die evaluering van pasiënte met grensgeval morfologie. Op grond van hierdie resultate, word die gebruik van die SB kleuringstegniek, bo die gebruik van RD en PAP, vir effektiewe en akkurate morfologie evaluering aanbeveel. Verdere ondersteuning vir die gebruik van die SB kleuringstegniek is die feit dat daar bevind is dat SB ‘n vinnige en eenvoudige metode is, wat toelaat vir maklike visualisering van sperme deur rekenaargesteunde sperm analise sisteme soos die Sperm Class Analyzer (SCA®). Die tweede doel van hierdie studie was om die konsentrasie, morfologie en die motiliteit van spermpopulasies te ondersoek, soos verkry tydens die voorbereiding van semen deur gebruik te maak van die PureSperm® digtheidsgradiënt en op-swem tegnieke. Die voorbereiding van semen is ‘n noodsaaklike stap in enige vrugbaarheidsbehandeling protokol, aangesien dit belangrik is dat die sperme wat deur hierdie prosesse verkry word oor die nodige morfologiese en motiliteit eienskappe beskik wat in staat is om die sukses van vrugbaarheidsbehandelings te verbeter. Huidiglik is die PureSperm® digtheidsgradiënt en op-swem tegnieke die mees algemeen gebruikte tegnieke in vrugbaarheidsklinieke. Alhoewel daar voldoende bewyse is wat voorstel dat elke tegniek effektief is vir die ekstraksie van sperme met beter kwaliteit vanuit semen, bly daar steeds onvoldoende bewyse wat daarop dui dat een van hierdie twee tegnieke beter is as die ander een. Huidige navorsing het getoon dat beide sperm voorbereidings metodes daarin geslaag het om sperme met normale morfologie en beter motiliteit te selekteer. Die opswem metode het ‘n spermpopulasie met beter motiliteit en verbeterde morfologie gelewer, soos getoets volgens die WGO kriteria, terwyl die PureSperm digtheidsgradiënt tegniek sperme met verbeterde morfologie, volgens beide die WGO en Tygerberg Streng Kriteria, en ‘n redelike verbetering in sommige motiliteits parameters geselekteer het. Hoewel die resultate wat verkry word via die op-swem metode voorstel dat dit die beste metode vir die gebruik tydens in vitro bevrugting sou wees, bly die baie lae konsentrasie van sperme wat met hierdie metode verkry word ‘n belangrike nadeel. Die PureSperm® skeidingstegniek laat egter toe vir die isolering van groter hoeveelhede sperme, wat waarskynlik meer voordelig sal wees vir bevrugtingsbehandelings wat meer sperme benodig. Gebaseer op hierdie bevindinge, word die gebruik van die PureSperm® digtheidsgradiënt tegniek aanbeveel, as gevolg van hierdie tegniek se vermoë om groot hoeveelhede goeie gehalte sperm te isoleer. Daar kan egter nogsteeds van op-swem metodes gebruik gemaak word tydens vrugbaarheidsbehandeling indien die semenmonster beskik oor ‘n hoë konsentrasie sperme met goeie beweeglikheid.

Thesis (MScMedSc) -- Stellenbosch University, 2001. / ENGLISH ABSTRACT: Osteopenia is a well documented complication of anorexia nervosa (AN). The pathogenesis of this bone loss is presently poorly defined in the literature. Pathogenetic mechanisms that have been implicated include certain nutritional factors, exercise abuse, hypogonadism, hypercortisolism and/or vitamin 0 deficiency. We studied, 59 Caucasian eating disorder patients aged 15-45yr. The eating disorder was classified by a single, qualified psychiatrist according to OSM IV R criteria as either anorexia nervosa (AN: n =25), bulimia nervosa (BN: n = 17) or eating disorder not otherwise specified (EONOS: n = 17). All patients were subjected to a detailed dietary and general history. We assessed the prevalence and severity (OEXA), the nature (osteocalcin, deoxypyridinoline) and site (vertebral versus hip) of osteopenla in these patients. he role of nutritional factors (energy intake, weight, height, BMI, plasma albumin, lipids), physical activity, hypercortisolemia (plasma and urinary free cortisol), vitamin 0 deficiency (plasma 250HD) and hypogonadism (amenorrhoea, E2, LH, FSH) in the pathogenesis of bone loss were also evaluated. Mild osteopenia (BMO decreased by more than 1SO below age-matched controls) was documented in 46% of the total study population, with more marked osteopenia (Z-Score < -2 SO) present in 15%. Both vertebral and hip osteopenia were documented. In the study population those patients with AN (Lumbar BMO (q/cm") = 0.869 ± 0.121) were most likely to develop osteoporosis, although a significant percentage of patients with BN (Lumbar BMO (q/crn") = 0.975 ± 0.16) and EONOS (Lumbar BMO (g/cm2) = 0.936 ± 0.10) were also osteopenic (29% and 35% respectively). Twenty four percent (24%) of the total patient population had a history of fragility fractures. These fractures were reported more commonly amongst patients with AN and EONOS (28% and29.4%). Fracture prevalence was however similar in patients with normal and low bone mass. Conventional risk factors were similar in patients with normal and low bone mass, except for a significantly longer duration of amenorrhoea (p = 0.009), a lower BMI (p = 0.0001) and greater alcohol consumption (p = 0.05) in the osteopenic patients. Nutritional parameters (S-albumin, protein, Ca, and P04 intakes), physical activity, as well as 25(OH) vitamin D levels were similar in AN and BN subjects, as well as in patients with a low versus normal BMD. Plasma and urine cortisol levels were also similar in these subgroups. With the exception of two patients with borderline osteopenia, significant bone loss was only documented in those patients with a past or current history of amenorrhoea. In the total patient population the duration of amenorrhoea was significantly (p<0.009) longer in patients with osteopenia versus those with a normal bone mass. A significant negative correlation between BMD (Z-Score) and duration of amenorrhoea was also documented in the total patient population (r = -0.4, P = 0.001) as well as in all three eating disorder groups (AN r - -0.4, P = 0.03; BN r = - 0.6, P = 0.008; EDNOS r = -0.6, P = 0.005). In the total patient population, those patients with amenorrhoea, had lower BMD and BMI values and lower estrogen levels compared to those with a normal menstrual cycle. We conclude that osteopenia commonly attends AN, as well as BN and EDNOS. Nutritional (with the exception of alcohol consumption) and mechanical factors as well as hypercortisolemia did not appear to contribute significantly to bone loss in this study population. Hypogonadism appeared to be the main cause of the bone loss observed in these patients. / AFRIKAANSE OPSOMMING: Osteopenie is In welbekende komplikasie van anorexia nervosa (AN). Die patogenese van hierdie beenverlies is swak in die huidige literatuur omskryf en nutrisiele faktore, 'n vita mien 0 gebrek, oormatige oefening, hiperkortisolemie en hipogonadisme word onder andere qeimpliseer. Vir die doel van die studie is In totaal van 59 Kaukasier eetsteurnis pasiente patients volledig ondersoek. Die tipe eetsteurnis is deur In enkel gekwalifiseerde psigiater volgens die DSM IV R kriteria geklassifiseer as anorexia nervosa (AN: n =25) of bulimia nervosa (BN: n = 17) of eetsteurnis nie anders gespesifiseer (EDNOS: n = 17). Elke pasient is ook aan In gedetailleerde dieet en algemene risikofaktor vraelys vir osteoporose onderwerp. Die voorkoms en graad (DEXA), die aard (osteokalsien, deoksipiridinolien) asook die tipe (werwelkolom/heup) osteopenie is ondersoek. Die rol van nutrisiele faktore (totale kalorie-inmame, gewig ,Iente LMI, plasma albumien, lipiede) en vitamien 0 gebrek, oefening, hiperkortisolemie (plasma en urinere kortisol) en hipogonadisme (amenoree, plasma E2, LH,FSH) in die patogenese van die beenverlies is ook evalueer. Matige osteopenie (BMD meer as 1 SO onder die van ouderdomskontrole) is in 46% van die totale pasientpopulasie gedokumenteer, met erge osteopenie (Z-Telling < -2) in 15%. Aantasting van beide werwelkolom en heup is aangetoon. In hierdie studiepopulasie kom osteopenie meer algemeen voor in die AN (Lumbaal BMD (g/cm2) = 0.869 ± 0.121) groep (64%) in vergelyking met BN (Lumbaal BMD (g/cm2) = 0.975 ± 0.16) (29%) en (EDNOS) (Lumbaal BMD (g/cm2) = 0.936 ± 0.10) (32%). Vier-en-twintig persent van die totale pasientpopulasie het In geskiedenis van frakture gehad. Frakture het meer algemeen in AN en EDNOS pasiente voorgekom (28% en 29%). Pasiente met 'n lae beenmassa was gekenmerk deur In betekenisvolle laer LMI (p = 0.0001), hoer alkolholverbruik (p = 0.05) en langer duurte van amenoree(p = 0.009). Nutrisiele parameters (s-albumien, protetene, Ca, P04 inname) oefening, asook 25(OH) vitamien 0 vlakke was soortgelyk in AN en BN pasiente. Hierdie parameters het ook nie verskil tussen pasiente met osteopenie en die met In normale BMD nie. Plasma en urinere vry kortisolvlakke was ook soortgelyk in hierdie twee groepe. Betekenisvolle beenverlies (met die uitsondering van twee pasiente met grenslyn osteopenie) het slegs voorgekom in pasiete met 'n huidige of In vorige geskiedenis van amenoree. In die totale pasientpopulasie was die duurte van amenoree (p< 0.009) betekenisvollanger in die pasiente met osteopenie. In Betekenisvolle negatiewe korrelasie tussen BMD (Z-Telling) en die duurte van amenoree in die toale pasient populasie (r = -0.4; P = 0.001) asook in al drie eetsteurnis groepe (AN: r = -0.4; P = 0.03; BN: r = -0.06; P = 0.008; EDNOS: r = - 0.6, P = 0.005) is aangetoon. In die groep as 'n geheel, het die amenoree pasiente In laer LMI, E2vlakke en BMD gehad in vergelyking met die pasiente met normale menses. Opsommend dus, kom osteopenie algemeen in pasiente met AN, asook BN en EDNOS voor. In Betekenisvolle bydrae van nutrisiele (met die uitsondering van alkoholinname) en meganiese faktore asook hiperkortisolemie tot been verlies, kon nie in hierdie tudie populasie gedemonstreer word nie. Hipogonadisme is as die hoofoorsaak van osteopenie in die pasiente qefdentifiseer.

Bibliography / Thesis (MMed (Pathology. Medical Microbiology))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Acinetobacter baumannii has emerged as one of the most troublesome nosocomial pathogens globally. This organism causes infections that are often extremely difficult to treat because of the widespread resistance to the major antibiotic groups. Colonization or infection with multidrugresistant A. baumannii is associated with the following risk factors: prolonged hospital stay, admission to an intensive care unit (ICU), mechanical ventilation, and exposure to broad spectrum antibiotics, recent surgery, invasive procedures, and severe underlying disease. A. baumannii has been isolated as part of the skin flora, mostly in moist regions such as axillae, groin and toe webs. It has also been isolated from the oral cavity and respiratory tract of healthy adults. Debilitated hospitalized patients have a high rate of colonization, especially during nosocomial Acinetobacter outbreaks. This organism is an opportunistic pathogen as it contains few virulence factors. Clinical manifestations of A. baumannii include nosocomial pneumonia, nosocomial bloodstream infections, traumatic battlefield and other wound infections, urinary tract infections, and post-neurological surgery meningitis. Fulminant community-acquired pneumonia has recently been reported, indicating that this organism can be highly pathogenic. The number of multidrug-resistant A. baumannii strains has been increasing worldwide in the past few years. Therefore the selection of empirical antibiotic treatment is very challenging. Antibiotic combinations are used mostly as empirical therapy in critically ill patients. One rationale for the use of combination therapy is to achieve synergy between agents. The checkerboard and time-kill methods are two traditional methods that have been used for synergy testing. These methods are labor intensive, cumbersome, costly, and time consuming. The E-test overlay method is a modification of the E-test method to determine synergy between the different antibiotics. This method is easy to perform, flexible and time efficient. The aim of this study was to assess the in vitro activity of different combinations of colistin, rifampicin, imipenem, and tobramycin against selected clinical strains of A. baumannii using the checkerboard and the E-test synergy methods. The MICs obtained with the E-test and broth microdilution method were compared. The results of the disk diffusion for imipenem and tobramycin as tested in the routine microbiology laboratory were presented for comparison. Overall good reproducibility was obtained with all three methods of sensitivity testing. The agreement of MICs between the broth dilution and E-test methods was good with not more than two dilution differences in MIC values for all isolates, except one in which the rifampicin E-test MIC differed with three dilutions from the MIC obtained with the microdilution method. However, the categorical agreement between the methods for rifampicin was poor. Although MICs did not differ with more than two dilutions in most cases, many major errors occurred because the MICs clustered around the breakpoints. The combinations of colistin + rifampicin, colistin + imipenem, colistin + tobramycin, rifampicin + tobramycin, and imipenem + tobramycin all showed indifferent or additive results by the E-test method. No results indicating synergy were obtained for all the above-mentioned combinations. There was one result indicating antagonistic effect for the combination of colistin + tobramycin. The results of the checkerboard method showed results indicating synergy in four of the six isolates for which the combination of colistin and rifampicin was tested. The other two isolates showed indifferent/additive results. All the other combinations showed indifferent/additive results for all isolates except isolate 30 (col + tob) and isolate 25 (rif + tob) which showed synergism. No antagonistic results were observed by the checkerboard method. When the results obtained with the E-test and checkerboard methods were compared, it was noted that for most antibiotic combinations an indifferent/additive result was obtained. However, for the colistin + rifampicin combination, the checkerboard method showed synergism for 4 of 6 isolates, whereas the E-test method showed indifference and an additive result in one. For the rifampicin + tobramycin, and colistin + tobramycin combinations, synergism was also shown with the checkerboard method in one isolate for each combination. The E-test method however showed an indifferent and additive result respectively. . The E-test method was found to be a rapid, reproducible, easy-to-perform, and flexible method to determine synergistic antibiotic activity. This study was however limited by low numbers of isolates. This might explain why no synergistic results were obtained with the E-test method and few synergistic results with the checkerboard method. Genotypic analysis using pulse-field gel electrophoresis (PFGE) may be considered in future studies to determine relatedness of the isolates which will facilitate the selection of different strains for synergy testing. Furthermore, clinical studies are needed to establish whether in vitro synergy testing is useful in the clinical setting and whether the results of synergy testing will have any bearing on the clinical outcome of patients infected with multidrug resistant A. baumannii. / AFRIKAANSE OPSOMMING: Acinetobacter baumannii het wêreldwyd as een van die mees problematiese nosokomiale patogene verskyn. Hierdie organisme veroorsaak infeksies wat dikwels baie moeilik is om te behandel weens wydverspreide weerstandigheid teen major antibiotikagroepe. Kolonisasie of infeksie met multi-weerstandige A. baumannii word geassosieer met die volgende riskofaktore: verlengde hospitaalverblyf, toelating tot ‘n intensiewe sorgeenheid (ICU), meganiese ventilasie, blootstelling aan breëspektrum antibiotika, onlangse chirurgie, indringende prosedures en ernstige onderliggende siekte. A. baumannii kan deel vorm van die normale velflora, veral in die axillae, inguinale area en tussen die tone. Dit is ook al vanuit die mondholte en die respiratoriese traktus van gesonde volwassenes geïsoleer. Verswakte gehospitaliseerde pasiënte word veral gekoloniseer gedurende nosokomiale Acinetobacter uitbrake. Hierdie organisme is ‘n opportunistiese patogeen en bevat min virulensie faktore. Kliniese manifestasies van A. baumannii sluit nosokomiale pneumonie, nosokomiale bloedstroom infeksies, troumatiese slagveld- en ander wondinfeksies, urienweginfeksies en meningitis wat volg op neurologiese chirurgie in. Fulminerende gemeenskapsverworwe pneumonie is onlangs beskryf en dui aan dat hierdie organisme hoogs patogenies kan wees. Die aantal multi-weerstandige A. baumannii stamme het wêreldwyd toegeneem oor die laaste paar jare. Daarom is die seleksie van empiriese antibiotiese behandeling ‘n uitdaging. Antibiotika kombinasies word meestal as empiriese behandeling in ernstige siek pasiënte gebruik. Die beginsel hiervan is om sinergistiese werking tussen agente te verkry. Die “checkerboard” en “time-kill” metodes is twee tradisionele metodes van sinergisme toetsing. Hierdie metodes is werksintensief, duur en tydrowend. Die E-toets sinergisme metode is gebaseer op die E-toets metode. Hierdie metode is maklik, buigbaar en tydseffektief. Die doel van hierdie studie was om die in vitro aktiwiteit tussen verskillende antibiotika kombinasies van colistin, rifampisien, imipenem, en tobramisien teen geselekteerde kliniese A. baumannii isolate te toets met die “checkerboard” en E-toets sinergisme toetsing metodes. Die minimum inhibitoriese konsentrasies (MIKs) verkry met die E-toets en “broth microdilution” metode is ook vergelyk. Die resultate van die skyfie diffusie metode (die metode wat in die roetiene mikrobiologie laboratorium gebruik word) vir imipenem en tobramisien word ook verskaf vir vergelyking van die resultate van verskillende sensitiwiteitsmetodes. In oorsig is goeie herhaalbaarheid van resultate verkry met al drie metodes van sensitiwiteitstoetsing. Die ooreenstemming van MIKs tussen die “broth dilution” en E-toets metodes was goed en resultate het met nie meer as twee verdunnings in MIK waardes verskil nie. Daar is een uitsondering waar die rifampisien E-toets MIK waarde met drie verdunnings van die MIK waarde verkry met die “microdilution” metode verskil. Die ooreenstemming tussen die sensitiwiteitskategorie resultate tussen die twee metodes was egter swak vir rifampisien. Alhoewel die MIKs in die meeste gevalle met nie meer as twee verdunnings in waarde verskil het nie, was daar baie major foute aangetoon omdat die MIKs rondom die breekpunte geval het. Die kombinasies van colistin + rifampisien, colistin + imipenem, colistin + tobramisien, rifampisien + tobramisien, en imipenem + tobramisien het oorwegend slegs matige interaksie met die E-toets metode getoon. Geen sinergisme is verkry met enige van die antibiotika kombinasies met hierdie metode nie. Daar was egter een resultaat wat antagonisme getoon het vir die kombinasie van colistin + tobramycin. Die resultate van die “checkerboard” metode het sinergisme getoon in vier van die ses isolate wat vir die kombinasie van colistin en rifampisien getoets was. Die ander twee isolate het slegs matige interaksie getoon. Al die ander kombinasies het ook slegs matige interaksie getoon, behalwe in isolaat 30 (col + tob) en isolaat 25 (rif + tob) waar die spesifieke kombinasies sinergisme getoon het. Geen antagonisme is waargeneem met die “checkerboard” metode nie. Met vergelyking van die E-toets en “checkerboard” metodes, is dit opmerklik dat vir die meeste van die antibiotika kombinasies slegs matige interaksie verkry is. Vir die colistin + rifampisien kombinasie toon die “checkerboard” metode egter sinergisme vir 4 uit 6 isolate, terwyl die E-toets metode slegs matige interaksie toon. Vir rifampisien + tobramisien, en colistin + tobramisien kombinasies is sinergisme getoon met die “checkerboard” metode in een isolaat vir elke kombinasie. Die E-toets metode het slegs matige interaksie getoon. Die E-toets sinergisme metode was vinnig, herhaalbaar en maklik om uit te voer. Hierdie studie word egter beperk deur lae getalle van isolate. Dit mag verklaar waarom geen sinergistiese resultate met die E-toets metode verkry is nie en die min sinergistiese resultate met die “checkerboard” metode. Genotipiese analiese met “pulse-field gel electrophoresis” mag in aanmerking geneem word in toekomstige studies om die verwantskap tussen isolate te bepaal wat die seleksie van verskillende stamme vir sinergisme toetsing sal vergemaklik. Verder, kliniese studies is nodig om te bepaal of in vitro sinergisme toetsing van waarde is en of die resultate van sinergisme toetsing ‘n rol speel in die kliniese uitkoms van pasënte geïnfekteer met multiweerstandige A. baumannii. / The National Health Laboratory Serivice

Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Throughout history disease entities have been described which match the description of diseases now known to be caused by hantaviruses; however these viruses were first identified as the aetiologic agent in 1976, the first species named Hantaan virus after the river near which its natural host, the rodent species Apodemus agrarius, was captured. Since then numerous species in the Hantavirus genus, family Bunyaviridae, have been found, with today more than 30 species worldwide being known. Hantaviruses are hosted by rodents from the Muridae and Cricetidae families and by shrews (insectivores) in the Soricidae family. There are two types of hantavirus disease, Haemorrhagic fever with renal syndrome (HFRS) in the Old World and Hantavirus cardiopulmonary syndrome (HCPS) in the New World. The first two African hantaviruses were identified in 2006 in Guinea, West Africa; Sangassou virus (SANGV) in a rodent, the African wood mouse (Hylomyscus simus), and Tanganya virus (TGNV) in Therese’s shrew (Crocidura theresae). In this study, rodents and shrews were trapped at localities in the Western Cape and Northern Cape provinces of South Africa, and in the southern regions of Namibia. RNA was extracted from their lungs and screened for hantavirus sequences by RTPCR, using degenerate primers designed to detect all members of the Hantavirus genus. In addition, an in-house IgG ELISA assay was set up, based on recombinant N antigen from Dobrava virus, DOB-rN, and Puumala virus, PUU-rN. The assay was used to screen patient sera collected in an anonymous convenience serological survey using residual serum samples left over from routine testing at NHLS laboratories in the Western Cape for hantavirus-specific antibodies. RNA from 576 animal specimens was screened by RT-PCR; no hantavirus genome was detected in any of the specimens. Sera from 161 patients were screened for hantavirus antibodies; 11.18% of the sera were reactive to DOB-rN, 4.97% against PUU-rN and 2.48% against both antigens. v Though no virus was detected in the animals screened, this does not necessarily mean that there are no hantaviruses present in Southern Africa. A previous seroepidemiological survey conducted in South Africa reported on the presence of hantavirus specific antibodies by IFA in two species of rodents trapped in the Western Cape and Northern Cape Aethomys namquensis and Tatera leucogaster. Our was the second known study in South Africa conducted that determined and proved the presence of hantavirus specific antibodies in humans. / AFRIKAANSE OPSOMMING: Dwarsdeur die geskiedenis was daar beskrywings van siektes wat ooreenstem met die beskrywing van hantavirus simptome, maar die eerste etiologiese oorsaak van die siekte is eers in 1976 geïdentifiseer en Hantaan virus genoem, vernoem na die rivier waar naby die gasheer, Apodemus agrarius, gevang is. Van daar af het die soektog na nuwe hantavirusse intensief gevorder en vandag is daar meer as 30 spesies wêreldwyd wat aan die Hantavirus genus, ’n lid van die Bunyaviridae familie, behoort. Knaagdiere van die Muridae en Cricetidae families, sowel as spitsmuise (insekvreters) in die Soricidae familie is gasheer vir hantavirusse. Twee tipes hantavirus siekte is bekend, hemorragische koors met nier sindroom (HFRS) in die Ou Wêreld en hantavirus kardiopulmonale sindroom in die Nuwe Wêreld. Die eerste twee Afrika hantavirusse is in 2006 in Guinee Wes-Afrika geïdentifiseer; Sangassou virus (SANGV) in ’n knaagdier, die Afrika hout muis (Hylomyscus simus) en Tanganya virus (TGNV) in Therese se spitsmuis (Crocidura theresae). In hierdie studie is knaagdiere en spitsmuise op verskeie plekke in die Wes- en Noord-Kaap provinsies, asook die Suide van Namibië, gevang. RNS is onttrek vanuit die longe en hantavirus volgordes is gesoek deur middel RT-PKR deur gebruik te maak van Pan-Hanta primers wat ontwerp is om alle lede van die Hantavirus genus op te spoor. ’n Self-ontwerpde IgG ELISA, gebasseer op rekombinante N antigeen van Dobrava virus, DOB-rN en Puumala virus, PUU rN, is opgestel en gebruik om pasiënt serum, verkry in ’n anonieme serologiese opname, te toets; oorblywende serum, na toetse uitgevoer is deur NHLS laboratoriums in die Wes-Kaap, is verkry en getoets vir hantavirus spesifieke teenliggaampies. RNS van 576 dier monsters is getoets deur middel van RT-PKR en geen hantavirus is in enige van die monsters geïdentifiseer nie. Serum van 161 pasiënte is getoets vir hantavirus teenliggaampies; 11.18% van die serum was reaktief teen DOB-rN, 4.97% teen PUU-rN en 2.48% teen albei antigene. Alhoewel geen virus in die diere geïdentifiseer is nie, beteken dit nie noodwendig dat geen hantavirusse in Suidelike-Afrika voorkom nie. ‘n Vorige sero-epidemiologiese opname wat in Suid-Afrika gedoen is het die teenwoordigheid van hantavirus spesifieke teenliggaampies in twee knaagdier spesies, Aethomys namquensis en Tatera leucogaster gevang in die Wes-en Noord-Kaap, gevind. Ons studie is die tweede studie bekend in Suid-Afrika uitgevoer, wat die teenwoordigheid van hantavirus spesifieke teenliggaampies bevind en bewys het.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Introduction: The discovery of the endothelium as a regulator of vascular tone, and the subsequent discovery of nitric oxide (NO) as the major endothelium-derived relaxing factor (EDRF), has opened up vast possibilities in the continued efforts to prevent and manage cardiovascular disease. Endothelial dysfunction (ED) is defined as reduced NO bioavailability and hence the reduced ability of the endothelium to maintain vascular homeostasis. ED represents the first, reversible step in the initiation of atherosclerotic disease and is thus regarded as a strong predictive tool of ischaemic heart disease (IHD). ED and its underlying mechanisms have been largely under-investigated in myocardial capillary-derived endothelial cells (cardiac microvascular endothelial cells, CMECs), and this study aimed to address this gap in the literature. Oleanolic acid (OA) is a bioactive triterpenoid derived from leaf extracts of African medicinal plants such as Syzigium cordatum (Water berry tree), and has been reported to elicit vasodilatory, hypoglycaemic and hypolipidaemic properties. However its effects particularly on CMECs and its putative role in reversing ED remain unclear, and this study aimed to investigate such effects. Aims: The aims of this study were to: (1) Establish an in vitro model of ED in cultured myocardial capillary-derived CMECs by developing protocols for the induction of ED. (2) Asses ED induction by measurement of the following biomarkers: (i) intracellular NO production, (ii) superoxide (O2-) production, (iii) nitrotyrosine expression and (iv) NADPH oxidase expression. (3) Investigate underlying cellular mechanisms of our ED model by measuring and comparing eNOS and PKB/Akt expression and activation in control and dysfunctional CMECs. (4) Investigate the effects of OA derived from leaf extracts obtained from Syzigium cordatum (Hochst.) [Myrtaceace], in both control and dysfunctional CMECs. Methods: (1) To induce ED, hyperglycaemia and inflammation were simulated by incubation with 25 mM glucose (24 hours) and 1 ng/ml TNF-á (24 hours) or 5 ng/ml TNF-á (6 and 24 hours) respectively. Reduced intracellular NO production was used as the main indicator of ED. NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide (PI) / Annexin V respectively. Cellular mechanisms were investigated by measurement of O2- levels via FACS analysis of DHE fluorescence, and measurement of total and activated PKB / Akt and eNOS, p22-phox, nitrotyrosine expression via Western blotting. (2) Effects of OA on CMECs were investigated by pre-treatment with 30 or 40 ìM OA for 5 and 20 min followed by NO production and cell viability measurements. To investigate the effects of OA on ED, CMECs were pre-treated with 40 ìM OA 1 hour prior ED induction followed by NO, cell viability, and eNOS expression / activation measurements. Results: (1) 25 mM glucose (24hours), 1 ng/ml TNF-á (24 hours) and 5 ng/ml TNF-á (6 hours) failed to induce ED as verified by an increase in NO production in the treated cells. A model of ED was successfully achieved by incubating CMECs with 5 ng/ml TNF-á (24 hours), as verified by a significant decrease in NO production. Investigations into cellular mechanisms underlying our TNF-á-induced ED model, showed that activated eNOS and PKB / Akt levels were reduced. Furthermore, O2- levels remained unchanged, however p22-phox (NADPH) expression was significantly increased suggesting oxidative stress. Nitrotyrosine levels (an oxidative / nitrosative stress marker and indirect measure of eNOS uncoupling) remained at control levels. (2) Investigations into the effects of OA on CMECs showed that 30 ìM OA increased NO production after 5 and 20 min of incubation whereas 40 ìM increased NO production after 20 min only. Pre-treatment with 40 ìM OA significantly reversed ED by restoring NO production back to control levels. Data from cellular mechanism investigations showed that 40 ìM OA significantly increased eNOS activation in both normal and dysfunctional CMECs. Cellular viability was not negatively affected by any of the above interventions. Discussion and Conclusions: Based on our findings, reduced activation of the PKB / Akt-eNOS pathway appears to be the primary mechanistic pathway of the TNF-á-induced model of ED. Though O2- levels remained at control levels, the significant increase in p22-phox is indicative of increased expression of the O2- producing enzyme, NADPH oxidase, thus suggesting oxidative stress. However, based on our nitrotyrosine expression data, there was no strong evidence of eNOS uncoupling in our ED model. OA significantly stimulated NO production in our model of CMECs. Furthermore, our findings showed that OA is able to reverse ED. The NO production stimulatory effects of OA in our cells appear to be achieved via the increased activation of eNOS. We have, for the first time as far as we are aware, developed a TNF-á-induced model of ED in myocardial capillary-derived endothelial cells. It appears that reduced activation of the PKB/Akt-eNOS pathway is the primary mechanism leading to decreased NO production in this model. However, we did find some evidence of elevated oxidative stress, which led us to believe that eNOS uncoupling cannot be excluded as a mechanism of ED in our model. In this study, we report for the first time convincing evidence that OA has powerful NO-increasing properties in myocardial capillary-derived CMECs. Our study also show novel data, which suggest that OA is able to reverse ED in this model. Follow-up investigations could shed more light on the exact mechanisms underlying OA.s effects in this model. / AFRIKAANSE OPSOMMING: Inleiding: Die ontdekking dat endoteel 'n reguleerder van vaskulêre tonus is, en die gevolglike ontdekking dat stikstofoksied (NO) die belangrikste endoteel-afgeleide verslappingsfaktor (EDRF) is, het verskeie moontlikhede in aangaande pogings om kardiovaskulêre siektes te voorkom en hanteer, ontsluit. Endoteel-disfunksie (ED), word gedefineer as verlaagde NO biobeskikbaarheid en dus 'n ingekorte vermoë van die endoteel om vaskulêre homeostase te handhaaf. ED verteenwoordig die eerste, omkeerbare stap in die ontstaan van aterosklerotiese siekte en word dus beskou as 'n sterk instrument waarmee isgemiese hartsiekte voorspel kan word. Studies oor ED en sy onderliggende meganismes, veral in miokardiale kapillêre-afgeleide endoteelselle (kardiale mikrovaskulêre endoteelselle, CMECs), word redelik afgeskeep in die literatuur, en hierdie studie het dit ten doel gehad om die gaping in die literatuur aan te spreek. Oleanoliese suur (OA) is 'n bio-aktiewe triterpenoïede wat gevind word in blaar ekstrakte van inheemse medisinale plante soos bv. Syzigium cordatum (Waterbessie boom). OA het bewese vasodilatoriese, hipoglukemiese en hipolipidemiese eienskappe. OA se effekte op CMECs, en sy moontlike rol in die omkering van ED, is egter onbekend, en hierdie studie het dit ten doel gehad om sulke effekte te ondersoek. Doelwitte: Die doelwitte van hierdie studie was: (1) Die vestiging van 'n in vitro model van ED in gekultuurde CMECs afkomstig van miokardiale kapillêre deur protokolle vir die induksie van ED te ontwikkel. (2) Die evaluering van ED induksie deur die volgende bio-merkers te meet: (i) intrasellulêre NO produksie, (ii) superoksied (O2-) produksie, (iii) nitrotirosien uitdrukking en (iv) NADPH oksidase uitdrukking. (3) Die ondersoek na onderliggende sellulere meganismes van ED in ons model deur die meting en vergelyking van eNOS and PKB/Akt uitdrukking en aktivering in kontrole en disfunksionele CMECs. (4) Ondersoek na die effekte van OA afkomstig van blaar ekstrakte verkry van Syzigium cordatum (Hochst.) [Myrtaceace], in beide kontrole en disfunksionele CMECs. Metodes: (1) Daar was gepoog om ED te induseer deur hiperglukemie en inflammasie te simuleer met onderskeidelik 25 mM glukose (24 uur) en 1 ng/ml TNF-a (24 uur) of 5 ng/ml (6 en 24 uur) inkubasie. Verlaagde intrasellulere NO produksie was ingespan as die hoof indikator van ED. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied (PI) / Annexin V onderskeidelik. Sellulere meganismes was ondersoek deur O2- vlakke via FACS analise van DHE fluoressensie te meet, asook die meting van totale en geaktiveerde PKB / Akt en eNOS, p22-phox, nitrotirosien uitdrukking via Western blot tegnieke. (2) Effekte van OA op CMECs was ondersoek deur vooraf-behandeling met 30 of 40 µM OA vir 5 en 20 min gevolg deur NO produksie en sellewensvatbaarheid metings. Resultate: (1) 25 mM glukose (24 uur), 1 ng/ml TNF-a (24 uur) and 5 ng/ml TNF-ƒaa (6 uur) kon nie daarin slaag om ED te induseer nie, soos blyk uit die verhoogde NO produksie waargeneem in die behandelde selle. 'n Model van ED was suksesvol verkry deur CMECs met 5 ng/ml TNF-a (24 uur) te inkubeer, soos waargeneem deur verlaagde NO produksie. Ondersoek na sellulere meganismes onderliggend tot ons TNF-a-geinduseerde ED model, het getoon dat geaktiveerde eNOS en PKB / Akt vlakke verlaag was. Verder is gevind dat O2- vlakke onveranderd gebly het hoewel p22-phox (NADPH) uitdrukking betekenisvol toegeneem het, wat 'n aanduiding van oksidatiewe skade is. Nitrotirosien vlakke (.n oksidatiewe / nitrosatiewe stres merker en indirekte maatstaf van eNOS ontkoppeling) het onveranderd rondom kontrole vlakke gebly. (2) Ondersoek na die effekte van OA op CMECs het getoon dat 30 µM OA tot verhoogde NO produksie na 5 en 20 min inkubasie gelei het, terwyl 40 µM slegs na 20 min NO-verhogende effekte gehad het. Vooraf behandeling met 40 µM OA het ED betekenisvol omgekeer deur NO terug na kontrole vlakke te laat herstel. Ondersoek na sellulere meganismes het getoon dat 40 µM OA eNOS aktivering betekenisvol verhoog het in beide normale en disfunksionele CMECs. Sellulere lewensvatbaarheid was nie negatief geaffekteer deur enige van bogeneemde ingrepe nie. Bespreking en afleidings: Gebaseer op ons bevindinge, blyk verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganistiese pad in ons TNF-a-geïnduseerde model van ED te wees. Alhoewel O2- vlakke rondom kontrole vlakke gebly het, was die betekenisvolle toename in p22-phox .n aanduiding van verhoogde uitdrukking van die O2- produserende ensiem, NADPH oksidase, wat dus suggererend van oksidatiewe stres was. Aan die ander kant was daar nie sterk bewyse van eNOS ontkoppeling in ons ED model nie, gebaseer op die nitrotirosien uitdrukking data. OA het duidelik NO produksie in ons model van CMECs gestimuleer. Verder wys ons resultate dat OA in staat is om ED om te keer. Die NO produksie-stimulerende effekte van OA in ons selle blyk die gevolg te wees van verhoogde aktivering van die PKB / Akt-eNOS pad. Ons het hier vir die eerste keer, sover ons bewus is, 'n TNF-a-geinduseerde model van ED in CMECs afkomstig van miokardiale kapillere gevestig. Dit blyk dat verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganisme was waardeur verlaagde NO produksie in ons model veroorsaak was. Ons het egter wel bewyse van verhoogde oksidatiewe stress gevind, wat ons laat glo dat eNOS ontkoppeling nie heeltemal as .n meganisme van ED in ons model uitgesluit kan word nie. In hierdie studie toon ons vir die eerste maal oortuigende bewyse dat OA kragtige NO-verhogende eienskappe in miokardiale kapillere-afgeleide CMECs het. Ons studie bring ook nuwe data na vore, wat suggereer dat OA in staat is om ED in hierdie model om te keer. Opvolgstudies sal meer lig kan werp op die onderliggende meganismes van OA in hierdie model.

Bibliography / Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Overview: Mycoplasmas and ureaplasmas are not routinely diagnosed and are under researched in South Africa. Prevalence, population shifts especially concerning genital flora and implications in infection or other conditions are unknown. Information pertaining to Mycoplasma pneumoniae in respiratory disease is similarly lacking. There is little information on antimicrobial susceptibilities and resistance development against Sexually Transmitted Infections (STI) syndromic management approaches. Aims: a) Elucidate mycoplasmal and ureaplasmal prevalence and contributing factors concerning cervical colonisation or preterm delivery in conjunction with HIV and Chlamydia trachomatis b) Investigate prevalence of M. pneumoniae in respiratory infections in conjunction with HIV, Mycobacterium tuberculosis and Pneumocystis jiroveci. c) Determine antimicrobial susceptibilities of mycoplasmas and ureaplasmas and analyse resistance genes. d) Assess the inter-generic transfer potential of resistance gene (tetM) between Ureaplasma spp. and Neisseria gonorrhea. Genital setting: The prevalence of genital mycoplasmas, ureaplasmas and Chlamydia on women attending their first prenatal visit, in conjunction with preterm labour or HIV status was investigated. For preterm labour (2003), 199 women were monitored for preterm delivery (<37 weeks); for colonisation and HIV (2005), 219 women were screened. Microbial detection was performed on DNA extracted from endocervical swabs employing PCR techniques. Colonisation was seen to be highest in the 14-20 year group from 2003. In women aged ±21 years, co-colonisation was 13% although there was a shift from co-colonisation with Mycoplasma hominis and Ureaplasma spp. in 2003 to other dual/triple combinations in 2005. Overall major trends from both collection periods were that the prevalence of Ureaplasma spp. tended to be higher in women ±26 years, whilst prevalence of C. trachomatis and M. hominis were lower. No association was evident between colonisation with M. hominis, U. urealyticum, Ureaplasma parvum and labour outcome. HIV status had no effect on the prevalence/co-colonisation of M. hominis, Ureaplasma spp. or C. trachomatis. Respiratory setting: Studies were conducted to determine the prevalence of community acquired atypical pneumonias in adults (M. pneumoniae and P. jiroveci) and neonates (mycoplasmas, ureaplasmas and Chlamydia trachomatis) in order to improve treatment management programmes in the Port Elizabeth region. Sputum specimens from 102 adult patients presenting with pneumonia/symptoms of pneumonia admitted to hospitals were assessed by PCR. Details of patient’s gender, age, HIV and Mycobacterium tuberculosis status were provided by the hospitals. Women were seen to be at high risk for community-acquired P. jiroveci colonisation. Overall, prevalence of P. jiroveci was 52.9% (54/102 patients). P. jiroveci was mainly associated with HIV (25/74) (P. jiroveci and HIV positive patients in patient sample for which clinical data and HIV status was available) and co-infection with M. tuberculosis was observed in 12 HIV cases and one HIV negative patient. No DHPS (20) or DHFR (17) resistance associated mutations were found in P. jiroveci. M. pneumoniae was detected in one patient. For prevalence studies (2007-2008) on atypical pneumonia in neonates, 69 endotracheal aspirates were obtained. PCR detection of M. hominis, U. urealyticum and C. trachomatis was performed and U. parvum detected in two specimens. Antibiotic susceptibilities and resistance genes: The following investigations on clinical isolates of U. parvum and U. urealyticum were conducted (i) antibiotic susceptibility profiles, (ii) detection of drug target gene mutations, or gene acquisitions and (iii) inter-generic resistance gene transfer potential to Neisseria gonorrhoeae. Culture techniques applied to 132 endocervical specimens provided 66 Ureaplasma cultures (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIC determinations to ofloxacin, erythromycin, tetracycline, doxycycline, azithromycin and josamycin were performed. Thirty-seven ureaplasma cultures were fully susceptible to all antibiotics tested; 21 showed intermediate resistance to erythromycin, azithromycin and ofloxacin; while seven were resistant to tetracycline, three of which were also resistant to doxycycline and one also resistant to azithromycin. Concerning ofloxacin resistance directed at quinolone resistance determining regions, a substitution of Ser83Leu in ParC was demonstrated in one intermediately-resistant Ureaplasma (MIC 4 µg/ml) while a triple substitution of Asp112Glu in GyrA along with Ala125Thr and Ala136Thr in ParC was found in six further intermediately-resistant strains. No mutations were found in strains with MICs 1 µg/ml. No mutations were detected in 23S rRNA operons, L4 or L22 proteins. TetM and int-Tn genes were found in seven tetracycline-resistant strains. On screening 59 tetracycline-susceptible and -intermediate strains, eleven whilst possessing an int-Tn gene lacked a large region of tetM and 48 only contained small regions of tetM. The tetM genes of the seven tetracycline-resistant strains were sequenced and comparisons performed against GenBank sequences of Neisseria gonorrhoeae, Streptococcus pneumoniae and U. urealyticum. For five strains tetM was seen to be highly mosaic in structure containing regions that were similar to those of the GenBank strains and others that were unique. In the tetM leader region, four hot spot recombination sites were identified that could certainly influence the formation of the mosaic structures, upstream insertion sequences/open reading frames and transposon regions that regulate expression. On characterising the int-Tn genes of the seven tetracycline-resistant strains, three types were present indicating transposons from different origins had integrated into ureaplasma genomes. Reciprocal tetracycline resistance gene transfer between ureaplasmas and N. gonorrhoeae were unsuccessful. However, low-level tetracycline resistance (MICs 4-8 µg/ml) was transferred to a U. parvum recipient from one U. urealyticum and three U. parvum donors that carried tetM with MICs 16-64 µg/ml. On tetM PCR analysis, tetM was not detected in the transformants. Conclusions: The importance of genital mycoplasmas, ureaplasmas and C. trachomatis in long term aetiologies requires further investigations, certainly in relation with syndromic management regimens that fail to reduce colonisation rates. The high prevalence of P. jiroveci, the presence of M. pneumoniae in cases of pneumonia and detection of U. parvum in two cases of neonatal pneumonia investigated emphasises that in the absence of definitive diagnoses, it is crucial to monitor treatment responses carefully, especially when first line antibiotic preferences are ß-lactams, in order to ensure adequate and informed delivery of medical care. The finding of transposon and/or tetM regions in all ureaplasmas investigated with or without full expression of tetracycline resistance, in conjunction with tetM gene diversity, certainly places ureaplasmas strongly in the picture for intra- and inter-generic exchange of antibiotic resistance genes. / AFRIKAANSE OPSOMMING: Oorsig: Mikoplasma en ureaplasma word nie roetineweg gediagnoseer nie en in Suid Afrika is nog min navorsing daaroor gedoen. Prevalensie, populasie verskuiwings, veral in genital flora, en die impliksies van infeksie en ander toestande is onbekend. Inligting rakende Mycoplasma pneumoniae in respiratoriese siekte is ook gebrekkig. Daar is min inligting beskikbaar rakende die antimikrobiale vatbaarheid en die ontwikkeling van weerstandigheid gesien teen die benadering tot sindromiese hantering van seksueel oordraagbare siektes. Doelwitte: a) Om inligting te verskaf oor die prevalensie van mikoplasma en ureaplasma en bydraende faktore betreffende voortydige kraam tesame met MIV en Chlamydia trachomatis. b) Ondersoek van die prevalensie van M. pneumoniae in respiratoriese infeksies tesame met MIV, Mycobacterium tuberculosis en Pneumocystis jiroveci. c) Bepaling van die antimikrobiale vatbaarheid van mikoplasma en ureaplasma en analisevan weerstandigheids gene. d) Bereken die inter-genetiese oordrag potensiaal van weerstandigheids gene (tetM) tussen Ureaplasma spp. en Naisseria gonorrhoeae. Genitale omgewing: Die prevalensie van genitale mikoplasma, ureaplasma en Chlamydia in vroue tydens hul eerste prenatale besoek, tesame met vroegtydige kraam en MIV status is ondersoek. In voortydige kraam (2003), is 199 vroue gemonitor vir voortydige kraam (<37 weke); vir kolonisasie en MIV (2005), is 219 vroue getoets. Mikrobiale toetsing is gedoen deur DNS te win vanaf endoservikale deppers met PKR tegnieke. Kolonisasie was die hoogste in die ouderdomsgroep 14.20 jaar, in 2003. In vroue van ±21 jaar was medekolonisasie 13% alhoewel daar en verskuiwing was van mede-kolonisasie met Mycoplasma hominis en Ureaplasma spp. in 2003 tot ander dubbel/trippel kombinasies in 2005. Die oorkoepelende tendens in altwee die tydperke van waarneming was dat die prevalensie van Ureoplasma spp. geneig was om hoër te wees in vroue ±26 jaar, terwyl prevalensie van C. trachomatis en M. hominis laer was. Geen assosiasie kon getoon word tussen koloniesasie met M. hominis, U. urealyticum, Ureaplasma parvum en uitkoms van kraam nie. MIV status het geen effek gehad op die prevalensie/mede-kolonisasie van M. hominis, Ureaplasma spp. of C. Trachomatis nie. Respiratories: Studies is gedoen om die prevalensie van gemeenskaps verworwe atipiese pneumonie in volwassenes (M. pneumoniae en P. jiroveci) en neonate (mikoplasma, ureaplasma en Chlamydia trachomatis) te bepaal om behandeling en hantering programme in die Port Elizabeth area te verbeter. Sputum monsters van 102 volwasse pasiënte wat presenteer het met pneumonie of simptome van pneumonie en wat tot hospitale toegelaat was, is ontleed. Besonderhede van die pasiënte se geslag, ouderdom, MIV en Mycobacterium tuberculosis status is deur die hospitale verskaf. PKR is gedoen met inleiers gerig teen die volgende gene: P. jiroveci vir die aantoning van mitokondriale groot subeenheid RNS en vir die analise van mutasies vir ko-trimoksasool weerstandigheid dihydropteroaat sintetase (DHPS) en dihydrofolaat reduktase (DHFR); M. pneumoniae vir die aantoning van P1 adhesien en 16S rRNS. Vroue het ‘n hoë risiko vir gemeenskapsverworwe P. jiroveci kolonisasie gehad. In die algemeen was die prevalensie van P. jiroveci 52.9% (54/102 pasiënte). P. jiroveci was hoofsaaklik geassosieerd met MIV (25/74) (P. jiroveci en MIV positiewe pasiënte in die pasiënt monster waarvoor daar kliniese data en MIV status bekend was) en mede-infeksie met M. tuberculosis is gesien in 12 MIV gevalle en een MIV negatiewe pasiënt. Geen DHPS (20) of DHFR (17) weerstandigheids geassosieerde mutasies is gevind in P. Jiroveci nie. M. pneumoniae was aangetoon in een pasiënt. Vir prevalensie studies (2007-2008) op atipiese pneumonie in neonate is 69 endotrageale aspirate verkry. PKR toetsing vir M. hominis, U. urealyticum en C. trachomatis is gedoen met ‘primers’ soos voorheen gepubliseer. Ureaplasma parvum is aangetoon in twee neonate met PKR met negatiewe kultuur resultate. Antibiotika sensitiwiteite en weerstandigheids gene: Die volgende toetse is gedoen op kliniese isolate van U. parvum en U. urealyticum (i) antibiotika sensitiwiteits profiele, (ii) aantoning van teiken geen mutasies, of geen aanwinste en (iii) potensiaal vir inter-generiese weerstandigheids geen oordrag na Neisseria gonorrhoeae. Kultuur tegnieke toegepas op 132 endoservikale monsters het 66 Ureaplasma kulture gelewer (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIK bepaling vir ofloksasien, eritromisien, tetrasiklien, doksisiklien, azitromisien en josamisien is gedoen. Sewe-en-dertig kulture was ten volle sensitief vir alle antibiotika wat getoets is; een-en twintig het intermediere weerstandigheid teenoor eritromisien, azitromisien en ofloksasien getoon, terwyl sewe weerstandig was vir tetrasiklien, drie daarvan was ook weerstandig vir doksisiklien. Wat betref ofloksasien weerstandigheid gemik teen kwinoloon weerstandigheids bepalende gebiede, is vervanging van Ser83Leu in ParC gedemonstreer in een intermedier weerstandige Ureaplasma (MIK 4 µml) terwyl en trippel vervanging van Asp112Glu in GyrA saam met Ala125Thr en Ala136Thr in ParC gevind is in ses ander intermedier weerstandige stamme. Geen mutasies is gevind in stamme met MIKs van MICs 1 µg/ml nie. Geeneen van die ureaplasma was weerstandig vir eritromisien/azitromisien nie en geen mutasies is gevind in 23S rRNA operons , L4 of L22 proteine nie. TetM en int- Tn gene is gevind in sewe tetrasiklien weerstandige stamme. 58 Tetrasiklien sensitiewe en .intermediere stamme is getoets, waarvan elf en int-Tn geen gekort het sowel as en groot deel van tetM, terwyl 48 slegs klein dele van TetM bevat het. Die tetM gene van die sewe tetrasiklein-werstandige stamme se geenvolgorde is bepaal en vergelykings is getref teenoor die GenBank volgordes van Neisseria gonorrhoeae, Streptococcus pneumoniae en U. urealyticum. In vyf stamme is gevind dat die tetM geen hoogs mosaiek in struktuur was met areas wat ooreenstem met die in GenBank stamme, en ander areas wat uniek is. In die tetM leier area, is vier ehot spot f herkombinasie areas geidentifiseer wat sekerlik die vorming van die mosaiiek strukture kon beinvloed, asook transposon areas wat geenuitdrukking bepaal. Met karakterisering van die int-Tn gene van die sewe tetrasikleinweerstandlige stamme, was drie tipes teenwoordig waarin transposons vanaf verskillende oorsprong aangedui was, geintegreerd met die ureaplama genome. Resiprokale tetrasiklien weerstandigheids geen oordrag tussen ureaplasma en n. gonorrhoea was nie suksesvol nie. Lae-vlak tetrasiklien weerstandigheid (MIK fs van 4 . 8 µg/ml) is wel suksesvol oorgedra na en U. parvum ontvanger vanaf een U. urealyticum en drie U. parvum ontvangers wat tetM gedra het met MIKs van 16-64 µg/ml. Met die analise van tetM met PKR, kon tetM nie aangetoon word in die transformante nie. Gevolgtrekkings: Die belang van genitale mykoplasma, ureaplasma en C. trachomatis in langtermyn etologie benodig verdere ondersoek, veral in die lig van die sindromiese behandeling regimes wat nie kolonisasie verminder nie. Die hoe prevalensie van P. jiroveci, die teenwoordigheid van M. pneumoniae in gevalle van pneumonie en die aantoning van U. parvum in twee gevalle van neonatale pneumonie benadruk dat, in die afwesigheid van en definitiewe diagnose, dit noodsaaklik is om respons tot behandeling sorgvuldig te moniteer, veral indien die eerste lyn antibiotika keuse ß-laktam antimikrobiale middels of kefalosporiene is, sodat behoorlike en ingeligde gesondheidsorg gelewer kan word. Die bevinding van transposon en/of tetM gebiede in alle ureaplasma wat ondersoek is met of sonder volle uitdrukking van tetrasiklien weerstandigheid, in samehang met tetM diversiteit, plaas verseker ureaplasma sterk in die prentjie vir intra- en inter-generiese uitruiling van antibiotika weerstandigheids gene. / Nelson Mandela Metropolitan University / National Research Foundation (NRF Thuthuka) / Medical Research Council

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.