Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Setting This study was conducted in the Tygerberg district, Cape Town, in the Western Cape, South Africa Background The evaluation of early tuberculosis (TB) treatment response is based on month 2 sputum culture status. This method of evaluation has a number of limitations: the test requires relatively advanced laboratory infrastructure and procedures, it takes several weeks to obtain results and is a relatively a poor marker at predicting treatment response. The discovery of potential host markers which reflect the efficacy of early treatment would be of great importance for clinical management of individual patients. The treatment failure would be detectable earlier than at week 8 of treatment. The duration of clinical trials of new anti-tuberculosis drugs may also be substantially reduced by such markers if these would be measurable earlier than at week 8 of therapy. Objectives 1) To evaluate diluted, 7-day whole blood cultures stimulated with live Mycobacterium tuberculosis (M.tb) for the presence of host markers of early TB treatment response 2) To evaluate an overnight, undiluted, M.tb antigen stimulated whole blood culture Quantiferon Gold In Tube (QFT-GIT) supernatants for host markers of early TB treatment response The study designs were as follows: In study one, baseline samples and samples from week 1, week 2 and week 4 of treatment from 30 cured TB patients were selected from a larger biomarker study, in which whole blood was stimulated with live M.tb or left unstimulated. Fifty seven host markers were measured in supernatants by multiplex cytokine arrays. In study two, baseline samples and samples from week 2 and week 8 of treatment from 19 cured TB patients were randomly selected from the placebo group in a micronutrient supplement study. QFT-GIT supernatants from these participants were assessed through multiplex cytokine arrays for levels of fifty seven host markers. All of the participants in both studies were Human Immunodeficiency Virus (HIV) negative. Changes in marker expression over time and between fast and slow responders to treatment were evaluated. Comparability between the two culture methods was assessed for markers that were evaluated in both studies. Results In study one, the majority of host markers showed significant changes over time in the unstimulated supernatants. Only GRO and IL-1beta changed significantly in an antigen-specific manner (background levels subtracted). No significant changes were observed between fast and slow responders. In study two, the majority of host markers showed significant changes over time in the unstimulated supernatants whereas only MDC and IL-4 changed during the observation period in antigen stimulated levels. Significant differences were observed between fast and slow responders at pre-treatment for IL-13 Ag-Nil and IL-1betaAg-Nil . Conclusion This study revealed, antigen-specific responses showed only limited potential for early TB treatment response monitoring, but may have potential in differentiating between treatment outcomes. Future investigations may have to include later time points during treatment as these were not included in the present assessment. The QFT-GIT samples do not appear to be equivalent to live M.tb stimulated 7-day whole blood assays. / AFRIKAANSE OPSOMMING: Instelling Die studie is uitgevoer in die Tygerbergdistrik, Kaapstad, Wes-Kaap, Suid-Afrika. Agtergrond Die evaluering van die respons op vroeë tuberkulose (TB) behandeling word gebaseer op die status van maand 2 sputum kulture. Hierdie evalueringsmetode het ‘n paar beperkinge: die toets benodig relatief gevorderde laboratorium infrastruktuur en prosedures, die toetsuitslae is eers na ‘n paar weke beskikbaar en dit is n relatiewe swak merker om repons op behandeling te voorspel. Die ontdekking van potensiële selfmerkers wat die doeltreffendheid van vroeë behandeling weerspieël sal van groot belang wees vir die kliniese bestuur van individuele pasiënte. Mislukking van die behandeling sal sodoende voor week 8 van behandeling waargeneem kan word. Die tydsduur van kliniese proewe van nuwe anti-tuberkulose medikasie mag ook baie verkort word met sulke merkers as dit voor week 8 van behandeling gemeet kan word. Doelwitte 1) Om verdunde, 7-dae oue volbloedkulture, met lewende Mikobakterium tuberkulosis (M.tb) gestimuleer, te evalueer vir die teenwoordigheid van vroeë TB behandeling respons selfmerkers. 2) Om die supernatant van oornag, onverdunde, M.tb antigeen gestimuleerde volbloedkulture Quantiferon Gold In Tube (QFT-GIT) vir vroeë behandeling respons selfmerkers te evalueer. Die studie-ontwerpe was soos volg: Met studie een is basislynmonsters en monsters verkry na week 1, week 2 en week 4 van behandeling van 30 geneesde TB-pasiënte geselekteer uit ‘n groter biomerkerstudie waarin die volbloed met lewende M.tb gestimuleer is of ongestimuleer gelaat is. Sewe-en-vyftig selfmerkers is in die supernatante gemeet deur middel van multipleks sitokine arrays. Met studie twee is basislynmonsters en monsters verkry na week 2 en week 8 van behandeling van 19 geneesde TB-pasiënte lukraak uit die plasebo-groep in ‘n mikrovoedingstowwe-aanvullingstudie geselekteer. Vlakke van 57 selfmerkers is in die QFT-GIT supernatante van hierdie deelnemers, deur middel van die multipleks sitokine arrays, bepaal. Al die deelnemers van beide studies was HIV negatief. Veranderinge in merker-uitdrukking oor tyd, asook tussen vinnige en stadige respons tot behandeling, is ge-evalueer. Die vergelykbaarheid van die twee kultuurmetodes is geassesseer ten opsigte van die ge-evalueerde merkers in albei studies. Resultate Met studie een het die meerderheid van die selfmerkers in die ongestimuleerde supernatante kenmerkende verandering oor tyd gewys. Slegs GRO en IL-1beta het aansienlik verander in die antigeenspesifieke wyse (agtergrond vlakke afgetrek). Geen kenmerkende veranderinge was waargeneem tussen die vinnige en stadige respons pasiënte nie. Met studie twee het die meerderheid van die selfmerkers aansienlike veranderinge oor tyd in die ongestimuleerde supernatante gewys, in vergelyking waar net die MDC en IL-4 veranderinge gedurende die observasie periode in antigeen gestimuleerde vlakke getoon het. Kenmerkende verskille is tussen die vinnige en stadige respons pasiënte in voorbehandeling vir IL-13 Ag-Nil en IL-1betaAg-Nil waargeneem. Gevolgtrekking Die studie bewys dat antigeenspesifieke response slegs beperkte potensiaal vir vroeë TB behandeling respons monitering het, maar mag die potensiaall vir onderskeidende behandeling uitkomste hê. Toekomstige ondersoeke sal dalk latere tydpunte gedurende die behandeling moet insluit aangesien dit nie in hierdie evaluasie ingesluit is nie. Die QFT-IT monsters verskyn nie as gelykwaardig met die lewendig M.tb gestimuleerde 7-dae volbloed toetse nie.

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.