Submitted in fulfillment of the requirements for the degree of Master’s of Technology: Radiography, Durban University of Technology, Durban, South Africa, 2015. / Introduction Tuberculosis remains a leading cause of death, second to the Human Immunodeficiency Virus. The risk of latent tuberculosis infection and active tuberculosis disease is a known occupational hazard. In South Africa, a high tuberculosis burden country, the potential of Mycobacterium tuberculosis transmission to health care workers is high. This includes diagnostic radiographers and other radiology staff working in radiology departments. Purpose of the Study This study aimed to investigate the association of demographic and occupational factors with latent tuberculosis infection in radiology staff in public sector hospitals of the eThekwini Health District. Methodology This cross-sectional study was conducted from 26 February 2013 to 07 June 2013. Quantitative methods were used to test for associations of demographic and occupational factors with latent tuberculosis infection in participants. A sample size of 181 participants for an estimated population of 340 radiology staff was recommended at the proposal stage. The study consisted of two phases; the questionnaire survey (phase one) and the administration of a two-step tuberculin skin test (phase two). Data was obtained with regard to demographics, occupational history, social behaviours, medical history; and family and home histories. Demographic and occupational associations with latent tuberculosis infection were made in relation to the size of the first tuberculin skin test induration. Frequency distributions were developed to describe data categories. Pearson’s and Spearman rho’ correlation coefficients were used to test for correlations between the independent variables. The chi-square test was used to determine associations between the categorical independent variables and the dependent variable. Bivariate analyses were performed using these tests. The multivariate analysis was performed using logistic and linear regression on the dependent variable. Results A total of 182 questionnaires were returned from approximately 280 radiology staff. At the outset, all doctors working in the radiology department had to be excluded due to numerous failed attempts to enlist their participation. Fifty-three (29.12 percent) participants were excluded from phase one of the study and a further thirteen participants were excluded from phase two. The total sample was 116 participants. Of the 116 participants, 86.2 percent tested positive for latent tuberculosis infection at the first step of the two-step testing method used. One (0.86 percent) participant went on to convert at the second step, testing positive at this level. Demographic associations with latent tuberculosis infection included age (older) as an associated factor. A significant demographic association with latent tuberculosis infection was the use of alcohol (p-value 0.033 on the multivariate analysis). Occupational associations with latent tuberculosis infection included longer durations of employment. The annual income (higher income earners) displayed significant associations with latent tuberculosis infection (p-value 0.048 on the multivariate analysis). It is necessary in this study to note that participants include support personnel (lower income earners) making up 37.8 percent of the study, diagnostic radiographers making up 48.3 percent; and radiography managers/assistant managers (highest income earners) making up 13.8 percent of the study. Conclusion and recommendations The risk of transmission of Mycobacterium Tuberculosis to health care workers is a known occupational hazard. This study has described the prevalence of latent tuberculosis infection in radiology staff, at district and regional hospitals within the eThekwini Health District. With 23.62 percent of all participants already having active TB disease and 86.2 percent of the tested group displaying positive results for latent tuberculosis infection, using the tuberculin skin tests, the need for tuberculosis screening is essential. The findings of this study will be used as a health improvement mechanism for stakeholders, having identified potential gaps in medical screening in healthcare in Kwa-Zulu Natal. This study makes recommendations for the early detection of active tuberculosis infection and the monitoring of health care workers that are latently infected, thus assisting in reducing the rate of conversion of latent tuberculosis infection to active tuberculosis disease in radiology staff. This reduces long-term exorbitant costs related to health care associated infections, such as tuberculosis. It also reduces rates of transmission and cross infection to both co-workers and already immunocompromised patients, helping to curb the overall epidemic in South Africa.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Setting Study conducted in Tygerberg, Cape Town in South Africa. Hypothesis Host biomarkers associated with the antimycobacterial immune response during active infection with M. tuberculosis and during anti-tuberculosis chemotherapy are indicative of bacterial killing in the host and can be used in models to predict eventual treatment outcome. Objectives 1. To investigate immune parameters that were selected in a biological context as biomarkers of the extent of disease and early response to anti-tuberculosis treatment. 2. To use selected immune parameters to characterise fast and slow responders to anti-tuberculosis therapy. Findings Evaluation of cytokine multiplex fluorescent bead-based immunoassays as a screening tool in the search for biomarkers The data showed that cytokine multiplex fluorescent bead-based immunoassays achieved acceptable recoveries to detect antigen-specific IFN- responses in whole blood supernatant making it attractive for biomarker screening. However, proper optimisation needs to be done and proper controls included when using these kits. Markers of extent of disease High levels of CRP at diagnosis were found to be associated with the presence of multiple cavities on chest X-rays. A high level of suPAR and sICAM-1 at diagnosis were associated with the extent of alveolar disease. Also significant were the associations between the level of granzyme B, LAG-3 at diagnosis and the size of the cavities. No significant associations were observed between sTNFRs or DR5 with the chest X-ray grading of tuberculosis disease. Early classification of fast and slow responders to anti-tuberculosis treatment After cross-validation classification, discriminant analysis (DA) and support vector machine (SVM) analysis of selected immune parameters (sICAM-1 CRP, granzyme B, suPAR, sTNFRs, LAG-3 and CD3dim/CD56+ (% of CD45+) resulted in a 75% to 100% correct classification of the fast responders and a 82% to 100% correct classification of the slow responders when using DA. For SVM, the correct classification of the fast responders ranged from 88% to 100%, and that for the slow responders ranged from 95% to 100%. Differential gene expression in fast and slow responders to treatment Direct comparison of fast and slow responders showed that IL-4 transcripts were significantly higher in the fast responders at week one after initiation of treatment when compared to slow responders. IL-42 was also differentially expressed. Although IL- was significantly up-regulated in both fast and slow responders after one week of treatment compared to diagnosis, IL- expression was more than two folds higher in slow responders than in fast responders. No significant differences between the fast and slow responders were observed in the expression of TGF-, TGF-RII, Foxp3 and GATA-3. Conclusion Predictive models for differential anti-tuberculous treatment responses combining host proteins are promising and should be included in larger prospective studies to find the optimal markers for inclusion into clinical trials of new drugs and for implementation into clinical practice. / AFRIKAANSE OPSOMMING: Ligging Studie onderneem in Tygerberg, Kaapstad, Suid-Afrika. Hipotese Gasheerbiomerkers wat verband hou met die antimikobakteriële immuunrespons tydens aktiewe infeksie deur M. tuberculosis en tydens teentuberkulose chemoterapie dui op bakteriële doding in die gasheer en kan in modelle gebruik word om die uiteindelike uitkoms van die behandeling te voorspel. Doelwitte 1. Om gekose immuunparameters in ’n biologiese konteks as biomerkers van die omvang van siekte en vroeë reaksie op behandeling te ondersoek. 2. Om gekose immuunparameters te gebruik om vinnige en stadige reageerders op teentuberkulosebehandeling te karakteriseer. Bevindings Evaluering van die sitokien veelvuldige fluoresseer-pêrelbaseerde immuuntoets (cytokine multiplex fluorescent bead-based immunoassays) as ’n siftingsinstrument in die soeke na biomerkers Die data het getoon dat die sitokien veelvuldige fluoresseer-pêrelgebaseerde immuuntoets in staat was om antigeenspesifieke IFN--respons te meet wat dit aanloklik maak vir biomerkersifting. Sorgvuldige optimering moet egter gedoen word en behoorlike beheer moet ingesluit word wanneer hierdie stelle gebruik word. Merkers van omvang van siekte Hoë vlakke van CRP by diagnose is getoon om verband te hou met die teenwoordigheid van veelvoudige holtes op die pasiënte se borskas x-strale. Hoë vlakke van suPAR en sICAM-1 by diagnose was assosieer met die omvang van alveolêre siekte. Die assosiasie tussen die vlakke van granzyme B, LAG-3 by diagnose en die grootte van die holtes was ook betekenisvol. Daar was geen betekenisvolle assosiasies toe sTNFRs of DR5 en die borskas x-straalgradering van tuberkulosesiekte nie. Vroeë klassifikasie van vinnige en stadige reageerders op teentuberkulosebehandeling Ná klassifikasie op grond van kruisstawing het diskriminant-analise (DA) en ondersteuningsvektormasjiene (SVM) van geselekteerde immuunparameters (sICAM-1 CRP, gransiem B, suPAR, sTNFRs, LAG-3 en CD3dim/CD56+ (% van CD45+)) gelei tot ’n 75% tot 100% korrekte klassifikasie van die vinnige reageerders met DA en ’n 82% tot 100% korrekte klassifikasie van stadige reageerders. Vir SVM het die korrekte klassifikasie van vinnige reageerders gewissel van 88% tot 100%, en vir stadige reageerders het dit gewissel van 95% tot 100%. Differensiële geenuitdrukking in vinnige en stadige reageerders op behandeling In vergelyking met die vlak by diagnose is die uitdrukkingsvlak van IL-4 in die vinnige reageerders betekenisvol opgereguleer met ’n faktor van 9.2 teen die eerste week ná die aanvang van behandeling, in kontras met die stadige reageerders. Daar was geen verskille tussen die vinnige en die stadige reageerders met betrekking tot die uitdrukking van TGF-, TGF-RII, Foxp3 en GATA-3 nie. Gevolgtrekking Voorspellende modelle vir differensiële tuberkulose behandelingsresponse wat gasheerproteïene kombineer, hou belofte in en behoort in groter prospektiewe studies ingesluit te word om die mees geskikte merkers te vind vir insluiting in kliniese proewe van nuwe middels en vir implementasie in kliniese praktyk.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Sequence analysis showed that the essential glnA1 gene of Mycobacterium tuberculosis might be closely related to an actinomycetes progenitor sequence and that this sequence may have undergone duplication into other non-essential GS encoding genes in some Actinobateria, notably the mycobacteria. Also, the M. tuberculosis glnA1 sequence remains conserved throughout the evolution of M. tuberculosis. It was also shown that glnA1 is widely expressed in M. tuberculosis infected human pulmonary tissue, where M. tuberculosis might be present in altered phenotypes. These results imply that glnA1 is under selective pressure against evolutionary change. At transcriptional level it was shown that M. tuberculosis glnA1 might be expressed from two alternate promoter sites, but that these promoter sites may not be controlled by environmental nitrogen (in the form of ammonium) variation. We also showed that M. tuberculosis GS is effectively exported by M. smegmatis to the cell wall, but that GS secretion into the exogenous environment does not occur. Also, evidence has been presented which suggested that M. tuberculosis GS might be modified at the C-terminus, probably as part of a mechanism that retains GS in the cytosol. This hypothesis was further substantiated where it was demonstrated that two GS isoforms of different size (short isoform in cytosol, longer isoform in cell wall) are present in M. bovis BCG. It is unknown what causes this modification, since it couldn’t be observed in M. smegmatis, but it was suggested that it might be through the action of a cis- or trans-acting element present in proximity of the M. tuberculosis glnA1 gene. It was also shown that a cluster of genes found immediately downstream of the M. tuberculosis glnA1 sequence might be regulated in an operonic fashion under conditions of elevated environmental nitrogen concentrations. Two of the genes (glnE and glnA2) in this operon arrangement have been previously shown to be involved in nitrogen and glutamine metabolism. The function of the other gene, Rv2223c, is unknown. It was shown that Rv2223c homologs are mostly found in the mycobacteria and that it may encode an exported protease. It was hypothesised that this sequence and its adjacently located progenitor sequence, Rv2224c, might be involved in M. tuberculosis GS mediated metabolism. It was showed that over-expression of Rv2223c and Rv2224c may be toxic to E. coli and mycobacterial hosts, such as M. smegmatis, but that inhibition of transcription of these genes may be fatal to M. bovis BCG and M. tuberculosis H37Rv. It was also shown that Rv2223c is widely expressed in M. tuberculosis infected human tissue, which was comparable to that of glnA1. The results presented in this study shed more light on the distribution and transcriptional regulation of GS in mycobacteria and has identified new genes that might be involved in GS regulation. These results may present new approaches to tuberculosis control and thereby contribute to alleviate the burden of the disease. / AFRIKAANSE OPSOMMING: Genetiese en proteien volgorde analise het aangedui dat die glnA1 (kodeer vir glutamien sintetase (GS), ‘n essentiele protein) geen van Mycobacterium tuberculosis die naaste verwant is aan ‘n actinomycetes voorloper volgorde wat duplikasie ondergaan het om die ander nieessensiele GS koderende gene in sommige Actinobakterieë te vorm, veral in die mikobakterieë. Voords het die glnA1 geen geneties gekonserveerd gebly gedurende die evolusie van M. tuberculosis. Dit is ook aangetoon dat volop transkribasie van die glnA1 geen voorkom in die M. tuberculosis geïnfekteerde pulmonêre weefsel waar M. tuberculosis moontlik mag voorkom. Op transkriptionele vlak is dit aangetoon dat die M. tuberculosis glnA1 geen vanaf twee onderskeie promotors uitgedruk mag word, maar dat hierdie twee promotors nie deur variasies in die konsentrasie van stikstof (in die vorm van ammonium) in die omgewing beïnvloed word nie. Daar is ook aangedui dat M. tuberculosis GS effektief deur M. smegmatis oor die selmembraan na die selwand vervoer word, maar dat daar nie GS sekresie na die ekstrasellulêre omgewing geskied nie. Ook is bewyse gevind dat M. tuberculosis GS modifikasie aan die C-terminus mag ondergaan wat waarskynlik dien om GS beweging uit die sitosol te verhoed. Hierdie hipotese is versterk deurdat twee isoforms van verskillende grootte (klein in sitosol en groter in die selwand) gevind is in M. bovis BCG. Dit modifikasie meganisme is onbekend, maar vind moontlik nie plaas in nie-patogeniese mikobakterieë soos M. smegmatis nie en mag moontlik deur cis- of trans-werkende elemente gefasiliteer word. Daar is aangedui dat ‘n groepering van vier gene lanksaan die glnA1 lokus in ‘n operoniese meganisme gereguleer mag word onder variërende konsentrasies van stikstof in die omgewing. Dit is bekend dat twee van die gene in die operon (glnE en glnA2) betrokke in stikstof en gultamien metabolisme is. Die funksie van die ander twee gene (Rv2223c en Rv2224c) is onbekend. Daar is aangetoon dat volgordes soortgelyk aan Rv2223c beperk is tot die mikobakterieë en dat die geen ‘n protease, wat moontlik gesekreteer word vanuit die sitosol, kodeer. Daar is aangetoon dat die oor-produksie van die Rv2223c en Rv2224c proteine toksies is vir E. coli en mikobakterieë, soos M. smegmatis, maar dat transkripsie inhibisie hierdie gene dodelik is vir M. tuberculosis H37Rv en M. bovis BCG. Daar is ook angedui dat die ekspresie van hierdie gene volop verspreid is in M. tuberculosis geïnfekteerde menslike weefsel, soortgelyk aan diè van glnA1. Die resultate vervat in hierdie studie werp meer lig op die verspreiding en transkiptionele regulasie van GS in mikobakteriee en nuwe gene is ontdek wat betrokke by GS regulasie mag wees. Hierdie resultate mag bydra tot nuwe maniere om tuberkulose te bekamp en daardeur die voorkoms van die siekte te beperk.

Thesis (M. A. (Communication Studies)) -- University if Limpopo, 2021 / This study was prompted by the number of people who die daily from tuberculosis (TB) in the study area. The study investigated the perceptions of Mokopane residents, and their level of understanding and knowledge of the disease, tuberculosis (TB). The aim of the study was to profile people’s perceptions of health beliefs, the causes of the increase and spread of TB and its prevention and their knowledge about TB awareness campaigns in the Mokopane area. Data was collected by means of both qualitative and quantitative methods. Ten (10) medical doctors who operate private practices in the Mokopane area were interviewed. Data was collected from four hundred and one (401) participants from both Sandsloot and Tshamahansi villages outside Mokopane. The major findings of this study showed that firstly residents have a firm belief that traditional methods cure TB; secondly, they are largely ignorant of TB, its prevention, and consequences; and lastly that there is a lack of TB related education.

Tuberculosis (TB) is one of the most deadly diseases worldwide. In the United States, TB disproportionately affects foreign-born individuals, individuals living in congregate settings, people with human immunodeficiency virus, and people who use illicit drugs. In 2005, a large homeless shelter outbreak in San Joaquin County resulted in 67 individuals diagnosed with TB with links to a homeless shelter. It is hypothesized that by using bed analysis to identify contacts that have been exposed to TB during this outbreak will allow for better identification of exposed high-risk individuals than screening alone. Demographics, exposure, screening, and QuantiFERON-Tuberculosis results were analyzed using bed assignments from the homeless shelter database and data from a homeless shelter screening (HSS) program. Individuals diagnosed with active TB disease were on average more likely to be identified through bed analysis than HSS, 95.08% versus 59.02%. Utilizing both bed analysis and HSS allows for improvement of identification and continuous testing of individuals exposed to TB.

Mycobacterium tuberculosis has caused a resurgence in pulmonary disease in both developed and developing countries in recent times, particularly amongst people infected with the human immunodeficiency virus. The disease has assumed epidemic proportions in South Africa and in the Eastern Cape Province in particular. Of further concern is the isolation of increasing numbers of multiply drug resistant strains. Knowledge of the genetic capability of this organism is essential for the successful development of novel antibiotics and vaccines in an attempt to bring the global pandemic under control. Measurement of the genetic diversity of the organism may significantly contribute to such knowledge, and is of vital importance in monitoring epidemics and in improving treatment and control of the disease. This will entail answering a number of questions related to the degree of genetic diversity amongst strains, to the difference between urban and rural strains, and between drug resistant and drug sensitive strains, and to the geographical distribution of strains. In order to establish such baseline information, RAPD profiling of a large population of isolates from the western and central regions of the Eastern Cape Province was undertaken. A smaller number of drug resistant strains from a small area of KwaZulu-Natal were also analysed, with a view to establishing the genetic difference between strains from the two provinces. Cluster analysis, analysis of molecular variance and Geographical Information Systems technology were used to analyse the RAPD profiles generated. An unexpectedly high degree of genetic diversity was detected in strains from both provinces. While no correlation was seen between genetic diversity and either urban-rural situation or geographical location, a small degree of population structure could be correlated with drug resistance in the Eastern Cape. Furthermore, a significant degree of population structure was detected between strains from the two provinces, although this was still within the parameters for conspecific populations. Future work is necessary to further characterise strains from rural areas of both provinces, as well as from the eastern region of the Eastern Cape in an attempt to pinpoint the cause of the separation of the provincial populations.

MPH / Department of Public Health

MSc (Chemistry) / Department of Chemistry / Identification of unique leads represents a significant challenge in drug discovery. This challenge is widely visible in neglected diseases such as tuberculosis, which is an infectious disease caused by bacillus Mycobacterium tuberculosis. The urgent need in search of new biological entities to fight back TB and drug resistant TB is a drive behind this project. Several specific synthetic protocols have been developed using 1,3,5-triazines due to the important biological properties which they display. The chemistry and an extensive spectrum of biological activities of s-triazines have been examined since several decades and this heterocyclic core has received emerging consensus. Hence, the aim of this project was to synthesize novel anti-TB drugs total with the usage of 1,3,5-triazine as a linker between known anti-TB drugs together with different types of amines. A total of 20 compounds were synthesized, 3 compounds were mono-substituted with an average yield of 75 %, 6 compounds were di-substituted with an average yield of 63 % and 11 compounds were tri-substituted with an average yield of 93 %. Out of 10 compounds which were analysed for biological activity 8 of which showed biological activity against M.smegmatis. Furthermore compound 26 which was hybridized with an amine and a known anti-TB drug inhibited better biological activity. In conclusion the influence of cyanuric chloride in combination with pyrrolidine and anti-TB drugs deserves further study. The newly synthesized compounds were characterized by IR, melting point, GC-MS, biological testing, 1H and 13C NMR. / NRF

This study investigated factors influencing the quality of data for the Tuberculosis (TB) control programme in Oshakati District in Namibia. A quantitative, cross-sectional descriptive survey was conducted using 50 nurses who were sampled from five departments in Oshakati State Hospital. Data was collected by means of a self-administered questionnaire. The results indicated that the majority (90%) of the respondents agreed that TB training improved correct recording and reporting. Sixty percent of the respondents agreed that TB trainings influenced the rate of incomplete records in the unit, while 26% of the respondents disagreed with this statement. This indicates that TB trainings influence the quality of data reported in the TB programme as it influences correct recording and completeness of data at operational level. Participants’ knowledge on TB control guidelines, in particular the use of TB records to, used to capture the core TB indicators influenced the quality of data in the programme. The attitudes and practises of respondents affected implementation of TB guidelines hence, influencing the quality of data in the programme. The findings related to the influence of the quality of data in the TB programme and its effect to decision-making demonstrated a positive relationship (p=0.0023) between the attitudes of study participant on the use of data collected for decision-making. Knowledge, attitudes and practice are the main factors influencing the quality of data in the TB control programme in Oshakati District. / Health Studies / M.A. (Public Health)

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Setting This study was conducted in the Tygerberg area of Cape Town in South Africa. Background A third of the world’s population is latently infected with Mycobacterium tuberculosis, and correlates of protection against progression to active disease urgently need to be identified to facilitate the development of an effective vaccine against the disease. The production of IFN-γ is recognised as an immune correlate of protection from tuberculosis, but other immune regulators have been implicated in playing a significant role in protective immunity. The aims of this project were three-fold: (i) to identify promising TB vaccine candidates by screening a panel of novel MTB antigens, by stimulating whole blood cultures in vitro with the novel proteins and quantifying the level of IFN-γ production, (ii) to identify other cytokines and chemokines that may be immune correlates of protection using the Luminex fluorescent bead-based technique and (iii) to compare the performance of the two techniques. Methods Antigen Screening study Whole blood of 57 adult and adolescent participants defined as latently infected individuals was stimulated with a panel of 78 novel TB-specific, DosR- or RD1-encoded antigens. The 7-day culture supernatants were used in IFN-γ ELISA to quantify the level of IFN-γ production. Luminex Assay study Whole blood culture supernatants of 15 HIV negative, TST positive adults were used in the Luminex LINCO 21-plex cytokine assay. This was done to determine which of 21 cytokines, that may be LTBI-associated cytokines, were produced after stimulation with 9 TB-specific recombinant antigens, and to quantify their level of expression. Results In the antigen screening study, it was found the majority of the 78 proteins tested were able to induce a positive IFN-γ response. The classic TB antigens were used as controls, and the frequency of responses was highest after stimulation with ESAT-6 and TesatCFP10 (80 – 85% of responders). Ten latency antigens elicited an IFN-γ response in 19 – 45% of participants, and five reactivation antigens stimulated a positive reaction in 15 – 48% of responders. The category of antigens that elicited the most frequent and highest responses overall was the resuscitation-promoting factors (Rpf). Over 30% of participants responded to all 5 Rpfs, and the level of responses were equally divided in the low and moderate-to-high levels, with an additional 5% of responses in the high (>1000pg/ml) range. In the Luminex study, the positive stimulant TesatCFP10 consistently induced expression of most cytokines. In addition latency antigens Rv1733c, Rv0569 and Rv2029c also induced moderate-to-high level cytokine expression. A Th1-biased cytokine profile was observed, with the preferential expression of pro-inflammatory and cell-mediated cytokines like IFN-γ, TNF-α, IP-10, MIP1-α and G-CSF being produced. Th2 cytokines IL-4, IL-5, IL-13 and eotaxin were very poorly expressed or were not expressed at detectable levels. A very strong induction of IL-6, IL-8 and MCP-1 was observed, but this cytokine/chemokine association suggested contamination of the recombinant antigens with bacterial endotoxins. Conclusion In this study of latently infected individuals, the pattern of response observed for both assays is largely a Th1-biased expression profile. The whole blood ELISA method is a well-established assay for quantifying IFN-γ in culture supernatants, and has proven to be effective here. This study has demonstrated, in humans with LTBI, immune recognition of these novel MTB-specific antigens as illustrated by the positive IFN-γ levels induced after stimulation. The multiplex technology is also a very versatile and sensitive assay, capable of detecting multiple analytes simultaneously in one sample. The multiplex has been valuable here in identifying some antigens as potential vaccine candidates, and a subset of cytokines as potential immune mediators and prognostic indicators in TB infection. / AFRIKAANSE OPSOMMING: Studie-area Hierdie studie was gedoen in die Tygerberg area van Kaapstad in Suid-Afrika. Agtergrond ‘n Derde van die wêreld se bevolking is latent geïnfekteer met Mycobacterium tuberculosis en korrelate van beskerming teen die siekte moet geïdentifiseer word om die ontwikkeling van ‘n effektiewe enstof te fasiliteer. Die produksie van IFN-γ is welbekend as ‘n immuunkorrelaat van beskerming teen tuberkulose (TB), maar ander immuunreguleerders speel ook ‘n belangrike rol in beskermende immuniteit. Die doelwitte van hierdie projek was drievoudig: (i) om belowende TB-entstof kandidate te identifiseer deur die sifting van ‘n paneel van nuwe MTB antigene mbv die in vitro stimulasie van volbloed kulture, ii) om ander sitokiene en chemokiene as immuunkorrelate van beskerming te identifiseer deur van die Luminex fluorescent bead-based tegniek gebruik te maak, en (iii) om die twee tegnieke te vergelyk op grond van hul prestasie as prognostiese of siftings metodes in latente infeksie. Metodes Antigeen siftings studie Volbloed van 57 volwasse en adolessente deelnemers, geïdentifiseer as latent geïnfekteerde individue, was gestimuleer met ‘n paneel van 78 nuwe TB-spesifieke DosR- or R-gekodeerde antigene. Die 7-dae kultuur supernatante was gebruik in ‘n IFN-γ ELISA om die hoeveelheid IFN-γ produksie the kwantifiseer. Luminex assay studie Volbloed kultuur supernatante van 15 HIV negatiewe, TST positiewe volwassenes was gebruik in die Luminex LINCO 21-plex cytokine assay. Dit was gedoen om die tipes en hoeveelheid ander LTBI-geassosieerde sitokienes te identifiseer wat geproduseer word na stimulasie met 9 TB-spesifieke rekombinante antigene. Resultate In die antigeen siftings studie is gevind dat die meerderheid van die 78 getoetste proteïene ‘n positiewe IFN-γ reaksie kon induseer. Vir die kontroles was die frekwensie van reaksies die hoogste na stimulasie met ESAT-6 en TesatCFP-10 (80 – 85% van reageerders). Tien latensie antigene was gereeld herken deur 19 – 45% van deelnemers en vyf reaktiverings-antigene het ‘n positiewe reaksie in 15 – 48% van reageerders gestimuleer. Die kategorie van antigene wat die meeste en hoogste response veroorsaak het, was die resusitasie-promoterende faktors (Rpf). Meer as 30% van deelnemers het op al 5 Rpfs gereageer en die vlak van reaksies was gelyk verdeel in die lae en matig-tot-hoog vlakke, met ‘n addisionele 5% van reaksies in die hoë (>1000pg/ml) reeks. In die Luminex studie het die positiewe stimulant TesatCFP-10 konsekwent die positiewe uitdrukking van die meeste sitokiene geïnduseer. Saam met dit het die latente antigene Rv1733c, Rv0569 en Rv2029c ook matige-toe-hoë vlakke van sitokien uitdrukking geïnduseer. ‘n Th1-gebaseerde sitokien profiel was waargeneem, met die begunstigde uitdrukking van pro-inflammatoriese en sel-gemedieerde sitokiene soos IFN-γ, TNF-α, IP-10, MIP1-α en G-CSF. Th2 sitokiene IL-4, IL-5, IL- 13 en eotaksien was of baie sleg uitgedruk of onder naspeurbare vlakke uitgedruk. ‘n Baie sterk induksie van IL-6, IL-8 en MCP-1 was waargeneem, maar hierdie sitokiene/chemokiene assosiasie stel moontlik kontaminasie van die rekombinante antigene met bakteriële endotoksiene voor. Samevatting Die reaksiepatroon wat in hierdie studie tussen die twee toetse waargeneem is, was grootliks ‘n Th1-gebaseerde uitdrukkingsprofiel vir latente infeksie met TB. Die volbloed ELISA metode is a betroubare gevestigde toets vir die kwantifisering van IFN-γ in kultuur supernatante, wat ook in hierdie studie bewys is om effektief te wees. Hierdie studie het gedemonstreer dat die nuwe TB-spesifieke antigene effektief positiewe IFN-γ response in mense met LTBI induseer. Die multipleks tegnologie is ook ‘n baie veelsydige en sensitiewe toets, wat in staat is om veelvoudige analite gelyktydig in een monster te kan opspoor. In hierdie studie was dit veral waardevol in die identifisering van ander moontlike antigene as prognostiese kandidate en sitokiene as immuunbemiddelaars in TB-infeksie.