Dissertations / Theses on the topic 'Tuberculosis markers'

ackground: The changes in body composition markers (weight, fat mass, lean mass, and BMI) over time can be associated with TB treatment outcome among HIV-infected patients. The aim of this study was to investigate whether changes in fat mass and lean mass were associated with the treatment response among patients with HIV infection and pulmonary tuberculosis. Materials and Methods: This was a prospective cohort study. Data from HIV-infected patients commencing TB therapy were analyzed. This included body weight measurement using bioimpedance equipment at baseline, one month, and two months after starting TB treatment. Results: The study was conducted in 125 patients, 17 patients (13.6%) died during treatment, of which 5 died during the first month of treatment, 4 during the second month and 8 after the second month. The group of patients with good response, increased their weight by 1.3 kg (p <0.001) at the end of the first month of TB treatment and 2.6 kg in the second month (p <0.001), and body fat increase was 1.2 Kg (p <0.001) and 2.3 kg (p <0.001), the first and second month respectively. The group of patients who died had lost 2.1 kg fat mass after the first month (p <0.001) and 3.7 kg in the second month (p <0.001). Conclusions: Our results show that the weight change during TB treatment (increased fat mass) helps us predict therapeutic response. Weight loss during the first month of starting therapy should be evaluated thoroughly to identify the probable cause of treatment failure.
Revisión por pares

Apesar das recomendações da Organização Mundial da Saúde, na reunião ministerial realizada em Amsterdam, Holanda em 2000, a tuberculose continua em ritmo crescente, atingindo principalmente os países em desenvolvimento e pessoas com o sistema imunológico comprometido, principalmente as infectadas pelo vírus da imunodeficiência adquirida. Os métodos utilizados no diagnóstico continuam os mesmos utilizados por muitos anos, cujas limitações impedem a ação rápida dos programas de saúde que buscam interromper a cadeia de transmissão da doença. Continuando uma linha de pesquisa do Laboratório de Soroepidemiologia e Imunobiologia do IMTSP, utilizando o método do Western Blotting, procuramos neste trabalho ampliar os conhecimentos da resposta imunológica de anticorpos em pacientes com tuberculose pulmonar, clinica e laboratorialmente definida, avaliando a participação das imunoglobulinas IgG, IgA e IgM, subclasses de IgG (IgG1 e IgG3) e a avidez da imunoglobulina IgG em amostras de soros colhidas no início e no final do tratamento. Nossos resultados mostraram que o melhor marcador imunológico foi a imunoglobulina IgG por apresentar melhor desempenho diagnóstico quando comparada com os resultados dos métodos microbiológicos e de dosagem de interferon gama, Quantiferon TB Gold. As frações protéicas que apresentaram melhor desempenho diagnóstico foram as de 38 e 30 KDa
Despite the recommendations of the World Health Organization, during the Ministerial meeting held in Amsterdam, Holland in 2000, tuberculosis continues at an important increasing rate, affecting mostly developing countries and people with severe compromised immune systems, especially those infected with human acquired immunodeficiency virus. The methods used for diagnosis are the same used for many years, whose limitations prevent the rapid action of countries health programs that seek to stop the chain of disease transmission. Continuing a line of research of the Laboratory of Seroepidemiology and Immunobiology of IMTSP using the method of Western blotting, in this study we tried to broaden the knowledge of the immune response of antibodies in patients with pulmonary tuberculosis, clinical and laboratory defined by evaluating the participation of IgG, IgA and IgM, IgG subclasses (IgG1 and IgG3) and immunoglobulin IgG avidity in serum samples collected in the beginning and end of treatment. Our results showed that the immunoglobulin IgG was best immunological marker when compared with the results of microbiological methods and determination of interferon gamma, QuantiFERON TB Gold. The proteins fractions that showed better diagnosis performance were 38 and 30 KDa

Tuberculosis in the number one infectious cause of death worldwide, but remains underappreciated as a cause of mortality in children and difficult to diagnose. Diagnostic difficulties of TB in children are a consequence of the non-specific clinical presentation, the different spectrum of disease in children and the paucibacillary nature of disease making microbiological confirmation challenging in many cases. Moreover, existing immunodiagnostic tests have important limitations, especially with regard to childhood TB: they lack sensitivity to rule out TB, and are unable to offer discrimination between contained infection and active stages of disease. Their limitations are emphasized in the youngest children that are at greatest risk of developing severe disseminated forms of TB. For that reason we developed, at the Laboratory of Vaccinology and Mucosal Immunity (LoVMI), several non-sputum based tests, that offer excellent diagnostic accuracy compared to commercialy available tests, for all forms of TB in children, in an early stage of infection. This research also provided insight in TB pathogenesis. The main results are summarized in a table (chapter General Discussion) and two algoritms (chapter Conclusions and Perspectives).
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008.
This series of studies includes both methodological analyses, aimed at furthering our understanding of, and improving the tools used in molecular epidemiology, and investigative projects which have used these tools to add to our knowledge of the M. tuberculosis epidemic. Using serial isolates from tuberculosis patients, we have investigated the evolutionary rate of the IS6110 RFLP pattern. In accordance with other studies, we determined a ½-life for this epidemiological marker of 10.69 years, confirming its appropriateness for this purpose. We also identified an initial, much higher apparent rate which we proposed was the result of pre-diagnostic evolution. In support of this, our investigations in the context of household transmission of M. tuberculosis revealed that IS6110-based evolution is closely associated with transmission of the organism, resulting in a strain population rate of change of 2.9% per annum. To accommodate evolution within estimates of transmission, we proposed that calculations incorporate the concept of Nearest Genetic Distance (cases most similar in RFLP pattern and most closely associated in time). We used this to create transmission chains which allowed for limited evolution of the IS6110 marker. As a result, in our study community, the estimated level of disease attributable to ongoing transmission was increased to between 73 and 88% depending on the Genetic Distance allowed. We identified the duration of a study as a further source of under-estimation of transmission. This results from the artefactual abridgement of transmission chains caused by the loss of cases at the temporal boundaries of a study. Using both real and simulated data, we showed that viewing a 12-year study through shorter window periods dramatically lowered estimates of transmission. This effect was negatively correlated with the size of a cluster. Various combinations of MIRU-VNTR loci have been proposed as an alternative epidemiological marker. Our investigations showed that, while this method yielded estimates of transmission similar to those of IS6110, there was discordance between the two markers in the epidemiological linking of cases as a result of their independent evolution. Attempting to compensate for this by allowing for evolution during transmission improved the performance of IS6110, but generally had a deleterious effect of that of MIRU-VNTR. However, this marker remains a valuable tool for higher phylogenetic analysis and we used it to demonstrate a correlation between sublineages of the Beijing clade and the regions in which they are found. We proposed that, either the host population had selected for a particular sublineage, or that specific sublineages had adapted to be more successful in particular human populations. We further explored the dynamics of the epidemic over a 12-year period in terms of the five predominant M. tuberculosis clades. We found that, while four of these clades remained relatively stable, the incidence of cases from the Beijing clade increased exponentially. This growth was attributed to drug-sensitive cases although drug-resistant Beijing cases also appeared to be more successful than their non-Beijing counterparts. Possible factors contributing to this clade’s success were a greater proportion of positive sputum smears and a lower rate of successful treatment.

Phase III trials for new tuberculosis treatment regimens require large numbers of participants and can take over five years to complete. A surrogate marker for poor outcome (failure at end of treatment or recurrence following successful treatment), the established endpoint in such trials, could shorten trial duration and reduce trial size. Culture results after two months of treatment have shown the most promise but, prior to this research, no formal evaluation had been performed. In this thesis, culture results during treatment are evaluated as prognostic and surrogate markers for poor outcome using data on 6974 patients from twelve tuberculosis treatment randomised controlled multi-arm trials conducted in East Africa and East Asia. A strong association was found between culture results during treatment and poor outcome. Nevertheless, culture results were not good patient-specific predictors of poor outcome with low sensitivities and specificities. Existing meta-analytic methods for evaluating surrogate markers are not wholly suited to this setting of multi-arm trials with binary true and surrogate endpoints. Extending these methods, the two month culture was found to be a good surrogate marker using data from Hong Kong trials and the three month culture was found to be a good surrogate marker using data from East African trials. These results are an indication that cultures during treatment do capture some of the treatment effect. Further work is needed in understanding the differences between the Hong Kong and East African trials. The meta-analytic methods for evaluating surrogate markers in this thesis included a graphical representation that permitted a clear visual evaluation of the surrogate. Methods developed in this thesis for modelling the relationship between the treatment effects on the true and surrogate endpoints were not satisfactory. The deficiencies were not overcome with the two extensions proposed. Further work is needed in developing a more appropriate model.

Tuberculosis (TB) is the leading cause of death from a single infectious agent worldwide and HIV-1 co-infection is the leading cause of susceptibility to tuberculosis. Sub-Saharan Africa has a high incidence of TB-HIV-1 coinfection and the risk of TB in HIV-1 infected people is increased at all stages of the infection. Antiretroviral treatment (ART) is the most effective way to reduce the risk of TB in HIV-1 co-infected people. By studying the protective, ART induced immune reconstitution in HIV infected individuals sensitised by Mycobacterium tuberculosis (Mtb), we can identify correlates of protection against tuberculosis in the form of transcriptomic, soluble or cellular biomarkers. This thesis focuses on characterising Mtb-specific reconstituting CD4 T cells as well as soluble and transcriptomic markers in HIV infected persons, sensitised by Mtb, by analysing samples collected longitudinally during 6 months of ART. Analysis of peripheral blood mononuclear cells by 14-colour flow cytometry revealed the proportion and numbers of central memory CD4+ T cells significantly expanded in HIV infected persons on ART, while the proportion and numbers of effector memory and terminally differentiated effector CD4+ T cells decreased significantly. Additionally we noted a significant decrease in the proportion of activated CD4+ T cells, and IL-2 single producing CD4+ T cells in HIV infected persons at 6 months of ART, while polyfunctional Mtb specific CD4+ T cells secreting IFN-γ, IL-2 and TNF-α simultaneously, proportionally increased. Analysis of soluble markers in the plasma of HIV infected persons revealed an overall decrease in pro-inflammatory cytokines during 6 months of ART. A significant decrease in IFN-γ, IL-1α, IL-1β, IL-6, IL-17A and TNF-α was observed, and concentrations of these cytokines fell towards those observed in HIV uninfected persons. Transcriptomic analyses of 30 genes normalized to 3 different housekeeping genes, showed an overall increase in the expression of T cell memory specific genes, illustrating the regeneration of the memory T cell pool in HIV infected adults on ART. Larger number of central memory specific genes showed increased expression when normalised to at least two housekeeping genes, as compared to effector memory specific genes. These results support the reconstitution of central memory CD4 T-cell specific response at 6 months of ART. Our data provides insight into the reconstituting immune response to latent TB infection in the context of HIV infection and identifies potential correlates of decreased susceptibility to TB. We also show decreasing soluble and cellular factors indicative of decreasing immune activation in HIV infected persons receiving ART.

Background: Development of ultra-short chemotherapy for tuberculosis (TB) is thwarted by drug-tolerant bacillary persistence and a lack of surrogate endpoints to predict outcome from early clinical studies. Characterising bacillary elimination amongst TB patients may provide important new information. Bacilli harbouring intra-cytoplasmic lipid bodies (LBs) may represent a drug-tolerant phenotype, responsible for delayed bacterial clearance. Methods: Malawian adults with pulmonary TB were treated with standard 6 month therapy. Two quantitative sputum culture methods were used to model bacillary elimination during the first 2 months; serial sputum colony counting (SSCC) and time to positivity (TTP) in BACTEC MGIT broth. Fluorescence microscopy was used to assess the proportion of LB positive bacilli on sputum smears. Plasma concentrations of anti-TB drugs were assayed at day 14 or 21. Patients were followed until one year post-treatment. Outcomes were defined as favourable (stable cure) or unfavourable (failure/relapse). The effect of microbiological and pharmacological factors on outcome was assessed. Results: 169 patients (59% with HIV co-infection) were recruited. Of 133 final outcomes, 15 (11%) were unfavourable. Partial likelihood non-linear mixed effects (NLME) modelling of SSCC data from 86 (64%) patients showed biphasic bacillary elimination; slow late-phase eradication of persisters was associated with unfavourable outcome (p=0.001). Linear mixed effects (LME) modelling of TTP data from 124 (93%) patients showed that a slower MGIT Bacillary Elimination Rate (MBER) was associated with unfavourable outcome (p=0.007). 28% (range 0-79%) of acid-fast bacilli in baseline sputum samples were LB positive. During the first month there was a trend towards higher LB counts in patients who went on to have unfavourable vs. favourable outcomes (p=0.085). Low plasma rifampicin and isoniazid concentrations were reported in 87% and 50% patients respectively. A lower isoniazid AUC(0-6hr) exposure was associated with unfavourable outcome (p=0.035). Conclusions: The two main findings were: 1. Modelling of bacillary elimination during the first 2 months of TB therapy predicted long-term outcome. The MBER, in particular, could be calculated for 93% of patients and should be considered as a surrogate marker for early clinical trials. 2. The observation of a higher proportion of LB positive bacilli in later sputum samples from patients with unfavourable outcomes suggests that these may be drug-tolerant persister cells, with negative implications for treatment efficacy.

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.

There is an ever-increasing need to enhance the capability of sensor technology for health, structural and environmental monitoring. One area of great concern is new strains of microbial organism and the spread of infectious diseases that requires rapid identification and detection in vivo and in vitro. Another area of major concern, worldwide, is the threat of chemical and biological terrorism. This points out onto necessity of improovement of existing and development of novel detection technologies based on nanomaterials. Nanophotonics-based sensors utilizing nanostructured multiple probes provide the ability for simultaneous detection of different biomedical and ecological objects as well as the ability for remote sensing where necessary. A useful future approach can utilize nanoscale optoelectronics with hybrid detection methods involving both photonics and electronics.

Tuberculosis (TB) is one of the top ten leading causes of death worldwide with millions of new TB cases reported every year. Understanding the genetic diversity of Mycobacterium tuberculosis (M. tuberculosis) is very crucial for rapid diagnosis and to reduce transmission of TB. Various diagnostic techniques, anti-tuberculosis reagents and vaccination are available, however, the disease is far from being eradicated (Brudey et al., 2006). Mycobacterium tuberculosis is classified into seven major lineages that are key to the most research areas. Recently, multidrug M. tuberculosis have been reported as the most dangerous strains that cause a life-threatening TB. However, the M. tuberculosis with modified virulence and transmissibility, particularly those that are caused by mutations leading to genetic variation and increased pathogenicity are highly reported (Zaychikova et al., 2015). Genetic markers such as variable number tandem repeats, insertion sequence element and direct repeats have been used to identify lineages. However, the techniques (such as spoligotyping, IS6110-RLFP and MIRU- VNTR) that use these genetic markers have a lot of drawbacks and some have low discriminatory power (Mikheecheva et al., 2017). Recently, single nucleotide polymorphisms (SNPs) are regarded as the most promising genetic markers for genotyping M. tuberculosis because they have low-level homoplasy and high discriminatory power (Zaychikova et al., 2015). The present study proposed that genotyping M. tuberculosis using polymorphisms in virulence genes may be an alternative approach to determine lineages and may help to detect the M. tuberculosis strains that are epidemiologically dangerous and have adapted to specific geographic regions. This study aimed to identify and evaluate a set of virulence gene SNPs as markers of M. tuberculosis strains circulating in the Tshwane region. A total of 150 susceptible and resistant M. tuberculosis cultures stored in Mycobacteria growth indicator tubes (MGIT) tubes were collected from May to October 2018 at the National Health Laboratory Service, Tshwane Academic Division (NHLS/TAD) to conduct this study. The DNA was extracted using hexadecyltrimethylammonium bromide (CTAB) method and spoligotyping was done to screen for M. tuberculosis lineages. The Beijing and LAM genotypes detected by spoligotyping were sequenced using the Illumina Miseq platform. The bioinformatic analysis of virulence genes in 56 genomes of M. tuberculosis belonging to Beijing and LAM genotypes was performed to detect lineage-specific SNPs markers. Of the 150 M. tuberculosis collected, 57.3% were susceptible M. tuberculosis strains while 42.7% were drug-resistant TB. Spoligotyping of 150 isolates resulted to 86.7% previously shared type (ST) and 13.3% orphans yielding a clustering rate of 63.3%. The Beijing family was found to be the most predominant lineage by 26.7%, followed by T family (16%), LAM (13.3%), East Africa Indian (EAI) (8.7%), S (6%), Manu (4.7%), H (4.7%), CAS (4.0%) and X3 (2.7%). The number of susceptible M. tuberculosis isolates per lineages was higher than drug-resistant TB with isolates detected as Beijing contributing 17.3% of all susceptible isolates, followed by isolates classified as orphans (10%), T family (9.3%), LAM family (8%) and CAS (2.67%). The association between anti-tuberculosis drug-resistant TB and lineages was found in EAI lineage (6.7%), Manu (4%) and S family (3.3%). The family with a high number of isolates which were drug-resistant TB was the EAI1-SOM sub-lineage belonging to the EAI family. This study successfully identified 29 Beijing and 6 LAM signature SNPs that can be used to classify clinical M. tuberculosis isolates. Within these signature SNPs, fadD28 (1521 C>T), eccCb1 (1479 G>A), pks5 (6210 G>A), and ponA2 (372 G>T) were identified in the Beijing strains and fadD28 (1392 C>G) within the LAM strains that were not reported in previous studies. Furthermore, this study detected the lineage-specific SNPs: mce3B (145 T>G), eccCb1 (1556 G>T), vapC12 (95 A>G) in Beijing BO/W148 and cyp125 (1076 T>C), mce3B (44 T>C), vapC25 (221 A>C), vapB34 (140 C>A) F15/LAM4/KZN sub-lineages which have been reported to be virulent and associated with drug resistance. This study showed a high genetic diversity of M. tuberculosis strains circulating within the Tshwane region. The Beijing lineage identified in this study was found to be more predominant than the rest of the identified genotypes. This study proposed the alternative method for genotyping M. tuberculosis strains using SNPs in virulence genes of M. tuberculosis. Observations from this study also highlight the advantage of using WGS technique over other genotyping methods such as IS6110-RFLP that has more drawbacks, as most genotypic methods discriminate M. tuberculosis strains using specific genes or regions in the genome of M. tuberculosis while WGS uses the complete genome of M. tuberculosis to determine different M. tuberculosis lineage.
Dissertation (MSc)--University of Pretoria, 2020.
The thesis/Dissertation is under embargo until September 2023.
National Research Foundation (NRF)
The thesis is under embargo until September 2022.
Medical Microbiology
MSc
Unrestricted

Les traitements simultanés des antituberculeux et de thérapie antirétrovirale (ARV) chez les patients co-infectés par le VIH et la tuberculose (TB) peut être compliqué en raison de la survenue du syndrome inflammatoire de reconstitution immunitaire associé à la TB (TB-IRIS) dont le diagnostic est basé sur les manifestations cliniques. La compréhension de l’immunopathologie de TB-IRIS est cruciale pour améliorer le diagnostic et la prise en charge des patients. L'immunité innée semble de plus en plus jouer un rôle dans le TB-IRIS. Dans la présente thèse de doctorat, j'ai étudié le rôle de l'immunité innée cellulaire, notamment des cellules NKT et γδ t, ainsi que l'implication des marqueurs soluble plasmatique : IL-1Ra, sCD14 et sCD163 liés à l’activation des monocytes/macrophages dans la survenue de l’iris chez les patients co-infectés par le VIH et TB au Cambodge.Les résultats ont montré que : 1/. Le TB-IRIS est associé a une forte activation des cellules γδ T et des sous populations γδ2+ avant l’initiation des ARV, 2/. Aucun des marqueurs IL-1Ra, sCD14 et sCD163 n’était prédictif de la survenue de l’iris. L'analyse longitudinale des taux plasmatiques d’ IL-1Ra pourrait être utile pour le diagnostic de l’iris et l’évaluation de la réponse au traitement antituberculeux. En conclusion, nos résultats révèlent l’association entre une activation importante de l’immunité innée et l’émergence de TB-IRIS dans la physiopathologie. De plus, nos données apportent des nouveaux éléments de l'iris et des marqueurs pour évaluer l'efficacité du traitement antituberculeux
Simultaneous anti-tuberculosis and antiretroviral (ARY) therapy in HIV and tuberculosis (TB) co-infected patients can be complicated due to the occurrence of TB-associated immune reconstitution inflammatory syndrome (TB-IRIS). The diagnosis test of TB-IRIS is not yet available and mainly based on clinical data. A better understanding of TB-IRIS immunopathology is crucial to improve diagnostic test and patients’ clinical outcomes. Innate immunity seems increasingly play a role in TB-IRIS. In the present doctoral thesis, is studied the role of cellular innate immunity, including NKT and γδ t cells, and as well as the implication of IL-1Ra, sCD14 and sCD163 plasma soluble markers related to the activation of monocytes/macrophages in the development of iris in HIV and TB co-infected patients in Cambodia. The results have shown that 1/. TB-IRIS is associated with a strong activation of γδ t cells and γδ2+ subset before initiation of ARY, 2/. None of IL-1Ra, sCD14 and sCD163 markers was predictive of the onset of iris. Longitudinal analysis of IL-1Ra plasma level could be useful for the diagnosis of the iris occurrence and for the evaluation of response to TB-IRIS In conclusion, our results reveal the association between important activation of innate immunity and the emergence of TB-IRIS in the physiopathology. In addition, our data provides new element of TB-IRIS and markers for evaluation of TB treatment efficacy

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: The immune response against M. tuberculosis is multifactorial, involving a network of innate and adaptive immune responses. Characterization of the immune response, a clear understanding of the dynamics and interplay of different arms of the immune response and the identification of infection-stage specific biomarkers are critical to allow the development of better tools for combating tuberculosis. In an attempt to identify such biomarkers, we studied pulmonary tuberculosis patients and their contacts in Addis Ababa, Ethiopia as part of EDCTP and BMGF funded tuberculosis projects by using multiplex techniques. We analysed 45 genes using the Multiplex Ligation Dependent Probe Amplification (MLPA) technique and the expression of IL-4δ2, BLR1, MARCO, CCL-19, IL7R, Bcl2, FcyR1A, MMP9, and LTF genes discriminate TB cases from their healthy contacts. FoxP3, TGFß1 and CCL-19 discriminate latently infected from uninfected contacts. Single genes predict with an area under the Receiver Operating Characteristic (ROC) curve of 0.68 to 0.85 while a combination of genes identified up to 95% of the different groups. Similarly, the multiplex analysis of cytokines and chemokines also showed that single or combinations of plasma cytokines and chemokines discriminate between different clinical groups accurately. The median plasma level of EGF, fractalkine, IFN-y, IL-4, MCP-3 and IP-10 is significantly different (p<0.05) in active tuberculosis and non active tuberculosis infection and the median plasma levels of IFN-y, IL-4, MCP-3, MIP-1ß and IP-10 were significantly different (p<0.05) before and after treatment. We also found a significant difference (p<0.05) in plasma levels of cytokines of patients infected with the different lineages and different families of the modern lineage. The plasma level of IL-4 was significantly higher in patients infected with lineage 3 (p<0.05) as compared to lineage 4 and the CAS familyinfected patients had a higher plasma level of IL-4 (P<0.05) as compared to patients infected with H and T families but there was no difference between H and T families. We identified genes and cytokines which had been reported from other studies in different settings and we believe that these molecules are very promising biomarkers for classifying active tuberculosis, latent infection, absence of infection and treated infection. These markers may be suitable for the development of clinically useful tools but require further validation and qualification in different populations and in larger studies.
AFRIKAANSE OPSOMMING: Die immuunrespons teen M. tuberculosis is multifaktoriaal en betrek ‘n netwerk van niespesifieke and spesifieke immuunresponse. Karakterisering van die immuunrespons, ‘n duidelike insig in die dinamika en tussenspel deur die verskillende arms van die immuunrespons en die identifikasie van spesifieke biomerkers is krities belangrik om die ontwikkeling van nuwe hulpmiddels teen tuberkulose te bevorder. In ‘n poging om sulke biomerkers te identifiseer het ons pulmonale tuberkulose pasiënte en hulle kontakte in Addis Ababa, Etiopië, as deel van die EDCTP en BMGF befondste tuberkulose projekte bestudeer met multipleks tegnieke. Ons het 45 gene analiseer met ‘Multiplex Ligation Dependent Probe Amplification (MLPA)’ en gevind dat die geenuitdrukking van IL-4•2, BLR1, MARCO, CCL-19, IL7R, Bcl2, Fc•R1A, MMP9, en LTF TB pasiënte van hulle kontakte onderskei. FoxP3, TGF•1 en CCL-19 onderskei tussen latent infekteerde en ongeïnfekteerde kontakte. Enkele gene voorspel met ‘n area onder die ‘Receiver Operating Characteristic (ROC)’ kurwe van 0.68 tot 0.85 terwyl die kombinasie van gene 95% van die verskillende groepe identifiseer. Soortgelyk het multipleks analise van sitokiene en chemokiene verskillende kliniese groepe akkuraat van mekaar onderskei. Die mediane plasmavlakke van EGF, fractalkine, IFN-•, IL-4, MCP-3 en IP-10 is beduidend verskillend (p<0.05) in aktiewe tuberkulose en nie-aktiewe tuberkulose infeksie en die mediane plasmavlak van IFN-•, IL-4, MCP-3, MIP-1• en IP-10 was beduidend verskillend voor en na behandeling. Ons het ook beduidende verskille (p<0.05) in plasmavlakke van sitokiene in pasiënte gevind wat infekteer is met verskillende stamme and verskillende families van die moderne stamme. Die plasmavlak van IL-4 was beduidend hoër in pasiënte wat infekteer is met stam 3 (p<0.05) teenoor stam 4 en die CAS familie-infekteerde pasiënte het ‘n hoër plasmavlak van IL-4 (p<0.05) teenoor pasiënte met H en T familie infeksie hoewel daar geen versikke was tussen die H en T families nie. Ons het gene en sitokiene identifiseer wat deur ander werkers onder verskillende omstandighede ook beskryf is en ons glo dat hierdie molekules baie belowende biomerkers is om aktiewe tuberkulose, latent tuberkulose, die afwesigheid van infeksie en behandelde infeksie van mekaar te onderskei. Hierdie merkers mag toepaslik wees vir die ontwikkeling van bruikbare kliniese hulpmiddele maar benodig verdere validasie en kwalifikasie in verskillende populasiegroepe en in groter studies.
Bill and Melinda Gates Foundation (BMGF)
European and Developing Countries Clinical Trials Partnership (EDCTP)
African European Tuberculosis Consortium (AE TBC).

The African buffalo (Syncerus caffer) is a member of one of Africa’s most well known tourist attractions and unique grouping of mammals – the ‘big five’. Historical records indicate that during the 19th century approximately 3 million African buffaloes inhabited almost the whole of sub-Saharan Africa. Several factors such as disease, habitat fragmentation, over-hunting and drought reduced the buffalo population to approximately 400 000 by 1990. The African buffalo is host to a variety of sub-acute diseases, such as bovine tuberculosis (BTB), foot-and-mouth disease (FMD) and corridor disease (CD). Disease is an important factor which influenced African buffalo populations throughout the continent and more specifically the Kruger National Park (KNP) and is largely responsible for the fact that buffaloes are restricted to enclosed areas with strict regulations imposed on their movement. The social organization of animals influences the distribution and spread of a disease - especially in the case of the African buffalo in the KNP. The emergence of BTB in the largest conservation area in South Africa (the KNP), threatens wild and domestic animals and humans who are in close proximity to the Park. The potential economic losses associated with this disease are excessive. The results presented in this thesis provide baseline information into the genetic status of sampled African buffaloes in the KNP, genetic relatedness between sampled individuals as well as BTB associations between sampled African buffaloes in the KNP, based on a limited dataset of 181 animals. Twelve microsatellite markers were used to evaluate 181 samples which were collected from 39 locations dispersed throughout the KNP. Specific population genetic parameters revealed information based on the intra and inter - relationships at the ‘per population’ level as well as at the ‘per prevalence group’ level. Evidence indicates a medium to high level of genetic diversity, a low to medium level of inbreeding (inbreeding coefficient (Fis) for each group ranges between 0.143 and 0.147) and a relatively high level of migration for buffaloes associated with each prevalence group. Pairwise relatedness estimates were determined between individuals, to reveal their level of relatedness (unrelated, full siblings, parent-offspring or half siblings), based on Queller and Goodnight’s (1989) coefficient of relatedness. Relatedness was determined on different levels, intra and interpopulation level, BTB infected and BTB uninfected group level as well as prevalence group levels. Evaluation of data based on these different levels and between different groups, painted an overall picture of the disease condition and genetic relatedness within and between sampled BTB infected and BTB uninfected buffaloes. Evidence indicated that the greater majority of our sampled African buffaloes (BTB infected or uninfected), were genetically unrelated (in terms of sibling and parent-offspring relationships), irrespective of their disease status. M. bovis infected buffaloes sampled and used in our study are not more closely related to each other than to uninfected buffaloes in the same population or prevalence group.
Dissertation (MSc)--University of Pretoria, 2010.
Production Animal Studies
unrestricted

Tuberculosis (TB) is one of the deadliest infectious diseases of our time, with 1.4 million deaths globally, recorded in 2010 (3800 deaths a day) by the World Health Organization (WHO). Currently, South Africa ranks third on the 2011 list of 22 high-burden TB countries in the world and it was estimated that each active-TB person could potentially infect 10–15 people annually. The WHO additionally reported that in the year 2009, 87% of all TB patients worldwide were successfully treated, with a treatment success rate of 74% reported for South Africa. Despite this however, non-adherence to anti-TB treatment is still a major issue, due to it resulting in a global increased prevalence of drug resistant TB and subsequently TB treatment failure. Treatment failure is thought to be caused by a number of factors, however, it still remains largely misunderstood. One aspect of this, that isn't clearly addressed in the literature, is the underlying variation in each patient, resulting in his/her varying reaction to the drug regimen, and hence it’s varying efficacy from one patient to the next. Furthermore, little is known about the underlying variation of the host to the primary TB infection or response to the TB disease state, and how some patients have more effective mechanisms for eliminating the infection, or recovering from the disease. Considering this, a metabolomics research study using GC×GC-TOFMS was conducted, in order to identify potential metabolite markers which may be used to better characterise the underlining mechanisms associated with poor treatment outcomes (treatment failure). The first aim was to evaluate the accuracy and efficiency of the methodology used, as well as to determine the capability and accuracy of the analyst to perform these methods. In order to evaluate the GCxGC-TOFMS analytical repeatability, one QC sample was extracted and injected repeatedly (6 times) onto the GC×GC-TOFMS. Similarly, the analyst's repeatability for performing the organic acid extraction and analyses was also determined, using 10 identical QC samples, which were extracted and injected separately. CV values were subsequently calculated from the collected and processed data as a measure of this. Of all the compounds detected from the 6 QC sample repeats used for GCxGC-TOFMS repeatability, 95.59% fell below a 50% CV value, and 93,7% of all the compounds analysed for analyst repeatability had a CV < 50. Subsequently, using the above metabolomics approach, in addition to a wide variety of univariate and multivariate statistical methods, two patient outcome groups were compared. A sample group cured from TB after 6 months of treatment was compared vs a sample group where treatment failed after the 6 month period. Using urine collected from these two patient groups at various time points, the following metabolomics comparisons where made: 1) at time of diagnosis, before any anti-TB treatment was administrated, 2) during the course of treatment, in order to determine any variance in these groups due to a varying response to the anti-TB drugs, 3) over the duration of the entire 6 months treatment regimen, in order to determine if differences exist between the two groups over time. A clear natural differentiation between the cured and failed outcome groups were obtained at time of diagnosis, and a total of 39 metabolites markers were subsequently identified. These metabolites were classified according to their various origins, and included (1) those associated with the presence of M. tuberculosis bacteria, (2) those resulting from an altered host metabolism due to the TB infection, and (3) metabolites of various exogenous origins. The detailed interpretation of these metabolites suggests that a possible underlying RCD or some sort of mitochondrial dysfunction may be present in the treatment failure group, which may also be induced through an external stimulus, such as alcohol consumption. We hypothesise that this may possibly result in a far greater severity to M. tuberculosis infection in this group, subsequently causing a reduced capacity for a successful treatment outcome, also considering the critical role of the mitochondria in the metabolism of anti-TB drugs. Furthermore, 20 metabolite markers were identified when comparing the two outcome groups during the treatment phase of this metabolomics investigation. A vast majority of these 20 metabolites were also identified as markers for time 0 (time of diagnosis). Additionally, metabolites associated with anti-TB drug induced side effects, were also found to be comparatively increased in the treatment failure group, indicative of more pronounced liver damage, accompanied by metabolites characteristic of a MADD metabolite profile, due to a deficient electron transport flavoprotein, confirming previous experiments done in rats. These side effects have also previously been implicated as a major contributor of poor treatment compliance, and ultimately treatment failure. Lastly, 35 metabolite markers were identified by time dependent statistical analysis and represented those metabolites best describing the variation between the treatment outcome groups over the entire study duration (from diagnosis, to week 26). This time dependent statistical analysis identified markers, using an alternative statistical approach, and confirmed previous findings and added in a better characterisation of treatment failure. Considering the above, we successfully applied a metabolomics approach for identifying metabolites which could ultimately aid in the prediction and monitoring of treatment outcomes. This additionally led to a better understanding and or characterisation of the phenomenon known as treatment failure, as well as the underlying mechanisms related to this occurrence.
MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013

O gênero Mycobacterium spp. compreende microrganismos saprófitas e patogênicos de interesse em saúde animal e humana. A espécie M. bovis, que causa tuberculose nos animais, é excretada através do leite de bovinos infectados e tem no consumo de leite cru e seus derivados uma importante via de transmissão para o homem, causando uma doença tão grave quanto à causada pelo M. tuberculosis. Como a doença nos animais é endêmica no Brasil e o queijo minas meia cura é normalmente fabricado com leite cru e muito apreciado pelo consumidor paulistano, amostras desse produto, obtidas em feiras-livres, foram analisadas quanto à ocorrência de micobactérias. As amostras foram descontaminadas pelo método HPC 1,5%, semeadas em meio Stonebrink-Leslie (incubadas a 37ºC/90 dias) e as colônias suspeitas, submetidas à reação de PCR TB multiplex e sequenciamento nucleotídico. Em 12% das amostras (16/133) foram isoladas 26 colônias de Mycobacterium spp., tendo sido identificadas 6 espécies, todas ambientais: Mycobacterium fortuitum, M. confluentis, M. elephantis, M. novocastrense, M. sphagni e M. arupense; 7 isolados, no entanto, permaneceram sem caracterização quanto à espécie. O M. fortuitum é um patógeno oportunista importante em saúde pública, sem que haja, entretanto, evidências de transmissão alimentar; o M. novocastrense, M. arupense e M. elephantis também têm sido consideradas espécies com potencial patogênico ao ser humano. Os resultados sugerem, tal como era esperado, que a frequência e a carga inicial de M. bovis em queijo Minas meia cura sejam baixas, mas se deve considerar que a metodologia empregada, por falta de outra específica, não privilegia a detecção em cenário de baixa carga inicial do agente acompanhada por alta carga contaminante. Sugerem também a necessidade de se avaliar a importância da transmissão alimentar de micobactérias não tuberculosas, especialmente para indivíduos imunossuprimidos.
Mycobacterium genus consists of saprophytic and pathogenic microorganisms of interest in animal and human health. M. bovis specie, which causes tuberculosis in animals, is excreted through the milk of infected cattle and the consumption of raw milk and its derivatives is an important route of transmission to humans, causing a disease as serious as the one caused by M. tuberculosis. Considering that the disease in animals is endemic in Brazil and that minas meia cura cheese cure is usually made from raw milk and much appreciated by paulistano consumer, samples of the product acquired in open markets, were analyzed for the occurrence of mycobacteria. The samples were decontaminated by the method HPC 1.5%, sown in Stonebrink-Leslie medium (incubated at 37 ° C/90 da ys) and the suspected colonies, submitted to PCR TB multiplex and nucleotide sequencing reaction. In 12% of samples (16/133) were isolated 26 colonies of Mycobacterium spp. and 6 species have been identified, all of them are environmental Mycobacterium fortuitum, M. confluentis, M. elephantis, M. novocastrense, M. sphagni and M. arupense; 7 isolates, however, remained without characterization for the specie. M. fortuitum is an important opportunistic pathogen in public health, without, however, evidence of being transmitted by food; M. novocastrense, M. arupense and M. elephantis species have also been considered potentially pathogenic to humans. The results suggest that the frequency and/or contamination load of M. bovis in meia cura Minas cheese is (are) low (s). We have to consider, however, that this result may have been influenced by the absence of an analytical method capable of identifying the agent in the food matrix in which a low load of microorganism is expected, accompanied by high load of contaminants. Also suggest the need to evaluate the possible importance of foodborne non-tuberculous mycobacteria, especially for immunocompromised individuals.

Tuberculosis (TB) is an important disease worldwide. Currently, one-third of the world’s population is infected with TB, and it is a leading cause of death among people living with HIV. Immediate but also accurate diagnosis is required for disease control, yet available diagnostics cannot do both simultaneously. Therefore, designing a technique that can diagnose the disease correctly in the shortest possible time is in great demand in order to stop its spread. Diffraction-based sensing is a novel technique for measuring of biomolecular interaction that has potential for disease diagnosis. In this study, diffraction-based sensing successfully demonstrated its usefulness for diagnostics of TB using recombinant TB antigen, or by detection of interferon-γ that is produced from white blood cells when the immune system activates. The feasibility of the technology was also evaluated in terms of providing real time observation, reducing diagnostic duration, and increasing sensitivity of detection.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2015.
Surface enhanced Raman spectroscopy (SERS) has emerged as a surface sensitive vibrational technique that leads to the enhancement of the Raman scattering molecules on or close to the surface of a plasmonic nanostructure. The enhancement is found to be in orders of 104 to 1015, which allows the technique to be sensitive enough to detect a single molecule. In this study, we report on the synthesis of different sizes of gold and silver nanoparticles (AuNPs and AgNPs) and gold nanorods (AuNRs). These are functionalized or co-stabilized with different stoichiometric ratios of HS-(CH2)11-PEG-COOH and alkanethiols (Raman reporters), i.e.; HS-(CH2)11-NHCO-coumarin(C), HS-(CH2)11-triphenylimidazole (TPI), HS- (CH2)11-indole (HSI), HS-(CH2)11-hydroquinone (HQ) to form mixed monolayer protected clusters (MMPCs). The alkanethiols were chosen as Raman reporters to facilitate the selfassembled formation of monolayers on the metal surface, thus resulting in stable MMPCs. The optical properties and stability of MMPCs were obtained using ultraviolet-visible (UVvis) spectrophometry and a zeta sizer. Size and shape of the as-synthesized nanoparticles were obtained using transmission electron microscopy (TEM). The tendency of thiolcapped nanoparticles to form self-assembled ordered superlattices was observed. Their Raman activities were evaluated using Raman spectroscopy, with the enhancement factor (EF) being calculated from the intensities of symmetric stretch vibrations of C-H observed in the region of about 2900 to 3000 cm-1 in all SERS spectra. In all four different alkanethiols (Raman reporters), smaller size metal nanoparticles (14 nm for AuNPs and 16 nm AgNPs) showed higher EF compared to 30 and 40 nm metal nanoparticles. The EF was observed to increase proportionally with stoichiometric ratios of alkanethiols from 1% iv | P a g e to 50%. The prepared MMPCs with small sizes were used as a SERS probe for the detection of malaria and tuberculosis biomarkers.

Nesta dissertação pretende-se perceber e identificar as principais alterações bioquímicas que ocorrem nas doenças infeciosas, nomeadamente na COVID-19, SIDA, tuberculose, infeções com Helicobacter pylori, Tinea corporis e toxoplasmose. Nesse sentido, foi alvo de análise o reconhecimento dos possíveis marcadores bioquímicos de cada doença, sendo realçada a forma como os mesmos podem ser utilizados no diagnóstico. Adicionalmente, foram identificados os tratamentos e fármacos empregues em cada patologia, bem como apresentadas as moléculas que se encontram disponíveis no mercado. Foi realizada uma revisão bibliográfica com o intuito de análise do estado atual em termos de conhecimento nesta área, reconhecendo as lacunas existentes e percebendo de que forma a ciência está a evoluir no desenvolvimento do tema. Assim, identificaram-se as alterações e os marcadores bioquímicos mais utilizados para o diagnóstico das doenças infeciosas. A proteína C reativa, interleucina-6, a procalcitonina, a lactato desidrogenase, os eletrolíticos, a urease e as imunoglobulinas integram alguns dos mais importantes. Concluiu-se que os biomarcadores, pelas potencialidades identificadas, devem ser utilizados na prática clínica e constituir parte integrante do diagnóstico das várias patologias. Entre as potencialidades são realçadas a capacidade para identificação de riscos e propensão para a ocorrência de uma doença, a capacidade para estratificar doentes e identificar a gravidade e/ou progressão de uma determinada patologia, previsão do prognóstico e aptidão para a monitorização de um determinado tratamento.
This dissertation aims to understand and identify the main biochemical alterations that occur in infectious diseases, namely in COVID-19, AIDS, tuberculosis, infections with Helicobacter pylori, Tinea corporis and toxoplasmosis. In this sense, the recognition of possible biochemical markers of each disease was highlighted with how they can be used in diagnosis. Additionally, the treatments and drugs used in each pathology were identified, as well as the molecules that are available in the market. A literature review was carried out in order to analyze the current state of knowledge in this area, the existing gaps and understanding how science is evolving in the development of the topic. Thus, the alterations and biochemical markers most used for the diagnosis of infectious diseases were identified. C reactive protein, interleucin-6, procalcitonin, lactate dehydrogenase, electrolytes, urease and immunoglobulins integrate some of the most important ones. It was concluded that biomarkers, due to the identified potentialities, should be used in clinical practice and be an integral part of the diagnosis of various pathologies. Among the potentialities, the capacity to identify risks and propensity to a disease occurrence, the capacity to stratify patients and to identify the severity and/or progression of a certain pathology, prognostic prediction and aptitude to monitor a certain treatment are highlighted.