Title Promoter Affiliations Abstract "Implications of the population structure of the Mycobacterium tuberculosis complex in Benin for tuberculosis presentation, diagnosis and outcome" "Dissou Affolabi, Leen Rigouts, Bouke de Jong" Mycobacteriology "The vast majority (95%) of global tuberculosis (TB) occurs in limitedresource countries [4], including Benin, a west-African country. In West-Africa, up to 40% of TB is caused by Mycobacterium africanum (MAF) [7,8], a species consisting of two distinct lineages within the Mycobacterium tuberculosis complex (MTBc). Recent results suggest that the MAF prevalence is decreasing in some countries, but not others [13,14]. The one and only study on strain diversity in Benin was conducted in 2006 [5]. Overall aim: Determine the population structure of the MTBc in Benin, and implications for tuberculosis presentation, diagnosis and outcome. Method: The proposed study is a prospective observational analytical cohort study, which builds on the nationwide system of continuous surveillance of drug-resistance already established in Benin. Sputa from 1490 (retreatment and new) TB patients will be collected from the 80 TB centers and shipped to the National Reference Laboratory for further laboratory analyses. Spoligotype analysis will be used to determine the MTBc lineage. Clinical characteristics of patients, outcome of TB treatment, and performance of various diagnostic tests will be determined for each MTBc lineage. The whole genome sequences of 30 MAF isolates from Benin will be analysed." "Consequences of Mycobacterium tuberculosis genetic diversity in the context of HIV co-infection for laboratory diagnosis of tuberculosis in Africa" "Inacio Mandomando, Frank G.J. Cobbelens, Bouke de Jong" Mycobacteriology "Objectives 1. To determine the variation and risk factors for drug-resistant tuberculosis in sub-Saharan Africa. 2. To evaluate the clinical utility of a novel molecular assay in various combinations with existing methods for diagnosis of HIV-related tuberculosis. 3. To determine the clinical predictors of mycobacteremia among HIV-infected smear- negative adults in a high prevalence HIV/TB setting in relation to the utility of a lateral flow urinary lipoarabinomannan assay. 4. To evaluate the comparative performance of urinary lipoarabinomannan assays and Xpert MTB/RIF in HIV-infected individuals with suspected tuberculosis in Uganda 5. To determine the phenotypic and genotypic diversity of Mycobacterium tuberculosis isolates from blood and sputum of the same HIV-infected adult TB patient. 6. To determine whether specific resistance-conferring mutations with known differential consequences for bacterial fitness differ in their ability to cause disease in HIV-infected versus non-HIV-infected patients." "Studies on multidrug resistance tuberculosis in Rwanda: Turning off the tap" "Jean-Baptiste Mazarati, Leen Rigouts, Bouke de Jong" "HIV and Tuberculosis, Mycobacteriology" "Tuberculosis control is challenged by the ongoing spread of multidrug-resistant tuberculosis (MDR-TB). Prompt initiation of appropriate treatment not only saves lives but also rapidly stops transmission; a vital goal of the “programmatic management of MDR-TB” (PMDT). In this PhD proposal, we intend to document the impact of PMDT interventions on the diagnostic- and treatment delays, and the resulting genotypic clustering of MDR-TB over a decade in Rwanda. In addition, we aim to characterize mutations associated with rifampicin resistance that are not detected by the currently implemented methods, and measure their prevalence stratified by patients’ treatment history and HIV infection. We will conduct a “before and after study” on the package of PMDT interventions, flanked by the 2005 and 2015 tuberculosis drug resistance surveys, including phylogeographic analysis of MDR-TB strains to study genotypic clustering in time and space, and will screen for ‘occult’ rifampicin resistance associated mutations. We anticipate to characterize key ingredients in the PMDT and potential approaches to strengthen the diagnostic pathway. These findings will likely inform TB control in Rwanda and elsewhere, by identifying circulating MDR-TB clones and likely strategies that may curb their ongoing spread." "Development of molecular diagnostic capabilities for the management of multidrug-resistant tuberculosis South Kivu" "Dirk Vogelaers" "Department of Internal medicine" "There are no known data regarding the prevalence and incidence of multidrug-resistant tuberculosis in the eastern Democratic Republic of Congo, which are necessary for the development and implementation of a national program to combat the epidemic. Through this project, diagnostic skills are developed to start a monitoring program in both North and South Kivu through advanced molecular biology (PCR)." "Innovate to reduce rifampicin-resistant tuberculosis in Rwanda and beyond" "Bouke de Jong" Mycobacteriology "Rifampicineresistente tuberculose (RR-TB) treft jaarlijks wereldwijd een half miljoen nieuwe patiënten. Het nationale TB-programma van Rwanda heeft van de bestrijding ervan een prioriteit gemaakt, wat heeft geleid tot een daling van het aantal gemelde RR-TB-gevallen. Voortbouwend op een vruchtbare samenwerking stellen wij nu voor om innovatieve onderzoeken uit te voeren die informatie zullen verschaffen voor verdere evidence-based controle-inspanningen. We hebben aangetoond dat de Xpert MTB/RIF valse resistentie detecteert wanneer de bacteriële belasting laag is, wat heeft geleid tot een herzien landelijk diagnostisch stroomschema. In moleculair-epidemiologische analyses die een kwart eeuw bestrijken, hebben we een virulente stam geïdentificeerd, de ""Rwanda RR-kloon"", die fitnesscompenserende mutaties heeft, en nog steeds de meerderheid van RR-TB in Rwanda veroorzaakt. In het huidige voorstel zullen we ten eerste de moleculair-epidemiologische analyse uitbreiden om de vraag ""wie is de volgende?"" te beantwoorden. Ten tweede zullen we met geavanceerde bioinformatica-analyses de omvang van de totale bacteriële populatie testen. Ten derde zullen we de diagnostiek optimaliseren voor de snelle detectie van RR-TB, ook door fenotypische tests, en specifiek de Rwanda RR kloon, door de connectiviteitssoftware van Xpert machines te integreren met een nieuw te ontwikkelen qPCR. Ten vierde zullen we een innovatieve case finding-aanpak testen om de overdracht van RR-TB verder te onderbreken. Door de samenwerking met collega's in het gebied van de Grote Meren te versterken, zijn we van plan om samen fundamentele inzichten te verschaffen in de dynamiek van de RR-TB-transmissie en informatie te verstrekken over optimale volgende stappen naar de bestrijding van RR-TB in Rwanda en elders." "Integrated Bovine Tuberculosis Diagnostics Approach: Bio-inspired Approaches" "Jan Paeshuyse" "Animal and Human Health Engineering (A2H)" "The overarching research question:""Can we generate a proof-of-concept for improved diagnostics based on naturally prevalent biomolecules?""More specifically, we will answer the following questions in:WP2: ""Can the heavy chain only antibodies directed against BTB in Alpacas be used as an affinity isolation/detection tool in BTB diagnostics?""WP3: ""Can the cell wall binding domains of endolysins isolated from Mycobacterium phage be used as an affinity isolation/detection tool in BTB diagnostics?" "Molecular Epidemiology of tuberculosis at Human-Animal interface in the pastoral areas of Ethiopia with subsequent development of improved diagnostic approach" "Jan Paeshuyse" "Animal and Human Health Engineering (A2H)" "Against the available diagnosis and treatment advances, tuberculosis remains a relentless disease that is one of the leading causes of death from a single infectious agent worldwide. More importantly, the highest burden of tuberculosis is attributed to low- and middle-income countries such as Ethiopia, where limited resources and high population density intersect. Since zoonotic mycobacteria have  numerous host ranges that serve as reservoirs, it can complicate the fight against animal and / or human tuberculosis and have negative consequences and have significant economic problems and threaten livelihoods. Human-to-animal transmission of Mycobacterium tuberculosis complex, including M. Tuberculosis, is well documented in many parts of the world, including Ethiopia, which could have an implication in the epidemiology and control of tuberculosis in human and animal populations. However, livestock farmers and other herdsmen and slaughterhouse workers can also contract the disease via contaminated air droplets via aerosols from infected animals with pulmonary tuberculosis. This is particularly important in Ethiopia, where tuberculosis is endemic, milk pasteurisation is limited and human-animal interaction is unavoidable. Reverse transmission of Mycobacterium tuberculosis complex and other atypical mycobacteria between camel and humans in areas of intense human-animal contact is one of the driving factors for the onset and maintenance of tuberculosis in environments such as camel-herding pastoral areas of Ethiopia. By endorsing the ''Global End TB Strategy'' by 2035, Ethiopia's National End TB Strategy aimed to eliminate tuberculosis epidemics by reducing tuberculosis-related deaths by 95% and by reducing incidental tuberculosis cases by 90% between 2015 and 2035. One of the huge obligations of this programme is to improve access and equitable tuberculosis services for vulnerable and marginalised populations where tuberculosis is concentrated and where most delays occur due to socio-economic and legal barriers. Moreover, research and innovations are also the top priorities that the program has highlighted to sharply bend the country's tuberculosis epidemic curve to meet its ambitious 2035 targets. On the other hand, as part of its livestock master plan, Ethiopia planned to increase its GDP by increasing the production and export of live animals and animal products, and also to meet the growing national demand for affordable animal protein. And camel production is focused on development to meet in the pastoral areas of Ethiopia, where camels are herded primarily as a livelihood and survival strategy of the pastoral communities. However, the sector is not contributing as expected to the national economy and the livelihoods of livestock farmers due to various constraints, diseases such as tuberculosis of which are huge barriers to livestock productivity, exports, food security and public health. Tuberculosis in dromedaries threatens the well-being and livelihoods of pastoral communities, in addition to having a significant economic impact through reduced meat and milk production and creating barriers to international trade in dromedaries and their products. It would therefore be very important to assess the occurrence of Mycobacteria infection at the human-animal interface in the  pastoral communities of camel rearing in Ethiopia, describe the type species and/or strain circulating among humans and camels, and relate the transmission risks while describing the epidemiology and transmission dynamics of Mycobacteria. Such a study is also essential in devising context-appropriate tuberculosis control and prevention interventions. However, the accuracy of diagnostic tests for tuberculosis infection used in animals, mainly camels, is often elusive when diagnosing tuberculosis, and yet there is no reliable diagnostic test for accurate early ante-mortem diagnosis of tuberculosis in dromedaries, indicating the need for exploration and development of a new diagnostic tool. Against this background, this project is designed to study the molecular epidemiology of tuberculosis at the human-animal interface and to assess the bidirectional zoonotic transmission of Mycobacteria species between humans and animals in the dromedary camel-rearing pastoral communities in Ethiopia, and the subsequent development of proof-of-concept for an improved diagnostic approach to animal and human tuberculosis using camelids, nanobodies and phage technology. Key words: Tuberculosis, Mycobacterium, Camel, Pastoral, Ethiopia, Diagnosis, Nanobodies" "Early diagnosis and care of tuberculosis through community-based active case-finding using rapid diagnostic tests in urban slums of Phnom Penh, Cambodia" "Bob Colebunders, Robert Colebunders" "Mycobacteriology, HIV and Tuberculosis" "Although globally tuberculosis (TB) incidence and prevalence is declining, the emergence of drugresistant tuberculosis is threatening tuberculosis control efforts. Limited access to rapid diagnostics and health care prevent early diagnosis and treatment of TB and continue to be a pressing issue among poor and vulnerable populations. Innovative approaches such as active case-finding are strongly advocated to overcome some of the barriers but little is known about its perception and acceptability in the community. More evidence is also needed on the best model to fit different epidemiological contexts and the impact active case-finding has on tuberculosis incidence. Moreover, the roll-out of the Xpert® MTB/RIF assay, a new rapid molecular test for the diagnosis of TB and rifampicin-resistant TB, solicits reflection on the best way to target this rapid diagnostic test in patients with suspected TB. It also highlights the need for rapid confirmatory drug susceptibility tests. In 2012, we piloted a community-based active case-finding (ACF) in low socio-economic communities of Phnom Penh, Cambodia. Key features are: community involvement, door-to-door screening and sputum collection, improved microscopy techniques, rapid diagnostic tests, facilitating access to tuberculosis services, and supporting adherence through individual follow-up. We will document the acceptability, feasibility, yield and impact of this community-based ACF strategy on drug-susceptible and drug-resistant tuberculosis case-detection. In addition, we will evaluate the optimal screening strategy of sputum samples in ACF. The last part of our research focuses on evaluating the role of thin layer agar as a rapid, affordable and simple drug susceptibility test in a clinical algorithm with or without Xpert® MTB/RIF." "Molecular epidemiological approach to understand the emergence and spread of drug resistant Mycobacterium tuberculosis strains in Peru" "Jorge Arevalo, Leen Rigouts" "University of Antwerp, Mycobacteriology" "Tuberculosis (TB) is a chronic infectious disease that remains a major health problem worldwide. It is more prevalent in underdeveloped and developing countries where it kills over 2 million people per year. One third ofthe world is thought to be infected by TB bacilli, and the World Health Organization estimates that nearly 1 billion additional people will become infected by M tuberculosis between 2000 and 2020 (36). In Latin America, Peru has the highest reported incidence rate of TB (160 per 100 000 population), with 18.5% of all cases occurring in the biggest district ofthe city ofUrna (San Juan de Lurigancho) (2). With the incorporation of the Directly Observed Therapy Shortcourse (DOTS) programme, Peru has become the model for TB control in Latin America. However, this DOTS success is threatened by the recent emergence of multidrug resistant (MDR) strains; defined as TB resistant to at least isoniazid (INH) and rifampicin (RMP), the two most powerful first-line anti-TB drugs. In 2005, the Technical Unit ofMDR-TB ofthe Peruvian Ministry ofHealth notified 1950 MDR-TB cases, among which 38% were from San Juan de Lurigancho (1). Drug resistance develops in the course of treatment and is the result of patients missing doses, doctors giving inappropriate treatment or patients failing to complete a course oftreatment. In addition, there is a new form ofTB caused by bacteria called extensively drug-resistant (XDR) TB, defined as MDR-TB being simultaneously resistant to one ofthe fluoroquinolones and one of the injectable second-line drugs (36). Diagnosis of drug-resistant TB can be achieved by in vitro drug-susceptibility testing after a positive culture. For some ofthe drugs molecular tools are available detecting mutations in relevant genes, such as the rpoB gene for detection ofRMPresistant TB. Previous sequencing studies revealed that the most common relevant rpoB mutations found in Peru were Ser531 followed by His526 mutations, and Ser315 as the most prevalent katG mutation (21,27). Nowadays studies are focusing on identifying specific mutations associated with second-line drugs resistance. Sequencing analysis has revealed two mutations in the rrs gene (A1401G and C1402T) associated with injectable drugs resistance (12) and two mutations in the gyrA gene (Ala-90 and Asp-94) associated to fluroquinolone resistance (34). Smear microscopy is the easiest, fastest and cheapest technique for laboratory-confirmed diagnosis of TB, but has a low detection rate. Cultivation followed by phenotypic identification and drug resistance testing is still the ""gold standard"", but has the disadvantage of taking a long time. Besides, biochemical characterization of isolates is not always conclusive and expensive. An alternative method for detection and/or identification comprises the amplification of specific loci by polymerase chain reaction (PCR). However, PCR-based systems lack sensitivity as compared to culture, partially due to the use of inadequate DNAextraction methods (26). So there still is a need for a rapid, simple and cost-effective diagnostic tool for detection of (drug-resistant) TB. The traditional view is that M tuberculosis strains are equally virulent. However, population-based genotyping has demonstrated that a small percentage of strains cause a disproportionately large number of cases, implying that some strains are spread more effectively than others. In the last two decades, epidemiological studies on TB have been assisted by the IS611O-based Restriction Fragment Length Polymorphism (RFLP) typing method (6,9, 13,15). Although IS611O-RFLP is considered as the gold standard typing method for TB, it also presents some disadvantages such as the labor-intensive manipulations and the complexity of comparing profiles (7, 16). Another typing technique described is based on the Variable Number ofTandem Repeats (VNTR) observed among TB-specific tandem repeats named Mycobacterial Interspersed Repetitive Units (MIRUs). These techniques have been also useful in studies on reinfection versus reactivation. Earlier, recurrent TB was systematically considered as a reactivation of the initial M tuberculosis strain, but DNA-fingerprinting revealed that reinfection with a new strain is possible as well. Despite their potential for genotyping and molecular epidemiology ofTB (29,30), these techniques are expensive for poor settings. So, for some specific purposes, it is better to do a first screening through the spoligotyping method which, in spite of a less discriminative power, is useful for the description of TB families. Also human factors playa role in the pathogenesis, evolution and outcome of disease. Involvement of human genetic heterogeneity in the susceptibility to TB has been suspected for many years (3). Although cytokines exhibit a low degree of genetic variations, an increasing number of association studies have implicated polymorphisms located on promoter regions or coding regions of cytokine genes as host factors influencing susceptibility to infectious diseases (4,14). The course ofM tuberculosis infection is regulated by two distinct T cell cytokine patterns. T helper 1 (Thl) cytokines, IL-2 and IFN-y, are associated with resistance to infection, whereas Th2 cytokines, IL-4 and IL-13, are associated with progressive disease (35). In addition, IL-l 0, one of the T-regulatory cytokines, seems to playa pivotal role during the chronic/latent stage of pulmonary TB, with increased production playing a potentially central role in promoting reactivation ofTB (33). Studies on TB patients with different ethnic backgrounds have shown conflicting evidence for 1NF-a (5,24,25) and IL-I0 -1082 (8, 18) polymorphisms association with TB. No association was found for TGF-~ (20). In contrast, published reports agree that IFN-y+874 allele A, associated with low IFN-y production, is associated with pulmonary TB (17). Finally there is also evidence that supports the association between polymorphism observed in IL12B (intron-2) and TB and between specific IL12B haplotypes and TB (32)." "Development and validation of Clinical Prediction Rules that address bottlenecks in tuberculosis diagnosis" "César Cabezas Sanchez, Patrick Van der Stuyft, Patrick Van der Stuyft" "The present project aims at the evaluation of Clinical Prediction Rules for Tuberculosis as diagnostic tools for decision-making ""on the spot"" in situations in which smears are negative but there is still an important clinical suspicion of active tuberculosis and the results of the cultures can't be awaited. The three most important scenarios have been chosen: first, isloation decision for hospitalized patients, second, decision of treatment initation in moderate-severely ill patients and third, decision of treatment initation in patients with tuberculosis meningitis suspicion.These Clinical Prediction Rules, under the format of clinical scores, will be validated in this project through formal field testing. In the items of clinical isolation decision and tuberculosis meningitis treamtent initation validation will be done prospectively and in the item of moderate-severe patients it will be performed using a previously created dataset from a study on smear-negative tuberculosis diagnosis and liquid culture media. This project will be accomplished in the context of ongoing collaborative research in which ITM has been participating together with the hospital in which the applicant is working at present."