Final Report Summary - TBSUSGENT (Sustaining research momentum over the coming decades: mentoring the next generation of researchers for tuberculosis)
Executive summary:
TBSUGENT is a European Community (EC) funded Seventh Framework Programme (FP7) project whose overall aim is to sustain investigator and scientific capacity in developing countries, where tuberculosis (TB) burden is the highest, namely South Africa and India. The specific objectives are:
1. to develop and manage a clinical-scientist fellowship programme focussed on activities encompassing poverty-related disease i.e. TB
2. to facilitate career progression of developing country clinical scientists through a programme offering scientific training and a network of mentors.
This will facilitate the career progression of many developing country clinical-scientists by offering scientific training and a network of mentors.
A summary of accomplishments in the different work packages (WPs) through student training is presented here:
1. Immunology: The following achievements have been realised, namely the cloning of a novel splice variant of the Th2 cytokine IL-4 i.e. IL4 Delta 2 and 3 and a study of its impact on mycobactericidal effector mechanisms, the novel observation of very high regulatory T-cell levels in non-converting extremely drug resistant tuberculosis (XDR TB) patients and a study of the mechanisms underpinning mycobacterial stasis and the first isolation and phenotypic characterisation of regulatory T-cells in BAL fluid of Tb patients, There are two papers currently being prepared for submission to the American Journal of Respiratory and Critical Care Medicine (AJRCCM) and Nature Medicine.
2. Quantitative T cell assays: There have also been important advances relating to the optimisation and development of T-cell assays, where the consortium has shown that T-cell assays using blood are not useful for the diagnosis of active TB in a high burden setting, however, they may have rule out value when combined with smear microscopy and Gram stain in the context of TB meningitis (Patel, AJRCCM, 2010). RD-1 ELISPOT assays can accurately be performed even when processing of samples is delayed and antigen-specific T cell responses are useful for the diagnosis of TB using BAL samples and for TB meningitis, but not pleural TB and when using induced sputum. Novel antigens like HBHA have also been evaluated. Some of these data have been supplied to the World Health Organisation (WHO) to facilitate policy decisions about the use of T cell assays in high burden settings (see Lenders et al., J Infect 2010, Dheda et al., Thorax 2009; Cashmore et al., PloS One 2010; Dheda et al., Eur Respir J 2009; Patel VB et al., AJRCCM, 2010).
3. TB diagnostics and biomarkers: a technology that has been successfully evaluated is urine lipoarabinomannan (LAM) antigen detection and the relevant findings have recently been published (Peter et al, Int J Tuberc Lung Dis, 2012, Curr Op in Pulm Med, 2010; Dheda et al., PLoS ONE 2010). A large study of sputum induction was completed which showed that health care worker instruction is the preferred modality of sample acquisition, over induced sputum, in a resource poor setting. A study of LED microscopy was completed (Whitelaw and Pater, ERJ, 2010) and several papers on Gene Xpert have been published (Theron et al., PloS ONE 2012, CID 2012, CID 2012, ERJ 2012, Lancet 2011, AJRCM 2011 ).
4. Clinical trial studies on XDR-TB are ongoing, with one important output describing the clinical outcomes of patients with XDR TB, which has had major programmatic policy implications (Dheda et al., Lancet 2010). Additional manuscripts by Shean K and Pieterson E et al (students in Cape Town) are now in preparation and will be submitted shortly. The clinical trials in India (smoking and TB and vitamin D supplementation) are now already in their advanced stages and will require a second no cost extension to be completed.
Several students have also received support from TBSUGENT biostatistics and epidemiology training. This training has enabled some of these students (and other members of the TBSUGENT consortium) to generate several manuscripts in this area (see Sohn et al., Exp Rev Anti-Infect Ther 2009; Parkash et al., Scand J Immunol 2009; Fontela et al., PLoS ONE 2009; Pai et al., Clinics Chest Med 2009; Minion et al., Int J Tuberc Lung Dis 2010; Christopher et al., PLoS ONE 2010).
Several training courses and seminars (biostatistics, diagnostics, immunology and drug-resistant TB) have been conducted by TBSUGENT partners - a number of which have occurred in Africa and focussed on addressing biostatistics and technical skills shortages. Those of note were diagnostics research courses (jointly run by UCT and McGill in South Africa), which were attended by internationals delegates and several noteworthy speakers.
In summary the activities thus far have facilitated training of several postgraduate students through higher degrees, coursework, seminars and courses. In addition infrastructural building undertaken through the grant has added to research capacity in Africa and India. There have been excellent scientific outputs thus far. The work is already having societal impact as the results of the T cell assay work, LED microscopy and Xpert have all be used for WHO endorsement of new diagnostics, which will enhance uptake by National TB Programmes. The work on XDR-TB have have generated several useful findings, such as defining treatment failure duration in XDR-TB, that will benefit TB programmes. The training of promising TB research leaders of the future will have a sustained impact in the field of TB many years to come. Thus, the outputs so far have exceeded expectations and it would be a useful exercise to see where many of these trainees are and what they have achieved five years from now. It is only then that the true impact of the grant will be realised.
Project context and objectives:
The project was sub divided into eight work packages (WPs):
WP1: Recruitment of fellows and assignment of project. Objectives of this WP:
1. to recruit at least six post-graduate students/ fellows from partner sites to enrol for a course of scientific training registrable for a post-graduate qualification (PhD or MD/masters)
2. to allocate a pre-specified project to the awardee
3. to structure the awarded project between the awardees institution and the host institution and to integrate relevant seminars and workshops (see WP7).
WP 2: Immunology and molecular biology course. Objectives of this WP:
1. to perform studies on the role of regulatory pathways (Th2, TH17 and Treg) in human tuberculosis (see work description in the TA for details of each individual study; these constituted three separate projects to choose from)
2. to correlate site-specific immunological parameters with clinical and laboratory variables
3. to undertake functional studies to test the effect of the different interventions (anti-IL-4, IL-4d2, anti-IL-17, anti-IL-10 and anti-TGF-b) on mycobacterial stasis/killing and host effector functions (cytokine production, apoptosis and proliferation)
WP3: T cell assays. Objectives of this WP:
1. to evaluate the performance outcomes of T cell assays for the diagnosis of tuberculosis in high burden settings
2. to modify existing assays and address specific drawbacks so that the IGRAs are better suited for use in high burden settings.
WP4: Diagnostics and markers of disease activity. Objectives of this WP:
1. to evaluate multiple novel and emerging technologies for TB diagnosis in high burden countries
2. to determine if feasibility and performance characteristics of the tests differ by human immunodeficiency (HIV) status and to identify the most promising candidate tests for populations with high HIV prevalence
3. to validate the utility and applicability of several biomarkers for the monitoring of treatment efficacy.
WP 5: Clinical trials programme. Objectives of this WP:
The clinical trials programme will focus on evaluation of the impact of a package of smoking-cessation interventions on TB and the impact of vitamin D on TB outcomes and others about pneumonia and sampling patients with TB . Another project focussed on studies of XDR TB. The XDR studies were piggy-backed on to existing studies being conducted in Cape Town, South Africa (SA).
WP 6: Epidemiology and bio-statistics. Objectives of this WP:
1. This WP is designed to develop proficiency and experience in epidemiology and bio-statistics relevant to TB. The specific objectives that will facilitate this aim are:To train selected fellows in epidemiology and biostatistics at Research Institute of McGill University Health Centre, Montreal, Canada.
2. to facilitate the data analyses components of the research projects that will be performed by the trainees
3. to facilitate manuscript preparation, to complete the research projects.
WP 7: Workshops, courses and seminars. Objectives of this WP:
1. to organise and hold subject-specific workshops, seminars to update current knowledge for laboratory staff e.g. newer diagnostic techniques, molecular biological techniques, basic record keeping, biobanks etc.
2. to hold workshops, seminars and day-update courses for technical staff and at each partner site e.g. modern technology such as real-time polymerase chain reaction (PCR), pharmacokinetics (PK) studies, LAMP, MODS, etc.
3. to hold workshops, seminars and day-update courses for research staff and clinicians eg MDR-TB, XDR-TB, newer TB drugs and treatment regimens, etc.
4. to hold practical workshops on project management, data entry and storage and accounting. Any level of staff may attend these workshops.
5. to hold workshops and seminars for all research-active junior staff and students registered for higher degrees e.g. MSc, PhD or diplomas (thus other students, apart those specified for this project, may attend). These will include writing skills, manuscript preparation, management skills, data analysis, collaborations, new lab techniques, good clinical practice (GCP), good laboratory practice (GLP)
6. to provide one day courses on capacity building activities.
WP8: Dissemination. Objectives:
1. to facilitate and promote knowledge dissemination to partners, the scientific community, professional organisations, lay public, commercial biotechnology and bio-informatics sector and research networks
2. to facilitate knowledge exploitation.
Project results:
WP1: Recruitment of fellows and assignment of project.
A total of 25 postgraduate students (16 PhD and 9 MSc students) across the seven beneficiaries have received financial support from TBSUGENT (either in the form of direct stipends or laboratory consumables, or blocks of training at specific sites) to date. Thus, several students have been trained in overseas laboratories including Cape Town, McGill and University College London (UCL).
Good progress was made to date in term of structuring projects across more than one institution. This was done when a clear scientific need existed for the visit. Anil Pooran and Tamryn Cashmore travelled from University of Cape Town (UCT) to UCL. UCT staff and students visited McGill for epidemiological and biostatistical training and Laurence Brunet and Daphne Ling have travelled to UCT to perform data analyses. Finally, Hridesh Mishra from AIIMS visited UCT for technical training in May 2010. There has thus been excellent technical networking and knowledge sharing across sites (this has often taken advantage of seminars held at that site).
WP2: Immunology and molecular biology course.
In terms of results towards specific objectives the following has been achieved:
1. IL4 Delta two and delta three have been cloned and used in co-culture experiments. IL4 Delta two and three cannot be purchased and the cloning of this novel cytokine will allow further novel experiments to proceed.
2. Malika Davids has made the novel observation of very high levels of regulatory T-cells in patients with XDR TB who do not, compared to those who convert and showed that Treg attenuate survival by a cell contact-independent mechanism. A manuscript is in preparation.
3. Alice Maredza has for the first time isolated regulatory T-cells from the BAL fluid and characterised their phenotype. She showed that Treg attenuate mycobacterial killing and a paper has now been submitted.
4. We therefore have achieved significant results for each of the three objectives with no deviations relating to the objectives of this WP occurring. Several manuscripts are currently in preparation.
WP 3: T cell assays
Significant results for this WP have been achieved:
1. it was shown that the T-cell assays are not useful for the diagnosis of active TB in a high burden setting
2. it was found that the enzyme-linked immunosorbent spot (ELISPOT) assays can accurately be performed even when processing of samples is delayed
3. it was found that they may have rule out value when combined with smear microscopy
4. ELISPOT RD1 assays are useful for the diagnosis of TB when using BAL fluid and CSF from TB meningitis patients, but not for pleural TB or when using induced sputum
5. it was shown that T cells correlate poorly with mycobacterial load and these assays were not suitable in treatment monitoring for TB
6. by contrast, unstimulated IFN-gamma performed well for TB diagnosis in the pleural and CSF compartments.
WP4: Diagnostics and markers of disease activity.
None of these objectives in India started on time because of the delays in receiving ethical approval from the various authorities for the commencement of the project. This delay effected all the downstream components of this WP due to be completed at both AIIMS and SVIMS (such as training, patient recruitment and sample collection). Great effort was made from by Profs Sharma, Alladi and Dheda to convince the review committee of the regulatory authority of India, i.e. the Heath Ministry Screening Committee (HMSC) of Indian Council of Medical Research (ICMR) for clearance of the project however the slow approval process inevitably caused project delays. The TBSUGENT EC project officer was informed of these delays as well as the remedial action to be taken on the part of the project coordinator (communicated on 15 December 2009 by email).
As of 2009, the ethical and HSMC approvals for the project as a whole were obtained and the field trial in India is currently ongoing. We obtained a six month no cost extension to complete these trials and now in the followup phase. We have asked for a second six month no cost extension which will allow for the completion of patient follow-ups. This is crucial for the students to complete their studies.
Remedial action
To minimise the impact of these delays (in India) on the project as a whole, certain activities (in addition to the visiting Indian student) started in Cape Town so as to take WP4 forward.
1. students involved are Jonny Peters, a PhD student, Laura Lenders, a Masters student upgrading to a PhD, as well as the visiting Indian student. The first technology evaluated was urine antigen detection by Jonny Peter. This was evaluated in outpatient TB suspects and also an ongoing study in hospitalised TB patients. The relevant findings have recently been published in the journal PLoS ONE. The third technology to be evaluated is light emitting diode (LED) microscopy. This is being done by Jonny Peters and a manuscript is in preparation. LED microscopy was found to be an accurate tool for the diagnosis of TB in HIV infected patients. This is a novel finding.
2. all the technologies listed in the relevant section of Annex I have also been investigated in terms of their feasibility and characteristics in HIV positive patients. Manuscripts here are currently being prepared.
3. the relevant student here, Laura Lenders, has taken these forward and evaluated T-cell assays at time points of zero, two and six months TB treatment. The samples have been banked for further analysis and the T-cell readouts are currently being analysed and a manuscript is being prepared.
Several papers have come from this WP as is seen later in the report.
WP5: Clinical trials programme
Significant results so far in this WP are as follows:
1. the outcomes of XDR TB were described, including predictors of outcome. These findings are important for policy making in high burden countries. See Dheda K et al: Early Treatment Outcomes of Extensively Drug-Resistant Tuberculosis in South Africa are poor regardless of HIV status. Lancet (IF = 28.41)
2. the Indian clinical trials have not yet started for reasons described earlier (see WP4 for explanation).
Other papers related to this WP have been published and are listed later in the report.
WP6: Epidemiology and bio-statistics
Two TBSUGENT-supported students have completed their MSc training at McGill to-date. A visiting scholar (Molebogeng Rangaka) from the University of Cape Town (participant five) is included here and is being co-supported by TBSUGENT (supervised by Prof Pai of McGill and Prof Wilkinson of UCT).
Seven TBSUGENT-supported courses and workshops related to TB epidemiology and bio-statistics have been organised to date by Prof Pai. These have been attended by TBSUGENT-supported students as well as many external trainees. A detailed list of these courses and workshops may be found in WP8.
Three McGill epidemiology and biostatistics trainees (all supervised by Prof Pai) have also made TBSUGENT-supported international field trips as part of their projects. The details of these visits (two of which were to the University of Cape Town in South Africa) are shown below.
Lastly, several McGill trainees have received support from TBSUGENT to attend external biostatistics and TB epidemiology courses.
WP7: Workshops, courses and seminars.
TBSUGENT funds have been used (in full or in part) to organise workshops and courses to date.
WP8: Dissemination
There has been excellent progress within this WP to date.
Potential impact:
TBSUSGENT has successfully added to much needed capacity development in countries such as South Africa and India. TB is an escalating concern in these countries as it targets poverty stricken communities with low levels of education. Furthermore HIV is highly concentrated in these communities, making it easier for TB to be acquired and spread. With funding from TBSUSGENT a variety of scientific-clinical studies related to diagnosing and managing TB were made possible. Several students and researchers from these countries were funded to complete postgraduate studies. The societal implication is clearly evident in the many research publications which have had an impact in guiding government health policies to better regulate and manage TB in these countries and globally. Furthermore these studies made possible for widespread testing, treatment and education of people during clinical trials. Workshops and seminars on new diagnostic methods, drug regimens, new methods of managing and treating TB in the face of HIV and drug resistance were addressed. TBSUSGENT assisted in creating new jobs and educating not only researchers and health care workers, but admin staff involved in the data capturing, project and financial management involved in several projects. TBSUSGENT has enriched lives in South Africa and India by supporting research that has furthered education and provided jobs that have positive widespread impact on TB healthcare.
WP1: Recruitment of fellows and assignment of project
As mentioned 25 postgraduate students (16 PhD and 9 MSc students) across the seven beneficiaries have received financial support from TBSUGENT (either in the form of direct stipends or laboratory consumables) to date, most of which have completed their studies.
WP2: Immunology and molecular biology course
Molecular and immunological studies related to the immune function of regulatory T cells in TB have been taken forward using laboratories at UCL and UCT, including the use of specialised equipment such a nine-colour flow cytometer (LSR 2), category three laboratories and a Rotorgene PCR platform for the real time PCR assays.
Excellent progress has been made in this work package with three students, Anil Pooran, Malika Davids and Alice Maredza, being involved. In the first project, a PhD student, Anil Pooran, is studying the effect of TH2-like cytokines on mycobacterial immunity. The splice variant of (IL4 Delta two) has been cloned and biological samples have been banked and the mycobacterial assays have been optimised. Further experiments are progressing. Malika Davids is looking at the immuno-phenotype of patients with XDR tuberculosis. She has intriguing findings showing a very high level of regulatory T-cells in certain sub-groups of XDR TB patients. She will now proceed with further experiments to determine whether this affects mycobactericidal immunity and then dissect out relevant mechanisms. Alice Maredza, a post-doctoral student, is studying the effect of regulatory T-cells in the different body compartments and their effect mycobactericidal immunity. Excellent progress has been made. T-regs have been isolated from the broncho alveolar lavarge fluid and their suppressive phenotype determined. Co-culture experiments have progressed.
WP3: T cell assays
Progress in this area has been excellent and achievements here have exceeded expectations. The majority of the work has been conducted at US where a total of 12 different mycobacterial antigens, including MTB heparin-binding-hemagglutinin (HBHA), MTB adrenodoxin oxidoreductase (FprA) and MAP-GSD, have been cloned and purified by students for use in enzyme-linked immuno sorbent assay (ELISA) experiments. The purpose of these experiments is to characterise the humoral response of people infected with TB or m. paratuberculosis, as well as animals infected by m. bovis or m. paratuberculosis. Monoclonal antibodies were thus raised against each antigen and sensitivities tested. A very good response was seen with 10 and 20 mg/ml of monoclonal antibody against the corresponding antigen. Their ability to bind native proteins in mycobacterial lysates was next assessed. Several types of monoclonal antibodies displayed poor reactivity with their corresponding proteins in crude lysates and were therefore excluded. Of note, antibodies against the FprA and MTB-HBHA antigens performed very well in all types of lysate tested and amongst other antibodies were raised to identify TB proteins directly in the blood and / or urine of infected patients.
The cross reactivity of these antibodies was also tested and most were shown to be able to recognise the same antigen across different mycobacterial strains.
In keeping with the capacity development nature of the grant, additional training in this area of research occurred at UCT. As part of ongoing research here, 500 TB suspects have been recruited from high burden settings. Additional tests for all the major T-cell assays, including T-Spot TB, QFT-GIT and MTB-HBHA were conducted here. Two Masters students from McGill (Laurence Brunet and Daphne Ling) have come down to UCT to analyse the required data and have also imparted transferable skills to African students. Leonardo Sechi of US and Madhu Pai of McGill have provided guidance and supervision for all students in this WP.
Various antigens have been cloned and purified with a view to test their efficacy in IGRAs. Workers at UCT have begun to validate these in human samples in order to address specific drawbacks of the interferon gamma release assays. In this regard, alternative antigens were tested to evaluate the efficiency of combining different antigens. MTB-HBHA was not found to be a suitable antigen to combine with the RD1 assays to improve the T-cell assays. Supernatants from all the work have been banked for proteomic and Luminex analysis in order to find additional biomarkers to improve existing assays.
There have been several notable publications in high impact factor journals from this work:
1. Dheda K, van Zyl-Smit RN, Meldau R, Meldau S, Symons G, Khalfey H, Govender N, Rosu V, Sechi LA, Maredza A, Semple PL, Whitelaw A, Wainwright H, Badri M , Dawson R, Bateman ED, Zumla A. Quantitative lung T cell responses aid the rapid diagnosis of pulmonary tuberculosis. Thorax 2009: 64: 847-853 (IF = 6.2)
2. Dheda K, van Zyl-Smit RN, Sechi LA, Badri M, Meldau R, Meldau S, Symons G, Semple L, Maredza A, Dawson R, Wainright H, Whitelaw A, Vallie Y, Raubenheimer P, Bateman ED, Zumla A. Utility of quantitative T cell responses versus unstimulated IFN-g for the diagnosis of pleural tuberculosis. Eur Respir J 2009 34: 1118?1126 (IF = 5.9)
3. Cashmore et al., Feasibility and Diagnostic Utility of Antigen-Specific Interferon Responses for Rapid Immunodiagnosis of Tuberculosis Using Induced Sputum. PLos One, (2010) (IF = 4.4)
4. Lenders et al. Comparison Of Same Day Versus Delayed Enumeration of TB-Specific T Cell Responses, Journal of Infection (2010), doi: 10.1016/j.jinf.2010.01.012 (IF = 4.1)
5. Patel VB, Singh R, Connolly C, Coovadia Y, Peer AK, Parag P, Kasprowicz V, Zumla A, Ndung'u T, Dheda K. Cerebrospinal T Cell Responses Aid the Diagnosis of Tuberculous Meningitis in a HIV and TB Endemic Population. Am J Respir Crit Care Med. 2010 (IF= 11.8)
6. Patel VB, Singh R, Connolly C, Kasprowicz V, Ndung'u T, Dheda K. Comparative utility of cytokine levels and quantitative RD-1-specific T cell responses for rapid immunodiagnosis of tuberculous meningitis. JCM, 2011 Nov;49(11):3971-6. (IF=4.16)
7. Patel VB, Singh R, Connolly C, Kasprowicz V, Zumla A, Ndungu T, Dheda K. Comparison of a clinical prediction rule and a LAM antigen-detection assay for the rapid diagnosis of TBM in a high HIV prevalence setting. PLoS One. 2010. (IF=4.41)
8. Patel VB, Bhigjee AI, Paruk HF, Singh R, Meldau R, Connolly C, Ndung'u T, Dheda K. Utility of a novel lipoarabinomannan assay for the diagnosis of tuberculous meningitis in a resource-poor high-HIV prevalence setting. Cerebrospinal Fluid Res. 2009 (IF=1.81)
WP4: Diagnostics and markers of disease activity
Despite the late start to the trials in India due to pending HSMC approval, Indian academics such as Prof Sharma of AIIMS have nonetheless continued to provide valuable intellectual input into experiments in this WP (see PloS One paper).
As part of the consortium UM contributed to the evaluation of different technologies for the detection of tuberculosis at its African Research Center, the NIMR-Mbeya Medical Research Programme and other African Partners. Michael Hoelscher participated in a LAM study conducted in Cape Town, in terms of advice in planning and data evaluation (see PloS One paper). In cooperation with UCT, UM conducted an evaluation of the GeneXpert on 325 TB suspected cases in Mbeya Tanzania, which was shown to have a sensitivity of 88.4 % in comparison to the gold standard, which is a combination of smear microscopy, solid and liquid TB culture from up to four sputum samples. This manuscript is currently in preparation.
The following paper has been published by the TBSUGENT consortium in this WP:
1. Dheda K, Davids V, Lenders L, Roberts T, Meldau R, Ling D, Brunet L, van Zyl-Smit RN, Peter J, Green C, Badri M, Sechi L, Sharma S, Hoelscher M, Dawson R, Whitelaw, A, Blackburn J, Pai M, Zumla A. Clinical Utility of a Commercial LAM-ELISA Assay for TB Diagnosis in HIV-Infected Patients Using Urine and Sputum Samples. PLoS ONE 2010 5(3): e9848. doi:10.1371/journal.pone.0009848 (IF = 4.4)
2. Peter JG, Theron G, van Zyl-Smit R, Haripersad A, Mottay L, Kraus S, Binder A, Meldau R, Hardy A, Dheda K. Diagnostic accuracy of a urine LAM strip-test for TB detection in HIV-infected hospitalised patients. 2012 Eur Respir J.
3. Peter JG, Cashmore TJ, Meldau R, Theron G, van Zyl-Smit R, Dheda K Diagnostic accuracy of induced sputum LAM ELISA for tuberculosis diagnosis in sputum-scarce patients. Int J Tuberc Lung Dis. 2012 Aug;16(8):1108-12.
4. Theron G, Peter J, Lenders L, van Zyl-Smit R, Meldau R, Govender U, Dheda K Correlation of mycobacterium tuberculosis specific and non-specific quantitative Th1 T-cell responses with bacillary load in a high burden setting. PLoS One. 2012;7(5):e37436.
5. Theron G, Peter J, Dheda K. Characteristics of Xpert MTB/RIF-Negative Patients With Pulmonary Tuberculosis. Clin Infect Dis. 2012 Aug;55(3):472-3
6. Theron G, Pooran A, Peter J, van Zyl-Smit R, Kumar Mishra H, Meldau R, Calligaro G, Allwood B, Sharma SK, Dawson R, Dheda K Do adjunct tuberculosis tests, when combined with Xpert MTB/RIF, improve accuracy and the cost of diagnosis in a resource-poor setting? Eur Respir J. 2012 Jul;40(1):161-8.
7. Whitelaw A, Peter J, Sohn H, Viljoen D, Theron G, Badri M, Davids V, Pai M, Dheda K. Comparative cost and performance of light-emitting diode microscopy in HIV-tuberculosis-co-infected patients. Eur Respir J. 2011 Dec;38(6):1393-7.
WP5: Clinical trials programme
The clinical trials undertaken were those by the PhD student Jonny Peters and by a second PhD student, Hoosain Khalfey. Given the late start of the Indian sites, the clinical trials plan has changed slightly. The Indian sites will now carry out clinical trials on smoking and TB and vitamin D as an intervention. The XDR TB studies have, however, gone ahead according to plan in South Africa. The MSc student Malika Davids has also been involved here, where she has looked at the typing of XDR strains using flow cytometry.
A prospective outcomes study was taken forward by a Masters student, Karen Shean, who is upgrading to PhD. This manuscript is published in The Lancet and describes the clinical outcomes of patients with XDR TB. A second clinical trial being undertaken by the student Jonny Peters is a randomised control trial of sputum induction for use in primary care and high burden settings. A third clinical trial is being undertaken by the student Hoosain Khalfey evaluating the effect of steroids in pneumonia in high burden settings, given that many patients with pneumonia present with tuberculosis, the project has a distinct tuberculosis-related component and hence the student was funded by TBSUGENT.
WP6: Epidemiology and bio-statistics
Several students have received guidance and training in manuscript preparation. Several manuscripts have resulted directly from this work package (WP):
1. Sohn H, Minion J, Albert H, Dheda K, Pai M. TB diagnostic tests: how do we figure out their costs? Exp Rev Anti-Infect Ther 2009;7(6):723-33. (no IF available)
2. Parkash O, Singh BP, Pai M. Regions of Differences Encoded Antigens as Targets for Immunodiagnosis of Tuberculosis in Humans. Scand J Immunol 2009; 70, 345?357. (IF = 2.9)
3. Fontela PS, Pai NP, Schiller I, Dendukuri N, Ramsay A, Pai M. Quality and Reporting of Diagnostic Accuracy Studies in TB, HIV and Malaria: Evaluation Using QUADAS and STARD Standards. PLoS One 2009;4(11): e7753. (IF = 4.4)
4. Pai M, Minion J, Sohn H, Zwerling A, Perkins MD. Novel and Improved Technologies for Tuberculosis Diagnosis: Progress and Challenges. Clinics Chest Med 2009;30:701-16. (IF = 1.86)
5. Minion J, Pai M. Bacteriophage assays for rifampin resistance detection in Mycobacterium tuberculosis: updated meta-analysis. Int J Tuberc Lung Dis 2010 (in press). (IF = 2.24)
6. Christopher DJ, Daley P, Armstrong L, James P, Gupta R, Premkumar B, Michael JS, Radha V, Zwerling A, Schiller I, Dendukuri N, Pai M. Tuberculosis infection among young nursing trainees in South India. PLoS One 2010;5(4): e10408. (estimated IF = 4.4)
7. Shenai S, Minion J, Vadwai V, Tipnis T, Shetty S, Salvi A, Udwadia Z, Pai M, Rodrigues C. Evaluation of LED based fluorescence microscopy for the detection of mycobacteria in a TB endemic region. Int J Tuberc Lung Dis 2010;15(4):483-8.
8. Minion J, Shenai S, Vadwai V, Tipnis T, Greenaway C, Menzies D, Ramsay A, Rodrigues C, Pai M. Fading of auramine-stained mycobacterial smears and implications for external quality assurance. J Clin Micro 2011;49(5):2024-26.
9. Minion J, Pai M, Menzies D, Ramsay A, Greenaway C. Comparison of LED and conventional fluorescence microscopy for detection of acid fast bacilli in a low-incidence setting. PLoS One 2011; 6(7): e22495.
10. Christopher DJ, James P, Daley P, Armstrong L, Isaac BT, Balmugesh T, Premkumar B, Zwerling A, Pai M. High annual risk of tuberculosis infection among nursing students in South India: a cohort study. PLoS One 2011; 6(10): e26199.
11. Denkinger C, Dheda K, Pai M. Guidelines on IGRAs for tuberculosis infection: concordance, discordance or confusion? Clin Microbiol Infect 2011;17:806-814.
12. Bhargava A, Pai M, Bhargava M, Marais B, Menzies D. Can social interventions prevent tuberculosis? The Papworth experiment (1918-1943) revisited. Am J Resp Crit Care Med 2012;186:442-449.
WP7: Workshops, courses and seminars
Funds have been used for a variety of training and workshops activities. These include for example, honoraria and travel costs, course materials, catering costs and venue fees.
WP8: Dissemination
Conference abstracts supported by TBSUGENT and acknowledged in the publications are listed here:
1. Ling D, et al. Abs. ID/Title: #4733 - Incremental Value of Interferon-Gamma Release Assays for Diagnosis of Active Tuberculosis in Smear-Negative Patients in a High-Burden Setting: A Multivariable Analysis. Accepted for oral presentation at the American Thoracic Society Conference, New Orleans, 16 May 2010.
2. Brunet L, et al. Abs. ID/Title: #2621 - Tobacco Smoking and Spectrum of Tuberculosis Infection and Disease in South African Patients with Suspected Tuberculosis. Accepted for oral presentation at the American Thoracic Society Conference, New Orleans, 18 May 2010.
3. J Peter, V Davids, L Lenders, M Badri, K Dheda,T Roberts, J Blackburn, D Ling, L Brunet, M Pai, A Whitelaw A Zumla - Clinical utility of a commercial LAM-ELISA assay for TB diagnosis in HIV-infected patients using urine and sputum samples. Accepted for oral presentation at the South African Tuberculosis conference, Durban, 1 to 4 June 2010.
4. Cashmore et al. - Feasibility and diagnostic utility of antigen-specific interferon-? responses for the rapid immunodiagnosis of TB using induced sputum. Accepted for oral presentation at the 2nd Global Symposium on IGRAs in Croatia, 30 May to 1 June, 2010.
5. van Zyl-Smit et al. The colliding epidemics of HIV, smoking, TB and COPD. International Union Against TB and Lung Disease World Conference, Cancun, Mexico, December 2009
6. van Zyl-Smit et al. Smoking Infection and Mortality. International Union Against TB and Lung Disease world conference, Cancun, Mexico, December 2009
7. van Zyl-Smit et al. The effects of tobacco smoke on pulmonary immunity. Combined SATS and Critical Care Society Congress, Sun City, August 2009
8. van Zyl-Smit et al. Cigarette smoke impairs macrophage immune responses to mycobacteria University of Cape Town Department of Medicine Research October 2009
9. van Zyl-Smit et al. Serial IGRA testing and the impact of TST. 2nd International Symposium on Interferon Gamma Release Assays, Dubrovnik, May 2009
10. van Zyl-Smit et al. Within- subject variability and Boosting of T-Cell interferon-? responses after tuberculin skin testing American Thoracic Society International Congress, San Diego, May 2009
11. van Zyl-Smit et al. Tobacco smoking and spectrum of tuberculosis infection and disease in South African patients with suspected tuberculosis ATS 2010
12. Shean K et al. MDR / XDR-TB: Challenges facing HCW?s within the health systems in which they work. National Academy of Sciences (NASs) and Institute of Medicine (IOM) Forum on Drug Discovery, Development and Translation to be held on 3 to 4 March 2010(MDRTB Pretoria Workshop)
13. Shean K et al. Outcomes in HIV co-infected versus uninfected patients with extensively drug-resistant tuberculosis. 40th Union World Conference on Lung Health, 3 to 7 December 2009, Cancun, Mexico
14. Shean K et al. ?South African patients with extensively drug-resistant tuberculosis have poor outcomes regardless of HIV status.? European Respiratory Society 19th Annual conference, Vienna, Austria, 12 to 16 September 2009.
In addition to the above, project information has been published on publically accessible sections of the TBSUGENT website. Certain deliverables, meeting minutes and reports from site visits have also been disseminated to project partners via password protected portions of the website as well as email.
Importantly, the South African National TB Programme has supported TBSUGENT activities throughout the project and has continuously been kept abreast of new developments through regular meetings.
Editorials in Tropical Medicine and International Health and the South African Medical Journal have been co-authored by site PIs Profs Zumla, Dheda and Hoelscher. These are targeted at policy makers as well as the scientific community at large and have highlighted specific themes in TB diagnostics and TB treatment in Southern Africa as well as World TB Day.
1. Grange J, Mwaba P, Dheda K, Hoelscher M, Zumla A. World TB Day 2010 ? New innovations are required for enhancing the global fight against Tuberculosis: the 'Captain of all these men of death' Trop Med and International Health 2010 15(3):274-276. (IF = 2.32)
2. Mwaba P, Dheda K, Zumla A. World TB Day 2010: Eradicating tuberculosis in sub-Saharan Africa needs effective and committed north-south partnerships. South African Medical Journal 2010 100(2):102-103. (IF = 0.79)
3. Zumla A, Huggett J, Dheda K, Green C, Kapata N, Mwaba P., Trials and tribulations of an African-led research and capacity development programme: the case for EDCTP investments. Trop Med Int Health. 2010 Apr;15(4):489-94. (IF = 2.32)
In addition the TBSUGENT grant has driven the ongoing capacity development activities of the UCL-UCT Collaborative initiative (Zumla and Dheda), which is endorsed by the Deans of the respective medical schools. There is ongoing networking and capacity development activities with other EU-related grants including TESA, TB-NEAT, ADAT, REMOX etc. Several of these grants are held or co-held by Prof Zumla at UCL who has provided excellent mentorship and development through the networking of several EU and African sites including the UCL-UNZA collaboration. Finally, there has been some local press coverage in South Africa regarding the broad area of work in which TBSUGENT falls. This has been in the form of a national Sunday Times article. Regular press releases have also been put out the UCT Lung Institute website (at http://www.lunginstitute.co.za).
List of websites:
http://www.tbsugent.org
TBSUGENT is a European Community (EC) funded Seventh Framework Programme (FP7) project whose overall aim is to sustain investigator and scientific capacity in developing countries, where tuberculosis (TB) burden is the highest, namely South Africa and India. The specific objectives are:
1. to develop and manage a clinical-scientist fellowship programme focussed on activities encompassing poverty-related disease i.e. TB
2. to facilitate career progression of developing country clinical scientists through a programme offering scientific training and a network of mentors.
This will facilitate the career progression of many developing country clinical-scientists by offering scientific training and a network of mentors.
A summary of accomplishments in the different work packages (WPs) through student training is presented here:
1. Immunology: The following achievements have been realised, namely the cloning of a novel splice variant of the Th2 cytokine IL-4 i.e. IL4 Delta 2 and 3 and a study of its impact on mycobactericidal effector mechanisms, the novel observation of very high regulatory T-cell levels in non-converting extremely drug resistant tuberculosis (XDR TB) patients and a study of the mechanisms underpinning mycobacterial stasis and the first isolation and phenotypic characterisation of regulatory T-cells in BAL fluid of Tb patients, There are two papers currently being prepared for submission to the American Journal of Respiratory and Critical Care Medicine (AJRCCM) and Nature Medicine.
2. Quantitative T cell assays: There have also been important advances relating to the optimisation and development of T-cell assays, where the consortium has shown that T-cell assays using blood are not useful for the diagnosis of active TB in a high burden setting, however, they may have rule out value when combined with smear microscopy and Gram stain in the context of TB meningitis (Patel, AJRCCM, 2010). RD-1 ELISPOT assays can accurately be performed even when processing of samples is delayed and antigen-specific T cell responses are useful for the diagnosis of TB using BAL samples and for TB meningitis, but not pleural TB and when using induced sputum. Novel antigens like HBHA have also been evaluated. Some of these data have been supplied to the World Health Organisation (WHO) to facilitate policy decisions about the use of T cell assays in high burden settings (see Lenders et al., J Infect 2010, Dheda et al., Thorax 2009; Cashmore et al., PloS One 2010; Dheda et al., Eur Respir J 2009; Patel VB et al., AJRCCM, 2010).
3. TB diagnostics and biomarkers: a technology that has been successfully evaluated is urine lipoarabinomannan (LAM) antigen detection and the relevant findings have recently been published (Peter et al, Int J Tuberc Lung Dis, 2012, Curr Op in Pulm Med, 2010; Dheda et al., PLoS ONE 2010). A large study of sputum induction was completed which showed that health care worker instruction is the preferred modality of sample acquisition, over induced sputum, in a resource poor setting. A study of LED microscopy was completed (Whitelaw and Pater, ERJ, 2010) and several papers on Gene Xpert have been published (Theron et al., PloS ONE 2012, CID 2012, CID 2012, ERJ 2012, Lancet 2011, AJRCM 2011 ).
4. Clinical trial studies on XDR-TB are ongoing, with one important output describing the clinical outcomes of patients with XDR TB, which has had major programmatic policy implications (Dheda et al., Lancet 2010). Additional manuscripts by Shean K and Pieterson E et al (students in Cape Town) are now in preparation and will be submitted shortly. The clinical trials in India (smoking and TB and vitamin D supplementation) are now already in their advanced stages and will require a second no cost extension to be completed.
Several students have also received support from TBSUGENT biostatistics and epidemiology training. This training has enabled some of these students (and other members of the TBSUGENT consortium) to generate several manuscripts in this area (see Sohn et al., Exp Rev Anti-Infect Ther 2009; Parkash et al., Scand J Immunol 2009; Fontela et al., PLoS ONE 2009; Pai et al., Clinics Chest Med 2009; Minion et al., Int J Tuberc Lung Dis 2010; Christopher et al., PLoS ONE 2010).
Several training courses and seminars (biostatistics, diagnostics, immunology and drug-resistant TB) have been conducted by TBSUGENT partners - a number of which have occurred in Africa and focussed on addressing biostatistics and technical skills shortages. Those of note were diagnostics research courses (jointly run by UCT and McGill in South Africa), which were attended by internationals delegates and several noteworthy speakers.
In summary the activities thus far have facilitated training of several postgraduate students through higher degrees, coursework, seminars and courses. In addition infrastructural building undertaken through the grant has added to research capacity in Africa and India. There have been excellent scientific outputs thus far. The work is already having societal impact as the results of the T cell assay work, LED microscopy and Xpert have all be used for WHO endorsement of new diagnostics, which will enhance uptake by National TB Programmes. The work on XDR-TB have have generated several useful findings, such as defining treatment failure duration in XDR-TB, that will benefit TB programmes. The training of promising TB research leaders of the future will have a sustained impact in the field of TB many years to come. Thus, the outputs so far have exceeded expectations and it would be a useful exercise to see where many of these trainees are and what they have achieved five years from now. It is only then that the true impact of the grant will be realised.
Project context and objectives:
The project was sub divided into eight work packages (WPs):
WP1: Recruitment of fellows and assignment of project. Objectives of this WP:
1. to recruit at least six post-graduate students/ fellows from partner sites to enrol for a course of scientific training registrable for a post-graduate qualification (PhD or MD/masters)
2. to allocate a pre-specified project to the awardee
3. to structure the awarded project between the awardees institution and the host institution and to integrate relevant seminars and workshops (see WP7).
WP 2: Immunology and molecular biology course. Objectives of this WP:
1. to perform studies on the role of regulatory pathways (Th2, TH17 and Treg) in human tuberculosis (see work description in the TA for details of each individual study; these constituted three separate projects to choose from)
2. to correlate site-specific immunological parameters with clinical and laboratory variables
3. to undertake functional studies to test the effect of the different interventions (anti-IL-4, IL-4d2, anti-IL-17, anti-IL-10 and anti-TGF-b) on mycobacterial stasis/killing and host effector functions (cytokine production, apoptosis and proliferation)
WP3: T cell assays. Objectives of this WP:
1. to evaluate the performance outcomes of T cell assays for the diagnosis of tuberculosis in high burden settings
2. to modify existing assays and address specific drawbacks so that the IGRAs are better suited for use in high burden settings.
WP4: Diagnostics and markers of disease activity. Objectives of this WP:
1. to evaluate multiple novel and emerging technologies for TB diagnosis in high burden countries
2. to determine if feasibility and performance characteristics of the tests differ by human immunodeficiency (HIV) status and to identify the most promising candidate tests for populations with high HIV prevalence
3. to validate the utility and applicability of several biomarkers for the monitoring of treatment efficacy.
WP 5: Clinical trials programme. Objectives of this WP:
The clinical trials programme will focus on evaluation of the impact of a package of smoking-cessation interventions on TB and the impact of vitamin D on TB outcomes and others about pneumonia and sampling patients with TB . Another project focussed on studies of XDR TB. The XDR studies were piggy-backed on to existing studies being conducted in Cape Town, South Africa (SA).
WP 6: Epidemiology and bio-statistics. Objectives of this WP:
1. This WP is designed to develop proficiency and experience in epidemiology and bio-statistics relevant to TB. The specific objectives that will facilitate this aim are:To train selected fellows in epidemiology and biostatistics at Research Institute of McGill University Health Centre, Montreal, Canada.
2. to facilitate the data analyses components of the research projects that will be performed by the trainees
3. to facilitate manuscript preparation, to complete the research projects.
WP 7: Workshops, courses and seminars. Objectives of this WP:
1. to organise and hold subject-specific workshops, seminars to update current knowledge for laboratory staff e.g. newer diagnostic techniques, molecular biological techniques, basic record keeping, biobanks etc.
2. to hold workshops, seminars and day-update courses for technical staff and at each partner site e.g. modern technology such as real-time polymerase chain reaction (PCR), pharmacokinetics (PK) studies, LAMP, MODS, etc.
3. to hold workshops, seminars and day-update courses for research staff and clinicians eg MDR-TB, XDR-TB, newer TB drugs and treatment regimens, etc.
4. to hold practical workshops on project management, data entry and storage and accounting. Any level of staff may attend these workshops.
5. to hold workshops and seminars for all research-active junior staff and students registered for higher degrees e.g. MSc, PhD or diplomas (thus other students, apart those specified for this project, may attend). These will include writing skills, manuscript preparation, management skills, data analysis, collaborations, new lab techniques, good clinical practice (GCP), good laboratory practice (GLP)
6. to provide one day courses on capacity building activities.
WP8: Dissemination. Objectives:
1. to facilitate and promote knowledge dissemination to partners, the scientific community, professional organisations, lay public, commercial biotechnology and bio-informatics sector and research networks
2. to facilitate knowledge exploitation.
Project results:
WP1: Recruitment of fellows and assignment of project.
A total of 25 postgraduate students (16 PhD and 9 MSc students) across the seven beneficiaries have received financial support from TBSUGENT (either in the form of direct stipends or laboratory consumables, or blocks of training at specific sites) to date. Thus, several students have been trained in overseas laboratories including Cape Town, McGill and University College London (UCL).
Good progress was made to date in term of structuring projects across more than one institution. This was done when a clear scientific need existed for the visit. Anil Pooran and Tamryn Cashmore travelled from University of Cape Town (UCT) to UCL. UCT staff and students visited McGill for epidemiological and biostatistical training and Laurence Brunet and Daphne Ling have travelled to UCT to perform data analyses. Finally, Hridesh Mishra from AIIMS visited UCT for technical training in May 2010. There has thus been excellent technical networking and knowledge sharing across sites (this has often taken advantage of seminars held at that site).
WP2: Immunology and molecular biology course.
In terms of results towards specific objectives the following has been achieved:
1. IL4 Delta two and delta three have been cloned and used in co-culture experiments. IL4 Delta two and three cannot be purchased and the cloning of this novel cytokine will allow further novel experiments to proceed.
2. Malika Davids has made the novel observation of very high levels of regulatory T-cells in patients with XDR TB who do not, compared to those who convert and showed that Treg attenuate survival by a cell contact-independent mechanism. A manuscript is in preparation.
3. Alice Maredza has for the first time isolated regulatory T-cells from the BAL fluid and characterised their phenotype. She showed that Treg attenuate mycobacterial killing and a paper has now been submitted.
4. We therefore have achieved significant results for each of the three objectives with no deviations relating to the objectives of this WP occurring. Several manuscripts are currently in preparation.
WP 3: T cell assays
Significant results for this WP have been achieved:
1. it was shown that the T-cell assays are not useful for the diagnosis of active TB in a high burden setting
2. it was found that the enzyme-linked immunosorbent spot (ELISPOT) assays can accurately be performed even when processing of samples is delayed
3. it was found that they may have rule out value when combined with smear microscopy
4. ELISPOT RD1 assays are useful for the diagnosis of TB when using BAL fluid and CSF from TB meningitis patients, but not for pleural TB or when using induced sputum
5. it was shown that T cells correlate poorly with mycobacterial load and these assays were not suitable in treatment monitoring for TB
6. by contrast, unstimulated IFN-gamma performed well for TB diagnosis in the pleural and CSF compartments.
WP4: Diagnostics and markers of disease activity.
None of these objectives in India started on time because of the delays in receiving ethical approval from the various authorities for the commencement of the project. This delay effected all the downstream components of this WP due to be completed at both AIIMS and SVIMS (such as training, patient recruitment and sample collection). Great effort was made from by Profs Sharma, Alladi and Dheda to convince the review committee of the regulatory authority of India, i.e. the Heath Ministry Screening Committee (HMSC) of Indian Council of Medical Research (ICMR) for clearance of the project however the slow approval process inevitably caused project delays. The TBSUGENT EC project officer was informed of these delays as well as the remedial action to be taken on the part of the project coordinator (communicated on 15 December 2009 by email).
As of 2009, the ethical and HSMC approvals for the project as a whole were obtained and the field trial in India is currently ongoing. We obtained a six month no cost extension to complete these trials and now in the followup phase. We have asked for a second six month no cost extension which will allow for the completion of patient follow-ups. This is crucial for the students to complete their studies.
Remedial action
To minimise the impact of these delays (in India) on the project as a whole, certain activities (in addition to the visiting Indian student) started in Cape Town so as to take WP4 forward.
1. students involved are Jonny Peters, a PhD student, Laura Lenders, a Masters student upgrading to a PhD, as well as the visiting Indian student. The first technology evaluated was urine antigen detection by Jonny Peter. This was evaluated in outpatient TB suspects and also an ongoing study in hospitalised TB patients. The relevant findings have recently been published in the journal PLoS ONE. The third technology to be evaluated is light emitting diode (LED) microscopy. This is being done by Jonny Peters and a manuscript is in preparation. LED microscopy was found to be an accurate tool for the diagnosis of TB in HIV infected patients. This is a novel finding.
2. all the technologies listed in the relevant section of Annex I have also been investigated in terms of their feasibility and characteristics in HIV positive patients. Manuscripts here are currently being prepared.
3. the relevant student here, Laura Lenders, has taken these forward and evaluated T-cell assays at time points of zero, two and six months TB treatment. The samples have been banked for further analysis and the T-cell readouts are currently being analysed and a manuscript is being prepared.
Several papers have come from this WP as is seen later in the report.
WP5: Clinical trials programme
Significant results so far in this WP are as follows:
1. the outcomes of XDR TB were described, including predictors of outcome. These findings are important for policy making in high burden countries. See Dheda K et al: Early Treatment Outcomes of Extensively Drug-Resistant Tuberculosis in South Africa are poor regardless of HIV status. Lancet (IF = 28.41)
2. the Indian clinical trials have not yet started for reasons described earlier (see WP4 for explanation).
Other papers related to this WP have been published and are listed later in the report.
WP6: Epidemiology and bio-statistics
Two TBSUGENT-supported students have completed their MSc training at McGill to-date. A visiting scholar (Molebogeng Rangaka) from the University of Cape Town (participant five) is included here and is being co-supported by TBSUGENT (supervised by Prof Pai of McGill and Prof Wilkinson of UCT).
Seven TBSUGENT-supported courses and workshops related to TB epidemiology and bio-statistics have been organised to date by Prof Pai. These have been attended by TBSUGENT-supported students as well as many external trainees. A detailed list of these courses and workshops may be found in WP8.
Three McGill epidemiology and biostatistics trainees (all supervised by Prof Pai) have also made TBSUGENT-supported international field trips as part of their projects. The details of these visits (two of which were to the University of Cape Town in South Africa) are shown below.
Lastly, several McGill trainees have received support from TBSUGENT to attend external biostatistics and TB epidemiology courses.
WP7: Workshops, courses and seminars.
TBSUGENT funds have been used (in full or in part) to organise workshops and courses to date.
WP8: Dissemination
There has been excellent progress within this WP to date.
Potential impact:
TBSUSGENT has successfully added to much needed capacity development in countries such as South Africa and India. TB is an escalating concern in these countries as it targets poverty stricken communities with low levels of education. Furthermore HIV is highly concentrated in these communities, making it easier for TB to be acquired and spread. With funding from TBSUSGENT a variety of scientific-clinical studies related to diagnosing and managing TB were made possible. Several students and researchers from these countries were funded to complete postgraduate studies. The societal implication is clearly evident in the many research publications which have had an impact in guiding government health policies to better regulate and manage TB in these countries and globally. Furthermore these studies made possible for widespread testing, treatment and education of people during clinical trials. Workshops and seminars on new diagnostic methods, drug regimens, new methods of managing and treating TB in the face of HIV and drug resistance were addressed. TBSUSGENT assisted in creating new jobs and educating not only researchers and health care workers, but admin staff involved in the data capturing, project and financial management involved in several projects. TBSUSGENT has enriched lives in South Africa and India by supporting research that has furthered education and provided jobs that have positive widespread impact on TB healthcare.
WP1: Recruitment of fellows and assignment of project
As mentioned 25 postgraduate students (16 PhD and 9 MSc students) across the seven beneficiaries have received financial support from TBSUGENT (either in the form of direct stipends or laboratory consumables) to date, most of which have completed their studies.
WP2: Immunology and molecular biology course
Molecular and immunological studies related to the immune function of regulatory T cells in TB have been taken forward using laboratories at UCL and UCT, including the use of specialised equipment such a nine-colour flow cytometer (LSR 2), category three laboratories and a Rotorgene PCR platform for the real time PCR assays.
Excellent progress has been made in this work package with three students, Anil Pooran, Malika Davids and Alice Maredza, being involved. In the first project, a PhD student, Anil Pooran, is studying the effect of TH2-like cytokines on mycobacterial immunity. The splice variant of (IL4 Delta two) has been cloned and biological samples have been banked and the mycobacterial assays have been optimised. Further experiments are progressing. Malika Davids is looking at the immuno-phenotype of patients with XDR tuberculosis. She has intriguing findings showing a very high level of regulatory T-cells in certain sub-groups of XDR TB patients. She will now proceed with further experiments to determine whether this affects mycobactericidal immunity and then dissect out relevant mechanisms. Alice Maredza, a post-doctoral student, is studying the effect of regulatory T-cells in the different body compartments and their effect mycobactericidal immunity. Excellent progress has been made. T-regs have been isolated from the broncho alveolar lavarge fluid and their suppressive phenotype determined. Co-culture experiments have progressed.
WP3: T cell assays
Progress in this area has been excellent and achievements here have exceeded expectations. The majority of the work has been conducted at US where a total of 12 different mycobacterial antigens, including MTB heparin-binding-hemagglutinin (HBHA), MTB adrenodoxin oxidoreductase (FprA) and MAP-GSD, have been cloned and purified by students for use in enzyme-linked immuno sorbent assay (ELISA) experiments. The purpose of these experiments is to characterise the humoral response of people infected with TB or m. paratuberculosis, as well as animals infected by m. bovis or m. paratuberculosis. Monoclonal antibodies were thus raised against each antigen and sensitivities tested. A very good response was seen with 10 and 20 mg/ml of monoclonal antibody against the corresponding antigen. Their ability to bind native proteins in mycobacterial lysates was next assessed. Several types of monoclonal antibodies displayed poor reactivity with their corresponding proteins in crude lysates and were therefore excluded. Of note, antibodies against the FprA and MTB-HBHA antigens performed very well in all types of lysate tested and amongst other antibodies were raised to identify TB proteins directly in the blood and / or urine of infected patients.
The cross reactivity of these antibodies was also tested and most were shown to be able to recognise the same antigen across different mycobacterial strains.
In keeping with the capacity development nature of the grant, additional training in this area of research occurred at UCT. As part of ongoing research here, 500 TB suspects have been recruited from high burden settings. Additional tests for all the major T-cell assays, including T-Spot TB, QFT-GIT and MTB-HBHA were conducted here. Two Masters students from McGill (Laurence Brunet and Daphne Ling) have come down to UCT to analyse the required data and have also imparted transferable skills to African students. Leonardo Sechi of US and Madhu Pai of McGill have provided guidance and supervision for all students in this WP.
Various antigens have been cloned and purified with a view to test their efficacy in IGRAs. Workers at UCT have begun to validate these in human samples in order to address specific drawbacks of the interferon gamma release assays. In this regard, alternative antigens were tested to evaluate the efficiency of combining different antigens. MTB-HBHA was not found to be a suitable antigen to combine with the RD1 assays to improve the T-cell assays. Supernatants from all the work have been banked for proteomic and Luminex analysis in order to find additional biomarkers to improve existing assays.
There have been several notable publications in high impact factor journals from this work:
1. Dheda K, van Zyl-Smit RN, Meldau R, Meldau S, Symons G, Khalfey H, Govender N, Rosu V, Sechi LA, Maredza A, Semple PL, Whitelaw A, Wainwright H, Badri M , Dawson R, Bateman ED, Zumla A. Quantitative lung T cell responses aid the rapid diagnosis of pulmonary tuberculosis. Thorax 2009: 64: 847-853 (IF = 6.2)
2. Dheda K, van Zyl-Smit RN, Sechi LA, Badri M, Meldau R, Meldau S, Symons G, Semple L, Maredza A, Dawson R, Wainright H, Whitelaw A, Vallie Y, Raubenheimer P, Bateman ED, Zumla A. Utility of quantitative T cell responses versus unstimulated IFN-g for the diagnosis of pleural tuberculosis. Eur Respir J 2009 34: 1118?1126 (IF = 5.9)
3. Cashmore et al., Feasibility and Diagnostic Utility of Antigen-Specific Interferon Responses for Rapid Immunodiagnosis of Tuberculosis Using Induced Sputum. PLos One, (2010) (IF = 4.4)
4. Lenders et al. Comparison Of Same Day Versus Delayed Enumeration of TB-Specific T Cell Responses, Journal of Infection (2010), doi: 10.1016/j.jinf.2010.01.012 (IF = 4.1)
5. Patel VB, Singh R, Connolly C, Coovadia Y, Peer AK, Parag P, Kasprowicz V, Zumla A, Ndung'u T, Dheda K. Cerebrospinal T Cell Responses Aid the Diagnosis of Tuberculous Meningitis in a HIV and TB Endemic Population. Am J Respir Crit Care Med. 2010 (IF= 11.8)
6. Patel VB, Singh R, Connolly C, Kasprowicz V, Ndung'u T, Dheda K. Comparative utility of cytokine levels and quantitative RD-1-specific T cell responses for rapid immunodiagnosis of tuberculous meningitis. JCM, 2011 Nov;49(11):3971-6. (IF=4.16)
7. Patel VB, Singh R, Connolly C, Kasprowicz V, Zumla A, Ndungu T, Dheda K. Comparison of a clinical prediction rule and a LAM antigen-detection assay for the rapid diagnosis of TBM in a high HIV prevalence setting. PLoS One. 2010. (IF=4.41)
8. Patel VB, Bhigjee AI, Paruk HF, Singh R, Meldau R, Connolly C, Ndung'u T, Dheda K. Utility of a novel lipoarabinomannan assay for the diagnosis of tuberculous meningitis in a resource-poor high-HIV prevalence setting. Cerebrospinal Fluid Res. 2009 (IF=1.81)
WP4: Diagnostics and markers of disease activity
Despite the late start to the trials in India due to pending HSMC approval, Indian academics such as Prof Sharma of AIIMS have nonetheless continued to provide valuable intellectual input into experiments in this WP (see PloS One paper).
As part of the consortium UM contributed to the evaluation of different technologies for the detection of tuberculosis at its African Research Center, the NIMR-Mbeya Medical Research Programme and other African Partners. Michael Hoelscher participated in a LAM study conducted in Cape Town, in terms of advice in planning and data evaluation (see PloS One paper). In cooperation with UCT, UM conducted an evaluation of the GeneXpert on 325 TB suspected cases in Mbeya Tanzania, which was shown to have a sensitivity of 88.4 % in comparison to the gold standard, which is a combination of smear microscopy, solid and liquid TB culture from up to four sputum samples. This manuscript is currently in preparation.
The following paper has been published by the TBSUGENT consortium in this WP:
1. Dheda K, Davids V, Lenders L, Roberts T, Meldau R, Ling D, Brunet L, van Zyl-Smit RN, Peter J, Green C, Badri M, Sechi L, Sharma S, Hoelscher M, Dawson R, Whitelaw, A, Blackburn J, Pai M, Zumla A. Clinical Utility of a Commercial LAM-ELISA Assay for TB Diagnosis in HIV-Infected Patients Using Urine and Sputum Samples. PLoS ONE 2010 5(3): e9848. doi:10.1371/journal.pone.0009848 (IF = 4.4)
2. Peter JG, Theron G, van Zyl-Smit R, Haripersad A, Mottay L, Kraus S, Binder A, Meldau R, Hardy A, Dheda K. Diagnostic accuracy of a urine LAM strip-test for TB detection in HIV-infected hospitalised patients. 2012 Eur Respir J.
3. Peter JG, Cashmore TJ, Meldau R, Theron G, van Zyl-Smit R, Dheda K Diagnostic accuracy of induced sputum LAM ELISA for tuberculosis diagnosis in sputum-scarce patients. Int J Tuberc Lung Dis. 2012 Aug;16(8):1108-12.
4. Theron G, Peter J, Lenders L, van Zyl-Smit R, Meldau R, Govender U, Dheda K Correlation of mycobacterium tuberculosis specific and non-specific quantitative Th1 T-cell responses with bacillary load in a high burden setting. PLoS One. 2012;7(5):e37436.
5. Theron G, Peter J, Dheda K. Characteristics of Xpert MTB/RIF-Negative Patients With Pulmonary Tuberculosis. Clin Infect Dis. 2012 Aug;55(3):472-3
6. Theron G, Pooran A, Peter J, van Zyl-Smit R, Kumar Mishra H, Meldau R, Calligaro G, Allwood B, Sharma SK, Dawson R, Dheda K Do adjunct tuberculosis tests, when combined with Xpert MTB/RIF, improve accuracy and the cost of diagnosis in a resource-poor setting? Eur Respir J. 2012 Jul;40(1):161-8.
7. Whitelaw A, Peter J, Sohn H, Viljoen D, Theron G, Badri M, Davids V, Pai M, Dheda K. Comparative cost and performance of light-emitting diode microscopy in HIV-tuberculosis-co-infected patients. Eur Respir J. 2011 Dec;38(6):1393-7.
WP5: Clinical trials programme
The clinical trials undertaken were those by the PhD student Jonny Peters and by a second PhD student, Hoosain Khalfey. Given the late start of the Indian sites, the clinical trials plan has changed slightly. The Indian sites will now carry out clinical trials on smoking and TB and vitamin D as an intervention. The XDR TB studies have, however, gone ahead according to plan in South Africa. The MSc student Malika Davids has also been involved here, where she has looked at the typing of XDR strains using flow cytometry.
A prospective outcomes study was taken forward by a Masters student, Karen Shean, who is upgrading to PhD. This manuscript is published in The Lancet and describes the clinical outcomes of patients with XDR TB. A second clinical trial being undertaken by the student Jonny Peters is a randomised control trial of sputum induction for use in primary care and high burden settings. A third clinical trial is being undertaken by the student Hoosain Khalfey evaluating the effect of steroids in pneumonia in high burden settings, given that many patients with pneumonia present with tuberculosis, the project has a distinct tuberculosis-related component and hence the student was funded by TBSUGENT.
WP6: Epidemiology and bio-statistics
Several students have received guidance and training in manuscript preparation. Several manuscripts have resulted directly from this work package (WP):
1. Sohn H, Minion J, Albert H, Dheda K, Pai M. TB diagnostic tests: how do we figure out their costs? Exp Rev Anti-Infect Ther 2009;7(6):723-33. (no IF available)
2. Parkash O, Singh BP, Pai M. Regions of Differences Encoded Antigens as Targets for Immunodiagnosis of Tuberculosis in Humans. Scand J Immunol 2009; 70, 345?357. (IF = 2.9)
3. Fontela PS, Pai NP, Schiller I, Dendukuri N, Ramsay A, Pai M. Quality and Reporting of Diagnostic Accuracy Studies in TB, HIV and Malaria: Evaluation Using QUADAS and STARD Standards. PLoS One 2009;4(11): e7753. (IF = 4.4)
4. Pai M, Minion J, Sohn H, Zwerling A, Perkins MD. Novel and Improved Technologies for Tuberculosis Diagnosis: Progress and Challenges. Clinics Chest Med 2009;30:701-16. (IF = 1.86)
5. Minion J, Pai M. Bacteriophage assays for rifampin resistance detection in Mycobacterium tuberculosis: updated meta-analysis. Int J Tuberc Lung Dis 2010 (in press). (IF = 2.24)
6. Christopher DJ, Daley P, Armstrong L, James P, Gupta R, Premkumar B, Michael JS, Radha V, Zwerling A, Schiller I, Dendukuri N, Pai M. Tuberculosis infection among young nursing trainees in South India. PLoS One 2010;5(4): e10408. (estimated IF = 4.4)
7. Shenai S, Minion J, Vadwai V, Tipnis T, Shetty S, Salvi A, Udwadia Z, Pai M, Rodrigues C. Evaluation of LED based fluorescence microscopy for the detection of mycobacteria in a TB endemic region. Int J Tuberc Lung Dis 2010;15(4):483-8.
8. Minion J, Shenai S, Vadwai V, Tipnis T, Greenaway C, Menzies D, Ramsay A, Rodrigues C, Pai M. Fading of auramine-stained mycobacterial smears and implications for external quality assurance. J Clin Micro 2011;49(5):2024-26.
9. Minion J, Pai M, Menzies D, Ramsay A, Greenaway C. Comparison of LED and conventional fluorescence microscopy for detection of acid fast bacilli in a low-incidence setting. PLoS One 2011; 6(7): e22495.
10. Christopher DJ, James P, Daley P, Armstrong L, Isaac BT, Balmugesh T, Premkumar B, Zwerling A, Pai M. High annual risk of tuberculosis infection among nursing students in South India: a cohort study. PLoS One 2011; 6(10): e26199.
11. Denkinger C, Dheda K, Pai M. Guidelines on IGRAs for tuberculosis infection: concordance, discordance or confusion? Clin Microbiol Infect 2011;17:806-814.
12. Bhargava A, Pai M, Bhargava M, Marais B, Menzies D. Can social interventions prevent tuberculosis? The Papworth experiment (1918-1943) revisited. Am J Resp Crit Care Med 2012;186:442-449.
WP7: Workshops, courses and seminars
Funds have been used for a variety of training and workshops activities. These include for example, honoraria and travel costs, course materials, catering costs and venue fees.
WP8: Dissemination
Conference abstracts supported by TBSUGENT and acknowledged in the publications are listed here:
1. Ling D, et al. Abs. ID/Title: #4733 - Incremental Value of Interferon-Gamma Release Assays for Diagnosis of Active Tuberculosis in Smear-Negative Patients in a High-Burden Setting: A Multivariable Analysis. Accepted for oral presentation at the American Thoracic Society Conference, New Orleans, 16 May 2010.
2. Brunet L, et al. Abs. ID/Title: #2621 - Tobacco Smoking and Spectrum of Tuberculosis Infection and Disease in South African Patients with Suspected Tuberculosis. Accepted for oral presentation at the American Thoracic Society Conference, New Orleans, 18 May 2010.
3. J Peter, V Davids, L Lenders, M Badri, K Dheda,T Roberts, J Blackburn, D Ling, L Brunet, M Pai, A Whitelaw A Zumla - Clinical utility of a commercial LAM-ELISA assay for TB diagnosis in HIV-infected patients using urine and sputum samples. Accepted for oral presentation at the South African Tuberculosis conference, Durban, 1 to 4 June 2010.
4. Cashmore et al. - Feasibility and diagnostic utility of antigen-specific interferon-? responses for the rapid immunodiagnosis of TB using induced sputum. Accepted for oral presentation at the 2nd Global Symposium on IGRAs in Croatia, 30 May to 1 June, 2010.
5. van Zyl-Smit et al. The colliding epidemics of HIV, smoking, TB and COPD. International Union Against TB and Lung Disease World Conference, Cancun, Mexico, December 2009
6. van Zyl-Smit et al. Smoking Infection and Mortality. International Union Against TB and Lung Disease world conference, Cancun, Mexico, December 2009
7. van Zyl-Smit et al. The effects of tobacco smoke on pulmonary immunity. Combined SATS and Critical Care Society Congress, Sun City, August 2009
8. van Zyl-Smit et al. Cigarette smoke impairs macrophage immune responses to mycobacteria University of Cape Town Department of Medicine Research October 2009
9. van Zyl-Smit et al. Serial IGRA testing and the impact of TST. 2nd International Symposium on Interferon Gamma Release Assays, Dubrovnik, May 2009
10. van Zyl-Smit et al. Within- subject variability and Boosting of T-Cell interferon-? responses after tuberculin skin testing American Thoracic Society International Congress, San Diego, May 2009
11. van Zyl-Smit et al. Tobacco smoking and spectrum of tuberculosis infection and disease in South African patients with suspected tuberculosis ATS 2010
12. Shean K et al. MDR / XDR-TB: Challenges facing HCW?s within the health systems in which they work. National Academy of Sciences (NASs) and Institute of Medicine (IOM) Forum on Drug Discovery, Development and Translation to be held on 3 to 4 March 2010(MDRTB Pretoria Workshop)
13. Shean K et al. Outcomes in HIV co-infected versus uninfected patients with extensively drug-resistant tuberculosis. 40th Union World Conference on Lung Health, 3 to 7 December 2009, Cancun, Mexico
14. Shean K et al. ?South African patients with extensively drug-resistant tuberculosis have poor outcomes regardless of HIV status.? European Respiratory Society 19th Annual conference, Vienna, Austria, 12 to 16 September 2009.
In addition to the above, project information has been published on publically accessible sections of the TBSUGENT website. Certain deliverables, meeting minutes and reports from site visits have also been disseminated to project partners via password protected portions of the website as well as email.
Importantly, the South African National TB Programme has supported TBSUGENT activities throughout the project and has continuously been kept abreast of new developments through regular meetings.
Editorials in Tropical Medicine and International Health and the South African Medical Journal have been co-authored by site PIs Profs Zumla, Dheda and Hoelscher. These are targeted at policy makers as well as the scientific community at large and have highlighted specific themes in TB diagnostics and TB treatment in Southern Africa as well as World TB Day.
1. Grange J, Mwaba P, Dheda K, Hoelscher M, Zumla A. World TB Day 2010 ? New innovations are required for enhancing the global fight against Tuberculosis: the 'Captain of all these men of death' Trop Med and International Health 2010 15(3):274-276. (IF = 2.32)
2. Mwaba P, Dheda K, Zumla A. World TB Day 2010: Eradicating tuberculosis in sub-Saharan Africa needs effective and committed north-south partnerships. South African Medical Journal 2010 100(2):102-103. (IF = 0.79)
3. Zumla A, Huggett J, Dheda K, Green C, Kapata N, Mwaba P., Trials and tribulations of an African-led research and capacity development programme: the case for EDCTP investments. Trop Med Int Health. 2010 Apr;15(4):489-94. (IF = 2.32)
In addition the TBSUGENT grant has driven the ongoing capacity development activities of the UCL-UCT Collaborative initiative (Zumla and Dheda), which is endorsed by the Deans of the respective medical schools. There is ongoing networking and capacity development activities with other EU-related grants including TESA, TB-NEAT, ADAT, REMOX etc. Several of these grants are held or co-held by Prof Zumla at UCL who has provided excellent mentorship and development through the networking of several EU and African sites including the UCL-UNZA collaboration. Finally, there has been some local press coverage in South Africa regarding the broad area of work in which TBSUGENT falls. This has been in the form of a national Sunday Times article. Regular press releases have also been put out the UCT Lung Institute website (at http://www.lunginstitute.co.za).
List of websites:
http://www.tbsugent.org