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TB NEAT Study B, Protocol Date 15 Dec 2010 Version: 1.0

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TB-NEAT Study B: Specificity of GeneXpert MTB/RIF® in culture-negative TB suspects Investigators of Study Site at NIMR-MMRP P.O. Box 2410 Hospital Hill Road Mbeya-Tanzania Programme Directors: Prof. Michael Hoelscher, MD Dr. Leonard Maboko, PhD NIMR - Mbeya Medical Research Programme (MMRP) P.O. Box 2410, Mbeya, Tanzania, Phone +255-25-2503364 Fax +255-25-250 3134 [email protected] [email protected]

Role: Prof. Hoelscher is the scientific Director of the NIMR – Mbeya Medical Research Programme. He is responsible for the overall coordination of study activities at the site. He is participating in study design, analysis and publication. Dr. Leonard Maboko is Director of the NIMR – Mbeya Medical Research Programme. He is responsible for the overall coordination of study activities at the site.

Principal Investigator (PI): Dr. Chacha Mangu NIMR - Mbeya Research Programme P.O. Box 419, Mbeya, Tanzania Phone +255-25-2502892 Fax +255-25-2503577 Email: [email protected]

Role: Dr. Mangu is an investigator in various TB research activities at NIMR-MMRP. He is also responsible for recruitment and patient contacts and treatment of patients during the study.

Co-Principal Investigator (Co-PI): Dr. Andrea Rachow NIMR - Mbeya Medical Research Programme P.O. Box 419, Mbeya, Tanzania Phone +255-25-2502892 Fax +255-25-2503577 [email protected] Co-Principal Investigator (Co-PI): Dr. Denis Ngatemelela NIMR - Mbeya Research Programme P.O. Box 419, Mbeya, Tanzania

Role: Andrea Rachow is leading the TB research activities at NIMR – MMRP She is responsible for the oversight and conduct of the study and is giving medical advice to the research team. She is participating in study design, protocol writing, analysis and publication. Role: Dr. Ngatemelela is an investigator in various TB research activities at NIMR-MMRP. He is also responsible for


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Phone +255-25-2502892 Fax +255-25-2503577 Email: [email protected]

recruitment and patient contacts and treatment of patients during the study

Co-Principal Investigator (Co-PI): Dr. Norbert Heinrich Department for Infectious Diseases & Tropical Medicine, Klinikum der LMU Leopoldstrasse 5, 80802 Munich, Germany Phone +49-89-218017601 Fax +49-89-338059 [email protected]

Role: Dr. Heinrich is coordinating TB diagnostic activities in the Department for Infectious Diseases and Tropical Medicine. He supports the investigators on site through inputs into study conduct, publication and gives logistical support. He is also participating in study-design and protocol writing.

Investigator: Gabriel Rojas-Ponce NIMR - Mbeya Research Programme P.O. Box 419, Mbeya, Tanzania Phone +255-25-2502892 Fax +255-25-2503577 Email: [email protected]

Role: Mr. Rojas-Ponce is the manager of the TB-laboratory at NIMR-MMRP. He will support and advice the other investigators in the study-activities and paper writing.

Investigator: Dr. Christof Geldmacher Department for Infectious Diseases & Tropical Medicine, Klinikum der LMU Leopoldstrasse 5, 80802 Munich, Germany Phone +49-89-218017601 Fax +49-89-338059 [email protected]

Role: Dr. Geldmacher works as a senior Immunologist and supervises the Immunology research activities conducted at MMRP. Dr. Geldmacher has conceived the TAM-IGRA assay and will participate in study design, analysis and publication.

Collaborator: Prof. Dr. Christian G. Meyer Bernhard Nocht Institute for Tropical Medicine Dept. Molecular Medicine Bernhard Nocht Str. 74 20359 Hamburg Collaborator: Prof. Dr. Christoph Lange Forschungszentrum Borstel Medizinische Klinik Borstel Fachkrankenhaus für Lungenerkrankungen, Infektionen und Allergien Parkallee 3523845 Borstel [email protected]

Role: Prof. Meyer will perform analysis of host genetic factors associated with TB disease. Role: Professor Lange will support the study site by training the site staff on bronchoscopy and on the processing of samples received from bronchoalveolar- lavage for further microbiological and immunological investigations


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1 Study Synopsis Protocol Title: Specificity of GeneXpert MTB/RIF® in culture-negative TB

suspects Primary Objective: To determine specificity of GeneXpert MTB/RIF® by analysing Xpert

positive, smear and culture negative TB suspects. Secondary Objectives: - To determine the contribution to disease by non-tuberculous

mycobacteria (NTM), and to describe new species of NTM found, - To determine Xpert performance in specimens other than sputum, - To evaluate blood culture for diagnosis of TB in the study

population, - To evaluate whether phenotypic and functional changes on MTB-

specific T cells can be used as a diagnostic marker for active TB including (TAM-IGRA assay)

- To generate information on infecting organisms by strain typing and their appearance in association with different levels of immunological suppression.

- To study genetic factors which play a role in susceptibility and resistance to TB

- To establish whether co-infection with other respiratory pathogens is a relevant factor in the disease of TB,

- To establish and evaluate an X-ray scoring system in Mbeya - To evaluate a lipoarabinomannan urine lateral flow test as a novel

diagnostic for TB

Study Design: A prospective analysis of TB suspects with a positive Xpert result, but failure to detect TB in initial gold standard sputum examinations.

Patient Population: Patients of at least 18 years of age, who have received a positive Xpert result in screening and have an initial negative smear, will be invited for study participation.

Study Duration: Overall duration of the study is anticipated to be 1,5 years. Anticipated study start (first patient in): 1Q 2011 Anticipated study completion (last patient out): 3Q 2013 Anticipated database lock: 3Q 2013

Primary Endpoint: Establishment of a definite, or at least a probable diagnosis on the target population, preferably by microbiological proof of TB, or proof of other diagnosis.


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Secondary Endpoints: • Exclusion or confirmation of the diagnosis of infection with non-tuberculous mycobacteria

(NTM) in patients with NTM growth in cultures. • Description of new species of NTM found in cultures. • Evaluation of performance of Xpert in specimens other than sputum. • Evaluation of blood culture in diagnosis of extra-pulmonary TB. • Evaluation of performance of TAM-IGRA in comparison with other diagnostic methods • Strain typing of infecting MTB and evaluation of correlation between infecting strain and

immunological status of host. • Evaluation of genetic host products which determine susceptibility or resistance to TB. • Establishment of co-infection with other respiratory pathogens by nucleic acid amplification

techniques and culture. • Establishment of a chest X-ray scoring system in Mbeya. • Determination of sensitivity and specificity of a urine lateral flow test to detect TB


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2 Abbreviations AFB Acid-Fast Bacilli AIDS Acquired Immune Deficiency Syndrome ATS American Thoracic Society CXR Chest x-ray DNA Desoxyribonucleic Acid DOT Directly Observed Treatment DTLC District TB and Leprosy Coordinator EIA Enzyme Immunoassay ELISA Enzyme-Linked Immunosorbent Assay EDCTP European & Developing Countries Clinical Trials Partnership FNAB Fine Needle Aspiration Biopsy GCP Good Clinical Practice GLP Good Laboratory Practice HIV Human Immunodeficiency Virus IGRA Interferon-γ Release Assay LAM Lipoarabinomannan LJ Loewenstein Jensen MGIT Mycobacteria Growth Indicator Tubes MMRP NIMR-Mbeya Medical Research Programme Mtb Mycobacterium tuberculosis complex NTLP National TB and Leprosy Programme NAAT Nucleid Acid Amplification Test PCR Polymerase Chain Reaction PTB Pulmonary Tuberculosis SOP Standard Operating Procedures TAM T-Cell Activation Markers TB Tuberculosis WHO World Health Organization Xpert GeneXpert MTB/RIF®


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3 Table of Contents 1 Study Synopsis .........................................................................................................4 2 Abbreviations ............................................................................................................6 3 Table of Contents......................................................................................................7 4 Background ...............................................................................................................9 5 Study Hypothesis ....................................................................................................10 6 Study Design...........................................................................................................10 7 Study Significance...................................................................................................11 8 Objectives ...............................................................................................................11

8.1 Primary Objective .............................................................................................11 8.2 Secondary Objectives.......................................................................................11

9 Study Endpoints ......................................................................................................12 9.1 Primary endpoint...............................................................................................12 9.2 Secondary endpoints ........................................................................................12

10 Methods ..................................................................................................................12 10.1 Study Site and Population.............................................................................12

10.1.1 Mbeya........................................................................................................12 NIMR-Mbeya Medical Research Programme TB- clinic is situated on the premises of Mbeya Referral Hospital (MRH) and the Regional Hospital. ...................................12

10.2 Inclusion criteria ............................................................................................13 10.3 Exclusion criteria ...........................................................................................13 10.4 Study Workflow .............................................................................................14

11 Study Procedures....................................................................................................21 11.1 Informed Consent..........................................................................................21 11.2 Clinical Interventions .....................................................................................21

11.2.1 Blood collection .........................................................................................21 11.2.2 Puncture/Fine Needle Aspiration/Biopsy ...................................................21 11.2.3 ECG...........................................................................................................22 11.2.4 Bronchoscopy............................................................................................22

11.3 Laboratory methods ......................................................................................22 11.3.1 Xpert® MTB/RIF Assay .............................................................................22 11.3.2 Sputum smear in study TB-laboratory .......................................................23 11.3.3 Loewenstein Jensen culture ......................................................................23 11.3.4 BACTEC® 960 / MGIT liquid culture .........................................................23 11.3.5 Blood culture for mycobacteria ..................................................................24 11.3.6 NAAT to identify NTM and/or other infectious agents ...............................24 11.3.7 Sputum collection and processing for RNA ...............................................24 11.3.8 Measurement of Mtb RNA extracted from sputum ..................................24 11.3.9 Speciation of mycobacteria by GenoType® Mycobacterium AS/CM/MTBC24 11.3.10 Sequencing and classification of mycobacteria (conducted at German TB reference centre in Borstel) .....................................................................................24 11.3.11 Sputum collection and processing for DNA with new PCR-based methods (e.g. Hain MTBDRplus and LAMP) other than Xpert:..............................................25 11.3.12 Blood collection for Serum-Marker.........................................................25 11.3.13 Analysis of genetic host factors relevant for resistance and susceptibility to TB (performed at Bernhard Nocht Institute, Hamburg, Germany)...........................26 11.3.14 QuantiFERON-TB Gold in-tube .............................................................26 11.3.15 TAM IGRA..............................................................................................26


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11.3.16 HIV testing .............................................................................................26 11.3.17 Stool DNA/RNA extraction and analysis ................................................26 11.3.18 Urine LAM lateral flow strip test (Determine TB®) .................................27 11.3.19 Sample storage and shipment ...............................................................27

8. Sample size calculations.............................................................................................27 12 9. Ethical Considerations ........................................................................................27

12.1 Ethical approval.............................................................................................27 12.2 Confidentiality................................................................................................27 12.3 Risks and Benefits from Study Participation .................................................28

13 Compensation to Patients .......................................................................................28 14 Data management and analysis..............................................................................28 15 Study Phases and timelines....................................................................................29 16 Study Budget...........................................................................................................30 17 Literature .................................................................................................................30 Figures and Tables Figure 1: Study outline. ...................................................................................................15 Table 1: Schedule of diagnostic sampling.......................................................................20 Table 2: Classification of patients according to clinical and laboratory results ...............29


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4 Background Tuberculosis (TB) is one of the world’s most important causes of mortality due to an infectious disease and continues to kill 1.8 million people annually.1 Despite several interventions including DOTS, global TB- incidence and –prevalence could not be reduced significantly. Furthermore, though several new diagnostic tools for TB-detection are on the market case detection rates are still low, especially in developing countries where the implementation of new tests was hampered by cost-, equipment- and training requirements. It is estimated that approximately 50% of patients with TB are not diagnosed and treated appropriately. The problem is compounded by the increasing prevalence of multi drug resistant (MDR) and extensively drug resistant (XDR) TB and the close association between TB and HIV infection.

Diagnostic tools introduced 100 years ago are still in routine use today and have become increasingly inaccurate in the face of the HIV-TB pandemic. Consequently, many patients with active TB remain undiagnosed and continue to spread the disease within the community. Thus, missed or delayed diagnosis results in ongoing transmission, mortality, and deleterious social and economic consequences. Currently, there is no available point-of-care test that allows early detection of active tuberculosis at the peripheral health clinic level. Lack of rapid, simple and accurate diagnostic tests at this level is a major hurdle in controlling the global burden of TB. A number of new TB diagnostic techniques has been developed and are undergoing performance testing in different stages.

Especially in sub-saharan Africa, the issue is further complicated by the abundance of non-tuberculous mycobacteria, which do occur both as environmental flora mainly in the soil and water, and as infectious agents to be found in disseminated opportunistic infection in HIV-positive patients with CD4 count below 50/µl but also as pulmonary infection in also non- immunocompromised individuals with underlying pulmonary diseases causing Tb-like symptoms2-4. Traditional staining techniques which rely on acid fastness are not able to discriminate these organisms from MTB in native smears or in smears done from positive cultures. Nucleic acid amplification techniques are necessary for speciation. In our experience in Tanzania, up to 30% of cultures yield NTM. Since many of the strains grown there have not been identified yet and further diagnostic work-up of the patient following the American Thoracic Society (ATS) guidelines on NTM is often not done, it is hard to classify a finding of NTM in culture as contamination or infection.

In 2009, Cepheid released the Xpert® MTB/RIF Assay, which is the only system able to deliver a diagnostic result directly from unprocessed samples by combining on-board preparation of the sample with real-time PCR in less than 2 hours5. Additionally, the Xpert® MTB/RIF Assay (Xpert) allows for simultaneous on-demand molecular testing for the detection of M.tb and RIF resistance. The GeneXpert™ system consists of a GeneXpert instrument, personal computer and disposable fluidic cartridges. The system combines cartridge-based sample preparation with amplification and detection in a fully integrated and automated nucleic acid analysis instrument. Xpert has been shown to be an accurate tool for the rapid diagnosis of tuberculosis in both smear-positive and smear-negative samples6 with a sensitivity of approximately 70% in smear negative culture positive TB in a large, recently published, study which was conducted by the Foundation for Innovative New Diagnostics (FIND)6. Our own data support this finding7. It is anticipated that WHO will soon


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endorse Xpert for use in the diagnostics of adult pulmonary TB based on cumulative data on Xpert performance. Despite these encouraging data, two important details regarding Xpert specificity still require clarification: Firstly, the ability of Xpert to discriminate between non-tuberculous mycobacteria (NTM) and M.tb in a patient sample has not yet been studied in a clinical setting. Preliminary data from testing 20 NTM laboratory strains showed no false positivity8. Unfortunately, in their large clinical demonstration study, FIND had excluded all patients with cultures yielding NTM only from their analysis. Secondly, due to the approach of screening symptomatic TB patients both with Xpert and standard methods, that was used by FIND and in our own study 7, a population of Xpert positive patients with negative cultures still poses a problem of interpretation. Since these patients are deemed to have TB based on clinical and radiological findings as well as treatment response, Xpert might have identified additional TB patients. However, without irrefutable proof of M.tb by a positive culture, there is still a level of uncertainty to classify these patients as a true TB cases. X-ray findings in TB can be caused by other infectious diseases, symptoms of a pulmonary infection caused by M.tb- or NTM can be similar and a resolution of symptoms on anti-TB treatment could be due to the fact that Rifampicin, which is part of standard anti-TB treatment, will also clear other respiratory infections apart from TB (and NTM-infection), since it is a potent antibiotic covering a wide range of respiratory pathogens9. Consequently, the group of culture negative patients in whom TB was diagnosed on clinical grounds alone, was also excluded from analysis in the FIND study6. Thereby, patients classified as TB without microbiological proof in these studies could well suffer from different respiratory conditions, and positive Xpert findings in such a group would thus be false positive. Our conclusion is that these patients need to be examined more closely, to determine the true nature of their disease beyond doubt. False positivity in Xpert would have a negative impact on the positive predictive value of a positive Xpert result. This would cast doubt about Xpert being the single diagnostic test to allow the decision about initiation of anti-TB treatment, and thus has to be clarified.

5 Study Hypothesis 1. Xpert positive, culture negative cases are true TB cases. 2. Xpert is not false positive in these cases, thus positive predictive value of a single

Xpert result is high.

6 Study Design The study is designed as a prospective analysis of TB suspects with a positive Xpert result, but failure to detect TB in initial gold standard sputum examinations. In the Mbeya region, an extensive evaluation project for Xpert in point of care settings is about to be launched. This project is funded by WHO under the TB REACH grant. Patients will receive a single Xpert- test in one spot sputum plus auramine smear for fluorescence microscopy in two sputa (spot and morning). This project is designed to comply with the Tanzanian National TB and Leprosy Programme (NTLP) diagnostic algorithm.


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Patients who will have positive Xpert but negative smear results within these diagnostic activities will be invited to participate in this study, if necessary through tracing. After giving consent, these patients will undergo an extensive clinical workup described under 10.4, with the aim of defining the clinical probability of (pulmonary) TB, primarily by enforcing a microbiological diagnosis of TB. In our experience, liquid cultures from sputum samples will turn positive for M.tb in most patients before day 12 (mean 8 days, median 9 days, unpublished data from Rachow et al.7). Therefore, at day 12, the patient with negative sputum cultures will be considered for bronchoscopy and bronchoalveolar lavage, anticipating that additional diagnostic measures are needed to proof TB or any other diagnosis. A separate information and consent procedure will be conducted for this procedure. Patients will be reviewed clinically by the study physicians, to timely detect dangerous conditions which will prompt anti- TB treatment initiation or any other anti- infectious therapy before completion of the diagnostic algorithm, e.g. suspected TB Meningitis. Should treatment initiation be deemed necessary, completion of the diagnostic algorithm may still be considered since TB treatment rarely leads to a culture conversion to negative before one month of treatment is completed10-11.

7 Study Significance A delayed diagnosis is one of the main reasons for the failure to control TB12. Xpert holds promise to change this situation. However, the performance of this device is not yet satisfactorily investigated. Especially the issue of specificity in Xpert positive, culture negative patients or in patients with growth of NTM remains to be fully clarified before a full implementation of Xpert as the single, decisive step towards TB treatment initiation. Above mentioned studies classify between 4% and 10% of positive Xpert-patients as culture negative6-7. In the study of Boehme at al., 29% of TB-suspects with a negative culture result who were treated on the ground of clinical findings were Xpert positive6, but these cases were excluded from analysis. Our study aims to establish whether these cases are false positives (e.g. cross reaction with other microorganisms/NTM which could cause TB-like symptoms) or whether they constitute TB cases which have not been detected by conventional diagnostics. This will improve certainty on the performance of this new device when used on programme level.

8 Objectives

8.1 Primary Objective

To determine specificity of GeneXpert MTB/RIF® in smear negative, culture negative TB suspects.

8.2 Secondary Objectives


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• To determine the contribution to pulmonary disease by non- tuberculous mycobacteria (NTM), and to describe new species of NTM found

• To determine Xpert performance in specimens other than sputum • To evaluate blood culture for diagnosis of TB in the study population • To evaluate whether phenotypic and functional changes on MTB-specific T cells can

be used as a diagnostic marker for active TB including (TAM-IGRA assay) • To generate information on infecting organisms by strain typing and their appearance

in association with different levels of immunological suppression. • To study genetic factors which play a role in susceptibility and resistance to TB. • To establish whether co-infection with other respiratory pathogens is a relevant factor

in the disease of TB, • To establish and evaluate an X-ray scoring system in Mbeya

9 Study Endpoints

9.1 Primary endpoint

Establishment of a definite, or at least a probable diagnosis on the target population, preferably by microbiological proof of TB, or proof of other diagnosis.

9.2 Secondary endpoints

• Exclusion or confirmation of the diagnosis of infection with non-tuberculous mycobacteria (NTM) in patients with NTM growth in cultures.

• Description of new species of NTM found in cultures. • Evaluation of performance of Xpert in specimens other than sputum. • Evaluation of blood culture in diagnosis of extra-pulmonary TB. • Evaluation performance of TAM-IGRA in comparison with other diagnostic methods • Strain typing of infecting MTB • Strain typing of infecting MTB and evaluation of correlation between infecting strain and

immunological status of host. • Evaluation of genetic host products which determine susceptibility or resistance to TB. • Establishment of co-infection with other respiratory pathogens by nucleic acid

amplification techniques and culture. • Establishment of a chest X-ray scoring system in Mbeya.

10 Methods

10.1 Study Site and Population

10.1.1 Mbeya

NIMR-Mbeya Medical Research Programme TB- clinic is situated on the premises of Mbeya Referral Hospital (MRH) and the Regional Hospital.

Located in south-west Tanzania, the Mbeya region has a high burden of TB- and HIV- infections with around 3.600 detected TB-cases in 2006 and an average HIV prevalence of 13% in people between 15 and 40 years.


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Outside South Africa, research centres are rare which are capable to conduct the complete scale of mycobacterial diagnosis (smear, solid and liquid culture, sensitivity testing, PCR) as well as multiparameter cellular immunological assays. The NIMR - Mbeya Medical Research Programme (MMRP), which has been built by LMU and collaboration partners, has a track record of high quality and comprehensive TB diagnostic studies (LAM, LAMP, ENOSE, several serological assays, TB-TrDNA, LED-microscopy) with more than 3,000 participants over the past 4 years. Currently MMRP is participating in the REMox TB treatment trial within the PanACEA consortium, and in the Europe AID funded ADAT (Active Detection of Active TB) in Children Study. MMRP has a fully equipped CAP-certified lab (College of American Pathologists) that is capable of performing clinical chemistry, immunology, and microbiology including HIV (Serology, Aptima HIV-RNA, viral load). The TB laboratory fulfils bio-safety level 3. The laboratory is equipped with state-of-the-art technology, including smear examinations (fluorescence and conventional microscopy), solid (Löwnstein-Jensen and Middlebrook 7H11 or 7H10) and liquid (two MGIT BACTEC 960) TB-culture, molecular methods (real time PCR (GeneXpert); GenoType® Mycobacterium AS/CM/MTBC/MTBDR; GenoType® MTBDRplus for parallel resistance testing) and a BACTEC drug susceptibility testing system. The TB laboratory is able to examine more than 5.000 samples per year. The MMRP Immunology Laboratory is a state-of-the-art laboratory with vast experience in polychromatic flow cytometry and other experimental platforms, such as the ELISPOT assay and thus is well equipped to study immunologic changes upon tuberculosis treatment initiation that may serve as early markers of treatment success or failure. MMRP has a proven track record for conducting and publishing high-quality cellular immunology research.

10.2 Inclusion criteria

• Able and willing to give informed consent for study participation, including HIV testing.

• TB-suspects with a positive result in Xpert screening, but no detection of AFB in smear microscopy

• Patient 18 years or above

10.3 Exclusion criteria

• Inability to give free, informed consent (e.g. prisoner, mentally impaired, less than 18 yr. old)

• Patient not meeting inclusion criteria


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10.4 Study Workflow


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Figure 1: Study outline for patient flow from screening, clinical workup, to more invasive

Day 1 (health center): spot sputum for microscopy and Xpert, Informed consent procedure for Xpert positive smear negative patients Day 2: (study clinic): signature on the informed consent; spot + morning

sputum for microscopy+ culture

Day 3‐7: Extensive workup in study clinic:

Clinical: questionnaire, examination, ECG (if required)

Imaging: Chest X‐ray (scoring system), ultrasound scan (abdomen/pleura)

Blood: HIV+ CD4, blood culture, Elispot, TAM‐IGRA, serum marker, blood culture

Sputum: spontaneous and induced sputum for culture, smear, repeat Xpert, Hain MTBDRplus,

mycobacterial RNA

Stool: PCR

Urine: LAM‐ELISA

Additional: Tap/Fine Needle Aspiration (if possible/indicated)

Cultures still negativeIntervention possible

Any smear AFB‐ positive or any M.tb‐positive culture: end of participation and referral to anti‐TB treatment

Any culture positive for M.tb: end of participation and referral to anti‐ TB treatment

Day 10‐12: Clinical: examination, review of available information

Bronchoscopy: bronchoalveolar secretion+lavage (microbiology,

immunology, PCR) brush biopsy

Sputum: last sample day after manipulation

Other: tap/biopsy (if possible/indicated)

Decision on TB treatment

Cultures still negative Intervention not possible

Day 10‐12: Clinical: examination, review of available information Decision on TB treatment

Month 1, 2, 6, 12: clinical follow up, repeated smear, culture, biopsy, x‐ray, ultra‐sound, BAL if indicated Month 6: repeat X‐ray in treated patients with initially abnormal X‐

ray

Any culture positive for M.tb: end of participation


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diagnostic tests as long as the cultures and other tests remain negative or immediate anti-TB-treatment is indicated. Day 1: Patients presenting to the collaborating outpatient clinics will receive symptom screening and, if suspected for TB, spot sputum will be collected for Xpert testing and smear microscopy. All Xpert positive patients who do not have positive sputum smears will receive study information and undergo informed consent procedure. They will be handed a sputum container for sampling their next day’s morning sputum. They will have time to consider about participation in the study overnight, and ask questions upon presentation at the study clinic in the following day. Day 2: At the study clinic, patients will finally sign their informed consent and will submit their early morning and spot sputum sample for ZN-staining. If stain result is negative, the sample will be inoculated for culture on liquid (MGIT) and solid (LJ) media. The patient will be invited to attend the next visit. Day 3-7: Patients will be informed about their smear result. Patients who had a positive smear (or culture) result have reached a study endpoint, and will be referred to the government health clinic for treatment initiation. Patients with negative smears will undergo intensive clinical and diagnostic workup. The following procedures will be performed: Clinical:

• clinical questionnaire • clinical examination • ECG optional

Imaging:

• Chest X-ray, application of scoring system? • Ultrasound of abdomen, pericardium, pleura, (lymph nodes optionally)

Blood testing:

• HIV- testing (pre- and post- test counseling) • CD4- count • Blood collection for serum markers, Quantiferon, TAM IGRA , and culture • • Full blood count and clotting tests optional

Sputum:

• Sputum collection (2 samples: 1 spot + 1 morning) for repeated smear and culture, repeated Xpert, and Hain MTBDRplus

• Collection of 3rd sputum after sputum induction

Stool: • Stool PCR

Additional:

• Fine needle aspiration of lesions or tapping of pleural effusion, ascites, lymph nodes, if reasonable and indicated, and after ruling out an intolerably high risk for bleeding


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Day 10-12: By this day, a sufficient interval has passed to receive a liquid culture result for most patients who will have positive liquid cultures for MTB. In our experience, liquid cultures will run positive in most patients before day 12 (mean 8 days, median 9 days). At day 10-12, patients with negative smear/culture results will be considered for bronchoscopy (and a post intervention sputum collection) after additional consenting. A safety laboratory including full blood count, clotting tests, chemistry and an ECG will be done to exclude patients with unacceptably increased risks for adverse events from the procedure. Furthermore anti TB- treatment initiation will be considered and discussed with the local governmental health clinic for all patients in this study phase. Month 1, 2, 6 and 12: A clinical follow up will be conducted for all patients without any microbiological proof of TB (liquid/solid M.tb- positive culture). Clinical examination will be conducted during follow-up. Upon decision of the attending study clinician, the workup of day 3-7 or parts of it will be repeated when reasonable and indicated. A repeated x-ray will be done in treated patients with initial abnormal x-ray findings. Anti TB- treatment initiation will be considered and discussed with the local governmental health clinic for all patients in this study phase who are still suspected of having TB.


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Sampling Scheme Before IC Within this Study

Day 1 Day 2 Days 3-7 Day 10-12 Month 1 Month 2 Month 6 Month 12

Sputum x 2x 1x x x x x

Induced Sputum 1x x x x x

Questionnaire x x x x x

Clinical Examination x x x x x

ECG optional optional optional optional optional

Stool x

X-ray x optional x optional

Ultrasound Scanning x optional optional optional optional

Tap, FNAB x optional optional optional optional Bronchoscopy: BAL, brush biopsy x optional optional optional

Blood:

HIV+cd4 3 ml

Blood culture 20 ml optional optional optional optional

Serological Markers 5 ml 5 ml 5 ml 5ml

Safety lab for tap, FNAB 5,7 ml (optional)

Immunological Assays 30ml 30ml 30ml 30ml Safety lab for bronchoscopy/biopsy 9.2 ml

Substudy: genetic susceptibility 5ml

Cumulative blood 68,7 ml 9,2 ml 5ml 5ml 5ml


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volume

Urine for LAM 90 ml optional optional optional Table 1: Schedule of diagnostic sampling


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11 Study Procedures

11.1 Informed Consent

Each participant will receive written information about the study, which will also be explained in the presence of a literate witness, if the patient is illiterate. Study information can be given by a study physician, assistant medical officer or clinical officer, who is sufficiently familiar with the study to answer the patient’s questions, and appointed with this task by the site investigator. For this purpose, the informed consent sheet will be provided in English and Swahili. It will be ensured that the consent form will be signed and dated by the patient and the consenting member of staff. In case of illiteracy, information will be given orally in the presence of an impartial literate witness, who will sign the informed consent sheet. An illiterate patient will have to thumbprint the consent sheet in addition. Signed or witnessed informed consent is mandatory for any study specific procedures to be performed on the patient. The researchers of this study are committed to the recommendations guiding physicians in biomedical research involving human subjects issued by the World Medical Association Declaration of Helsinki, as last updated in 2008 in Seoul [20].

11.2 Clinical Interventions

Some the below described sampling procedures, clinical and laboratory assessments may be omitted or done later on stored samples, should clinical and/or laboratory capacity be insufficient.

11.2.1 Blood collection

Blood will be taken from patients after informed consent was received via venous puncture performed according to the site SOP for this procedure. The maximal total amount of blood taken at day 3-7 (68,7 ml) and the purpose of each aliquot are indicated in the sampling scheme (table 1) above. Although 68,7 ml blood is a relevant amount, this volume is required to perform all tests which are indicated and reasonable. However, blood drawing of 68,7 ml does not pose any risk to the patients. On day 10-12, 9,2 ml of blood are required for safety laboratory. During follow up (month 2, 6, 12) 5 ml of blood will be collected for measurement of serological markers.

11.2.2 Puncture/Fine Needle Aspiration/Biopsy

There might be clinical signs of (extra-pulmonary) TB in patients who have ascites, pleural effusion, abnormal neurological signs or abnormal lymph nodes. To further elaborate on these symptoms a puncture, fine needle aspiration biopsy (FNAB) or biopsy can be necessary to establish a definite diagnosis. Safety laboratory will be taken to establish platelet levels above 50.000/µl and coagulation parameters within acceptable ranges, before intervention. The procedure will be performed by a trained person under sterile conditions. General anesthesia may be required for biopsy. The


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further investigation of received specimens will be performed in the TB laboratory (smear, culture, PCR) or pathology department of MRH.

11.2.3 ECG

A standard 12-lead electrocardiography will be done mainly in patients with a clinical impression of more severe illness, and with abnormalities in cardiovascular clinical examination. The purpose of this examination is to identify individuals with a high risk for complications during intervention, and to build capacity in Mbeya regarding ECG registration and analysis.

11.2.4 Bronchoscopy

Patients negative in all smears and cultures by day 10-12 will be considered for fiberoptic bronchoscopy, in order to obtain additional specimens for microbiology, cytology and immunology, and a macroscopic impression of bronchial lesions. Diagnostics: Bronchoscopy will be done to harvest bronchoalveolar lavage fluid, and bronchial epithelial biopsy using a brush. Further investigation of received specimens will be performed in the TB-laboratory and pathology department, if available (smear, culture, PCR, cytology). Mononuclear cells will be isolated from bronchoalveolar lavage and analysed for TB-specific T cells using the TAM-IGRA. The day after the procedure, a sputum sample will be collected for smear and culture. Safety: This procedure will be done only after specific, additional informed consent is signed by the patient. To ensure patient safety, clotting test (INR, PTT), full blood count and transaminases (AST and ALT) will be done, to identify and exclude patients with an increased risk for bleeding and other complications. Vital signs (blood pressure, heart rate, oxygen saturation, ECG) will be taken to identify and exclude patients with increased risk of complications from sedation or bronchoscopy. This procedure will be performed by trained medical doctors only. Patients will receive mild sedation and be constantly monitored throughout the procedure for oxygen saturation, heart rate and blood pressure until the sedation wears off. In case of any emergency or complication, MMRP TB ward will be able to supply supplementary oxygen and circulatory support. Further support is available in MRH intensive care unit.

11.3 Laboratory methods

11.3.1 Xpert® MTB/RIF Assay

This assay will be performed in the out- patients clinic/health centre and the TB-laboratory of study clinic. The Xpert® MTB/RIF assay is based on multiplex, nested real-time PCR. A series of molecular beacons is used to simultaneously detect the presence of M.tb and to diagnose rifampicin resistance as a surrogate marker for MDR disease. Species-specific primers allow amplification of the M.tb rpoB core region. Nested PCR is used in order to increase the sensitivity of the assay. Up to six target sequences can be detected simultaneously with the six color assay: one of the six molecular beacon probes was


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designed to detect DNA of the sample processing control Bacillus globigii; the other five molecular beacons are designed to hybridize to overlapping segments of the rpoB core region – the amplicon. The GeneXpert machine and the corresponding computer will be located in a secure designated area within the peripheral health clinic or the TB-laboratory. The pre-treatment of the sample will be according to the manufacturer’s instructions: Sample treatment buffer is added to sputum samples and a defined volume of this mixture is then transferred to the sample chamber of the cartridge. The cartridge is then inserted in the GeneXpert™ (see addendum 1). From this point on, all steps are automated. A semi-quantitive positive result or a negative result for M.tb is displayed by the PC connected to the PCR-machine and can be saved electronically. All cartridges and reagents will be safely stored and discarded if expired.

11.3.2 Sputum smear in study TB-laboratory

After Ziehl-Neelsen staining, the slides are examined with the 100x objective using oil-immersion. Representative areas of the slides are read until either 300 fields are read or Mycobacteria are found. The smear is scored as follows: Number of acid fast Bacilli Result 0 negative 1-9/100 fields record number of AFB 10-99/100 fields 1+ 1-10/field 2+ > 10/field 3+ Optional auramine stains will be prepared for fluorescence microscopy for screening. Every positive result will be confirmed with stand Ziehl- Neelsen-smear and results will be graded if positive as indicated above.

11.3.3 Loewenstein Jensen culture

LJ media are examined weekly for a total of 56 days. The week of first culture growth is reported. Colonies suspicious for mycobacteria are examined by Ziehl-Neelsen staining. The number of colonies is reported as follows: Number of AFB colonies Result 0 negative 1-19 record number of colonies 20-100 1+ 101-200 2+ 201-500 3+ >500 4+

11.3.4 BACTEC® 960 / MGIT liquid culture

Positive MGIT-culture is confirmed by Ziehl-Neelsen staining. The duration of incubation until positive result is recorded.


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11.3.5 Blood culture for mycobacteria

The amount of 20 ml venous blood will be divided and inoculated into 2 appropriate culture bottles for culturing with an automated blood culture system. Positive cultures will be confirmed by Ziehl-Neelsen staining and, if AFB-positive, speciation will be performed.

11.3.6 NAAT to identify NTM and/or other infectious agents

To identify other infecting agents besides TB, small aliquots of sputum samples or of decontaminated sputa will be analysed by nucleic acid amplification techniques (NAAT). This may comprise sequencing e.g. of the 16S region of strains of bacteria grown in culture, like non-tuberculous mycobacteria, and sequencing of native patient material using novel techniques, e.g. pyrosequencing of 16S sequences. Further, detection of a limited number of pathogens may be done using multivalent PCRs which are able to detect several sequences in one assay. This will be done outside of Tanzania, at a site yet to be identified.

11.3.7 Sputum collection and processing for RNA

A subset of the sputa will not be decontaminated. The volume of each collection will be estimated by comparison with an empty sample container that has been calibrated with known volumes of water. The sputa will be digested by mixing with an equal volume of 1:10 Sputasol (Oxoid) in a screw-capped container and vortex mixed for 20 seconds. Following digestion, 4 volumes of 5M guanidine thiocyanate (GTC) will be added to the sample in order to preserve the RNA. Samples will be left at room temperature for 2 hours and then transferred to -80°C. Samples will be thawed in batches and the RNA extracted, using the RNApro blue kit (MP Biomedicals).

11.3.8 Measurement of Mtb RNA extracted from sputum

RNA extracted from sputum will be used to detect the expression of Mtb genes using realtime quantitative RT-PCR technology. Mtb genes initially selected are 16S, sigA, rpoB, 85B, ARC and carD. Other genes may also be included but they will be Mtb relevant. A non-Mtb internal control gene will be spiked into the samples prior to RNA extraction and this will be detected in a similar way to that of the Mtb genes in order to control for the effects of inhibition.

11.3.9 Speciation of mycobacteria by GenoType® Mycobacterium AS/CM/MTBC

At the MMRP (Mbeya), identification of the mycobacteria species is performed from positive solid and/or liquid cultures using DNA-STRIP® technology which allows detecting and differentiating of M. tuberculosis complex and 13 non - tuberculous mycobacteria species. Further differentiation of species by PCR and 16S sequencing will be performed if GenoType CM does not provide a conclusive result.

11.3.10 Sequencing and classification of mycobacteria (conducted at German TB reference centre in Borstel)

For phylogenetic classification, all isolated M. tuberculosis complex strains will be genotyped with various methods. Isolation of genomic DNA (total DNA) for genotyping and IS6110-DNA-fingerprint-analysis will be performed according to standard protocols.


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Following the isolation of the total DNA and digestion of the DNA with PvuII, separation of the DNA fragments will be performed in an agarose-gel. Transfer of the fragments to a nylon membrane is performed by vacuum blot and Southern hybridisation with a DIG-labeled IS6110-PCR-fragment as a gene probe.

Spoligotyping-Analysis will be performed following PCR-amplification of the „direct repeat“(DR)-region with biotin-labelled primers hybridisation of the PCR-Products is performed against 43 immobilised oligonucleotides by „reverse-line-blot“.

For MIRU-VNTR genotyping twenty-four loci were amplified by PCR. Analyses are performed using multiplex PCRs, Rox-labeled MapMarker 1000 size standard (BioVentures, Inc, Murfreesboro, USA) and the ABI 3130 XL sequencer with 16 capillaries (Applied Biosystems, Foster City, U.S.A). Sizing of the PCR fragments and assignment of the various VNTR alleles were done using a customized GeneMapper software package (Applied Biosystems). The molecular typing data will be analyzed with the Bionumerics software (version 6.1; Applied Maths, Sint-Martens-Latem, Belgium) as instructed by the manufacturer. Calculation of the similarity of IS6110-band patterns will be performed by the use of the DICE coefficient. Similarities of spoligotyping and MIRU-VNTR patterns will be calculated by using the categorical coefficient. A dendrogram will be generated using the unweighted pair group method with arithmetic averages (UPGMA). Minimum Spanning Tree analysis based on MIRU-typing data will be done by using the categorical coefficient. Identification of MTBC genotypes will be carried out by using the MIRU-VNTRPlus database (Allix-Béguec, C., D. Harmsen, T. Weniger, P. Supply, and S. Niemann. 2008.) For the cluster analysis, a cluster will be defined as a minimum of two strains harbouring identical genotype pattern from different patients belonging to the study population. Additional analysis e.g. of resistance genes will be done by PCR and sequencing of target regions. Whole genome sequencing of particular strains might be done by established protocols using the Solexa genome analyzer.

11.3.11 Sputum collection and processing for DNA with new PCR-based methods (e.g. Hain MTBDRplus and LAMP) other than Xpert:

A subset of each sputum will not be decontaminated. The volume of 1ml will be used for PCR. The fresh sputa will be digested and further processed according to the requirements of the respective PCR-methods. The new PCR-methods are easy to handle kits which will be applied according to manufacturer´s instructions.

11.3.12 Blood collection for Serum-Marker

5ml blood will be collected, and serum stored to analyse serum markers for TB treatment response. Candidate markers will be chosen from the literature with the aim of selecting TB specific markers. A time course of such a marker typical for TB during treatment would help in diagnostic classification of patients. Candidate markers are Neopterin, ICAM-1 and soluble CD2510. Analysis of these may be done on stored materials after a patient has completed follow-up, and may be limited to cases of diagnostic uncertainty.


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11.3.13 Analysis of genetic host factors relevant for resistance and susceptibility to TB (performed at Bernhard Nocht Institute, Hamburg, Germany)

An amount of 5 ml venous blood of patients with proofed TB will be collected and shipped for further genetic investigation by chromosomal analysis of known hot spots which are linked with clinical resistance or susceptibility to tuberculosis.

11.3.14 QuantiFERON-TB Gold in-tube

The QuantiFERON®-TB Gold IT test (Cellestis, Victoria, Australia) is an in vitro test for Cell Mediated Immune (CMI) responses to peptide antigens that simulate mycobacterial proteins. These proteins, ESAT-6, CFP-10 and TB7.7, are absent from all BCG strains and from most non-tuberculosis mycobacteria with the exception of M. kansasii, M. szulgai and M. marinum. Specimen obtained from BAL (1ml) is collected into each of the QuantiFERON®-TB Gold blood collection tubes, which include a Nil Control tube, TB Antigen tube, and an optional Mitogen tube (positive control). The tubes should be incubated at 37°C as soon as possible and at least within 16 hours of collection. Following a 16 to 24 hour incubation period, the tubes are centrifuged, the plasma is removed and the amount of IFN-γ (IU/mL) measured by ELISA.

11.3.15 TAM IGRA

T cell activation markers (TAM) are cell surface and intracellular proteins that can help to discriminate latent from active TB. The TAM-IGRA is an experimental approach that evaluates the expression of T cell maturation markers on Mycobacterium tuberculosis-specific T cells using poly-chromatic Flow cytometry. PBMC will be stimulated with a peptide pool containing Early secretory antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) and additionally with PPD. Subsequently, the exact phenotype and functionality of MTB-specific T cells will be analysed after staining of T cell maturation markers and the antigen-specific production of various cytokines with fluorochrome labeled antibodies.

11.3.16 HIV testing

All patients will be initially screened with an HIV rapid assay (e.g. HIV ½ STAT-PAK); rapid test results are then confirmed with an ELISA (e.g. Behring Enzygnost ELISA). In case of discrepancy of those tests, a nucleic acid confirmation strategy (e.g.viral load) will be used. In addition, CD4 cell count will be carried out in all HIV positive patients.

11.3.17 Stool DNA/RNA extraction and analysis

DNA extracted from stool will be tested using different quantitative/semi-quantitative PCR techniques. RNA extracted from stool will be used to detect the expression of Mtb genes using real-time quantitative RT-PCR technology. Different extraction techniques will be tested, as previously published studies have emphasized the importance of eliminating PCR inhibitors with stool samples. A non-Mtb internal control gene will be spiked into the samples prior to RNA extraction and this will be detected in a similar way to that of the Mtb genes, in order to control the effects of inhibition.


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11.3.18 Urine LAM lateral flow strip test (Determine TB®)

Patients will be required to give a sample of urine (90ml) for testing and storage. The sample will be a mid-stream urine sample collected into a sterile urine pot. The Determine TB® urine LAM lateral flow strip test will be performed in accordance with the manufacturers instructions using 60μl of urine per patient. Tests will be read at 25 min and graded as positive, negative or indeterminate.

11.3.19 Sample storage and shipment

Sample storage will be performed according to GCP/GLP. The researchers who manage the human specimen data, and other investigators who have access to it, are legally and ethically obligated to protect data that is considered private information. Information regarding the specimens will be recorded by the investigator in such a manner that subjects cannot be identified directly or through identifiers linked to the subjects.

12 Sample size calculations Mbeya: Based on data accumulated from the disctrict “Mbeya Urban”, we expect 400 Xpert positive patients per year. In our experience, 10% of Xpert positive patients in our setting will be culture negative, thus 40 Xpert positive, culture negative patients are expected. Xpert false positivity rate in our target population will depend on the rate of a definite diagnosis of “non-TB” to be established, for which a rough guess would be 30%.The rate of false positivity in our target population can then be determined with a 95% confidence interval of 30% ± 12,7%.

13 Ethical Considerations

13.1 Ethical approval

Each site will obtain local Institutional Review Board (IRB) approval and national IRB approval where appropriate before initiation of study related procedures. All patients will complete an informed consent. The form will be translated into local languages. Patients will be informed that the samples will be stored for 10 years and that may be analysed with and by collaborators where appropriate. The regulations of the declaration of Helsinki, in its last version as amended in Seoul 2008, will be heeded.

13.2 Confidentiality

All patient information will be treated in a strictly confidential manner and will be linked to a unique ID number. CRF information will be entered in a password protected database, for which a frequently updated backup must be available. A second data entry will be made to eliminate potential data entry errors.


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13.3 Risks and Benefits from Study Participation

Patients and their community will directly benefit from ‘being found’ with TB – a disease with significant attendant morbidity and mortality. Patients may potentially benefit from access to improved TB diagnostic technologies; a decreased diagnostic and treatment initiation time and close follow-up from the study staff in addition to the standard clinic care. There are some diagnostic methods planned in the study like biopsy, puncture and/or bronchoalveolar lavage which bear a potential risk for the patients, but are routinely conducted for diagnosis of TB in settings with less resource constraints. The patient will be informed about these procedures, their risks and benefits. Only upon unconcerned signature of the procedure specific free, informed consent, these interventions will be performed. The investigator will ensure that the condition of the patient allows such investigations. Tapping of effusions and biopsy are standard procedures if extra-pulmonary or lymph node TB is suspected. These investigations can lead to a definite diagnosis with consecutive treatment which is a benefit for the patient on the long run. Bronchoalveolar lavage is not a standard procedure in many countries to diagnose pulmonary TB. However the sensitivity and specificity of many laboratory methods (smear, culture, PCR) can be massively improved in specimen which was obtained directly from the brochoalveolar system.

14 Compensation to Patients Patients who are required to be transported to the clinic and undergo diagnostic testing will be reimbursed 10.000 TSH per visit if they are required to follow-up for study purposes. Should any patient suffer adverse events from study related procedures, treatment will be provided free of charge.

15 Data management and analysis Based on results from investigations and clinical judgement, patients will be classified into the following categories based on available clinical and laboratory data:

Group Diagnosis Criteria (Example)

A , Confirmed TB

≥ 1 culture positive for M.tb complex

B Clinical TB Clinical judgement, positive smear with negative culture, suggestive histology without species determination, TB treatment response


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C non-TB patients Proof of alternative diagnosis, sustained recovery after antibiotic treatment and all smears and cultures negative

E NTM disease NTM infection rather than colonization determined by ATS criteria, NTM isolated from sterile body site.

F Indeterminate Insufficient data for classification

Table 2: Classification of patients according to clinical and laboratory results Case report forms (CRFs), as well as an Access database for on-site double data entry, will be developed and supervised by the on-site data managers. Query and report functions will allow basic real-time data analysis, e.g. for identification of cases requiring follow-up. Any changes on the case report form or in the clinical or laboratory procedures will be documented and signed. Changes in the database will be tracked automatically. At the end of the enrolment phase, preliminary data sets will be analysed under the supervision of TB NEAT statisticians, with the final analysis being completed when final culture results and follow-up results are available. All study documentation will be stored on site for at least 10 years. During the close-out visit, random control of at least 10% between laboratory result forms, case report forms and database fields will ensure quality of data.

16 Study Phases and timelines Feasibility: Mbeya: Patients will be screened for TB in 2 health facilities with standard microscopy and Xpert MTB/RIF-assay. According to our experience and documented case detection rate of those facilities, the following numbers of patients in categories are expected in 12 months: 420 Xpert positive cases 150 Xpert positive, smear negative cases for informed consent and day 3-7 workup; 42 Xpert positive cases in whom day 1+2 cultures remain negative 20 cases with negative cultures after extensive day 3-7 workup 10 cases with negative cultures after bronchoscopy and biopsies Only the last category of patients will receive clinical follow-up for 12 months. Study plan, site infrastructure development and ethics: 6 months Finalisation of protocol Ethics approvals Site development (Xpert machine set-up and consumable- and equipment for broncoscopy acquisition, training)


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Enrolment phase: 12 months: Enrolment of 150-200 smear negative patients out of which 45-70 patients remain culture negative

Follow-up phase for suspected TB cases without microbiological proof: up to 12 months Data analysis and publication: 4-6 months

17 Study Budget The budget calculated for one study site at NIMR-MMRP in Mbeya, Study B, calculation for the whole study duration

Infrastructure Upgrade Establishment of facility/infrastructure/training for bronchoscopy, € 20.000 Study Conduct Project management € 5.000Clinical Supplies/Patient Compensations € 13.000Consumables laboratory € 30.000Personnel costs € 80.000Local and overseas travel costs € 2.000

Capacity Building € 10.000 Grand total € 160.000

18 Literature 1. WHO. Global tuberculosis control : surveillance, planning, financing WHO report

2009. Geneva: World Health Organisation; 2009. Report No.: WHO/HTM/TB/2009.411.


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2. Chilima BZ, Clark IM, Floyd S, Fine PE, Hirsch PR. Distribution of environmental mycobacteria in Karonga District, northern Malawi. Appl Environ Microbiol. 2006 Apr;72(4):2343-50.

3. Buijtels PC, van der Sande MA, Parkinson S, Verbrugh HA, Petit PL, van Soolingen D. Isolation of non-tuberculous mycobacteria at three rural settings in Zambia; a pilot study. Clin Microbiol Infect. 2009 Oct 14.

4. Hatherill M, Hawkridge T, Whitelaw A, Tameris M, Mahomed H, Moyo S, et al. Isolation of non-tuberculous mycobacteria in children investigated for pulmonary tuberculosis. PLoS ONE. 2006;1:e21.

5. Xpert MTB/RIF [package insert]. Cepheid, Sunnyvale, CA, 300-7810 Rev. A, April 2009.

6. Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med. 2010 Sep 9;363(11):1005-15.

7. Andrea Rachow AZ, Norbert Heinrich, Gabriel Rojas-Ponce, Bariki Mtafya, Klaus Reither, Elias N. Ntinginya, Justin O`Grady, Jim Higget, Keertan Dheda, Catharina Boehme, Mark Perkins, Elmar Saathoff and Michael Hoelscher. Rapid and accurate detection of Mycobacterium Tuberculosis in sputum samples by Xpert MTB/RIF Assay- A clinical validation study.

8. Helb D, Jones M, Story E, Boehme C, Wallace E, Ho K, et al. Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. J Clin Microbiol. 2010 Jan;48(1):229-37.

9. Schaaf SH, Zumla AI. Tuberculosis. 1 ed. London: Elsevier; 2009. 10. Wallis RS, Doherty TM, Onyebujoh P, Vahedi M, Laang H, Olesen O, et al.

Biomarkers for tuberculosis disease activity, cure, and relapse. Lancet Infect Dis. 2009 Mar;9(3):162-72.

11. Weiner M, Burman W, Vernon A, Benator D, Peloquin CA, Khan A, et al. Low isoniazid concentrations and outcome of tuberculosis treatment with once-weekly isoniazid and rifapentine. Am J Respir Crit Care Med. 2003 May 15;167(10):1341-7.

12. Keeler E, Perkins MD, Small P, Hanson C, Reed S, Cunningham J, et al. Reducing the global burden of tuberculosis: the contribution of improved diagnostics. Nature. 2006 Nov 23;444 Suppl 1:49-57.

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