Project Coordinator: A. Rachow MD
In collaboration with FIND-Diagnostics (Geneva), Cepheid (USA), and University College London (UCL), new diagnostics for simple and rapid detection of TB and drug resistance are being developed and evaluated.
Currently, the portfolio in evaluation comprises PCR-based methods directly from smear positive sputum or positive culture, such as GeneXpert® and Hain MTBDRplus®, and the further evaluation of IFN-y release assays (IGRAs) for their performance in detection of childhood TB, and as a markers for treatment course.
In another project, transrenally excreted mycobacterial DNA is evaluated as biomarker for TB treatment course.
As a gold standard, culture on solid and liquid media is done in our state-of-the art TB laboratory, followed by molecular species determination.
LMU Department for Infectious Diseases and Tropical Medicine provides study design and study coordination within the NIMR-MMRC collaboration, and manages the TB Laboratory through its team members.
Transrenal DNA detection for TB Diagnosis
Coordinator: K. Reither MD, Msc
Background / Purpose:
Tuberculosis (TB) is a global threat to public health. The lack of accurate, rapid, inexpensive and convenient tests for tuberculosis still hinders patient management and disease control. The potential diagnostic capability of cell-free transrenal DNA (Tr-DNA), short DNA fragments which pass the renal barrier, has been demonstrated in infectious diseases, including TB, HIV, and malaria.
The main objectives of the project are to evaluate the potential of transrenal DNA detection in urine specimen for diagnosis of tuberculosis, to optimize and simplify the assay and the specimen preparation, and to explore the suitability of the new technology in a developing world setting. The overall goal will be to obtain the knowledge to develop a robust, accurate and rapid procedure to diagnose TB on the basis of Tr-DNA.
The research project on Tr-DNA is a co-operative venture of 7 participating institutions providing technical and scientific expertise. The project comprises 7 work packages. The project time line is 3 years.
Work package 1 and 2 will take place at the Mbeya Medical Research Center (MMRC / Mbeya, Tanzania) and the University of Zambia (UNZA / Lusaka, Zambia). In this part of the project 900 TB suspected patients and 250 healthy controls will be recruited and followed-up. An established highly sensitive detection assay, using hemi-nested PCR, will be evaluated performing sensitivity and specificity assessment. Furthermore, a bio bank of urine samples from a cohort of patients with symptoms of tuberculosis will be build up.
In the following work packages a small European cohort of TB confirmed patients will be recruited,and a urine bio bank of these clearly defined TB positive patients will be established. The samples will be utilized for optimisation by comparing different extraction procedures for the purification of DNA from urine assessing both the yield of DNA and the removal of nucleic acid amplification inhibitors. In addition, an assessment of potential storage conditions to prevent the degradation of target DNA will be performed. A comprehensive assessment of the biological characteristics of Tr-DNA will be carried out. Moreover, a project database facilitating the interaction and scientific discussion between partners to maximise the final outputs will be established.
European Commission, DG XII
(1) Spaxen Italia Ssrl, Rome, Italy (coordinating partner).
(2) University College London (UCL), London, UK.
(3) University of Zambia (UNZA), Lusaka, Zambia.
(4) Department of Infectious Diseases & Tropical Medicine, Ludwig-Maximilians-University (LMU), Munich, Germany.
(5) The Mbeya Medical Research Center (MMRC), Mbeya, Tanzania.
(6) Istituto Nazionale per le Malattie Infettive L. Spallanzani (INMI), Rome, Italy.
(7) Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland.
Coordinatior: K. Reither, MD, Msc
Background / Purpose:
The lack of accurate, rapid and convenient tests for tuberculosis (TB) still hinders patient management and control of this deadly disease.
In most parts of the developing world, the microscopy smear test is the only affordable and feasible method to detect TB. However, the sensitivity and specificity of this test are highly variable and the procedures are very time- and labor-intensive. Other technologies, as for instance culture or molecular biological methods, are expensive and technically very difficult and thus are not applicable on a large scale in resource poor countries.
One of the technologies which might fulfill the requirements for a new diagnostic tool for TB is the electronic nose. It mimics the human olfactory system using chemical sensors which recognize patterns in the volatiles of samples. Advantages of the electronic nose, among others, are the simple sample preparation and the fact that no reagents or other expensive consumables are required. Additionally, results are available very quickly, which would facilitate the prevention of transmission of the disease and speed up treatment initiation.
The purpose of this project is the preclinical and clinical evaluation of an electronic nose for diagnosing pulmonary tuberculosis. The sampling method, as well as data reproducibility, sensitivity and specificity of the technology will be assessed and improved.
The evaluation of the electronic nose is carried out in six different phases. Three of these phases take place at the Mbeya Medical Research Center:
Phase II: pre-clinical study
Phase III: training set
Phase IV: feasibility evaluation
The objectives of these phases are to test and assure clinical and operational data reproducibility on site, as well as to validate the sampling protocol, data capture software and analysis capacity. Furthermore, the diagnostic potential of an electronic nose for pulmonary tuberculosis in different types of specimen, and the sensitivity and specificity of the method are evaluated.
Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland
(1) KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands.
(2) Scensive Technologies Ltd., Normanton, United Kingdom.
(3) Department of Infectious Diseases & Tropical Medicine, Ludwig-Maximilians-University (LMU), Munich, Germany.
(4) The Mbeya Medical Research Center (MMRC), Mbeya, Tanzania.
(5) Foundation for Innovative New Diagnostics (FIND) / World Health Organization (WHO), Geneva, Switzerland
Rapid Direct Antigen Test for Tuberculosis
Coordinator: C. Boehme PhD
Lab Coordinator: M. Gerhardt PhD
Lab Coordinator: S. Dufke
The main purpose of the proposed project is the development and clinical evaluation of a rapid direct antigen test for diagnosis of pulmonary tuberculosis using an immunochromatographic test format. One major obstacle in the fight against tuberculosis is the lack of an inexpensive, easy to use and sensitive method to detect TB infected patients. The only method that is affordable in many parts of the developing world is the microscopy smear test. It is used to detect TB-patients and monitor their treatment success. Unfortunately this test is labour intensive and is mainly used to detect “productive, open”, pulmonary TB. In addition, the sensitivity depends dramatically on the quality of the test performers. More advanced methodologies such as culture and PCR are technically difficult, not that reliable and therefore not widespread in rural Africa.
Together with a company that developed a prototype rapid direct antigen immunoassay detecting the presence of Mycobacterium tuberculosis in clinical specimens such as urine, blood and sputum, we want to explore the sensitivity and specificity in an African clinical setting. Immunological assays detecting directly TB antigens or antibodies against TB have been identified as most promising candidates to substitute and complement smear testing.
In a first clinical evaluation the LAM assay has proven to have a high sensitivity and specificity in 231 patients with suspected pulmonary tuberculosis (TB) and 103 healthy volunteers were screened with standard TB tests and with the new LAM-ELISA. Of 132 patients with confirmed pulmonary mycobacterial disease (positive sputum culture), 106 were positive using the LAM-ELISA (sensitivity 80.3%). In comparison, the sensitivity of acid-fast bacilli (AFB) sputum microscopy was 62.1% (82 of 132 confirmed cases).
Of the 231 patients, 17 were both culture- and AFB-negative, but had typical radiographic signs of pulmonary mycobacterial infection and did not respond to antibiotic treatment. Of these17 patients, 13 (76.5%) had positive LAM-ELISA test results. To define the specificity of the assay, urine samples from 103 healthy volunteers were also screened using LAM-ELISA. All but one had an optical density below the cut-off (specificity 99%).
Of interest was a significant correlation between the level of microscopic density of mycobacteria in sputum and LAM antigen concentration in urine (χ2=8.44). The LAM-ELISA is a field-adapted tool that can improve screening standards in countries with a high incidence of TB.
We are currently conducting a much larger final phase III evaluation in TB suspect patients in Mbeya.
This research is jointly funded by Chemogen Inc., Portland, USA, FIND (Foundation for new innovative diagnostics), Geneva, CH and University of Munich, Germany
Partners involved in the project are the Mbeya Regional TB & Leprosy Program, the Dept. of Infectious Diseases and Tropical Medicine at the LMU, Munich and Chemogen Inc, Portland, ME, USA, FIND, Geneva, CH