Improving diagnosis of extrapulmonary tuberculosis by implementation of a sensitive and specific assay in routine tuberculosis diagnostics

Lack of better diagnostic tools is one of the major obstacles towards proper tuberculosis (TB) management and control. TB outside lungs (extrapulmonary) accounts for approximately 14-40 % of all TB infections and poses special diagnostic challenges. The routinely used methods are slow (culture takes 6-8 weeks) and fail to accurately diagnose many cases. This leads to diagnostic delay, improper treatment, increased morbidity, and mortality. At University of Bergen, we have developed a rapid, robust, sensitive, and specific antigen detection test to diagnose TB from various patient samples including fluids, aspirates, and biopsies by using immunochemistry. The test is shown to be as sensitive as the more technically demanding nested-PCR, but its robustness makes it suitable for implementation in the low-resource high TB endemic settings. The main objective of this project was to, 1) further improve the test by generation of functional monoclonal antibody, 2) implement and validate the test in routine diagnostic settings in high and low TB and HIV burden countries, 3) compare the assay with the WHO endorsed Xpert MTB/RIF automated molecular assay (Xpert). Hospitals implementing WHO endorsed DOTS strategy and equipped with a pathology laboratory were selected from each of these five sites: Tanzania and Zanzibar with high TB and HIV, India and Pakistan with high TB low HIV, and Norway with low TB and HIV. Results: 1) Work on the development of a functional monoclonal antibody faced several challenges. After four attempts we generated 3 clones and the purified monoclonal antibodies. These antibodies, despite very good ELISA results, do not work with immunohistochemistry on the formalin-fixed tissues. Therefore, we focused on the reproduction of functional polyclonal antibody in large quantity keeping in mind the large-scale use of this test. We have made a single batch of functional anti-MPT64 antibodies in large volume. The sensitivity and specificity of these new antibodies are 87% and 80% respectively by validation on a small number (n=75) of formalin fixed paraffin embedded clinical biopsies. These antibodies are being tested on the larger patient material in India and Pakistan. These antibodies are made available for public use through a non-profit company, Ximbio (https://ximbio.com/reagent/157698/anti-mpt64-antibody2), in collaboration with the technology transfer platform, VIS, (https://www.visinnovasjon.no/). 2 & 3) The work on implementation of the test (by using the previous anti-MPT64 antibodies) and comparison with Xpert assay, in routine diagnostic settings at the tertiary care hospitals in Zanzibar, Tanzania Mainland, Pakistan and Norway, has been completed. Data analysis for validation of the test, feasibility of implementation, diagnostic delay, and cost effectiveness analysis has been completed for Zanzibar, and ongoing for other sites. The results till now show there is a significant delay in the diagnosis and treatment of extrapulmonary TB which leads to increased morbidity, mortality, and economic burden. The MPT64test is robust, implementable in the routine diagnostics in low-resource settings, and the sensitivity and specificity are better than the routine tests as well as Xpert assay. This test can contribute towards timely and better diagnosis of TB particularly in the high-TB endemic settings. The MPT64 test with the new polyclonal antibodies has comparable sensitivity but somewhat lesser specificity as compared to the previous antibody. However, these results are based on a smaller material, and result from larger material are expected to give more precise validation of the new antibodies. Outcome: The results from this project have been published as seven original papers in the peer-reviewed international journals, one manuscript is under revision after comments from the reviewers, and several manuscripts are under construction. The project will result in six PhD, and three master theses. The supervision of master- and doctoral candidates, and the training provided to the technical staff at the laboratories in the low-resource settings is expected to lead to the academic and technical capacity building. Through the availability of the anti-MPT64 antibodies, the test can be used by any laboratory for diagnosis of TB. Collaborating institutions: Norway: University of Bergen, Haukeland University Hospital, Oslo University Hospital, Stavanger University Hospital. Tanzania: National Institute for Medical Research, Muhimbili National Hospital, Mbeya Zonal Referral Hospital Zanzibar: Mnazi Mmoja Hospital India: Ujjain Charitable Hospital & Research Center Pakistan: National & Provincial Tuberculosis Control Programme, Gulab Devi Chest Hospital, Shaukat Khanum Memorial Cancer Hospital & Research Center, Social and Health Inequalities Network. This project is funded by GLOBVAC and is part of the EDCTP2-Program supported by the European Union.

The MPT64test is robust, better than the routine tests, and implementable in the routine diagnostics. This test can contribute towards timely and better diagnosis of TB particularly in the high-TB endemic settings. The results have been published as seven original papers, and several manuscripts are under construction. The project will result in six PhD, three master theses, and technical capacity building. Through the availability of the antibodies, the test can be used by any laboratory. Impact: TB related research is highly prioritized among the major global stakeholders (WHO/StopTB Partnerships) as an effort to reduce poverty and improve the economic and social well-being. Lack of accurate diagnostic tools has been identified as one of the major hindrances towards global TB control. The proposed MPT64test will contribute towards achieving a healthier society by improving the health of children, mothers and adults who are the most economically productive age-group and caregivers.

Extrapulmonary tuberculosis (TB) accounts for approximately 14-40 % of all TB infections, and diagnosis is difficult due to paucibacillary nature of the disease. We have developed a rapid, robust, sensitive and specific immunochemistry-based assay to diag nose extrapulmonary TB by detection of the secreted mycobacterial antigen MPT64 from fluids, aspirates and biopsies. Using Mycobacterium tuberculosis complex specific nested-PCR as gold standard the sensitivity and specificity of the assay is up to 95%, s ignificantly better than acid-fast staining and culture (sensitivity of 12% and 22% respectively). The assay can differentiate between pathological and atypical mycobacteria and performs equally well in HIV co-infected patients. Lower costs and robustness makes it suitable for implementation in TB endemic countries. The main objective of this project is to improve the assay by producing a functional monoclonal antibody, implementation and validation of the assay in routine diagnostic settings, and compare the assay with the WHO endorsed Xpert MTB/RIF automated molecular assay. A hospital implementing WHO endorsed DOTS strategy and a pathology laboratory will be selected from each of the 5 sites; Tanzania, Zanzibar, India, Pakistan, and Norway. All consecu tive TB suspects (minimum 400 cases) will be enrolled for one year. Clinical data and samples will be collected. The assay will be performed in addition to the routine diagnostic work-up on all samples. This cohort will be followed up from diagnosis to th e completion of treatment. Response to treatment will define a TB case. Interviews, pre-designed questionnaire, and data from two comparable control hospitals from each site will be used to measure the outcomes which includes validation of the test, feasi bility of implementation, increase in case detection of adults and childhood TB, and cost effectiveness analysis for future scale-up. This assay has a great potential for improving the diagnosis of extrapulmonary TB.