Rapid tests for multidrug resistant tuberculosis in low income settings

Tuberculosis (TB) is at epidemic levels in the resource-limited settings (RLSs) due to HIV/AIDS, poverty and insufficient TB control programmes. These factors are also contributing to TB drug resistance. Patients with multidrug drug resistant tuberculosis (MDR-TB) do not respond to first line drugs. These patients require unique drug regimens, making it necessary to routinely screen for MDR-TB. Screening for MDR-TB with the Lowenstein-Jensen proportion method (LJPM), which is common in the RLSs is a very slow process taking 2-3 months. More rapid tests suitable for RLSs are urgently needed. In this thesis, a comparison of the technical and operational performance of several rapid tests for MDR-TB was done, and the most optimal tests for RLSs are proposed.

In paper I, a meta-analysis of rapid tests for direct detection of MDR-TB was conducted. The direct nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) and Genotype® MTBDRplus (GT-DRplus) were highly sensitive and specific, and far more rapid than the conventional indirect drug susceptibility testing (DST).

In paper II, the NRA, MODS, Mycobacterium Growth Indicator Tube (MGIT 960), GT-DRplus, Alamar blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and resazurin assays were compared head-to-head for indirect detection of MDR-TB at the National Tuberculosis Reference Laboratory (NTRL) Kampala. The NRA, MGIT 960, GT-DRplus and MODS were the most sensitive and specific tests, with significantly shorter time to results compared to the LJPM.

In paper III, the direct NRA and MODS assays were compared at the NTRL on sputum specimens from consecutive re-treatment TB patients. Interpretable results were obtained in over 90% of the samples with both assays. The median days to results were 10 with the NRA and 7 with MODS. The direct NRA was more sensitive and specific, and was cheaper.

In paper IV, the sensitivity, specificity, time to results (TTR) and reproducibility of the direct GTDRplus against the MGIT 960 was assessed. Sensitivity and specificity were 100% and 96% for detection of rifampicin resistance; 81%, and 100% for isoniazid resistance; and 92%, and 96%, for MDR-TB, respectively. The TTR was 1-3 days, and concordance of results between the Molecular Laboratory at Makerere University and the FIND Diagnostics Laboratory was 98%.

In paper V, we applied spoligotyping to study the clustering rate and predominant genotypic strains of 99 MDR-TB strains isolated from patients in Kampala. Eighty-three percent of the strains occurred in clusters, and the T2 lineage was the largest single cluster.

Conclusion. The direct NRA and the GT-DRplus appear to be the most appropriate tests for MDR-TB in RLSs. The NRA being the cheapest test can be applied where resources are extremely limited, while the ultra rapid but commercially available GT-DRplus can be used where resources permit.

List of scientific papers

I. Bwanga F, Hoffner S, Haile M, Joloba ML. (2009). "Direct susceptibility testing for multi drug resistant tuberculosis: a meta-analysis" BMC Infect Dis 9: 67
https://pubmed.ncbi.nlm.nih.gov/19457256

II. Bwanga F, Joloba ML, Haile M, Hoffner S. (2010). "Evaluation of seven tests for the rapid detection of multidrug-resistant tuberculosis in Uganda." Int J Tuberc Lung Dis 14: 890-895
https://pubmed.ncbi.nlm.nih.gov/20550774

III. Bwanga Freddie, Melles Haile, Sven Hoffner, Emmanuel Ochom, Moses L. Joloba. (2010). "Direct Nitrate Reductase Assay versus Microscopic Observation Drug Susceptibility for rapid detection of MDR-TB in Uganda." (Manuscript)

IV. Albert H, Bwanga F, Mukkada S, Nyesiga B, Ademun JP, Lukyamuzi G, Haile M, Hoffner S, Joloba M, OBrien R. (2010). "Rapid screening of MDR-TB using molecular Line Probe Assay is feasible in Uganda." BMC Infect Dis 10: 41
https://pubmed.ncbi.nlm.nih.gov/20187922

V. Bwanga Freddie, William George Muyombya, Sven Hoffner, Melles Haile, Benon Asiimwe, David Kateete, Fred Katabazi, Jennifer Asiimwe, Maria Wijkander, Moses L Joloba. (2010). "High clustering of MDR-TB strains in Kampala, Uganda: Predominance of the T2 lineage." (Manuscript)