Tuberculosis : Diagnosis and drug susceptibility testing where resources are scarce

University dissertation from Stockholm : Karolinska Institutet, Department of Public Health Sciences

Abstract: Background: Tuberculosis remains a major global public health problem. Not surprisingly, most cases of this disease occur in poor countries and an increasing number of patients harbor drug - resistant bacteria. The cornerstone of bacteriological diagnosis of tuberculosis is direct sputum smear microscopy. This method is rapid, inexpensive and specific, however, sensitivity is discouragingly low. Regarding drug susceptibility testing, the methods available today are either cheap and slow or fast but too costly to be applicable in most high incidence areas. The aim of this investigation was to improve detection and drug susceptibility testing of Mycobacterium. tuberculosis with special emphasis on settings with scarce resources. Methods: We evaluated an improved method for sputum microscopy, which is based on liquefaction of the sample with household bleach, followed by its concentration by centrifugation prior to staining and microscopy. The bleach microscopy method was compared to standard direct microscopy in Honduras. Then, we performed a literature review in search for all studies that have compared the bleach method to the direct method in low- and middle-income countries. We further sent out questionnaires to key persons in national tuberculosis control programs in order to investigate the knowledge and opinions about this alternative method. We also developed and evaluated a new method for rapid and inexpensive drug susceptibility testing, which is based on the ability of M. tuberculosis to reduce nitrate to nitrite. The performance of this method, the Nitrate Reductase Assay, for drug susceptibility testing was compared to the standard BACTEC 460 liquid culture system. Moreover, we evaluated the performance of a commercial automatic culture system called BacT/ALERT 3D for primary detection of M. tuberculosis in clinical samples and for drug susceptibility testing. Results: We found that the bleach method could improve sensitivity with 3 7 %, Arith unchanged specificity, in Honduras. In the scientific literature, we found 19 studies that had compared the bleach microscopy method with the direct smear. In 15 out of these there was a significant improvement ranging from 7-253 % of the proportion of positive tests using the new method. We received answers from 84 key persons in 69/85 included countries (81 %). Thirty-six key persons thought that the bleach method could increase case detection in their countries, 40 did not know and five thought it could not. Furthermore, recommendations from the World Health Organization or the International Union Against Tuberculosis and Lung Disease, as well as studies in their own countries, were factors that would make the key persons promote the bleach microscopy method for routine use. When tested in 57 M. tuberculosis strains, the Nitrate Reductase Assay showed equivalent susceptibility results to the BACTEC method for isoniazid and rifampicin, and had a similar turn-around time. The BacT/ALERT technique, which was tested on 2659 clinical specimens, detected M. tuberculosis at a similar rate as when cultured on standard Löwenstein-Jensen medium. Concerning drug susceptibility testing, this new technique showed a lesser sensitivity in detecting drug resistance compared to the BACTEC method in 50 M. tuberculosis strains. Conclusions: The bleach microscopy method can clearly improve case detection of tuberculosis and key persons in national tuberculosis control programs are interested in this technique. I my own opinion, the World Health Organization should recommend its evaluation and introduction for routine use. The Nitrate Reductase Assay might become a suitable option for rapid and inexpensive drug susceptibility testing of rifampicin and isoniazid (the two most important antituberculosis drugs) if it proves successful in field studies. The BacT/ALERT 3D system is a valid alternative for primary isolation but should be further developed before it can be used for drug susceptibility testing. The heavy cost of the apparatus and substrates limits, however, the applicability where resources are scarce.

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