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We examined the feasibility of using DNA extracted from stained sputum smears for the detection of rifampin and isoniazid resistance with the commercial MTBDR plus assay from Hain Lifescience GmbH, Nehren, Germany. Overall sensitivity was initially low (70.0%) but increased to 96.7% when a multiplex PCR preamplification step was added. We then tested stored Mycobacterium tuberculosis -positive stained smears prepared from 297 patients' sputum samples. Species identification and drug susceptibility testing (DST) had been performed at the Institut Pasteur de Madagascar. Overall, the performance of the MTBDR plus assay applied to slide DNA was similar to that obtained in other studies with DNA extracted from clinical specimens. With the ready availability of stained smears in routine diagnostic laboratories and their easy transport and storage at room temperature, this approach should be useful for optimizing the treatment of multidrug-resistant tuberculosis and for conducting resistance surveys aimed at identifying hot-spot regions and breaking chains of transmission.

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop tuberculosis (TB), though many may become latently infected. More precise measurement of the human immune response to M. tuberculosis infection may help us understand this difference and potentially identify those subjects most at risk of developing active disease. Gamma interferon (IFN-γ) production has been widely used as a proxy marker to study infection and to examine the human immune response to specific M. tuberculosis antigens. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses and clinical outcome in human tuberculosis is still poorly understood. We therefore evaluated the antigen-specific IFN-γ production responses in peripheral blood mononuclear cells from two cohorts of subjects recruited in Antananarivo, Madagascar, from 2004 to 2006 and examined the influence of the infecting M. tuberculosis strains on this response. The cohorts were sputum-positive index cases and their household contacts. Clinical strains isolated from the TB patients were typed by spoligotyping. Comparison of the IFN-γ responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-γ responses than “ancient” strains, like East African-Indian (EAI) strains, in index cases and their household contacts. These results suggest that new strains may have evolved to induce a host response different from that of ancient strains. These findings could have important implications in the development of therapeutic and diagnostic strategies.

The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis , from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis . Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS 6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains ( Mycobacterium africanum , Beijing, M. bovis , EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.

We read with great interest the letter previously published by M. P. Grobusch et al. (1). Evaluation of diagnostic tests is indeed a topical problem, especially in developing countries. We agree with the authors' remark that the rapid test (AMRAD/ICT) could be considered only as an adjunct test to standard techniques of tuberculosis (TB) diagnosis. However, we would like to comment on the last conclusion drawn by the authors. The reported study was realized in a European hospital, and the specificity observed was very good. This would not be always the case in developing countries, particularly in tropical areas where people are permanently in contact with various pathogens and develop cross-reacting antibodies responsible for poor specificity. Therefore, it is important that every new test and, particularly, every new serodiagnostic assay be validated with the controls of the country where the test will be applied. Furthermore the low sensitivity of the AMRAD/ICT assay (50%) will result in false-negative results for many patients. Thus, although the specificity is excellent, the negative predictive value of the test will be unacceptably low even in countries with a high prevalence of TB. Therefore, we do not believe that such a test would be suitable as a first-line method of diagnosis. Sputum smear microscopy has been proven to be very useful for diagnosis of contagious TB cases in developing countries where diagnostic tools such as radiology are missing. Still, a serological test for the diagnosis of paucibacillary and extrapulmonary TB would be of interest if it were more sensitive and faster and could be used on large series of samples. Also, keep in mind that serodiagnostic tests necessitate blood sample collection and disposable materials that are not readily available in most countries with a high prevalence of human immunodeficiency virus or hepatitis. In resource-poor areas, all these factors have to be taken into account when evaluating the efficacy and cost of a rapid test.