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. 2002 Nov;70(11):6509–6511. doi: 10.1128/IAI.70.11.6509-6511.2002

ESAT-6 and CFP-10: What Is the Diagnosis?

Nico C Gey van Pittius 1,*, Robin M Warren 1, Paul D van Helden 1
PMCID: PMC130415  PMID: 12379740

In a recent issue of Infection and Immunity, Geluk and coworkers (7) reported the observation that human T cells from both Mycobacterium leprae- and Mycobacterium tuberculosis-sensitized individuals recognize the M. leprae ESAT-6 orthologue. This has prompted us to comment on the potential use of these antigens as diagnostic markers, as well as to comment on the use of the misnomer “M. tuberculosis specific” when referring to these antigens.

The use of the designation “M. tuberculosis specific” has grown steadily over the last few years as the number of studies on the potential use of ESAT-6 and CFP-10 as diagnostic markers for M. tuberculosis infection has increased. Contrary to common belief (e.g., see references 1 to 3, 6, and 12), the secreted M. tuberculosis ESAT-6 and CFP-10 T-cell antigens are not M. tuberculosis specific (8). Despite earlier evidence to the contrary (9), orthologues of ESAT-6 and CFP-10 are present in the genomes of M. leprae and even the distantly related, nonpathogenic, fast-growing, environmental mycobacterium M. smegmatis (8). This is further supported by results dating back to 1995, which showed that the genes for these proteins are also present in other pathogenic mycobacteria (M. africanum, M. kansasii, M. marinum, and M. szulgai [9, 14] and M. bovis [9]) as well as the slow-growing nonpathogenic mycobacterium M. gastri (5) and the fast-growing nonpathogenic environmental species M. flavescens (9).

This raises a question concerning the potential use of these antigens as diagnostic markers. We have previously suggested that the presence of orthologues of ESAT-6 in other mycobacterial species may influence the use of ESAT-6 as a diagnostic marker for M. tuberculosis infection (8). The results presented by Geluk and coworkers (7) that show significant cross-reactivity between the M. tuberculosis ESAT-6 and its orthologue from M. leprae support our viewpoint. The investigators came to the conclusion that this significant cross-reactivity indicates low specificity and has implications for its use as a diagnostic tool in areas where both tuberculosis and leprosy are endemic.

The similarities between the M. tuberculosis ESAT-6 and CFP-10 proteins and their orthologues in M. smegmatis are 80 and 71%, respectively (Fig. 1), whereas M. leprae ESAT-6 shares a much lower amino acid sequence similarity with M. tuberculosis ESAT-6 of around 63%. Therefore, it is likely that these proteins also share epitopes that may result in cross-reactive T-cell responses. Furthermore, given the evolutionary history of the mycobacteria (13) and the presence of ESAT-6 and CFP-10 in M. smegmatis and other mycobacterial species, it is plausible that these genes would be present in the genomes of most other environmental mycobacteria. The homology between ESAT-6 and CFP-10 of the many environmental mycobacterial strains phylogenetically more closely related to M. tuberculosis may even be higher than that between the antigens of M. tuberculosis and M. smegmatis (Fig. 1). We believe that there is an urgent need to study the extent of amino acid sequence similarity between the ESAT-6 and CFP-10 proteins of different pathogenic and nonpathogenic environmental mycobacteria, as well as the influence of secreted ESAT-6 and CFP-10 from environmental mycobacteria on the T-cell responses from M. tuberculosis-infected individuals. Gamma interferon production in response to ESAT-6 and CFP-10 from environmental mycobacteria by peripheral blood mononuclear cells from infected patients has, to our knowledge, not been studied. This is surprising, given the fact that numerous studies have already been done on the use of these antigens as diagnostic tools (see, for example, references 1 to 3 and 12). Results are still needed which indicate that the host cellular immune response is able to distinguish between the ESAT-6 and CFP-10 proteins secreted from either environmental mycobacteria or M. tuberculosis.

FIG. 1.

FIG. 1.

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Alignment of the protein sequences of the ESAT-6 and CFP-10 orthologues from M. tuberculosis (M. tb.) and M. smegmatis (M. smeg.). Although studies have indicated the presence of multiple T-cell epitopes scattered throughout the ESAT-6 protein sequence (10, 11, 15), the positions of predominantly recognized epitopes are underlined (4, 10, 11, 12, 15). Asterisks indicate identical amino acid residues; colons and dots indicate conserved and semiconserved substitutions, respectively, according to their physiochemical criteria. aa, amino acid.

It is possible that the promising results obtained with ESAT-6 and CFP-10 in industrialized countries may be of less benefit to people living in developing countries where environmental mycobacteria are present in large amounts and where the real need for these tests lies.

REFERENCES

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Infect Immun. 2002 Nov;70(11):6509–6511.

Authors' Reply

A Geluk 1, T H M Ottenhoff 1,*, S M Arend 2, P Andersen 3, T M Doherty 3

We read with interest the letter of Dr. Gey van Pittius et al. in response to our recent paper (6). Both the ESAT-6 and CFP-10 antigens have over the years been analyzed extensively for their distribution in mycobacterial species (13), and their diagnostic relevance has been discussed (11). As Dr. Gey van Pittius et al. state, thus far homologous genes have been detected in a number of species outside the Mycobacterium tuberculosis complex. As a consequence, the expression of these antigens in other mycobacteria may in theory confound specific diagnosis of Mycobacterium tuberculosis and Mycobacterium bovis infections.

The question, however, is what consequences does this have for clinical and epidemiological practice in tuberculosis control. The presence of ESAT-6 and CFP-10 homologues in other species, for example, does not seem to confound the detection of M. tuberculosis-associated specific responses in the large number of studies conducted by different groups over the last 5 years (1-3, 9, 10). At present it remains unknown whether these genes are truly expressed in nonpathogenic as opposed to pathogenic mycobacteria and if the amino acid identity observed is enough to trigger a highly specific T-cell response. Particularly important for this discussion is the observation that T-cell responses to ESAT-6 and CFP-10 are apparently associated with active ongoing infection and as such have prognostic protential (5, 14). Therefore, T-cell responses to these antigens are presumably not associated with exposure to nonpathogenic strains such as M. smegmatis and M. scrofulaceum. Even in highly sensitive enzyme-linked immunospot assays that detect single ESAT-6-positive T cells, control individuals were negative (8). This conclusion is also supported by the many studies conducted in cattle where these reagent are highly specific indicators of ongoing M. bovis infection although cattle must be exposed daily to nonpathogenic mycobacteria from soil and natural water sources (4, 12, 14). That (rare) clinical infection with the two pathogenic strains M. marinum and M. kansasii, on the other hand, actually can trigger ESAT-6- and CFP-10-specific T-cell responses was recently convincingly demonstrated (S. M. Arend, K. E. van Meijgaarden, K. de Boer, E. Cerdá de Palou, D. van Soolingen, T. H. M. Ottenhoff, and J. T. van Dissel, submitted for publication). The same holds true for M. leprae as discussed above (6). While some caution may therefore be needed in the immunodiagnosis of clinical tuberculosis since infections with these three pathogens cannot be excluded by ESAT-6- and CFP-10-based tests, in practice only infections with M. kansasii may, though rarely, pose a differential diagnostic problem.

We agree with Dr. Gey van Pittius et al. that there is an urgent need for good diagnostic tools in the developing world. We assume that the major complicating factor for the application of reagents such as ESAT-6 and CFP-10 in the diagnosis of tuberculosis in countries of endemicity, however, is not the presence of environmental mycobacteria but the enormous reservoir of latent human tuberculosis (7).

Thus, despite the fact that antigens such as ESAT-6 and CFP-10 are not restricted to M. tuberculosis, they hold promise for the specific detection of M. tuberculosis infection.

REFERENCES

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