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. 1999 Jun;37(6):2111–2112. doi: 10.1128/jcm.37.6.2111-2112.1999

Rapid Immunochromatographic Assay for Diagnosis of Tuberculosis: Antibodies Detected May Not Be Specific

Roger Freeman 1,2, John Magee 1,2, Anne Barratt 1,2, Janice Wheeler 1,2, Michael Steward 1,2, Maureen Lee 1,2, Nigel Piggott 1,2
PMCID: PMC85054  PMID: 10383257

We read with interest the letter by Grobusch et al. (3) on a rapid immunochromatographic assay for the diagnosis of tuberculosis but wish to present evidence that this test may not be as specific as they portrayed it to be.

It was long thought that the 38-kDa antigen of Mycobacterium tuberculosis was confined to organisms of the M. tuberculosis complex (MTBC) (1). As suggested in the letter of Grobusch et al., it has therefore been assumed that detection of antibody to the 38-kDa antigen implies infection (although not necessarily disease) with M. tuberculosis, M. africanum, M. bovis, or M. microti. The lack of antibody development after M. bovis BCG vaccination has been ascribed to the lower expression of the 38-kDa antigen in this variant (9).

However, Thangaraj and colleagues reported in 1996 (8) the detection of genes encoding the 38-kDa antigen in a strain of M. intracellulare and the expression of the antigen in this species. They speculated that the possession of a homologue of the M. tuberculosis 38-kDa antigen by M. intracellulare might be associated with virulence, since M. intracellulare has the ability to cause disease in immunocompetent hosts, in distinction to M. avium, which does not possess the antigen. Thangaraj et al. did not detect the 38-kDa antigen or the appropriate gene sequence in M. avium, M. paratuberculosis, M. smegmatis, M. fortuitum, M. chelonei, M. leprae, M. kansasii, M. marinum, or M. vaccae. M. malmoense was not included in these investigations.

We have produced a recombinant 38-kDa antigen in Escherichia coli as a histidine-tagged fusion protein (7) and used this to raise a panel of 15 mouse monoclonal antibodies (MAbs) by using standard hybridoma technology (6). Selection was based on reactivity with the recombinant antigen by enzyme-linked immunosorbent assay and/or Western blotting of an M. tuberculosis lysate.

To further assess the specificity of the MAbs, they were screened against lysates of M. avium, M. intracellulare, M. kansasii, M. malmoense, M. vaccae, and M. xenopi. Upon immunoblotting, 2 of the 15 MAbs (7 and 29) reacted with a 38-kDa protein in M. intracellulare and 2 MAbs (1 and 7) reacted with a 38-kDa protein in M. malmoense (Fig. 1). All remaining MAbs were negative against all lysates. Twenty-one of 23 M. malmoense lysates examined by immunoblotting consistently reacted with both MAbs. The two isolates of M. malmoense not expressing the 38-kDa antigen are also atypical when examined by other taxonomic methods, including random amplified polymorphic DNA analysis (5).

FIG. 1.

FIG. 1

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Western blot of mycobacterial lysates with murine MAb 7 to recombinant 38-kDa antigen of M. tuberculosis. Lane M, low-molecular-mass standards (in kilodaltons; Bio-Rad); lanes 1 to 6; lysates of M. tuberculosis, M. avium, M. malmoense, M. kansasii, M. intracellulare, and M. xenopi, respectively. Note the presence of the 38-kDa antigen in M. tuberculosis, M. malmoense, and M. intracellulare.

Our results confirm the observations of Thangaraj et al. in relation to M. intracellulare and strongly suggest that M. malmoense also expresses a homologue of the 38-kDa antigen of M. tuberculosis. M. malmoense is a recognized primary pathogen of immunocompetent children and the elderly, and it has been estimated to cause up to 10% of tuberculosis-like pulmonary disease in the United Kingdom and other northwest European countries (2, 4).

We have yet to proceed with studies to detect the gene sequence in M. malmoense which encodes for the antigen. If such a sequence is found it will extend the argument of Thangaraj et al. (8) that possession of the antigen may be associated with virulence, demonstrating the presence of a common immunodominant antigen in the three species of mycobacteria (MTBC organisms, M. intracellulare, and M. malmoense) which cause disease in immunocompetent individuals. These findings may have important implications for vaccine studies.

However, it is also clear from our results and those of Thangaraj et al. that the detection of antibodies which react with the 38-kDa antigen of M. tuberculosis cannot be taken to indicate infection or disease due to MTBC organisms alone, since such antibodies may also be evoked by infection or disease with M. intracellulare or M. malmoense.

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J Clin Microbiol. 1999 Jun;37(6):2111–2112.

AUTHOR’S REPLY

Martin P Grobusch 1

Wheeler and colleagues provide interesting in vitro data suggesting that homologues of the M. tuberculosis 38-kDa antigen found in M. intracellulare and M. malmoense also lead to antibody production in humans. However, clinical data to support their findings are lacking so far. If, as speculated by Thangaraj et al. (1-4), mycobacterial virulence is enhanced by the possession of a homologue of the M. tuberculosis 38-kDa antigen, skepticism arises from the lack of demonstration of the antigen and the appropriate gene sequence, at least for M. kansasii, another species well capable of causing tuberculosis-like disease in immunocompetent individuals (1-2).

The sera of immunocompetent individuals investigated in our study (1-3) cited by Wheeler et al. were from patients with clinical signs and symptoms suggesting tuberculosis, and in all of those who were antibody positive, diagnosis was confirmed by culture—thus showing that in our small collective seropositivity indicated not merely the presence of the antigen (infection) but overt disease caused by M. tuberculosis. In one case, specimens growing non-M. tuberculosis mycobacteria stemmed from an immunocompetent seronegative patient with tuberculosis-like pulmonary disease caused by M. kansasii. Unfortunately there was no case of M. malmoense infection among our patients, which would have given at least anecdotal evidence for or against the notion of Wheeler et al. As reported, Cole et al. (1-1) and Zhou et al. (1-5) found specificities of 92 to 93% for the rapid immunochromatographic assay in large trials performed in China, but isolation of mycobacteria other than M. tuberculosis in those cases labelled false positive were not reported.

Further research should aim to elucidate whether the 38-kDa antigen homologues identified in various mycobacteria are a common feature of species capable of causing tuberculosis-like disease, and diagnostic trials should allow a judgement on whether human antibody response to the 38-kDa antigen should serve as an indicator for mycobacterial disease requiring treatment, rather than only for tuberculosis, in the nonimmunocompromised patient.

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