Overlapping peptides of Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 offer increased specificity over the purified protein derivative skin test when they were used in an ex vivo enzyme-linked immunospot (ELISPOT) assay for gamma interferon detection for the diagnosis of M. tuberculosis infection from recent exposure. We assessed whether equivalent results could be obtained for a fusion protein of the two antigens and whether a combined readout would offer increased sensitivity in The Gambia. We studied the ELISPOT assay results for 488 household contacts of 88 sputum smear-positive tuberculosis (TB) cases. The proportions of subjects positive by each test and by the tests combined were assessed across an exposure gradient, defined according to sleeping proximity to a TB case. Eighty-eight (18%) subjects were positive for CFP-10 peptides, 148 (30%) were positive for ESAT-6 peptides, 161 (33%) were positive for both peptides, and 168 (34%) were positive for the fusion protein; 188 (39%) subjects had either a positive result for a peptide or a positive result for the fusion protein. There was reasonable agreement between the peptide and the protein results (kappa statistic ⴝ 0.78) and no significant discordance (P ⴝ 0.38). There was a strong correlation between the fusion protein and combined peptide spot counts (r ⴝ 0.9), and responses to the peptide and the proteins all increased significantly according to M. tuberculosis exposure. The proportion of subjects positive for either the pool of peptides or the fusion protein offered maximum sensitivity, being significantly higher than the proportion of subjects positive for ESAT-6 peptides alone (P ⴝ 0.007). A fusion protein of ESAT-6 and CFP-10 is equivalent to overlapping peptides for the diagnosis of latent M. tuberculosis infection. Use of a combination of peptides and fusion protein offers improved sensitivity.
The diagnosis of childhood active tuberculosis (aTB) and latent Mycobacterium tuberculosis (M. tuberculosis) infection (LTBI) remains a challenge, and the replacement of tuberculin skin tests (TST) with commercialized gamma interferon (IFN-␥) release assays (IGRA) is not currently recommended. Two hundred sixty-six children between 1 month and 15 years of age, 214 of whom were at risk of recent M. tuberculosis infection and 51 who were included as controls, were prospectively enrolled in our study. According to the results of a clinical evaluation, TST, chest X ray, and microbiological assessment, each children was classified as noninfected, having LTBI, or having aTB. Long-incubation-time purified protein derivative (PPD), ESAT-6, and CFP-10 IGRA were performed and evaluated for their accuracy in correctly classifying the children. Whereas both TST and PPD IGRA were suboptimal for detecting aTB, combining the CFP-10 IGRA with a TST or with a PPD IGRA allowed us to detect all the children with aTB with a specificity of 96% for children who were positive for the CFP-10 IGRA. Moreover, the combination of the CFP-10 IGRA and PPD IGRA detected 96% of children who were eventually classified as having LTBI, but a strong IFN-␥ re- sponse to CFP-10 (defined as >500 pg/ml) was highly suggestive of aTB, at least among the children who were <3 years old. The use of long-incubation-time CFP-10 IGRA and PPD IGRA should help clinicians to quickly identify aTB or LTBI in young children.
Regarding the ESAT-6/CFP-10 ICA, all of the 10 false- negative and 7 false-positive cultures were assayed again 1 week after the initial assay. In addition, since M. kansasii, M. szulgai, and M. marinum were reported to secrete ESAT-6 and CFP-10, cultures of these NTM species might show cross- reactivity with the ESAT-6/CFP-10 ICA (22). Thus, the other 16 M. kansasii-containing cultures (9 type 1 and 7 type 2) and the 1 M. szulgai-containing culture that were ESAT-6/CFP-10 ICA negative also underwent another strip assay 1 week after the initial assay. During this week, these cultures remained in the BACTEC MGIT 960 system for incubation. The results showed that, of the 10 false-negative MTC cultures, 9 were positive but 1 was still negative with the repeat strip assay. The method of Bakshi et al. (1) was then used to further identify whether these 10 MTC cultures contained M. bovis, M. tuber- culosis, or other MTC members. The results indicated that the culture that was negative in both initial and repeat assays contained M. bovis, while the other nine cultures negative with the initial but positive with the repeat assay contained M. tuberculosis.
NTM in cattle in Great Britain. Only a small percentage of mycobacterial isolates obtained from the tissues of cattle slaughtered under the test and slaughter control policy in Great Britain are NTM (ca. 0.8% of total isolates from 2004 to 2006). The majority of these NTM isolates belong to the M. avium/M. intracellulare (MAI) complex (for example, 54% of the NTM isolates in Great Britain from 2004 to 2006). The remaining NTM isolates remained unclassified or comprised a number of different mycobacterial species. Recently, the spe- cies of 46 non-MAI NTM isolates (5 from cows with visible lesions typical of BTB and 41 from cattle presenting without visible lesions postmortem) were determined by using the Hain genotype system (22). The results indicated that 22/46 of the non-MAI NTM isolates tested were M. kansasii, including iso- lates from two of five of the lesioned animals. Genotyping revealed that all M. kansasii isolates found in these cattle were subtype I or II, which could be isolated from humans in a study in Holland (4). The other species found were M. celatum (5/ 46); M. nonchromogenicum (5/46); M. gordonae (5/46); M. szul- gai (4/46); M. fortuitum (2/46); and M. scrofulaceum, M. inter- medium, and M. shimodei (1/46 each). These results do not constitute the findings of a representative study of NTM iso- lates in Great Britain. They do demonstrate, however, that M. kansasii can be isolated from cattle tissues. As stated above, apart from M. kansasii, M. gordonae and M. szulgai were also isolated, and these species also contain genes for ESAT-6 homologues (4) and most likely also genes for CFP-10 homo- logues. Therefore, these three NTM species can potentially express these cross-reactive antigens. M. kansasii isolates have also been identified in an analysis of NTM cattle isolates from Northern Ireland, although the dominant NTM species were part of the MAI complex (46%) and the M. terrae complex (39%), with 8% of the isolates being M. kansasii (20). TABLE 1. Peptides used in this study
The “gold standard” for the diagnosis for TB is a combina- tion of clinical and radiologic examination and microbiological tests, both microscopic and cultural (the last one can take up to 8 weeks, and in 10 to 20% of cases Mycobacterium tuberculosis is not successfully cultured) (1). The tuberculin skin test (TST), using purified protein derivative (PPD), is largely utilized for both diagnosis and screening. However, the greatest drawback of PPD is its broad cross-reactivity with antigens (Ags) derived from several mycobacterial species, e.g., attenuated M. bovis bacillus Calmette-Gue´rin (BCG) used for vaccination, greatly decreasing the specificity of the TST (10). Moreover, 10 to 25% of TB patients have a negative TST result, and this per- centage increases up to 50% in patients with advanced disease or with immunodeficiency due to HIV coinfection (12, 20, 21). M. tuberculosis infection evokes a strong cell-mediated im- mune response, and detection of specific T cells might be a means to detect infection (17, 39). However, the problem of the assays currently used is the lack of well-defined specific Ags. Recently, two secretory, low-molecular-mass proteins, early secreted antigenic target 6 (ESAT-6) and culture filtrate pro- tein 10 (CFP-10) have been identified, and the corresponding genes have been coded (7, 38). These genes belong to the RD-1 genomic region of the M. tuberculosis complex, being absent in all vaccine strains of BCG and in environmental
An alternative approach to using recombinant proteins is the application of synthetic peptides derived from antigens such as those described above. Synthetic peptides have the advantages of lower production costs and easier standardization and qual- ity control, and they carry no risk of infection since they are fully chemically synthesized. Encouragingly, we have recently provided proof of principle that the development of peptide- based reagents is a feasible approach by demonstrating that a pool of seven peptides derived from ESAT-6, MPB70, MPB83, and MPB64 differentiates between infected and BCG-vacci- nated cattle. However, further improvement to the sensitivity of this peptide-based reagent was required before the levels of sensitivity would be acceptable for the implementation of testing and slaughter-based strategies based on such reagents (50, 51). The objective of the present study was to develop an im- proved peptide-based diagnostic reagent. Since MPB70 and MPB83 have shown promise as candidates for DNA-based subunit vaccination (9, 34, 52), we decided to concentrate our efforts on developing diagnostic reagents based on the antigens ESAT-6 and CFP-10. ESAT-6 is a well-studied antigen found in short-term culture filtrates of pathogenic mycobacteria of the M. tuberculosis complex. It has been shown to be able to discriminate between infected and BCG-vaccinated guinea pigs and humans (16, 28, 45, 47) and also to discriminate between M. bovis-infected cattle and cattle sensitized with en- vironmental mycobacteria (39). CFP-10 has been also identi- fied in the low-molecular-mass fraction of culture filtrate. The genes encoding CFP-10 and ESAT-6 are adjacent on the ge- nome and are transcribed together. Both encode small ex- ported proteins and share some degree of homology at the DNA level (5). Both are therefore members of the so-called ESAT-6 family of small mycobacterial proteins (5, 11, 46). Like ESAT-6, CFP-10 is recognized by M. tuberculosis-infected guinea pigs and humans but not by individuals vaccinated with BCG (2, 45, 49). In addition, it is also recognized by lympho-
Purified PCR products were subjected to two-step digestion with Xho I/Hind III and Not I/Xho I restriction enzymes for ESAT-6 and CFP-10, respectively. The purified, digested PCR products were ligated into pcDNA3.1(+). Standard techniques for these steps such as plasmid DNA preparation, ligation, competent cell preparation, and transformation were followed as described previously (24). Competent E. coli TOP10 cells (Invitrogen, Carlsbad, CA) were transformed with pcDNA3.1+/ESAT-6 and pcDNA3.1+/CFP-10 using a transformation kit (Fermantas, Lithuania). Ampicillin-resistant cultures were grown in LB medium containing 50 µl/ml of ampicillin at 37 o C with shaking until OD at 600 nm = ~ 0.5. The fidelity of the transformants was verified by PCR using ESAT-6 and CFP-10-specific primers that had previously been used for amplification. The plasmids were purified and restriction enzyme-digested. After electrophoresis on a 1.5% agarose gel, the purified plasmids were sequenced (Seq Lab, microgen).
on CD4 T cells after stimulation with the antigens tuberculin pu- rified protein derivative (PPD), 6-kDa early secretory antigenic target (ESAT-6) and 10-kDa culture filtrate protein (CFP-10), and HBHA by using ICS. We quantified the responding cells as both a proportion of CD4 cells and the absolute number of CD4 cells circulating in the blood, to determine if there were particular com- binations of surface markers and cytokine staining that could dis- criminate subjects with active from those with latent TB. FIG 1 Flow cytometric gating strategy. Representative flow cytometric plots for an individual subject are shown to detail the gating strategy used. The top row details the CD154 ⫹ TNF- ␣ ⫹ region for cells gated positive for CD3 and CD4 and negative for the dump channel (Live/Dead fixable dye positive and CD14 ⫹ CD16 ⫹ CD19 ⫹ ), stimulated with PPD or with no stimulus. The middle row details cytokine and CD27 staining on the PPD-stimulated cells positive for CD154 and TNF- ␣ . The bottom row shows CD27 and CXCR3 staining on the total CD4 T cells (left) and the PPD-stimulated CD154 ⫹ TNF- ␣ ⫹ cells (right).
Secreted proteins of M. tuberculosis have been reported as rich source of immunogens (8). In the last decades, these proteins have gained specific attention as virulence factors, vaccine and diagnostic candidates (3, 9-13). Although there are highly immunogenic proteins belonged to the 23-membered Esx family in mycobacterial culture filtrate, ESAT-6 and CFP- 10 are absent from all Mycobacterium bovis BCG vaccine strains (14) and induce potent Th 1 responses
At present we cannot predict who or how many of the ESAT-6- or CFP-10-responsive non-TB patients will develop TB. We have previously shown an association between ESAT-6 responsiveness and later progression to TB in a group of healthy exposed contacts (9). In the present study, 10 QFT- RD1 responsive non-TB patients did not develop active TB within the observation period of almost 2 years, but 1 patient with pneumonia and pericarditis, who was initially categorized as a non-TB patient, progressed clinically and developed mil- iary TB after having received intensive immunosuppressive treatment. The patient who developed TB was QFT-RD1 pos- itive when presenting with symptoms 3 months before pro- gressing to miliary TB, but at that time active TB was not found despite intensive investigations (24). This case suggests that RD- 1-responsive patients may be at risk of progressing to active TB during immune suppression. The use of immune suppression with biological agents like anti-tumor necrosis factor alpha antibodies is increasing, and reactivation of LTBI is one of the most feared side effects (14). Screening for LTBI with the QFT-RD1 test may turn out to be extremely useful in this situation.
hemoagglutinin (mitogen-positive control), or the specific antigens (ESAT-6 and CFP-10) in separate wells. The number of IFN-␥-releasing T cells in each well was then quantified using an automated ELISPOT imager. Results were ex- pressed as the number of spot-forming cells (SFCs). A positive antigen-specific result is defined as a well containing at least six SFCs more than the negative control. The presence of a satisfactory reaction (⬎20 SFCs) to the mitogen- positive control demonstrates T-cell function and also validates the assay result. An indeterminate result was reported when high background levels prevented interpretation or when less than 20 SFCs were detected in the positive control wells. No personal identifiers or clinical histories were available to laboratory personnel. After the blood samples for the T-SPOT.TB assay were collected,
A new whole-blood interferon-gamma (IFN- ␥ ) assay has been approved by the U.S. Food and Drug Administration for the detection of LTBI and is commercially available as QuantiFERON-TB-GOLD (37). This assay detects in vitro cell-mediated immune responses to TB infection by quantify- ing the amount of the IFN- ␥ that is released in the plasma of whole blood incubated overnight with mixtures of overlapping peptides spanning the sequence of the early-secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10) (QuantiFERON-TB-GOLD; Cellestis Ltd., Carnegie, Victoria, Australia). These antigens are the products of a genomic region (RD1) that is present in all M. tuberculosis and pathogenic M. bovis strains but absent in all BCG vaccine strains and nearly all MOTT (6, 25). Consequently, IFN- ␥ assays utilizing ESAT-6 and CFP-10 either as recombinant antigens (5, 7, 38) or mixtures of overlapping peptides (8, 27, 37) have been shown to be significantly more specific than TST. In addition, these assays offer several advantages over TST, including the need for only a single patient contact, simultaneous measurement of a subject’s immune reactivity to mitogen (phytohemagglutinin) and nil control (normal saline) antigens, elimination of subjectivity in administering the test or reading the result, and the availability of test results within 24 h. IFN- ␥ assays utilizing ESAT-6 and CFP-10 antigens have been studied in the general population in several countries * Corresponding author. Mailing address: Department of Family
Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN- ␥ )-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental non- tuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis- infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF- ␣ ), IFN- ␥ , and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding re- sponses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN- ␥ and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN- ␥ responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.
The mycobacterial immunodominant ESAT-6 and CFP-10 antigens are strongly recognizable in tuberculosis-infected cattle, and they do not elicit a response in cattle without infection. In addition, they are absent in most environmental mycobacterial spe- cies, and therefore, their use can be an alternative to purified protein derivative (PPD) tuberculin in the development of a more specific skin diagnostic test in cattle. The aim of the current study was to assess the potential of an ESAT-6 and CFP-10 (E6-C10) protein cocktail in a skin test format in naturally tuberculosis-infected and paratuberculosis-infected cattle. We also included MPB83 as a third component in one of the protein cocktail preparations. The protein cocktail was tested at different dose con- centrations (5, 10, and 15 g per protein). The best skin response to the E6-C10 protein cocktail was obtained with 10 g. Subse- quently, this concentration was tested in 2 herds with high and low bovine tuberculosis prevalence, the latter with paratubercu- losis coinfection. Our data show that the E6-C10 cocktail allows identification of an important proportion of animals that PPDB is not able to recognize, especially in low-prevalence herds. The protein cocktail did not induce reactions in tuberculosis-free cattle or in paratuberculosis-infected cattle. Addition of MPB83 to the protein cocktail did not make any difference in the skin reaction.
CSF (5ml) by lumbar puncture and heparinised peripheral venous blood (10ml) were collected from each of these patients at the time of the initial testing, at admission. Samples for ELISPOT assay were processed within 6 hours of collection. If collected overnight, they were kept in an air-conditioned room and sent to the lab for processing in the morning. All CSF samples were also subjected to estimation of sugar, protein content and to microscopy of centrifuged CSF with Gram, Ziehl-Neelsen and India ink stains, with PCR for mycobacterium tuberculosis (using IS6110 primers), and bacterial, mycobacterial and fungal culture.
cell counts was determined. The median value of these ratios for both assays, RD1 peptides and proteins, was sig- nificantly higher in the active TB group than that in the control group (Table 2). Then we explored the possibility of using this approach to better identify the patients with active TB. We used the ratio data to perform a receiver- operator characteristic (ROC) analysis, using the groups with and without active disease as comparator groups (Figure 4A–B). Significant results for area under the curve (AUC) analysis were obtained for the sum of ESAT-6 and CFP-10 peptides (AUC, 0.88; P = 0.0009; 95% CI, 0.72–1.0). Interestingly, a ratio higher than 0.21 predicted active TB with 100% sensitivity (95% CI, 81%–100%) and 80% specificity (95% CI, 44%–97%) (Figure 5A). In addition when considering the sum of ESAT-6 and CFP- 10 proteins, (AUC, 0.77; P = 0.01; 95% CI, 0.57–0.97), a ratio higher than 0.22 predicted active TB with 89% sensi- tivity (95% CI, 81%–100%) and 70% specificity (95% CI, 34%–93%) (Figure 5B). No statistically significant differ- ence was found between the AUC of the RD1 peptides and protein assay (p = 0.4), although the accuracy of the RD1 peptide test was higher.
could enhance the DTH reaction and improve the sensitivity (13/ 14) of the skin test based on the cocktail of CFP-10 and ESAT-6 (6, 14). These data indicated that the M. bovis-specific proteins used as stimuli in the skin test are promising. However, thus far there has not been a large-scale clinical assessment in the field to dem- onstrate feasibility and validation in a double-blind testing (6, 15). In this study, we chose three Mycobacterium-specific proteins, namely, TB10.4, MPT63, and Rv3872 (Table 1), to supplement CFEP-10 and ESAT-6 in order to strengthen the sensitivity of the skin test. TB10.4 is encoded by esxH, which exists in both Myco- bacterium tuberculosis complex and Mycobacterium avium com- plex (MAC), and can induce higher levels of IFN- ␥ in TB patients than ESAT-6 and CFP-10 (18, 19). Thus, TB10.4 has potential to enhance the sensitivity of the skin test but may cause a loss of specificity. MPT63 and Rv3872 can induce a high level of IFN-␥ release in TB patients but not in MAC-infected persons or healthy ones (20–22), so the addition of MPT63 and Rv3872 may increase the sensitivity of the skin test and not sacrifice specificity. Accord- ing to the characteristics of these proteins, a CFP-10/ESAT-6/ TB10.4 protein cocktail and a CFP-10/ESAT-6/Rv3872/MPT63 protein cocktail were prepared and used as stimuli in the skin test. Our present study was to assess the efficiency of protein cocktail- based skin tests in a total of 1,097 cattle, with the tuberculin skin test and IFN- ␥ release assay as references.
pects. In addition, high sensitivity is also crucial in order to identify residual infections. The IFN- ␥ assay is accepted to be more sensitive than the skin test (12, 34), thus indicating that it would be beneficial to use the assay as a primary screening test. Its level of specificity in regions with a low incidence of bTB has been reported to be too low with the standard PPD- based assay; however, by use of specific antigens, the assay may be adapted to provide a highly specific and sensitive screening test, which could then be used as a stand-alone test or in conjunction with other tests (9, 29). There has been a long search for antigens having potential as diagnostic markers of bTB (1, 10, 14, 29). In a recent study, Rv3615c stimulated responses in M. bovis-infected animals not responding to ESAT-6 and CFP-10 (four of seven animals). Diagnostics for bTB require a panel of antigens; so far, no single antigen has been identified which could be used as an efficient diagnostic immunoreagent. The cellular response to individual antigens is known to vary between animals and to change over time during the course of infection. These findings may be related to the turnover of mycobacterial growth in vivo (data suggest that there are intervals of 5 to 7 weeks between peaks of cellular activity) or to the sequestration of reactive clones, resulting in periodical recognition in peripheral blood (33). Similar mech- anisms may also explain the one bTB-positive animal which was not detected with the combination of OmpATb, ESAT-6, and CFP-10 in our study. This animal had been tested repeat- edly with the IFN- ␥ assay before our study: 5 months before our sampling, it was positive with PPD and OmpATb but negative with ESAT-6 and CFP-10, and 8 months before our sampling, it was negative with the PPD-based IFN- ␥ assay (alternative antigens were not tested at that time point).
Antigens. Bovine and avian PPDs were supplied by the VLA Tuberculin Production Unit. Histidine-tagged recombinant proteins were expressed in Esch- erichia coli cells and purified by nickel affinity chromatography. MPB70, MPB83, ESAT-6, and CFP-10 were supplied by Lionex Diagnostics and Therapeutics GmbH (Germany), and Rv3615c was produced at the Agri-Food and Biosciences Institute (Belfast, Northern Ireland). Rv3615c is referred to in the current paper using the M. tuberculosis genome annotation (http://genolist.pasteur.fr /TubercuList/). In the M. bovis genome, it is annotated as Mb3645c (http: //genolist.pasteur.fr/BoviList/). All synthetic peptides used in the study were synthesized by solid-phase F-moc (9H-fluoren-9-yl-methoxycarbonyl) chemistry and supplied at a purity of ⬎90% (Pepceuticals Ltd., United Kingdom). The identity of each peptide was confirmed by mass spectrometry. For ESAT-6 and CFP-10, 16-mer peptides with 8-amino-acid overlaps of the complete protein sequences were used (21 peptides in total). For MPB83, a single 20-mer peptide that had previously been shown to encode a bovine T-cell epitope (p195-214) was used (24), and similarly, for Rv3615c, three bovine T-cell epitopes encoding 20-mer peptides were used (p65-84, p73-92, and p84-103) (18).
presumptive for M. tuberculosis complex, evoking further diag- nostic and epidemiological investigations and action. The use of defined antigens (e.g., early secretory antigenic target-6 [ESAT-6] and culture filtrate protein-10 [CFP-10]) generally enhances the differentiation of immune responses to M. tuber- culosis complex organisms from those elicited by nontubercu- lous mycobacteria (2, 5, 8, 33, 34, 36, 40, 45). The esat-6 and cfp-10 genes are located in region of difference 1, an area of the virulent M. tuberculosis complex genome not present in the vaccine strain M. bovis bacille Calmette-Gue ´rin (BCG) and most other nontuberculous mycobacteria. Unfortunately, the esat-6 and cfp-10 genes as well as the esat-6 family genes TB10.4, TB10.3, and TB12.9 are also present in M. kansasii and are of significant homology (88% to 90% nucleotide homology and ⬎ 95% amino acid homology) to the respective genes within M. tuberculosis complex organisms (2, 18, 19, 21, 37, 39). Additionally, M. kansasii contains a gene encoding MPB83 (41), a cell surface immunodominant antibody target used for the “specific” diagnosis of M. bovis infection of cattle (28) and several other wildlife reservoirs (e.g., the Eurasian badger  and white-tailed deer ). It may be anticipated that sensiti- zation and/or infection with M. kansasii would result in anti- body and cell-mediated responses that potentially confound the interpretation of TB tests based on either specific antigens (i.e., ESAT-6, CFP-10, or MPB83) or complex antigens (e.g., tuberculins, whole-cell sonicates, or culture filtrates). Indeed, humans with clinical disease resulting from M. kansasii infec- tion have detectable IFN- ␥ concentration responses to recom- binant and peptide mixes of ESAT-6 and CFP-10, albeit at a much lower rate and level than M. tuberculosis-infected indi- viduals (4). The impact of M. kansasii sensitization without the induction of clinical disease on TB diagnostic tests, however, is unclear.