Field application of serodiagnostics to identify elephants with tuberculosis prior to case confirmation by culture

Three serologic methods for antibody detection in elephant tuberculosis (TB), the multiantigen print immunoassay (MAPIA), ElephantTB STAT-PAK kit, and DPP VetTB test, were evaluated using serial serum samples from 14 captive elephants infected with Mycobacterium tuberculosis in 5 countries. In all cases, serological testing was performed prior to the diagnosis of TB by mycobacterial culture of trunk wash or tissue samples collected at necropsy. All elephants produced antibody responses to M. tuberculosis antigens, with 13/14 recognizing ESAT-6 and/or CFP10 proteins. The findings supported the high serodiagnostic test accuracy in detecting infections months to years before M. tuberculosis could be isolated from elephants. The MAPIA and/or DPP VetTB assay demonstrated the potential for monitoring antimycobacterial therapy and predicting TB relapse in treated elephants when continuously used in the posttreatment period. History of exposure to TB and past treatment information should be taken into consideration for proper interpretation of the antibody test results. Data suggest that the more frequent trunk wash culture testing of seropositive elephants may enhance the efficiency of the TB diagnostic algorithm, leading to earlier treatment with improved outcomes.     

Highly Accurate Antibody Assays for Early and Rapid Detection of Tuberculosis in African and Asian Elephants

Tuberculosis (TB) in elephants is a reemerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current methods for screening and diagnosis rely on trunk wash culture, which has serious limitations due to low test sensitivity, slow turnaround time, and variable sample quality. Innovative and more efficient diagnostic tools are urgently needed. We describe three novel serologic techniques, the ElephantTB Stat-Pak kit, multiantigen print immunoassay, and dual-path platform VetTB test, for rapid antibody detection in elephants. The study was performed with serum samples from 236 captive African and Asian elephants from 53 different locations in the United States and Europe. The elephants were divided into three groups based on disease status and history of exposure: (i) 26 animals with culture-confirmed TB due to M. tuberculosis or Mycobacterium bovis, (ii) 63 exposed elephants from known-infected herds that had never produced a culture-positive result from trunk wash samples, and (iii) 147 elephants without clinical symptoms suggestive of TB, with consistently negative trunk wash culture results, and with no history of potential exposure to TB in the past 5 years. Elephants with culture-confirmed TB and a proportion of exposed but trunk wash culture-negative elephants produced robust antibody responses to multiple antigens of M. tuberculosis, with seroconversions detectable years before TB-positive cultures were obtained from trunk wash specimens. ESAT-6 and CFP10 proteins were immunodominant antigens recognized by elephant antibodies during disease. The serologic assays demonstrated 100% sensitivity and 95 to 100% specificity. Rapid and accurate antibody tests to identify infected elephants will likely allow earlier and more efficient treatment, thus limiting transmission of infection to other susceptible animals and to humans.Tuberculosis (TB) in captive elephants has been recognized as a reemerging zoonotic disease since at least the 1960s (22, 24, 26). In the past decade, growing numbers of elephant TB cases due to Mycobacterium tuberculosis or Mycobacterium bovis have been reported, presumably as a result of increased surveillance (11, 16, 25, 28). North American populations of African (Loxodonta africana) and Asian (Elephas maximus) elephants are declining, while captive breeding is historically poor. Efforts to maintain a self-sustaining captive population are hindered by TB-related issues. Many infected elephants may never exhibit clinical signs, whereas others with progressive disease may do so only in the terminal stages, thus making it difficult to recognize TB early (22, 24). Shedding of organisms during the preclinical period results in environmental contamination and presents a high risk of transmission to humans, elephants, and other mammals (11, 20, 27, 31).The diagnostic value of the only existing antemortem testing method (i.e., culture of trunk wash samples) officially recommended by the United States Department of Agriculture (USDA) is limited by poor accuracy, slow turnaround time for sample processing, variable specimen quality, and sample acquisition logistics (11, 16, 25). Antibody detection assays have shown promising potential for identification of elephants infected with M. tuberculosis or M. bovis (10, 16). The new version of the Guidelines for the Control of Tuberculosis in Elephants 2008 (4) was recently approved by the United States Animal Health Association TB Committee. The document, including certain serologic tests, along with trunk wash culture for routine surveillance, is currently under USDA review to be adopted. This study describes the use of three novel serologic techniques (the ElephantTB Stat-Pak kit, multiantigen print immunoassay [MAPIA], and the dual-path platform [DPP] test) designed for early and accurate detection of TB in elephants.     

Antigens of Mycobacterium tuberculosis recognized by antibodies during incipient, subclinical tuberculosis

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of approximately 12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (approximately 90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.     

PrimaTB STAT-PAK assay, a novel, rapid lateral-flow test for tuberculosis in nonhuman primates

Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP against Mycobacterium tuberculosis to identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated with M. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP.     

Antigens of Mycobacterium tuberculosis Recognized by Antibodies during Incipient, Subclinical Tuberculosis

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of ~12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (~90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.     

Antibody responses in reindeer (Rangifer tarandus) infected with Mycobacterium bovis

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.     

Genomic approach to identification of Mycobacterium bovis diagnostic antigens in cattle

Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10(6) CFU) and a low (10(4) CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individual antigens varied greatly over time. However, combining the results for ESAT-6, CFP-10, and TB27.4, possibly supplemented with TB10.4, gave sensitivities at different infection stages close to those obtained with M. bovis purified protein derivative. Importantly, while responsiveness to ESAT-6 and CFP-10 correlated strongly for individual samples, the same was not the case for ESAT-6 and TB27.4 responsiveness. The results suggest that combinations of specific antigens such as these have great potential in development of optimized diagnostic systems for bovine tuberculosis.     

Development of new vaccines and diagnostic reagents against tuberculosis

Tuberculosis (TB) is a major infectious disease problem with one-third of the world population infected, 8 million people developing the active disease and 2 million dying of TB each year. The attenuated Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only available vaccine against TB. However, the trials conducted in different parts of the world have shown that this vaccine doe not provide consistent protection against TB. The purified protein derivative (PPD) of Mycobacterium tuberculosis is the commonly used reagent for the diagnosis of TB. However, PPD lacks specificity because of the presence of antigens crossreactive with M. bovis BCG and other mycobacteria. The studies to identify M. tuberculosis antigens and epitopes as candidates for new protective vaccines and specific diagnostic reagents against TB have led to the identification and characterization of several major antigens of M. tuberculosis including heat shock proteins (hsp) and secreted antigens present in the culture filtrate (CF) of M. tuberculosis. Some of these antigens have shown promise as new candidate vaccines (hsp60, Ag85 and ESAT-6, etc.) and specific diagnostic reagents (ESAT-6 and CFP10, etc.) for TB. Moreover, in the mouse model of TB, vaccination with DNA-hsp60 has immunotheraputic effects and helps in eradication of persisters. In addition, identification of proper adjuvant and delivery systems has shown the promise to overcome the problem of poor immunogenicity associated with subunit and peptide based vaccines. More recently, the comparison of the genome sequence of M. tuberculosis with M. bovis BCG and other mycobacteria has led to the identification of M. tuberculosis-specific genomic regions. Evaluation of these regions for encoding proteins with immunological reactivity can lead to the identification of additional antigens of M. tuberculosis useful as new vaccines and reagents for specific diagnosis of TB.     

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovis BCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.     

Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.     

Comparison of Antigen-Specific T-Cell Responses of Tuberculosis Patients using Complex or Single Antigens of Mycobacterium tuberculosis

We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-γ (IFN-γ) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (rGroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis . The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M . tuberculosis , M . tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well as the vaccine strain, Mycobacterium bovis bacillus Calmette–Guérin (BCG). In addition, M . tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN-γ secretion showed that the most frequently recognized antigen was ESAT-6, followed by MPT59, GroES, MPB70, MPT64, DnaK, GroEL and PstS. The frequency of ESAT-6 responders, as measured both by proliferation (18/19) and secretion of IFN-γ (16/19) was comparable to the results obtained with whole-cell M . tuberculosis , MT-CF and M . bovis BCG. We also observed that most of the high responders to complex antigens recognized all of the antigens tested (covariation), demonstrating that the repertoire of human T-cell specificities induced by natural infection is directed towards several unrelated culture filtrate as well as somatic-derived protein antigens. In conclusion, the results obtained suggest that the cellular immune response in humans is directed against several important target antigens of M . tuberculosis and that some antigens, such as ESAT-6, are recognized by a high number of individuals. Such antigens represent candidates to be used for development of specific diagnostic reagents or in subunit vaccines.     

Association of strong immune responses to PPE protein Rv1168c with active tuberculosis

Accurate diagnosis of tuberculosis (TB) infection is critical for the treatment, prevention, and control of TB. Conventional diagnostic tests based on purified protein derivative (PPD) do not achieve the required diagnostic sensitivity. Therefore, in this study, we have evaluated the immunogenic properties of Rv1168c, a member of the PPE family, in comparison with PPD, which is routinely used in the tuberculin test, and Hsp60 and ESAT-6, well-known immunodominant antigens of Mycobacterium tuberculosis. In a conventional enzyme immunoassay, the recombinant Rv1168c protein displayed stronger immunoreactivity against the sera obtained from patients with clinically active TB than did PPD, Hsp60, or ESAT-6 and could distinguish TB patients from Mycobacterium bovis BCG-vaccinated controls. Interestingly, Rv1168c antigen permits diagnosis of smear-negative pulmonary TB as well as extrapulmonary TB cases, which are often difficult to diagnose by conventional tests. The immunodominant nature of Rv1168c makes it a promising candidate to use in serodiagnosis of TB. In addition, our studies also show that Rv1168c is a potent T-cell antigen which elicits a strong gamma interferon response in sensitized peripheral blood mononuclear cells obtained from TB patients.     

Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle.

Tuberculosis continues to be a worldwide health problem for both humans and animals. The development of improved vaccines and diagnostic tests requires detailed understanding of the immune responses generated and the antigens recognized during the disease. This study examined the T-cell response which develops in cattle experimentally infected with Mycobacterium bovis. The first significant T-cell response was found 3 weeks after the onset of infection and was characterized by a pronounced gamma interferon (IFN-gamma) response from peripheral blood mononuclear cells directed to antigens in culture filtrates. Short-term culture filtrate (ST-CF) was separated into molecular mass fractions and screened for recognition by T cells from experimentally infected and field cases of bovine tuberculosis. Cattle in the early stages of experimental infection were characterized by strong IFN-gamma responses directed predominantly toward the lowest-mass (<10-kDa) fraction of ST-CF, but cattle in later stages of experimental infection (16 weeks postinfection) exhibited a broader recognition of antigens of various molecular masses. Field cases of bovine tuberculosis, in comparison, preferentially recognized low-mass antigens, characteristic of animals in the early stages of infection. The major T-cell target for this dominant IFN-gamma response was found to be the secreted antigen ESAT-6. This antigen was recognized strongly by the majority of field cases of bovine tuberculosis tested. As ESAT-6 is unique to pathogenic mycobacterial species, our study suggests that ESAT-6 is an antigen with major potential for vaccination against and specific diagnosis of bovine tuberculosis.     

Plasma antibody profiles as diagnostic biomarkers for tuberculosis

Two billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), worldwide. Ten million to 20 million of the infected individuals develop disease per year. TB is a treatable disease, provided that it is diagnosed in a timely manner. The current TB diagnostic methods are subjective, inefficient, or not cost-effective. Antibody-based blood tests can be used efficiently and cost-effectively for TB diagnosis. A major challenge is that different TB patients generate antibodies against different antigens. Therefore, a multiplex immunoassay approach is needed. We have developed a multiplex panel of 28 M. tuberculosis antigen-coated microbeads. Plasma samples were obtained from over 300 pulmonary TB patients and healthy controls in a country where TB is endemic, Pakistan. Multiplex data were analyzed using computational tools by multivariate statistics, classification algorithms, and cluster analysis. The results of antibody profile-based detection, using 16 selected antigens, closely correlated with those of the sputum-based diagnostic methods (smear microscopy and culture) practiced in countries where TB is endemic. Multiplex microbead immunoassay had a sensitivity and specificity of approximately 90% and 80%, respectively. These antibody profiles could potentially be useful for the diagnosis of nonpulmonary TB, which accounts for approximately 20% of cases of disease. Since an automated, high-throughput version of this multiplex microbead immunoassay could analyze thousands of samples per day, it may be useful for the diagnosis of TB in millions of patients worldwide.     

PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family

Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.     

Immunological crossreactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis

Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette-Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP-10 in M. leprae (ML0050) is identified and characterized. Interferon-gamma production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP-10 of M. leprae is a potent antigen that crossreacts with CFP-10 of M. tuberculosis at the T-cell level. This crossreactivity has implications for the use of CFP-10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.     

Antigens of Mycobacterium tuberculosis expressed during preclinical tuberculosis: serological immunodominance of proteins with repetitive amino acid sequences

Four antigens of Mycobacterium tuberculosis that are expressed in vivo after aerosol infection but prior to the development of clinical tuberculosis (TB) in rabbits were identified by immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained 5 weeks postinfection. Three of the proteins identified, PirG (Rv3810), polymorphic GC-repetitive sequence (PE-PGRS; Rv3367), and proline-threonine repetitive protein (PTRP) (Rv0538), have multiple tandem repeats of unique amino acid sequences and have characteristics of surface or secreted proteins. The fourth protein, MtrA (Rv3246c), is a response regulator of a putative two-component signal transduction system, mtrA-mtrB, of M. tuberculosis. All four antigens were recognized by pooled sera from TB patients and not from healthy controls, confirming their in vivo expression during active infection in humans. Three of the antigens (PE-PGRS, PTRP, and MtrA) were also recognized by retrospective preclinical TB sera obtained, prior to the clinical manifestation of TB, from human immunodeficiency virus-TB patients, suggesting that they are potential candidates for devising diagnostic tests for active, preclinical TB.     

Specific delayed-type hypersensitivity responses to ESAT-6 identify tuberculosis-infected cattle

Human and bovine tuberculosis have long been detected by skin testing with purified protein derivative (PPD), a complex mix of partly denatured mycobacterial antigens with suboptimal specificity. In the present study, skin tests based on ESAT-6, a recombinantly produced antigen highly specific for tuberculosis infection, were investigated. Although ESAT-6 was strongly recognized in vitro and induced high levels of gamma interferon, initial investigations demonstrated that higher doses of ESAT-6 than of PPD were needed to induce substantial delayed-type hypersensitivity reactions. Also, the kinetics of the skin test response differed for the two reagents; PPD showed maximal response at 72 h, but the response to ESAT-6 often peaked later at 96 h. Tests based on an optimized strategy (400 micro g of ESAT-6 measured between 72 and 96 h), in cattle infected with Mycobacterium bovis (n = 22) and animals sensitized by exposure to environmental mycobacteria showed ESAT-6 to have a promising diagnostic potential (sensitivity, 82%; specificity, 100%; optimal cutoff, 3 mm), compared with PPD (sensitivity, 86%; specificity, 90%; optimal cutoff, 4 mm). Larger investigations are required to refine cutoff points for any new diagnostic test, but the present results indicate great potential for skin tests based on specific antigens for accurate in vivo diagnosis of tuberculosis.     

PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a potential B-cell antigen used for serological diagnosis to distinguish vaccinated controls from tuberculosis patients

Proteins encoded by a 9.5-kb DNA segment, termed the region of difference (RD), of Mycobacterium tuberculosis have been demonstrated to be important in bacterial virulence, vaccine development and the design of diagnostic reagents. This study evaluated the immunogenic properties of Rv3425, a member of the PPE family of proteins, encoded by an open reading frame found in RD11 of M. tuberculosis, in comparison with two other well-known antigens, the early secreted antigen target 6 (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). RT-PCR demonstrated that Rv3425 mRNA is expressed in liquid culture by M. tuberculosis H37Rv. When tested in a conventional ELISA in the form of a His-tagged recombinant protein, Rv3425 revealed a statistically significant antigenic distinction between healthy bacille Calmette-Guérin (BCG)-vaccinated controls and tuberculosis (TB) patients (p <0.0001). The anti-IgG response to recombinant Rv3425 was almost equal to that for CFP-10, and was higher than that for ESAT-6. The results highlight the immunosensitive and immunospecific nature of Rv3425, which shows promise for use in the serodiagnosis of TB.