The relationship between specific antibody profiles and tuberculosis (TB) state was investigated by measuring serum antibody levels to six Mycobacterium tuberculosis antigens in human subjects grouped into four diagnostic categories: active disease, inactive (past) tuberculosis, latent infection without radiographic chest abnormalities, and infection free. Statistical data analyses showed that the latter two groups were serologically indistinguishable and that active tuberculosis and inactive tuberculosis were characterized by different antibody profiles. Antibodies to the 38-kDa antigen, alanine dehydrogenase, and Rv2626c were associated with active TB, while antibodies to the 16-kDa antigen, ferredoxin A, and ESAT-6 were associated with inactive TB. Thus, the targets of the immune response vary with tuberculosis state. The correlation between bacterial antigen production and infection stage was investigated in mice infected with M. tuberculosis by bacterial transcription profiling. It was found that levels of transcripts encoding the six M. tuberculosis antigens varied during infection. Together, the data indicate that antigen composition of tubercle bacilli varies with stage of infection and that immunoprofiling can distinguish between tuberculosis states.

Leprosy can be a devastating chronic infection that causes nerve function impairment and associated disfigurement. Despite the recent reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. The diagnosis of leprosy is currently based on the appearance of clinical signs and requires expert clinical, as well as labor-intensive and time-consuming laboratory or histological, evaluation. For the purpose of developing an effective, simple, rapid, and low-cost diagnostic alternative, we have analyzed the serologic antibody response to identify Mycobacterium leprae proteins that are recognized by leprosy patients. More than 100 recombinant antigens were analyzed in a protein array format to select those with discriminatory properties for leprosy diagnosis. As expected, multibacillary leprosy patients recognized more antigens with stronger antibody responses than paucibacillary leprosy patients. Our data indicate, however, that multibacillary patients can be distinguished from paucibacillary patients, and both of these groups can be segregated from endemic control groups. We went on to confirm the diagnostic properties of antigens ML0405 and ML2331 and the LID-1 fusion construct of these two proteins by enzyme-linked immunosorbent assay. We then demonstrated the performance of these antigens in rapid test formats with a goal of developing a point-of-care diagnostic test. A serological diagnostic test capable of identifying and allowing treatment of leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

The aim of the study was to evaluate serological correlates of active tuberculosis and of response to antituberculosis treatment in a cohort of HIV-negative patients with pulmonary tuberculosis studied at diagnosis and during treatment at the Service de Pneumo-Phtisiologie, Centre Hospitalier-Universitaire Ignace Deen, Conakry, Republic of Guinea. Two similar cohorts of HIV-negative healthy households of patients and healthy community controls were included in the study. Plasma samples were obtained from 168 untreated tuberculosis patients, 167 healthy household controls, and 168 healthy community controls. Serial plasma samples were also obtained from the tuberculosis patients at 2 and 8 months after initiation of chemotherapy. IgG antibody levelswere measured by an enzyme-linked immunosorbent assay (ELISA) using ten purified M. tuberculosis antigens. ELISA results were analysed by comparing geometric means of data. Of the ten antigens tested, five (14kDa Ag, 19kDa Ag, AlaDH, MS, and MPT83) elicited similar antibody responses in untreated TB patients and controls. In contrast, levels of three antibodies (ESAT-6, LAM, and 38kDa Ag) were higher in untreated TB patients than in household or community controls (p < 0.0001). Levels were higher in untreated patients than in community controls also for the anti-Rv2626c antibody (p = 0.0001) and, at a lower significance level, for the anti-FdxA antibody (p < 0.025). Antibody levels against ESAT-6 and Rv2626c decreased during therapy, while antibody levels to the 38 kDa antigen and LAM increased during therapy; FdxA antibody levels did not vary with treatment. Neither severity of presentation nor chest X-ray patterns affected levels of these antibodies before treatment. In contrast, after the 8-month therapeutic course, patients who presented with moderate/severe disease had higher levels of anti-ESAT-6, anti-FdxA, and anti-38kDa antibodies than those of patients with mild disease onset. Patients with bilateral lung lesions had significantly higher anti-38kDa and anti-LAM levels, both at diagnosis and after 8-month treatment, than patients with lesions involving only one lung. Antibodies to alanine dehydrogenase and malate synthetase measured at initiation of treatment were higher in tuberculosis patients who subsequently failed therapy than in those who were cured. The main conclusions ofthe study are: a) plasma levels of antibodies to a number of M. tuberculosis represent serological correlates of active disease; b) these correlates are affected in an antigen-specific fashion by anti-tuberculosis treatment; c) particular serological markers may be predictive of treatment outcome.

Antibodies against Mycobacterium tuberculosis antigens were detected by enzyme-linked immunosorbent assay in cerebrospinal fluid (CSF) samples obtained from 442 patients with tuberculous meningitis (TBM) and 102 control patients. Antibodies were found in the CSF of 87% of patients with clinical (culture-negative) TBM, 72% of patients with culture-positive TBM, and 65% of patients with autopsy-proven TBM. That anti-M. tuberculosis antibodies were detected in the CSF of patients with clinically diagnosed cases more frequently than in patients with culture-positive cases suggests that the detection of antibodies in CSF tends to decrease as bacillary load increases. Of the patients with clinical TBM who were coinfected with human immunodeficiency virus (HIV), 70% exhibited anti-M. tuberculosis antibody in CSF, which suggests that antibody responses in this group were substantially weaker than those in HIV-negative patients with clinical TBM. Some groups showed a stronger response to certain antigens, which suggests that antigen recognition patterns may be specific for the stage of disease.

Previous work in our laboratory showed that the ESAT-6 protein of Mycobacterium tuberculosis and Mycobacterium bovis induces strong antibody responses in a large proportion (ϳ90%) of experimentally or naturally infected nonhuman primates. Here, the antibody response to ESAT-6 in tuberculous monkeys was characterized at the epitope level by measuring antibodies to overlapping, synthetic peptides spanning the ESAT-6 sequence. The antibody response against the COOH-terminal portion of the protein was the strongest in both experimentally and naturally infected animals. Moreover, these antibodies became detectable the earliest during experimental infection, suggesting an ordered expansion of ESAT-6-specific B-cell clones in the course of infection. The data support use of synthetic peptides in lieu of the full-length ESAT-6 protein in diagnostic antibody detection assays.Tuberculosis (TB) not only is one of the human plagues but also affects a variety of animals, with great consequences to wildlife, agriculture, and zoo keeping (4, 9, 10). Moreover, TB in captive nonhuman primates greatly affects research endeavors. Outbreaks of TB in research monkey colonies, even when only suspected, are economically costly. The losses are caused not only by the elevated costs of animal losses but also by the expenses associated with disrupted research, lost time, and sometimes delayed release of new products into the market. As a result, strict control guidelines have been implemented (2). However, effective TB control requires accurate diagnostic methods, and the current method for diagnosing TB in living nonhuman primates, i.e., the tuberculin skin test, has limitations (8). A new method, which is based on detection of gamma interferon in whole blood (5), is still being evaluated. Thus, we have begun to characterize the antibody response to Mycobacterium tuberculosis antigens to evaluate the prospect of serologic methods for the diagnosis of TB in monkeys.In previous work members of our group showed that 6-kDa early secretory antigenic target (ESAT-6), a low-molecularweight protein secreted by virulent M. tuberculosis and Mycobacterium bovis (6, 12), induced strong antibody responses in Ͼ90% of experimentally infected monkeys (1). An outbreak of M. bovis infection in a nonhuman primate research facility provided the opportunity to characterize the antibody response to ESAT-6 in naturally infected macaques. We found that almost 90% of the animals exhibiting TB lesions at necropsy had anti-ESAT-6 immunoglobulin G (IgG) antibody (7). These studies strongly imply that an antibody detection assay utilizing ESAT-6 has a place in the diagnosis of TB in nonhuman primates. The present study was conducted to further characterize the IgG antibody response to ESAT-6 at the epitope level in experimentally infected and naturally infected nonhuman primates.Three groups of nonhuman primates were included in the study. The first comprised 11 animals (4 cynomolgus macaques, 5 rhesus macaques, and 2 African Green monkeys) infected experimentally w...

Summaryobjective Microbiological identification of Mycobacterium tuberculosis is insensitive and slow, and clinical distinction of tuberculous meningitis (TBM) from other subacute or chronic meningoenchephalitides (SACM) is difficult. Successful use of highly specific M. tuberculosis serological assays on cerebrospinal fluid has been reported, but their performance for diagnosis in a tuberculosis endemic country where they would be of most value is unclear. We sought to determine the biological basis for the uncertainty in interpretation of antibody detection in the CSF of TBM patients.methods We identified prospectively 46 adults with SACM and explored the concordance between TBM diagnosis and detection of highly specific M. tuberculosis antibodies in CSF. The source of antibodies in CSF was explored by evaluating the correlation between antibody titres in CSF with those in serum, or with the albumin quotient. Intrathecal IgG synthesis was assessed by the IgG index.results Positive antibody titres were more frequent among TBM patients (76%), but were also present in individuals with other SACM (59%). A positive correlation between antibody titres in CSF with those in serum, or with the albumin quotient, supported the leakage of antibodies from plasma to CSF through an increased blood-brain barrier permeability. Intrathecal IgG synthesis was only detected in 35% of the TBM cases.conclusion Plasma antibodies likely synthesized in response to previous tuberculosis infections were a major source of mycobacterial antibodies in CSF due to leakage through an impaired blood-brain barrier. Interpretation of mycobacterial antibodies in CSF of adults for TBM, however specific, must take into account the contribution of antibodies from plasma, and hence, has questionable use for diagnosis.