Association of Micronutrients with Tuberculosis Development in HIV Infected Patients.

Human immunodeficiency virus (HIV) infection associated with weakened immune system due to decreased CD4 T cell count favors development of tuberculosis. Effector immune responses are also associated with micronutrient status due to their prominent role in maintaining immune functions. Micronutrient deficiencies are quite common among HIV patients that further result into compromised immunity thus making the conditions even more favorable for mycobacteria to establish disease. So, current study was designed to assess association of different micronutrients with development of TB in HIV patients. Micronutrient levels were measured in asymptomatic HIV patients who were monitored for the development of TB during follow up period (incident TB) within one month to one year and also in symptomatic microbiologically confirmed HIV-TB patients. Among various micronutrients assessed, levels of ferritin were found to be significantly increased (p < 0.05) with significant decreased zinc (p < 0.05) and selenium (p < 0.05) levels in incident TB group as well as in HIV-TB subjects compared to asymptomatic HIV patients who did not develop TB in the follow up period. Importantly, increased levels of ferritin and decreased levels of selenium were significantly associated with development of tuberculosis in HIV patients.

Evaluation of immunodominant peptides of in vivo expressed mycobacterial antigens in an ELISA-based diagnostic assay for pulmonary tuberculosis.

Non-sputum-based biomarker assay is urgently required as per WHO's target product pipeline for diagnosis of tuberculosis. Therefore, the current study was designed to evaluate the utility of previously identified proteins, encoded by in vivo expressed mycobacterial transcripts in pulmonary tuberculosis, as diagnostic targets for a serodiagnostic assay. A total of 300 subjects were recruited including smear+, smear- pulmonary tuberculosis (PTB) patients, sarcoidosis patients, lung cancer patients and healthy controls. Proteins encoded by eight in vivo expressed transcripts selected from previous study including those encoded by two topmost expressed and six RD transcripts (Rv0986, Rv0971, Rv1965, Rv1971, Rv2351c, Rv2657c, Rv2674, Rv3121) were analyzed for B-cell epitopes by peptide arrays/bioinformatics. Enzyme-linked immunosorbent assay was used to evaluate the antibody response against the selected peptides in sera from PTB and controls. Overall 12 peptides were selected for serodiagnosis. All the peptides were initially screened for their antibody response. The peptide with highest sensitivity and specificity was further assessed for its serodiagnostic ability in all the study subjects. The mean absorbance values for antibody response to selected peptide were significantly higher (p<0.001) in PTB patients as compared to healthy controls; however, the sensitivity for diagnosis of PTB was 31% for smear+ and 20% for smear- PTB patients. Thus, the peptides encoded by in vivo expressed transcripts elicited a significant antibody response, but are not suitable candidates for serodiagnosis of PTB.

Mycobacterium tuberculosis transcriptome in intraocular tuberculosis.

Introduction. Intraocular tuberculosis (IOTB) is a significant cause of visual morbidity in tuberculosis (TB)-endemic countries. Although Mycobacterium tuberculosis (M. tb) has been detected in both the retinal pigment epithelial (RPE) cells and in the intraocular fluid (IOF) in some cases, IOTB is paucibacillary in the vast majority of patients. As a result, M. tb pathogenesis in the ocular compartment is poorly defined.Hypothesis. The transcriptional profile of M. tb in the ocular compartment will differ from those of M. tb in environments that represent earlier stages of infection.Aim. Our aim is to shed light on the pathogenesis of M. tb in a clinically relevant but challenging environment to study.Methodology. Whole-genome microarray analysis was performed on M. tb grown in an IOF model (artificial IOF; AIOF) over 6 days against reference log phase bacteria grown in 7H9. Results were compared to published M. tb transcriptomes in other physiologically relevant environments, e.g. RPE cell line.Results. M. tb replicates slowly in AIOF. Genes involved in active replication and aerobic respiration as well as lipid metabolism were either downregulated or not differentially expressed. Yet, M. tb in AIOF downregulated genes of the DosR regulon, indicating the suppression of dormancy, similar to M. tb in RPE cells. This transcriptional profile is distinct from the active and virulent transcriptomes of M. tb in alveolar epithelial cells and blood.Conclusion. M. tb likely acquires a non-invasive and quiescent phenotype, between active infection and dormancy, upon reaching an extrapulmonary niche, i.e. the ocular environment.

Evolution of Antibodies to Epitopes of PE_PGRS51 in the Spectrum of Active Pulmonary Tuberculosis.

BACKGROUND: Intrafamily homology has impeded correlation of expression of individual PE_PGRS proteins with stage of tuberculosis (TB). We investigated the in vivo expression of PE_PGRS51, which has 3 unique regions. METHODS: Sera from patients across the spectrum of TB were used to screen peptide arrays spanning PE_PGRS51. RESULTS: Antibodies against a subset of conserved "core epitopes" within PE/PGRS domains are elicited during early TB. The epitope repertoire expands to adjacent regions with disease progression. Antiunique region antibodies appear only during cavitary TB. CONCLUSIONS: Elicitation of antiunique region antibodies can serve as markers for in vivo expression of PE_PGRS proteins.

Mycobacterium tuberculosis Primary Infection and Dissemination: A Critical Role for Alveolar Epithelial Cells.

Globally, tuberculosis (TB) has reemerged as a major cause of morbidity and mortality, despite the use of the Mycobacterium bovis BCG vaccine and intensive attempts to improve upon BCG or develop new vaccines. Two lacunae in our understanding of the Mycobacterium tuberculosis (M. tb)-host pathogenesis have mitigated the vaccine efforts; the bacterial-host interaction that enables successful establishment of primary infection and the correlates of protection against TB. The vast majority of vaccine efforts are based on the premise that cell-mediated immunity (CMI) is the predominating mode of protection against TB. However, studies in animal models and in humans demonstrate that post-infection, a period of several weeks precedes the initiation of CMI during which the few inhaled bacteria replicate dramatically and disseminate systemically. The "Trojan Horse" mechanism, wherein M. tb is phagocytosed and transported across the alveolar barrier by infected alveolar macrophages has been long postulated as the sole, primary M. tb:host interaction. In the current review, we present evidence from our studies of transcriptional profiles of M. tb in sputum as it emerges from infectious patients where the bacteria are in a quiescent state, to its adaptations in alveolar epithelial cells where the bacteria transform to a highly replicative and invasive phenotype, to its maintenance of the invasive phenotype in whole blood to the downregulation of invasiveness upon infection of epithelial cells at an extrapulmonary site. Evidence for this alternative mode of infection and dissemination during primary infection is supported by in vivo, in vitro cell-based, and transcriptional studies from multiple investigators in recent years. The proposed alternative mechanism of primary infection and dissemination across the alveolar barrier parallels our understanding of infection and dissemination of other Gram-positive pathogens across their relevant mucosal barriers in that barrier-specific adhesins, toxins, and enzymes synergize to facilitate systemic establishment of infection prior to the emergence of CMI. Further exploration of this M. tb:non-phagocytic cell interaction can provide alternative approaches to vaccine design to prevent infection with M. tb and not only decrease clinical disease but also decrease the overwhelming reservoir of latent TB infection.

Transcriptional signatures of Mycobacterium tuberculosis in mouse model of intraocular tuberculosis.

BACKGROUND: Studies on human intraocular tuberculosis (IOTB) are extremely challenging. For understanding the pathogenesis of IOTB, it is important to investigate the mycobacterial transcriptional changes in ocular environment. METHODS: Mice were challenged intravenously with Mycobacterium tuberculosis H37Rv and at 45 days post-infection, experimental IOTB was confirmed based on bacteriological and molecular assays. M. tuberculosis transcriptome was analyzed in the infected eyes using microarray technology. The identified M. tuberculosis signature genes were further validated and investigated in human IOTB samples using real-time polymerase chain reaction. RESULTS: Following intravenous challenge with M. tuberculosis, 45% (5/12) mice showed bacilli in the eyes with positivity for M. tuberculosis ribonucleic acid in 100% (12/12), thus confirming the paucibacillary nature of IOTB similar to human IOTB. M. tuberculosis transcriptome in these infected eyes showed significant upregulation of 12 M. tuberculosis genes and five of these transcripts (Rv0962c, Rv0984, Rv2612c, Rv0974c and Rv0971c) were also identified in human clinically confirmed cases of IOTB. CONCLUSIONS: Differentially expressed mycobacterial genes identified in an intravenously challenged paucibacillary mouse IOTB model and presence of these transcripts in human IOTB samples highlight the possible role of these genes for survival of M. tuberculosis in the ocular environment, thus contributing to pathogenesis of IOTB.

Transcriptional Profile of Mycobacterium tuberculosis in an in vitro Model of Intraocular Tuberculosis.

Background: Intraocular tuberculosis (IOTB), an extrapulmonary manifestation of tuberculosis of the eye, has unique and varied clinical presentations with poorly understood pathogenesis. As it is a significant cause of inflammation and visual morbidity, particularly in TB endemic countries, it is essential to study the pathogenesis of IOTB. Clinical and histopathologic studies suggest the presence of Mycobacterium tuberculosis in retinal pigment epithelium (RPE) cells. Methods: A human retinal pigment epithelium (ARPE-19) cell line was infected with a virulent strain of M. tuberculosis (H37Rv). Electron microscopy and colony forming units (CFU) assay were performed to monitor the M. tuberculosis adherence, invasion, and intracellular replication, whereas confocal microscopy was done to study its intracellular fate in the RPE cells. To understand the pathogenesis, the transcriptional profile of M. tuberculosis in ARPE-19 cells was studied by whole genome microarray. Three upregulated M. tuberculosis transcripts were also examined in human IOTB vitreous samples. Results: Scanning electron micrographs of the infected ARPE-19 cells indicated adherence of bacilli, which were further observed to be internalized as monitored by transmission electron microscopy. The CFU assay showed that 22.7 and 8.4% of the initial inoculum of bacilli adhered and invaded the ARPE-19 cells, respectively, with an increase in fold CFU from 1 dpi (0.84) to 5dpi (6.58). The intracellular bacilli were co-localized with lysosomal-associated membrane protein-1 (LAMP-1) and LAMP-2 in ARPE-19 cells. The transcriptome study of intracellular bacilli showed that most of the upregulated transcripts correspond to the genes encoding the proteins involved in the processes such as adherence (e.g., Rv1759c and Rv1026), invasion (e.g., Rv1971 and Rv0169), virulence (e.g., Rv2844 and Rv0775), and intracellular survival (e.g., Rv1884c and Rv2450c) as well as regulators of various metabolic pathways. Two of the upregulated transcripts (Rv1971, Rv1230c) were also present in the vitreous samples of the IOTB patients. Conclusions:M. tuberculosis is phagocytosed by RPE cells and utilizes these cells for intracellular multiplication with the involvement of late endosomal/lysosomal compartments and alters its transcriptional profile plausibly for its intracellular adaptation and survival. The findings of the present study could be important to understanding the molecular pathogenesis of IOTB with a potential role in the development of diagnostics and therapeutics for IOTB.

Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients.

Pulmonary tuberculosis, the disease caused by Mycobacterium tuberculosis, still retains a top rank among the deadliest communicable diseases. Sputum expectorated during the disease continues to be a primary diagnostic specimen and also serves as a reservoir of bacteria. The expression pattern of mycobacteria in sputum will lead to an insight into bacterial adaptation at the most highly transmissible stage of infection and can also help in identifying newer diagnostic as well as drug targets. Thus, in the present study, a whole genome microarray of Mycobacterium tuberculosis was used to elucidate the transcriptional profile of mycobacteria in the sputum samples of smear positive pulmonary tuberculosis patients. Overall, the mycobacteria in sputum appeared to be in a low energy and low replicative state as compared to in vitro grown log phase M. tb with downregulation of genes involved in ATP synthesis, aerobic respiration and translational machinery. Simultaneously, downregulation was also seen in the genes involved in secretion machinery of mycobacteria along with the downregulation of genes involved in the synthesis of phthiocerol dimycocerosate and phenol glycolipids. In contrast, the majority of the genes which showed an upregulation in sputum mycobacteria were of unknown function. Further identification of these genes may provide new insights into the mycobacterial behavior during this phase of infection and may help in deciphering candidates for development of better diagnostic and drug candidates.

Understanding dissemination of Mycobacterium tuberculosis from the lungs during primary infection.

Understanding how inhaled Mycobacterium tuberculosis achieves dramatic replication and crosses the alveolar barrier to establish systemic latent infection, before adaptive immunity is elicited in humans, is limited by the small infecting inoculum carried in aerosol droplets (1-5 µm diameter) and the inability to identify the time of infection. M. tuberculosis is believed to disseminate via infected macrophages. However, like other invasive bacterial pathogens, M. tuberculosis could also cross the barrier directly using adhesins and toxins. An in vitro alveolar barrier mimicking the gas-exchange regions of the alveolus was devised comprising monolayers of human alveolar epithelial and endothelial cells cultured on opposing sides of a basement membrane. Migration of dissemination-competent strains of M. tuberculosis, and dissemination-attenuated M. tuberculosis and Mycobacterium bovis mutant strains lacking adhesin/toxin ESAT-6 and adhesin HBHA were tested for macrophage-free migration across the barrier. Strains that disseminate similarly in vivo migrated similarly across the in vitro alveolar barrier. Strains lacking ESAT-6 expression/secretion were attenuated, and absence of both ESAT-6 and HBHA increased attenuation of bacterial migration across the barrier. Thus, as reported for other bacteria, M. tuberculosis utilizes adhesins and toxins for macrophage-independent crossing of the alveolar barrier. This in vitro model will allow identification and characterization of molecules/mechanisms employed by M. tuberculosis to establish systemic latent tuberculosis infection during primary infection.

Transcriptional Profiling of Mycobacterium tuberculosis Replicating in the Human Type II Alveolar Epithelial Cell Line, A549.

Alveolar epithelial cells outnumber alveolar macrophages by ~500 fold and increasing evidence suggests Mycobacterium tuberculosis may replicate dramatically in these cells during the initial weeks of infecting the lung [1,2]. Here, we report in experimental detail the transcriptional profiling of Mycobacterium tuberculosis replicating at 72 hr post-infection in the human type II alveolar epithelial cell line, A549, as compared to Mycobacterium tuberculosis growing logarithmically in laboratory broth culture [2]. All resulting transcriptional profiling data was deposited to the Gene Expression Omnibus (GEO) database under the accession number GSE58466.

Transcriptional profile of Mycobacterium tuberculosis replicating in type II alveolar epithelial cells.

Mycobacterium tuberculosis (M. tb) infection is initiated by the few bacilli inhaled into the alveolus. Studies in lungs of aerosol-infected mice provided evidence for extensive replication of M. tb in non-migrating, non-antigen-presenting cells in the alveoli during the first 2-3 weeks post-infection. Alveoli are lined by type II and type I alveolar epithelial cells (AEC) which outnumber alveolar macrophages by several hundred-fold. M. tb DNA and viable M. tb have been demonstrated in AEC and other non-macrophage cells of the kidney, liver, and spleen in autopsied tissues from latently-infected subjects from TB-endemic regions indicating systemic bacterial dissemination during primary infection. M. tb have also been demonstrated to replicate rapidly in A549 cells (type II AEC line) and acquire increased invasiveness for endothelial cells. Together, these results suggest that AEC could provide an important niche for bacterial expansion and development of a phenotype that promotes dissemination during primary infection. In the current studies, we have compared the transcriptional profile of M. tb replicating intracellularly in A549 cells to that of M. tb replicating in laboratory broth, by microarray analysis. Genes significantly upregulated during intracellular residence were consistent with an active, replicative, metabolic, and aerobic state, as were genes for tryptophan synthesis and for increased virulence (ESAT-6, and ESAT-6-like genes, esxH, esxJ, esxK, esxP, and esxW). In contrast, significant downregulation of the DevR (DosR) regulon and several hypoxia-induced genes was observed. Stress response genes were either not differentially expressed or were downregulated with the exception of the heat shock response and those induced by low pH. The intra-type II AEC M. tb transcriptome strongly suggests that AEC could provide a safe haven in which M. tb can expand dramatically and disseminate from the lung prior to the elicitation of adaptive immune responses.

Development of a POC test for TB based on multiple immunodominant epitopes of M. tuberculosis specific cell-wall proteins.

The need for an accurate, rapid, simple and affordable point-of-care (POC) test for Tuberculosis (TB) that can be implemented in microscopy centers and other peripheral health-care settings in the TB-endemic countries remains unmet. This manuscript describes preliminary results of a new prototype rapid lateral flow TB test based on detection of antibodies to immunodominant epitopes (peptides) derived from carefully selected, highly immunogenic M. tuberculosis cell-wall proteins. Peptide selection was initially based on recognition by antibodies in sera from TB patients but not in PPD-/PPD+/BCG-vaccinated individuals from TB-endemic settings. The peptides were conjugated to BSA; the purified peptide-BSA conjugates striped onto nitrocellulose membrane and adsorbed onto colloidal gold particles to devise the prototype test, and evaluated for reactivity with sera from 3 PPD-, 29 PPD+, 15 PPD-unknown healthy subjects, 10 patients with non-TB lung disease and 124 smear-positive TB patients. The assay parameters were adjusted to determine positive/negative status within 15 minutes via visual or instrumented assessment. There was minimal or no reactivity of sera from non-TB subjects with the striped BSA-peptides demonstrating the lack of anti-peptide antibodies in subjects with latent TB and/or BCG vaccination. Sera from most TB patients demonstrated reactivity with one or more peptides. The sensitivity of antibody detection ranged from 28-85% with the 9 BSA-peptides. Three peptides were further evaluated with sera from 400 subjects, including additional PPD-/PPD+/PPD-unknown healthy contacts, close hospital contacts and household contacts of untreated TB patients, patients with non-TB lung disease, and HIV+TB- patients. Combination of the 3 peptides provided sensitivity and specificity>90%. While the final fully optimized lateral flow POC test for TB is under development, these preliminary results demonstrate that an antibody-detection based rapid POC lateral flow test based on select combinations of immunodominant M. tb-specific epitopes may potentially replace microscopy for TB diagnosis in TB-endemic settings.

Specific amplification of gene encoding N-terminal region of catalase-peroxidase protein (KatG-N) for diagnosis of disseminated MAC disease in HIV patients.

Disseminated Mycobacterium avium-intracellulare complex (MAC) infection is considered as severe complication of advanced HIV/AIDS disease. Currently available various laboratory investigations have not only limited ability to discriminate between MAC infection and tuberculosis but are also laborious and time consuming. The aim of this study was, therefore, to design a molecular-based strategy for specific detection of MAC and its differentiation from Mycobacterium tuberculosis (M. tb) isolated from the blood specimens of HIV patients. A simple PCR was developed based on the amplification of 120-bp katG-N gene corresponding to the first 40 amino acids of N-terminal catalase-peroxidase (KatG) protein of Mycobacterium avium that shows only ~13% sequence homology by clustal W alignment to N-terminal region of M. tb KatG protein. This assay allowed the accurate and rapid detection of MAC bacteremia, distinguishing it from M. tb in a single PCR reaction without any need for sequencing or hybridization protocol to be performed thereafter. This study produced enough evidence that a significant proportion of Indian HIV patients have disseminated MAC bacteremia, suggesting the utility of M. avium katG-N gene PCR for early detection of MAC disease in HIV patients.

Transcriptional profiling of Mycobacterium tuberculosis replicating ex vivo in blood from HIV- and HIV+ subjects.

Hematogenous dissemination of Mycobacterium tuberculosis (M. tb) occurs during both primary and reactivated tuberculosis (TB). Although hematogenous dissemination occurs in non-HIV TB patients, in ∼80% of these patients, TB manifests exclusively as pulmonary disease. In contrast, extrapulmonary, disseminated, and/or miliary TB is seen in 60-70% of HIV-infected TB patients, suggesting that hematogenous dissemination is likely more common in HIV+ patients. To understand M. tb adaptation to the blood environment during bacteremia, we have studied the transcriptome of M. tb replicating in human whole blood. To investigate if M. tb discriminates between the hematogenous environments of immunocompetent and immunodeficient individuals, we compared the M. tb transcriptional profiles during replication in blood from HIV- and HIV+ donors. Our results demonstrate that M. tb survives and replicates in blood from both HIV- and HIV+ donors and enhances its virulence/pathogenic potential in the hematogenous environment. The M. tb blood-specific transcriptome reflects suppression of dormancy, induction of cell-wall remodeling, alteration in mode of iron acquisition, potential evasion of immune surveillance, and enhanced expression of important virulence factors that drive active M. tb infection and dissemination. These changes are accentuated during bacterial replication in blood from HIV+ patients. Furthermore, the expression of ESAT-6, which participates in dissemination of M. tb from the lungs, is upregulated in M. tb growing in blood, especially during growth in blood from HIV+ patients. Preliminary experiments also demonstrate that ESAT-6 promotes HIV replication in U1 cells. These studies provide evidence, for the first time, that during bacteremia, M. tb can adapt to the blood environment by modifying its transcriptome in a manner indicative of an enhanced-virulence phenotype that favors active infection. Additionally, transcriptional modifications in HIV+ blood may further accentuate M. tb virulence and drive both M. tb and HIV infection.

LipC (Rv0220) is an immunogenic cell surface esterase of Mycobacterium tuberculosis.

We have reported previously the identification of novel proteins of Mycobacterium tuberculosis by the immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained from M. tuberculosis-infected rabbits at 5 weeks postinfection. In this study, we report the further characterization of one of these antigens, LipC (Rv0220). LipC is annotated as a member of the Lip family based on the presence of the consensus motif "GXSXG" characteristic of esterases. Although predicted to be a cytoplasmic enzyme, we provide evidence that LipC is a cell surface protein that is present in both the cell wall and the capsule of M. tuberculosis. Consistent with this localization, LipC elicits strong humoral immune responses in both HIV-negative (HIV-) and HIV-positive (HIV+) tuberculosis (TB) patients. The absence of anti-LipC antibodies in sera from purified protein derivative-positive (PPD+) healthy subjects confirms its expression only during active M. tuberculosis infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 of which map to the exposed surface of the modeled LipC protein. The recombinant LipC (rLipC) protein also elicits proinflammatory cytokine and chemokine responses from macrophages and pulmonary epithelial cells. rLipC can hydrolyze short-chain esters with the carbon chain containing 2 to 10 carbon atoms. Together, these studies demonstrate that LipC is a novel cell surface-associated esterase of M. tuberculosis that is highly immunogenic and elicits both antibodies and cytokines/chemokines.

Commercial serological tests for the diagnosis of active pulmonary and extrapulmonary tuberculosis: an updated systematic review and meta-analysis.

BACKGROUND: Serological (antibody detection) tests for tuberculosis (TB) are widely used in developing countries. As part of a World Health Organization policy process, we performed an updated systematic review to assess the diagnostic accuracy of commercial serological tests for pulmonary and extrapulmonary TB with a focus on the relevance of these tests in low- and middle-income countries. METHODS AND FINDINGS: We used methods recommended by the Cochrane Collaboration and GRADE approach for rating quality of evidence. In a previous review, we searched multiple databases for papers published from 1 January 1990 to 30 May 2006, and in this update, we add additional papers published from that period until 29 June 2010. We prespecified subgroups to address heterogeneity and summarized test performance using bivariate random effects meta-analysis. For pulmonary TB, we included 67 studies (48% from low- and middle-income countries) with 5,147 participants. For all tests, estimates were variable for sensitivity (0% to 100%) and specificity (31% to 100%). For anda-TB IgG, the only test with enough studies for meta-analysis, pooled sensitivity was 76% (95% CI 63%-87%) in smear-positive (seven studies) and 59% (95% CI 10%-96%) in smear-negative (four studies) patients; pooled specificities were 92% (95% CI 74%-98%) and 91% (95% CI 79%-96%), respectively. Compared with ELISA (pooled sensitivity 60% [95% CI 6%-65%]; pooled specificity 98% [95% CI 96%-99%]), immunochromatographic tests yielded lower pooled sensitivity (53%, 95% CI 42%-64%) and comparable pooled specificity (98%, 95% CI 94%-99%). For extrapulmonary TB, we included 25 studies (40% from low- and middle-income countries) with 1,809 participants. For all tests, estimates were variable for sensitivity (0% to 100%) and specificity (59% to 100%). Overall, quality of evidence was graded very low for studies of pulmonary and extrapulmonary TB. CONCLUSIONS: Despite expansion of the literature since 2006, commercial serological tests continue to produce inconsistent and imprecise estimates of sensitivity and specificity. Quality of evidence remains very low. These data informed a recently published World Health Organization policy statement against serological tests. Please see later in the article for the Editors' Summary.

Antibodies against immunodominant antigens of Mycobacterium tuberculosis in subjects with suspected tuberculosis in the United States compared by HIV status.

The immunodominance of Mycobacterium tuberculosis proteins malate synthase (MS) and MPT51 has been demonstrated in case-control studies with patients from countries in which tuberculosis (TB) is endemic. The value of these antigens for the serodiagnosis of TB now is evaluated in a cross-sectional study of pulmonary TB suspects in the United States diagnosed to have TB, HIV-associated TB, or other respiratory diseases (ORD). Serum antibody reactivity to recombinant purified MS and MPT51 was determined by enzyme-linked immunosorbent assays (ELISAs) of samples from TB suspects and well-characterized control groups. TB suspects were diagnosed with TB (n = 87; 49% sputum microscopy negative, 20% HIV(+)) or ORD (n = 63; 58% HIV(+)). Antibody reactivity to MS and MPT51 was significantly higher in U.S. HIV(+)/TB samples than in HIV(-)/TB samples (P < 0.001), and it was significantly higher in both TB groups than in control groups with latent TB infection (P < 0.001). Antibody reactivity to both antigens was higher in U.S. HIV(+)/TB samples than in HIV(+)/ORD samples (P = 0.052 for MS, P = 0.001 for MPT51) but not significantly different between HIV(-)/TB and HIV(-)/ORD. Among U.S. HIV(+) TB suspects, a positive anti-MPT51 antibody response was strongly and significantly associated with TB (odds ratio, 11.0; 95% confidence interval, 2.3 to 51.2; P = 0.002). These findings have implications for the adjunctive use of TB serodiagnosis with these antigens in HIV(+) subjects.

Potential role for ESAT6 in dissemination of M. tuberculosis via human lung epithelial cells.

ESAT6 has recently been demonstrated to cause haemolysis and macrophage lysis. Our studies demonstrate that ESAT6 causes cytolysis of type 1 and type 2 pneumocytes. Both types of pneumocytes express membrane laminin, and ESAT6 exhibits dose-dependent binding to both cell types and to purified human laminin. While minimal ESAT6 was detected on the surface of Mycobacterium tuberculosis grown in vitro, exogenously provided ESAT6 specifically associated with the bacterial cell surface, and the bacterium-associated ESAT6 retained its cytolytic ability. esat6 transcripts were upregulated approximately 4- to approximately 13-fold in bacteria replicating in type 1 cells, and approximately 3- to approximately 5 fold in type 2 cells. In vivo, laminin is primarily concentrated at the basolateral surface of pneumocytes where they rest on the basement membrane, which is composed primarily of laminin and collagen. The upregulation of esat6 transcripts in bacteria replicating in pneumocytes, the specific association of ESAT6 with the bacterial surface, the binding of ESAT6 to laminin and the lysis of pneumocytes by free and bacterium-associated ESAT6 together suggest a scenario wherein Mycobacterium tuberculosis replicating in pneumocytes may utilize surface ESAT6 to anchor onto the basolateral laminin-expressing surface of the pneumocytes, and damage the cells and the basement membrane to directly disseminate through the alveolar wall.