Serum Of Tuberculosis Patients Research Articles

Effectiveness of in-house developed Sandwich ELISA for antigen detection of tubercular antigen in resource constraint setting

Background: Around 26% of World’s tuberculosis (TB) cases are in India. In resource constrained settings, it is difficult to use diagnostic tests such as nucleic acid amplification technique and mycobacterial culture due to their complexity and expensiveness. Developing an assay using polyclonal antibody may help to solve this problem. Aims and Objectives: On the basis of this, we worked to detect tubercular antigen by developing Sandwich ELISA from the serum of TB patients. Materials and Methods: For this study, 50 cases, 50 disease controls, and 30 healthy control subjects were taken. Commercially available reagents were used for the development of Sandwich ELISA. The data were statistically analyzed with the help of software SPSS version 16.0. Results: Our effectively developed test had 82% area under the ROC curve. About 81% (n=35) patients of pulmonary TB cases with non-HIV infection detected by our Sandwich ELISA and 57% cases with the coinfection of HIV-TB. Developed test had 70.91% positive predictive value and 75.56% negative predictive value. Conclusion: We successfully developed Sandwich ELISA for tubercular antigen detection and to increase the specificity of the Sandwich ELISA, as per the WHO guidelines, further work is needed for the detection of tubercular antigen.

Effectivity of in-house developed sandwich ELISA for antigen detection in resource constraint settings

Introduction: Around 26% of World’s Tuberculosis (TB) cases are in India. In resource constrained settings, it is difficult to use diagnostic tests like nucleic acid amplification technique and mycobacterial culture due to their complexity and expensiveness.Developing an assay using polyclonal antibody may help to solve this problem. On the basis of that we worked to detect tubercular antigen developing Sandwich ELISA from the serum of tuberculosis patients. Materials & Methods: For the study 50 cases, 50 disease controls, 30 healthy control subjects were taken. Commercially available reagents were used for the development of Sandwich ELISA. The data was statistically analyzed with the help of software SPSS version 16.0. Results: Our effectively developed test had 82% area under the ROC curve. 81% (n=35) patients of PTB cases with non-HIV infection detected by our Sandwich ELISA and 57% cases with the co-infection of HIV-TB. Developed test had 70.91% positive predictive value and 75.56% negative predictive value. Conclusion: We successfully developed Sandwich ELISA for tubercular antigen detection. To increase the sensitivity and specificity of the Sandwich ELISA, as per the WHO guidelines, further work is needed.

Sex-Dependent Differential Expression of Lipidic Mediators Associated with Inflammation Resolution in Patients with Pulmonary Tuberculosis.

There is a sex bias in tuberculosis’s severity, prevalence, and pathogenesis, and the rates are higher in men. Immunological and physiological factors are fundamental contributors to the development of the disease, and sex-related factors could play an essential role in making women more resistant to severe forms of the disease. In this study, we evaluated sex-dependent differences in inflammatory markers. Serum samples were collected from 34 patients diagnosed with pulmonary TB (19 male and 15 female) and 27 healthy controls (18 male and 9 female). Cytokines IL2, IL4, IL6, IL8, IL10, IFNγ, TNFα, and GM-CSF, and eicosanoids PGE2, LTB4, RvD1, and Mar1 were measured using commercially available immunoassays. The MDA, a product of lipidic peroxidation, was measured by detecting thiobarbituric-acid-reactive substances (TBARS). Differential inflammation patterns between men and women were observed. Men had higher levels of IL6, IL8, and TNFα than women. PGE2 and LTB4 levels were higher in patients than healthy controls, but there were no differences for RvD1 and Mar1. Women had higher RvD1/PGE2 and RvD1/LTB4 ratios among patients. RvD1 plays a vital role in resolving the inflammatory process of TB in women. Men are the major contributors to the typical pro-inflammatory profile observed in the serum of tuberculosis patients.

Down-regulation of GAS5 has diagnostic value for tuberculosis and regulates the inflammatory response in mycobacterium tuberculosis infected THP-1 cells

ObjectiveThis study aimed to investigate the expression of long non-coding RNA (lncRNA) growth arrest-special transcript 5 (GAS5) in the serum of tuberculosis (TB) patients and discuss the mechanism of GAS5 in TB by establishing an in-vitro TB cell model. MethodsSerum expressions of GAS5 and miR-18a-5p were determined by quantitative real-time PCR (qRT-PCR). The effects of GAS5 on macrophage cell viability and the inflammatory response after MTB infection were assessed by CCK-8 and ELISA. Luciferase reporter gene assay was applied to delve into the potential target gene of GAS5. ResultsThe expression of GAS5 in TB patients was down-regulated, while miR-18a-5p was up-regulated, and the serum inflammatory factors were negatively correlated with the expression level of GAS5. MTB infection induced significant upregulation on the cell viability and inflammatory response but the acceleration effect could be rescued by GAS5-overexpression. Meanwhile, miR-18a-5p was recognized as the target gene of GAS5. ConclusionThis study indicated that the expression level of GAS5 in the serum of TB patients was decreased, while in the cells infected with MTB, the down-regulated GAS5 might develop a role in facilitating the cell vitality and the inflammatory response by adsorbing miR-18a-5p in the form of molecular sponge.

Lipoarabinomannan as a Point-of-Care Assay for Diagnosis of Tuberculosis: How Far Are We to Use It?

Tuberculosis (TB) is still a severe public health problem; the current diagnostic tests have limitations that delay treatment onset. Lipoarabinomannan (LAM) is a glycolipid that is a component of the cell wall of the bacillus Mycobacterium tuberculosis, the etiologic agent of TB. This glycolipid is excreted as a soluble form in urine. The World Health Organization has established that the design of new TB diagnostic methods is one of the priorities within the EndTB Strategy. LAM has been suggested as a biomarker to develop diagnostic tests based on its identification in urine, and it is one of the most prominent candidates to develop point-of-care diagnostic test because urine samples can be easily collected. Moreover, LAM can regulate the immune response in the host and can be found in the serum of TB patients, where it probably affects a wide variety of host cell populations, consequently influencing the quality of both innate and adaptive immune responses during TB infection. Here, we revised the evidence that supports that LAM could be used as a tool for the development of new point-of-care tests for TB diagnosis, and we discussed the mechanisms that could contribute to the low sensitivity of diagnostic testing.

Significance of the chemokine CXCL10 and human beta-defensin-3 as biomarkers of pulmonary tuberculosis

The biomarker significance of IL-35, chemokines (CXCL9 and CXCL10) and human beta-defensins (hBD2 and hBD3) was determined in pulmonary tuberculosis (TB) of 105 Iraqi patients; 37 had active disease, 41 had multi-drug resistant (MDR) PTB and 27 had a relapse of TB. A control sample of 79 healthy persons was also included. Serum levels of markers were assessed using enzyme-linked immunosorbent assay kits. Kruskal-Wallis test together with Dunn-Bonferroni post hoc test revealed significance differences between patients and controls in levels of IL-35, CXCL9, CXCL10 and hBD3, while hBD2 showed no significant difference. Receiver operating characteristic analysis demonstrated that CXCL10 and hBD3 were the most significant markers in predicting TB, particularly active disease. Logistic regression analysis proposed the susceptibility role of CXCL10 in TB. Gender- and age-dependent variations were also observed. Spearman's rank correlation analysis showed different correlations between markers in each group of patients and controls. In conclusion, CXCL10 was up-regulated in serum of TB patients, while hBD3 showed down-regulated level. Both serum proteins are possible candidate biomarkers for evaluation of TB progression, particularly in active disease.

Expression of ORAI1 and STIM1 genes in blood of patients with pulmonary tuberculosis.

This study aimed to detect the expression level of ORAI1 and STIM1 genes in blood of patients with bilateral pulmonary tuberculosis (TB) in comparison with the control group. Both genes encode proteins providing store-operated Ca2+ entry (SOCE) into the cells, including immune cells, to activate transcriptional factors for producing cytokines and inflammation-restricting proteins. The study included 45 patients with confirmed TB, aged 20 to 86, and 35 volunteers, aged from 21 to 73, without active TB infection. The expression of ORAI1 and STIM1 genes in blood was performed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the referent gene. Inflammation was assessed by levels of interferon γ (IFN-γ) and interleukin 18 (IL-18) in serum (ELISA method). The results showed lower expression of ORAI1 in blood and higher levels of IFN-γ and IL-18 in serum of TB patients than that of the control group and no differences in expression of the STIM1 gene. It indicates some impairment in the SOCE mechanism of immune cells, which is associated with TB.

MiRNAs and exosomes in tuberculosis: Their decisive role in the fate of TB

Aim and objectives: Tuberculosis (TB) is one of the most lethal global infectious diseases. Despite the availability of much higher levels of technology in health and medicine, tuberculosis still remains a serious global health problem. Mycobacterium tuberculosis has the capacity for prolonged survival inside macrophages by exploiting host metabolic and energy pathways and perturbing autophagy and apoptosis of infected cells. Results: The mechanism (s) underlying this process are not completely understood but evidence suggests that mycobacteria subvert the host miRNA network to enable mycobacterial survival. The role of miRNAs in TB immune escape mechanisms and the potential for miRNA-based TB therapeutics has been described. Further validation studies are required to (i) elucidate the precise effect of TB on host miRNAs, (ii) determine the inhibition of mycobacterial burden using miRNA-based therapies and (iii) identify novel miRNA biomarkers that may prove useful in TB diagnosis and treatment monitoring. Circulating exosomes have been used as diagnostic biomarkers in various diseases. Recently, the serum-derived exosomes were isolated from TB patients and expression of miR-484, miR-425, and miR-96 was examined. Conclusion: They found that the expression of miR-484, miR-425, and miR-96 were significantly increased in serum of TB patients which correlated with the TB infection level. In conclusion it is well stablished that exosomal miRNAs have diagnostic potential in active tuberculosis. The diagnostic power may be improved when combined with conventional diagnostic markers.

Serum Exosomal miRNAs Are Associated with Active Pulmonary Tuberculosis.

Introduction Tuberculosis (TB) remains a major threat to human health. Due to the limited accuracy of the current TB diagnostic tests, it is critical to determine novel biomarkers for this disease. Circulating exosomes have been used as diagnostic biomarkers in various diseases. Objective of the Study In this pilot study, we examined the expression of miRNAs as biomarker candidates for the diagnosis of TB infection. Methods Serum-derived exosomes were isolated from TB patients and matched control subjects. The expression of miR-484, miR-425, and miR-96 was examined by RT-PCR methods. Results The expression of miR-484, miR-425, and miR-96 were significantly increased in serum of TB patients which correlated with the TB infection level. A receiver operating characteristic (ROC) curve analysis showed the diagnostic potency of each individual serum exosomal miRNA with an area under the curve (AUC) = 0.72 for miR-484 (p < 0.05), 0.66 for miR-425 (p < 0.05), and 0.62 for miR-96 (p < 0.05). Conclusion These results demonstrate that exosomal miRNAs have diagnostic potential in active tuberculosis. The diagnostic power may be improved when combined with conventional diagnostic markers.

Complement Component C1q as Serum Biomarker to Detect Active Tuberculosis.

Background: Tuberculosis (TB) remains a major threat to global health. Currently, diagnosis of active TB is hampered by the lack of specific biomarkers that discriminate active TB disease from other (lung) diseases or latent TB infection (LTBI). Integrated human gene expression results have shown that genes encoding complement components, in particular different C1q chains, were expressed at higher levels in active TB compared to LTBI.Methods: C1q protein levels were determined using ELISA in sera from patients, from geographically distinct populations, with active TB, LTBI as well as disease controls.Results: Serum levels of C1q were increased in active TB compared to LTBI in four independent cohorts with an AUC of 0.77 [0.70; 0.83]. After 6 months of TB treatment, levels of C1q were similar to those of endemic controls, indicating an association with disease rather than individual genetic predisposition. Importantly, C1q levels in sera of TB patients were significantly higher as compared to patients with sarcoidosis or pneumonia, clinically important differential diagnoses. Moreover, exposure to other mycobacteria, such as Mycobacterium leprae (leprosy patients) or BCG (vaccinees) did not result in elevated levels of serum C1q. In agreement with the human data, in non-human primates challenged with Mycobacterium tuberculosis, increased serum C1q levels were detected in animals that developed progressive disease, not in those that controlled the infection.Conclusions: In summary, C1q levels are elevated in patients with active TB compared to LTBI in four independent cohorts. Furthermore, C1q levels from patients with TB were also elevated compared to patients with sarcoidosis, leprosy and pneumonia. Additionally, also in NHP we observed increased C1q levels in animals with active progressive TB, both in serum and in broncho-alveolar lavage. Therefore, we propose that the addition of C1q to current biomarker panels may provide added value in the diagnosis of active TB.

Identification of M. tuberculosis antigens in the sera of tuberculosis patients using biomimetic affinity chromatography in conjunction with ESI-CID-MS/MS.

The profiling of abnormally-expressed proteins in host cells using mass spectrometry (MS) analysis is a classical approach for screening disease-associated biomarkers in clinical diagnosis. However, few pathogen-specific antigens can currently be detected in serum using this proteomic approach, since these are very low-abundant proteins that are easily masked by host high-abundant proteins. Identification of pathogen-specific antigens in the sera of tuberculosis patients is crucial for the clinical diagnosis of this infectious disease, especially in immune-compromised patients. In the present study, two biomimetic affinity chromatography (BiAC) media, At-23 and A115-94, were selected from a library of BiAC media and used to selectively fractionate Albumin and Immunoglobulin from sera, respectively, prior to MS analyses. Each fraction was collected and screened against the proteomic database of M. tuberculosis complex. Three antigens, FbpA, FbpB and BfrB, were identified with two distinct peptides in BiAC-fractionated sera from tuberculosis patients, which were confirmed by Western blotting. Moreover, the identification of pathogen-specific antigens in sera by BiAC in conjunction with ESI-CID-MS/MS represents a promising strategy for the discovery of disease-associated biomarkers in other diseases.

Changes of Zinc Copper and Cu/Zinc ratio and impaired nutritional status in tuberculosis patients of Urmia, Northwest Iran

Malnutrition is observed frequently in patients with pulmonary tuberculosis (TB), but their nutritional status, especially of micronutrients, is still poorly documented. The objective of this study was to investigate the nutritional status of patients with TB compared with that of healthy controls in Urmia, Iran. In a case-control study, 52 patients aged 17–86 y with untreated TB were compared with 58 healthy controls selected from neighbors of the patients. Anthropometric, clinical, serum biochemical and micronutrient status data were collected. Compared to controls, TB patients had significantly lower concentrations of serum albumin, Total iron-binding capacity, ferritin, and calcium (p<0.05). Serum zinc concentrations were significantly lower in patients than controls (p= 0.04). The average serum levels of copper and zinc in healthy volunteers were 3.90±2.17 and 17.04±2.85 µg/dL, respectively. In tuberculosis patients, serum copper and zinc levels were 5.03±2.93 µg/dL and 16.46±3.17 µg/dL, respectively. Patients showed significantly higher serum copper level than controls (p= 0.01). The serum Cu/Zn ratio was significantly higher in the serum of TB patients, (0.30 vs 0.23, P=0.05). In conclusion, the nutritional status of patients with TB was poor compared with healthy subjects. Low level of serum Zn in patients with tuberculosis signifies importance of nutritional assessments, in particular micronutrients, for better management of TB.

MiR-20b inhibits mycobacterium tuberculosis induced inflammation in the lung of mice through targeting NLRP3

Tuberculosis (TB) is one of immune-related disorders. Although dysregulation of miRNAs has been implicated in innate and adaptive immune response, the specific effect of miR-20b on TB has not been documented. In this study, downregulation of miR-20b and upregulation of NLRP3 were observed in macrophages of TB patients, increased levels of inflammatory factors secretion were observed in serum of TB patients. Next, we established a TB mice model by using M. tuberculosis infection and intravenous injection of miR-20b mimic. HE staining showed a denser lymphocytic infiltration and extensive tissue necrosis in the lung of TB mice. Lesions in the TB mice displayed a higher bacterial load by plating of lung homogenates. At the same time, lessened miR-20b and activated NLRP3/caspase-1/IL-1β pathway were observed in TB mice. MiR-20b mimic alleviated the inflammatory response and deactivated the NLRP3/caspase-1/IL-1β pathway in TB mice. Subsequently, we found that miR-20b induced M2 macrophage polarization by using flow cytometry. In addition, luciferase reporter assay confirmed that miR-20b directly bind to the 3′-UTR of NLRP3 and regulated its expression negatively. Further, we found that miR-20b mitigated the inflammation and pyroptosis in alveolar epithelial cells co-cultured with macrophages. Our results indicate that miR-20b could alleviate the inflammatory response in TB mice via targeting the NLRP3/caspase-1/IL-1β pathway, which provides a novel potential molecular mechanism of miR-20b therapy for TB.

Expression of YKL-40 and MIP-1a proteins in exudates and transudates: biomarkers for differential diagnosis of pleural effusions? A pilot study.

BackgroundYKL-40 is an extracellular matrix glycoprotein with a significant role in tissue inflammation and remodeling. MIP-1a has chemotactic and pro-inflammatory properties, and is induced by YKL-40 in several lung disorders. The aim of this study was to determine the levels of YKL-40 and MIP-1a in blood serum and pleural fluids of various pulmonary diseases, and to evaluate their potential role as differential diagnosis biomarkers.MethodsWe recruited 60 patients (age: 62.5 ± 20.6 years) with pleural effusions: 49 exudates and 11 transudates (T). Exudates were further classified based on the underlying disease: ten with tuberculosis (TB), 13 with lung cancer (LCa), 15 with metastatic cancer (MCa) of non-lung origin and 11 with parapneumonic (PN) effusions. YKL-40 and MIP-1a levels were measured by ELISA.ResultsPleural YKL-40 levels (ng/ml) were similar among all patient groups (TB: 399 ± 36, LCa: 401 ± 112, MCa: 416 ± 34, PN: 401 ± 50, T: 399 ± 42, p = 0.92). On the contrary, YKL-40 was significantly lower in the serum of TB patients (TB: 58 ± 22, LCa: 212 ± 106, MCa: 254 ± 140, PN: 265 ± 140, T: 229 ± 123, p < 0.001). Pleural MIP-1a protein levels (ng/ml) were statistically lower only in patients with LCa (TB: 25.0 ± 20.2, LCa: 7.3 ± 6.0, MCa: 16.1 ± 14.9, PN: 25.4 ± 27.9, T: 18.5 ± 7.9, p = 0.012), a finding also observed in serum MIP-1a levels (TB: 17.1 ± 7.6, LCa: 9.4 ± 7.0, MCa: 28.7 ± 28.7, PN: 33.3 ± 24.0, T: 22.9 ± 8.7, p = 0.003).ConclusionsOur data suggest that both YKL-40 and MIP-1a, particularly in serum, could prove useful for the differentiation of pleural effusions in clinical practice, especially of TB or LCa origin. However, large-scale studies are needed to validate these findings.

Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection

The lack of effective and accurate diagnostic tools contributes to the high prevalence of tuberculosis (TB) worldwide. The current serodiagnostics for TB are inadequate mainly due to lack of TB-specific antigens with highly accurate diagnosis. In the current study, we aimed to identify novel diagnostic antigens using glutathione S-transferase (GST)-fusion protein technique. We determined the reactivity of these recombinant proteins arrayed in solution and on GSH-immobilized microplates with TB patient sera. Of 409 TB proteins produced, ninety-two yielded seropositive reactions, fourteen including eight novel proteins showed strong immunoreactivity. Further, six were selected and constructed as a multiple-antigen combination set through analysis of various combinations. A comparative study of the multiple-antigen combination set and a commercially available kit revealed that the combination set showed 66.3% (95% CI 60.5–71.8) sensitivity, which was significantly higher than that of the commercial kit [31.6% (95% CI 26.3–37.3)]. The specificity of both methods was similar at 89.6% (95% CI 83.3–95.4) and 90.6% (95% CI 83.0–95.6), respectively. This study provides a set of novel diagnostic protein markers with great potential for the development of novel diagnostic tools for active TB.

Exposure of Monocytes to Lipoarabinomannan Promotes Their Differentiation into Functionally and Phenotypically Immature Macrophages.

Lipoarabinomannan (LAM) is a lipid virulence factor secreted by Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis. LAM can be measured in the urine or serum of tuberculosis patients (TB-patients). Circulating monocytes are the precursor cells of alveolar macrophages and might be exposed to LAM in patients with active TB. We speculated that exposing monocytes to LAM could produce phenotypically and functionally immature macrophages. To test our hypothesis, human monocytes were stimulated with LAM (24–120 hours) and various readouts were measured. The study showed that when monocytes were exposed to LAM, the frequency of CD68+, CD33+, and CD86+ macrophages decreased, suggesting that monocyte differentiation into mature macrophages was affected. Regarding functionality markers, TLR2+ and TLR4+ macrophages also decreased, but the percentage of MMR+ expression did not change. LAM-exposed monocytes generated macrophages that were less efficient in producing proinflammatory cytokines such as TNF-α and IFN-γ; however, their phagocytic capacity was not modified. Taken together, these data indicate that LAM exposure influenced monocyte differentiation and produced poorly functional macrophages with a different phenotype. These results may help us understand how mycobacteria can limit the quality of the innate and adaptive immune responses.

English

Tuberculosis (TB) is a major health problem in the developing world. 9 million new cases of TB and three million deaths are report- ed every year around the globe. The management of tuberculosis in the regions of developing countries urgently needs an efficient diagnostic test. DevR (also called DosR) is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. The similarity in sequences of DevR in Mycobacterium smegmatis and Mycobacterium tuberculosis has been already proved and hence My- cobacterium smegmatis is used as a model in present study for studying and evaluating the immunogenic properties of antigen DevR. Elimi- nation of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic methods do not qualify for use in a developing country due to lack of either desired accuracy and cost. In the present study ELISA was used to evaluate the diagnostic potential of an immuno-dominant antigen DevR from 36 BCG vaccinated and 12 non-vaccinated TB patients. 48 samples were collected from healthy control subjects. We have evaluated the diagnosis of tuberculosis, based on detection of anti-IgG antibodies in the TB patient's sera. The mean IgG antibody levels in sera of both BCG vaccinated (n=36) and non-vaccinated (n=12) patients with tuberculosis were significantly higher compared to the healthy donors (p<0.05). The results suggest that DevR could be possible candidate biomarker for effective use in the serodiagnosis of persistant tuberculosis infections.

Interleukin 24 as a novel potential cytokine immunotherapy for the treatment of Mycobacterium tuberculosis infection

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a major global health problem. Interleukin 24 (IL-24) is a novel tumor suppressor and a unique member of the IL-10 family of cytokines. However, the in vivo immunological consequences of this cytokine's activity during Mtb infection are still unknown. We found that IL-24 concentration was significantly decreased in the sera of TB patients, and Mtb infection suppressed IL-24 expression of human peripheral blood mononuclear cells (PBMCs) in vitro. Furthermore, we used a mouse infection model utilizing the virulent Mtb H37Rv strain to demonstrate that the administration of exogenous IL-24 had a protective effect against the bacteria. We found that IL-24 could activate human CD8 + T cells, driving CD8 + T cells to produce interferon-γ (IFN-γ) and counteract TB. This activity was found to be dependent on early involvement of neutrophils in the mouse model. IL-24 strongly stimulated IFN-γ production mainly by signaling through the IL-24 receptors of human CD8 + T cells. IL-12 secretion from neutrophils in response to IL-24 treatment might be a minor factor in activating human CD8 + T cells to secrete IFN-γ. Suppression of IL-24 expression by Mtb infection might enhance susceptibility to infection and promote the development of chronic TB. This new information could potentially stimulate the development of a new cytokine-based immunotherapeutic approach using IL-24 to stimulate immunity against TB.

Nonspecificity of 35 kDa protein: A proposed marker for the differential diagnosis of M. avium infection in the Indian population

The subunit vaccine strategies and development of various diagnostic reagents for Mycobacterium avium infection relies on the presence of secreted, species-specific mycobacterial antigens. The M. avium 35 kDa protein has been suggested as a candidate for vaccine/diagnostic reagent, specifically for M. avium infection. The present study was conducted to evaluate the diagnostic specificity of the M. avium 35 kDa protein in the Indian population. Culture filtrate proteins were isolated by growing the bacilli in modified Youman's medium. The 35 kDa protein was purified by high-resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blast search was carried out. Western blotting was performed with either monoclonal antibody CS-38 or serum samples of tuberculosis (TB) patients. The 35 kDa-specific immunoglobulin G antibody titer was estimated in the sera of TB patients and healthy individuals by indirect enzyme-linked immunosorbent assay (ELISA). Despite the absence of gene for the 35 kDa protein, the sera of TB patients and TB patient's contacts nonspecifically recognize it. Of 109 TB patients tested, the sera of 84 patients in ELISA (percentage recognition = 87.5%) and 27 of 29 TB patients tested in western immunoblotting (percentage recognition = 93.10%) recognized the M. avium 35 kDa protein, while with sera of TB patient's contacts, the recognition was 50%. Contrary to Western studies, the M. avium 35 kDa protein does not seem to be a good candidate for the specific diagnosis of M. avium infection in the Indian population.

Rapid immunodiagnostic assays for Mycobacterium Tuberculosis infection

Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensitivity and specificity that can be used in the field and in the laboratory and that can be formatted for use with multiple species. This would help to obtain definitive early diagnosis of TB. The present study was developed to determine the role of using early secreted antigenic target-6 (ESAT-6) in immunodiagnosis of Mycobacterium tuberculosis. Methods: Serum samples were obtained from TB infected patients and normal healthy controls. Two rapid immunodiagnostic assays (Enzyme-linked immunosorbent assay (ELISA) and Immunoblotting) were performed. Results: The sensitivity of immunoblotting assay was 100%; however, ESAT-6 antigen was not able to discriminate between patients and normal controls. Application of direct ELISA using ESAT-6 antigen yielded 97.6% sensitivity and 75% specificity for the diagnosis of TB infection. Conclusion: In conclusion, the detection of antibodies against ESAT-6 antigen in the sera of TB patients by direct ELISA could be used as a preliminary assay for diagnosis of human M. tuberculosis infection. A combination of the ELISA with either radiological or microscopic examination is required to overcome the low specificity of the assay for negative results.