Fig 6 - uploaded by Dr.Ram Pramod Tiwari
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Structure of: (a) Gram-negative, (b) Gram-positive bacterial cell wall and (c) M. tuberculosis (Acid Fast) cell wall.
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Tuberculosis is a chronic granulomatous disease caused by tubercle
bacteria known as Mycobacterium tuberculosis and is transmitted from
infectious persons through aerosol. It is a facultative intra-cellular organism
that is readily phagocytosed, but is resistant to intra-cellular killing by
macrophase. It grows intracellularly and can remain dorman...
Context in source publication
Context 1
... cell wall of M. tuberculosis is similar to that of other Mycobacteria because it has a thickness of about 20nm as seen through an electron microscope, different than other Gram-negative and Gram-positive bacteria (Fig. 6a, b, c), it also appears to consist of an inner, electron dense layer surrounded by an outer electron transparent layer ( Payne et al. ...
Citations
... A similar approach with liposome formulation has been devised wherein a cocktail of antiglycolipid antibodies were coated on the card for enhanced specificity, sensitivity, and shelf life of the diagnostic reagent [52]. Based on the previous reports, 38 kDa, 19 kDa, and 16 kDa, and HSP 71 proteins are very effective as immunogens [53]. The immunodiagnostic reagents developed in the past had a variable specificity and sensitivity in the diagnosis of tuberculosis hence the cocktail of antigens was targeted for developing diagnostics of enhanced sensitivity and specificity [54]. ...
Introduction: Tuberculosis (TB) is still one of the major global health threats and delayed diagnosis or misdiagnosis continues to fuel the global epidemic. The conventional diagnostic approaches have shortcomings that might hinder the process of diagnosis of the disease and ultimately affect the prognosis. Area covered: We emphasize on the process of the synthesis of liposomes, its physicochemical properties affecting the formulation and their utilization in the field of molecular diagnostics for TB. The review also sheds a light on other nanoparticle-based molecular diagnostic approaches for TB. Despite the advent of science, we are yet to have a diagnostic tool that is simple, rapid, sensitive, and specific, and most importantly, one that enables us to demarcate patients with active tuberculosis from those with quiescent lesions, prior vaccination, or other diseases. Expert opinion: The utility of liposomes for diagnostic purposes has been attempted so as to overcome the challenges posed by conventional diagnostic tools for TB. Through this review, we present insights into liposome formulation and selection processes, various studies that report the use of liposome-based diagnostic tools for TB, as well as the limitations associated with the same that can be improvised to make the technology more efficient. ARTICLE HISTORY
... More specifically, M.tuberculosis arose from a soil bacterium that evolved to infect cows, and then transferred to humans about the time of animal domestication. M.tuberculosis and M. leprae both grow remarkably slow in which M.tuberculosis doubles its population every [18][19][20][21][22][23][24] hours, while M. leprae doubles its population about every 14 days. This extremely long generation time probably contributes to the chronic nature of both diseases. ...
Tuberculosis (TB) is a communicable disease primarily affecting lungs but can disseminate to other parts of the body and causes mild to severe damage. The disease is caused by transmission of aerosolized droplets of M.tuberculosis. Tuberculosis kills approximately 2 million people annually worldwide and the epidemic is spreading is assuming alarming proportion. Around 8 million new cases of tuberculosis are registered every year. In India the disease kills nearly 7 lakh people and 3 million new cases annually. Diagnosis of tuberculosis is primarily based on X-ray examination and / or sometimes on symptoms that are often confusing and misleading. While infectious cases are frequently missed, some people are mistakenly diagnosed with TB and inappropriately treated. The burgeoning pool of infection and the resulting risk of becoming infected with this pool are tremendous. Hence there is an urgent need for development of cost effective, sensitive and specific diagnostic tool to correctly and timely diagnose the patients and initiate effective chemotherapy for effective cure.
Despite an enormous amount of search since the time of Koch, we still have no simple, sensitive & specific test that will differentiate most or all patients with active tuberculosis from those with quiescent lesions, previous vaccination or other diseases or even from those who are completely healthy (Grange et al., 1990). Current procedures for identifying infected persons are limited by a lack of sensitivity and delay in reporting the diagnosis of Mycobacterium tuberculosis.
Conventional techniques for the diagnosis of tuberculosis like microscopic evaluation of Mycobacteria using either ZN (Ziehl-Nelsen) or flurochrome stained smears is insensitive which can detect acid fast bacilli only when there are > 104 Mycobacteria/ ml are present. Culture on solid medium is not only labor intensive but also too slow for clinical usefulness takes more than three weeks and often longer for microscopy negative samples. Still in many cases, diagnosis cannot be confirmed at the time of presentation (Nunn et al., 1994)
Radiometric liquid (BACTEC) and biphasic (MB chek) culture system have improved both the recovery rates & speed of isolation, but these systems are very costly and require expert workers and established facilities (Vareldzis et al., 1994). The other commonly used procedure is ‘Mantoux test’ which doesn’t make a conclusive evidence of active disease, while negative tests do not exclude it. Positive tuberculin test may be due to active tuberculosis, past infection, past BCG vaccination or sensitization by environmental Mycobacterium.
Recently, with the advent of recombinant DNA technology several workers have developed kits based on polymerase chain reaction (PCR) for the accurate and rapid diagnosis of tuberculosis (Miller et al., 1994). But the application of PCR based diagnosis is beset with the need for expertise and is also very costly. One PCR tests costs around $15 (Bennedsen et al., 1996).
Many workers have attempted to isolate the species-specific antigen for use in diagnostic tests, but this task has proved very difficult, because of two reasons Firstly, specific antigenic determinants often occur on the same protein molecule as shared antigen, therefore, making it impossible for purification even by affinity chromatography based on binding with specific antibody. Secondly, a given determinant may be present on a range of molecules of differing physiochemical properties. Thus preparative techniques based on such difference (gel-filtration & Ion exchange chromatography) have not proved very useful (Grange et al., 1988). Further due to the antigenic diversity in mycobacterial diseases, especially in tuberculosis, no single antigen can cover all the positive cases.
Several antigens of Mycobacterium tuberculosis have been found to be useful in the serodiagnosis of the clinical disease (Charpin et al., 1990; Craud et al., 1990; Laszlo et al., 1992). A number of glycolipids, phospholipids, and sphingolipids have been reported to be antigenic (Goren et al., 1979). The species and type species specific lipids are present on the Mycobacterial cell surface including phenolic glycolipids, dimycocerosates of phthiocerols and lipooligosaccharides. As most of them are surface exposed, most of them are supposed to be in face-to-face flight with body’s defense mechanism (Ortalo- Magne et al., 1996). Trehalose based glycolipids are found in a variety of structural forms in the lipids of Mycobacteria and related bacteria. Serologically active glycolipids extracted from Mycobacterium bovis BCG have been described (Reggiardo et al, 1975). Thus glycolipids have been studied and found as potential serological marker, when tested with various group of specimens obtained from tuberculosis infected individuals.
The method commonly employed to detect the presence of antigen or antibody in the patient sample are ELISA / western blot etc. ELISA system though very practical and sensitive but it is not always available in countries with high prevalence of tuberculosis especially in the peripheral health units, where most of the tuberculosis cases are diagnosed. Also by using rapid test method of tuberculosis detection is considerably simplified. Thus a more simple and specific and sensitive test is required that can detect both multibacillary and paucibacillary infection clumping all IgG, IgA and IgM antibody classes, which have an additional advantage in detecting maximum number of tuberculosis infections. Furthermore since, no single antigen reagent gives 100 %sensitivity due to antigenic diversity, however; future research should identify the best combination of antigens for the serodiagnosis of tuberculosis (Bothamley et al., 1995).
Present study was undertaken with the aim to develop a simple, sensitive, specific and cost effective diagnostic tool (TB antibody/ TB antigen detection kit) for screening patients with pulmonary and extra-pulmonary tuberculosis from those-of non-tuberculosis, of other common infections and-of normal healthy controls have been developed.
Keeping in view we identified a pool of antigens to devise appropriate system for diagnosis of tuberculosis. The selection of antigen was based on finding that mycobacterial lipids/hydrophobic components when used together could provide suitable serodiagnosis of tuberculosis. Glycolipids can be used in the conventional ways for coating on various solid surfaces so as to observe antigen-antibody interactions taking place.
The serological response of purified Mycobacterial glycolipid antigens were examined by liposome agglutination assay. The assay was able to detect the presence of very low antiglycolipid antibody concentrations in the infected individuals when tested with 1813 samples from various group. Out of which 604 samples have been included for sensitivity study where as 1209 samples were enrolled for specificity study. The serum from the tuberculosis patient group having significant concentration of antiglycolipid antibody than uninfected control subjects with 94% sensitivity and 98.3% specificity have been found. Glycolipids of M.tuberculosis H37Rv antigens were isolated (Figure 1), purified and characterized (Figure 2). These purified antigens after interchelation with liposome particles (Figure 3, Right first bottle, and principle of TB Screen test), specifically bound to antiglycolipid antibodies present in sera of tuberculosis patients, resulting in formation of blue colored agglutination (Figure 4, 5). The strengths of agglutination vary depending upon the rate of infection leads variable concentration of antibodies in infected individuals. This protocol clearly differentiates normal healthy controls and BCG vaccinated subjects from those of active tuberculosis sufferers. The resultant diagnostic tool so called herein (TB Screen test) “ TB antibody detection kit” is economical and rapid (4 minutes) as compared to currently available products and can be utilized for mass screening of heavily afflicted population (Tiwari et al., 2005).
Additionally, we have also attempted to develop a agglutination based assay for the detection of M.tuberculosis glycolipid antigen in cerebrospinal fluids (CSF) of patients with active tubercular meningitis, processed tissue-biopsy specimens for other extra- pulmonary tuberculosis and serum samples for pulmonary/extra-pulmonary tuberculosis cases. A liposomal agglutination card test (TB/M Card test) “TB-antigen detection kit” was developed for this purpose. This assay was developed to facilitate the recognition and binding of affinity purified anti-mycobacterial glycopolid antibodies raised in rabbits. They were coupled to the surface of activated liposome particles (0.2-0.4µm) with the conjugation of Phosphatidyl ethanolamine (PE) and in presence of EDAC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), when these Liposomes were used for the assessment of specimen, were bind to the antigens present in the samples and agglutination like reaction were formed (Figure 1B, and principle of TB/M card test) and could be interpreted within 4 minutes. The assay proved to be a simple and quick rapid diagnostic test with superior sensitivity and specificity compared to conventional AFB smear and culture methods. A total of 1276 biological fluids viz., CSF from 428 patients of active tuberculosis meningitis, 216 sera of clinically confirmed (smear/culture) pulmonary cases, 72 specimen of extra pulmonary, 86 samples of MDR tuberculosis, 126 patients with other respiratory diseases, 115 patients with non-respiratory diseases, 58 samples of BCG vaccinated healthy children and 175 samples of healthy subjects were included in this study. The “TB-antigen detection kit” test showed 95.76% sensitivity and 93% specificity.
Thus in brief we have developed two, simple cost effective sensitive and specific test (TB antibody detection and TB antigen detection kit) for the detection of either antibody or antigen in the patient samples. Both the tests were found to be more sensitive and specific than the other serological tests in the market (Tiwari at al., 2005) additionally, the test is rapid and can be easily performed without any special setup.
In Conclusion, TB-antibody detection kit (Tiwari et al., 2005a, TB Screen test Indian Patent application No. IPR/4.18.4/03026/2004, Allotment No.226/Del/2004, International patent application No. PCT/IN2005/000063, World intellectual
property copy right No.PCT-WO2005080987-A1, European application No. W02005IN0006320050221, Priority No. IN2004E0022620040219, International Patent classification No.G01N33/539;GOIN33/532;GOIN33/549, dated 1st Sept.2005 in association with NRDC Govt. of India) and TB-antigen detection kit could able to investigate and examine of the active tuberculosis sufferers in developing as well as developed countries where prevalence of disease is more and helpful for epidemiological studies and disease control programs of various government agencies. Thus further transmission of infection in the community can be checked by mass/routine screening as alternate diagnostic products (e g. AFB) and effective chemotherapy can be started.
