International Journal of Pharma and Bio Sciences DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS BY ELISA AND REAL TIME PCR ABSTRACT

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1 Research Article Biotechnology International Journal of Pharma and Bio Sciences ISSN DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS BY ELISA AND REAL TIME PCR MAYS NOORI FALIH Microbiology Department Indian Academy Degree College, Hennur cross, hennur main road. Bangalore ABSTRACT Until now there has been little effort to improve techniques for diagnosing tuberculosis hence control of tuberculosis is hampered by slow, insensitive diagnostic methods. The effective treatment of tuberculosis will be one of the greatest public health interventions. In this study we assessed the performance of Real Time PCR and ELISA. IgG antibodies to MTb are investigated by ELISA in chronic infections. Also, M. tuberculosis DNA is extracted from samples and amplified using Real Time Amplification and detected using fluorescent reporter dye probes specific for M. tuberculosis. We found a 100% match of the results for MTB detection. Future research on this topic is certainly needed, and this research would be benefited from the increased detection capacities of both Real Time PCR and ELISA. KEYWORDS: M. tuberculosis, ELISA, RT-PCR, Diagnosis MAYS NOORI FALIH Microbiology Department Indian Academy Degree College, Hennur cross, hennur main road. Bangalore *Corresponding author B

2 INTRODUCTION Tuberculosis is a common and deadly infectious disease caused by mycobacterium, mainly Mycobacterium tuberculosis which attacks the lungs but can also affect other systems. M. tuberculosis is an intracellular pathogen that is highly adapted to human. Its major host cell reservoir is the mononuclear phagocyte (monocytes and macrophages). Given its mechanisms it has adapted strategies to manipulate the host cell response during and after its entry into the macrophage 1. Over one-third of the world's population has been infected by the TB bacterium, and new infections occur at high rate. India is one of the high TB burden countries accounting for one fifth of the global incidence of TB and tops the list of 22 high TB burden countries 2. India is one such region where M. tuberculosis has remained in equilibrium with the population, resulting in an area of tuberculosis endemicity 3, 4. One of the most important factors influencing the current TB epidemic in resource-limited settings is poverty, which is closely related to malnutrition, crowded living conditions, lack of access to free or affordable health care services that can facilitate the transmission of tuberculosis 5. Not everyone infected develops the fullblown disease; however, one in ten latent infections progress to active TB disease, which, if left untreated, kills more than half of its victims. The outermost components of the M. tuberculosis cell wall, predominately lipids and carbohydrates, are the first to contact host molecular constituents and play a major role in facilitating host cell recognition and modulation of host responses 6. According to international standards, tuberculosis diagnosis must be confirmed either by bacteriology or by histology studies, but the bacteriological methods do not always allow detecting M. tuberculosis in people affected with pulmonary tuberculosis. Early diagnosis of tuberculosis makes effective treatment possible and increases the probability of clinical outcome owing to quite effective anti-tuberculosis therapy; however the tuberculosis diagnosis has certain difficulties. Smear stain technique is a rapid and cheap method, but it has low sensitivity, not high specificity, and cannot differentiate TB from other mycobacterium. The application of molecular biology methods allow to overcome the difficulties in the diagnosis of M. tuberculosis. An estimated 1.5 million TB serological tests are performed in India every year 7.The commercially available serological tests showed that the diagnostic sensitivities of these tests with patients with active tuberculosis ranged from as low as 16 per cent and maximum up to to 57 per cent 8. The result from a serological test using the ELISA could be available within hours, and the result using an immune chromatographic assay format, within minutes 9. The real-time PCR assay has demonstrated high performance and could be used in a range of low- and middle-income countries 10. In this study the Real Time PCR and ELISA method were used for diagnosis of M. tuberculosis in clinical specimens. MATERIALS AND METHODS ELISA The ELISA was performed as per the manufacturer s instruction for detection of IgG antibodies to MTb in two-step incubation procedure. Briefly, polystyrene microwell strips are pre-coated with affinity purified MTb antigens including A60 antigen complex. The wells are washed and rabbit anti-human IgG antibodies (anti-igg) conjugated to horseradish peroxidase (HRP) are added. Then Chromogen solutions containing Tetramethylbenzidine (TMB) and urea peroxide are added to the wells and after stopping reaction the amount of color intensity was measured. In the same way other antibodies (IgA and IgM) were detected and measured. Calculation of Cut-off value (C.O.) = *Nc (*Nc = the mean absorbance value for three negative controls.) Real Time PCR M. tuberculosis DNA is extracted from samples and amplified using Real Time Amplification and detected using fluorescent reporter dye probes specific for M. tuberculosis. Internal Control (IC) serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition. IC is B

3 Int J Pharm Bio Sci 2013 July; 4(3): (B) detected in a channel other than the M. tuberculosis DNA. MTB Real-TM contains UDG-Enzyme which is added to the reaction mix. Since deoxyuridine triphosphate (dutp) is only present in amplicon while deoxythymidine triphosphate (dttp) is present in MTB DNA the use of UDG enzyme degrades only amplicons generated from previous runs avoiding possibility of amplicon contamination. UDG is active at room temperature during Mastermix preparation, while during amplification is inactive, not affecting the correct and wanted experiment s amplicon. RESULTS AND DISCUSSION IgG antibodies to MTb are investigated in chronic infections. The ELISA is used for qualitative detection of IgG antibodies to M. tuberculosis in human serum or plasma. It is intended for diagnosing and monitoring of patients related to infection by M. tuberculosis and other Mycobacteria. Sputum and blood samples were obtained from Patients and the raw data for the ELISA test was evaluated. The O.D. values for ELISA were shown in table 1. Table 1 O.D. Values The results are calculated by relating each sample s optical density (OD) value to the Cutoff value (C.O.) of the plate. If the Cut-off reading is based on single filter plate reader, the results should be calculated by subtracting the Blank well OD value from the print report values of samples and controls. On the other hand, low levels of IgG in an inactive disease state indicates the non-evacuation of intra cellular infectious pockets with possible progression of the disease to active at later state. The test results with parameters were showed in table 2 Table 2 The test parameters for ELISA The fluorescence data was showed in figure 1. Samples (5.6%) were positive on ELISA and real-time PCR tests. Seven samples were positive on all tests (4.9%). Six samples (4.2%) were positive on real-time PCR alone. Prevalence of Sputum MTB infection, estimated sensitivities for solid culture, liquid culture and real-time PCR were 0.72, 0.65 and B

4 0.72, respectively. Estimated serum ELISA sensitivity was 0.26, and specificity was When data from the two herds were analyzed separately, no significant differences (p > 0.05) in the TAGS estimated sensitivities and specificities of solid culture, liquid culture and real-time PCR were found. Accuracy of each of the tests, based on the above calculated sensitivities, specificities, and prevalence were 0.91 (solid culture), 0.93 (liquid culture), 0.90 (real-time PCR) and 0.82 (serum ELISA). Figure 1 Fluorescence for positive sample, IC and tested samples Test sensitivity for culture methods and realtime PCR, as well as test accuracy, are comparable. although not as accurate as the other tests evaluated, continues to be a useful test because of its rapid turn-around. Now, with real-time PCR, more accurate results can be available as fast as for ELISA. The realtime PCR test for detection is comparable to culture methods, and gives fast results for better decision making. Since ELISA as a clinical test is most commonly used in the screening of animals, we wanted to see that how it performs in relation to pathology and other tests conducted on tissues. The PCR assay in the present study was found to be 100% sensitive in the detection of MTB genome as compared to ELISA in the diseased state. REFERENCES 1. Flynn JL, Chan J, Immunology of tuberculosis. Annu Rev Immunol, 19: , (2001). 2. World Health Organisation Geneva, WHO Report on Global Tuberculosis Control: Epidemiology, Strategy, Financing (2010). 3. Coleman PG, Perry BD, Woolhouse ME, Endemic stability a veterinary idea applied to human public health. Lancet, 357: , (2001). 4. Rothschild BM, Laub R, Hyperdisease in the late Pleistocene: validation of an early 20th century hypothesis. B

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