Introduction Background of the study

Transcription

1 1 Introduction Tuberculosis (TB) is one of the leading cause of morbidity and mortality worldwide, affecting one-third of world population. Geographically, the incidence is much higher in Southeast Asia (India and China together account nearly 40% of the global TB cases) 1-2. Further, the condition in India is aggravated due to the emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis. Indeed, the MDR strains are those strains of M. tuberculosis, which exhibit resistance to both rifampicin (RIF) and isoniazid (INH) with or without resistance to other first line drugs 3. The outcome of treatment of patients harboring such strains of M. tuberculosis is poor and mortality rate is also high. In addition, their chance of being cured is very low, requires significant expenditure of health care resources, and remains infectious for a prolonged period and more likely to infect others 4. Background of the study Several rapid and inexpensive tests to detect drug resistance in M. tuberculosis have been developed recently 5-20.Unfortunately; such tests have been evaluated only in a few low-resource countries 17-20, but not so far TB laboratories in India. The colorimetric methods employing the oxidation-reduction indicators like resazurin and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Alamar blue and 2,3,5 tri nitro phenyl tetrazolium chloride (TTC) have been successfully used to determine minimum inhibitory concentrations (MICs) of rifampicin and several other antitubercular drugs 5-9,11and However, all the colorimetric assays were carried out employing limited number of clinical specimens. Therefore, the workers recommend that the validation of these assays to be affirmed using large number of clinical specimens. Among the colorimetric assays investigated, the Resazurin microtiter assay (REMA), which works on the principle of actively metabolizing bacteria to reduce the resazurin, which in turn

2 2 produces a change in color from blue to pink. The Middlebrook 7H9 broth with OADC enrichment (oleic acid, albumin, dextrose, and catalase) and PANTA antibiotic mixture (inhibits bacterial and fungal contamination) used as culture media in this assay. The resazurin assay can be conducted in micro centrifuge tubes or micro titer assay plates as well. Recommended concentration of drugs are added to the each wells containing Middlebrook 7H9 broth followed by the addition of a suspension of mycobacterial culture to be tested. Then these plates are covered and sealed in plastic bags and incubated at 37 0 C. After 7 days of incubation, resazurin solution is added to control wells followed by overnight incubation at 37 0 C. A change in color from blue (oxidized state) to pink (reduced) indicate the growth of bacteria, and then add resazurin solution to other wells also. The MIC is defined as the lowest concentration of drug that prevented this change in colour as well. 9, 16, The MTT assay utilizes the ability of mitochondrial dehydrogenase enzyme of living cells to reduce MTT (an yellow colored dye) to an insoluble purple MTT-formazan crystals which, after solubilization (by adding 20% sodium dodecyl sulfate in 50% N,N-dimethylformamide), can be measured spectrophotometrically. The MTT assay can be conducted in micro centrifuge 5, 11, 12, tubes or micro titer plates also using the same methodology of REMA assay and 15. On the other hand, the Microplate Alamar Blue Assay (MABA) utilizes the principle of the alamar blue oxidation-reduction reaction, which in turn is the measure of cell viability or cellular growth 14. However,the TTC assay utilizes the principle of MTT assay, in which the insoluble TTC formazan crystals are formed as a red ring at the bottom of the tube. Moreover, the TTC assay has advantages over other colorimetric assays; other assays need to be examined regularly with addition of the particular reagents for the detection of mycobacterial growth. However, TTC assay indicate growth of bacteria with production of red colored

3 3 formazan ring at the bottom of the tube without the addition of the supplementary reagents 8. The microscopic observation drug susceptibility (MODS) assay is a non colorimetric assay, which is performed in sterile 24-well plates. This method employs Middlebrook 7H9 broth with OADC enrichment and PANTA antibiotic mixture (inhibits bacterial and fungal contamination) as the culture media. In this assay, the particular anti tubercular drugs and the decontaminated specimen to be tested is added in to each well containing Middlebrook 7H9 broth. Then cover and seal the plates in plastic bags and incubate at 37 C.After third day onwards of incubation, start observing the drug-free control wells using an inverted-light microscope (40 X magnification). The growth of Mycobacterium is indicated as serpentine clusters of bacteria and the drug susceptibility results can be interpreted on the same day when the distinct growth visualized in control wells. In this assay, a sample is considered susceptible if growth is visible in the drugfree well but not in the drug-containing well while a sample is considered resistant if both the drug-free and the drug-containing wells are showing visible growth 17, (For more details; The nitrate reductase assay (NRA), which is based on the ability of M. tuberculosis to reduce nitrate to nitrite. In this assay, each LJ media slants are incorporated with potassium nitrate (KNO 3 ) and the particular anti tubercular drug to be tested. For each assay, inoculate each LJ slant with undiluted test inoculums (i.e. three LJ slants with KNO3 but without anti tubercular drugs and the other LJ slants with KNO 3 and the drug to be tested). Then, incubate these slants at 37 0 C for 7 days. After incubation, add 0.5 ml mixture of three reagents (1 part 50% concentrated hydrochloric acid, 2 parts 0.2% sulfanilamide and 2 parts 0.1% N-1- naphthylenthylenediamine dihydrochloride) to one drug-free control tube. If its color changes to pink, then test the LJ tubes with drugs on the same day. In this assay, an isolate is considered resistant if there is a color change in the drug

4 4 containing LJ slant greater than in the growth control. In case, the drug-free control tube are not showing any color change or remained same, subsequently remaining tubes should be incubated and repeat the procedure on day s 10 th and 14 th 13, 17 respectively. Need for the study: The majority of the mycobacterial clinical laboratories in India are experiencing intricacy in obtaining drug susceptibility information for M. tuberculosis isolates due to financial or technical reasons. Moreover, treatment of tuberculosis without the knowledge of susceptibility information increases the risk of treatment failure, the spread of resistant strains and the development of resistance to additional drugs as well 14. However, the commonly used traditional mycobacterial drug susceptibility testing methods such as proportion method (PM), absolute concentration and resistance ratio methods require several weeks of incubation to give the results In addition, these results are qualitative in nature and not quantitative, therefore often making the result clinically irrelevant.however, the currently expending BACTEC radiometric system (Becton Dickinson, Sparks, MD, USA) has the advantage of being more rapid than the proportion method but requires the use of radioisotopes and can be costly to perform 5,7. Although other commercial tests like the MGIT (mycobacterial growth indicator tube) and molecular tools such as the INNOLiPA Rif.TB (line probe assay; Innogenetics, Ghent, Belgium) have been developed, but are also expensive and impractical for routine use in low resource settings 28. The detection of drug resistant TB and other mycobacterial infections in the early stage of disease is utmost important to trim down the incidence. Therefore, it is necessary for clinical microbiology laboratories to detect rapidly and identify mycobacteria from human clinical material. However, the available literatures suggest that the current techniques such as MGIT and BACTEC and molecular methods, which are widely used, in fact are not cost effective and affordable for

5 5 most of the clinical microbiology laboratories in developing countries 29. Hence the current study is undertaken to evaluate the performance of two rapid methods, Direct MODS and direct TTC assay, with indirect proportion method (using LJ media) on Ziehl-Neelsen smear positive sputum specimens. Moreover, to our knowledge, this is the first study, which will investigate the performance of Direct MODS and direct TTC assay with conventional indirect proportion method (using LJ media) directly on Ziehl-Neelsen smear positive sputum specimens.

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