1. Multicentric evaluation of a sensitive smear Microscopy technique for detection of AFB in sputum
Principal Investigator:Dr.N.Selvakumar
Funding: Indian Council of Medical Research
The objective of the present study is to evaluate in a multicentric laboratory setting a highly sensitive method of smear microscopy developed in Dr. Jaya S. Tyagi's laboratory at AIIMS. By this method (USP smear microscopy) specimens with a bacillary load as low as ~300-400 bacilli per ml can be reproducibly reported as positive. The smear method is essentially a component of a multipurpose method that is applicable on both pulmonary and extra-pulmonary specimens and also compatible with culture and PCR techniques. This method is highly likely to identify a significant number of patients who go undetected by the direct method of smear microscopy thereby contributing to improved disease control.
Sputum samples collected from Otteri TB Hospital are transported to the TRC Bacteriology laboratory and processed within 4 to 6 hrs. A maximum of 8 – 10 samples are processed per day. After receiving the sputum sample direct smear is made. Later each sample is divided into two portions of approximately equal volume. One portion is processed by Modified Petroff's method and the other portion by the USP method for culture. The intake is continuing and about 800 samples are processed.
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2. Use of Phagebiotics for growing tubercle bacilli in liquid medium from sputum processed by Chitin and Petroff's methods
Principal Investigator: Dr.Vanaja Kumar
Funding: Indian Council of Medical Research
Phage cocktail (Phagebiotics) substituting the use of antibiotics to control the overgrowth of normal flora in sputum samples has been established and it forms a novel, bio-friendly approach to tackle the problem of non-mycobacterial contaminants. It is essential to know the effect of phagebiotics on the growth and retrieval of M. tuberculosis to use it in rapid diagnostic assays. The present approach is aimed at evaluating the effect of phagebiotics on sputum samples processed by Chitin and modified Petroff's methods for the early and better recovery of tubercle bacilli on Lowenstein-Jensen (LJ) medium. A total of 120 sputum samples were collected. Culturing M. tuberculosis after processing by modified Petroff's method is considered as gold standard. Culture results of chitin direct, chitin-phagebiotics and Petroff's-phagebiotics were compared with Petroff's culture. Sensitivity and specificity of Petroff's-phagebiotics was 89% and 76% respectively while that of chitin direct method was 84% and 87%, respectively. Only 38% of the real positives were picked up by chitin- phagebiotics while the specificity was as high as 96%. Chitin being acidic, sputum deposits processed with chitin had a pH of ~5.5 after the addition of phagebiotics while those processed with 4% NaOH were at ~8.5. Overnight incubation of sputum deposits in acidic pH probably lead to the killing of tubercle bacilli. Sputum processing with chitin resulted in moderately better retrieval of tubercle bacilli while overnight incubation with phagebiotics reduced the sensitivity much further. Reduction in pH and dilution of reaction mixture by the addition of phagebiotics lead to the reduction in number of viable bacilli. Further studies are planned with neutralized deposits.
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3. Standardization of second line anti-TB drug susceptibility testing
Principal Investigator: Dr.Ranjani Ramachandran
The global situation with respect to tuberculosis has worsened with the emergence of MDR TB. Various studies have shown that MDR -TB can be cured by a combination of second-line drugs under DOTS-Plus. With the spread of MDR TB, there is an increasing demand for drug susceptibility test (DST) for second line anti-TB drugs. Since the critical concentration for second line drugs have not been completely established, the present study is focusing on evaluating the critical concentration of some of the second line drugs namely Kanamycin, Ethionamide, Capreomycin, Amikacin, PAS, Pyrazinamide. The aims are (1) To Standardize DST of Second line drugs using standard conventional methods and automated systems and other rapid phenotypic methods (2) To compare the results obtained by automated and phenotypic rapid methods with that of standard conventional methods.
Clinical isolates from the patients attending the TRC clinic will be selected for this experiment. Selection of isolates are based on their first line drug susceptibility pattern, where 50% of them are MDR and 25% are polyresistant to first line drugs and 25% fully susceptible to all drugs. Isolates from freshly subcultured onto LJ medium will be used for setting up DST after randomization. The following methods for DST will be used viz: LJ, agar, liquid medium, automated systems and colorimetric methods.
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4. Microscopic Observation Drug Susceptibility (MODS) assay as a rapid low cost test for detection and drug Susceptibility of Mycobacterium tuberculosis in HIV-TB and Non HIV-TB Individuals.
Principal Investigator: Dr.Ranjani Ramachandran.
Funding: Indian Council of Medical Research
In the last decade, there has been dramatic resurgence in the incidence of tuberculosis (TB) throughout the world. The situation is compounded with increasing HIV infections. India is estimated to have 3.5 million HIV patients and about 1.8 million of them are co-infected with TB. Most cases occur in developing, resource poor countries, where the expensive automated systems (MGIT 960 and BACTEC 460) are not feasible. To curb the transmission of infection early detection of MDR-TB using a low cost rapid test is the need of the hour. This study aims to evaluate MODS for the detection and drug susceptibility of M. tuberculosis in sputum samples from patients with suspected pulmonary TB in HIV positive and non- HIV individuals and to compare the results of MODS with conventional methods and automated systems to detect cross contamination and relative cost. The sputum samples referred to TRC will be processed for AFB culture as per standard protocol. The remaining sputum sediment following will be inoculated into 7H9 broth with and without drug and periodically examined for cord formation under inverted microscope. DST is performed for the following drugs: Streptomycin, Isoniazid, Rifampicin, Ethambutol, Kanamycin, Ethionamide and Oflaxocin. The results of MODS after decoding will be subjected to statistical analysis.
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5. Tetrazolium Micro Plate Assay (TEMA) - Rapid Colorimetric Method and Thin Layer Agar (TLA) For Determination Of DST of M. tuberculosis
Principal Investigator: Dr.Ranjani Ramachandran
Funding : Indian Council of Medical Research
To control the spread of MDR-TB, rapid and reliable method for early diagnosis and rapid drug susceptibility test (DST) is the need of the hour. There are various rapid methods available for DST, which includes, automated system using BACTEC 460 and MGIT 960 (Becton and Dickenson) and Molecular methods such as INNO-LiPa (Line probe assay) that are expensive and are impractical for routine use. In the last few years much attention has been posed towards colorimetric assays such as Nitrate Reductase Assay (NRA) using solid media, MABA and TEMA (A rapid calorimetric method based on the principle of reduction of MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5 diphenyl tetrazolium bromide), which are quite rapid and cost effective and easy to perform.
The aims of this study are (i) To standardize and evaluate TEMA and TLA (Thin Layer Agar) (ii)To determine susceptibility of M.tuberculosis for first and second line drugs and
(iii) To compare the TEMA and TLA results with that of conventional methods.
After incubation readings will be taken on days 3, 5, 7 and10 respectively under an inverted light microscope. Susceptibility pattern can be determined by detecting microcolonies in drug free and drug containing plates. Percentage of resistance will be calculated. After completion of the experiments, the strains will be decoded and results will be analyzed using appropriate statistical method.130 cultures from patients referred to TRC will be selected for the study and randomized by the study statistician.
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