Combination of commercially available molecular assays and culture based methods in diagnosis of tuberculosis and drug resistant tuberculosis

ABSTRACT Early diagnosis of tuberculosis is of major clinical importance. Among 4733 clinical specimens collected from 3363 patients and subjected to Ziehl-Neelsen microscopy, 4109 were inoculated onto Löwenstein-Jensen slants and 3139 in Bactec/9000MB. Polymerase Chain Reaction (PCR) was performed in 3139 specimens, whereas, a genotypic assay was directly applied in 93 Mycobacterium tuberculosis complex PCR-positive for isoniazid and rifampicin resistance detection specimens (GenoType MTBDRplus). Recovered M. tuberculosis isolates (64) as well as, 21 more sent from Regional Hospitals were tested for antimycobacterial resistance with a phenotypic (manual MGIT-SIRE) and a genotypic assay (GenoType MTBDRplus). PCR in the clinical specimens showed excellent specificity (97.4%) and accuracy (96.8%), good sensitivity (70.4%), but low positive predictive value (40.3%). MGIT-SIRE performed to M. tuberculosis did not confer a reliable result in 16 isolates. Of the remaining 69 isolates, 15 were resistant to streptomycin, seven to isoniazid, seven to ethambutol and five to rifampicin. GenoType MTBDRplus correctly detected isoniazid (seven) and rifampicin-resistant M. tuberculosis strains (five), showing an excellent performance overall (100%). Susceptibility results by the molecular assay applied directly to clinical specimens were identical to those obtained from recovered isolates of the corresponding patients. Combining molecular and conventional methods greatly contribute to early diagnosis and accurate susceptibility testing of tuberculosis.

Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

Evaluation of crystal violet decolorization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.

Dormancy models for Mycobacterium tuberculosis: A minireview

Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs.

Whole-body magnetic resonance imaging in children: state of the art / Ressonância magnética de corpo inteiro em pediatria: estado da arte

Whole-body imaging in children was classically performed with radiography, positron-emission tomography, either combined or not with computed tomography, the latter with the disadvantage of exposure to ionizing radiation. Whole-body magnetic resonance imaging (MRI), in association with the recently developed metabolic and functional techniques such as diffusion-weighted imaging, has brought the advantage of a comprehensive evaluation of pediatric patients without the risks inherent to ionizing radiation usually present in other conventional imaging methods. It is a rapid and sensitive method, particularly in pediatrics, for detecting and monitoring multifocal lesions in the body as a whole. In pediatrics, it is utilized for both oncologic and non-oncologic indications such as screening and diagnosis of tumors in patients with genetic syndromes, evaluation of disease extent and staging, evaluation of therapeutic response and post-therapy follow-up, evaluation of non neoplastic diseases such as multifocal osteomyelitis, vascular malformations and syndromes affecting multiple regions of the body. The present review was aimed at describing the major indications of whole-body MRI in pediatrics added of technical considerations.

A avaliação de corpo inteiro em crianças era classicamente realizada com radiografias simples, cintilografia e tomografia por emissão de pósitrons combinada ou não à tomografia computadorizada, estes com a desvantagem de exposição à radiação ionizante. A ressonância magnética de corpo inteiro (RMCI), associada ao desenvolvimento de técnicas metabólicas e funcionais como difusão, trouxe a vantagem de uma avaliação global do paciente pediátrico sem os riscos da radiação ionizante habitualmente presente nos métodos radiológicos convencionais. A RMCI é um método rápido e sensível, com aplicação especial na área de pediatria na detecção e no monitoramento de lesões multifocais no corpo como um todo. Em pediatria, esta técnica é utilizada tanto em oncologia - no diagnóstico e rastreamento de tumores em pacientes portadores de síndromes genéticas, na avaliação da extensão de doenças e estadiamento oncológico, na avaliação da resposta terapêutica e no seguimento pós-terapêutico - como em lesões não neoplásicas - osteomielite multifocal, malformações vasculares e síndromes que comprometam múltiplas regiões do corpo. Esta revisão tem como objetivo mostrar as principais indicações do exame na população pediátrica e técnica de realização.

Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area

The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.

Assessment of phagosomes infected with Mycobacterium tuberculosis as a vaccine candidate against tuberculosis.

The present study describes a novel and simple vaccination strategy that involve culturing of M. tuberculosis in the macrophage cells. Isolation of phagosome from macrophage (cell line J774) infected with M. tuberculosis (H37) and M. bovis (BCG) at early and late phase of infection was done ensuing the identification and characterization of these phagosome. In vitro study of apoptosis induced by phagosome infected with (H37) and (BCG) was performed. The vaccine candidate with H37 MOI- 1:10 at 3 h, MOI- 1:20 at 1, 1.5, 2.5 and 3 h and BCG MOI- 1:20 at 3.5 h showed percentage apoptosis as 38.64, 39.93, 34.66, 22.56,34.59 and 37.81% respectively. The results designates that macrophages provide cellular niche during infection and illustrate considerable immunogenic property. Novel antigens expressed or secreted by H37 in infected macrophages can provide evidence to be a successful vaccine candidate as it endures enhanced immune response than BCG.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Performance comparison between the mycobacteria growth indicator tube system and Löwenstein-Jensen medium in the routine detection of Mycobacterium tuberculosis at public health care facilities in Rio de Janeiro, Brazil: preliminary results of a pragmatic clinical trial / Comparação do desempenho do sistema mycobacteria growth indicator tube e meio Löwenstein-Jensen na detecção de rotina de Mycobacterium tuberculosis em unidades do sistema único de saúde no Rio de Janeiro: resultados preliminares de um ensaio clínico pragmático

In view of the fact that the World Health Organization has recommended the use of the mycobacteria growth indicator tube (MGIT) 960 system for the diagnosis of tuberculosis and that there is as yet no evidence regarding the clinical impact of its use in health care systems, we conducted a pragmatic clinical trial to evaluate the clinical performance and cost-effectiveness of the use of MGIT 960 at two health care facilities in the city of Rio de Janeiro, Brazil, where the incidence of tuberculosis is high. Here, we summarize the methodology and preliminary results of the trial. (ISRCTN.org Identifier: ISRCTN79888843 [http://isrctn.org/]).

Em razão da recomendação da Organização Mundial da Saúde sobre o uso do sistema mycobacteria growth indicator tube (MGIT) 960 para o diagnóstico de tuberculose e da falta de evidências sobre o impacto clínico de sua incorporação em sistemas de saúde, um ensaio clínico pragmático está sendo conduzido para avaliar o desempenho clínico e a relação custo-efetividade do MGIT 960 em duas unidades do Sistema Único de Saúde na cidade do Rio de Janeiro, que tem uma elevada incidência de tuberculose. Apresentamos aqui, de forma sintética, o método e resultados preliminares do ensaio. (ISRCTN.org Identifier: ISRCTN79888843 [http://isrctn.org/]).

Diagnostic role of MGIT culture of BAL samples in sputum smearnegative pulmonary tuberculosis.

Background: In view of the diagnostic difficulties associated with sputum- negative pulmonary TB (PTB), we aimed at exploring if bronchoalveolar lavage (BAL) samples can be subjected to smear- microscopy and rapid mycobacterial culture (by Mycobacterial Growth Indicator Tube (MGIT) method) to achieve improved diagnosis of this condition. Methods: Patients presenting with clinico-radiological features suggestive of pulmonary tuberculosis and whose sputum smears were negative for acid- fast bacilli (AFB) or who could not expectorate sputum were prospectively enrolled in this study. BAL samples collected from them were subjected to smear- microscopy for AFB and micro-MGIT culture. BAL samples were also inoculated on Lowenstein- Jensen (LJ) slants. Results: A total of 105 patients (74 males) were recruited in the study, with a mean (±SD) age of 51 (± 15) years. The diagnosis of PTB was made in 52 patients on the basis of clinico- radiological presentation, with or without microbiological confirmation. Thirty- four patients (65.4 %) had microbiologically confirmed PTB. Of them, AFB were detected in 12 BAL samples, while culture- positivity was noted in 24 and 27 patients by the LJ and MGIT methods respectively. Intertest agreement between the LJ and MGIT methods was found to be significant (ê= 0.655; p= <0.001). However, the mean time to positivity was significantly lower for the MGIT method than for the LJ method (p= <0.001). Conclusion: Examination of BAL samples by smear- microscopy and micro-MGIT culture can, therefore, provide a rapid and definitive diagnosis of PTB in sputum- negative patients.

Rapid culture diagnosis of tuberculous lymphadenitis from a tertiary care centre in an endemic nation: Potential and pitfalls.

In spite of low sensitivity and specificity, standard diagnostic algorithm recommends fine needle aspiration cytology (FNAC) and direct microscopic screening for acid-fast bacilli (AFB) for the routine diagnosis of tuberculous lymphadenopathy (LNTB). In this study, the diagnostic utility of liquid broth based automated culture (BacT/ALERT 3D) technique was assessed in comparison with conventional techniques in 89 clinically suspected tubercular lymphadenitis patients. 60% (n = 53) were positive by FNAC and 38.4% (n = 34) demonstrated AFB in smear examination. BacT/ALERT yielded isolation in 43.1% (n = 38) aspirates, confirming tubercular aetiology. We also found six paediatric culture-positive cases which showed negative outcome by both FNAC and smear. Thus, we conclude that culture by BacT/ALERT, may be used for faster yield of Mycobacteria in LNTB, especially in children. Additionally, this could also be used as a platform for further differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria (NTM) infection and for testing of anti-tubercular chemotherapeutic agents whenever drug resistance is suspected.

Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of extrapulmonary tuberculosis.

Purpose: Extrapulmonary tuberculosis (EPTB) is emerging problem in developing and developed countries. The diagnosis of EPTB in its different clinical presentations remains a true challenge. IS6110-based polymerase chain reaction (PCR) is used for rapid identification and positivity rate of the Mycobacterium tuberculosis complex in clinical isolates of different sites of EPTB. The present study was carried out to study the prevalence of M. tuberculosis complex in clinical isolates of EPTB at tertiary care centres in Lucknow. Materials and Methods: Seven hundred fifty-six specimens were collected from the suspected cases of EPTB which were processed for Mycobacteria by Ziehl Neelson (ZN) staining and BACTEC culture. All the specimens were also processed for IS6110-based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of the M. tuberculosis complex. Results: Of these 756 specimens, 71(9.3%) were positive for acid fast bacilli (AFB) by ZN staining, 227(30.1%) were positive for mycobacteria by BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 165 (20.7%) isolates. We found a significant difference in sensitivities of different tests (P<0.05). Conclusions: This study reveals the positivity of M. tuberculosis complex in clinical isolates of EPTB case in tertiary care hospitals in Northern India. 72.7% of M. tuberculosis complex was confirmed by IS6110-PCR in culture isolates from different sites of EPTB. The high prevalence of the M. tuberculosis complex was seen in lymph node aspirate and synovial fluid. However, utility of PCR may play a potentially significant role in strengthening the diagnosis of EPTB especially targeting IS6110.

performance of tofu-whey as a liquid medium in the propagation of Mycobacterium tuberculosis strain H37Rv

To investigate the performance of "tofu-whey liquid medium" for the propagation of Mycobacterium tuberculosis [MTB] strain H37Rv. Two hundred micro liters [200 micro l] of 1 McFarland standard [1 mg/ml-bacillary suspension] were inoculated into different batches of tofu-whey liquid medium. Each series contained three trials of test [tofu-whey liquid medium] and control media [Middlebrook 7H9 medium]. Turbidity was measured within three weeks of inoculation using a nephelometer. The combinations of various tofu-whey liquid culture media were as follows; T1 [tofu-whey + ADC + glycerol + Potassium sulfate + Magnesium citrate + Sodium glutamate]; T2 [tofu-whey + ADC + glycerol + Potassium sulfate + Magnesium citrate]; T3 [tofu-whey + ADC + glycerol + Potassium sulfate]; T4 [tofu-whey + ADC + glycerol]; T5 [tofu-whey + ADC]; and T6 [tofu-whey only]. In all test and control liquid culture media, the multiplication of M. tuberculosis was documented under light and fluorescence microscopy. Of various tofu-whey medium used, T1 demonstrated the most potential for MTB propagation. The increased turbidity reading represented by the value in "unit drop of% transmittance" was higher [25 scores] in the T1 tofu-whey medium, compared with the T6 tofu-whey medium [8 scores]. The overall growth was significantly better in Middlebrook 7H9 culture media, although by the third day of incubation, the bacillary growth was superior in the T1 tofu-whey culture media. Sub-cultures in Lowenstein-Jensen [L-J] medium yielded between 87% [47 of 54] and 89% [48 of 54] recovery rate with between 7% and 13% contamination rate with coagulase-negative staphylococci. Tofu-whey media can be used as an economical alternative to Middlebrook 7H9 in resource-limited settings

Diagnóstico bacteriológico de tuberculosis renal: experiencia del Laboratorio Regional de Tuberculosis de la provincia de Córdoba

Dada la considerable incidencia de tuberculosis renal entre enfermos con tuberculosis pulmonar, nos propusimos estudiar la frecuencia de esta asociación en pacientes atendidos en centros de salud públicos y privados de Córdoba a lo largo del período 1997-2009. Se tomó en consideración la incidencia según el sexo y las especies del complejo Mycobacterium tuberculosis identificadas. El análisis de 948 muestras de orina de 383 pacientes indicó tuberculosis renal en 24 casos (6,3 %), con presencia mayoritaria de Mycobacterium tuberculosis (95,8 %) y presencia de Mycobacterium bovis en 4,2 % de los casos. La asociación tuberculosis renal-tuberculosis pulmonar activa se encontró en 6 casos. En esta investigación quedó demostrada la importancia del cultivo seriado de muestras de orina y la conveniencia de cultivar en medios sólidos y líquidos. Asimismo, el aislamiento de Mycobacterium bovis pone de relieve la importancia de usar el medio Stonebrink junto con el medio de Lowenstein-Jensen. El medio líquido no tuvo un aporte significativo al diagnóstico de tuberculosis renal; sin embargo, el cultivo de muestras seriadas aumentó la sensibilidad de la detección.

Bacteriological diagnosis of renal tuberculosis: an experience at the Regional Tuberculosis Laboratory in Córdoba province, Argentina. Given the incidence of renal tuberculosis in patients suffering of pulmonary tuberculosis, we seek to study both the frequency of this association in diagnosed cases of renal tuberculosis and the Mycobacterium tuberculosis complex species that were identified (period 1997-2009), observing its incidence by sex, demonstrating the importance of serial culture of urine samples and evaluating the convenience of using solid and liquid media. The analysis of urine samples from 383 patients indicated renal tuberculosis in 24 cases; in most cases, (95.8 %) Mycobacterium tuberculosis complex species prevailed, whereas the presence of Mycobacterium bovis accounted for 4.2 % of the cases. The association of pulmonary and renal tuberculosis was found in 6 cases. The isolation of Mycobacterium bovis indicates the importance of including Stonebrink medium along with Lowenstein- Jensen medium. The liquid medium made no significant contribution to the diagnosis of renal tuberculosis, but indeed, cultivating serial samples increases sensitivity.

Effects of culture filtrates of endophytic fungi obtained from Piper aduncum L. on the growth of mycobacterium tuberculosis

Substances that inhibit the growth of Mycobacterium tuberculosis could potentially be used as antibiotics. These substances could also be added to test culture media to improve the speed of tuberculosis diagnosis. The aim of this work was to investigate the influence of culture filtrates of endophytic fungi isolated from P. aduncum L. on the growth of M. tuberculosis. To achieve this objective, the following methodology was used: a) endophytic fungi were isolated from the leaves and stems of P. aduncum L.; b) the isolated fungi were submitted to submerged bioprocessing; c) culture filtrates from the bioprocess were assayed to evaluate their effect on the growth of M. tuberculosis. We isolated 315 fungal types, which represented 85 morphologies, from different parts of P. aduncum L. The bioassays were performed on 82 culture filtrates and 6 plant extracts and resulted in the detection of 1 culture filtrate that stimulated the growth of M. tuberculosis and 15 that inhibited microbial growth. None of the phytochemical extracts had an effect on the growth of M. tuberculosis. In conclusion, we observed that the endophytic fungi isolated from P. aduncum L. (Piperaceae) produced extracellular metabolites (present in the culture filtrate) that affect the growth of M. tuberculosis. These compounds have the potential to be used as antimicrobials or in the diagnosis of tuberculosis.

Growth kinetics of mycobacterium tuberculosis measured by quantitative resazurin reduction assay: a tool for fitness studies

We standardized a method to evaluate the growth kinetics of Mycobacterium tuberculosis by measuring quantitatively the reduction of resazurin by spectrophotometry. Growth curves and the rate of growth of twenty-one M. tuberculosis clinical isolates were determined. The method showed technical simplicity and is inexpensive to assess the fitness of each isolate.

Comparison of a novel bilayered medium with the conventional media for cultivation of Mycobacterium tuberculosis.

Background & objectives: There is resurgence of tuberculosis in recent years in spite of availability of comprehensive multidrug therapy. Conventional culture media require a long time for the appearance of growth of Mycobacterium tuberculosis, while the other methods are expensive. Hence, a rapid low cost and safe bilayered medium was developed for early growth and sensitivity testing of M. tuberculosis and the results were compared with those on Lowenstein Jensen medium, Middlebrook 7H10 and Kirchner’s liquid media. Methods: A specially designed bilayered medium, consisting of a lower layer of Lowenstein Jensen medium without malachite green and a top layer of Middlebrook 7H 10 medium with added antibiotics and antifungal agents was prepared. Sputum from clinically suspected cases of tuberculosis, pleural fluid and pus samples were inoculated on the bilayered medium along with the inoculation on other conventional media after proper decontamination and concentration of the samples. Antibiotic sensitivity pattern was determined against a few rapidly growing control and test strains by disc diffusion technique and the results could be recorded by 3 to 7 days. Results: Statistically significant (P< 0.001) isolation rate was obtained on this bilayered medium when compared with the other three media, being 81.7 per cent growth by 7 days. Antibiotic sensitivity test could be recorded by 3 days in case of the rapidly growing strains on this medium, and by 7 days in case of M. tuberculosis strains. Interpretation & conclusions: Bilayered medium produced rapid growth earliest by 48 h, higher isolation rates were achieved as compared to the other conventional media and drug sensitivity testing could also be carried out successfully. Thus, the bilayered medium can be used for obtaining early culture report.