Search

Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guerin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ m) and the frequency (f) for a crossover frequency (f xo1) test are decided to detect the change of σ m-f xo1 in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f xo1. Finally, the parameters of the electrophysiology of BCG cells, C envelope and σ cyto, are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells.

An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6)-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.

A rapid, accurate tuberculosis diagnostic tool that is compatible with the needs of tuberculosis-endemic settings is a long-sought goal. An immunofluorescence microtip sensor is described that detects Mycobacterium tuberculosis complex cells in sputum in 25 minutes. Concentration mechanisms based on flow circulation and electric field are combined at different scales to concentrate target bacteria in 1 mL samples onto the surfaces of microscale tips. Specificity is conferred by genus-specific antibodies on the microtip surface. Immunofluorescence is then used to detect the captured cells on the microtip. The detection limit in sputum is 200 CFU mL(-1) with a success rate of 96%, which is comparable to PCR.

Rapid detection of tuberculosis (TB) has been critically demanded over the last century. To detect TB, numerous methods screening Mycobacterium tuberculosis (MTB) have been developed. However, the methods still have challenges of rapid and specific enrichment of MTB. In this study, we present a novel specific enrichment method of MTB using a microfabricated tip. Through our simulation study, a wavy-shaped microtip is designed to enhance capturing efficiency of bacteria. Using an optimized tip, bacteria are attracted by dielectrophoresis and captured by affinity binding and capillary action. In experiment, a surrogate marker of MTB, Bacilli Calmette-Guerin (BCG), is enriched with the micromachined tip. When a microtip decorated with capturing antibodies is used, BCG cells spiked in saliva are detected at the concentration of 5×105 CFU/mL within 20 minutes. The tip enrichment system demonstrates the potential for rapid, culture-free detection of MTB in a raw sample.

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low cost detection of Mycobacterium Tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing- and rinsing steps are presented with use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU/mL, comparable to a more labor-intensive fluorescence detection method reported previously.

Successful long-term preservation of Mycobacterium tuberculosis (MTB) cells or sputum specimens is very important for sample transport, biobanking, research and the development of new drugs, vaccines, biomarkers, and diagnostics. In this work, M . bovis Bacillus Calmette–Guerin (BCG) and M. tuberculosis H37Ra were used as models of MTB complex strains to study cryopreservation of MTB complex cells in diverse sample matrices (phosphate buffer saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum). Efficacy of cryopreservation was quantified by microbiological culture. Results showed that among the variables tested, slow cooling rate was the most critical factor for the successful cryopreservation of Mycobacterium tuberculosis complex cells. Preincubation of frozen/thawed MTB cells in 7H9 broth before culturing did not help the cells repair cryoinjury. Cell inactivation by fast cooling was not associated with a compromised cell envelope, as indicated by the comparison results of microbiological culture and the BacLight live/dead staining. After cryopreservation, the MTB cell surface antigen can still function well for the MTB diagnosis as biomarker. For the Point-of-Care (POC) diagnosis of MTB, an occupationally safe (biosafe) sputum liquefaction protocol and a semi-automated antibody-based microtip immunofluorescence sensor were developed. Sputum was treated with a synergistic chemical–thermal protocol that included moderate concentrations of NaOH and detergent at 60 °C for 5–10 min. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. The biosafe sputum liquefaction method, cryopreservation protocol, and the microtip device make it possible to diagnose the tuberculosis with high feasibility, sensitivity and accuracy.