Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS)

Abstract

BackgroundMultiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long “time-to-detection” (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable.MethodsLiving bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical “scans” taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension’s baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria.ResultsOur TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively.ConclusionOur culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.

Direct-from-sputum rapid phenotypic drug susceptibility test for mycobacteria

“…We have previously demonstrated the use of m-EIS to detect mycobacteria in decontaminated sputum samples: both on the basis of detecting their proliferation (a process taking ~36 hours for M . bovis BCG ) [ 49 ], and a novel “detection by death” approach [ 52 ]. Micro-wells dedicated to implementing these approaches may be incorporated into the same microfluidic cassette to bring together a portable, sensitive, and affordable device to detect M .…”

“…The data collected by the impedance analyzer is in the form of resistance (R) and reactance (X) values of the bacterial suspension at 128 logarithmically equi-spaced frequencies (w) between 1 KHz and 100 MHz. As described by us previously [49] the R and X vs. ω data is fitted to the equivalent electrical circuit shown in Fig 5 using a commercially available software package (ZView™). The software accepts as an input the Z vs. ω data and provides an estimate for all the circuit parameters, including the "bulk capacitance" (our parameter of interest that provides a measure of charges stored in the interior of the suspension (away from the electrodes)).…”

“…Future work will incorporate (a) Real-world sample (human sputum with M. tuberculosis cells), (b) self-contained and sealable microfluidic cassettes with multiple wells, each containing chosen amounts of candidate drugs pre-loaded into 10μL "wells", into which suspensions containing growth media, mycobacteria and MNPs can be loaded, and (c) integrated electronics to keep track of multiple wells in parallel. We have previously demonstrated the use of m-EIS to detect mycobacteria in decontaminated sputum samples: both on the basis of detecting their proliferation (a process taking~36 hours for M. bovis BCG) [49], and a novel "detection by death" approach [52]. Micro-wells dedicated to implementing these approaches may be incorporated into the same microfluidic cassette to bring together a portable, sensitive, and affordable device to detect M. tuberculosis in sputum samples, and simultaneously determine the multidrug resistance profile of the pathogen, all within 3 days of sputum collection.…”

Reactivation of latent tuberculosis through modulation of resuscitation promoting factors by diabetes

“…Latent tuberculosis mouse model was developed according to the method of Zhang et al Briefly, BALB/c mice (5–6 weeks, either sex) were infected with BCG through aerosolization (O.D. of 0.5 at 600 nm ~ 5 × 10 6 CFU/ml) 42 . After 6 weeks of immunization with BCG, animals were infected with M. tuberculosis H37Rv (O.D.…”

Overview of Non Tuberculosis Mycobacterial Lung Diseases

“…BACTEC MGIT 960 system is a fully automated, nonradiometric system that is suitable for the detection of growth of TB and other mycobacteria with the shorter detection time ~2 weeks [108]. The recently introduced, microchannel electrical impedance spectroscopy (m-EIS) has ability to detect M. smegmatis with initial loads of 1000 CFU/ml within 20 h, while commercial BACTEC MGIT 960 system need 41.7 h for the same [109].…”