(241f) Rapid Culture-Based Detection of Mycobacteria Using Electrical Impedance Spectroscopy (EIS) Measurements

Tuberculosis (TB) is a bacterial infection which commonly affects the pulmonary system. According to a WHO’s Global TB report 2014, in 2013, about 9 million people were detected with TB and about 1.5 million people died. However, if detected early, TB is treatable. Treating TB is possible if the TB mycobacterium in the clinical samples can be detected rapidly.

Till date, culture based detection of TB mycobacteria is the most common mode of detection and is the current gold standard. This method is cheap and reliable. However, it is a time consuming procedure and may take anywhere from 7-56 days as pathogens like Mycobacterium species are slow growing organisms (having a doubling time of ~24 hours vs ~20mins for Escherichia coli). Here we show the feasibility of using multi-frequency EIS based to detect TB mycobacterium in a significantly shorter amount of time.

To conduct our in vitro time-to-detection (TTD) study, Mycobacterium bovis (an analog) was used instead of Mycobacterium tuberculosis. M. bovis was cultured in modified Middlebrook 7H9 mycobacteria media at 37C. It was also grown in Lowenstein-Jensen plates. For our study, three different concentrations of the cultured M. bovis were seeded in the growth media: low (having 2-5 CFU/ml), medium (having 100-200 CFU/ml and high (having ~3000 CFU/ml). In addition, a control sample (with no bacteria) was also studied. One set of samples (control, low, medium and high concentrations) were also sent to the University of Missouri Hospital’s microbiology laboratory for detection using the current state of the art automated culture-based system; viz. the BACTEC MGIT^TM 960 from Becton Dickinson. The various concentration samples and control samples were all incubated under similar conditions of 37C and periodically small amounts of aliquots were withdrawn from it and put on a 3D printed microfluidic channel to analyze the bulk capacitance of the suspension using multi-frequency EIS measurements. The microfluidic channel is designed to have a thin capillary channel with two gold electrodes inserted in it such that the distance between the electrodes is 1 cm. During EIS measurement, AC voltage is applied to the electrodes and the bulk capacitance of the suspension resulting from the charges that accumulated in the bacterial cells is recorded at multiple frequencies ranging from 1 KHz to 100MHz.

For the control samples, it is observed that subsequent bulk capacitance readings remain statistically unchanged from the baseline reading. On the other hand, bulk capacitance recorded for samples containing Mycobacteria, show gradual increase in the value, and become statistically significantly different from the baseline reading when the concentration of the suspension reaches 10³-10⁴ CFU/ml. Plate counts of the aliquots withdrawn are done to get the concentration values. Using our method, the TTD for high concentration samples were about 60-70 hours (< 3 days), medium concentration samples were about 84-108 hours (< 5 days) and for low concentration samples were about 132-156 hours (< 7 days). In contrast, the TTD obtained for the same samples from the hospitals were about 5-6 days for high concentrations. TTD for the samples with medium and low concentrations is greater than 40 days (we are still awaiting results).

Hence we have demonstrated a potential technique which has much lower TTD than the current automated systems. Less TTD implies faster clinical response resulting in improved patient response.