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

Abstract

Background: The spread of multi-drug resistant tuberculosis (MDR-TB) is a leading global public-health challenge. Because not all biological mechanisms of resistance are known, culture-based (phenotypic) drug-susceptibility testing (DST) provides important information that influences clinical decision-making. Current phenotypic tests typically require pre-culture to ensure bacterial loads are at a testable level (taking 2-4 weeks) followed by 10-14 days to confirm growth or lack thereof.

Methods and findings: We present a 2-step method to obtain DST results within 3 days of sample collection. The first involves selectively concentrating live mycobacterial cells present in relatively large volumes of sputum (~2-10mL) using commercially available magnetic-nanoparticles (MNPs) into smaller volumes, thereby bypassing the need for pre-culture. The second involves using microchannel Electrical Impedance Spectroscopy (m-EIS) to monitor multiple aliquots of small volumes (~10μL) of suspension containing mycobacterial cells, MNPs, and candidate-drugs to determine whether cells grow, die, or remain static under the conditions tested. m-EIS yields an estimate for the solution "bulk capacitance" (Cb), a parameter that is proportional to the number of live bacteria in suspension. We are thus able to detect cell death (bactericidal action of the drug) in addition to cell-growth. We demonstrate proof-of-principle using M. bovis BCG and M. smegmatis suspended in artificial sputum. Loads of ~ 2000-10,000 CFU of mycobacteria were extracted from ~5mL of artificial sputum during the decontamination process with efficiencies of 84% -100%. Subsequently, suspensions containing ~105 CFU/mL of mycobacteria with 10 mg/mL of MNPs were monitored in the presence of bacteriostatic and bactericidal drugs at concentrations below, at, and above known MIC (Minimum Inhibitory Concentration) values. m-EIS data (ΔCb) showed data consistent with growth, death or stasis as expected and/or recorded using plate counts. Electrical signals of death were visible as early as 3 hours, and growth was seen in < 3 days for all samples, allowing us to perform DST in < 3 days.

Conclusion: We demonstrated "proof of principle" that (a) live mycobacteria can be isolated from sputum using MNPs with high efficiency (almost all the bacteria that survive decontamination) and (b) that the efficacy of candidate drugs on the mycobacteria thus isolated (in suspensions containing MNPs) could be tested in real-time using m-EIS.

Fig 1. Timeline of available approaches to obtaining drug susceptibility information for TB.

(A) GeneXpert genotypic approach that only identifies Rifampicin susceptible pathogens. If RIF-resistant, the sample is channeled to path B. (B) Traditional culture based phenotypic approach (MGIT etc.) (C) Microscopic Observation of Drug Susceptibility (MODS). (D) Proposed approach (isolation using MNPs + growth/death/ stasis assay using EIS).

Fig 2. Experimental protocol.

(A) the preparation of artificial sputum with mycobacteria + Gram-positive and Gram-negative bacteria (B) the digestion/decontamination (C) Addition of MNP and allowing them to bind to the mycobacteria (D) Isolation of the mycobacterium into a pellet and its resuspension (E) Periodic electrical assay using m-EIS.

Fig 3. Real-world clinical patient sputum sample processing protocol.

Schematic of proposed approach involving isolation of mycobacteria using MNPs to bypass the preculture, allowing the assay of growth/death using m-EIS measurements to be completed quickly.

Fig 4. Schematic and electrical circuit model representation of microchannel.

(a) Electrical Model of an aqueous suspension in contact with metal electrodes. The equation relates the real (in-phase) and imaginary (out-of-phase) components of the measured impedance (Z) and how they vary as a function of frequency and model parameters (Re, Ce, Rb and Cb) (b) schematic, and (c) picture of microfluidic cassette.

Fig 5. Data analysis using the program ZView®.

The left side shows the graphical fit-line to the Resistance (Z’) and Reactance (Z”) which is fit to the circuit of choice (top right). The fit results in an estimate of the bulk capacitance (circled, bottom right).

Fig 6. Decontamination and extraction results.

Data showing decontamination and extraction of viable mycobacteria from synthetic sputum using RapiPREP-TB TM beads for (A) M. smegmatis (n = 3) and (B) M. bovis BCG (n = 2) (>95% of cells viable after de-contamination is collected to the pellet).

Fig 7. Bulk capacitance changes over time for M. smegmatis.

Change in the value of the measured bulk capacitance (Cb) as a function of time for M. smegmatis cultures with and without Magnetic Nano-particles (MNPs) when exposed to cidal, static, and ineffective antibiotics. All changes are normalized to the baseline (time t = 0) value. (n = 5 at each data point).

Fig 8. Bulk capacitance changes over time for M. bovis BCG.

Change in the value of the measured bulk capacitance (Cb) as a function of time for M. bovis BCG cultures with and without Magnetic Nano-particles (MNPs) when exposed to cidal, static, and ineffective antibiotics. All changes are normalized to the baseline (time t = 0) value. (n = 5 at each data point).