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-ofprinciple using M. bovis BCG and M. smegmatis suspended in artificial sputum. Loads of2 000-10,000 CFU of mycobacteria were extracted from~5mL of artificial sputum during the decontamination process with efficiencies of 84%-100%. Subsequently, suspensions con-taining~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.

QMAC-DST for Rapid Detection of Drug Resistance in Pulmonary Tuberculosis Patients: A Multicenter Pre–Post Comparative Study

“…These tests also facilitate the rapid detection of drug resistance but are typically limited to first-line anti-TB drugs, are labor-intensive, and may pose safety concerns. They also depend on the skill of laboratory personnel for result interpretation and are not efficient for processing large sample volumes [22][23][24][25]. QMAC-DST addresses several limitations of traditional pDST by offering rapid TAT, the ability to test various first-and second-line drugs, high throughput, reduced labor intensity, effectiveness regardless of inoculum size, enhanced safety with sealing film and a locking lid, along with an agarose matrix embedding the MTB and elimination of errors due to the MTB tracking failure [12][13][14][15].…”

Evolution of tuberculosis diagnostics: From molecular strategies to nanodiagnostics

“…It can be done by observing the viability or metabolic inhibition in a medium containing anti-tuberculosis drugs [ 75 ]. The susceptibility can be determined from various technical standpoints such as macroscopic observation in drug-free or in the media-containing drug [ 76 ]. The metabolic activity can be another parameter to test their susceptibility.…”