Use of a phage-based assay for phenotypic detection of mycobacteria directly from sputum

Abstract

The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of Mycobacterium tuberculosis isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplification of phage occurs. The aims of the present study were to optimize the speed and sensitivity of the assay and reduce its cost for developing countries by using an M. tuberculosis-spiked sputum model with (i). identification of inhibitory components of sputum and optimization of decontamination methods; (ii). simplification of the washing and development steps; (iii). reduction of the use of high-cost components, e.g., oleate-albumin-dextrose-catalase (OADC) supplement; and (iv). optimization of virucide treatment. The following results were obtained. (i). An inhibitory factor in sputum which could be removed by treatment of the sample with sodium dodecyl sulfate or NaOH decontamination was identified. (ii). A microcentrifuge-based approach with thixotropic silica as a bedding and resuspension agent was developed as an alternative to conventional centrifugation medium exchange. The yield was increased 228-fold, with increased speed and reduced cost. (iii). At present, after extracellular inactivation of phage, the ferrous ammonium sulfate (FAS) virucide is sequestered by dilution with an expensive supplement, OADC. Sodium citrate with calcium chloride was found to be a cost-effective after treatment with the FAS protectant and offered greater protection than OADC. Kinetic-lysis experiments indicated that an infection time of 1 to 3 h prior to FAS addition was optimal. (iv). Amplification of the signal (which corresponded to the burst size) was shown by allowing lysis prior to plating in a spiked medium model (up to 20-fold) and a spiked sputum model (up to 10-fold). A liquid culture detection method capable of detecting approximately 60 viable M. tuberculosis organisms in 1 ml of sputum was developed. Taken together, these improvements support the routine application of the assay to sputum specimens.

FIG. 1.

Schematic flow representation of sputum processing and PhaB assay execution by the methods published by McNerney et al. (12) (a) and suggested on the basis of the findings of this study (b). D29, infection of mycobacteria with mycobacteriophage D29; FAS, inactivation of extracellular D29 with the virucide FAS following infection of mycobacteria; OCG, OADC, calcium chloride, glycerol 7H9 medium supplement; CC, 7H9 medium supplemented with sodium citrate and calcium chloride. Steps 1 to 5 (a) and steps 1 to 4 (b) relate to washes after sputum decontamination and neutralization with medium involving a number of centrifugation steps, which result in the resuspension of mycobacteria in 7H9-OCG prior to overnight incubation at 37°C (see Materials and Methods). Steps 6 to 10 in the scheme in panel a refer to the collection of a 0.2-ml aliquot for D29 infection (step 6), 3.5 h of incubation at 37°C prior to addition of FAS (step 7), 10 min of incubation prior to FAS inactivation by the addition of 1 ml of 7H9-OGC (step 8), 4 h of incubation at 37°C to enable lytic burst (step 9), and plating of a 0.1-ml aliquot with 7H9-OCG-agar and M. smegmatis sensor cells (step 10). Steps 5 to 7 in the scheme in panel b refer to D29 infection (step 5), 2 h of incubation at 37°C prior to addition of FAS (step 6), and 10 min of incubation prior to FAS inactivation by plating with 7H9 medium supplemented with sodium citrate, calcium chloride, agar, and M. smegmatis sensor cells (step 7). Plaques result on the sensor-cell lawn when mycobacteria harboring D29 lyse following plating.

FIG. 2.

(a) Ability of heat treatment to remove activity inherent in sputum inhibitory to the PhaB assay and M. tuberculosis survival after heat treatment. One milliliter of mixed, smear-negative sputum or 7H9-10% (vol/vol) OCG was spiked with M. tuberculosis either before (open bars) or after (solid bars) heat treatment (slashed bars, medium spiked with M. tuberculosis before heat treatment). After immersion in a water bath at different temperatures for 10 min and cooling to room temperature, an equal volume of 0.25% (vol/vol) NaOH-0.5% (vol/vol) N-acetyl cysteine was added to the samples. These were incubated at room temperature for 20 min, prior to centrifugation at 3,000 × g for 20 min. The supernatants were discarded, and after washing with 10 ml of 7H9 and a centrifugation step, the samples were resuspended in 7H9-10% (vol/vol) OCG for overnight recovery, prior to application of the PhaB assay (see Materials and Methods). (b) A plate after incubation of a mixture of M. smegmatis and phage D29. The plate was overlaid with filter paper squares bearing sputum (filter paper a), a sputum fraction washed with dH₂O (filter paper b), a sputum fraction washed with 0.05% (wt/vol) SDS-1% (wt/vol) NaCl (filter paper c), dH₂O (filter paper d), or 0.05% (wt/vol) SDS-1% (wt/vol) NaCl (filter paper e). This experiment was designed to demonstrate the ability to fractionate the activity of sputum inhibitory to the assay upon washing with SDS solution. The soaked filter paper squares were overlaid on a plate with a mixture of M. smegmatis and D29 containing the MBBacT antibiotic cocktail. M. smegmatis growth zones (translucent zone, e.g., within filter paper c) against a background of confluent lysis correspond to inhibition of D29 infection (see Materials and Methods).

FIG. 3.

(a) Effect of TS addition to 2-ml microcentrifuge tubes during processing of 0.5 ml of M. tuberculosis (TB)-spiked sputum prior to application of the PhaB assay. An equal volume of 2% (wt/vol) NaOH-1.45% (wt/vol) trisodium citrate · 2H₂O was used for processing, followed by application of the microcentrifugation method with TS. The lines above the bars are standard errors. (b) Bar chart comparing the yields from the PhaB assay after processing of 0.5 ml of sputum specimens spiked with M. tuberculosis by the universal method with 30 ml of supernatant (slashed bars) compared with the yields after processing by the method with a 2-ml microcentrifuge tube with TS (solid bars) prior to application of the PhaB assay (open bars, M. tuberculosis spiked with 7H9-10% [vol/vol] OCG). Specimens were processed with an equal volume of 2% (wt/vol) NaOH-1.45% (wt/vol) trisodium citrate · 2H₂O, as outlined in Materials and Methods. +, positive control (7H9-10% [vol/vol] OCG spiked with M. tuberculosis with no prior processing); −, negative control (unspiked mixed sputum).

FIG. 4.

(a) Effects of various OCG contents in the 7H9 used for overnight recovery following mock NaOH treatment (treatment with an equal volume of 2% [wt/vol] NaOH-1.45% [wt/vol] trisodium citrate · 2H₂O) of 0.5 ml of unspiked (−) or M. tuberculosis-spiked (+) 7H9 on the PhaB assay. The samples were then processed by the microcentrifugation protocol with TS prior to overnight incubation at 37°C in 7H9 supplemented with different OCG concentrations, as indicated. The asterisk indicates the control that received no NaOH treatment. (b) Effect of TS on performance of the PhaB assay with old cultures. A 6-month-old suspension from an LJ medium slope (in 7H9-10% [vol/vol] OCG) was applied directly to the assay or was applied following overnight recovery (o/n rec.) incubation at 37°C in the presence or absence of TS, as indicated. −, unspiked medium; +, M. tuberculosis-spiked medium; the lines above the bars indicate standard errors.

FIG. 5.

(a) Yields from the PhaB assay by using various infection times prior to addition of virucide and plating. Aliquots from an agitated culture of 1 ml of M. tuberculosis-spiked 7H9-10% (vol/vol) OCG at 37°C were taken at different time points after addition of phage D29. These were treated with FAS for 10 min and then plated (see Materials and Methods). (b) Time courses of lytic yield from the PhaB assay after phage D29 infection under different conditions (see Materials and Methods). At the indicated times, 0.5 ml of sputum was processed by the addition of 0.5 ml of 2% (wt/vol) NaOH-1.45% (wt/vol) trisodium citrate · 2H₂O, followed by the microcentrifugation method with TS. Symbols: +, unspiked sputum with TS and OCG; ○, unspiked sputum with TS and CC; ▾, medium with OCG spiked with M. tuberculosis; ×, medium spiked with M. tuberculosis with CC; •, medium with TS and OCG spiked with M. tuberculosis; *, sputum with TS and OCG spiked with M. tuberculosis; and ▵, sputum with TS and CC spiked with M. tuberculosis, where medium refers to 7H9-10% (vol/vol) OCG, CC is 5 ml of 7H9-8 mM trisodium citrate-10 mM calcium chloride, and OCG is 5 ml of 7H9-10% (vol/vol) OCG added after FAS addition. Overnight recovery was allowed for each sample prior to application of the PhaB assay. Phage D29 was added at time zero. FAS was added after 2 h of infection. Aliquots were plated at different time points after further incubation at 37°C.