Bacterial strains, culture conditions, and clinical samples.
The Mycobacterium bovis BCG Pasteur and Mycobacterium smegmatis Mc2155 strains used in all experiments were a kind gift of William Jacobs, Jr., Howard Hughes Medical Institute, Bronx, NY. M. tuberculosis strain H37Rv was obtained from the American Tissue Culture Collection (ATCC), Manassas, VA. Mycobacteria were cultured on 7H10 solid medium (BD Diagnostic Systems, Sparks, MD) supplemented with 0.5% glycerol and 10% albumin-dextrose-catalase with sodium chloride (Middlebrook ADC Enrichment; BD Diagnostic Systems). Each 4-ml mycobacterial growth indicator tube (MGIT) (BD Diagnostic Systems) was supplemented with 0.5 ml MGIT oleic acid-albumin-dextrose-catalase (OADC) and 0.1 ml PANTA antibiotic (BD), supplied with the kit. The Middlebrook 7H9 liquid medium (BD Diagnostic Systems) used in the BioSampler studies was supplemented with 10% ADC (BD Diagnostic Systems) and 0.05% Tween 80 (Sigma-Aldrich, St. Louis, MO). M. bovis BCG (ATCC 35743; TMC 1028) was acquired from the ATCC by the independent testing service (Microbiotest, Sterling, VA).
BCG organisms were inoculated into Middlebrook 7H9 liquid medium and incubated at 37 ± 2°C with gentle agitation for 15 to 25 days or until a pellicle was formed. This material was used as the inoculum for the quantitative tuberculocidal test as follows. Solid-medium cultures were wrapped in aluminum foil, incubated at 37°C for up to 6 weeks, and checked periodically for growth. MGITs were cultured and analyzed in a Bactec MGIT 960 automated culture system (BD) according to the manufacturer's recommendations, and the number of days between MGIT inoculation and a positive culture (time-to-positive days [TpD]) was recorded for each sample. Negative cultures were discarded after 6 weeks. M. tuberculosis was confirmed in all positive MGIT cultures by the observation of a positive AFB smear and a positive immunochromatographic lateral-flow assay for MPB64 (Capilia TB Neo) performed according to the manufacturer's instructions (Tauns Laboratories, Inc., Shizuoka, Japan). Solid cultures on 7H10 plates were identified as contaminated when the colonies present did not have the typical morphological appearance of M. tuberculosis, and representative colonies were confirmed to be Capilia TB Neo test negative. Liquid cultures that were indicated as positive by the MGIT 960 system were identified as contaminated if both the Capilia TB Neo test and the AFB smear from the culture medium were negative.
The origins of the sputum samples varied in accordance with study objectives. Analytical studies that required the addition of known numbers of BCG CFU into tuberculosis-negative sputum were performed with sputa obtained from patients at New Jersey Medical School—UMDNJ University Hospital, Newark, NJ, who were not suspected of having tuberculosis, as described previously (14
), and/or with sputum pools from confirmed negative patients obtained from Montefiore Medical Center, Bronx, NY. Sputum samples for the clinical SR killing studies were obtained from patients being evaluated for tuberculosis at Hinduja Hospital, India, between January and February 2009. The Hinduja patients were required to have symptoms suggestive of tuberculosis and grade 2+ to 3+ AFB smears on prescreening (where 2+ represents 1 to 9 AFB per field and 3+ represents 10 or more AFB per field). Patients were excluded if they had received any antituberculosis therapy within the past 60 days. Sputum samples were collected prospectively from each patient, frozen, and then sent to a laboratory in the United States for SR killing studies. Sputum samples were obtained only after approval by the Hinduja and UMDNJ institutional review boards.
Detection of viable aerosols.
As there is no standardized reference method for detecting bioaerosol contamination from new devices or during procedures, we aimed to detect both total and culturable aerosol generation under simulated conditions. Aerosolization studies during smearing and pipetting were performed in a 6-foot (2-m) class II biological safety cabinet (BSC-2) (NU-425-600; NuAire, Plymouth, MN). The GeneXpert aerosolization studies were performed in a 4-foot (1.2- m) BSC-2 (NU-425-400; NuAire) with the airflow turned off and the sash pulled down into an incomplete airtight seal. The limit-of-detection (LOD) (2
) studies were performed inside a custom-built aerosol generation and sampling chamber based on the design of a chamber successfully used for similar aerosol studies (15
). This was essentially a closed wind tunnel kept under negative pressure, which permitted safe aerosolization and sampling of infectious particles. The chamber was operated inside the biological safety cabinet to control the large numbers of infectious BCG that were aerosolized during the experiment. For all aerosol studies, viable-aerosol production was measured using both a single-stage N-6 viable Andersen cascade impactor (Thermo Scientific, Waltham, MA) and a BioSampler (SKC Inc., Eighty Four, PA) loaded with 20 ml Middlebrook 7H9 liquid medium. The Andersen impactor was operated at calibrated vacuum flow rates of 28.3 liters per minute, and the BioSampler was operated at 12.8 liters per minute. Before sampling, the airflow rates for all samplers were calibrated with a DryCal DC-lite Calibrator (SKC Inc., Eighty Four, PA). The aerosol sampling time for each study is noted below. The BioSampler's collection liquid was plated in triplicate on 7H10 agar plates. All the agar plates were incubated at 37°C for up to 5 weeks for CFU counts. The total airborne bacterial concentrations were expressed per cubic meter of air.
The total airborne concentration (CFU/m3) for the Andersen impactor, CANDERSEN, was calculated as follows: CANDERSEN = 1,000 × Ntotal/Qsts, where Ntotal is the total number of CFU on the agar plate, Qs is the sampling flow rate per minute, and ts is the sampling time (min).
The total airborne concentration (CFU/m3) for the BioSampler, CBIOSAMPLER, was calculated as follows: CBIOSAMPLER = 1,000 × (Ntotal/Qsts) × (VLIQ/VPLIQ), where VLIQ is the volume of liquid left after collection and VPLIQ is the volume of liquid used for plating.
The total numbers of particles present in the BSCs before, during, and after the sample preparation steps were also studied as a second measure of potentially infectious aerosol generation. Particles were detected using an aerodynamic particle sizer spectrometer (APS model 3321; TSI Inc., St. Paul, MN).
Limits of detection of the aerosol samplers.
7H9 medium was spiked with log dilutions of BCG ranging from 10 to 104 CFU/ml. Approximately 0.8 ml of each mixture was then aerosolized using a Sparging Liquid Aerosol Generator (CH Technologies, Inc., Westwood, NJ) over 10 min within the custom-built aerosol generation and sampling chamber. Total aerosol particle counts within the chamber were measured using an APS Spectrometer. Aerosol collection was begun after the total aerosol particle counts were stable and then continued for 1 min using the Andersen impactor and for 15 min using the BioSampler.
Aerosolization during AFB smear and Xpert MTB/RIF assay setup.
Pooled human sputa from nontuberculous patients were spiked with BCG at a final concentration of 5 × 108 CFU/ml of sputum. SR was added to the BCG-sputum mixture at an SR/sputum ratio of 2:1. The “incorrect use of SR” was simulated by loading the SR-sputum mixture immediately into Xpert MTB/RIF cartridges without performing the recommended 15-min SR-sputum incubation. Instead, after SR introduction, the SR-sputum container was briefly shaken, and then the sputum was immediately pipetted in and out of three Xpert MTB/RIF cartridges for 15 min, simulating the loading of at least 30 cartridges. All pipetting steps used the disposable plastic pipettes included in the Xpert MTB/RIF test kit. The “correct use of SR” was simulated by briefly shaking the SR-sputum mixture and incubating the mixture for 15 min at room temperature before pipetting the mixture in and out of three Xpert MTB/RIF cartridges for 15 min as described above. AFB smear preparation was performed by smearing a loopful (10 μl) of the BCG-spiked sputum (without SR) onto three microscope slides for 10 min, simulating the preparation of 20 sputum AFB slides. Smears were made using disposable plastic loops. Aerosol collection using the Andersen impactor and BioSampler was begun at the start of each procedure and continued for 2 min and 10 min, respectively.
Air sampling during automated sample processing.
The production of viable aerosols generated by the GeneXpert instrument during sample-processing stages in Xpert MTB/RIF cartridges was studied by loading Xpert MTB/RIF cartridges with several different sample types and measuring viable-aerosol production during an assay run. Cartridges were loaded with one of three sample types: (i) water spiked with 2 ml of 5 × 108 BCG CFU/ml without SR treatment, (ii) 2 ml of sputum spiked with BCG (5 × 108 CFU/ml) treated for 15 min with SR at a 2:1 ratio, or (iii) 2 ml of sputum spiked with M. smegmatis (7 × 108 CFU/ml) treated for 15 min with SR at a 2:1 ratio. For each sample type studied, the GeneXpert instrument was loaded with three cartridges and the air was sampled for 2 and 15 min using two Andersen samplers and a BioSampler, respectively, during the Xpert MTB/RIF assay run. The first Andersen sampler was operated simultaneously with the start of the BioSampler, and the second Andersen sampler was operated at the 13th minute of the BioSampler's run time. Each assay was run three times, individually, for a total of nine cartridges tested under each condition.
SR killing studies.
Fifty M. tuberculosis-positive sputum samples collected from the Hinduja study were thawed and vortexed for 1 min. Five of the samples did not have the required minimum 3-ml volume and were discarded. The remaining 45 samples were thoroughly mixed with a pipette and then split into three portions of 1 ml each. The three portions were randomly assigned as follows. The first 1-ml portion was decontaminated by a standard NALC-NaOH (N-acetyl-l-cysteine-NaOH) method, using a MycoPrep kit (BD Diagnostic Systems) according to the manufacturer's instructions and resuspended in 700 μl of phosphate-buffered saline (PBS), pH 7.4. A 200-μl volume of each sample was inoculated onto 7H10 plates, and 500 μl was inoculated into MGITs supplemented with PANTA and OADC according to the manufacturer's instructions. Sputum samples from six tuberculosis-negative patients were also split, and 1 ml was cultured as described above. A second and a third 1-ml portion of each sputum sample was treated with SR at an SR/sputum ratio of 2:1 or 3:1, respectively. The SR-sputum mixtures were briefly shaken and incubated at room temperature for 15 min. A 1.5-ml volume of each sample was then transferred to a 50-ml centrifuge tube, which was filled to the top with PBS and centrifuged for 20 min at 4,000 × g. The supernatant was then discarded, and the remaining pellet was resuspended in 700 μl of PBS. Two hundred microliters of each sample was inoculated onto 7H9 medium, and 500 μl was inoculated into an MGIT. Positive growth in the MGIT was confirmed by both Capilia TB-Neo assay (Tauns, Shizuoka, Japan) and by AFB smear examination; 7H10 colonies were confirmed for M. tuberculosis bacilli by Capilia TB-Neo assay only.
SR stability at varied temperatures.
SR was stored under different conditions for at least 3 months. The killing efficiency of SR under each storage condition was evaluated in a manner similar to that described above. Briefly, negative-control sputum was spiked with 109 CFU/ml BCG cells, and SR was added at the recommended 2:1 ratio (SR/sputum) to the sample. The preparation was shaken for 5 s, incubated for 15 min at ambient temperature, and again shaken manually for 5 s. PBS was added to make up the volume to 45 ml and mixed well, and the tube was centrifuged for 15 min at 4,000 × g. The pellet was resuspended in 1 ml of fresh PBS, and 100 μl was plated on 7H10 plates in triplicate. Four individual experiments were run to evaluate the average killing efficiency of these stored SR. The average log kill was expressed as follows: log10(average initial CFU/ml) − log10(average test CFU/ml).
Quantitative tuberculocidal tests performed by an independent test service.
Testing was performed by Microbiotest (Sterling, VA). BCG organisms were macerated with a tissue grinder at 0 to 4°C, and the homogenized suspension was diluted with 7H9 medium to a concentration between 107 and 108 CFU/ml, as determined by spectrophotometric methods. Two milliliters of BCG suspension was added to 4.7 ml of sputum and vortexed to mix them, and then the mixture was added to 13.3 ml of SR in sterile glass tubes. Duplicate preparations were made. After incubation for 15 min, aliquots of the mixture were removed and mixed with aliquots of sterile saline to make 10-fold serial dilutions. Duplicates of each dilution were filtered through a 47-mm-diameter cellulose nitrate membrane filter with a pore size of 0.45 μm, and the filters were washed with sterile saline. The filters were aseptically transferred to the surface of a Middlebrook 7H11 plate and incubated at 37 ± 2°C for 15 to 25 days. Colonies were counted using a dissecting microscope with lateral lighting, and the average number of CFU/ml was calculated. Initial counts of BCG organisms were determined by mixing 2 ml of M. bovis BCG suspension with 18 ml of sterile saline and serially diluting 1-ml aliquots with sterile saline blanks. These control samples were processed similarly to the test samples, and the plates were incubated along with the test plates. BCG colonies were confirmed by AFB staining and colony morphology. The log reduction in viability was calculated as explained above: log10(average initial CFU/ml) − log10(average test CFU/ml).
Stability of samples after prolonged SR treatment.
M. tuberculosis-negative sputum samples were spiked with M. tuberculosis strain H37Rv at a final concentration of 60 CFU/ml. Negative controls were run for each sputum pool and at every time point. Samples were incubated at room temperature with SR at a 2:1 ratio for 15 min, 5 h, 8 h, 24 h, 3 days, and 7 days, and the Xpert MTB/RIF assay was performed in a GeneXpert system according to the manufacturer's instructions. Assay amplicons were assessed for mutations by DNA sequencing as described previously (C. C. Boehme, P. Nabeta, D. Hillemann, M. Nicol, S. Shennai, F. Krapp, J. Allen, R. Tahirli, R. Blakemore, R. Rustomjee, A. Milovic, M. Jones, S. M. O'Brien, D. H. Persing, S. Ruesch-Gerdes, E. Gotuzzo, C. Rodrigues, D. Alland, and M. D. Perkins, submitted for publication).
Statistical analysis by t test was performed using Sigmaplot version 8.0 (Systat Software Inc., San Jose, CA), and Fisher's exact t test was performed using SAS version 9.1 (SAS, Cary, NC).