Containment of bioaerosol infection risk by the Xpert MTB/RIF assay and its applicability to point-of-care settings

Abstract

The recently introduced Xpert MTB/RIF assay (Xpert) has point-of-care potential, but its capacity for biohazard containment remained to be studied. We compared the bioaerosols generated by the Xpert assay to acid-fast bacillus (AFB) microscope slide smear preparation. The Xpert assay sample treatment reagent (SR) was also studied for its sterilizing capacity, stability, and effect on assay sensitivity after prolonged treatment. During the preparation of AFB smears, sputum samples spiked with Mycobacterium bovis BCG at 5 × 10(8) CFU/ml produced 16 and 325 CFU/m(3) air measured with an Andersen impactor or BioSampler, respectively. In contrast, neither the sample preparation steps for the Xpert assay nor its automated processing produced any culturable bioaerosols. In testing of SR sterilizing capacity, clinical sputum samples from strongly smear-positive tuberculosis patients treated with SR at a 2:1 ratio eliminated Mycobacterium tuberculosis growth in all but 1/39 or 3/45 samples cultured on solid or liquid medium, respectively. These few unsterilized samples had a mean 13.1-day delay in the time to positive culture. SR treatment at a 3:1 ratio eliminated growth in all samples. SR retained a greater than 6-log-unit killing capacity despite storage at temperatures spanning 4 to 45°C for at least 3 months. The effect of prolonged SR sample treatment was also studied. Spiked sputum samples could be incubated in SR for up to 3 days without affecting Xpert sensitivity for M. tuberculosis detection and up to 8 h without affecting specificity for rifampin resistance detection. These results suggest that benchtop use of the Xpert MTB/RIF assay limits infection risk to the user.

FIG. 1.

Particles generated during manual sample preparation. Total airborne particles were measured for AFB smear preparation (A) or pipetting and cartridge loading (B). Measurements were first taken either with the BSC airflow on or with the BSC airflow off and the sash closed as much as possible before and during each sample preparation procedure. Each time point represents the mean of three experiments. The error bars indicate ±1 standard deviation.

FIG. 2.

Particles generated during automated sample processing. Total airborne particles were measured during the sample-processing portion of the Xpert MTB/RIF assay, which is performed automatically by the GeneXpert instrument. During each sample-processing run, the GeneXpert was loaded with three cartridges containing water spiked with 5 × 10⁸ CFU/ml BCG (A), sputum spiked with 5 × 10⁸ CFU/ml BCG and then treated with SR (B), or sputum spiked with 7 × 10⁸ CFU/ml M. smegmatis and treated with SR (C). Measurements were first taken with the BSC airflow off and the sash closed as much as possible before and after or during each sample-processing run. Each time point represents the mean of three experiments. The error bars indicate ±1 standard deviation.

FIG. 3.

SR killing activity in strongly smear-positive clinical sputum samples from tuberculosis patients. Forty-five clinical tuberculosis sputum samples were decontaminated with either SR/sputum ratios of 2:1 or 3:1 or Mycoprep (BD, Sparks, MD) as indicated. The washed samples were then cultured on 7H10 agar plates or in MGITs. The number of sputum samples culture positive for M. tuberculosis (MTB), culture negative for M. tuberculosis, or contaminated by other bacteria is shown for each culture type.

FIG. 4.

SR stability study in sputa spiked with 60 CFU/ml of M. tuberculosis H37Rv. Sputum spiked with 60 CFU/ml M. tuberculosis was treated with a 2:1 SR-to-sample ratio and incubated for up to 7 days. Samples were tested by Xpert MTB/RIF assay at 15 min (n = 59), 5 h (n = 145), 8 h (n = 38), 24 h (n = 118), 3 days (n = 20), and 7 days (n = 11) (n represents the number of replicates run at each time point). The percentage of positives detected at each time point was plotted. Linear regression curve fitting (r² = 0.6) was performed, and 95% upper and lower confidence intervals were determined using SigmaPlot 8.0. Fisher's t test at a P value of >0.05 showed no significant difference at different time points.