Improved contamination control for a rapid phage-based rifampicin resistance test for Mycobacterium tuberculosis

Abstract

A prospective study was conducted of the rapid FASTPlaque-Response test for determination of rifampicin resistance in Mycobacterium tuberculosis with and without the addition of an antimicrobial supplement containing nystatin, oxacillin and aztreonam (NOA) to control specimen-related contamination. A total of 631 smear-positive sputum specimens was tested. The age of specimens ranged from 0 to 21 days. The NOA antimicrobial was effective at controlling contamination, with 4.1 % of specimens contaminated when the NOA antimicrobial supplement was used compared with 13.9 % contamination without NOA. Overall levels of interpretability of the test with NOA were 87.8 % with specimens of ¡3 days and 79.0 % for all specimens. This compared with 70.1 and 73.8 % readable results, respectively, from conventional culture-based drug susceptibility testing (DST). Sensitivity, specificity and overall accuracy of the FASTPlaque-Response test for rifampicin resistance were 98.1, 96.3 and 96.6 %, respectively, for all specimens with NOA, and 93.2, 96.3 and 95.9 % without NOA, when compared with resolved conventional DST results. Inclusion of the NOA supplement reduced contamination, increased the number of interpretable results and did not adversely affect the performance of the FASTPlaque-Response test. Thus, the use of NOA improves the robustness of the test, facilitating its wider implementation.

Face Mask Sampling for the Detection of Mycobacterium tuberculosis in Expelled Aerosols

“…To our surprise, we found that this appeared to be an effective means of releasing mycobacterial cells from the mask and pfu counts well in excess of control values were readily obtained (OM assay, see figure 1). We also determined that decontamination with NaOH/NALC reduced counts by at least 3-fold (data not shown) and that assay contamination could be prevented with the combination of nystatin, oxacillin and aztreonam (NOA) recommended by Mole and colleagues [17].…”

A Rapid Growth-Independent Antibiotic Resistance Detection Test by SYBR Green/Propidium Iodide Viability Assay

“…Despite the high sensitivity achieved by the MBT-ASTRA, as the authors noted, it seems unlikely that MBT-ASTRA will be used in clinical laboratories due to its lack of automated readout ( 12 ). Additionally, a luciferase phage system which was developed to detect rifampicin resistance in mycobacteria in 2 days has contamination issues ( 31 ). In contrast, the SYBR Green I/PI assay we developed can be used for ultra-rapid DST for not only fast-growing organisms but also for slow-growing organisms like M. tuberculosis in a high-throughput manner such as in 96-well or 384-well plate format, has low detection limits, background noise and more quantitative, and is extremely cost-effective.…”

Rapid Clinical Bacteriology and Its Future Impact

“…Interestingly, the system was never widely accepted in clinical microbiology, nor was it expanded to other bacterial applications. Two of the major problems were laboratory contamination with bacteriophage [120] and possible phage resistance of certain bacterial strains. So irrespective of the sound biological principle of this approach, this system is unlikely to change our AST practice.…”