Comparison of quantitative techniques including Xpert MTB/RIF to evaluate mycobacterial burden.
BACKGROUND
Accurate quantification of mycobacterial load is important for the evaluation of patient infectiousness, disease severity and monitoring treatment response in human and in-vitro laboratory models of disease. We hypothesized that newer techniques would perform as well as solid media culture to quantify mycobacterial burden in laboratory specimens.


METHODS
We compared the turn-around-time, detection-threshold, dynamic range, reproducibility, relative discriminative ability, of 4 mycobacterial load determination techniques: automated liquid culture (BACTEC-MGIT-960), [(3)H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative PCR(Xpert -MTB/RIF) using serial dilutions of Mycobacterium bovis and Mycobacterium tuberculosis H37RV. Mycobacterial colony-forming-units(CFU) using 7H10-Middlebrook solid media served as the reference standard.


RESULTS
All 4 assays correlated well with the reference standard, however, bioluminescence and uracil assays had a detection threshold ≥1×10(3) organisms. By contrast, BACTEC-MGIT-960 liquid culture, although only providing results in days, was user-friendly, had the lowest detection threshold (<10 organisms), the greatest discriminative ability (1 vs. 10 organisms; p=0.02), and the best reproducibility (coefficient of variance of 2% vs. 38% compared to uracil incorporation; p=0.02). Xpert-MTB/RIF correlated well with mycobacterial load, had a rapid turn-around-time (<2 hours), was user friendly, but had a detection limit of ~100 organisms.


CONCLUSIONS
Choosing a technique to quantify mycobacterial burden for laboratory or clinical research depends on availability of resources and the question being addressed. Automated liquid culture has good discriminative ability and low detection threshold but results are only obtained in days. Xpert MTB/RIF provides rapid quantification of mycobacterial burden, but has a poorer discrimination and detection threshold.
Lung Infection and Immunity Unit, Division of Pulmonology and UCT Lung Institute, Department of Medicine, University of Cape Town, Cape Town, South Africa.
Richard N
RN
van Zyl-Smit
Anke
A
Binder
Richard
R
Meldau
Hridesh
H
Mishra
Patricia L
PL
Semple
Grant
G
Theron
Jonathan
J
Peter
Andrew
A
Whitelaw
Suren K
SK
Sharma
Robin
R
Warren
Eric D
ED
Bateman
Keertan
K
Dheda
D015169:Colony Count, Microbial / Q000379:methods*; D049449:Luminescence; D009163:Mycobacterium bovis / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction