Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology.
Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System's MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay's buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.
Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry, New Jersey, Newark, New Jersey 07103, USA.
Danica
D
Helb
Martin
M
Jones
Elizabeth
E
Story
Catharina
C
Boehme
Ellen
E
Wallace
Ken
K
Ho
JoAnn
J
Kop
Michelle R
MR
Owens
Richard
R
Rodgers
Padmapriya
P
Banada
Hassan
H
Safi
Robert
R
Blakemore
N T Ngoc
NT
Lan
Edward C
EC
Jones-López
Michael
M
Levi
Michele
M
Burday
Irene
I
Ayakaka
Roy D
RD
Mugerwa
Bill
B
McMillan
Emily
E
Winn-Deen
Lee
L
Christel
Peter
P
Dailey
Mark D
MD
Perkins
David H
DH
Persing
David
D
Alland
D000293:Adolescent; D000328:Adult; D000368:Aged; D000995:Antitubercular Agents / Q000494:pharmacology*; D001431:Bacteriological Techniques / Q000379:methods*; D024881:Drug Resistance, Bacterial; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000302:isolation & purification; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000379:methods; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology; D014454:Uganda; D014744:Vietnam; D055815:Young Adult
D000995:Antitubercular Agents; D012293:Rifampin