Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.
Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.
Department of Medicine, New Jersey Medical School, Newark, New Jersey, USA chakraso@njms.rutgers.edu.
Soumitesh
S
Chakravorty
Department of Medicine, New Jersey Medical School, Newark, New Jersey, USA.
Sandy S
SS
Roh
Cepheid Inc., Sunnyvale, California, USA.
Jennifer
J
Glass
Department of Medicine, New Jersey Medical School, Newark, New Jersey, USA.
Laura E
LE
Smith
Cepheid Inc., Sunnyvale, California, USA.
Ann Marie
AM
Simmons
Cepheid Inc., Bothell, Washington, USA.
Kevin
K
Lund
Cepheid Inc., Bothell, Washington, USA.
Sergey
S
Lokhov
Henan Provincial Chest Hospital, Zhengzhou, Henan Province, China.
Xin
X
Liu
Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institutes of Biomedical Sciences and Institute of Medical Microbiology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Peng
P
Xu
Sino-US International Research Center of Tuberculosis, Zhengzhou, China.|Henan Public Health Clinical Center, Zhengzhou, China.
Guolong
G
Zhang
Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, Maryland, USA.|Institute of Infectious Disease and Molecular Medicine, Department of Pathology, University of Cape Town, Cape Town, South Africa.
Laura E
LE
Via
Sino-US International Research Center of Tuberculosis, Zhengzhou, China.
Qingyu
Q
Shen
Henan Provincial Chest Hospital, Zhengzhou, Henan Province, China.
Xianglin
X
Ruan
Henan Provincial Chest Hospital, Zhengzhou, Henan Province, China.
Xing
X
Yuan
Sino-US International Research Center of Tuberculosis, Zhengzhou, China.
Hong Zhu
HZ
Zhu
Cepheid Inc., Bothell, Washington, USA.
Ekaterina
E
Viazovkina
Department of Medicine, New Jersey Medical School, Newark, New Jersey, USA.
Shubhada
S
Shenai
Department of Medicine, New Jersey Medical School, Newark, New Jersey, USA.
Mazhgan
M
Rowneki
Department of Microbiology, International Tuberculosis Research Center, Changwon, Gyeongsnag, Republic of Korea.
Jong Seok
JS
Lee
Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, Maryland, USA.|Institute of Infectious Disease and Molecular Medicine, Department of Pathology, University of Cape Town, Cape Town, South Africa.
Clifton E
CE
Barry
Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institutes of Biomedical Sciences and Institute of Medical Microbiology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
Qian
Q
Gao
Cepheid Inc., Sunnyvale, California, USA.
David
D
Persing
Cepheid Inc., Sunnyvale, California, USA.
Robert
R
Kwiatkawoski
Cepheid Inc., Sunnyvale, California, USA.
Martin
M
Jones
Cepheid Inc., Bothell, Washington, USA.
Alexander
A
Gall
Department of Medicine, New Jersey Medical School, Newark, New Jersey, USA.
David
D
Alland
D000483:Alleles; D000583:Amikacin / Q000494:pharmacology; D000995:Antitubercular Agents / Q000494:pharmacology*; D057205:Automation, Laboratory / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D054908:Extensively Drug-Resistant Tuberculosis / Q000175:diagnosis* / Q000382:microbiology; D024841:Fluoroquinolones / Q000494:pharmacology; D005798:Genes, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D007612:Kanamycin; D000583:Amikacin; D007538:Isoniazid
10-color assay; XDR-TB; point-of-care test; three-phase PCR