Rapid detection and monitoring therapeutic efficacy of Mycobacterium tuberculosis complex using a novel real-time assay.
We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 nonrespiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was < or =35 and the ratio of real-time RT-PCR and real-time PCR load was > or =1.51. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including nonrespiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, PR China.
Li Juan
LJ
Jiang
Wen Juan
WJ
Wu
Hai
H
Wu
Son Sik
SS
Ryang
Jian
J
Zhou
Wei
W
Wu
Tao
T
Li
Jian
J
Guo
Hong Hai
HH
Wang
Shui Hua
SH
Lu
Yao
Y
Li
D000995:Antitubercular Agents / Q000494:pharmacology / Q000627:therapeutic use; D016903:Drug Monitoring; D005798:Genes, Bacterial / Q000235:genetics; D020459:Genes, rRNA / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008198:Lymph Nodes / Q000382:microbiology; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D010996:Pleural Effusion / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000188:drug therapy* / Q000382:microbiology*
D000995:Antitubercular Agents