Monitoring therapeutic efficacy by real-time detection of Mycobacterium tuberculosis mRNA in sputum.
BACKGROUND
Current laboratory methods for monitoring the response to therapy for tuberculosis (TB) rely on mycobacterial culture. Their clinical usefulness is therefore limited by the slow growth rate of Mycobacterium tuberculosis. Rapid methods to reliably quantify the response to anti-TB drugs are desirable.


METHODS
We developed 2 real-time PCR assays that use hydrolysis probes to target DNA of the IS6110 insertion element and mRNA for antigen 85B. The nucleic acids are extracted directly from concentrated sputum samples decontaminated with sodium hydroxide and N-acetyl-L-cysteine. We prospectively compared these assays with results obtained by sputum mycobacterial culture for patients receiving anti-TB therapy.


RESULTS
Sixty-five patients with newly diagnosed TB and receiving a standardized first-line anti-TB drug regimen were evaluated at week 2 and at months 1, 2, and 4 after therapy initiation. Both the DNA PCR assay (98.5% positive) and the mRNA reverse-transcription PCR (RT-PCR) assay (95.4% positive) were better than standard Ziehl-Neelsen staining techniques (83.1%) for detecting M. tuberculosis in culture-positive sputum samples. The overall agreement between culture and mRNA RT-PCR results for all 286 sputum samples was 87.1%, and compared with culture, the mRNA RT-PCR assay's diagnostic sensitivity and specificity were 85.2% and 88.6%, respectively. For monitoring efficacy of therapy, mRNA RT-PCR results paralleled those of culture at the follow-up time points.


CONCLUSIONS
The continued presence of viable M. tuberculosis according to culture and results obtained by RT-PCR analysis of antigen 85B mRNA correlated clinically with resistance to anti-TB drugs, whereas the DNA PCR assay showed a high false-positive rate. This mRNA RT-PCR assay may allow rapid monitoring of the response to anti-TB therapy.
Mdivani
Nino
N
Georgian Foundation against Tuberculosis and Lung Diseases, Tbilisi, Georgia, USA.
Li
Haijing
H
Akhalaia
Maka
M
Gegia
Medea
M
Goginashvili
Leila
L
Kernodle
Douglas S
DS
Khechinashvili
George
G
Tang
Yi-Wei
YW
D000328:Adult; D000995:Antitubercular Agents / Q000627:therapeutic use*; D005189:False Positive Reactions; D005260:Female; D006801:Humans; D008297:Male; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000188:drug therapy* / Q000382:microbiology*
D000995:Antitubercular Agents; D012329:RNA, Bacterial; D012333:RNA, Messenger