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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: clinchem.2009.124396v1 55/9/1694    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Mdivani, N. Articles by Tang, Y.-W. PubMed PubMed Citation Articles by Mdivani, N. Articles by Tang, Y.-W.

Monitoring Therapeutic Efficacy by Real-Time Detection of Mycobacterium tuberculosis mRNA in Sputum

¹ Georgian Foundation against Tuberculosis and Lung Diseases, Tbilisi, Georgia; ² Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN; ³ National Center of Tuberculosis and Lung Diseases, Tbilisi, Georgia; ⁴ Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN.

^aAddress correspondence to this author at: Molecular Infectious Disease Laboratory, Vanderbilt University Hospital, 4605 TVC, Nashville, TN 37232-5310. Fax +615-343-8420; e-mail yiwei.tang{at}vanderbilt.edu.

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.

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