This proposal addresses RFA-AI-10-003 "Partnerships for Biodefense" and brings together academic researchers, industry, MDR TB clinicians, BSL3 Labs, and field sites to develop a faster, quantitative, second line drug susceptibility test (DST) for multiple and extensively drug-resistant tuberculosis. The emergence of drug resistant TB, a Category C threat, has led the ominous situation of a highly transmissible and in some instances untreatable disease. Several Tb DST platforms are in use yet all have significant constraints, particularly turnaround time. We feel that we must persevere with a phenotypic culture based method, since direct molecular testing will remain poorly defined for second line drugs, but we have shown that can improve existing liquid methods with an earlier 3 day quantitative PCR-based readout that measures DNA amplification in the setting of drug. By using high-throughput small-volume microtiter plates, our method can yield both an accurate rapid qualitative result and a quantitative "inhibition index" akin to the MIC - a measurement that is clinically needed but rarely available for Tb. Because biosafety of MDR/XDR Tb for laboratory personnel is of utmost importance, we will then transform this assay into a novel-design, closed- system, disposable, qPCR-capable microfluidic chip. This microfluidic chamber can accurately meter the number of bacilli fed into each well, minimizing one area of poor reproducibility, and can also measure growth in situ by other parameters beyond qPCR, including a newly-invented DNA detection technique we term pinwheel. Thus the qPCR assay development and chip project will benefit from but are not tied to each other's success. The goal is an improved DST platform for second line drugs that yields faster information to better treat and decrease transmission of MDR/XDR Tb. NARRATIVE Drug susceptibility testing for MDR and XDR Tb is slow and fraught with technical difficulties. This proposal will develop a rapid, quantitative PCR-based diagnostic to detect susceptibility or resistance of Tb to second line drugs in a microplate format within 3 days. The assay will be adapted to a closed-system disposable chip to enhance biosafety for lab personnel and permit DNA quantification by our new 'pinwheel'approach. The results will be immediately usable by MDR Tb clinicians to better treat and decrease transmission of MDR/XDR Tb.

Public Health Relevance

Drug susceptibility testing for MDR and XDR Tb is slow and fraught with technical difficulties. This proposal will develop a rapid, quantitative PCR-based diagnostic to detect susceptibility or resistance of Tb to second line drugs in a microplate format within 3 days. The assay will be adapted to a closed-system disposable chip to enhance biosafety for lab personnel and permit DNA quantification by our new 'pinwheel' approach. The results will be immediately usable by MDR Tb clinicians to better treat and decrease transmission of MDR/XDR Tb.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI093358-04
Application #
8648998
Study Section
Special Emphasis Panel (ZAI1)
Program Officer
Jacobs, Gail G
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Virginia
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Foongladda, Suporn; Klayut, Wiphat; Chinli, Rattapha et al. (2014) Use of mycobacteriophage quantitative PCR on MGIT broths for a rapid tuberculosis antibiogram. J Clin Microbiol 52:1523-8
Pholwat, Suporn; Stroup, Suzanne; Gratz, Jean et al. (2014) Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis. Tuberculosis (Edinb) 94:20-5
Banu, Sayera; Rahman, S M Mazidur; Khan, M Siddiqur Rahman et al. (2014) Discordance across several methods for drug susceptibility testing of drug-resistant Mycobacterium tuberculosis isolates in a single laboratory. J Clin Microbiol 52:156-63
Mpagama, Stellah G; Ndusilo, Norah; Stroup, Suzanne et al. (2014) Plasma drug activity in patients on treatment for multidrug-resistant tuberculosis. Antimicrob Agents Chemother 58:782-8
Zhdanova, Svetlana; Heysell, Scott K; Ogarkov, Oleg et al. (2013) Primary multidrug-resistant Mycobacterium tuberculosis in 2 regions, Eastern Siberia, Russian Federation. Emerg Infect Dis 19:1649-52
Mpagama, Stellah G; Heysell, Scott K; Ndusilo, Nora D et al. (2013) Diagnosis and interim treatment outcomes from the first cohort of multidrug-resistant tuberculosis patients in Tanzania. PLoS One 8:e62034
Heysell, Scott K; Thomas, Tania A; Sifri, Costi D et al. (2013) 18-Fluorodeoxyglucose positron emission tomography for tuberculosis diagnosis and management: a case series. BMC Pulm Med 13:14
Pholwat, Suporn; Stroup, Suzanne; Foongladda, Suporn et al. (2013) Digital PCR to detect and quantify heteroresistance in drug resistant Mycobacterium tuberculosis. PLoS One 8:e57238
Mpagama, Stellah G; Houpt, Eric R; Stroup, Suzanne et al. (2013) Application of quantitative second-line drug susceptibility testing at a multidrug-resistant tuberculosis hospital in Tanzania. BMC Infect Dis 13:432
Pholwat, Suporn; Ehdaie, Beeta; Foongladda, Suporn et al. (2012) Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J Clin Microbiol 50:754-61

Showing the most recent 10 out of 11 publications