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)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI093358-02
Application #
8245684
Study Section
Special Emphasis Panel (ZAI1-LG-M (J3))
Program Officer
Jacobs, Gail G
Project Start
2011-04-01
Project End
2016-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$787,280
Indirect Cost
$196,817
Name
University of Virginia
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Foongladda, S; Banu, S; Pholwat, S et al. (2016) Comparison of TaqMan(®) Array Card and MYCOTB(TM) with conventional phenotypic susceptibility testing in MDR-TB. Int J Tuberc Lung Dis 20:1105-12
Pholwat, Suporn; Stroup, Suzanne; Heysell, Scott et al. (2016) eis Promoter C14G and C15G Mutations Do Not Confer Kanamycin Resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 60:7522-7523
Heysell, Scott K; Ahmed, Shahriar; Ferdous, Sara Sabrina et al. (2015) Quantitative drug-susceptibility in patients treated for multidrug-resistant tuberculosis in Bangladesh: implications for regimen choice. PLoS One 10:e0116795
Ndusilo, Norah D; Heysell, Scott K; Mpagama, Stellah G et al. (2015) Improvement in plasma drug activity during the early treatment interval among Tanzanian patients with multidrug-resistant tuberculosis. PLoS One 10:e0122769
Pholwat, Suporn; Liu, Jie; Stroup, Suzanne et al. (2015) Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. MBio 6:e02273
Heysell, Scott K; Pholwat, Suporn; Mpagama, Stellah G et al. (2015) Sensititre MycoTB plate compared to Bactec MGIT 960 for first- and second-line antituberculosis drug susceptibility testing in Tanzania: a call to operationalize MICs. Antimicrob Agents Chemother 59:7104-8
Foongladda, Suporn; Klayut, Wiphat; Pholwat, Suporn et al. (2015) Comparison and development of pyrazinamide susceptibility testing methods for tuberculosis in Thailand. Diagn Microbiol Infect Dis 83:270-3
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
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
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

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