Speaker Details

Biography

Mariana Piuri, PhD, Assistant Professor, University of Buenos Aires, Adjunct Investigator CONICET, Argentina. She graduated with a M. Sc. In Biology from the School of Exact and Natural Sciences (FCEN), University of Buenos Aires (UBA) in 1998. She got her PhD in Biological Chemistry at the same institution in 2003 working on the role of the proteolytic system during osmotic stress in Lactobacillus casei under supervision of Dr. Sanchez-Rivas. She was a postdoctoral fellow and Postdoctoral Research Associate in the laboratory of Dr. Graham Hatfull at University of Pittsburgh, Pittsburgh, USA between 2003-2009 focusing in the study of general biology of Mycobacteriophages and its application as diagnostic tools for Mycobacterium tuberculosis. In 2010, she was appointed Assistant Researcher of CONICET at the Department of Biological Chemistry, FCEN, UBA where she started her lab working on the use of bacteriophages as genetic tools and for diagnosis of human pathogens. Her work has been published in high-quality international journals in microbiology and she has been an invited speaker in several international meetings, especially in Bacteriophage, Tuberculosis and Lactic Acid Bacteria field. Since 2015 she is an Assistant Professor in Microbiology and Biotechnology at the same institution.

Abstract

Fluorophages for rapid TB-diagnosis in sputum samples and phenotypic drug susceptibility testing

Fluorophages for rapid TB-diagnosis in sputum samples and phenotypic drug susceptibility testing Tuberculosis (TB) is a major cause of human mortality with 9 million new cases and nearly two million deaths annually; approximately two billion people are infected with the causative agent, Mycobacterium tuberculosis (M.tb). The emergence of resistant strains has become a serious public health problem worldwide complicating treatment and control of the disease. The World Health Organization (WHO) estimates that 40% of tuberculosis cases go undiagnosed and consequently not treated. In 2016, WHO recommended the use of rapid molecular tests to speed up tuberculosis drug susceptibility testing (DST) although due to the cost of equipment and supplies, Ziehl Neelsen staining of Mycobacterium spp. in sputum, with subsequent culture to determine viable bacilli and DST using the proportion method is often the method of choice. Culture methodology is laborious and takes 3-6 weeks to report the presence of viable mycobacteria in the sample and a few additional weeks for DST. Fluoromycobacteriophages (or Fluorophages) are reporter mycobacteriophages containing a fluorescent gene. These phages are a simple and rapid mean of revealing the metabolic state of M.tb cells, and therefore their response to antibiotics. We have constructed a new Fluorophage, mCherrybombφ, with higher sensitivity and less time to detection of signal in M. tb. Using this improved Fluorophage we have developed a simple microscopy-based methodology for detection of viable Mycobacterium spp. and phenotypic determination of rifampicin resistance within just 3-5 days from sputum sample collection. Fluorophage methodology is compatible with regularly used protocols in clinical laboratories for TB diagnosis and paraformaldehyde fixation after infection reduces biohazard risks with sample analysis by fluorescence microscopy. Using clinical isolates of pre-XDR and XDR-TB strains, we tested mCherrybombφ for extended DST and we compared the antibiotic resistance profile with those predicted by whole genome sequencing. Our results emphasize the utility of a phenotypic test for M. tuberculosis extended DST. We had also set up the conditions for infection of pure cultures in a multiwell format in the presence of increasing concentrations of drugs. The effect of the drug was evaluated monitoring the appearance of fluorescence as a function of time using a fluorimeter. The concentration that inhibited 90% of fluorescence in the absence of drug was reported as the minimal inhibitory concentration (MIC) for that drug. Using a fluorimeter, a complete DST of M. tb could be done from pure culture in 6 or 30 hs (when pre-incubation with the drug was required). We found a good correlation between the MIC values obtained with this technique and the proportion method used as gold standard for TB. Overall, we have developed a simple and inexpensive assay for rapid detection and determination of rifampicin resistance of M.tb in sputum samples. This new method could facilitate therapy and prevent the spread of drug-resistant strains in low resource settings. Finally, we had optimized the conditions for an automated phenotypic assay to test in a short time susceptibility of pure cultures to different drugs used for TB treatment. The implementation of this standardized methodology will also contribute to the development of a rapid, easy and high sensitive method for HTS of new anti TB drugs.