Reporter Fluoromycobacteriophages for Rapid TB - Diagnosis in Sputum Samples and Screening of New Antitubercular Drugs

CECILIA LATINI; MARIO MATTEO; SUSANA POGGI; LILIANA RONDÓN; FLORENCIA PAYASLIAN; SERGIO NEMIROVSKY; MARIANA PIURI; ESTEFANÍA URDÁNIZ; FELIPE JAWORSKI; GRAHAM HATFULL

Olympia

Congreso; 23rd Biennial Evergreen International Phage Meeting; 2019

Evergreen State College

WHO estimates that 40% of tuberculosis cases go undiagnosed and consequently are 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. Fluoromycobacteriophages are reporter mycobacteriophages containing a fluorescent gene. These phages are a simple and rapid mean of revealing the metabolic state of M. tuberculosis cells, and their response to antibiotics. We constructed a new Fluoromycobacteriophage, mCherrybombφ, with higher sensitivity and less time to detection of signal in M. tuberculosis. We developed a simple microscopy-based methodology for detection of viable Mycobacterium spp. and phenotypic determination of rifampicin resistance in 3-5 days from sputum sample collection compatible with regularly used protocols in clinical laboratories for TB diagnosis. Paraformaldehyde fixation after infection reduces biohazard risks. We tested mCherrybombφ for extended DST of clinical isolates of pre-XDR and XDR-TB strains and we compared the antibiotic resistance profile with those predicted by whole genome sequencing emphasizing the utility of a phenotypic test for M. tuberculosis.We had also set up the conditions for infection of pure cultures in a multiwell format in the presence of increasing concentrations of drugs monitoring the appearance of fluorescence as a function of time using a fluorimeter. 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 have also adapted this technique to a 384 well format for HTS of new anti TB drugs finding a good correlation between our results and standard methods used in the pharmaceutical industry. 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 adaption of this methodology also contributed to the development of a rapid, easy and high sensitive method for HTS of new anti-TB drugs.