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

URDÁNIZ, ESTEFANÍA; SOSA, EZEQUIEL J.; NEMIROVSKY, SERGIO; PIURI, MARIANA; PAYASLIAN, FLORENCIA; DO PORTO, DARÍO F.; HATFULL, GRAHAM F.; RONDÓN, LILIANA; MATTEO, MARIO; TURJANSKI, ADRIAN G.; POGGI, SUSANA

Congreso; XXIV Congreso Latinoamericana de Microbiología; 2019

Despite substantial advances in diagnosis and treatment of tuberculosis (TB), this disease remains a significant global health threat. The WHO estimates that 40% of tuberculosis cases go undiagnosed and consequently not treated. Ziehl Neelsen staining of Mycobacterium spp. in sputum, with subsequent culture 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 -reporter phages containing a fluorescent reporter gene ? have potential for rapid diagnosis and DST of tuberculosis clinical isolates. Recently, we described the construction of a new fluoromycobacteriophage, mCherrybombΦ, which express mCherrybomb gene in mycobacteria. This phage is thermosensitive and at 37o C does not lyse cells allowing rapid detection of fluorescent mycobacteria by fluorescence microscopy, fluorimetry or flow cytometry. Also, it can rapidly and easily reveal the metabolic state of M. tuberculosis and consequently report its response to antibiotics. Furthermore, fluorescence microscopy is available in many clinical laboratories, and fixation of cells with paraformaldehyde after infection reduces biohazard concerns. Here, we report evaluation of the new mCherrybombΦ for detection of M. tuberculosis and rifampicin resistance in 283 sputum samples from presumptive TB patients in Buenos Aires city using fluorescence microscopy. We also evaluated the use of p-nitrobenzoic acid for selective inhibition of members of the M. tuberculosis complex to distinguish from atypical or non-tuberculous mycobacteria. When we evaluated the performance of mCherrybombΦ compared to the reference method the sensitivity and specificity reached values of 91.98 and 98.96%, respectively. Additionally, we established conditions for determination of susceptibility to other antibiotics that discriminate between multi-drug resistant TB (MDR-TB); extremely-drug resistant TB (XDR- TB), or pre-XDR. We did whole genome sequencing of XDR-TB strains from this study to identify the genetic determinants of antibiotic resistance. We found some discrepancies between genotypic and phenotypic results and for some antibiotic resistant clinical isolates we were not able to identify any known gene mutations that could explain the observed phenotype. These results underscore the importance of rapid methods that evaluate phenotypic resistance in clinical M. tuberculosis isolates. Our research has been funded by several international and local agencies including: NIH, CONICET, and ANPCyT.