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

LILIANA RONDÓN; ESTEFANÍA URDÁNIZ; CECILIA LATINI; FLORENCIA PAYASLIAN; MARIO MATTEO; EZEQUIEL J SOSA; DARÍO FERNÁNDEZ DO PORTO; ADRIÁN G TURJANSKI; SERGIO NEMIROVSKY; GRAHAM F HATFULL; SUSANA POGGI; MARIANA PIURI

Congreso; XXIV Congreso Latinoamericano de Microbiología; 2018

Sociedad Argentina de Microbiología

Despite substantial advances in diagnosis and treatment of tuberculosis (TB), this disease remains a significant globalhealth threat. The WHO estimates that 40% of tuberculosis cases go undiagnosed and consequently not treated. ZiehlNeelsen staining of Mycobacterium spp. in sputum, with subsequent culture is often the method of choice. Culturemethodology is laborious and takes 3-6 weeks to report the presence of viable mycobacteria in the sample and afew 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 constructionof a new fluoromycobacteriophage, mCherrybombΦ, which express mCherrybomb gene in mycobacteria. This phage isthermosensitive and at 37oC does not lyse cells allowing rapid detection of fluorescent mycobacteria by fluorescencemicroscopy, fluorimetry or flow cytometry. Also, it can rapidly and easily reveal the metabolic state of M. tuberculosisand consequently report its response to antibiotics. Furthermore, fluorescence microscopy is available in manyclinical laboratories, and fixation of cells with paraformaldehyde after infection reduces biohazard concerns. Here, wereport evaluation of the new mCherrybombΦ for detection of M. tuberculosis and rifampicin resistance in 283 sputumsamples from presumptive TB patients in Buenos Aires city using fluorescence microscopy. We also evaluated the useof p-nitrobenzoic acid for selective inhibition of members of the M. tuberculosis complex to distinguish from atypicalor non-tuberculous mycobacteria. When we evaluated the performance of mCherrybombΦ compared to the referencemethod the sensitivity and specificity reached values of 91.98 and 98.96%, respectively. Additionally, we establishedconditions 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 strainsfrom this study to identify the genetic determinants of antibiotic resistance. We found some discrepancies betweengenotypic and phenotypic results and for some antibiotic resistant clinical isolates we were not able to identify anyknown gene mutations that could explain the observed phenotype. These results underscore the importance of rapidmethods that evaluate phenotypic resistance in clinical M. tuberculosis isolates.