Fluoromycobacteriophages Can Detect Viable Mycobacterium tuberculosis and Determine Phenotypic Rifampicin Resistance in 3-5 Days From Sputum Collection.

Liliana Rondón,Mariana Piuri,Sergio Nemirovsky,Cecilia Latini,Ezequiel J Sosa,Estefanía Urdániz,Darío F Do Porto,Susana Poggi,Mario Matteo,Graham F Hatfull,Adrian G Turjanski,Florencia Payaslian

doi:10.3389/fmicb.2018.01471

Abstract

The World Health Organization (WHO) estimates that 40% of tuberculosis (TB) cases are not diagnosed and treated correctly. Even though there are several diagnostic tests available in the market, rapid, easy, inexpensive detection, and drug susceptibility testing (DST) of Mycobacterium tuberculosis is still of critical importance specially in low and middle-income countries with high incidence of the disease. In this work, we have developed a microscopy-based methodology using the reporter mycobacteriophage mCherrybombϕ for detection of Mycobacterium spp. and phenotypic determination of rifampicin resistance within just days from sputum sample collection. Fluoromycobacteriophage 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. We have also set up conditions for discrimination between M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains by addition of p-nitrobenzoic acid (PNB) during the assay. Using clinical isolates of pre-XDR and XDR-TB strains from this study, 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. The many attributes of mCherrybombΦ suggests this could be a useful component of clinical microbiological laboratories for TB diagnosis and since only viable cells are detected this could be a useful tool for monitoring patient response to treatment.

Highlights

There are substantial advances in diagnosis and treatment of tuberculosis (TB), but this disease remains a significant global health threat (Sulis et al, 2016)
Using fluorescence microscopy for detection, we evaluated the use of p-nitrobenzoic acid (PNB) for selective inhibition of members of the M. tuberculosis complex (MTBC) (Sharma et al, 2010; Shakoor et al, 2010) to distinguish MTBC from atypical or non-tuberculous mycobacteria (NTM)
We evaluated the performance of mCherrybombφ to detect the presence of Mycobacterium spp. and determination of RIF resistance in the remnant of 283 sputum samples from presumptive TB patients, treatment-naive, previously treated for TB or under antibiotic treatment for TB

Summary

INTRODUCTION

There are substantial advances in diagnosis and treatment of tuberculosis (TB), but this disease remains a significant global health threat (Sulis et al, 2016). The current fluoromycobacteriophages are derived from phage TM4, a broad host range phage that can infect several Mycobacterial species including, M. tuberculosis, M. bovis, M. smegmatis, and atypical Mycobacteria (Ford et al, 1998) They can rapidly and reveal the metabolic state of M. tuberculosis and report its response to antibiotics (Piuri et al, 2009; Rondón et al, 2011), and have potential advantages over existing phage-based TB diagnostic methods. Whole genome sequencing (WGS) of XDRTB strains identified in this study allowed comparison between phenotypic and genotypic results, and underscore the importance of rapid methods that evaluate phenotypic resistance in clinical isolates

MATERIALS AND METHODS

RESULTS

C Mutation

DISCUSSION