MPT64 secretion as a viability-associated immunoassay readout for rapid phenotypic drug susceptibility classification of Mycobacterium tuberculosis : a proof-of-concept study
Yoshiro Murase, Rikiya Takeuchi, Yoshiharu Ohtaki, Makiko Hosoya, Akio Aono, Kinuyo Chikamatsu, Yuta Morishige, Yuriko Igarashi, Joseph Edwin L Bascuña, Gel Anne Marie V Atienza, Angeline Grace A Sedusta, Catherine Ann P L Sacopon, Ramón P Basilio, Midori Kato-Maeda, Satoshi MitaraiAbstract
Objectives
To evaluate MPT64 secretion as a viability-associated readout for rapid phenotypic drug susceptibility testing (pDST) of Mycobacterium tuberculosis.
Methods
We evaluated MPT64-pDST in 130 M. tuberculosis strains: clinical isolates, WHO quality assessment strains and in vitro-selected resistant mutants (for linezolid only). We assessed the diagnostic performance of MPT64-pDST for determining susceptibility to rifampicin, isoniazid and moxifloxacin. As a secondary objective, we explored its performance for linezolid and bedaquiline. MPT64 was quantified by chemiluminescent enzyme immunoassay, and susceptibility was inferred from Log2 fold change relative to drug-free controls. Reference standards were minimum inhibitory concentration testing for rifampicin, isoniazid, linezolid and bedaquiline and MGIT-based DST for moxifloxacin. Performance was evaluated using receiver operating characteristic analysis, full-dataset Youden-optimal candidate cutoffs and 2000-iteration stratified bootstrap out-of-bag (OOB) internal validation.
Results
Using drug-specific full-dataset Youden-optimal candidate cutoffs, MPT64-pDST showed high categorical agreement with reference DST, with no false-resistant classifications for rifampicin, isoniazid or moxifloxacin. Resistance-detection sensitivity was 100% for rifampicin, 98% for isoniazid and 90% for moxifloxacin, and out-of-bag bootstrap estimates were comparable to the full-dataset estimates. Exploratory analyses for linezolid demonstrated separation between susceptible and resistant mutants. The single bedaquiline-resistant isolate was clearly distinguishable from 39 bedaquiline-susceptible isolates.
Conclusions
These findings provide proof of concept that the immunoassay-based MPT64-pDST can enable rapid phenotypic susceptibility classification for rifampicin, isoniazid and moxifloxacin. Further validation in larger isolate collections, including isolates with borderline phenotypes, is warranted.