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Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study.

S Vijay(1,2,3) N L H Bao(1) D N Vinh(1) L T H Nhat(1) D D A Thu(1) N L Quang(1) L P T Trieu(1) H N Nhung(1) V T N Ha(1) P V K Thai(6) D T M Ha(6) N H Lan(6) M Caws(4) G E Thwaites(1,2) B Javid(5) N T T Thuong(1,2)

1:Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam; 2:Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom; 3:Theoretical Microbial Ecology, Friedrich Schiller University, Jena, Germany; 4:Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom; 5:Division of Experimental Medicine, University of California, San Francisco, California, USA; 6:Pham Ngoc Thach Hospital, Ho Chi Minh City, Vietnam

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. Therefore, we investigated the rifampicin tolerance of M. tuberculosis, with or without isoniazid-resistance. Rifampicin survival fraction was determined by minimum duration of killing assay in isoniazid susceptible (n=119) and resistant (n=84) isolates. The longitudinal isoniazid-resistant isolates were analyzed for rifampicin tolerance based on collection time from patients and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis survival by 90% (minimum duration of killing-MDK90) increased from 1·23 (95%CI 1·11; 1·37) and 1·31 (95%CI 1·14; 1·48) to 2·55 (95%CI 2·04; 2·97) and 1·98 (95%CI 1·69; 2·56) days, for isoniazid-susceptible and resistant respectively, during 15 to 60 days of incubation respectively. Increase in MDK90 time indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log10-fold survival fraction enabled classification of tolerance as low, medium or high and revealed isoniazid-resistance association with increased tolerance with faster growth (OR=2·68 for low vs. medium, OR=4·42 for low vs. high, P-trend=0·0003). The high tolerance in longitudinal isoniazid-resistant isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Our study reveals that isoniazid resistance is associated with higher tolerance with growth fitness. Furthermore, rifampicin treatment may select isoniazid-resistant isolate microvariants with higher rifampicin tolerance, with survival potential similar to multi-drug resistant isolates. These findings suggest that isoniazid-resistant tuberculosis needs to be evaluated for rifampicin tolerance or needs further improvement in treatment regimen.

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