Targeted sequencing from cerebrospinal fluid for rapid identification of drug-resistant tuberculous meningitis.

Mortality from tuberculous meningitis (TBM) remains around 30%, with most deaths occurring within 2 months of starting treatment. Mortality from drug-resistant strains is higher still, making early detection of drug resistance (DR) essential. Targeted next-generation sequencing (tNGS) produces high read depths, allowing the detection of DR-associated alleles with low frequencies. We applied Deeplex Myc-TB-a tNGS assay-to cerebrospinal fluid (CSF) samples from 72 adults with microbiologically confirmed TBM and compared its genomic drug susceptibility predictions to a composite reference standard of phenotypic susceptibility testing (pDST) and whole genome sequencing, as well as to clinical outcomes. Deeplex detected Mycobacterium tuberculosis complex DNA in 24/72 (33.3%) CSF samples and generated full DR reports for 22/24 (91.7%). The read depth generated by Deeplex correlated with semi-quantitative results from MTB/RIF Xpert. Alleles with <20% frequency were seen at canonical loci associated with first-line DR. Disregarding these low-frequency alleles, Deeplex had 100% concordance with the composite reference standard for all drugs except pyrazinamide and streptomycin. Three patients had positive CSF cultures after 30 days of treatment; reference tests and Deeplex identified isoniazid resistance in two, and Deeplex alone identified low-frequency rifampin resistance alleles in one. Five patients died, of whom one had pDST-identified pyrazinamide resistance. tNGS on CSF can rapidly and accurately detect drug-resistant TBM, but its application is limited to those with higher bacterial loads. In those with lower bacterial burdens, alternative approaches need to be developed for both diagnosis and resistance detection.

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

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. There is limited understanding of antibiotic tolerance in clinical isolates of M. tuberculosis. Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. In-vitro rifampicin survival fractions determined by minimum duration of killing assay in isoniazid susceptible (n=119) and resistant (n=84) M. tuberculosis isolates. Rifampicin tolerance was correlated with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. 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 surviving fraction 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 IS and IR 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 identifies a range of rifampicin tolerance and 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. It is made available under a CC-BY 4.0 International license.

FLASH-TB: an Application of Next-Generation CRISPR to Detect Drug Resistant Tuberculosis from Direct Sputum.

Offering patients with tuberculosis (TB) an optimal and timely treatment regimen depends on the rapid detection of Mycobacterium tuberculosis (Mtb) drug resistance from clinical samples. Finding Low Abundance Sequences by Hybridization (FLASH) is a technique that harnesses the efficiency, specificity, and flexibility of the Cas9 enzyme to enrich targeted sequences. Here, we used FLASH to amplify 52 candidate genes probably associated with resistance to first- and second-line drugs in the Mtb reference strain (H37Rv), then detect drug resistance mutations in cultured Mtb isolates, and in sputum samples. 92% of H37Rv reads mapped to Mtb targets, with 97.8% of target regions covered at a depth ≥ 10X. Among cultured isolates, FLASH-TB detected the same 17 drug resistance mutations as whole genome sequencing (WGS) did, but with much greater depth. Among the 16 sputum samples, FLASH-TB increased recovery of Mtb DNA compared with WGS (from 1.4% [IQR 0.5-7.5] to 33% [IQR 4.6-66.3]) and average depth reads of targets (from 6.3 [IQR 3.8-10.5] to 1991 [IQR 254.4-3623.7]). FLASH-TB identified Mtb complex in all 16 samples based on IS1081 and IS6110 copies. Drug resistance predictions for 15/16 (93.7%) clinical samples were highly concordant with phenotypic DST for isoniazid, rifampicin, amikacin, and kanamycin [15/15 (100%)], ethambutol [12/15 (80%)] and moxifloxacin [14/15 (93.3%)]. These results highlighted the potential of FLASH-TB for detecting Mtb drug resistance from sputum samples.

Most-Probable-Number-Based Minimum Duration of Killing Assay for Determining the Spectrum of Rifampicin Susceptibility in Clinical Mycobacterium tuberculosis Isolates.

Accurate antibiotic susceptibility testing is essential for successful tuberculosis treatment. Recent studies have highlighted the limitations of MIC-based phenotypic susceptibility methods in detecting other aspects of antibiotic susceptibilities in bacteria. Duration and peak of antibiotic exposure, at or above the MIC required for killing the bacterial population, has emerged as another important factor for determining antibiotic susceptibility. This is broadly defined as antibiotic tolerance. Antibiotic tolerance can further facilitate the emergence of antibiotic resistance. Currently, there are limited methods to quantify antibiotic tolerance among clinical M. tuberculosis isolates. In this study, we develop a most-probable-number (MPN)-based minimum duration of killing (MDK) assay to quantify the spectrum of M. tuberculosis rifampicin susceptibility within subpopulations based on the duration of rifampicin exposure required for killing the bacterial population. MDK90-99 and MDK99.99 were defined as the minimum duration of antibiotic exposure at or above the MIC required for killing 90 to 99% and 99.99% of the initial (pretreatment) bacterial population, respectively. Results from the rifampicin MDK assay applied to 28 laboratory and clinical M. tuberculosis isolates showed that there is variation in rifampicin susceptibility among isolates. The rifampicin MDK99/99.99 time for isolates varied from less than 2 to 10 days. MDK was correlated with larger subpopulations of M. tuberculosis from clinical isolates that were rifampicin tolerant. Our study demonstrates the utility of MDK assays to measure the variation in antibiotic tolerance among clinical M. tuberculosis isolates and further expands clinically important aspects of antibiotic susceptibility testing.