ABSTRACT
Tuberculous meningitis (TBM) is a severe form of tuberculosis with a high mortality rate. The factors associated with TBM pathogenesis are still unclear. Using comparative whole-genome sequence analysis we compared Mycobacterium tuberculosis (Mtb) isolates from cerebrospinal fluid of TBM cases (n = 73) with those from sputum of pulmonary tuberculosis (PulTB) patients (n = 220) from Thailand. The aim of this study was to seek genetic variants of Mtb associated with TBM. Regardless of Mtb lineage, we found 242 variants that were common to all TBM isolates. Among these variants, 28 were missense SNPs occurring mainly in the pks genes (involving polyketide synthesis) and the PE/PPE gene. Six lineage-independent SNPs were commonly found in TBM isolates, two of which were missense SNPs in Rv0532 (PE_PGRS6). Structural variant analysis revealed that PulTB isolates had 14 genomic regions containing 2-3-fold greater read depth, indicating higher copy number variants and half of these genes belonged to the PE/PPE gene family. Phylogenetic analysis revealed only two small clusters of TBM clonal isolates without support from epidemiological data. This study reported genetic variants of Mtb commonly found in TBM patients compared to PulTB patients. Variants associated with TBM disease warrant further investigation.
Subject(s)
Bacterial Proteins/genetics , Genotype , Mycobacterium tuberculosis/genetics , Tuberculosis, Meningeal/microbiology , Tuberculosis, Pulmonary/microbiology , Adult , Female , Humans , Male , Middle Aged , Phylogeny , Polyketide Synthases/genetics , Polymorphism, Single Nucleotide , Tuberculosis, Meningeal/genetics , Tuberculosis, Pulmonary/genetics , Whole Genome Sequencing , Young AdultABSTRACT
New fluoroquinolones (FQs) have been shown to be more active against drug-resistant Mycobacterium tuberculosis strains than early FQs, such as ofloxacin. Sitafloxacin (STFX) is a new fluoroquinolone with in vitro activity against a broad range of bacteria, including M. tuberculosis This study aimed to determine the in vitro activity of STFX against all groups of drug-resistant strains, including multidrug-resistant M. tuberculosis (MDR M. tuberculosis), MDR M. tuberculosis with quinolone resistance (pre-XDR), and extensively drug-resistant (XDR) strains. A total of 374 drug-resistant M. tuberculosis strains were tested for drug susceptibility by the conventional proportion method, and 95 strains were randomly submitted for MIC determination using the microplate alamarBlue assay (MABA). The results revealed that all the drug-resistant strains were susceptible to STFX at a critical concentration of 2 µg/ml. Determination of the MIC90s of the strains showed different MIC levels; MDR M. tuberculosis strains had a MIC90 of 0.0625 µg/ml, whereas pre-XDR and XDR M. tuberculosis strains had identical MIC90s of 0.5 µg/ml. Common mutations within the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB did not confer resistance to STFX, except that double mutations of GyrA at Ala90Val and Asp94Ala were found in strains with a MIC of 1.0 µg/ml. The results indicated that STFX had potent in vitro activity against all the groups of drug-resistant M. tuberculosis strains and should be considered a new repurposed drug for treatment of multidrug-resistant and extensively drug-resistant TB.
Subject(s)
Antitubercular Agents/pharmacology , Extensively Drug-Resistant Tuberculosis/microbiology , Fluoroquinolones/pharmacology , Mycobacterium tuberculosis/drug effects , Tuberculosis, Multidrug-Resistant/microbiology , DNA Gyrase/genetics , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , ThailandABSTRACT
The early detection of drug-resistant tuberculosis (TB) strains is of utmost importance for patient management and effective TB control programs. This study aimed to demonstrate the performance of direct drug susceptibility testing (DST) performed in our laboratory in the past 11 years. The direct DST was performed on Middlebrook 7H10 medium using isoniazid (INH) and rifampicin (RIF), and the results were compared with those obtained from indirect DST (gold standard). The direct DST was performed with 15,598 smear-positive sputum samples, of which 11,284 (72%) yielded reportable results. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated and revealed 89%, 99%, 95%, and 99%, respectively, for RIF and 90%, 98%, 93%, and 97%, respectively, for INH. Direct DST results could be reported within 1 month after sample processing. This method was also shown to be suitable for use in resource-limited countries, particularly in settings with high numbers of TB cases.
