ABSTRACT
The most widely discussed antibiotic-resistant tuberculosis strains ("W" and "B0/W148", "CAO") belong to L2/Beijing Lineage and are characterized by IS6110 insertion sequences at the NTF locus. We present a high-throughput, microbead-based method, called NTF-RINT for detection of IS in NTF and Rifampicin and Isoniazid Typing. This method provides tuberculosis diagnostic confirmation, screens for the so-called modern L2/Beijing sublineage and detects mutations involved in resistance to Rifampicin (RIF) and Isoniazid (INH).
Subject(s)
Bacteriological Techniques , DNA Transposable Elements , DNA, Bacterial/genetics , Drug Resistance, Multiple, Bacterial/genetics , High-Throughput Nucleotide Sequencing , Mutation , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/diagnosis , DNA Mutational Analysis , Genotype , Humans , Kazakhstan/epidemiology , Molecular Epidemiology , Mycobacterium tuberculosis/pathogenicity , New York City/epidemiology , Phenotype , Polymerase Chain Reaction , Population Surveillance , Predictive Value of Tests , Reproducibility of Results , Tuberculosis, Multidrug-Resistant/epidemiology , Tuberculosis, Multidrug-Resistant/genetics , VirulenceABSTRACT
Tuberculosis remains the world's leading cause of death from an infectious agent, and is a serious health problem in Papua New Guinea (PNG) with an estimated 36,000 new cases each year. This study describes the genetic diversity of Mycobacterium tuberculosis among tuberculosis patients in the Balimo/Bamu region in the Middle Fly District of Western Province in PNG, and investigates rifampicin resistance-associated mutations. Archived Ziehl-Neelsen-stained sputum smears were used to conduct microbead-based spoligotyping and assess genotypic resistance. Among the 162 samples included, 80 (49.4%) generated spoligotyping patterns (n = 23), belonging predominantly to the L2 Lineage (44%) and the L4 Lineage (30%). This is consistent with what has been found in other PNG regions geographically distant from Middle Fly District of Western Province, but is different from neighbouring South-East Asian countries. Rifampicin resistance was identified in 7.8% of the successfully sequenced samples, with all resistant samples belonging to the L2/Beijing Lineage. A high prevalence of mixed L2/L4 profiles was suggestive of polyclonal infection in the region, although this would need to be confirmed. The method described here could be a game-changer in resource-limited countries where large numbers of archived smear slides could be used for retrospective (and prospective) studies of M. tuberculosis genetic epidemiology.
Subject(s)
DNA, Bacterial/genetics , Genetic Variation , Genotype , Mycobacterium tuberculosis/genetics , Sputum/microbiology , Tuberculosis , Adult , Female , Humans , Male , Molecular Epidemiology , Papua New Guinea/epidemiology , Prospective Studies , Tuberculosis/epidemiology , Tuberculosis/geneticsABSTRACT
Several diagnostic tests are being developed to detect drug resistance in tuberculosis. In line with previous developments detecting rifampicin and isoniazid resistance using microbead-based systems (spoligoriftyping and TB-SPRINT), we present here an assay called TB-EFI detecting mutations involved in resistance to ethambutol, fluoroquinolones and the three classical injectable drugs (kanamycin, amikacin and capreomycin) in Mycobacterium tuberculosis. The proposed test includes both wild-type and mutant probes for each targeted locus. Basic analysis can be performed manually. An upgraded interpretation is made available in Excel 2016®. Using a reference set of 61 DNA extracts, we show that TB-EFI provides perfect concordance with pyrosequencing. Concordance between genotypic resistance and phenotypic DST was relatively good (72 to 98% concordance), with lower efficiency for fluoroquinolones and ethambutol due to some untargeted mutations. When compared to phenotypical resistance, performances were similar to those obtained with Hain MTBDRsl assay, possibly thanks to the use of automatized processing of data although some mutations involved in fluoroquinolone resistance could not be included. When applied on three uncharacterized sets, phenotype could be predicted for 51% to 98% depending on the setting and the drug investigated, detecting one extensively drug-resistant isolate in each of a Pakistan and a Brazilian set of 91 samples, and 9 XDR among 43 multi-resistant Kazakhstan samples. By allowing high-throughput detection of second-line drugs resistance and of resistance to ethambutol that is often combined to second-line treatments, TB-EFI is a cost-effective assay for large-scale worldwide surveillance of resistant tuberculosis and XDR-TB control.
