Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

Abstract

e As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n ‫؍‬ 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n ‫؍‬ 162 for rpoB gene sequencing and n ‫؍‬ 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n ‫؍‬ 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.

Validation of a Microbead-Based Format for Spoligotyping of Legionella pneumophila

“…As the number of L. pneumophila genome sequences will increase and the spacer catalog is likely to concomitantly expand, more knowledge obtained through the use of data-mining softwares such as Sipina (http: //eric.univ-lyon2.fr/ϳricco/sipina.html) and/or Weka (http: //www.cs.waikato.ac.nz/ml/weka/) to select the most informative spacers will become feasible and necessary, similar to what has been done for the M. tuberculosis model (23). New spacers can be added to the previous set of 42 spacers in a new spoligotyping format that would extend LP-SPOL applicability to all ST1 isolates, all Paris pulsotype isolates, or eventually to other worldwide clinically predominant L. pneumophila sequence types harboring one CRISPR locus.…”

Simplified Microarray System for Simultaneously Detecting Rifampin, Isoniazid, Ethambutol, and Streptomycin Resistance Markers in Mycobacterium tuberculosis

“…However, the number of known genes and mutations conferring resistance to first-and second-line drugs greatly exceeds the multiplexing capacity of these platforms, which may limit their clinical efficacy in the treatment and control of multidrug-resistant or extensively drug-resistant TB. Planar and suspension microarrays are well suited to address the multiple-gene, multiple-mutation challenge of diagnosing drug-resistant TB (4)(5)(6)(7)(8)(9)(10)(11)(12), but clinical adoption of microarray technology is hampered by poor reproducibility (13)(14)(15), complex workflows, and/or extensive user subjectivity and involvement in image and data analysis (16). In order to translate microarrays into efficacious TB diagnostics at the point of need, it is therefore necessary to simplify user interaction with the technology while retaining the ability to detect multiple genes and multiple mutations in a timely manner.…”

Novel laboratory diagnostic tests for tuberculosis and their potential role in an integrated and tiered laboratory network

“…Newer molecular platforms such as the GeneDrive system by Epsitem, UK and BectoneDickinson, the TrueLab by Mobio, India, the Loopamp by Eiken, Japan can further simplify molecular testing for TB and drug resistant TB and can be the first point of care systems for further decentralization of testing to health care center level or to mobile diagnostic units similarly to advanced POC flow cytometry testing for HIV viral load. Other molecular platforms such as Multiplex Ligationdependent Probe Amplification or the recently developed TB-SPRINT assay enable the integration of molecular drug resistance testing and strain typing for molecular epidemiology that may well offer both individual and community benefit using a single test [13,14]. However, the instrumentation and laboratory requirements of these assays do not allow their decentralization to the lower levels of a tiered diagnostic network at present [13,14].…”

“…Other molecular platforms such as Multiplex Ligationdependent Probe Amplification or the recently developed TB-SPRINT assay enable the integration of molecular drug resistance testing and strain typing for molecular epidemiology that may well offer both individual and community benefit using a single test [13,14]. However, the instrumentation and laboratory requirements of these assays do not allow their decentralization to the lower levels of a tiered diagnostic network at present [13,14].…”