Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS

Judith Schlanderer; Harald Hoffmann; Jan Lüddecke; Andrey Golubov; Wolfgang Grasse; Elisabeth V. Kindler; Thomas A. Kohl; Matthias Merker; Christoph Metzger; Vanessa Mohr; Stefan Niemann; Claudia Pilloni; Sara Plesnik; Bijendra Raya; Bhawana Shresta; Christian Utpatel; Roland Zengerle; Markus Beutler; Nils Paust

doi:10.1039/D3LC00783A

Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS†

Judith Schlanderer,

^a Harald Hoffmann,^b Jan Lüddecke,^ac Andrey Golubov,^d Wolfgang Grasse,^e Elisabeth V. Kindler,^e Thomas A. Kohl,

^f Matthias Merker,^f Christoph Metzger,^e Vanessa Mohr,^f Stefan Niemann,^f Claudia Pilloni,^d Sara Plesnik,^d Bijendra Raya,^g Bhawana Shresta,^g Christian Utpatel,^f Roland Zengerle,^ac Markus Beutler^d and Nils Paust

*^ac

Author affiliations

* Corresponding authors

^a Hahn-Schickard, 79110 Freiburg, Germany
E-mail: Nils.Paust@Hahn-Schickard.de
Tel: +49 761 203 73245

^b SYNLAB Gauting SYNLAB Human Genetics Munich, 82131 Gauting, Germany

^c Laboratory for MEMS Applications, IMTEK – Department of Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany

^d WHO supranational Tuberculosis Reference Laboratory, IML red, 82131 Gauting, Germany

^e gerbion GmbH & Co KG, 70806 Kornwestheim, Germany

^f Molecular and Experimental Mycobacteriology, Forschungszentrum Borstel, 23845 Borstel, Germany

^g German Nepal Tuberculosis Project (GENETUP), Nepal Anti-Tuberculosis Association (NATA), Kalimati, Nepal

Abstract

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml⁻¹, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

This article is part of the themed collection: Andreas Manz – Pioneer, Mentor, Friend

Lab on a Chip

Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS†

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Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS

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