Towards a Future of Rapid, Low-Cost, Multiplexed Detection of Antimicrobial Resistance Markers for Tuberculosis and Other Pathogens.

Abstract

By 2050, antimicrobial resistance (AMR)3 will put an estimated 10 million lives at risk each year (1). The discovery of new antibiotics has slowed in recent years, and the prospect of a return to the preantibiotic era, in which simple medical procedures become life-threatening, has triggered various efforts to improve antimicrobial stewardship to halt the development of further AMR. A key element of antimicrobial stewardship is the use of diagnostic procedures that inform antibiotic prescribing. This is typically achieved using phenotypic drug susceptibility testing by microbiological culture, the gold standard method for evaluating the drug susceptibility profile of a given clinical isolate. However, this is time-consuming, usually taking days. Molecular tests, in which nucleic acid sequences of a specific pathogen, strain, or drug resistance-conferring mutation are detected, are significantly faster, typically yielding results in hours. Nucleic acid detection tests are already used to guide antibiotic usage in laboratory settings for well-characterized resistance-associated mutations. For example, the Cepheid Xpert MTB/RIF nucleic acid amplification test detects the presence of genes specific to Mycobacterium tuberculosis, in addition to rifampicin resistance-conferring mutations in the rpoB gene (2). Such diagnostic tests can provide a rapid guide for clinicians when prescribing antibiotics. However, the multiplexing capabilities of multicolor fluorophore-based diagnostics are limited by the need for fluorophores with sufficient spectral resolution that can be used in a homogenous reaction. Base calling resolution also limits the use of molecular diagnostics for the detection of single-nucleotide polymorphisms (SNPs), which is required for the detection of AMR in some pathogens, such as M. tuberculosis. Next-generation sequencing technologies are not limited by multiplexing capabilities and base calling resolution and have been used to diagnose infections and identify AMR. The time requirements and cost of next-generation sequencing have fallen markedly in recent years, increasing their clinical utility. In …