Implications of direct amplification for measuring antimicrobial resistance using point-of-care devices

Abstract

Antimicrobial resistance (AMR) is recognized as a global threat to human health. Rapid detection and characterization of AMR is a critical component of most antibiotic stewardship programs. Methods based on amplification of nucleic acids for detection of AMR are generally faster than culture-based approaches but they still require several hours to more than a day due to the need for transporting the sample to a centralized laboratory, processing of sample, and sometimes DNA purification and concentration. Nucleic acids-based point-of-care (POC) devices are capable of rapidly diagnosing antibiotic-resistant infections which may help in making timely and correct treatment decisions. However, for most POC platforms, sample processing for nucleic acids extraction and purification is also generally required prior to amplification. Direct amplification, an emerging possibility for a number of polymerases, has the potential to eliminate these steps without significantly impacting diagnostic performance. This review summarizes direct amplification methods and their implication for rapid measurement of AMR. Future research directions that may further strengthen the possibility of integrating direct amplification methods with POC devices are also summarized.

Microfluidic centrifugation assisted precipitation based DNA quantification

“…Detection of amplified DNA is often based on measurement of turbidity, 12 fluorescence after staining with a detection dye 13 or absorbance. 14 Commercially available instruments for DNA quantification can be broadly divided into three categories: instruments that measure turbidity (e.g.…”

On-filter direct amplification of Legionella pneumophila for rapid assessment of its abundance and viability

“…Amplification without DNA extraction and purification, referred to as direct amplification has been shown for several organisms and polymerases and reviewed recently (Williams et al, 2017) but to our knowledge, its performance for L. pneumophila was not yet validated. It is well known that at low cell abundance, recovery losses impact the lower detection limit.…”

Isothermal assay targeting class 1 integrase gene for environmental surveillance of antibiotic resistance markers

“…While the measurements in this study were done with DNA extracted from environmental samples, one key advantage of isothermal amplification is the potential for direct amplification with minimal DNA extraction and purification (Williams et al, 2017). Ultimately, genetic testing outside of a specialized laboratory should allow for sample-in-results-out with automation or minimal sample preparation even when quantitative abundance data is desired.…”

“…However, more rapid and simplified measurement techniques may increase broader monitoring/surveillance of intI1 as a marker of ARG abundance or anthropogenic activity. Isothermal amplification, such as loop mediated isothermal amplification (LAMP) with Bst polymerase provides a more simplistic approach because it allows amplification with minimal sample processing requirements (Williams et al, 2017) and is less influenced by PCR inhibition (Koloren et al, 2011; Stedtfeld et al, 2016a). Furthermore, constant temperature (Notomi et al, 2000) and high amplicon yields (Mori et al, 2001), allows quantification of abundance with relatively simpler devices (e.g.…”