Daniel Weiß1,2, Sascha Braun1,2, Stefan Monecke1,2,4, Olaf Piepenburg3, Oliver Nentwhich3, Ralf Ehricht1,2 (1Alere Technologies GmbH, Germany; 2InfectoGnostics e.V; 3TwistDx Ltd., Babraham, United Kingdom;4Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Germany)
Introduction
The worldwide emergence and rapid dissemination of multidrug resistant bacteria have reached alarming levels. This includes a rise in infections with enterobacteria that produce extended spectrum beta-lactamases (ESBL) and/or that are fluoroquinolone resistant. For that reason, carbapenems are commonly viewed as a last reliable option for a presumptive therapy, or as last line of therapy for infections with otherwise resistant bacteria. However, the increasing use of carbapenems leads to a selective pressure favoring an acquisition of resistance toward these compounds. Recent reports have shown that carbapenemase-producing bacteria are rapidly spreading worldwide. Conventional methods for detection of carbapenemase-positive bacteria in native patient samples are time consuming. Rapid molecular identification of carbapenemase genes in such samples could be helpful for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact on the selection of an appropriate initial treatment and could therefore be of great benefit for ICU patients. Isothermal molecular identification methods could become a suitable tool for this task, especially in point-of-care settings, because these methods combine high speed with excellent sensitivity and specificity. Furthermore, they can be used in any environment without a need for trained technicians or sophisticated laboratories. For this purpose we developed a prototype assay to analyze samples of different reference strains containing different carbapenemase genes using RPA (Recombinase Polymerase Amplification) as amplification method and microarrays for sequence specific detection of the amplicons.
Figure 1: Worldwide dissemination of carbapenemases in Acinetobacter spp (Bae, Jeong, and Lee; Korean J Clin Microbiol. 2012 Mar;15(1):1-8. Korean)
Materials and Methods RPA (Recombinase Polymerase Amplification) RPA is a rapid isothermal amplification method, which requires less than ten minutes at a temperature ranging between 37°C and 42°C for a complete amplification run. Therefore, RPA does not rely on sophisticated, and expensive, instruments. The RPA reaction uses three core proteins. The first enzyme is a recombinase (T4 usvX) that binds to primers and forms filaments which are able to recombine to homologous DNA. The second enzyme, a single-stranded DNA binding protein (T4 gp32), prevents dissociation of the primers by binding to the displaced DNA strand. The third enzyme is a strand-displacing polymerase (Bsu), which opens the DNA double helix and amplifies the DNA starting from the 3’ end of the bound primers. For establishing a RPA based model assay we selected three carbapenemase genes (blaKPC, blaVIM, blaOXA-48) for single and multiplex reactions. In a first step, the bacterial DNA was exponentially amplified using RPA. The reaction was initiated using magnesium acetate and it was performed at 42°C for twenty minutes. Labeling was achieved by the use of 5´-biotin-coupled reverse primers. Afterwards, labeled amplicons were hybridized without further purification and specifically detected with an oligonucleotide microarray (Braun et al. 2014). The sensitivity of the entire assay was determined using dilution series of reference DNA samples from different strains in a range of 101 to 107 genomic equivalents.
DNA-Labeling by RPA with BiotinLabeled Reverse Primers
Hybridization of the Labeled DNA
Analysis of the Microarray Signals
Figure 2: Scheme of the RPA cycle (TwistDx.co.uk)
Results In a first set of experiments with this assay, various low copy number DNA preparations of different reference strains containing the carbapenemase genes blaKPC, blaVIM and/or blaOXA-48 alone and in combinations were analyzed. First analyses of all target genes showed a good sensitivity in single (table 1) and multiplex reactions (table 2). Specific signals of all target genes were detected on the microarray with templates comprising as few as 101 genomic equivalents in the single reactions and as few as 102 genomic equivalents in multiplex reactions. Compared to blaVIM-1 and blaVIM-2, reactions for blaOXA-48 and blaKPC showed a slightly better sensitivity. The good sensitivity of the multiplex RPA reactions for each target gene suggests that the primers did not interfere with each other. strain
target
template DNA [ge] 107 106 105 104 103 102 101 100 negative control Carb146 OXA-48 hp_oxa_613 0.87 0.86 0.86 0.86 0.87 0.87 0.87 0.00 0.00 Carb64 KPC hp_kpc4_611 0.87 0.87 0.86 0.86 0.87 0.87 0.36 0.00 0.00 DSM24600 VIM-1 hp_vim-264 0.85 0.85 0.86 0.86 0.85 0.78 0.00 0.01 0.00 DSM24599 VIM-2 hp_vim-264 0.86 0.86 0.86 0.85 0.84 0.45 0.07 0.00 0.00 Table 1: Summary of microarray signals of the single RPA primer amplification of each target gene to test the sensitivity for different amounts of genomic equivalents (ge) of template DNA positive signal
probe name
ambiguous signal
negative signal
strain
target
probe name
template DNA [ge] 107 106 105 104 103 Carb146 oxa48 hp_oxa_613 0.86 0.86 0.85 0.86 0.86 Nord1 oxa48 hp_oxa_613 0.87 0.86 0.85 0.86 0.86 Carb22 KPC-2 hp_kpc4_611 0.86 0.86 0.86 0.86 0.84 Carb2 KPC-3 hp_kpc4_611 0.86 0.86 0.87 0.86 0.34 DSM24600 VIM-1 hp_vim-264 0.86 0.86 0.86 0.85 0.29 DSM24599 VIM-2 hp_vim-264 0.85 0.85 0.86 0.85 0.00 Table 2: Summary of multiplex RPA microarray signals to test the sensitivity for each genomic equivalents (ge) of template DNA
102 0.85 0.00 0.02 0.00 0.16 0.18 target
101 100 negative control 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 gene with different amounts of
Discussion Our first results showed that in general the multiplex amplification using RPA followed by hybridization on microarrays showed a good sensitivity and specificity. The sensitivity and speed of the isothermal amplification strategy allows a future development of a point-of-care device for the detection of clinically important carbapenemase genes in native patient samples.
Author: Daniel Weiß, R&D, Alere Technologies GmbH, Jena, Germany. e-Mail:
[email protected] Download: http://alere-technologies.com/fileadmin/ Media/Paper/Poster/CARB_RPA_Poster_ DGHM2015.pdf
