Anti-‐Microbial Resistance

Transcription

Anti-‐Microbial Resistance Landscape Analysis of the State of AMR Testing Technologies Heinz Reiske, PhD Table of Contents Table of Contents ................................................................................................................................. 2 List of Abbreviations ............................................................................................................................ 3 Executive Summary .............................................................................................................................. 4 Introduction ......................................................................................................................................... 5 Rationale .................................................................................................................................................. 5 Methodology and conflicts of interest ..................................................................................................... 5 Anti-‐microbial resistance ......................................................................................................................... 5 Section 1: Commercially Available Technology Landscape .................................................................. 10 Molecular Systems ................................................................................................................................. 10 Performance Attributes of Molecular Systems .................................................................................................. 10 Commercially Available Technology Landscape .................................................................................. 26 Phenotypic-‐based systems ..................................................................................................................... 26 Chromogenic agars ............................................................................................................................................ 26 Immunoassays .................................................................................................................................................... 28 ID/AST Platforms ................................................................................................................................................ 28 Performance Attributes of Phenotypic Systems ................................................................................................ 30 Commercially Available Technology Landscape .................................................................................. 32 Differentiators ........................................................................................................................................ 32 Genotypic vs. Phenotypic: .................................................................................................................................. 32 Differentiators among Molecular Tests and Platforms ...................................................................................... 33 Laboratory Developed Tests (LDTs) -‐ Technology Landscape .............................................................. 34 Prototype Assay Landscape ................................................................................................................ 36 Emerging and Technology Landscape ................................................................................................. 42 Appendix A: Food Safety Testing ........................................................................................................ 47 References ......................................................................................................................................... 48 2 | P a g e List of Abbreviations AMR Antimicrobial Resistance MRSA Methicillin-‐resistant Staphylococcus aureus bla beta-‐lactamase NASBA Nucleic acid sequence based amplification CLSI NC Negative Control PBP Penicillin Binding Protein CPE Clinical and Laboratory Standards Institute Co-‐amplification at Lower Denaturation temperature PCR Carbapenemase Producing Enterobacteriaceae PC Positive Control CRE Carbapenem-‐resistant Enterobacteriaceae PCR Polymerase Chain Reaction DHFR Dihydrofolate Reductase PoC Point of Care DHPS Dihydropteroate Synthase QC Quality Control EIA Enzyme Immunoassay qPCR Quantitative PCR ESBL Extended-‐spectrum Beta Lactamase European Committee on Antimicrobial Susceptibility Testing RT-‐PCR Reverse Transcription PCR SARS Severe Acute Respiratory Syndrome FDA Food and Drug Administration VISA FIND Foundation for Innovative New Diagnostics VRE Vancomycin-‐intermediate Staphylococcus aureus Vancomycin-‐resistant Enterococcus FISH Fluorescent in situ Hybridization VRSA Vancomycin-‐resistant Staphylococcus aureus FRET Fluorescence Resonance Energy Transfer WHO World Health Organization GDH Glutamate Dehydrogenase XDR-‐TB Extensively drug-‐resistant Tuberculosis gDNA Genomic DNA GDP Gross domestic product HIV Human Immunodeficiency Virus HRM High-‐resolution melt IC Internal Control ID/AST Identification / Antimicrobial Susceptibility Testing IFU Instructions for Use inc Incubate LDT Laboratory Developed Test LIM Laboratory Information Management LIS Laboratory Information System LoD MALDI-‐
TOF-‐MS MAMA-‐PCR Limit of Detection Matrix-‐assisted laser desorption/ionization-‐Time of flight-‐Mass Spectroscopy Mismatch Amplification Mutation Assay PCR MDR Multidrug Resistant MDR-‐TB Multidrug-‐resistant Tuberculosis Mfr Manufacturer MIC Minimum Inhibitory Concentration Methicillin-‐resistant Coagulase-‐negative Staphylococcus COLD-‐PCR EUCAST MR-‐CoNS 3 | P a g e Executive Summary Background: The spread of AMR is a critical are of concern as it negates the ability of physicians to control the spread of infections that lead to patient morbidity and in some cases death. The economic impact of AMR is felt in the increased health costs due to complications of infections and increased efforts applied to infection control. AMR is spread in part due to the misuse of antibiotics in human patients and in livestock, which has provided the selective pressure for various antibiotic resistance mechanisms to proliferate among their host organisms. The ability to treat infections with β-‐lactams, vancomycin and methicillin are curtailed by the spread of resistance genes such as blaNDM-‐1, vanA and mecA respectively. Combating the spread of AMR will depend on better utilization of antimicrobials in patient treatment and animal husbandry as well as better infection control procedures and diagnostics. Rationale: The aim of this report was to review the technology landscape devoted to AMR diagnostics. This report details diagnostic systems and products detecting the high priority drug resistant organisms listed in table 1. Commercially available molecular systems, phenotypic systems are discussed as are offerings in the form of LDTs and prototype tests described in peer-‐
reviewed literature. Finally, future technology is described with a particular emphasis on how it can be deployed in resource-‐
limited environments. Findings: A majority of commercially available molecular tests focus on detecting C. difficile, MRSA and VRE (i.e. vancomycin resistance markers, not VRE per se). A few platforms also offer tests for CRE and MTB. The characteristics of molecular testing platforms range in time-‐to-‐result from less than 1 hour to 5-‐8 hours. Multiplex potential varies from 4 distinct targets (i.e. 4 distinct detection labels) to tens of targets per reaction. Workflow also varies for characteristics such as ease of use and system integration from complete non-‐integrated manual set-‐up to complete sample-‐to-‐result instruments where the end-‐user only adds sample to an assay consumable. Selection of a given platform will depend on the user needs in a given environment. LDT menu parallels that of commercially available molecular systems and LDTs are also facing the uncertainty associated with changes in governance in the US and EU. In light of this and the availability of commercially available platforms with desired testing menu, testing facilities may limit their LDT development activity in favor of bringing commercial systems on line. Phenotypic systems also fit the target organism pattern seen with their molecular counterparts, but these assays require far less skill and infrastructure to operate. In addition they tend to be rapid tests. While not as sensitive as molecular tests, phenotypic tests may have utility in patient screening with molecular or routine microbiology being used in reflex testing. Prototype assays are more diverse with respect to their target organism profiles. In addition the selected studies do show adaptability to low-‐resource environments with respect to equipment requirements and assay readouts. Future technology lists some interesting concepts for instrument-‐free amplification (112, 113) and seemingly “low tech” methods for higher multiplexing capabilities (Toehold RNA switches on filter paper). Application of these could lead to the development of molecular tests and the benefits they provide without the requirement for expensive laboratory equipment and infrastructure. There is a broad and varied landscape of technology available for AMR detection, which lends itself to deployment in a variety of environments and contexts. While gaps still remain in test menu (e.g. ESBL, MDR-‐TB, XDR-‐TB, Plasmodium, etc.) and platform simplification, it is foreseeable that advances over the next 5 – 10 years will fill these gaps and allow better use of diagnostics to combat AMR. 4 | P a g e Introduction Rationale The aim of this report is to identify and describe current and future technologies that are or can be utilized for antimicrobial resistance (AMR) testing. Methodology and conflicts of interest Heinz Reiske prepared this report with support from FIND in Geneva. The material was selected from a review of publically available information in scholarly publications, regulatory documents and commercial websites. This report is provided for information only and is not intended to endorse or recommend any commercial products or services. Anti-‐microbial resistance In 1967, the Surgeon General of the United States of America, William Stewart, was quoted, “The time has come to close the book on infectious diseases. We have basically wiped out infection in the United States.” Since that time, the world has witnessed the emergence and spread of numerous pathogens, including HIV, SARS, MRSA and MDR-‐TB. AMR is an area of global concern as it threatens the ability of clinicians to control a constantly expanding collection of bacteria, viruses, fungi and parasites that can cause morbidity and mortality in patients, and cause significant public health problems. AMR arises in part by the inappropriate and unnecessary use of antimicrobial drugs in patient treatment and animal husbandry, which provides the selective pressure enabling the proliferation of resistant organisms. For example, patients are often given antibiotics when suffering viral infections and livestock are given antibiotics to treat infections and promote growth. Misuse of antibiotics in animal husbandry creates a reservoir of AMR organisms that are transmitted to humans via direct contact with the colonized animals or food-‐borne (1 -‐ 4). Resistant organisms now render previously treatable conditions untreatable and lead to a host of economic problems such as increased health care costs and impacts to GDP. Absent effective antimicrobials, infection control also becomes more difficult with patients remaining infectious for longer, which makes more likely the spread of AMR organisms (2-‐4). Multidrug-‐resistant organisms or “superbugs” are a particular area of concern. These are organisms that have acquired resistance to multiple antimicrobials -‐ in some cases to multiple drugs of different classes. For instance, multidrug-‐resistant Enterobacteriaceae acquire genes through horizontal transfer of resistance genes encoding β-‐
lactamases. One such gene, blaNDM-‐1, confers resistance to all β-‐lactam agents except aztreonam (5). Vancomycin resistant MRSA strains have also been isolated (6). These strains (known as VRSA) are resistant to methicillin and other penicillin class drugs as well as the glycopeptide vancomycin, which is typically recommended for the treatment of MRSA infections. Combating AMR will require the concerted actions of scientists, clinicians, pharmacists and policy makers worldwide. What are the strategies that might be utilized to combat AMR? Clearly, the development of new anti-‐
microbial therapies is one, but their development may lag versus the ongoing evolution of drug resistance. It is therefore critical to utilize existing anti-‐microbials more precisely and appropriately. Public health recommendations, including those provided by WHO, counsel the use of anti-‐microbials only when they are truly needed and also using the right anti-‐microbials to treat a given illness. In addition the potential misuse of anti-‐microbials in clinical settings, abuse of anti-‐microbials in food 5 | P a g e production is well described (1). Avoiding antibiotic abuse in food production can potentially remove livestock as a reservoir of AMR organisms and prevent transmission of these to humans. Knowledge of AMR mechanisms are needed to provide the basis to select appropriate diagnostic tools and subsequent treatments. AMR can be both intrinsic and acquired. For example, gram negative bacteria are intrinsically resistant to vancomycin due to the fact that vancomycin is too large to penetrate the cell well of Gram negative bacteria (7). Additionally, the Gram positive VRE become resistant to vancomycin when certain Enterococcus species acquire a mobile element containing an operon encoding proteins that inhibit the ability of vancomycin to penetrate into the cytoplasm (8). These represent two different mechanisms of resistance to the same antibiotic. The former might be identified by a simple Gram stain, while the later can be detected by PCR amplification of resistance markers (e.g. vanA or vanB) present on the operon. Some Staphylococcus aureus strains exhibit reduced sensitivity (VISA) or outright resistance to vancomycin (VRSA), but do not harbor any of the vancomycin resistance genes (vanA, vanB, vanC, etc.). Studies characterizing these strains have shown that prominent phenotypic features relate to properties of the cell wall and that the level of resistance is variable among strains (9). Attempts to identify a genetic basis for resistance suggest that multiple mutations, changes in gene expression and multiple pathways are responsible (9). A genotypic-‐based VISA/VRSA diagnostic would be difficult to design and develop given that the molecular mechanisms are not understood. For this reason and the variability in resistance levels, a phenotypic-‐based test would be better suited for VISA/VRSA detection. Another basis for acquired resistance is through target altering mutations; for instance, Mycobacterium tuberculosis strains harboring point mutations in the rpoB gene that inhibit rifampicin binding to the mutant β subunit of RNA polymerase confers resistance to rifampicin (10 -‐12). Commercially available phenotypic (e.g. culture-‐based, immunoassay) and genotypic (molecular diagnostic) AMR detection technologies exist to detect AMR organisms. Additional and newer technologies are under development for AMR detection with the potential for commercialization in the next 3-‐5 years. Table 1 provides a list of AMR organisms commonly cited by various government and non-‐government organizations as being of significant concern. This report will highlight those AMR detection technologies aimed at detecting the organisms listed in Table 1. Table 1: AMR Organisms (2,3) Organism Drug-‐resistance Mechanism Carbapenem-‐resistant Enterobacteriaceae (CRE) (also known as Carbapenemase Producing Enterobacteriaceae (CPE)) Carbapenems Extended β-‐lactamase producing Enterobacteriaceae (ESBLs) Extended-‐spectrum (third generation) cephalosporins and monobactams Plasmid-‐borne and chromosomal genes encoding four classes of β-‐lactamase enzymes; each class contains multiple varieties β-‐lactamase enzymes cleave the β-‐lactam ring of carbapenems (13) Plasmid-‐borne and chromosomal genes encoding β-‐
lactamase enzymes; genes encoding these enzymes possess mutations that confer catalytic activity enabling the protein to hydrolyze third generation cephalosporins and monobactams (14, 15) 6 | P a g e Table 1: AMR Organisms (2,3) Organism Drug-‐resistance Escherichia coli Third-‐generation cephalosporins, fluoroquinolones Klebsiella pneumonia Third-‐generation cephalosporins and carbapenems Methicillin-‐resistant Staphylococcus aureus (MRSA) Methicillin Non-‐typhoidal Salmonella Fluoroquinolones Drug-‐resistant Salmonella typhi Ciprofloxacin Drug-‐resistant Shigella species Fluoroquinolones Streptococcus pneumonia Penicillin and erythromycin, other clinically relevant drugs Mechanism β-‐lactamases and PBPs provide resistance to cephalosporins (15) Fluoroquinolone resistance occurs due to mutations in the gyrA and gyrB genes (DNA gyrase) as well as the parC and parE genes (topoisomerase IV) that reduce the affinity of fluoroquinolone binding to DNA gyrase and topoisomerase IV respectively. Overexpression of the AcrAB (efflux pump) also contributes to resistance; resistance variable between strains and among different fluoroquinolones (16) Acquisition of β-‐lactamase enzymes that hydrolyze cephalosporins and carbapenems PBPs provide resistance to cephalosporins (15) Penicillin binding protein 2A encoded by mecA gene binds to and sequesters penicillin class antibiotics, including methicillin and oxacillin, preventing their action in inhibiting cell wall synthesis (17) Fluoroquinolone resistance occurs due to mutations in the gyrA and gyrB genes (DNA gyrase) as well as the parC and parE genes (topoisomerase IV) that reduce the affinity of fluoroquinolone binding to DNA gyrase and topoisomerase IV respectively. (16) Overexpression of the AcrAB (efflux pump) also contributes to resistance; resistance variable between strains and among different fluoroquinolones (16) Mutations in gyrase and topoisomerase IV genes (target modification) prevent ciprofloxacin binding (16) Fluoroquinolone resistance occurs due to mutations in the gyrA and gyrB genes (DNA gyrase) as well as the parC and parE genes (topoisomerase IV) that reduce the affinity of fluoroquinolone binding to DNA gyrase and topoisomerase IV respectively. Overexpression of the AcrAB (efflux pump) also contributes to resistance; resistance variable between strains and among different fluoroquinolones (16) Acquisition of PBP genes that bind to and sequester penicillins and prevent their ability to inhibit cell wall synthesis; multiple PBP genes are known, resistance levels are variable. (18, 19) Ribosomal methylation by rRNA adenine N-‐6-‐
methyltransferase, encoded by the ermB gene modifies the drug target preventing its action (19) 7 | P a g e Table 1: AMR Organisms (2,3) Organism Drug-‐resistance Drug-‐resistant Neisseria gonorrhoeae Third-‐generation cephalosporins, sulfonamides, penicillin, tetracycline Multidrug-‐resistant Acinetobacter Penicillins, cephalosporins, fluoroquinolones and aminoglycosides Drug-‐resistant Campylobacter Macrolide and/or fluoroquinolones Drug-‐resistant Candida Fluconazole Vancomycin-‐resistant Enterococcus (VRE) Vancomycin Multidrug-‐resistant Pseudomonas aeruginosa β-‐lactams, carbapenems, aminoglycosides, and fluoroquinolones Multidrug-‐resistant Mycobacterium tuberculosis (MDR-‐TB) Isoniazid and rifampicin Mechanism PBPs sequester and bind to penicillins β-‐lactamases hydrolyze both cephalosporins and penicillins Mutations in mtrR promoter, MtrR repressor gene involved in resistance to tetracycline, penicillin and macrolides Alterations to ribosomal protein S10 confers tetracycline resistance Alterations to dihydropteroate synthase confer sulfonamide resistance (20, 21) Transferases modify aminoglycosides by acetylation, adenylation and phosphorylation (22) β-‐lactamases cleave β-‐lactams (15) Efflux pump for multiple antibiotics including fluoroquinolones (16) Mutations in the gyrase gene confer fluoroquinolone resistance. (16) Mutations in ribosomal genes confer macrolide resistance (22) ERG11 mutations prevent binding of fluconazole to 14 α-‐demethylase (23) Efflux pumps (CDR and MDR genes) (24) Ligases encoded by vanA, vanB and vanC genes catalyze the formation of a D-‐alanine – D-‐Lactate (vanA and vanB) and D-‐alanine – D-‐serine (vanC) and replace the D-‐Alanine-‐D-‐Alanine dipeptide in peptidoglycan synthesis, which reduces vancomycin binding affinity enabling cell wall synthesis (8) Transferases modify aminoglycosides by acetylation, adenylation and phosphorylation (22) β-‐lactamases cleave β-‐lactams (15) Efflux pump for multiple antibiotics including fluoroquinolones (16) Mutations in the katG gene (catalase peroxidase) render the protein unable to activate the isoniazid pro-‐drug Mutations in the rpoB gene (β-‐subunit of RNA polymerase) prevent rifampicin binding and confer resistance (10, 11, 12) 8 | P a g e Table 1: AMR Organisms (2,3) Organism Drug-‐resistance Extensively drug-‐resistant Mycobacterium tuberculosis (XDR-‐TB) Drugs for treating MDR-‐TB and fluoroquinolones and second-‐line drugs Clostridium difficile May not be antibiotic resistant, but C. difficile infections linked to treatment of other resistant organisms Chloroquine and sulfadoxine-‐
pyrimethamine combination Plasmodium falciparum Mechanism Mechanisms described for MDR-‐TB as well as mutations in gyrA and gyrB confer resistance to fluoroquinolone A1401G mutation in the rrs gene coding for 16S rRNA associated with resistance to second-‐line drugs (10, 11, 12) Long-‐term treatment with antibiotics kill off intestinal flora that inhibit C. difficile; treatment of C. difficile infection with vancomycin provides selective pressure for VRE (25) Mutations in the gene encoding chloroquine resistance transporter (CRT) leads to moderate resistance; this coupled with mutations in the gene encoding the multidrug resistance p-‐glycoprotein pump (MDR) confers high levels of resistance to chloroquine. Multiple mutations in the dihydrofolate reductase (DHFR) gene confers moderate and high level resistance to pyrimethamine Multiple mutations in the dihydropteroate synthase (DHPS) gene confers moderate resistance to sulfadoxine (26) 9 | P a g e Section 1: Commercially Available Technology Landscape Molecular Systems Molecular systems are those that detect nucleic acid markers either through enzymatic amplification of targets (e.g. PCR, NASBA) followed by detection with labeled probes or in some cases through direct hybridization of labeled probes (e.g. FISH) without target amplification. Commercially available molecular systems and technologies were analyzed based on workflow, chemistry and the pathogens targeted by each product. In addition, the required instrumentation and ancillary equipment, as well as performance characteristics for commercially available products are described. The following were used to describe the mechanism and required equipment of commercially available technology and are summarized in Table 2: •
•
•
Workflow / Sample types Instrumentation Chemistry Workflow can be defined as automated, where the end-‐user obtains a test result by using a platform that integrates nucleic acid extraction, amplification (if applicable) and detection. The user is not required to manually prepare final reagent formulations or pre-‐process clinical samples beyond simple dilution steps. Automated platforms are either fully or partially integrated. In the latter case, the end-‐
user may have to transfer processing intermediates between instrument modules (e.g. assembled PCRs from liquid handling module to a thermal cycler). Fully automated and integrated “sample to answer” platforms also exist where the end-‐user simply adds the sample to a consumable containing all of the reagents needed for nucleic acid extraction, amplification and detection. Following sample addition, the consumable is loaded onto the instrument and processed without additional manipulation by the end-‐
user. Lastly, instrument software (in both fully and partially integrated platforms) provides results interpretation and can potentially be interfaced with a LIM/LIS system. Workflow can also be defined as manual, where the end-‐user must manually assemble reagent components in order to extract and detect nucleic acids. The amplification reactions are then loaded onto a thermal cycler and the user must interpret the results based on instructions provided by the manufacturer. Performance Attributes of Molecular Systems The performance attributes for AMR assays available on each instrument platform are summarized in Table 2. In general, assays detecting specific antibiotic resistance markers are listed. Assays detecting toxigenic Clostridium difficile are available on several platforms, and while the analytes (tcdA and tcdB) are not involved in antibiotic resistance per se, the colonization and development of C. difficile associated disease is related to antibiotic treatment for other infections (25). 10 | P a g e Critical attributes for the developing world include affordability, sensitivity and specificity, ease of use, rapid time to result and minimal requirements for equipment – including that required for storage (27, 28). Another concern for diagnostic testing in the developing world is the quality of diagnostic tests (27, 28) in terms of performance as well as robustness, including reagent stability during shipping and storage. Detailed comparisons of these critical attributes are described in the Differentiators section. Cost of selected platforms can be found in Appendix B. Molecular tests that detect antibiotic resistance markers such as mecA or vanA may imply the detection of drug resistance; these tests can also imply drug susceptibility based on the absence of detection. For instance, detection of a S. aureus marker absent the detection of mecA in a blood sample would imply susceptibility to methicillin, while detection of mecA would imply resistance to methicillin. These results would be obtained much quicker than those obtained with standard microbiology techniques. However, caution should be taken in interpreting test results this way as alternative mechanisms of resistance may be present. For instance, VISA is resistant to vancomycin despite not harboring the vanA or vanB genes. Thus a vanA or vanB negative clinical sample where VISA was present would be misinterpreted as being susceptible to vancomycin. Indeed the intended usages of molecular tests detecting resistance markers instruct the end-‐user to confirm results using other means (e.g. AST of isolates). 11 | P a g e !
Table&2:&Platform&Mechanisms&and&Required&Equipment&
Platform&/&Mfr.&
QIAGEN®!/!
QIAsymphony®!
system!
Cepheid®!/!
GeneXpert®!system!
Workflow&
Automated;!partially!
integrated!;!user!transfers!
assembled!PCR!plate!to!
thermal!cycler;!software!
provides!results!interpretation!
!
Sample!types:!!
• Liquid!or!soft!stool!for!C.#
difficile!assay!
• Rectal!or!perianal!swab!for!
vancomycin!resistance!
Fully!integrated!and!
automated;!user!adds!sample!
to!consumable!and!loads!
instrument;!multi;module!
versions!allow!on;demand!
usage;!software!provides!
results!interpretation!
!
Sample!types:!
• Rectal!swabs!for!vanA!
difficile!
Chemistry&
Instrumentation&and&ancillary&
equipment&
qPCR!and!RT;qPCR!amplification!
of!nucleic!acids;!detection!using!
fluorescently;labeled!
oligonucleotide!probes!
!
Run!QC!by!external!PC!and!NC!
(included!in!each!run),!IC!(added!
to!each!sample)!
QIAsymphony®!SP/AS:!liquid!
handling!platform!that!performs!all!
pipetting!steps!for!extraction!and!
PCR!assembly.!!!
!
Rotor;Gene®!Q:!real;time!PCR!
platform,!capable!of!melt!analysis!
!
Vortex!mixer!and!micropipettes!
interfaced!PC!
!
Assay!QC!by!external!PC!and!NC!
(run!separately),!IC!(added!to!
each!sample)!
GeneXpert®:!Self;contained,!
cartridge;based!nucleic!acid!
purification,!real;time!PCR!
amplification!and!detection.!!
Instruments!available!in!range!of!
random!access!modules!
•
GeneXpert®!I!
•
GeneXpert®!II!
•
GeneXpert®!IV!
•
GeneXpert®!XVI!
•
GeneXpert®!Infinity;48s!
•
GeneXpert®!80!
!
Micropipettes,!interfaced!PC!
*Time!to!result!is!approximate!and!based!on!publically!available!information!
!
Clinical&performance&or&Method&Comparison&
artus®!VanR!QS;RGQ!Kit!(29)&
&
Rectal&or&perianal&swabs:&
Sensitivity!93%!(95%!CI:!86–96)!
Specificity!92%!(95%!CI:!88–95)!
artus®!C.#difficile!QS;RGQ!MDx!kit!(30)&
&
Reference&method&is&enriched&culture:&
Sensitivity!90%!(95%!CI:!83;94)!
Specificity!97%!(95%!CI:!96;98)!
!
Reference&method&is&direct&culture:&
Sensitivity!99%!(95%!CI:!94;100)!
Specificity!97%!(95%!CI:!95;98)!
Analytes&and&time&
to&result*&&
VRE!resistance!
markers:!
vanA!
vanB!
!
IC!sequence!also!
detected!
!
TTR:!5;8!hrs.!
C.#difficile#toxin!A!and!
B!genes#
tcdA!
tcdB!
!
IC!sequence!also!
detected!
!
TTR:!5;8!hrs.!
Xpert®!vanA!Assay!(31)&
&
Reference&method&is&direct&culture:!
%!Positive!agreement:!98.4%!
%!Negative!agreement:!92.4%!
!
Reference&method&is&enriched&culture:&
%!Positive!agreement:!86.5%!
%!Negative!agreement:!93.5%!
VRE!resistance!
marker:!
vanA!
!
IC!sequence!also!
detected!
!
TTR:!<!1!hr.!
Xpert®!C.#difficile!Assay!(32)&
&
Reference&method&is&direct&culture:&
Sensitivity:!98.79%!!
Specificity:!90.82%!
C.#difficile#toxin!B!
gene#
tcdB!
!
IC!sequence!also!
detected!
!
TTR:!<!1!hr.!
!
!
!
Table&2:&Platform&Mechanisms&and&Required&Equipment&(continued)&
Platform&/&Mfr.&
Cepheid®!/!
GeneXpert®!system!
(continued)!
Workflow&
!
Sample!types:!
•! Liquid!or!soft!stool!for!C.#
difficile!
Chemistry&
!
each!sample)!
*Time!to!result!is!approximate!and!based!on!publically!available!information!!
equipment&
•
GeneXpert®!I!
•
GeneXpert®!II!
•
GeneXpert®!IV!
•
GeneXpert®!XVI!
•
•
GeneXpert®!80!
!
Analytes&and&time&
to&result*&&
Xpert®!C.#difficile/Epi!Assay!(33)&
&
Direct&culture&and&REA&(used&for&typing)&
Detection:!
Sensitivity:!98.76%!
Typing:!
%!Pos!Agreement:!100%!
%!Neg!Agreement:!97.61%!
!
Direct&culture&and&PCR&ribotyping&
Detection:!
Specificity:!90.86%!!
Typing:!
!
!
Direct&culture&and&PFGE&(used&for&typing)&
Detection:!
Typing:!
!
Reference&culture&and&REA&(used&for&typing)&
Detection:!
Typing:!
%!Pos!Agreement:!96.51%!
!
Reference&culture&and&PCR&ribotyping&(used&to&type&C"difficile&as&the&
027/NAP1/BI&strain)&
Detection:!
Typing:!
!
Reference&culture&and&PFGE&(used&to&type&C"difficile&as&the&027/NAP1/BI&
strain)&
Detection:!
Typing:!
C.#difficile!toxin!B!and!
binary!toxin!genes!
and!a!deletion!in!the!
tcdC!gene!at!
nucleotide!117!!!The!
binary!toxin!gene!and!
deletion!in!the!tcdC!
are!utilized!for!
presumptive!
identification!of!the!C!
difficile!as!the!
027/NAP1/BI!strain!
#
tcdB!
cdtB!
tcdC!Δ117!
!
IC!sequence!also!
detected!
!
TTR:!<!1!hr.!
!
13!|!P a g e !
!
!
Platform&/&Mfr.&
Cepheid®!/!
GeneXpert®!system!
(continued)!
Workflow&
!
Sample!types:!
• Nasal!swabs!for!MRSA!
tests!
• Blood!culture!for!MRSA!
blood!culture!assay!
Chemistry&
!
each!sample)!
equipment&
•
GeneXpert®!I!
•
GeneXpert®!II!
•
GeneXpert®!IV!
•
GeneXpert®!XVI!
•
•
GeneXpert®!80!
!
!
Analytes&and&time&
to&result*&
Xpert!MRSA!(34)&
&
Assay&against&Reference&culture:&
!
Assay&against&Direct&culture:&
Sensitivity:!94.3%!
Specificity:!93.2%!
SCCmec!/!attB!
junction!
!
IC!sequence!also!
detected!
!
TTR:!<!1!hr.!
Xpert®!SA!Nasal!Complete!Assay!(35)&
&
MRSA:&&
Sensitivity:!91.9%!(95%!CI:!86.8;95.5)!
Specificity:!97.9%!(95%!CI:!97.2;98.4)!
!
Staphylococcus&aureus:&
Sensitivity:!93.3%!(95%!CI:!91.1;95.1)!
Specificity:!90.5%!(95%!CI:!89.1;91.8)!
Staphylococcal!protein!A!
gene,!
methicillin/oxacillin!
resistance!gene!and!the!
junction!sequence!
between!the!SCCmec!
and!Staphylococcal!
chromosome!
!
spaA!
mecA!
SCCmec!/!attB!junction!
!
IC!sequence!also!
detected!
!
TTR:!<!1!hr.!
Xpert®!MRSA/SA!Blood!Culture!Assay!(36)!
!
MRSA:!
%!Pos!Agreement:!98.1%!(95%!CI:!93.3;99.8)!
%!Neg!Agreement:!99.6%!(95%!CI:!98.7;99.9)!
#
Staphylococcus#aureus:!
%!Pos!Agreement:!99.6%!(95%!CI:!97.7;100)!
%!Neg!Agreement:!99.5%!(95%!CI:!98.4;99.9)!
Staphylococcal!
protein!A!gene,!
resistance!gene!and!
the!junction!
sequence!between!
the!SCCmec!and!
Staphylococcal!
chromosome!
!
spaA!
mecA!
SCCmec!/!attB!
junction!
!
IC!sequence!also!
detected!
!
TTR:!<!1!hr.!
14!|!P a g e !
!
!
Platform&/&Mfr.&
Cepheid®!/!
GeneXpert®!system!
(continued)!
bioMérieux®!/!
NucliSENS!easyQ®!
Workflow&
Chemistry&
!
Sample!types:!
• Soft!tissue!swab!for!MRSA!
SSTI!assay!
• Sputum!for!MTB!assay!
!
each!sample)!
Manual,!user!performs!manual!DNA!
extraction!and!adds!crude!lysate!to!
chemistry!for!restriction!digestion!on!
incubator;!this!mixture!is!
subsequently!transferred!from!the!
incubator!and!combined!with!
amplification!chemistry!and!loaded!
onto!the!analyzer;!software!provides!
result!interpretation!
!
Sample!types:!
• Nasal!swab!for!MRSA!assay!
• Rectal!swab!or!stool!for!KPC!
Isothermal!NASBA!amplification!
and!detection!using!
fluorescently!labeled!molecular!
beacons!
!
to!each!sample)!
equipment&
•
GeneXpert®!I!
•
GeneXpert®!II!
•
GeneXpert®!IV!
•
GeneXpert®!XVI!
•
•
GeneXpert®!80!
!
easyQ®!Incubator!for!DNA!
extraction!
easyQ®!Analyzer!for!nucleic!acid!
amplification!and!detection!
!
Vortex!mixer!(with!specific!tube!
adaptor),!microcentrifuge,!
micropipettes,!interfaced!PC!and!
printer!
Analytes&and&time&
to&result*&
Xpert®!MRSA/SA!SSTI!Assay!(37)&
&
Samples&from&patients&with&no&antibiotic&use:&
MRSA!
%!Pos!Agreement:!93.8%!(95%!CI:!88.6;97.1)!
%!Neg!Agreement:!97.3%!(95%!CI:!94.7;98.8)!
!
Staphylococcus#aureus#
%!Pos!Agreement:!95.7%!(95%!CI:!92.2;97.9)!
%!Neg!Agreement:!89.5%!(95%!CI:84.6;93.3)!
!
Samples&from&patients&with&unknown&antibiotic&use:&
MRSA!
%!Pos!Agreement:!94.0%!(95%!CI:!83.5;98.7)!
%!Neg!Agreement:!97.3%!(95%!CI:!93.3;99.3)!
!
Staphylococcus#aureus#
%!Pos!Agreement:!96.9%!(95%!CI:!91.2;99.4)!
%!Neg!Agreement:!88.3%!(95%!CI:!80.5;93.8)!
!
Samples&from&patients&with&known&antibiotic&use:&
%!Pos!Agreement:88.0%!(95%!CI:!!75.7;95.5)!
%!Neg!Agreement:!92.4%!(95%!CI:!87.0;96.0)!
%!Pos!Agreement:95.2%!(95%!CI:!88.3;98.7)!
%!Neg!Agreement:!76.4%!(95%!CI:!67.9;83.6)!
Staphylococcal!
protein!A!gene,!
resistance!gene!and!
the!junction!
sequence!between!
the!SCCmec!and!
Staphylococcal!
chromosome!
!
spaA!
mecA!
SCCmec!/!attB!
junction!
!
IC!sequence!also!
detected!
!
TTR:!<!1!hr.!
Xpert®!MTB/RIF!Assay!(38)&
&
Assay&against&culture&reference&method&on&AcidQFast&Bacillus&smear&
positive&samples&
Sensitivity:!99.7%!(95%!CI:!98.4!;!99.9)!
Specificity:!98.5%!(95%!CI:!91.9!–!99.7)!
!
Assay&against&culture&reference&method&on&AcidQFast&Bacillus&smear&
negative&samples&
Sensitivity:!76.1%!(95%!CI:!67.6!;!82.9)!
Specificity:!98.8%!(95%!CI:!97.5!–!99.4)!
!
Assay&against&drugQsusceptibility&testing&
Sensitivity:!94.7%!(95%!CI:!75.4;99.1)!
Specificity:!99.0%!(95%!CI:!97.5;99.6)!
MTB;complex!DNA!
and!the!genetic!
mutations!associated!
with!rifampin!(Rif)!
resistance!
!
Core!region!of!rpoB#
!
IC!sequence!also!
detected!
!
TTR:!<!2!hrs.!
NucliSENS!easyQ®!MRSA!Assay!(39)!
!
Sensitivity:!95.8%!(95%!CI:!91.1!;!98.4)!
Specificity:!96.8%!(95%!CI:!95.5!;!97.7)!!
SCCmec!cassette!junction!
and!the!mecA!gene!
!
IC!sequence!also!detected!
!
TTR:!3;4!hrs.!
NucliSENS!easyQ®!KPC!assay!(40)!
!
Assay!against!routine!phenotypic!determination:!
Sensitivity:!93.3%!(95%!CI:!77.9–99.2)!
Specificity:!99.0%!(95%!CI:!98.0–99.6)!
blaKPC!gene!
!
IC!sequence!also!
detected!
!
TTR:!3;4!hrs.!
!
15!|!P a g e !
!
!
Platform&/&Mfr.&
TM
Quidel®!/!Lyra !
direct!real;time!PCR!
assays!
Quidel®!/!
AmpliVue®!
Meridian!
Bioscience®!/!
illumigene®!
molecular!diagnostic!
system!
Workflow&
Manual,!user!prepares!a!crude!
extract!of!the!clinical!sample!
and!adds!this!to!the!
assembled!PCR;!the!PCR!plate!
or!tubes!are!loaded!on!to!the!
real;time!PCR!instrument;!
user!manually!interprets!the!
results!based!on!output!data!
supplied!by!real;time!PCR!
instrument!software!
!
Sample!types:!
difficile!
extract!of!the!clinical!sample!by!
heating!a!diluted!specimen!and!
adding!this!to!lyophilized!
amplification!reagents;!the!
assembled!reaction!is!loaded!onto!a!
heat!block!for!isothermal!
amplification;!the!reaction!is!then!
loaded!into!a!cassette!that!provides!a!
visual!display!of!reaction!products;!
user!manually!interpret!results!
!
Sample!type:!
• Liquid!or!soft!stool!for!C.#difficile!
extract!of!the!clinical!sample!by!
heating!a!diluted!specimen!and!
adding!this!to!lyophilized!
amplification!reagents;!the!
assembled!reaction!is!loaded!onto!
the!instrument!for!isothermal!
amplification!and!detection;!
integrated!instrument!software!
interprets!results!
!
Sample!type:!
• Liquid!or!soft!stool!for!C.#difficile!
Chemistry&
equipment&
!
(run!individually),!IC!(added!to!
each!sample)!
7500!Fast!Dx!(Applied!
Biosystems®)!
TM
QuantStudio !(Life!Technologies)!
TM
SmartCycler!II !(Cepheid)!
!
Microcentrifuge,!micropipettes,!
interfaced!PC!
Isothermal!HDA!amplification!of!
DNA!using!biotinylated!
oligonucleotides;!detection!
using!AmpliVue®!cassette!
providing!visual!display!of!
analyte!detection!
!
each!sample)!
Assay!cassette!for!visualization!of!
results!
!
Micropipettes;!heat!blocks!for!
sample!lysis!and!isothermal!
incubation!
Isothermal!LAMP,!amplification!
products!detected!based!on!
change!in!optical!density!of!
reaction!
!
each!sample)!
TM
illumipro;10 !instrument!for!
LAMP!and!reaction!product!
detection!
!
Micropipettes;!heat!blocks!for!
sample!lysis;!vortex!mixer!
Analytes&and&time&
to&result*&
Lyra!Direct!C.#difficile!Assay!(41)&
&
Assay&against&cell&cultureQbased&cytotoxicity&assay;&PCR&on&7500&Fast&Dx:&
Sensitivity:!94.3!%!(95%!CI:!87.4!–!97.5)!
Specificity:!94.2%!(95%!CI:!91.9!–!95.8)!
!
Assay&against&enriched&toxigenic&bacterial&culture;&PCR&on&7500&Fast&Dx:&
Sensitivity:!88.9!%!(95%!CI:!82.2!–!93.3)!
Specificity:!98.9%!(95%!CI:!97.6!–!99.5)!
!
TM
Assay&against&direct&bacterial&culture;&PCR&on&QuantStudio :&
Sensitivity:!93.3%!(95%!CI:86.9!–!96.7)!
Specificity:!93.4%!(95%!CI:!91.3!–!95.0)!
!
TM
Assay&against&enriched&toxigenic&bacterial&culture;&PCR&on&QuantStudio :&
Sensitivity:!87.3%!(95%!CI:!81.1!–!91.6)!
Specificity:!98.7%!(95%!CI:!97.5!–!99.4)!
!
TM
Assay&against&direct&bacterial&culture;&PCR&on&SmartCycler&II :&
Sensitivity:!89.7%!(95%!CI:81.5!–!94.5)!
Specificity:!93.3%!(95%!CI:!91.0!–!95.1)!
!
TM
Assay&against&enriched&toxigenic&bacterial&culture;&PCR&on&SmartCycler&II :&
Sensitivity:!82.4%!(95%!CI:!74.8!–!88.1)!
Specificity:!97.9%!(95%!CI:!95.4!–!98.3)!
C.#difficile#toxin!A!and!
toxin!B!genes#
tcdA#
tcdB!
!
IC!sequence!also!
detected!
!
TTR:!<!2!hrs.!
AmpliVue®#C.#difficile!Assay!(42)!
!
Sensitivity:!93.6%!(95%!CI:!97.3!–!96.9)!
Specificity:!94.1%!(95%!CI:!92.1!–!95.6)!
C.#difficile#toxin!A!
gene#
tcdA#
!
IC!sequence!also!
detected!
!
TTR:!<!2!hrs.!
Illumigene®!C.#difficile!DNA!Amplification!Assay!(43)&
&
Assay&against&cytotoxic&bacterial&culture:&
Sensitivity:!95.2%!(95%!CI:!89.2!–!97.9)!
Specificity:!95.3%!(95%!CI:!93.2!–!96.7)!
!
Assay&against&cytotoxic&bacterial&culture&(pediatric&samples):&
Sensitivity:!93.3%!(95%!CI:78.7!–!98.2)!
Specificity:!96.3%!(95%!CI:!92.2!–!98.3)!
C.#difficile#toxin!A!
gene#
tcdA#
!
IC!sequence!also!
detected!
!
TTR:!<!2!hrs.!
!
16!|!P a g e !
!
!
Platform&/&Mfr.&
Workflow&
Chemistry&
equipment&
Analytes&and&time&
to&result*&
LightCycler®!MRSA!Advanced!Test!(44)&
&
Assay&against&direct&chromogenic&culture&
%!Pos!Agreement:!95.2%!(95%!CI:!91.1!;!97.8)!
%!Neg!Agreement:!96.4%!(95%!CI:!95.2!;!97.4)!
!
Assay&against&second&FDAQcleared&nucleic&acid&amplification&test:&
%!Pos!Agreement:!71.7%!(95%!CI:!65.9!–!77.0)!
%!Neg!Agreement:!98.2%!(95%!CI:!97.2!;!98.9)!
!
Assay&against&broth&culture:&
%!Pos!Agreement:!89.8%!(95%!CI:!84.8!;!93.5)!
%!Neg!Agreement:!96.8%!(95%!CI:!95.6!;!97.7)!
Automated!and!Manual!
depending!on!assay:!
Manual:!User!prepares!a!
crude!lysate!and!assembles!
the!PCR!(manual)!PCR!plates!is!
loaded!onto!the!real;time!PCR!
instrument!
!
Automated!sample!
preparation!where!user!
transfers!assembled!PCR!to!
thermal!cycler;!instrument!
software!interprets!the!results!
!
Sample!types:!
• Perianal!swabs!for!VRE!
!
to!each!sample)!
MagNA!Lyser:!for!crude!lysate!
preparation!
!
MagNA!Pure:!Automated!liquid!
handling!that!performs!all!
PCR!assembly.!
!
LightCycler®!real;time!PCR!
platform!
!
Micropipettes,!microcentrifuge,!
vortex!mixer,!heat!block!and!
interfaced!PC!
Roche!/!cobas®!
4800!system!
Automated;!partially!
integrated!;!user!transfers!
assembled!PCR!plate!to!
thermal!cycler;!software!
!
Sample!types:!
!
to!each!sample)!
cobas®!x!480!instrument:!liquid!
handling!platform!that!performs!all!
PCR!assembly.!
!
cobas®!z!480!analyzer:!real;time!
PCR!platform!
!
Vortex!mixer!and!micropipettes;!
interfaced!PC!
cobas®!MRSA/SA!Test!(46)&
&
MRSA:&
Sensitivity:!93.1%!(95%!CI:!88.1!;!96.1)!
Specificity:!97.5%!(95%!CI:!96.8!;!98.0)!
"
S."aureus&(MSSA):&
Sensitivity:!93.5%!(95%!CI:!89.9!;!95.9)!!
Specificity:!92.9%!(95%!CI:!90.9!;!94.5)!
ELITech!
Semi;automated,!user!adds!
sample!to!EASYMAG®!
instrument!for!DNA!extraction!
and!manually!assembles!the!
PCR;!PCR!plate!is!loaded!onto!
the!real;time!PCR!instrument;!
the!user!manually!opens!run!
file!to!analyze!results!with!
supplied!software!
!
Sample!types:!
qPCR!amplification!and!
detection!using!fluorescently!
labeled!MGB!probes!
!
(included!in!each!run);!IC!(added!
to!each!sample)!
EASYMAG®!Instrument!
(bioMérieux®)!for!DNA!extraction!
!
7500!Fast!Dx!(Applied!
Biosystems®)!for!nucleic!acid!
amplification!and!detection!
!
Vortex!mixer,!microcentrifuge,!
micropipettes!and!interfaced!PCs!
MRSA/SA!ELITe!MGB®!(47)&
&
MRSA:!
Sensitivity:!92.3%!(95%!CI:!88.08;95.16)!
Specificity:!95.2%!(95%!CI:!94.32;95.87)!
&
MSSA:!
Sensitivity:!96.1%!(95%!CI:!94.48;97.25)!Specificity:!95.1%!(95%!CI:!
94.16;95.89)!
Roche!/!LightCycler®!
480!
Right!extremity!(RE)!
of!the!SCCmec/orfX#
junction!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
vanA,!vanB!genes!
(assay!differentiates!
vanB!from!vanB2/3!
subtypes)!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
Right!extremity!(RE)!
of!the!SCCmec/orfX#
junction!
!
cpe!gene!of!
Staphylococcus!
aureus!(species;
specific!marker)!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
VRE!RUO!(45)&
&
Assay&against&Enterococcosel&screening&using&6&ug/mL&vancomycin:&
Sensitivity:!100%!(95%!CI:!59–100)!!
Specificity:!97%!(95%!CI:!94–98)!
!
Assay&against&Enterococcosel&screening&using&8&ug/mL&vancomycin:&&
Sensitivity:!100%!(95%!CI:!69–100)!!
Specificity:!95%!(95%!CI:!93–97)!
Staphylococcus!aureus!
ldh1!gene!(species;
specific!marker)!and!
mecA!gene!(methicillin!
resistance)!
!
IC!sequence!also!
detected!
!
TTR:!3;5!hrs.!depending!
on!batch!size!
!
17!|!P a g e !
!
!
Platform&/&Mfr.&
Workflow&
Chemistry&
equipment&
AdvanDx!/#
XpressFISH®!
Manual,!user!performs!
fixation!and!probe!
hybridization!steps!followed!
by!slide!mounting!for!
fluorescent!microscopy;!user!
manually!interpret!results!
!
Sample!type:!
• Blood!culture!smears!
Fluorescently!labeled!PNA!FISH!
probes!
!
Sample!slides!contain!separate!
PC!and!NC!wells!adjacent!to!
sample!well!to!monitor!process!
Fluorescent!microscope!
!
AdvanDx!Microscope!Filter,!Dual!
Band!Filter!Set!+!Holder!!
!
AdvanDx!SlideStation–10!
!
AdvanDx!10uL!Pipette!!
!
TM
QuickFISH !Mixing!Station!
Great!Basin!
Scientific!/!Portrait!
Analyzer!
instrument;!software!provides!
!
Sample!type:!
•! Liquid!or!soft!stool!for!C.#
difficile!
Isothermal!HDA!of!target!
nucleic!acids;!biotinylated!
oligonucleotides!binding!to!anti;
biotin/HRP!conjugates;!
colorimetric!substrate!within!
macroarray!
!
Run!QC!by!IC!(added!to!each!
sample)!
Portrait!Analyzer:!Self;contained!
single!module,!cartridge;based!
nucleic!acid!purification,!
isothermal!HDA!and!array;based!
detection!
!
Micropipettes,!vortex!mixer,!
interfaced!PC!
Luminex®!/!xTAG®!
User!performs!manual!pre;
treatment!of!samples,!
followed!by!automated!
nucleic!acid!extraction.!!The!
user!manually!assembles!PCRs!
and!then!transfers!to!
analyzer;!analyzer!software!
interprets!the!results!
!
Sample!types:!
• Stool!or!stool!in!Cary!Blair!
medium!
PCR!amplification!using!
biotinylated!primers!and!
primers!containing!the!universal!
xTAG®!tag!sequence.!!!
EASYMAG®!Instrument!for!
extraction!(bioMérieux®)!
!
Thermal!cycler!
!
Luminex®!100/200™!and!MAGPIX®!
instruments!(analyzer)!
!
interfaced!PC!
!
Analytes&and&time&
to&result*&
mecA!XpressFISH®!(48)!
!
Sensitivity!
98.7%!(95%!CI:!95.4;99.6)!
Specificity!
99.5%!(95%!CI:!97.0;99.9)!
mecA!gene!
!
IC!sequence!also!
detected!
!
TTR:!<!1!hr.!(staining!
portion!only;!longer!
time!required!for!
blood!culture)!
Portrait!Toxigenic!C.#difficile!Assay!(49)&
&
Assay&against&reference&culture&and&cytotoxicity&assay:&
Sensitivity:!98.0%!(95%!CI:!92.4!–!99.6)!
Specificity:!90.9%!(95%!CI:!87.7!–!93.3)!
gene#
tcdB#
!
IC!sequence!also!
detected!
!
TTR:!<!2!hrs.!
xTAG®!Gastrointestinal!Pathogen!Panel!(GPP)!(50)!
!
NOTE:"Only"C."difficile"(tcdA,"tcdB)"performance"data"included"in"
summary!
!
%!Pos!Agreement:!93.9%!(95%!CI:!87.9!–!97.0)!
%!Neg!Agreement:!89.0%!(95%!CI:!86.9!–!90.8)!
Panel!of!
gastrointestinal!
pathogen!markers,!
including!the!C.!
difficile!toxin!A!and!B!
genes!
tcdA#
tcdB#
!
IC!sequence!also!
detected!
!
TTR:!5!hrs.!
18!|!P a g e !
!
!
Platform&/&Mfr.&
Nanosphere!/!
Verigene®!
Workflow&
Automated,!user!loads!
instrument!with!assay!
consumables!and!chemistry!
and!adds!sample!to!
consumable;!following!sample!
processing!and!PCR!
amplification,!user!transfers!
slide!from!test!cartridge!to!a!
reader;!instrument!software!
interprets!results!
!
Sample!types:!
• Blood!cultures!for!Gram!
negative!and!positive!
assays!
Chemistry&
Target!DNA!hybridization!to!
microarray;based!
oligonucleotides!with!
visualization!based!on!gold!
nanoparticle!oligonucleotide!
probes!
!
each!sample)!
equipment&
Processor!SP!for!sample!
preparation!and!hybridization!
reaction!
!
Verigene®!Reader!for!hybridization!
complex!detection!
!
Micropipettes!
Verigene®!Gram;Negative!Blood!Culture!Nucleic!Acid!Test!(BC;GN)!
(51)!
!
NOTE:"Only"performance"data"pertaining"to"antibiotic"resistance"
markers"included"in"summary&
&
OXA:!
%!Pos!Agreement:!95.3%!(95%!CI:!86.9;99.0)!
%!Neg!Agreement:!99.9%!(95%!CI:!99.5;100)!
!
CTXQM:&
%!Pos!Agreement:!98.7%!(95%!CI:!95.4;99.8)!
%!Neg!Agreement:!99.9%!(95%!CI:!99.5;100)!
!
KPC:&
%!Pos!Agreement:!100%!(95%!CI:!93.1;100)!
%!Neg!Agreement:!100%!(95%!CI:!99.7;100)!
!
NDM:&
%!Pos!Agreement:!100%!(95%!CI:!91.4;100)!
%!Neg!Agreement:!100%!(95%!CI:!99.7;100)!
!
IMP:&
%!Pos!Agreement:!100%!(95%!CI:!92.6;100)!
%!Neg!Agreement:!100%!(95%!CI:!99.7;100)!
!
VIM:&
%!Pos!Agreement:!100%!(95%!CI:!91.4;100)!
%!Neg!Agreement:!100%!(95%!CI:!99.7;100)!
Verigene®!Gram;Positive!Blood!Culture!Nucleic!Acid!Test!(BC;GP)!
(52)!
!
"
mecA:"
%!Pos!Agreement:!94.2%!(95%!CI:!91.5;96.3)!
%!Neg!Agreement:!98.2%!(95%!CI:!97.1;98.9)!
!
vanA:"
%!Pos!Agreement:!94.2%!(95%!CI:!85.8;98.4)!
%!Neg!Agreement:!99.8%!(95%!CI:!99.4;99.9)!
!
vanB:"
%!Pos!Agreement:!100%!(95%!CI:!29.2;100)!
%!Neg!Agreement:!100%!(95%!CI:!99.7;100)!
!
Analytes&and&time&
to&result*&
Panel!of!Gram!
negative!bacteria!and!
antibiotic!resistance!
markers,!including:!
blaCTX;M!
blaOXA;48!
blaNDN!
blaIMP!
blaOXO;23!
blaVIM!
blaKPC!
(Resistance!markers!
to!carbapenems)!
!
IC!sequence!also!
detected!
!
TTR:!<!3!hrs.!
Panel!of!Gram!
positive!bacteria!and!
mecA#
vanA#/#vanB#
to!methicillin!and!
Vancomycin!
respectively)!
!
IC!sequence!also!
detected!
!
TTR:!<!3!hrs.!
19!|!P a g e !
!
!
Platform&/&Mfr.&
Workflow&
Nanosphere!/!
Verigene®!
(continued)!
Automated,!user!loads!
instrument!with!assay!
consumables!and!chemistry!
and!adds!sample!to!
consumable;!following!sample!
processing!and!PCR!
amplification,!user!transfers!
slide!from!test!cartridge!to!a!
reader;!instrument!software!
interprets!results!
!
Sample!types:!
difficile!
FilmArray®!system!/!
Biofire®!
automated;!user!loads!sample!
and!rehydrates!reagents!on!
cartridge;!this!assembly!
subsequently!loaded!onto!the!
!
Sample!types:!
difficile!
Chemistry&
Target!DNA!hybridization!to!
microarray;based!
oligonucleotides!with!
visualization!based!on!gold!
nanoparticle!oligonucleotide!
probes!
!
each!sample)!
Nested!PCR!and!RT;PCR;!
detection!using!fluorescent!
intercalation!dye!within!
separate!array!module!(1!target!
per!well!within!array)!
equipment&
Analytes&and&time&
to&result*&
Processor!SP!for!sample!
preparation!and!hybridization!
reaction!
!
Verigene®!Reader!for!hybridization!
complex!detection!
!
Micropipettes!
Verigene®!Clostridium!difficile!Nucleic!Acid!Test!(CDF)!(53)&
&
Assay&against&direct&culture&and&PCR&ribotyping&(percent&
agreement&for&hyper&virulent&strain&027):&
Sensitivity:!98.7%!(95%!CI:!99.5!;!99.8)!
Specificity:!87.6%!(95%!CI:!85.9!;!89.1)!
%!Pos!Agreement:!97.5%!(95%!CI:!86.8!;!99.9)!
%!Neg!Agreement:!97.8%!(95%!CI:!97.0!;!98.4)!
!
Assay&against&enriched&culture&and&PCR&ribotyping&(percent&
agreement&for&hyper&virulent&strain&027):&
Sensitivity:!91.8%!(95%!CI:!87.9!;!94.8)!
Specificity:!92.5%!(95%!CI:!91.1!;!93.7)!
%!Pos!Agreement:!91.4%!(95%!CI:!81.0!;!97.1)!
%!Neg!Agreement:!98.5%!(95%!CI:!97.8!–!99.0)!
!
Assay&against&direct&culture&with&biQdirectional&sequencing&and&
biQdirectional&sequencing(percent&agreement&for&hyper&virulent&
strain&027):!
Sensitivity:!98.7%!(95%!CI:!95.5!;!99.9)!
Specificity:!87.5%!(95%!CI:!85.8!;!89)!
%!Pos!Agreement:!97.7%!(95%!CI:!87.7!;!99.9)!
%!Neg!Agreement:!97.8%!(95%!CI:!97.0!;!98.4)!
!
Assay&against&enriched&culture&with&biQdirectional&sequencing&and&
biQdirectional&sequencing(percent&agreement&for&hyper&virulent&
strain&027):!
Sensitivity:!91.9%!(95%!CI:!88.1!;!94.9)!
Specificity:!92.5%!(95%!CI:!99.1!;!93.7)!
%!Pos!Agreement:!93.7%!(95%!CI:!84.5!;!98.2)!
%!Neg!Agreement:!98.7%!(95%!CI:!98.1!;!99.2)!
C.#difficile!toxin!A,!B!
and!binary!toxin!
genes!and!a!deletion!
in!the!tcdC!gene!at!
nucleotide!117!!!The!
binary!toxin!gene!and!
deletion!in!the!tcdC!
are!utilized!for!
presumptive!
identification!of!C!
difficile!PCR!ribotype!
027!strains!
#
tcdA#
tcdB!
cdtB!
tcdC!Δ117!
!
IC!sequence!also!
detected!
!
TTR:!<!3!hrs.!
FilmArray®!Gastrointestinal!(GI)!Panel!(54)!
!
NOTE:"Only"C."difficile"(tcdA,"tcdB)"performance"data"included"in"
summary!
!
Assay!against!alternative!PCR!and!bi;directional!sequencing:!
%!Pos!Agreement:!98.8%!(95%!CI:!95.7;99.9)!
%!Neg!Agreement:!97.1!%!(95%!CI:!96.0;97.9)!
Panel!of!
gastrointestinal!
pathogen!markers,!
including!the!C.!
difficile!toxin!A!and!B!
genes!
tcdA#
tcdB#
!
IC!sequence!also!
detected!
!
TTR:!1;2!hrs.!
FilmArray®:!Self;contained!single!
module,!cartridge;based!nucleic!
acid!purification,!amplification!and!
detection!
!
Interfaced!PC!and!bar!code!reader!
!
20!|!P a g e !
!
!
Table&2:&Platform&Mechanisms&and&Required&Equipment&(continued)!
Platform&/&Mfr.&
Workflow&
Chemistry&
equipment&
FilmArray®!system!
(continued)!
automated;!user!loads!sample!
and!rehydrates!reagents!on!
cartridge;!this!assembly!
subsequently!loaded!onto!the!
!
Sample!types:!
• Blood!cultures!!
Nested!PCR!and!RT;PCR;!
detection!using!fluorescent!
intercalation!dye!within!
separate!array!module!(1!target!
per!well!within!array)!
FilmArray®:!Self;contained!single!
acid!purification,!amplification!and!
detection!
!
Interfaced!PC!and!bar!code!reader!
Hain!Lifescience!/!
FluoroType®!
User!performs!manual!
extraction!and!PCR!assembly.!!
PCR!plate!loaded!into!the!real;
time!PCR!instrument;!
software!interprets!results!
!
Sample!types:!
• Nasal,!throat,!skin,!wound!
swabs!
qPCR!amplification!and!
detection!using!fluorescently!
labeled!probes!
!
(included!in!each!run);!IC!(added!
to!each!sample)!
FluoroCycler®!!
!
interfaced!PC!
*Time!to!result!is!approximate!based!on!publically!available!information!!
!
Analytes&and&time&
to&result*&
FilmArray®!Blood!Culture!Identification!(BCID)!panel!(55)!
!
&
Assay&against&PCR/sequencing&directly&from&blood&culture:&
mecA!(all!Staphylococcus)!
%!Pos!Agreement:!98.4%!(95%!CI:!96.8!–!99.3)!
%!Neg!Agreement:!98.3!(95%!CI:!96.0!–!99.4)!
!
vanA/B#
%!Pos!Agreement:!100%!(95%!CI:!94.4!–!100)!
%!Neg!Agreement:!100%!(95%!CI:!94.6!–!100)!
!
KPC!(Enterobacteriaceae)!
%!Pos!Agreement:!100%!(95%!CI:!91.0!–!100)!
%!Neg!Agreement:!100%!(95%!CI:!99.3!–!100)!
!
Assay&against&PCR/sequencing&of&blood&culture&isolates:&
mecA!(all!Staphylococcus)!
%!Pos!Agreement:!98.9%!(95%!CI:!97.5!–!99.6)!
%!Neg!Agreement:!87.9%!(95%!CI:!83.9!;!91.3)!
!
vanA/B#
%!Pos!Agreement:!100%!(95%!CI:!94!–!100)!
%!Neg!Agreement:94.4%!(95%!CI:!86.2!–!98.4)!
!
KPC!(Enterobacteriaceae)!
%!Pos!Agreement:!100%!(95%!CI:!91.0!–!100)!
%!Neg!Agreement:!100%!(95%!CI:!99.2!–!100)!
FluoroType®!MRSA!(56)&
&
Assay&against&culture&Q&study&#1&across&two&hospitals:&
Sensitivity:!100%!
Specificity:!99.2%!
!
Assay&against&culture&–&study&#2&comparing&to&routine&testing:&
Sensitivity:!100%!
Specificity:!96.1%!
Panel!of!Gram!
positive/negative!
bacteria,!yeast!and!
mecA#
vanA#/#vanB#
blaKPC#
to!methicillin,!
vancomycin,!and!
carbapenems!
respectively)!
!
IC!sequence!also!
detected!
!
TTR:!1;2!hrs.!
SCCmec;orfX!junction!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
21!|!P a g e !
!
!
Platform&/&Mfr.&
Hain!Lifescience!/!
GenoQuick®!
Workflow&
Manual!lysis!and!DNA!
amplification!
!
hybridization!reaction!with!
lateral!flow!readout!(gold)!
!
Sample!type:!!
• Nasal!swab!
Chemistry&
biotinylated!primers!
!
PCR!product!detection!using!
gold;binding!streptavidin;
conjugated!probe;!lateral;flow!
dipstick!used!to!visualize!
products!
!
IC!present!on!dip;stick!
equipment&
Thermal!cycler!
!
micropipettes,!hybridization!trays!
and!incubators!
Analytes&and&time&
to&result*&
SCCmec;orfX!junction!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
GenoQuick®!MRSA!(57)!
!
Sensitivity:!57%!
Specificity:!100%!
MRSA;specific!
sequence!
(SCCmec/ofrX!
junction)!
!
IC!sequence!also!
detected!
!
TTR:!4!hrs.!
Species;specific!
markers!for!E.#
faecalis,#E.#faecium,#
E.#gallinarum#and!E.#
caseliflavus!
Vancomycin!
resistance!markers:!
vanA,#vanB,#vanC1,#
vanC2/3!
!
IC!sequences!also!
detected!
!
TTR:!21!hrs.!
GenoType!MRSA!Direct!(58)!
!
Assay!against!culture:!
Sensitivity:!94.59%!!
Hain!Lifescience!/!
DNA!STRIP!
Manual!or!semi;automated!
lysis!and!DNA!amplification.!
!
colorimetric!assay!readout.!!
End;user!manually!interpret!
results!based!on!
manufacturer’s!instructions!
!
Sample!types:!!
• Nasal!swab!for!MRSA!
• Blood!culture!for!Gram!
positive!/!negative!assay!
• Rectal!swab!for!
Enterococcus!feasible;!
bacterial!suspension!of!
isolates!
!
streptavidin;HRP!conjugated!
probes,!followed!by!incubation!
with!colorimetric!substrate;!
nitrocellulose!strip!pre;coated!
with!hybridization!probe!used!
to!visualize!products!
!
IC!present!on!pre;coated!
nitrocellulose!strips!
Thermal!cycler!
For!some!assays:!GenoLyse®!or!
GenoXtract®!instruments!for!DNA!
extraction!
!
and!incubators!
GenoType!Enterococcus!(59)!
!
Out!of!105!isolates!tested,!with!various!vancomycin!resistance!
genotypes!(i.e.!vanA,!vanB!or!vanC),!100%!were!correctly!
identified!by!the!GenoType!Enterococcus!test!vs.!99%!correctly!
identified!using!VITEK!(bioMérieux)!!
GenoType!BC!grampositive!/!GenoType!BC!gramnegative!(two!
separate!test!strips)!(60)!
!
NOTE:"Only"vancomycin"and"methicillin"resistance"markers"data"
included"in"summary!
!
99%!concordance!with!Gram!positive!cocci!determined!to!be!
resistant!to!vancomycin!or!methicillin.!
!
Sequences!
corresponding!to!Gram!
positive!cocci!and!Gram!
negative!rods!as!well!as!
resistance!markers!to!
vancomycin!and!
methicillin:!
vanA,#vanB,#vanC1,#
vanC2/3,#mecA#
#
Internal!control!
sequences!also!detected!
!
TTR:!5!hrs.!
22!|!P a g e !
!
!
Platform&/&Mfr.&
Hain!Lifescience!/!
DNA!STRIP!
(continued)!
Workflow&
!
results!based!on!
!
Sample!types:!
• Sputum,!MTB!culture!
material!
Chemistry&
!
!
equipment&
Thermal!cycler!
extraction!
!
and!incubators!
!
Analytes&and&time&
to&result*&
GenoType!MTBDRplus#(61)&
&
Assay&against&culture&and&DST&(rifampicin&resistance)&from&smearQ
positive&samples:&
Extraction!by!GenoLyse!(Hain)!
Sensitivity:!100%!
Specificity:!92.3%!
Extraction!by!GenoXtract!(Hain)!
Sensitivity:!96.0%!
Specificity:!100%!
!
Assay&against&culture&and&DST&(rifampicin&resistance)&from&smearQ
negative&samples:&
Sensitivity:!96.0%!
Specificity:!93.0%!
Sensitivity:!86%!
Specificity:!100%!
!
Assay&against&culture&and&DST&(isoniazid&resistance)&from&smearQ
positive&samples:&
Sensitivity:!96.7%!
Specificity:!87.5%!
Sensitivity:!100%!
Specificity:!100%!
!
Assay&against&culture&and&DST&(isoniazid&resistance)&from&smearQ
negative&samples:&
Sensitivity:!96.7%!
Specificity:!90.0%!
Sensitivity:!90.6%!
Specificity:!71.4%!
Mycobacterium!
species;specific!
markers!as!well!as!
mutations!in!the!rpoB!
gene!and!mutations!
in!the!katG!genes!and!
inhA!gene!promoter!
for!resistance!to!
rifampicin!and!
isoniazid!respectively!
!
IC!sequences!also!
detected!
!
TTR:!5!hrs.!
23!|!P a g e !
!
!
Platform&/&Mfr.&
Hain!Lifescience!/!
DNA!STRIP!
(continued)v!
Workflow&
!
results!based!on!
!
Sample!types:!
• Sputum,!MTB!culture!
material!
Chemistry&
!
!
equipment&
Thermal!cycler!
extraction!
!
and!incubators!
GenoType!MTBDRsl&
&
Study&#1&(62)&Q&assay&against&DST&using&the&MGIT®&system&(BD):&
Fluoroquinolones!resistance:!
Aminoglycosides!resistance:!
Sensitivity:!100%!
Specificity:!100%!
Ethambutol!resistance:!
!
Study&#2&(63)&Q&assay&against&DST&using&the&agar&proportion&method&or&the&
MGIT®&system&(BD):&
Fluoroquinolones!resistance:!
Sensitivity:!85.1%!
Specificity:!100%!
Aminoglycosides!resistance:!
Sensitivity:!43.2%!;!84.2%!
Specificity:!97.3%!;!100%!
Ethambutol!resistance:!
Sensitivity:!56.2%!
Specificity:!100%!
TM!
BD!MAX MRSA!Assay!(64)!
!
Sensitivity:!93.0%!(95%!CI:!87.9%,!96.0%)!
Specificity:!95.9%!(95%!CI:!94.8%,!96.7%)!
TM
TM
BD !/!BD!MAX !
automated;!user!loads!
sample,!reagent!cartridges!
and!assay!consumables!on!to!
the!instrument;!software!
!
Sample!types:!
• Nasal!swab!for!MRSA!
• Rectal!swab!for!CRE!
!
each!sample)!
TM
BD!MAX :!Self;contained!single!
acid!purification,!real;time!PCR!
amplification!and!detection!!
!
Vortex!mixer!and!micropipettes,!
interfaced!PC!
!
BD!MAX™!MRSA!XT#Assay!(65)&
&
Assay&against&direct/enriched&culture:&
Sensitivity:!93.1%!(95%!CI:!88.1!;!96.1)!
Specificity:!97.5%!(95%!CI:!96.8!;!98.1)!
!
Assay&against&direct&culture:&
%!Pos!Agreement:!96.5%!(95%!CI:!92.0!;!98.5)!
%!Neg!Agreement:!96.9%!(95%!CI:!96.1!;!97.6)!
TM
BD!MAX !CRE!Assay!(66)&
&
Assay&against&selective&culture&and&alternative&PCR:&
%!Pos!Agreement:!100%!(3/3)!
%!Neg!Agreement:!94.6%!(226/239)!
!
Contrived&samples&using&known&strains:&
18/18!detected!
!
Contrived&negative&samples:&
0/3!detected!
Analytes&and&time&
to&result&&
MTB!sequence,!
mutations!in!the!gyrA!
gene!for!resistance!to!
fluoroquinolones,!
mutations!in!the!rrs!
gene!for!resistance!to!
aminoglycosides!and!
mutations!in!the!
embA,!embB!and!
embC!genes!for!
resistance!to!
ethambutol!
!
IC!sequences!also!
detected!
!
TTR:!5!hrs.!
SCCmec#cassette!at!
orfX#junction!and!
mecA#gene!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
SCCmec#cassette!at!
orfX#junction!mecA,#
mecC#genes!!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
Carbapenemase!
genes!blaKPC,!!
blaOXA;48!or!blaNDM!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
24!|!P a g e !
!
!
Platform&/&Mfr.&
TM
TM
BD !/!BD!MAX !
Abbott!/!IRIDICA!
Workflow&
automated;!user!loads!
sample,!reagent!cartridges!
and!assay!consumables!on!to!
the!instrument;!software!
!
Sample!types:!
difficile!
User!adds!sample!and!
transfers!between!modules!
for!nucleic!acid!extraction;!
PCR!and!ESI/MS;!instrument!
software!provides!results!
interpretation.!
!
Sample!type:!
• Direct!blood!specimen!
Chemistry&
!
each!sample)!
PCR!amplification!of!targets!
followed!by!amplicon!detection!
and!identification!via!ESI/MS!
*Time!to!result!is!approximate!based!on!publically!available!information!
equipment&
TM
BD!MAX :!Self;contained!single!
acid!purification,!real;time!PCR!
amplification!and!detection!!
!
Vortex!mixer!and!micropipettes,!
interfaced!PC!
Bead!beater!and!extraction!
module!(sample!prep)!
Thermal!cycler,!de;salter,!mass!
spectrometer!
!
Micropipettors!and!interfaced!PC!
TM
BD!MAX !StaphSR!Assay!(67)&
&
MRSA:!
Sensitivity:!93.1%!(95%!CI:!88.1%,!96.1%)!
Specificity:!97.5%!(95%!CI:!96.8%,!98.1%)!
!
Staphylococcus#aureus:#
Sensitivity:!92.0%!(95%!CI:!89.7!;!93.9)!
Specificity:!93.1%!(95%!CI:!91.8!;!94.2)!
!
MRSA:!
%!Pos!Agreement:!96.5%!(95%!CI:!92.0,!98.5)!!
%!Neg!Agreement:!96.9%!(95%!CI:!96.1,!97.6)!
!
Staphylococcus#aureus:#
%!Pos!Agreement:!95.1%!(95%!CI:!93.1,!96.6)!
%!Neg!Agreement:!90.9%!(95%!CI:!89.5,!92.1)!
TM
BD!MAX !Cdiff!Assay!(68)&
&
Sensitivity:!87.7%!(95%!CI:!83.6!–!91.0)!
Specificity:!96.8%!(95%!CI:!95.8!;!97.6)!
!
%!Pos!Agreement:!96.5%!(95%!CI:!93.0!;!98.3)!
%!Neg!Agreement:!92.7%!(95%!CI:!91.4!;!93.9)!
!
Assay&against&an&FDAQcleared&PCR&test:&
%!Pos!Agreement:!99.1%!(95%!CI:!94.9!;!99.8)!
%!Neg!Agreement:!97.4%!(95%!CI:!95.7!;!98.4)!
!
Assay&against&a&second&FDAQcleared&PCR&test:&
%!Pos!Agreement:!95.5%!(95%!CI:!92.1!;!97.5)!
%!Neg!Agreement:!98.8%!(95%!CI:!97.9!;!99.3)!
BAC/BSI!(69)!
Sensitivity:!88%!
Specificity:!63%!
Analytes&and&time&
to&result&&
SCCmec#cassette!at!
orfX#junction,!mecA,#
mecC#genes,!nuc!
gene!as!a!S.#aureus!
marker!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
gene#
tcdB!
!
IC!sequence!also!
detected!
!
TTR:!2;3!hrs.!
Panel!of!over!780!
bacterial!and!fungal!
agents;!includes!
resistance!markers!
mecA,!vanA,!vanB!
and!kpc#
#
IC!sequences!also!!
TTR:!8!hrs.!
25!|!P a g e !
!
!
Commercially+Available+Technology+Landscape+
Phenotypic+based0systems0
Phenotypic,based!systems!detecting!AMR!organisms!were!analyzed!based!on!workflow,!chemistry!and!
the!analytes!detected!by!each!product.!!Phenotypic!systems!can!be!further!subdivided!into!those!that!
are! culture,based! and! require! expansion! of! viable! organisms! or! AMR! isolates! or!
biochemical/immunoassay!based!where!the!test!detects!AMR!associated!antigens!and!metabolites.!!In!
addition,! platforms! developed! for! microbial! ID/AST! were! also! described.! ! The! following! were! used! to!
describe!the!mechanism!and!required!equipment!of!commercially!available!phenotypic,based!systems:!
•
•
•
Workflow!
Equipment!
Chemistry!
Summaries! are! presented! below! describe! chromogenic! agars,! immunoassays! (e.g.! agglutination! tests,!
rapid!C.%difficile!tests)!and!ID/AST!platforms.!!
Chromogenic+agars+
Chromogenic!agars!directly!identify!AMR!bacteria!based!on!a!color,forming!reaction!corresponding!to!a!
specific! organism.! ! Typically,! a! colorless,! soluble! substrate! is! enzymatically! cleaved! by! the! target!
organism! and! results! in! a! colored,! insoluble! metabolite! that! precipitates! with! the! colony.! ! Different!
substrates!are!used!to!detect!different!pathogens.!
Several! commercially! available! media! targeting! AMR! pathogens! are! listed! below! in! table! 3.! ! The! end,
user!simply!plates!the!clinical!sample!and!incubates!for!24!–!48!hours!and!observes!the!plate!to!see!if!
colonies! corresponding! to! the! target! organism! grow.! ! Required! equipment! consists! of:! micropipettes,!
vortex!mixers!and!incubators.!
Table&3:&Chromogenic&agars&
Mfr.&
Assay&
Target&
organism&
TM
TM
BD !
BBL !
CHROMagar®!
MRSA!II!(70)!
MRSA!
TM
BBL !
TM
CHROMagar !VRE!
(71)!
Bio,Rad®!
TM
MRSAselect !(72)!
VRE!
MRSA!
Performance&
Test&against&Cefoxitin&disk:&
%!Pos!Agreement:!92.0%!(95%!CI:!86.7!,!95.7)!
%!Neg!Agreement:!99.9%!(95%!CI:!99.5!,!100)!
!
Test&against&culture&sites&1&&&2:&
%!Pos!Agreement:!92.0%!(95%!CI:!84.8!,!96.5)!
%!Neg!Agreement:!98.8%!(95%!CI:!97.8!,!99.5)!
!
Test&against&culture&site&3:&
%!Pos!Agreement:!90.2%!(95%!CI:!78.6!,!96.7)!
%!Neg!Agreement:!98.9%!(95%!CI:!96.8!,!99.8)!
Test&against&BEAV&culture,&48&hr&incubation:&
Sensitivity:!99.1%!(95%!CI:!95.2–100)!!
Specificity:!94.8%!(95%!CI:!92.2–96.8)!
Sensitivity:!91.7%!(95%!CI:!87.3!,!94.7)!
Specificity:!99.4%!(95%!CI:!98.5!,!99.8)!
!
!
!
Table&3:&Chromogenic&agars&
Mfr.&
Assay&
Target&
organism&
TM
ChromID !MRSA!
(73)!
MRSA!
TM
ChromID !VRE!
(74)!
VRE!
TM
ChromID !CARBA!
(75)!
CRE!
bioMérieux®!
TM
ChromID !ESBL!
(76)!
ESBL!
TM
Thermo!
Scientific!
ChromID !!
C.%difficile!(77)!
C.%difficile%
Spectra™!MRSA!
(78)!
MRSA!
TM
Spectra !VRE!(79)!
TM
Brilliance !CRE!
(80)!
VRE!
CRE!
TM
Brilliance !ESBL!
(81)!
ESBL!
Performance&
24&hr&incubation:&&
%!Pos!Agreement:!94.22%!(95%!CI:!90.90!,!96.60)!
%!Neg!Agreement:!97.20%!(95%!CI:!95.96!,!98.15)!
24&hr&incubation:&&
97.1%!(95%!CI:!94.7!,!98.6)!
99.7%!(95%!CI:!99.1!,!99.9)!
!
48&hr&incubation:&&
96.9%!(95%!CI:!94.5!,!98.4)!
99.7%!(95%!CI:!99.1!,!99.3)!
Sensitivity:!96.5!%!
Specificity:!91.2!%!!
24&hr&incubation:&
Sensitivity:!88.0%!(95%!CI:!72.3!–!95.3)!
Specificity:!94.0%!(95%!CI:!92.5!–!95.9)!
!
48&hr&incubation:&
Sensitivity:!94.0%!(95%!CI:!80.0!–!98.4)!
Specificity:!90.5%!(95%!CI:!88.0!–!92.4)!
24&hr&incubation:&&
Sensitivity:!58.3%!(95%!CI:!40.8,74.5)!
Specificity:!99.3%!(95%!CI:!96.3,100)!
!
48&hr&incubation:&&
Sensitivity:!100%!(95%!CI:!90.3,100)!
Sensitivity:!90.6%!(95%!CI:!84.7,94.8)!
Test&against&standard&culture:&&
%!Pos!Agreement:!96.2%!(95%!CI:!91.9–98.6)!
%!Neg!Agreement:!100%!(95%!CI:!99.2–100)!
&
Test&against&PBP2’&agglutination:&&
%!Pos!Agreement!:!97.4%!(95%!CI:!93.4–99.3)!!
%!Neg!Agreement!:!99.0%!(95%!CI:!97.4–99.6)!
&
Test&against&oxacillin&MIC:&&
%!Pos!Agreement:!96.2%!(95%!CI:!91.9–98.6)!!
%!Neg!Agreement:!100%!(95%!CI:!99.2–100)!
Assay&against&standard&culture&methods,&performance&for&E.#faecium&
and&E.#faecalis&isolates:&&
E.%faecium:%
%!Pos!Agreement:!99.0%!(95%!CI:!96.3–99.9)!
%!Neg!Agreement:!99.8%!(95%!CI:!98.7–100)!
E.%faecalis:%
%!Pos!Agreement:!100%!(95%!CI:!88.1–100)!
%!Neg!Agreement:!100%!(95%!CI:!99.4–100)!
Overall!sensitivity:!94%!(against!all!isolates)!!
Sensitivities!per!carbapenemase!gene:!
• 100%!for!blaKPC,!blaNDM!and!blaGIM!
• 90%!for!blaVIM!
• 84!%!for!blaOXA,48!
Specificity:!71%!!
24&hr&incubation:&
Sensitivity:!94.9%!(95%!CI:!92.1–97.8)!
Specificity:!95.1%!(95%!CI:!94.1–96.1)!
27!|!P a g e !
!
!
Immunoassays+
Table! 4! lists! representative! immunoassays! relevant! to! AMR.! ! Immunoassays! rely! upon! monoclonal!
antibodies! against! AMR! markers,! such! as! C.% difficile! toxin! A! or! the! MRSA! /! MR,CoNS! penicillin! binding!
protein.!!Readouts!are!manually!interpreted!using!consumables!provided!by!the!manufacturer.!!All!tests!
listed! below! are! rapid! and! require! only! ancillary! laboratory! equipment! (e.g.! micropipettes! or! transfer!
pipettes).!
Table&4:&Immunoassays&
Assay&/&Mfr.&
Target&
organism&
Penicillin!Binding!Protein!(PBP2f)!
Latex!Agglutination!Test!/!Thermo!
Scientific!(Oxoid)!(82)!
MRSA!
Clearview®!Exact!PBP2a!Test!/!
TM
Alere !(83)!
MRSA!
C.!DIFF!QUIK!CHEK!COMPLETE®!
TM
test!/!Alere !(84)!
C.%difficile%
Xpect®!Clostridium!difficile!Toxin!
A/B!Test!/!Thermo!Scientific!
(Oxoid)!(85)!
C.%difficile%
ImmunoCard®!Toxins!A&B!/!
Meridian!Bioscience®!(86)!
C.%difficile%
Mechanism&/&Chemistry&
Kit!reagents!and!consumables!provided!
for!bacterial!cell!lysis!and!reaction;!latex!
particles!coated!with!mAb!against!PBP!
agglutinate!in!presence!of!PBP;!end,user!
visually!observes!agglutination!against!
positive!and!negative!controls!
Kit!reagents!and!consumables!provided!
for!bacterial!cell!lysis!and!reaction;!latex!
particles!coated!with!mAb!against!PBP!
agglutinate!in!presence!of!PBP;!end,user!
visually!observes!agglutination!against!
positive!and!negative!controls!
Sample!is!loaded!into!assay!consumable!
using!a!transfer!pipette.!!Lateral!flow!
type!test!utilizing!antibodies!against!C.%
difficile!glutamate!dehydrogenase!and!
toxins!A!and!B.!!The!end,user!mixes!
diluent!and!conjugate!reagents!and!
manually!adds!the!sample.!!The!end,
user!manually!interprets!the!results!
based!on!the!IFU!
using!a!transfer!pipette.!!Lateral!flow!
type!test!utilizing!antibodies!against!C.%
difficile!glutamate!dehydrogenase!and!
toxins!A!and!B.!!The!end,user!mixes!
diluent!and!conjugate!reagents!and!
manually!adds!the!sample.!!The!end,
user!manually!interprets!the!results!
based!on!the!IFU!
test!and!control!wells!using!a!transfer!
pipette.!!Lateral!flow!type!test!utilizing!
antibodies!against!C.%difficile!toxins!A!
and!B.!!The!end,user!mixes!diluent!and!
conjugate!reagents!and!manually!adds!
the!sample.!!The!end,user!manually!
interprets!the!results!based!on!the!IFU!
Performance&
Sensitivity!and!specificity!>95%!against!
various!culture!methods!!
Sensitivity!and!specificity!>98%!against!
various!culture!methods!!
Test&against&bacterial&culture:&&
Sensitivity!90.5%!(95%!CI:!85.7!,!93.9)!
Specificity!93.1%!(95%!CI:!91.2!,!94.7)!
!
Test&against&tissue&cultureKbased&cytotoxin&
assay:&&
Sensitivity!87.8%!(95%!CI:!81.4!,!92.3)!
Specificity!99.4%!(95%!CI:!98.6!,!99.7)!
Test&against&cytotoxin&assay:&
Sensitivity:!86.3%!(95%!CI:!79.8!–!91.3)!
Specificity:!96.2%!(95%!CI:!94.5!–!97.5)!
Assay&against&cytotoxin&assay,&prospective&
samples:&&
Sensitivity:!93.1%!(95%!CI:!87.1,98.9)!
Specificity:!98.9%!(95%!CI:!97.6,100)!
!
Assay&against&cytotoxin&assay,&
retrospective&samples:&&
Sensitivity:!100%!(95%!CI:!N/A)!
Specificity:!98.4%!(95%!CI:!96.4,99.6)!
ID/AST+Platforms+
ID/AST!platforms!are!automated!and!enable!to!the!end,user!to!identify!bacterial!isolates!and!determine!
susceptibility! to! numerous! antimicrobial! drugs! within! a! single! run.! ! These! platforms! are! used! to! guide!
28!|!P a g e !
!
!
treatment! decisions! and! are! often! used! in! concert! with! pathogen! detection! assays.! ! Workflow,!
chemistry!and!required!equipment!are!presented!in!table!5!for!fully!automated!ID/AST!instruments.!
Table&5:&Automated&ID/AST&systems&
Platform&/&
Workflow&/&Chemistry&
Mfr.&
User!dilutes!isolate!and!sets!up!the!
instrument!along!with!a!consumable!
pre=loaded!with!reagents!for!specific!
organisms!and!drug!resistance.!!
Diluted!culture!isolate!and!
consumable!are!loaded!onto!the!
instrument;!liquid!handling,!
incubation!and!growth!monitoring!
based!on!light!absorption!are!fully!
automated.!!Instrument!software!
provides!organism!ID!and!drug!
susceptibility.!
VITEK®!/!
bioMérieux!
Microscan!
WalkAway®!/!
Siemens!
User!dilutes!isolate!and!uses!
consumable!and!pipetting!device!
provided!by!the!manufacturer!to!
inoculate!a!microtitre!plate!pre,
loaded!with!reagents!for!specific!
organisms!and!drug!resistance.!!
Inoculated!microtitre!plate!is!loaded!
onto!the!instrument;!incubation!and!
growth!monitoring!based!on!turbidity!
are!fully!automated.!!Instrument!
software!provides!organism!ID!and!
drug!susceptibility.!
Required&
Equipment*&
VITEK!instrument;!
interfaced!PC!and!
bar!code!scanner.!
Microscan!WalkAway!
instrument!
interfaced!with!PC!
and!bar!code!
scanner.!!!
Drugs&that&can&be&tested&for&susceptibility**&
Amikacin!
Amoxicillin!
Ampicillin!
Aztreonam!
Cefalotin!
Cefazolin!
Cefepime!
Cefotaxime!
Cefotetan!
Cefoxitin!
Cefpodoxime!
Ceftazidime!
Ceftizoxime!
Ceftriaxone!
Cefuroxime!
Ciprofloxacin!
Clavulanic!Acid!
Amikacin!
Amoxicillin!
Amoxicillin/K!
Clavulanate!
Ampicillin!
Ampicillin/!
Sulbactam!
Azithromycin!
Aztreonam!
Cefazolin!
Cefepime!
Cefixime!
Cefotaxime!
Cefoxitin!
Cefpodoxime!
Ceftaroline!
Ceftazidime!
Ceftriaxone!
Cefuroxime!
Cephalothin!
Chloramphenicol!
Ciprofloxacin!
Clarithromycin!
Clavulanate!
Clindamycin!
Colistin!
Clindamycin!(new!formulation)!
Daptomycin!
Doripenem!
Doxycycline!
Ertapenem!
Erythromycin!
Gentamicin!
Gentamicin!High!Level!Synergy!
Imipenem!
Inducible!Clindamycin!
Resistance!(ICR)!
Levofloxacin!
Linezolid!
Meropenem!
Moxifloxacin!!
Nalidixic!Acid!
Nitrofurantoin!
Daptomycin!!
Doripenem!
Ertapenem!
Erythromycin!
Fosfomycin!
Fusidic!Acid!
Gentamicin!
Gentamicin!Synergy!Screen!
Imipenem!
Inducible!Clindamycin!Test!
Kanamycin!
Kanamycin!Synergy!Screen!
Levofloxacin!
Lincomycin!
Linezolid!
Mecillinam!
Meropenem!
Minocycline!
Moxifloxacin!
Mupirocin!
Nalidixic!Acid!
Netilmicin!
Nitrofurantoin!
Norfloxacin!
Ofloxacin!
Oxacillin!
Norfloxacin!
Oxacillin!
Piperacilllin!
Rifampicin!
Streptomycin!High!Level!
Synergy!
Sulbactam!
Sulfamethoxazole!
Tazobactam!
Tetracycline!
Ticarcillin!
Tigecycline!
Tobramycin!
Trimethoprim/!
Sulfamethoxazole!
Vancomycin!
Penicillin!
Piperacillin!
Piperacillin/Tazobactam!
Pristinamycin!
Rifampin!
Streptomycin!
Synercid!
Teicoplanin!
Tetracycline!
Ticarcillin!
Ticarcillin/K!Clavulanate!
Tigecycline!
Tobramycin!
Trimethoprim!
Trimethoprim/Sulfameth
oxazole!
Vancomycin!
*Ancillary!equipment!such!as!incubators,!micropipettors,!vortex!mixers,!etc.!are!required!for!organism!isolation!from!patient!sample.!
**Not!a!complete!list!of!manufacturer!offerings!
29!|!P a g e !
!
!
Table&5:&Automated&ID/AST&systems&
Platform&/&
Workflow&/&Chemistry&
Mfr.&
Sensititre!ARIS®!
2X!/!Thermo!
Scientific!
User!dilutes!isolate!and!measures!
culture!density!with!a!nephalometer!
supplied!by!the!manufacturer.!!An!
electronic!pipette!used!to!inoculate!a!
microtitre!plate!pre,loaded!with!
reagents!for!specific!organisms!and!
drug!resistance.!!Inoculated!
microtitre!plate!is!loaded!onto!the!
instrument;!incubation!and!growth!
monitoring!are!fully!automated.!!
Instrument!software!provides!
organisms!ID!and!drug!susceptibility.!
Required&
Equipment*&
Sensititre!ARIS!2X!
instrument!
interfaced!with!PCR!
and!bar!code!
scanner.!!Electronic!
pipette!and!
nephalometer!for!
microtitre!plate!
inoculation.!
!
Drugs&that&can&be&tested&for&susceptibility**&
Amikacin!
Amoxicillin/!
clavulanic!acid!
Ampicillin!
Ampicillin/!
Sulbactam!
Azithromycin!
Aztreonam!
Cefazolin!
Cefepime!
Cefotaxime!
Cefotaxime/!
Clavulanic!Acid!
Cefotetan!
Cefoxitin!
Cefoxitin!Screen!
Cefpodoxime!
Ceftaroline!
Ceftazidime!
Ceftazidime/!
Clavulanic!Acid!
Ceftriaxone!
Cefuroxime!
Cephalothin!
Ampicillin!
Azithromycin!
Cephalosporins!
Chloramphenicol!
Ciprofloxacin!
Clarithromycin!
Clindamycin!
Erythromycin!
Gatifloxacin!
Gentamycin!
Glycopeptides!
Grepafloxacin!
Levofloxacin!
Linezolid!
Lomefloxacin!
Macrolides!
Chloramphenicol!
Ciprofloxacin!
Clindamycin!
Colistin!
Daptomycin!
Doripenem!
Doxycycline!
D,Test!
Ertapenem!
Erythromycin!
Florfenicol!
Gatifloxacin!
Gentamicin!
Imipenem!
Levofloxacin!
Linezolid!
Meropenem!
Metronidazole!
Mezlocillin!
Minocycline!
Moxifloxacin!
Nalidixic!Acid!
Nitrofurantoin!
Oxacillin!
Moxifloxacin!
Norfloxacin!
Ofloxacin!
Oxacillin!
Pefloxacin!
Penicillin!G!
Quinipristin/Dalfopristin!
Rifampin!
Streptomycin!
Tetracycline!
Trimethoprim/!
Sulfamethoxazole!
Trovafloxacin!
Vancomycin!
Penicillin!
Piperacillin!
Piperacillin/Tazobactam!
Polymyxin!B!
Quinupristin/dalfopristin!
Rifampin!
Streptomycin!
Sulfamethoxazole!
Telavancin!
Telithromycin!
Tetracycline!
Ticarcillin/Clavulanic!Acid!
Tigecycline!
Tobramycin!
Trimethoprim/!
Sulfamethoxazole!
Vancomycin!
!
User!dilutes!isolate!and!
measures!culture!density!with!a!
nephalometer!supplied!by!the!
Phoenix!
manufacturer.!!The!diluted!
Automated!
culture!is!poured!into!a!
Microbiology!
TM
Phoenix !
consumable!pre,loaded!with!
System!interfaced!
Automated!
reagents!specific!for!the!
with!PC!and!bar!
Microbiology!
organism!or!drug!resistance.!!The!
code!scanner.!!
System!/!BD!
consumable!is!then!loaded!onto!
Vortex!mixer!and!
the!instrument;!incubation!and!
nephalometer!for!
growth!monitoring!are!fully!
culture!dilution.!
automated.!!Instrument!software!
provides!organisms!ID!and!drug!
susceptibility.!
User!inoculates!MGIT!medium!
!
!
culture!tube!supplemented!with!
different!antibiotics,!with!growth!
being!indicative!of!drug!
TM
BACTEC !
resistance.!!Inoculated!culture!
BACTEC!MGIT!960! Rifampin!
Isoniazid!
TM
MGIT !960!
tube!is!loaded!onto!the!
System!interfaced!
Ethambutol!
System!
instrument;!incubation!and!
with!PC.!
Streptomycin!
fluorometric,based!growth!
monitoring!are!fully!automated.!!
Instrument!software!interprets!
results.!
*Ancillary!equipment!such!as!incubators,!micropipettors,!vortex!mixers,!etc.!are!required!for!organism!isolation!from!patient!sample.!
**Not!a!complete!list!of!manufacturer!offerings!
!
Performance+Attributes+of+Phenotypic+Systems+
The! performance! attributes! for! phenotypic,based! AMR! assays! are! summarized! above! in! tables! 3! ,! 5.!!
Tests!that!detect!specific!resistant!organisms!are!listed,!and!also!included!are!ID/AST!systems,!which!can!
30!|!P a g e !
!
!
be! considered! complimentary! to! those! tests! aimed! at! detecting! pathogens! and! are! not! necessarily!
aimed!at!providing!treatment!decisions.!
As! with! molecular! assays,! the! same! critical! attributes! for! the! developing! world! apply! to! phenotypic,
based!tests:!quality!affordability,!sensitivity!and!specificity,!ease!of!use,!rapid!time!to!result!and!minimal!
equipment!and!storage!requirements!(27,!28).!!Detailed!comparisons!of!these!critical!attributes!as!they!
relate!to!phenotypic!vs.!molecular!tests!are!also!described!in!the!Differentiators!section.!
!
!
31!|!P a g e !
!
!
Commercially+Available+Technology+Landscape+
Differentiators0
Genotypic+vs.+Phenotypic:+
Chromogenic!agars!of!the!type!presented!in!Table!3!are!designed!to!detect!target!organisms!in!a!single!
step! from! mixed! flora! clinical! sample! ,! akin! to! molecular! tests.! ! Table! 6! highlights! strengths! and!
weaknesses!of!chromogenic!agars!relative!to!molecular!tests.!!One!advantage!of!chromogenic!agars!is!
that!they!can!detect!organisms!for!which!the!design!of!a!molecular!test!would!be!difficult.!!For!instance,!
the! numerous! variations! and! number! of! targets! encompassed! by! ESBL! Enterobacteriaceae! would!
require! multiple! primer/probe! combinations,! while! a! phenotypic! test! need! only! provide! a! single!
selection!method!(growth!medium!and!antibiotic!concentration).!!In!addition,!while!AMR!mechanisms!
that!cannot!easily!be!linked!to!a!species!using!molecular!test!can!be!linked!by!the!single,step!growth!and!
identification!of!viable!organisms!on!chromogenic!agar!plates.!!For!example,!detecting!the!vanA!or!vanB!
gene! usually! infers! VRE,! it! is! possible! that! the! resistance! marker! could! be! harbored! by! a! different!
species;!this!occurs!somewhat!frequently!with!vanB!detection,!which!is!often!found!in!non,Enterococcus!
gut!anaerobes!(i.e.!not!Enterococci)!(87,!88).!!!
In!general,!the!analytical!sensitivity!(Limit!of!Detection)!of!molecular!tests!is!expected!to!be!lower!(more!
sensitive)!than!chromogenic!agar;!organism!viability!is!also!not!required!for!molecular!detection,!which!
may!be!an!advantage!if!clinical!sample!handling!and!storage!is!a!concern.!!The!time!to!result!is!faster!
with! molecular! tests! and! with! some! platforms,! manual! recording! of! results! would! not! be! required.!!
Likewise! more! comprehensive! information! can! be! provided! by! molecular! tests! if! the! mechanism! of!
resistance!is!known.!!For!example,!molecular!tests!for!MRSA!can!be!designed!that!specifically!detect!the!
mecC!variant!of!mecA!(89).!!Differentiating!these!with!chromogenic!agar!is!not!likely!to!be!possible!using!
a! single! antibiotic! concentration,! but! is! easily! done! with! allele,specific! probes! labeled! with! different!
fluorophores.!!While!specific!strain!typing!and!epidemiological!studies!would!be!required!to!determine!
the!prevalence!of!different!strains!for!infection!control!purposes,!the!detection!of!unique!markers!such!
as!mecC!serves!as!a!starting!point.!
Immunoassays! have! slightly! lower! sensitivities! compared! to! molecular! tests! and! chromogenic! agars,!
however!the!examples!provided!in!Table!4!are!rapid!and!relatively!easy!to!use,!which!makes!them!ideal!
for! more! resource! challenged! environments.! ! If! the! goal! of! testing! is! for! screening! patients,! then!
immunoassays! have! great! value! in! that! negative! patients! can! be! rapidly! identified,! with! additional!
testing! being! applied! to! the! fraction! of! patients! testing! positive.! ! Table! 6! highlights! the! strengths! and!
weaknesses!of!immunoassays!compared!with!molecular!tests!and!chromogenic!agars.!
Table&6:&Comparison&of&phenotypic&tests&to&molecular&tests&
Attribute&
Molecular&test&
Chromogenic&agar&
Sensitivity!
In!general!>90%!sensitivity!
In!general!>90%!sensitivity!(after!
48!hr.!inc.)!
Immunoassay&
Slightly!lower!than!molecular!
tests!and!chromogenic!agars,!
with!most!assays!being!>90%!
sensitive,!some!>85%!
sensitive!
32!|!P a g e !
!
!
Table&6:&Comparison&of&phenotypic&tests&to&molecular&tests&
Attribute&
Molecular&test&
Specificity!
In!general!>90%!specificity!
Time!to!result!
<!8!hours!depending!on!platform!
Moderate!to!high!complexity!
depending!on!platform;!some!
platforms!more!difficult!than!
chromogenic!agar!plates!
Ease!of!use!
Reagent!robustness!
Cold!chain!required!for!most!
platforms!
Chromogenic&agar&
In!general!>90%!specificity!(after!
48!hr.!inc.)!
24!–!48!hours!
Basic!micro!techniques;!visual!
results!interpretation!and!
manual!recording!of!results!
Refrigeration!of!culture!plates,!
shorter!shelf!life!
Immunoassay&
In!general,!>90%!specific!
Rapid,!<!1!hour!
Basic! laboratory! skills;! visual!
results! interpretation! is!
straightforward!
Refrigeration!
of!
most!
reagents,! some! reagents! and!
consumables!can!be!stored!at!
room!temperature!
!
Differentiators+among+Molecular+Tests+and+Platforms+
Below! is! a! link! to! a! slide! deck! sorted! by! platform! that! lists! the! strengths! and! weaknesses! of! the!
molecular!assays!listed!in!Table!2.!!The!following!attributes!are!included!in!the!analysis:!
•
•
•
•
•
•
Ease!of!use,!including!pre/post,processing!requirements!
System!automation!and!integration,!including!requirements!for!data!analysis!
Time!to!result!
Analyte!profile!/!AMR!menu!
Equipment/resource!requirements!
Storage!requirements!
[Molecular!testing!overview]!
Below!is!a!link!to!a!sortable!spreadsheet!provided!as!a!supplement!for!comparing!different!platforms!
and!assays.!
[Sortable!platform!worksheet]!
!
!
33!|!P a g e !
!
!
Laboratory+Developed+Tests+(LDTs)+D+Technology+Landscape+
LDTs,!colloquially!referred!to!as!“home!brews”,!are!assays!developed!and!verified!for!their!performance!
characteristics! by! a! clinical! testing! laboratory.! ! The! FDA! has! declared! that! it! has! the! legal! right! and!
jurisdiction! to! regulate! LDTs.! However,! to! date! it! has! only! in! rare! occurrence! utilized! its! oversight!
mandate.!This!is!in!the!processes!of!changing!and!the!FDA!has!proposed!(90)!that!LDTs!be!regulated!in!a!
risk! based! manner! and! that! clinical! labs! utilizing! LDTs! maintain! a! quality! system! similar! to! what! is!
currently! required! under! 21CR! Part! 820! and! ISO13485.! Until! oversight! is! initiated! clinical! laboratories!
have! self,regulated! themselves! via! following! CLIA! regulations! in! which! it! is! stated! that! performance!
characteristics!must!be!verified!by!the!clinical!laboratory!and!that!processes!must!be!in!place!to!ensure!
the!consistent!performance!of!the!LDT.!As!the!wording!in!the!CLIA!regulations!is!vague,!the!manner!in!
which!clinical!laboratories!oversees!their!LDTs!varies!from!laboratory!to!laboratory.!!EU!rules!state!that!
LDTs! meet! the! definition! of! an! IVD,! but! exemptions! are! made! for! certain! LDTs! –! those! that! are!
manufactured! and! utilized! in! the! same! health! institution! without! being! transferred! to! another! entity.!!
Similar! to! the! US! FDA,! EU! rules! are! limiting! the! scope! of! this! exemption! to! those! institutions! in!
compliance!with!ISO!15189!or!an!equivalent!recognized!standard!(91).!
In!light!of!these!pending!changes!in!LDT!governance,!the!availability!of!cleared!IVD!kits!becomes!more!
critical! in! combating! AMR.! ! A! search! of! the! Infectious! Diseases! Test! Directory! provided! by! AMP! (92)!
shows!that!several!LDTs!targeting!C.%difficile,!VRE!and!MRSA!are!available!and!summarized!in!Table!8.!!A!
test!is!also!available!for!Plasmodium!resistance!using!a!nested!PCR!to!detect!DHFR!for!pyrimethamine!
resistance.!!A!test!registry!review!of!major!testing!laboratories!shows!that!the!LDT!availability!parallels!
that!of!commercially!available!assays!(Table!9).!
Table&8:&LDTs&addressing&AMR&in&AMP&directory&
Organisms&
Tests&
PCR/gel!electrophoresis!,!multiplexed!real,time!PCR!
and!PCR!followed!by!PFGE!(typing)!detecting!mecA!
MRSA!
and!S.%aureus!markers!from!patient!swabs!and!
isolates!
PCR/gel!electrophoresis!and!real,time!PCR!assays!
Vancomycin,resistant!Enterococcus!(VRE)! detecting!vanA!and!vanB;!some!tests!also!detect!
vanC!(subtypes!1,!2,!and!3)!
Clostridium%difficile%
Real,time!PCR!tests!detecting!toxin!A!and!B!genes!
Nested!PCR!targeting!DHFR!for!pyrimethamine!
Plasmodium%falciparum%
resistance!
!
Table&9:&LDTs&addressing&AMR&available&at&major&testing&labs&
Organisms&
Tests&
MRSA!
mecA!gene!detection!
Vancomycin,resistant!Enterococcus!(VRE)! vanA/vanB!gene!detection!
tcdB!gene!detection,!can!also!be!a!reflex!test!
Clostridium%difficile%
following!positive!toxin!A/B!(EIA)/GDH!result!
MDR,TB!
Sequencing!to!ID!resistance!genes!
CRE!
PCR!detecting!blaKPC!and!blaNDM!
!
34!|!P a g e !
!
!
The!majority!of!lab!testing!that!addresses!AMR!is!conducted!via!routine!microbiology!using!disk!diffusion!
and!MIC!testing,!following!the!clinical!cutoffs!set!by!CLSI!(note:!EUCAST!guidelines!utilized!in!the!manner!
in!the!EU).!!Both!techniques!are!highly!dependent!on!the!skill!and!expertise!of!the!operator!and!require!
the!ability!to!culture!and!isolate!the!AMR!organism.!
Some! challenges! associated! with! disk! diffusion! include! data! interpretation! –! double! zones,! zones! with!
colonies! and! the! difficulty! distinguishing! between! a! mixed! bacterial! population! and! a! resistant! sub,
population.! ! Disk! diffusion! is! also! not! reliable! for! all! species! (e.g.% Staphylococcus! and! vancomycin!
susceptibility).!!Data!interpretation!is!perhaps!more!straightforward!when!conducting!MIC!testing,!but!
skill!is!required!when!generating!the!inoculum!density!of!the!isolate.!
Regardless!of!the!method,!interpretation!can!only!be!based!on!published!guidelines.!!If!a!CLSI!or!EUCAST!
guideline!does!not!exist!for!a!given!organism!or!antimicrobial,!then!the!laboratory!must!develop!its!own!
cutoffs.! ! Absent! guidelines! or! commercial! tests,! the! benefit! of! lab! defined! breakpoints! is! to! alleviate!
shotgun!approaches!to!treatment!that!may!result!in!the!over!use!of!antibiotics!and!hence!selection!of!
resistant!organisms.!
!
35!|!P a g e !
!
!
Prototype+Assay+Landscape+
A! literature! search! was! conducted,! focusing! on! the! organisms! listed! in! table! 1,! particularly! the! more!
difficult!targets!for!which!no!commercially!available!assay!or!LDT!are!available.!!Recently!(last!5!years)!
published! assays! that! are! rapid! (molecular! or! other! technology! that! delivers! results! in! <8! hours)! were!
selected! from! the! literature! search.! ! A! description! of! technology! and! chemistry,! as! well! as! pertinent!
performance!attributes!are!summarized!in!Table!10.!!
Table&10:&Prototype&assays&
Target&organism(s)&
MDR!Acinetobacter!and!
Pseudomonas!(93)!
Campylobacter!(94)!
Chemistry&and&Equipment&
Two!module!(i.e.!one!sample!
tested!in!two!tubes)!PCR!using!a!
touch!down!thermal!protocol.!!
Each!module!is!multiplex!
detection!of!carbapenemase!
genes!(resistance!markers);!PCR!
products!resolved!using!gel!
electrophoresis.!
!
Analytes!detected:!
Module!1:!blaNDM,1,!blaOXA,181/48,!
blaVIM,!blaIMP,!blaKPC!
Module!2:!blaOXA,23,!blaOXA,24,!
blaOXA,51,!blaOXA,58!
!
Standard!molecular!biology!
laboratory!equipment!including!
thermal!cycler!and!gel!imaging!
equipment!
MAMA,PCR!to!detect!(gel,based)!
a!single!C!to!T!point!mutation!in!
the!gyrA!gene,!which!confers!
ciprofloxacin!resistance.!
!
Required:!Standard!molecular!
biology!laboratory!equipment!
including!thermal!cycler!and!gel!
imaging!equipment.!
!
Boil!lysis!method!used!to!extract!
DNA.!
Performance&attributes&
Concordance!compared!to!
routine!microbiology!for!ID!and!
susceptibility!testing:!
97%!(86/88)!for!detection!of!
carbapenemases!
98%!(51/52)!for!metallo,beta!
lactamases!
100%!concordance!(between!all!
three!methods)!identifying!
resistance!in!C.%jejuni!isolates!
tested!by!this!assay,!MIC!testing!
and!direct!sequencing!of!the!
gyrA!gene.!
!
89/100!identified!correctly!as!
resistant,!11/100!identified!
correctly!as!sensitive!
36!|!P a g e !
!
!
Target&organism(s)&
Candida!(95)!
Escherichia%coli!(96)!
DNA!extracted!from!whole!blood!
using!QIAGEN®!!DNeasy®!kit.!
!
PCR!amplification!of!targets!
followed!by!hybridization!of!
amplicons!with!probes!labeled!
with!the!electroactive!compound!
ferrocene!and!detection!using!
differential!pulse!voltammetry.!
!
Species,specific!probes!linked!to!
detecting!drug!susceptible!and!
drug!resistant!Candida!species.!
!
including!thermal!cycler;!
equipment!required!for!
electrochemical!analysis.!
Series!of!singleplex!reactions!
amplifying!resistance!marker!
genes:!
Tetracycline!resistance:!tet(A),!
tet(B)!
Streptomycin!resistance:!aadA1!
Trimethoprim:!dfrA1!
Quinolones:!qnr!
Gentamicin:!aac(3),IV]!
Sulfonamides:!sul1!
Beta,lactams:!blaSHV,!blaCMY!
Erythromycin:!ere(A)!
Chloramphenicol:!catA1,!cmlA!
!
including!thermal!cycler!and!gel!
imaging!equipment!
LoD!is!10!CFU/mL!of!whole!blood!
Assay!correlates!well!to!
susceptibility!testing!performed!
using!routine!microbiology.!
!
Samples!taken!from!poultry!
identifying!potential!reservoir!of!
AMR!organisms!
37!|!P a g e !
!
!
Target&organism(s)&
MRSA!(97)!
Neisseria%gonorrhea%(98)%
Streptococcus!pneumoniae!(99)!
Oligo!probes!specific!for!mecA!
attached!to!gold!disk!electrodes;!
fragmented!gDNA!(no!PCR!
amplification)!hybridization!to!
probes!measured!using!
electrochemical!impedance!
spectroscopy!
!
Technology!could!be!applied!to!
PoC!systems!
Real,time!PCR!assay!(TaqMan!
probes)!specific!for!the!penA!
gene!of!N%gonorrhea!H401!
ceftriaxone,resistant!strain!!
!
Assay!performed!on!Rotor,
Gene®!real,time!PCR!instrument!
(QIAGEN®!)!
Real,time!PCR!using!a!standard!
curve!to!quantitate!S.%
pneumoniae!by!detecting!the!
lytA!gene!using!FRET!probes.!!
Gel,based!multiplex!PCR!to!
detect!penicillin!resistance!
markers:!pbp1a,!pbp2b,!pbp2x;!
macrolide!resistance!markers:!
ermB!and!mefA%
!
DNA!extracted!using!QIAamp®!
DNA!blood!minikit!(QIAGEN®!)!
!
Assay!performed!using!
LightCycler!real,time!PCR!
platform!(ROCHE)!
!
Also!required:!standard!
molecular!biology!laboratory!
equipment!including!thermal!
cycler!and!gel!imaging!
equipment!
Femtomolar!concentrations!of!
target!nucleic!acids!possible.!
!
Assay!can!detect!down!to!104!
cells!/!mL!
Dynamic!range!over!9!logs;!
specific!for!H401!strain;!no!
detection!of!non,gonococcal!
strains!
High!sensitivity!and!specificity!
for!pneumococci!and!could!
detect!drug!resistance!in!both!
clinical!S.%pneumoniae!strains!
and!sputum!samples!
38!|!P a g e !
!
!
Target&organism(s)&
Salmonella%(100)!
Plasmodium%(101)!
“Popcorn!shaped”!Gold,
conjugated!monoclonal!
antibodies!targeted!against!
Salmonella!typhimurium!DT104!
antigens!(S.%typhimurium!DT104!
is!multi,drug!resistant).!
!
Gold,conjugated!antibodies!
cross,link!bacteria,!which!results!
in!a!color,forming!reaction!for!
detection!
!
Rapid!assay,!simple!readout!
High!pure!PCR!template!
preparation!kit!(version!20;!
Roche!diagnostics,!GmbH,!
Germany)!used!to!extract!
parasite!DNA!from!dried!blood!
spots!on!filter!paper.!
!
SYBR!green!I!real,time!PCR,!
where!region!of!18S!rRNA!gene!
amplified!that!allows!species!
identification!based!on!melt!
peak!analysis.!
!
Real,time!PCR!performed!on!
Exicycler!96TM!
Specific!to!S.%typhimurium!DT104!
and!not!other!strains!of!
Salmonella!or!non,Salmonella!
species.!
PCR!assay!detected!more!mixed!
infections!when!compared!to!
microscopy;!mixed!infections!
could!lead!to!drug,resistant!
strains!
39!|!P a g e !
!
!
Target&organism(s)&
MDR,TB!(102,!103)!
XDR,TB!(104)!
Assay&#1:&
Gel,based!two,step!nested!PCR!
targeting!rpoB!and!katG!genes!
(primary!PCR);!secondary!PCR!
specific!for!mutations!associated!
with!resistance!to!rifampicin!and!
isoniazid.!(102)!
!
Also!required:!standard!
molecular!biology!laboratory!
equipment!including!thermal!
cycler!and!gel!imaging!
equipment!
!
Assay&#2&
COLD,PCR!followed!by!HRM!(LC!
Green!as!the!fluorescent!dye)!to!
detect!rpoB!mutations!within!
background!of!wild!type!TB!
gDNA.!
!
Amplification!on!SmartCycler®!
(Cepheid),!HRM!on!LightCycler®!
(Roche)!
Real,time!PCR!using!dual,labeled!
probes!in!a!triplex!reaction,!
where!probes!are!labeled!with!
different!fluorophores!to!
differentiate!mutations!in!the!
gyrA,!rrs!genes!and!the!promoter!
of!eis!that!correspond!to!
resistance!to!fluoroquinolones!
and!second,line!injectable!
agents!(capreomycin,!amikacin!
and!kanamycin).!
!
Detection!is!based!on!melt,curve!
analysis,!where!specific!melt,
peaks!correspond!to!specific!
mutations!or!wild,type.!
!
Amplification!and!melt!analysis!
performed!on!Bio,Rad®!CFX96!
real,time!PCR!instrument.!
rpoB!and!katG!mutations!
detected!in!phenotypically!
resistant!isolates!at!sensitivities!
of!93%!and!83%!respectively!
(Assay!#1).!
!
Sensitivity:!95.2%,!detection!
down!to!5%!of!mutation!in!WT!
background!(Assay!#2).!
100%!concordance!between!
assay!and!sequencing!on!109!
clinically!resistant!isolates!
!
LoD:!100!copies!gDNA!/!µL!
!
40!|!P a g e !
!
!
With! the! exception! of! MRSA! (commercial! products! and! LDTs),! Plasmodium! (LDTs)! and! MDR,TB!
(commercial!products!and!LDTs),!no!commercially!available!or!LDT!exists!to!detect!the!organisms!listed!
in!Table!10.!
The!molecular!tests!can!be!run!as!published!in!lower!resource!environments!or!improved!by!porting!to!
automated!platforms!and!converting!gel!based!PCR!tests!to!real,time!PCR!tests.!!The!MRSA!and!Candida!
tests!using!label,free!detection!have!the!potential!to!be!used!in!novel!PoC!systems!by!circumventing!the!
challenges!associated!with!fluorescent,based!detection!systems.!!!
The! one! non,PCR! assay! identified! detects! a! strain! of! Salmonella! known! to! be! drug,resistant.! ! The!
advantages!of!this!assay!are!the!rapid!time!to!results,!simple!readout!and!potential!adaptability!to!a!low!
resource! environment.! ! Additional! monoclonal! antibodies! must! be! raised! against! other! antigens!
associated!with!drug!resistance!to!expand!the!menu!of!this!type!of!assay.!!The!prototype!assays!listed!in!
Table!10!fill!a!gap!in!the!LDT!and!commercial!testing!menu!for!AMR.!
!
!
41!|!P a g e !
!
!
Emerging+and+Technology+Landscape+
Newly!emerging!technologies!and!established!technologies!not!traditionally!used!in!AMR!detection!that!
have!the!potential!for!commercialization!are!summarized!in!table!11!below.!!An!emphasis!was!placed!on!
how!each!technology!could!be!utilized!to!support!testing!needs!in!the!developing!world.!
Table&11:&Emerging&Technology&
Technology&
Description&
PoC!nucleic!acid!
amplification!platforms!
Several!manufacturers!offer!
fully!integrated!and!fully!
automated!instruments!for!
nucleic!acid!amplification!
and!detection!(LAMP,!
qPCR).!!CLIA!waiver!has!
been!granted!for!an!
Influenza!test!that!runs!on!
TM
the!Alere !i!instrument.!!
This!isothermal!
amplification,based!assay!is!
complete!in!less!than!15!
minutes!(105)!
Gaps&filled&by&technology&
Adaptability*&
These!systems!can!
potentially!be!ruggedized!
and!battery!powered!to!
enable!their!use!in!resource,
limited!environments.!!In!
addition,!those!devices!with!
ambient!temperature!
stabilized!reagents!(in!
consumable)!avoid!the!need!
for!cold!chain!during!
transport!and!storage.!!Thus,!
PoC!platforms!are!a!potential!
solution!to!bringing!
molecular!tests!to!resource,
limited!environments.!
Adaptable!
Arrays!contain!nucleic!acid!
specific!capture!and!
detection!probes!that!can!
be!formatted!on!flat!
surfaces!or!microparticles!
suspended!in!solution.!
AMR!resistance!targets!are!
!
numerous!and!microarrays!
Several!array!platforms!are! offer!a!rapid!(<!8!hours)!
Nucleic!acid!microarrays!
Challenging!
available,!but!the!
comprehensive!means!of!
instrumentation!is!
detection!that!cannot!be!
expensive!and!requires!
matched!by!PCR.!
highly!skilled!operators.!!
Despite!this,!assays!
detecting!MDR,TB!and!ESBL!
have!been!developed!
(reviewed!in!106)!
NextGen!sequencing!
platforms!are!commercially!
Can!provide!phylogenetic!
available,!but!require!high!
information!and!be!an!aid!in!
skill!and!are!expensive!to!
tracking!for!infection!control!
Next!Generation!
operate.!!In!addition!to!the!
purposes.!!WGS!used!to!track! Challenging!
(NextGen)!Sequencing!
chemistry!and!hardware,!
the!spread!of!MRSA!in!a!high!
data!analysis!and!storage!
transmission!environment!
can!be!a!challenge!to!
(107)!
resource!limited!areas!
(reviewed!in!106).!
*Adaptability#refers#to#the#ease#at#which#emerging#technology#could#be#adapted#for#use#in#the#developing#world#
within#3<5#years.#
42!|!P a g e !
!
!
Technology&
Description&
MADLI,TOF,MS!(Mass!
Spec)!
Sample!is!ionized!and!
passed!through!mass!
spectrometer!for!analysis.!!
MALDI,TOF,MS!instruments!
are!commercially!available!
and!are!utilized!for!AMR!
detection!and!can!be!
applied!to!determining!
susceptibility!(reviewed!in!
106).!
!
The!instrumentation!is!
expensive!and!requires!
highly!skilled!operators!
Adaptability*&
Can!provide!detailed!
phylogenetic!information!
down!to!sub,species!level!
and!detect!multiple!
biomarkers!(e.g.!DNA,!
proteins,!and!metabolites)!in!
a!single!sample!in!a!single!
run!rapidly.!
!
Results!are!delivered!in!
minutes.!
Challenging!
FISH!is!simple!and!rapid;!
probe!design!and!production!
Use!of!labeled!probes!to!
is!simple!compared!to!
detect!genetic!targets!in!a!
multiplexed!real,time!PCRs!
variety!of!sample!types.!!
or!antibody!production!for!
Samples!are!fixed!and!the!
agglutination!assays.!!Thus,!
probes!are!hybridized!in%situ!
FISH!occupies!a!mid,ground!
!
Moderately!
FISH!
between!the!high!sensitivity!
Microscopic!analysis!is!used!
challenging!
of!PCR!and!simplicity!of!
for!detection.!
agglutination!tests.!
!
!
An!intermediate!level!of!skill!
Hybridization!ovens!and!
is!required!for!this!
microscopes!would!be!well!
technique!(reviewed!in!106)!
suited!for!laboratories!in!
resource,limited!areas!
Novel!expression!vector!
packages!inside!a!bioactive!
particle!that!can!introduce!
the!vector!into!host!
bacteria;!luciferase!
Single!platform!with!
expression!driven!by!species! potential!to!address!large!
TM
Smarticles !
Challenging!
specific!promoter!on!vector! menu!of!antimicrobials!and!
and!expressed!in!live!host!
tie!results!to!clinical!decisions!
cell;!addition!of!anti,
microbials!identifies!AMR!
and!susceptibility!
simultaneously!(108).!
Single!bacterial!cells!are!
combined!with!antibiotics!in!
Platform!is!high!throughput!
picodroplets!and!incubated.!!
Sphere!Fluidics!
enabling!rapid!susceptibility!
Challenging!
Resistant!bacteria!will!grow!
testing.!
and!be!optically!detected!
within!the!droplet!(109)!
within#3<5#years.#
43!|!P a g e !
!
!
Technology&
Description&
Adaptability*&
Toehold!RNAs!are!synthetic!
RNAs!that!conceal!ribosome!
binding!sites!through!a!
hairpin!structure.!!Part!of!
the!hair,pin!is!
complementary!to!a!specific!
This!technology!could!be!
target!sequence,!that!when!
applied!to!paper,based!
present,!unzips!the!hair,pin!
arrays!and!be!utilized!
and!exposes!the!ribosome!
similarly!to!DNA!microarrays!
binding!sequence.!!The!
in!the!sense!that!multiple!
ribosome!binding!sequence!
AMR!resistance!markers!can! Moderately!
Toehold!RNA!switches!
can!be!upstream!from!a!
be!assayed!in!one!test.!
challenging!
sequence!encoding!an!
!
enzyme!such!as!luciferase!
The!ability!to!manufacture!
that!can!be!utilized!as!a!
and!deploy!paper,based!
reporter.!(110)!
arrays!could!be!valuable!in!
!
resource,limited!settings.!
A!prototype!diagnostic!was!
created!using!freeze,dried!
cell!extracts!and!toe,hold!
RNAs!specific!for!Ebola!virus!
sequences!(111)!on!a!paper,
based!array!format.!
within#3<5#years.#
44!|!P a g e !
!
!
Technology&
Description&
Adaptability*&
Isothermal!amplification!
methods!for!detecting!AMR!
are!commercially!available;!
however!these!platforms!
have!the!same!limitations!in!
their!application!to!
resource,limited!
environments!–!cost!of!
instrumentation,!skill!of!the!
operator,!and!the!
Applying!instrument,free!
laboratory!infrastructure!
solutions!to!isothermal!
needed!to!support!their!use.!!!
amplification!methods!
!
provides!the!speed!and!
One!critical!advantage!of!
sensitivity!of!PCR!without!the!
Instrument,free!
isothermal!amplification!
requirement!for!
Adaptable!
isothermal!method!
methods!over!PCR!is!no!
instrumentation!and!a!
requirement!for!thermal!
laboratory!infrastructure!that!
cycling.!!This!means!that!any!
can!be!challenging!to!
means!of!reaching!and!
produce!in!resource,limited!
maintaining!the!
environments!
amplification!temperature!
can!be!used!to!amplify!
target!DNA.!
!
LAMP!has!been!conducted!
without!a!powered!
temperature!block!using!the!
heat!generated!by!the!
exothermic!hydration!of!
calcium!oxide!(112)!
Use!of!pH!indicators!that!
allow!visualization!(color)!of! Has!value!in!obviating!the!
amplification!reactions,!
need!for!detection!
including!isothermal!LAMP!
equipment,!even!simple!
Visual!detection!of!nucleic!
and!strand!displacement!
spectrophotometer!and!
Adaptable!
acid!amplification!
amplification!as!well!as!PCR.!! could!be!combined!with!
Method!conducted!on!both! other!instrument,free!
DNA!and!RNA!templates!
solutions.!
(113)!
within#3<5#years.#
Some! of! the! technologies! listed! in! Table! 11,! such! as! paper,based! diagnostics! and! instrument,free!
isothermal! amplification,! are! ideal! for! low! resource! environments! in! the! sense! that! they! would! not!
require!cold!chain!transport!and!storage,!power,!or!advanced!laboratory!infrastructure.!
Other! technologies! like! MALDI,TOF! mass! spec! and! the! novel! AST! platforms! by! GeneWEAVETM! and!
Sphere!Fluidics!clearly!fill!a!gap!in!AMR!diagnostics!and!may!even!be!utilized!in!the!developing!world!as!
is.! ! What! might! make! these! platforms! attractive! to! low! resource! environments! is! the! potential! for!
45!|!P a g e !
!
!
customization!and!the!wide!breadth!of!testing!they!can!do.!!For!instance,!susceptibility!testing!could!be!
accomplished!with!the!GeneWEAVETM!platform!to!the!extent!that!given!antibiotics!are!available!to!the!
end!user.!!This!is!a!limitation!of!commercial!ID/AST!platforms!that!rely!on!the!manufacturer!to!provide!
consumables!dedicated!to!specific!organisms!and!antibiotics!to!run!on!the!instrument.!!MALDI,TOF!can!
be!similarly!utilized!and!provides!far!more!comprehensive!data.!
The!emerging!technologies!do!indeed!fill!gaps!in!current!testing!methodologies!and!their!adaptation!has!
the! potential! to! not! only! improve! AMR! testing! but! also! aid! in! infection! control,! which! is! critical! to!
combating!AMR!and!reversing!the!spread!of!these!pathogens.!
!
!
46!|!P a g e !
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!
Appendix+A:+Food+Safety+Testing+
Food!safety!testing!must!be!part!of!the!solution!for!combating!AMR!as!AMR!can!develop!through!the!
overuse! of! antibiotics! in! livestock! and! also! the! contamination! of! food.! ! In! both! cases! humans! can! be!
exposed!to!AMR!organisms.!
Traditional! microbiology! methods! have! been! widely! used! to! test! animals! and! food! for! microbial!
contamination! and! could! be! extended! to! look! for! AMR! specifically.! ! As! with! human! infectious! disease!
diagnostics,! molecular! testing! and! novel! technology! is! being! exploited! –! at! times! in! place! of! routine!
microbiology,!especially!when!rapid!turnaround!times!are!required.!!As!with!other!screening!paradigms!
for!testing,!molecular!results!may!prompt!reflex!testing!to!specific!microbiology!testing.!!Table!12!lists!
several!examples.!
Table&12:&Food&Safety&Testing&Platforms&
Mfr.&
3MTM!
BioFireTM!
DuPont®!Nutrition!and!Health!
mericon!Real,time!PCR,!QIAGEN®!
Roka®!Bioscience!ATLAS®!System!
Sample!6!Bioillumination!Platform™!
Technology*&
3M™! Molecular! Detection! System! uses! isothermal!
amplification! combined! with! bioluminescence! detection;!
assay! kits! formatted! in! pre,dispensed! reagent! packs! for! the!
lysis! of! enrichment! cultures! and! addition! of! sample! into!
reactions.!!Single!instrument,!small!footprint!!
R.A.P.I.D.! BioDetection! System,! incorporates! LightCycler®!
instrumentation! for! amplification! and! detection;! freeze,
dried! reagents! and! sample,to,answer! platform.! ! Instrument!
is! also! field,deployable! and! manufacturer! offers! assays! for!
food!and!water!testing.!
BAX®!System;!uses!real,time!PCR!to!detect!food!pathogens.!!
Assay!kits!formatted!in!pre,dispensed!reagent!packs!for!the!
lysis! of! enrichment! cultures! and! addition! of! sample! into!
reactions.!
mericon! kits! come! with! reagents! required! for! enrichment!
cultures,! DNA! purification! and! PCR.! ! Assays! are! run! on! the!
Roto,Gene®!Q.!
User! performs! enrichment! culture! step! off,deck! and!
transfers! culture! aliquot! to! transfer! tube! loaded! with! lysis!
buffer.! ! This! lysate! is! loaded! onto! the! ATLAS®,! which! is! an!
integrated! sample,to,answer! platform! performing! nucleic!
acid! purification! and! transcription! mediated! amplification!
(isothermal)!and!ribosomal!RNA!targets.!
Technology! uses! engineered! bioparticles! that! specifically!
“infect”! target! pathogens.! ! The! bioparticles! then! introduce!
reporter! gene! sequences! into! the! host! cell;! detection! is!
bioluminescence! based! using! a! reader! provided! by! the! mfr.!!
No! enrichment! culture! steps! required! and! the! process! can!
be!completed!in!6!hrs.!
*Information% gathered% from% manufacturer% websites.% % Please% note% that% the% products% described% above% are% not%
intended%for%clinical%diagnostics.%
47!|!P a g e !
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Anti-‐Microbial Resistance

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