Bacterial typing idse14_wm

The introduction of new molecular technologies into the clinical

microbiology setting is transforming clinical diagnostic testing and

laboratory operations. The rapid adoption of these technologies

is providing microbiological diagnostic information with turnaround

times measured in hours rather than days, as with traditional, culture-

based techniques. An increasing body of information is already

demonstrating that the earlier availability of actionable diagnostic

bacterial and viral testing results can improve patient outcomes.

Available molecular diagnostic technologies now include nucleic acid amplification, mass spectrometry (MS), and next-generation sequencing (NGS)–based approaches. Increasingly, many laboratories are eval-uating their operations to identify opportunities to employ molecular testing approaches and to poten-tially reduce or limit their current reliance on high-cost, labor-intensive, culture-based diagnostic testing to only those applications where it is deemed necessary.1-4 As clinical microbiologists explore and define the balance between molecular diagnostic testing and culture-based techniques, antibiotic stewardship, infection control, and public health practitioners are also experimenting with

ways these technologies can affect their work. This arti-cle will focus on how these new molecular-based tests are being applied to infection control practice. It will introduce each of these options sequentially, highlight some commercially available assays in each category—with a bias toward FDA-approved tests—and provide some evidence for how infection control practitioners have successfully implemented them into their practice.

Nucleic Acid Amplification ApproachesNucleic acid amplification–based diagnostic testing

was the first molecular technology to be applied to clin-ical laboratory operations, and it has the longest track

New Molecular Diagnostic Technologies: Opportunities for Infection Control?

BRIAN CURRIE, MD, MPHVice President, Medical ResearchDivision of Infectious DiseasesMontefiore Medical CenterAssistant Dean for Clinical Research at Montefiore Medical CenterProfessor of Clinical MedicineAlbert Einstein College of MedicineBronx, NY

Dr. Currie reported that he has no relevant financial

interests to disclose.

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Table 1. FDA-Approved Molecular Diagnostic Tests for Nosocomial Pathogens

Device, Manufacturer Assay Type Sample Type TargetPathogen

Turn- aroundTime

High-ThroughputPlatform

BD GeneOhmBD Diagnostics

Real-time PCR Direct swab C. difficile, MRSA, S. aureus, VRE

1.5 h Yes

GeneXpertCepheid

Real-time PCR Direct swab C. difficile, MRSA, S. aureus, VRE

1 h Yes

Meridian IllumigeneMeridian Bioscience

IsothermicDNA amplification

Direct swab C. difficle 1 h No

Prodesse ProGastroProdesse

Real-time PCR Direct swabSample requires pre-preparation DNA extraction

C. difficle 3-4 h No

Nuclisens Easy QbioMérieux

Real-time PCR Direct swabSample requires pre-preparation DNA extraction

MRSA 2 h No

Roche LightCyler MRSAAdvanced TestRoche Molecular Systems

Real-time PCR Direct swab MRSA 2 h No

FilmArrayBiofire

Real-time PCR Direct swab C. difficle via panel 1 h No

C. difficle, Clostridium difficle; MRSA, methicillin-resistant Staphylococcus aureus; PCR, polymerase chain reaction; VRE, vancomycin-resistant enterococci

record of application to infection control practice. The technology continues to be best exemplified by the introduction of real-time multiplex polymerase chain reaction (PCR) assays that have demonstrated mark-edly superior sensitivity and specificity for detection of targeted pathogens compared with culture techniques or nonmolecular antigen tests.3,5 When optimized to run from direct swab samples without sample pre-prep-aration or DNA extraction, they offer turnaround times of 1 to 1.5 hours. Coupled with the development of high-throughput platforms capable of running multiple dif-ferent assays simultaneously, these approaches began to represent attractive alternatives for clinical microbi-ology laboratories to use in the diagnosis of both bac-terial and viral infections.

Early in the introduction of these assays, infection control practitioners quickly identified that their use could profoundly affect the ability to successfully iden-tify and rapidly isolate potentially infectious patients. The attributes of these assays provided support for point-of-care (POC) decision making for newly admit-ted and hospitalized patients, as clinically indicated. Not only could POC decision making optimize the isola-tion of patients who require it, it could also prevent the

unnecessary isolation of patients who do not, reduc-ing the costs associated with isolation and optimizing the use of relatively scarce resources, such as single-patient isolation rooms and antiviral drugs. Additionally, these assays also represented a way to rapidly identify patients who are asymptomatically colonized with tar-geted organisms, thus greatly facilitating the conduct of hospital-based prevalence surveys and active surveil-lance programs designed to reduce the prevalence of targeted organisms.

Commercially available assays initially focused on the detection of the etiologies of most hospital-acquired infections with individually tailored assays for specific pathogens, but available products have quickly expanded to include symptom-based panels (ie, poten-tial detection of any of the most common infectious causes of diarrhea or upper respiratory symptoms) or panels that screen for asymptomatic carriage of any number of multidrug-resistant (MDR) nosocomial bac-teria that could potentially be associated with rectal colonization of hospitalized patients. To accommodate the needs associated with newly emerging pathogens such as carbapenemase-producing Klebsiella pneu-moniae (carbapenem-resistant Enterobacteriaceae

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Table 2. Real-Time CRE PCR Assays in Development

Assay Name/Manufacturer

Target Genes

BD MAX CRE RUO assayBD Diagnostics

KPC, NDM OXA

Acuita MDRO Gene TestOpGen

KPC, NDM, OXA VIM, IMP, also detects CTX-M, Van A

NuclisSENS Easy Q KPC TestbioMérieux

KPC only

Xpert Carba–RCepheid

KPC, NDM, VIM, OXA, IMP

Check-MDR Carba testCheck-Points

KPC, NDM, OXA, VIM, IMP

eazyplex SuperBug CREAmplex

KPC, NDM, OXA, VIM

CRE, carbapenem-resistant Enterobacteriaceae; PCR, polymerase chain reaction

[CRE]), new assays are being developed.Table 1 summarizes the characteristics of a number of

commercially available, FDA-approved, real-time PCR products that have focused on the detection of com-mon nosocomial bacterial pathogens. FDA-approved assays are available for Staphylococcus aureus, meth-icillin-resistant S. aureus (MRSA), vancomycin-resis-tant enterococci (VRE), and Clostridium difficile. The BD GeneOhm and GeneXpert devices continue to be the most commonly deployed real-time PCR platforms because they work from direct swab samples, have a broader library of available assays, and are available as high-throughput platforms. Currently available kits for the detection of gram-negative nosocomial pathogens remain somewhat limited due in large part to the fact that gram-negative resistance gene targets are much harder to characterize.3,5-7 Given the emergence of CRE as a significant public health threat, and the press-ing need for an accurate diagnostic test, manufactur-ers have focused on the development of real-time CRE PCR assays.3,7

CRE carbapenem resistance is caused by a com-plex group of genes—KPC, NDM, OXA, VIM, and IMP—all of which encode for carbapenemases.3,7 These genes share less than 60% sequence homology with each other, and numerous genetic variants occur within each group. They present a significant challenge for the development of real-time CRE PCR assays.3,7 Despite these obstacles, a number of assays are currently com-mercially available in the United States and Europe, on a research use-only basis (Table 2). All are optimized to use direct patient swab samples with rapid turnaround times of 50 to 120 minutes. Preliminary performance data suggests that all tests have been able to detect their stated targets with high sensitivity and specific-ity.8-13 Only 2 of the assays are available for use in the United States—the BD MAX CRE assay, which has FDA research use-only status, and the Acuitas MDRO Gene Test, which is only available via a company-owned cen-tral laboratory that is Clinical Laboratory Improvement Amendment approved and offers 24-hour turnaround.

Still, the application of CRE-specific, multiplex real-time PCR assays as infection control tools has been established and these efforts are quite illustrative of how this technology can potentially affect practice. Although they are primarily based on the use of “home-grown” applications or prototype assays to date, these technologies have been used by infection control prac-titioners for sentinel surveillance and periodic large-scale, hospital-wide prevalence surveys. Demonstration projects have explored the use of these assays for POC rapid screening of emergency room patients who are about to be admitted to the hospital for asymptomatic CRE rectal colonization.14 Coupled with rapid institu-tion of contact isolation precautions, this approach may prove effective in addressing the significant influx of CRE-colonized patients from outside acute and chronic care facilities in areas already endemic for CRE. Per-haps most significantly, these assays have been used to

successfully drive active surveillance programs for CRE, which trigger rapid initiation of contact isolation pre-cautions for all CRE-positive patients. This approach is effective in reducing the prevalence of CRE in individ-ual hospital units and across institutions. When applied across all ICUs in a New York-based health care system, this approach resulted in a sustainable 52.7% reduction in CRE prevalence.15 A similar approach has been dem-onstrated to contain a hospital-wide CRE outbreak.16

Although these results are quite promising, wide-spread adoption of active surveillance strategies for CRE, or for any MDR nosocomial pathogens for that matter, will continue to depend on the extent to which these technologies are accepted by clinical microbiol-ogy laboratories. The increased cost of testing, the lim-ited capacity of most laboratories to run the assays, and the current lack of FDA-approved products continue to serve as barriers to laboratory implementation.

Mass Spectrometry ApproachesMass spectrometry applications for bacterial identifi-

cation were first reported in 1975, and are based on the fact that small molecules can be harvested from lyoph-ilized bacteria that produce distinct MS “signatures,” enabling taxonomic distinction of some bacteria.2 How-ever, it was not until the later development of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that the characteriza-tion of macromolecules became feasible. Subsequent work has established that application of the technique

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Figure. Schematic of MALDI-TOF MS process.

MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Spectrum

Detector

Vacuum Flight Tube

Laser

Crystalized matrix with analytes

Spectrum created

Detection

Separation: TOF(no electric field)

Acceleration through electrostatic field

Matrix-assisted laser desorption ionization

to whole bacterial cells can produce characteristic and reproducible spectra, largely based on proteins used to reliably identify bacteria to the genus and species lev-els within minutes.2,17

The MALDI-TOF MS process is summarized in the Figure. Microbial samples are deposited on a target plate and a matrix solution that crystalizes with the sample and lyses the cells is added. The samples are then vaporized and ionized by short laser bursts. Ion-ized particles pass through an electrostatic field and are accelerated through a vacuum. The time it takes for each particle to reach a detector, the time of flight, depends on the mass and charge of the particle and is used to create a spectral profile. Identification of the organism is accomplished with software that automat-ically compares the obtained profile with a reference library and turnaround time is measured in minutes.2,17,18

In general, MALDI-TOF MS has been demonstrated to outperform traditional culture techniques for identifi-cation, and is extremely rapid.18

Two MALDI-TOF systems are commercially avail-able—the MALDI Biotyper (Brucker Daltonics) and the VITEK MS (bioMérieux). The bioMérieux product was approved by the FDA in 2013 and the MALDI Biotyper was approved by the FDA for research use only in 2014. Published literature suggests that both systems have comparable performance characteristics.19 Although the systems present a significant initial outlay for the cost of equipment, subsequent analysis of samples is very inex-pensive.2,3 Studies have demonstrated that this technol-ogy can be applied directly to positive blood cultures and can be used to optimize appropriate antibiotic ther-apy.2,18,21-24 Additional protocols seek to optimize the direct application to urine samples without preculture.2,24

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Table 3. Commercially Available NGS Platforms

Illumina Platform (Illumina)

454 genome sequencer (Roche Applied Science)

SOLiD platform (Life Technologies)

Benchtop applications

MiSeq (Illumina)

Ion Torrent platforms (Life Technologies)

Micro SEQ (Life Technologies)

NGS, next-generation sequencing

The use of MALDI-TOF MS for the detection of anti-biotic resistance is currently an area of intense inves-tigation.2,20,24,25 Several different types of assays are in development for the detection of MDR bacteria. One approach is to analyze drug metabolites via MS after bacteria are subjected to short incubation in the pres-ence of antibiotics.24,25 A second approach incorpo-rates incubation of samples in stable isotopes of carbon and nitrogen and the presence of antibiotics and deter-mines drug resistance by the characteristic shift in pro-file peaks.20 A third approach seeks to identify enzymes related to antibiotic resistance via direct detection of their signature MS peaks.25 Protocols for MRSA, VRE, and β-lactamases, including the carbapenemases of CRE, are in development and require further validation.3,25

There is also interest in the use of MALDI-TOF MS for strain typing of bacteria as an epidemiologic tool to assist infection control investigations.2,24,26 Successful application of MALDI-TOF MS for bacterial strain typ-ing remains problematic, and it has been argued that the current technology is not capable of providing the level of resolution necessary to accomplish the task.2,24

Conversely, it has been suggested that strict adher-ence to sample preparation to reduce MALDI-TOF MS profile variations that are attributed to differences in sample culture conditions; improved lysis of samples to provide enhanced detection of minor peaks in gen-erated profiles currently missed; enhanced libraries to improve resolution; greater inclusion of other nonpro-tein macromolecules into the MS analysis; enhanced use of biometrics-enabled analytic tools; and more detailed evaluation of specific profile peaks may yet allow for bacterial strain typing.26,27 Even if achievable, there appears to be consensus that strain-typing capability will only be bacterium-specific.26 Despite these obsta-cles, MALDI-TOF MS applications are useful strain-typ-ing tools in the investigation of a VRE outbreak and for a Streptococcus pneumoniae conjunctivitis out-break.28,29 However, these applications required com-plex sample preparation and the creation of specific novel spectral libraries.

Given the requirement for pure culture samples for analysis and the current capacity of existing commer-cially available equipment, the primary affect of MALDI-TOF MS on infection control and hospital epidemiology practice will result from the earlier identification of patients with clinical infections caused by targeted nos-ocomial pathogens and more timely institution of iso-lation protocols. In the near future, this capability may be enhanced to include earlier identification of clinical isolates of MDR nosocomial pathogens. Future investi-gations may provide for the development of some spe-cies-specific strain-typing technology, but it is still not clear how and if they would become integrated into clinical microbiology laboratory operations. Because MALDI-TOF MS cannot be optimized to run directly from patient samples, other than blood or urine, it sub-sequently will not have utility as a tool for real-time active surveillance.

Next-Generation Sequencing ApproachesNucleic acid sequencing approaches have rapidly

evolved from first-generation capillary electrophore-sis-based Sanger methods with the application of NGS technology.1,29,30 Contrary to prior approaches to whole-genome sequencing, NGS approaches include frag-menting DNA into libraries of smaller segments that are then subjected to massively parallel sequencing. The subsequent shorter “reads” are then reassembled using a known reference genome as a scaffold (resequenc-ing) or by using overlapping regions (de novo assem-bly). The approach has revolutionized whole-genome sequencing by providing rapid high-throughput results at a relatively low cost. Initially applied to human genomic targets, the process is being adapted for use in the clinical microbiology laboratory for bacterial spe-ciation, identification of antibiotic resistance, and as a bacterial strain-typing method in support of epidemi-ologic investigations.1,30-32 Current commercially avail-able, widely used sequencing products are summarized in Table 3, and notably, now include 3 lower-cost bench-top options.

Adapting NGS to bacterial strain typing presents several challenges. First, there are only a limited num-ber of reference genomes available, so de novo assem-bly frequently is required. Shorter read lengths can result in a higher number of sequence gaps and can effect data quality. Some NGS platforms offer paired-end sequencing protocols, which allows sequencing to progress from both ends of a DNA fragment and results in enhanced alignment of the reads. This prob-lem has also been addressed by the use of publicly available short-read software algorithms that can pro-vide enhanced assembly. A third option is the use of targeted sequencing, where only a subset of genes or defined regions of interest are sequenced.1 Both rese-quencing and targeted sequencing will require the cre-ation of comparative genome libraries by laboratory staff.






Next-generation sequencing has demonstrated util-ity as a strain-typing tool in support of outbreak inves-tigations at institutions with exceptional laboratory support. NGS was used to investigate recurrent epi-sodes of MRSA colonization of infants in a neonatal ICU (NICU) over a 6-month period.33,34 The investiga-tion was able to demonstrate MRSA transmission in the NICU, as well as between mothers in a postnatal ward and in the community. MRSA carriage by a staff mem-ber was ultimately identified as the source of the pro-longed outbreak in the NICU.

Similarly, whole-genome sequencing was used as a key adjunct epidemiologic tool during an investigation of a prolonged hospital outbreak of CRE that persisted despite initiation of enhanced infection control inter-ventions.35 Data from the analysis was able to recon-struct a transmission map that established 3 different lines of patient-to-patient CRE transmission that ema-nated from a single known CRE-infected patient who had been discharged 3 weeks before the hospital iden-tified the first clinical case of the outbreak. The analysis provided actionable insights that were used to facilitate subsequent control of nosocomial transmission.

Whole-genome sequencing was used during another hospital-wide CRE outbreak investigation to charac-terize infection chains occurring in the surgical ICU.16 Analysis was able to establish that transmission was not related to staff colonization. An additional investigation used whole-genome sequencing as a strain-typing tool to evaluate 1,223 cases of C. difficile infection over a 5-year period from a single community.36 Analysis was able to establish that patient-to-patient transmission only accounted for slightly more than one-third of the cases. A large proportion of infections involved geneti-cally unique strains, suggesting exposure to asymptom-atic human or environmental sources as the etiology of these infections. The authors concluded that the use of rapid benchtop sequencing could provide nearly real-time C. difficile strain typing and could prove to be an important adjunct tool for institutional infection con-trol practices.

Although NGS whole-genome sequencing shows great promise as an adjunct infection control tool for epidemiologic investigations, it is currently restricted to those institutions that have access to exceptional laboratory support far beyond the current capabil-ity of most clinical microbiology laboratories. Even in these settings, it has been difficult to take full advan-tage of the near real-time capability of the technology. Given the exponential explosion of genome sequencing technology in the past 5 years, it is not unreasonable to expect that low-cost benchtop NGS whole-genome sequencing platforms will be optimized for clinical lab-oratory use in the near future. The routine availability of near real-time strain typing will profoundly influence infection control practice.

SummaryThe past several years have seen the introduction of

significant molecular-based diagnostic technologies into the clinical microbiology laboratory. Options for real-time PCR testing have expanded to include syn-dromic and gene-resistance panels, new assays are being developed for newly emergent pathogens, such as CRE, and high-throughput platforms have been intro-duced. MALDI-TOF MS and NGS platforms have been introduced and are increasingly being adopted by clini-cal microbiologists and reference laboratories.

Although designed and marketed primarily as diag-nostic tools, antibiotic stewardship, infection control, and public health practitioners are already experimenting with how these technologies can improve their practice. The infection control literature now includes an expand-ing portfolio of reports describing successful use of both real-time PCR testing and NGS as adjunct tools for hos-pital epidemiology. Real-time PCR testing continues to represent an alternative tool for POC screening to opti-mize initiation of isolation protocols and to drive effec-tive active surveillance intervention strategies. It is also an attractive tool for rapid surveillance surveys. NGS is an effective near real-time bacterial strain-typing tool for the investigation of outbreaks. However, continued development of NGS technologies will be necessary to incorporate them into routine clinical microbiology lab-oratory operations. The utility of MALDI-TOF MS as an infection control tool remains uncertain.

There are significant barriers to adoption of real-time PCR testing and NGS technologies. Both are currently associated with a relatively high cost per sample ana-lyzed. Although real-time PCR has largely been opti-mized for clinical microbiology use, NGS applications will need to become less complex. The widespread adop-tion and availability of these technologies for infection control use is also largely dependent on their appeal and acceptability to clinical laboratories for application to routine diagnostic testing. For instance, it is still not clear how NGS will find a niche in the clinical diagnostic arena. Alternatively, some medical centers may elect to support the use of these technologies for infection con-trol activities, independent of their utility as clinical diag-nostic tools. Continued research in the infection control arena demonstrating high-impact results from access to real-time PCR and NGS may facilitate this process.

References

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