Multiparametric technologies for the diagnosis of syndromic infections

Clinical Microbiology Newsletter Vol. 34, No. 20

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October 15, 2012

Multiparametric Technologies for the Diagnosis of Syndromic Infections Luc Bissonnette, Ph.D. and Michel G. Bergeron, O.Q., M.D., FRCPC, Département de microbiologie-infectiologie et d’immunologie, Faculté de médecine, Université Laval, and Centre de recherche en infectiologie de l’Université Laval, Axe infectiologie et immunologie, Centre de recherche du CHUQ, Quebec City, Quebec, Canada

Abstract The introduction of molecular diagnostics in the management of infectious diseases provides clinical microbiology with tools allowing a faster counterattack in the fight against disease-causing microbes. However, in light of the multi- and polymicrobial origin of infections and frequency of antimicrobial resistance, most currently approved tests impose a limitation on clinicians, essentially due to the number of microbes a single test can identify. For many life-threatening conditions, where the balance of host-microbe interactions could dramatically shift if an inappropriate or untimely course of treatment is initiated, it would be strategically beneficial if the laboratory could detect and identify the etiology/etiologies of the infection faster and more accurately. This article reviews currently available clinical molecular microbiology tests for the diagnosis of infectious syndromes developed on multiparametric detection platforms, such as multiplex real-time PCR, molecular hybridization, nucleotide sequencing, mass spectrometry, and integrated fluidic systems.

Introduction Despite significant advances in medicine and sanitation in the 20th century, infectious diseases are still the number one cause of mortality worldwide, causing approximately 25% of recorded deaths annually, a number expected to decrease slightly over the next 20 years due principally to an increase in noncommunicable diseases. Humans are susceptible to infection by a wide variety of microbial pathogens, and the diagnosis of some infections is complicated, since there is considerable over-

Corresponding author: Luc Bissonnette, Ph.D., Département de microbiologie-infectiologie et d’immunologie, Faculté de médecine, Université Laval, and Centre de recherche en infectiologie de l’Université Laval, Axe infectiologie et immunologie, Centre de recherche du CHUQ, 2705 Laurier Blvd., Québec City (Québec) Canada G1V 4G2. Tel.: 418-656-4141 ext. 48748. Fax: 418-654-2715. E-mail: [email protected]

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lap in symptoms and clinical presentation, despite differences in the causes. Therefore, accurate microbial identification is one of the most crucial steps of disease management. For a physician, the presence of an organism – bacterium, fungus, or parasite – in a sample collected from an ill patient suspected of having an infection is usually sufficient to initiate a course of action supported by an antimicrobial regimen; however, the initial rates of inadequate empiric antimicrobial treatment are reported to range from 15 to 30% (1). This empiric approach to antimicrobial therapy is not always straightforward and is often complicated by (i) a delay in appropriate treatment (2-5), perhaps caused by the length of the diagnostic cycle; (ii) antimicrobial resistance (4,5); (iii) lack of antibiotic development for treating multidrugresistant organisms (6); (iv) drug shortages (7); (v) polymicrobial infections; (vi) pathogenic biofilms (3,8,9); and (vii) the onset of opportunistic infec© 2012 Elsevier

tions, either in immunosuppressed individuals or after a primary antimicrobial course, which may provide an advantage to an unsuspected pathogen. The management of a febrile, potentially infected patient initiates a diagnostic cycle consisting of several timeconsuming steps. With traditional phenotypic microbiology methods, it is generally accepted that the time to complete the laboratory’s analytical phase of this cycle is the most important temporal limitation. Delays in pre- and post-analytical phases, such as sample transport, batching practices, and transmission of results, increase the turnaround time of the test(s), thereby decreasing the chances of recovery

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or survival of the patient (10). Since 2000, molecular diagnostic tools, such as polymerase chain reaction (PCR), real-time PCR, RNA amplification methods, molecular hybridization-based technologies, and nucleic acid sequencing, have been gradually approved as adjuncts to or replacements for traditional culture-based methods (11,12). Molecular methods have impacted infectious disease management by accelerating the microbial detection/ identification phase of the laboratory diagnostic cycle from greater than 24 to 48 hours, which is generally required by phenotypic (culture) methods, to 1 to 2 hours (1,5). Phenotypic or genotypic determination of drug resistance may require even more time due to additional time required to detect various resistance mechanisms. Once available, this information may lead to re-evaluation of therapeutic option(s). However, for a large number of cases, the time effectiveness provided by using molecular microbiological methods has already improved the chances for patient survival. In the absence of rapid tests, infections are empirically managed, essentially driven by clinical experience and local (microbiological) knowledge. This approach has some merit but has been associated with overuse of broad-spectrum antibiotics with subsequent development of antimicrobial multidrug resistance and emergence of hospitalacquired infections. Empiric management is especially unsuccessful for viral diseases, as exemplified by McGeer (13), who showed that 89% of patients diagnosed with influenza had receivedinappropriate antibacterial therapy upon admission to a Canadian hospital. A few years before the timid entrance of molecular technologies in clinical microbiology, the marketing of largecapacity automated systems for analy-

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tical chemistry and microbiology stimulated the emergence of centralized laboratories performing a wide spectrum of biochemical, microbiological, and genetic analyses (10). Although this model has logistic, operational, and economic merits, the approach has serious limitations in terms of timeliness and communication of results when infectious diseases are considered. Because the critical window for the appropriate management of a life-threatening infection is (conservatively) less than 6 hours, the well-being of patients should be the primary goal. Preferably, this window should be less than 1 hour, as most clinicians will have already initiated their “empiric therapy.” For infectious diseases, we believe that hospitals, and perhaps clinics, should have access to specialized satellite molecular diagnostic laboratories offering a 24/7 comprehensive menu of tests based on multiparametric detection technologies (14,15). The advent of simple, Clinical Laboratory Improvement Amendments (CLIA)-waived, point-of-care devices and tests will support this diagnostic perspective. Bloodstream infections and sepsis Sepsis is the perfect example of a life-threatening infection for which the critical time window for appropriate management is estimated to be less than 6 hours, where every hour gained to initiate proper antimicrobial therapy significantly increases the probability of patient survival. With a mortality rate of 35% (~50% in intensive care units), which translates into 200,000 deaths per year (600 per day) in the United States alone, sepsis is the 10th leading cause of mortality in industrialized countries (8,16). Sepsis management can be severely complicated by its multiple microbial etiologies; in fact, 20 to 25 microbes are responsible for approximately 75% of sepsis cases. To further complicate

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matters, it was reported that in almost 25% of candidemia cases, the infection was polymicrobic (8). Blood culture is the gold standard method for diagnosing bloodstream infections. However, the overall process generally requires more than 24 hours – often 48 to 72 hours – a lengthy time during which infection can progress, with a severe or deadly outcome if inadequate antimicrobial therapy is initiated (2,16,17). Diagnosis of bloodstream infections would be advanced significantly if multiparametric detection could be applied to whole blood; however, this approach is complicated by low bacterial loads found in blood specimens. This limitation presents major challenges in terms of adequate blood volume and preparation. In order for rapid diagnosis to occur, microbial nucleic acids will need to be efficiently detected among massive amounts of human DNA, as well as hemoglobin and lactoferrin, known PCR inhibitors. Indeed, infected blood (20- to 30-ml volume) from adults may contain less than 1 CFU/ml. In young children, this value can exceed 100 CFU/ml, but the blood sample volume available for testing from a neonate might be only 1 ml (18). Finally, multiparametric detection could also be extremely useful for the detection of pathogens that are fastidious or uncultivable in blood culture medium. Respiratory tract infections The human respiratory tract is highly susceptible to infection by a wide variety of viruses and bacteria and, to a lesser extent, yeasts and filamentous fungi. Children are especially affected by viruses causing lower respiratory tract infections (19,20). An analysis of four studies of the etiology of pneumonia in hospitalized children revealed that mixed viral and bacterial infections were detected in 23 to 33% of cases

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(20). In addition, health care-associated pneumonia is the second most common hospital-acquired infection, associated with ~25% of all infections in intensive care units, accounting for more than 50% of antibiotic prescriptions (19). In the context of empiric antimicrobial therapy, respiratory tract infections are highly problematic, since a significant proportion of infections are caused by viruses (which do not respond to antibiotics). Clearly, diagnostic procedures enabling better coverage of associated pathogens should result in a more rational use of antibiotics (21). Urinary tract infections Urinary tract infections are among the most common bacterial infections. It is estimated that 1 million nosocomial cases occur annually in the U.S. (22). In 2010, it was estimated that the high incidence of urinary tract infections translates into annual direct and indirect costs of $2.3 billion in the U.S. The diagnosis of uropathogens is usually accomplished by performing quantitative urine cultures, along with an analysis of urinary symptoms. Positivity is established above a given threshold, usually 1,000 CFU/ml. Although the most frequently encountered bacterial pathogens, such as Escherichia coli, are a primary focus, several other (fastidious or unculturable) bacterial pathogens have been discovered by culture and culture-independent methods (23), providing further evidence for the polymicrobial nature of urinary tract infections. Urine testing may represent another opportunity to exploit molecular microbiology to better understand, diagnose, and treat these infections, especially in the context of increasing antimicrobial resistance. Hospital-acquired infections Hospital-acquired (nosocomial) infections (HAIs) have become a major concern in health care facilities. In the U.S., it is estimated that more than 1.7 million HAIs per year lead to 100,000 deaths and to astronomical direct costs of $6.5 billion in health care expenditures (24). HAIs mainly include catheter-associated bloodstream infections, urinary tract infections, ventilator-associated pneumonia, and surgical site infections, with case fatality rates of 12.3, 2.3, 14.4, and 2.8%, respectively, according to data collected in U.S. hospitals in 2002 Clinical Microbiology Newsletter 34:20,2012

(25). Considering that HAIs are the second most common infection among hospitalized patients in the U.S. and that inappropriate initial antimicrobial therapy has been associated with decreased survival of patients, the use of rapid multiparametric molecular diagnostics could be highly significant.

Multiparametric Detection Technologies – Increasing the Probability of Getting the Right Answer By enabling the detection of a broader range of microbes present in a complex clinical sample, multiparametric technologies have the potential to impact the morbidity and mortality of infectious diseases, decrease health care costs, and reduce the selective pressure exerted on microbes by administration of unnecessary or inappropriate antimicrobial agents. On a simpler scale, it has already been demonstrated that use of molecular diagnostic methods for the detection of methicillin-resistant Staphylococcus aureus (MRSA) can reduce MRSA-associated mortality rates, length of stay of patients with MRSA bacteremia, and mean hospital costs (26). The impact of molecular diagnostics on the mortality rate and cost-effectiveness of sepsis management is also supported by a mathematical prediction model (1). The thesis defended in this article is that for assays with similar costs (multiplex real-time PCR), or possibly lower (mass spectrometry [MS]), or even slightly higher (microarray hybridization or pyrosequencing), multiparametric detection technologies can identify a broader range of microbial pathogens, and well within the critical window of 6 to 24 hours, so that an appropriate antimicrobial treatment can be initiated more rapidly. Faster implementation of appropriate therapy could lead to improved patient management and survival. With the exception of integrated fluidic systems, our discussion is restricted to commercially available systems that simultaneously detect or identify at least 4 microbial targets. Multiplex real-time PCR Although molecular microbiological methods to identify organisms in positive blood culture bottles are faster than current culture-based procedures, PCRbased detection of bloodborne pathogens could be even faster and thus more © 2012 Elsevier

significant if detection was achieved directly from blood. While real-time PCR generally detects less than six genetic targets, Roche Molecular Diagnostics has opened the field for a new brand of multiparametric PCR in the form of the LightCycler SeptiFast test (Roche Molecular Diagnostics, Pleasanton, CA), a multiplex assay designed to detect 25 pathogens directly from blood in less than 6 hours by combining real-time PCR and highly specific melting point analysis. SeptiFast was shown to have a higher positive detection rate than conventional blood culture (27,28). This diagnostic procedure will become a useful tool for the evidence-based treatment of critically ll patients (27,28). SeptiFast was also used to successfully detect pathogens in purulent body fluids (29). However, the usefulness of SeptiFast might be limited by its number of microbial targets, the specificity of primers and probes, and the variability of genetic targets (27). Two multiplex real-time PCR-based platforms, SeptiFast and SeptiTest (Molzym, Bremen, Germany), are assays that detect a wide spectrum of bloodborne pathogens directly from blood in less than 12 hours. Of note, the SeptiTest uses real-time PCR to determine if blood contains bacteria or fungi, but confirmation of identification must be performed by nucleotide sequencing. The overall performance of the Prove-it Sepsis test (Mobidiag, Helsinki, Finland), in terms of its sensitivity, specificity, and speed, suggests that it could provide earlier management of sepsis (30). There is no doubt that the introduction of multiplex real-time PCR applied to the diagnosis of infectious diseases could positively impact patient management and lead more quickly to appropriate antimicrobial treatment. As noted later in this article, the closed-tube configuration of multiplex real-time PCR in a clinical laboratory has methodological advantages over molecular hybridization platforms. However, greater impact of the technology would be observed if batching practices were minimized to provide a result to the treating physician as rapidly as possible. Molecular hybridization The multiparametric detection and identification of microorganisms present in an infected biological sample can be accomplished by hybridizing 0196-4399/00 (see frontmatter)

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amplicons generated by multiplex PCR amplification to an array of capture probes that can discriminate speciesspecific polymorphisms (19,31) (Table 1). The microarray format allows costeffective interrogation of a sample for the presence of a “syndrome-associated” microbial panel, thereby increasing the probability of identifying the diseasecausing microorganism over individual tests. The first molecular hybridization platform approved by the FDA for infectious disease diagnostics was xTAG RVP Fast (Luminex Molecular Diagnostics, Austin, TX) performed on the xTag platform. This test for diagnosing viral respiratory tract infections has two research-use-only (RUO) equivalents, ResPlex (Qiagen, Venlo, The Netherlands) and MultiCode-PLx (Eragen Biosciences, Madison, WI; now a Luminex company) (32). Compared to conventional (bi-dimensional) microarrays, the xTAG platform is a tridimensional array consisting of microbeads, each with a specific probe to capture labeled amplicons. The microbeads contain a mixture of fluorescent dyes, which increases the specificity of the assay during flow cytometric analysis, conferring an easier means of individually validating each lot of functionalized microbeads. In a recent study using the Luminex xTAG platform for the diagnosis of viral respiratory infections, Dundas and colleagues (33) determined that this multiparametric detection technology was more costeffective than direct immunofluorescence assays and culture for the detection of respiratory viruses; the turnaround time of the xTAG RVP Fast test is estimated to be less than 8 hours for 96 samples. A strategic advantage of the xMAP/ xTAG technology lies in its versatility, in the sense that novel combinations of functionalized microbeads can be assembled after being individually validated. In contrast, conventional microarrays have a more fixed format. For in vitro diagnostics, the RVP Fast test (GenMark Diagnostics, Carlsbad, CA) is FDA approved. Another assay, the eSensor XT-8 system (GenMark Diagnostics), operates cartridges that contain a microarray of oligonucleotide probes immobilized on gold-plated electrodes. These probes capture targetor disease-specific amplicons by molec162

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ular hybridization; detection is accomplished by the formation of an electrochemical circuit following the hybridization of ferrocene-labeled oligonucleotides to a region of the amplicon overhanging the capture probe. It is our opinion that molecular hybridization is a highly advantageous strategy if applied to detection of multiple etiological agents to better guide the physician in diagnosis and therapy. Theoretically, it could be possible to develop a comprehensive “universal” microbial panel on the xTAG platform to detect a greater variety of pathogens from different biological sample types. In addition to the Conformité Européene (CE)-marked gastrointestinal microbial panel developed by Luminex, independent teams have developed multiplex assays for bacterial vaginosis, yeasts, and gastrointestinal pathogens on this platform (34-36). Such methodological advances cannot be realized without efforts to develop sample-adapted preparation methods that optimize the extraction of microbial nucleic acids. For example, SIRS-Lab GmbH (Jena, Germany) has developed LOOXSTER, a kit that specifically concentrates bacterial DNA from a mixture containing eucaryotic DNA by binding bacterial DNA to a protein-based column matrix; washes of the crude DNA extract eliminates unbound DNA (more than 90% of eucaryotic DNA) and inhibitors before the elution of bacterial DNA. Mass spectrometry MS is a powerful analytical method that provides mass-to-charge ratio (m/z) profiles of ionized macromolecule fragments generated by the laser-induced pulverization of a protein or nucleic acid sample, followed by a charge- and/or mass-based separation of fragments or bases (37). Fragmentation of macromolecules is accomplished by either MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) or ESI (electrospray ionization). For rapid bacterial and fungal identification, MALDI-TOF has been applied to cost-effectively analyze the products derived from a pure culture isolate of positive blood or urine culture in less than 2 hours, although the analytical phase may require only a few minutes (38-40). Currently, only the MALDI Biotyper (Bruker Daltonics Inc., Billerica, MA) is CE marked for in vitro diagnos© 2012 Elsevier

tics in Europe. The VITEK MS from bioMérieux (Marcy l’Étoile, France) is currently only available as an RUO method. Another strategy, PCR/ESI-MS, is based on the determination of the molecular weight and base composition (A, C, G, and T) of up to eight PCR-generated amplicons (17,37). This technology, commercially available in the form of the PLEX-ID instrument (Abbott Ibis Biosciences, Carlsbad, CA), recently received CE marking (Table 2). With regard to conventional clinical microbiology, MS will easily compete with other post-culture microbial identification schemes, as long as the spectrum generated from a biological sample can be analyzed against a comprehensive and up-to-date library of reference spectra. This application of the technology will provide considerable advantages in terms of speed and cost and significantly contribute to improved management of bloodstream and other infections. Although the diagnostic answer will be delivered to the treating physician at best 18 to 24 hours after collection of blood, detection of pathogens directly from blood using MS has also been reported (17). Nonetheless, implementation of MS will constitute a giant leap forward in the management of sepsis. Nucleotide (pyro)sequencing and next-generation sequencing TRUGENE HIV-1 (Siemens Healthcare Diagnostics, Tarrytown, NY) and ViroSeq HIV-1 (Abbott Molecular, Des Plaines, IL) were the first personalized medicine tests developed for the management of an infectious disease, HIVAIDS (Table 3). Following multiplex PCR amplification of the reverse transcriptase and protease genes, nucleotide sequencing, and interrogation of an antiviral resistance mutation database, these methods enable the adjustment of antiviral treatment to the genotypic resistance profile of the circulating virus population . MicroSeq (Applied Biosystems [now a Life Technologies company], Foster City, CA) and PyroMark ID (QIAGEN, Venlo, The Netherlands) are nucleic acid sequencing and pyrosequencing platforms that genotype post-PCR amplicons of evolutionarily conserved genes, such as rRNA genes, to identify microbes by interrogating databases of reference microorganisms (41). Pyrosequencing, Clinical Microbiology Newsletter 34:20,2012

Table 1. Molecular hybridization platforms and mass spectrometry systems for multiparametric diagnosis of infectious syndromes Commercially available platform or test

Description, applications, and regulatory status

Molecular hybridization

xTAG RVP Fast and RVP Fast 2.0 tests (Luminex Molecular Diagnostics; www.luminexcorp.com) xTAG GPP (Luminex Molecular Diagnostics; www.luminexcorp.com) eSensor Respiratory Viral Panel (GenMark Diagnostics; www.genmarkdx.com) INNO-LiPA (Innogenetics NV; www.innogenetics.com)

GenoType (Hain Lifescience; www.hainlifescience.com) Prove-it Sepsis (Mobidiag; www.mobidiag.com)

CLART Technology (Genomica; www.genomica.es) EntericBio (www.entericbio.com) INFINITI RVP Plus (Autogenomics; www.autogenomics.com) ResPlex (Qiagen; www.qiagen.com)

ArrayTube (Identibac; www.identibac.com) VYOO/LOOXSTER (SIRS Lab; www.sirs-lab.com)

• Simultaneous detection of multiple viral types and subtypes from nasopharyngeal swabs from individuals suspected of respiratory tract infections • Multiplex PCR and hybridization on tri-dimensional array (microbeads) • xTAG RVP Fast is FDA 510(k)a • Simultaneous detection of viral, bacterial, and protozoan parasites causing gastrointestinal diseases • CE IVDb • Multiplex RT-PCR amplification and electrochemical detection of molecular hybridization for the detection of common RNA and DNA respiratory viruses • Test submitted to FDA for approval in December 2011, but eSensor XT-8 is 510(k) • Hepatitis C virus genotyping test in the pipeline. • Multiplex PCR amplification followed by strip-based molecular hybridization and colorimetric detection • Multiparametric assays for detection/identification or rifampin susceptibility of mycobacteria and genotyping or subtyping of hepatitis B and C viruses • CE IVD • Multiplex PCR amplification and molecular hybridization tests for wide spectrum of bacterial targets, antimicrobial resistance, and toxin markers • CE IVD • Multiplex PCR amplification and microarray hybridization for detection of 60 bacteria, 13 fungi, and methicillin and vancomycin resistance genes • Two formats: TubeArray or StripArray • CE IVD • Multiplex PCR amplification and detection/identification of microbial pathogens (several panels available) on low-density microarrays • CE IVD • Detection of 4 fecal pathogens (enriched by culture) by multiplex PCR and hybridization • CE IVD • RT-PCR and microarray hybridization in an automated platform for the detection of 25 respiratory viruses • RVP Plus test is RUOc but INFINITI Analyzer is FDA 510(k) cleared • Multiplex analysis of respiratory pathogens by molecular amplification and hybridization on tri-dimensional array (xMAP technology) • RUO • Multiplex PCR amplification and molecular hybridization platform for bacterial and viral detection, antimicrobial resistance and virulence factors, and influenza virus typing • RUO • Rapid detection of sepsis pathogens (34 bacteria, 7 fungi, and 5 resistance gene alleles) from whole blood by multiplex PCR coupled to microarray hybridization • LOOXSTER protein used to preferentially bind bacterial and fungal DNA • RUO

Mass spectrometry

MALDI Biotyper (Bruker Daltonics; www.bdal.com) VITEK MS (bioMérieux; www.biomerieux-industry.com/id) Micromass MALDI micro MX (Waters; www.waters.com) PLEX-ID (Abbott Ibis Biosciences; www.ibisbiosciences.com) a b c

• • • • • • • • • •

Post-culture microbial identification by MALDI-TOF Possible sample types include pure culture, positive blood cultures, and urine CE IVD Post-culture microbial identification by MALDI-TOF on Shimadzu ARAMIS instrument Possible sample types include pure culture, positive blood cultures, and urine Post-culture microbial identification by MALDI-TOF Possible sample types include pure culture, positive blood cultures, and urine Post-PCR microbial identification by PCR-ESI Identification by interrogation of reference spectra CE IVD

FDA, cleared by U.S. Food and Drug Administration for in vitro diagnostics. CE IVD, Conformité Européenne marking for in vitro diagnostics. RUO, research use only.

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a real-time sequencing technology that enables much faster identification of microbes and determination of antimicrobial resistance mutations from clinical samples or cultures (42,43), is the core technology of the 454 genome sequencers, which became the first next-generation instruments. While classical chain termination (Sanger method) sequencing was used for a large number of microbial genomesequencing projects, the explosion of metagenomics was catalyzed by a phenomenal increase in the speed (with decreased costs) of high-throughput nucleic acid de novo sequencing technologies and bioinformatics software for genome assembly and annotation (44,45). Perhaps, MS has the potential to replace multiplex PCR or microarrays for the rapid identification of microbial pathogens from blood cultures or isolated colonies, but nucleotide sequencing remains the only strategy to efficiently characterize new pathogens or novel mutations that may reduce the probability of detection of a pathogen, especially if destabilizing polymorphisms arise in amplification primers or detection/capture probe binding regions (17,44,46). Recent events, such as the E. coli O104 foodborne outbreak in 2011 in Germany, demonstrated that de novo massive parallel sequencing may render the identification of an unknown pathogen in a matter of hours (47), thereby speeding up the response from public health authorities. The promises of personalized medicine and the race to the “$1,000 genome” have catalyzed a technological hype that has led to the development of several next-generation sequencing platforms (Table 2) designed to (i) decipher a human exome or genome in a matter of days, (ii) discover mutations associated with rare genetic disorders (48), and/or (iii) determine the pharmacogenetic profile of an individual or of cancer tumors in order to guide treatment and dosage or prevent adverse drug reactions (49). These applications will be the main elements of an emerging model called “precision medicine” (50). Integrated fluidic systems One obstacle to implementing multiparametric detection technologies in clinical laboratories is the complexity of test protocols and instruments that 164

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require skilled and highly trained technologists, sometimes an increased number of technical steps, and strict instrument operational requirements for adequate reagent and test performance. To alleviate these drawbacks, technology developers are investing efforts to bring to market automated instruments or fluidic devices that integrate highly specialized nucleic acid-based tests, not necessarily multiparametric for the time being (51). A class of molecular microbiology tools aimed at the point-of-care market was pioneered by Cepheid’s GeneXpert system (Cepheid, Sunnyvale, CA) and was categorized by the FDA as “moderate complexity” under the CLIA in 2007. Since then, the Xpert cartridge has been redesigned so that the diagnostic procedure can be performed closer to the patient, ideally at the point of care. Other integrated fluidic systems, such as the BD MAX (BD Diagnostic Systems, Sparks, MD), FilmArray (Idaho Technologies, Salt Lake City, UT), and Liat (IQuum, Marlborough, MA) have tests approved by the FDA, while the Unyvero (Curetis AG, Holzgerlingen, Germany), TruDiagnosis (Akonni Biosystems, Frederick, MD), Biocartis (Lausanne, Switzerland), and VereID (Veredus Laboratories Pte Ltd, Singapore, Singapore) systems are commercialized but are not yet approved for in vitro diagnostics in the U.S. (Table 3). The single most complicated and challenging task in designing an integrated microfluidic system for infectious diseases diagnostics is sample pretreatment and preparation. Special challenges are found in the context of an infectious syndrome, such as sepsis, where the microbial load can be as low as 1 CFU/ml, and the volume of the clinical sample is quite large (1 to 30 ml); thus, microbial targets are surrounded by an ocean of human DNA and amplification process inhibitors. There is no doubt that technology developers will eventually successfully address these challenges and bring extraction methods to clinical environments with sophisticated instruments capable of rapid diagnostics at the point of care from complex and/or large-volume samples. However, in the meantime, commercial systems, such as those listed in Table 3, generally accept samples, such as swabs or nucleic acid extracts, prepared externally. © 2012 Elsevier

The implementation of multiparametric technologies – advantages and challenges In the last 10 years, molecular diagnostics has been continually transformed in clinical microbiology, catalyzed by the regulatory acceptance of real-time PCR closed-tube tests that are capable of accelerating the management of infectious diseases (12). However, despite performances similar or superior to that of gold standard culturebased methods, the market penetration of molecular diagnostics has been hampered by cost, regulatory, and reimbursement issues (52). The management of infectious syndromes definitely needs adaptable multiparametric detection platforms that are capable of accelerating the diagnosis so that more patients can survive a bloodstream, respiratory tract, or hospitalacquired infection (5,21). Logically, a more comprehensive coverage of clinically relevant microorganisms should enable more timely identification of pathogens, better targeted management of infections, and better utilization of antimicrobials, so that medicine can finally regain some control over multidrug resistance. Significant progress in this area will be accompanied by a smaller economic burden on patients and their families and to health care providers, who are sometimes helpless witnesses of human suffering and death. In terms of regulatory approval, which should be applicable to all infectious syndromes, “The Infectious Diseases Society of America does not believe that regulatory clearance (approval) |of a new diagnostic test should require that the test validates the presence of pneumonia or another variant of respiratory tract infection. Instead, the focus of a clinical trial should be the accuracy of the new molecular method in the detection of a given bacterial or viral organism… Given the laboratory result, the physician integrates all the clinical data and determines whether pneumonia is present and whether the identified organism(s) is/are a likely etiology” (21). Accordingly, we believe that the regulatory approval of the highly adaptable xTAG RVPFast test by the FDA constiClinical Microbiology Newsletter 34:20,2012

Table 2. Nucleotide sequencing and next-generation sequencing platforms Commercially available platform or test

Description, applications, and regulatory status

Nucleotide (pyro)sequencing

TRUGENE HIV-1 (Siemens Healthcare; www.medical.siemens.com) ViroSeq HIV-1 (Abbott Molecular; www.abbottmolecular.com) BlackLight Sepsis kit (BlackBio; www.blackbio.eu)

MicroSeq rDNA identification system (Applied Biosystems; www.appliedbiosystems.com) PyroMark ID (Qiagen; www.pyrosequencing.com)

• Genotyping of the HIV-1 reverse transcriptase and protease genes by nucleotide sequencing to identify mutations associated with antiviral resistance • FDA 510(k)a • Identification of bacteria from clinical samples or blood culture by multiplex PCR complemented by pyrosequencing • DNA is extracted using BlackLight cards • CE IVDb • Microbial (bacteria and fungi) identification based on PCR amplification and nucleotide sequencing of small ribosomal RNA gene (rDNA) • RUOc • Microbial identification and resistance mutation scanning by pyrosequencing of amplicons generated by PCR or reverse transcriptase-PCR (RT-PCR) • RUO

Next-generation sequencing

Genome Sequencer FLX+ and Junior systems (Roche Diagnostics; www.my454.com)

HiSeq, Genome Analyzer II, and MiSeq systems (Illumina; www.illumina.com)

5500 Series Genetic Analysis Systems (Applied Biosystems; www.appliedbiosystems.com)

HeliScope single-molecule sequencer (Helicos Biosciences; www.helicosbio.com)

PacBio (Pacific Biosciences; www.pacificbiosciences.com)

Ion PGM and Ion Proton sequencers (Ion Torrent; www.iontorrent.com)

GridION platform and MinION device (Oxford Nanopore Technologies; www.nanoporetech.com)

• • • • • • • • • • • • • • • •

• • • • • • • • • • • • • • • • •

Highly massive pyrosequencing technology Template amplification by emulsion PCR New reagent formulation enable read lengths of up to 1,000 bases; 85% of reads are >500 bases Estimated throughput of up to 0.8 gigabases/day/instrument RUO Sequencing by synthesis (SBS) technology of DNA templates immobilized in a flow cell Template amplification by bridge PCR Single or paired reads of 100-150 bases Estimated throughput of up to 36 gigabases/day/instrument RUO Also known as SOLiD system (sequencing by oligonucleotide ligation and detection) Template amplification by emulsion PCR Maximum read length of 75 bases Estimated throughput of up to 300 gigabases/day/instrument RUO Direct sequencing of single DNA or RNA molecules by a proprietary form of sequencing by synthesis in which labeled bases are sequentially added to nucleic acid templates captured on a flow cell No template amplification Maximum read length of 55 bases Estimated throughput of up to 40 gigabases/day/instrument RUO SMRT (single-molecule real-time) sequencing in zero-mode waveguides Base incorporation determined by transit time of fluorescent pyrophosphate in nanowell No template amplification Read length of 1,000-10,000 bases per nanowell Estimated throughput of >0.2 gigabase/day/instrument RUO Proprietary semiconductor technology that detects the pH change when a proton (H+) is released during incorporation of a nucleotide Target DNA is immobilized on beads Maximum read length of 100 bases Estimated throughput up to 1 gigabase/day/instrument RUO Nanopore-based system for the direct, real-time electronic analysis of single molecules, including DNA and RNA Marketing planned for 2012

a

FDA, cleared by U.S. Food and Drug Administration for in vitro diagnostics. b CE IVD, Conformité Européenne marking for in vitro diagnostics. c RUO, research use only.

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tutes a pivotal event encouraging broader use of multiparametric technologies in clinical microbiology. Cost-effectiveness studies have shown (or will support) development, validation, regulatory approval, and implementation of more versatile platforms and tests (53). Operationally, multiplex real-time PCR, MALDI-TOF, and integrated fluidic systems are technologies that follow the road paved by the rapid closed-tube real-time PCR assays, pioneered by Infectio Diagnostic (IDI) Inc. (now BD Diagnostics GeneOhm, Québec City, QC, Canada). This approach reduced the risk of laboratory cross-contamination by amplified genetic material, in contrast to molecular hybridization and nucleotide-sequencing platforms. This means that the implementation of the latter technologies in clinical microbiology should be performed under strict operational guidelines to guarantee the precision and clinical effectiveness of the diagnostic result. The rapid emergence of clinical genomic sequencing in personalized medicine should thus provide an opportunity to upgrade, renovate, integrate, and/or build adequate molecular biology infrastructures within health care facilities, usable for both human genetics and molecular microbiology and offering a comprehensive menu of high-utility tests 24/7. To put it bluntly, a blood sample, infected or not, is still a blood sample.

Conclusions Multiparametric detection technologies do come with a price in human resources and qualified staff, but in the context of improving the outcome and elevated morbidity and mortality rates of syndromic infections, it must be noted that in 2008, the average cost of the top 0.5% most expensive hospital stays exceeded $500,000 (30.5% of those admitted for septicemia died; [54]), i.e., several times the cost of purchasing and maintaining a multiparametric instrument for a full year. Bringing the microbial identification phase of the infectious disease diagnostic process from 24 to 72 hours to less than 6 hours while significantly augmenting its accuracy, will undoubtedly facilitate patient management and better guide the choice of an antimicrobial regimen to ultimately improve the rate of survival of patients. It is anticipated 166

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that clinicians may be initially overwhelmed by the result interpretation phases, but as with the empirical management practices that evolved from clinical experience and risk analysis strategies, we believe that specialists will rapidly adapt to the new methodologies. References 1. Lehmann, L.E. et al. 2010. Cost and mortality prediction using polymerase chain reaction pathogen detection in sepsis: evidence from three observational trials. Crit. Care 14:R186. 2. Kumar, A. et al. 2009. Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock. Chest 136:12371248. 3. Lin, J.-N. et al. 2010. Characteristics and outcomes of polymicrobial bloodstream infections in the emergency department: a matched case-control study. Acad. Emerg. Med. 17:1072-1079. 4. Micek, S.T., R.M. Reichley, and M.H Kollef. 2011. Health care-associated pneumonia (HCAP): empiric antibiotics targeting methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa predict optimal outcome. Medicine 90:390-395. 5. Nicolau, D.P. 2011. Current challenges in the management of the infected patient. Curr. Opin. Infect. Dis. 24(Suppl.1): S1-S10. 6. Jabes, D. 2011. The antibiotic R&D pipeline: an update. Curr. Opin. Microbiol. 14:564-569. 7. Griffith, M.M. et al. 2012. The impact of anti-infective drug shortages on hospitals in the United States: trends and causes. Clin. Infect. Dis. 54:684-691. 8. Klotz, S.A. et al. 2007. Polymicrobial bloodstream infections involving Candida species: analysis of patients and review of the literature. Diagn. Microbiol. Infect. Dis. 59:401-406. 9. Rogers, G.B. et al. 2010. Revealing the dynamics of polymicrobial infections: implications for antibiotic therapy. Trends Microbiol. 18:357-364. 10. Schimke, I. 2009. Quality and timeliness in medical laboratory testing. Anal. Bioanal. Chem. 393:1499-1504. 11. Bissonnette, L. and M.G. Bergeron. 2006. The next revolution in the molecular theranostics of infectious diseases: microfabricated systems for personalized medicine. Expert Rev. Mol. Diagn. 6:433-450. 12. Association for Molecular Pathology. FDA-cleared/approved molecular diag-

© 2012 Elsevier

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

nostics tests. (http://www.amp.org/ FDATable/FDATable.doc; last accessed February 2012). McGeer, A. 2007. Antiviral therapy and outcomes of influenza requiring hospitalization in Ontario, Canada. Clin. Infect. Dis. 45:1568-1575. Bissonnette, L. and M.G. Bergeron. 2010. Diagnosing infections – current and anticipated technologies for point-of-care diagnostics and home-based testing. Clin. Microbiol. Infect. 16:1044-1053. Cohen-Bacrie, S. et al. 2011. Revolutionizing clinical microbiology laboratory organization in hospitals with in situ point-of-care. PLoS One 6:e22403. Daniels, R. 2011. Surviving the first hours in sepsis: getting the basics right (an intensivist’s perspective). J. Antimicrob. Chemother. 66:ii11-ii23. Ecker, D.J. et al. 2010. New technology for rapid molecular diagnosis of bloodstream infections. Expert Rev. Mol. Diagn. 10:399-415. Paolucci, M., M.P. Landini, and V. Sambri. 2010. Conventional and molecular techniques for the early diagnosis of bacteraemia. Int. J. Antimicrob. Agents 36S:S6-S16. Endimiani, A. et al. 2011. Are we ready for novel detection methods to treat respiratory pathogens in hospital-acquired pneumonia? Clin. Infect. Dis. 52(Suppl. 4):S373-S383. Pavia, A.T. 2011. Viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis. Clin. Infect. Dis. 52(S4):S284-S289. Infectious Diseases Society of America. 2011. An unmet need: rapid molecular diagnostics tests for respiratory tract infections. Clin. Infect. Dis. 52(S4): S384-S385. Foxman, B. 2010. The epidemiology of urinary tract infection. Nat. Rev. Urol. 7:653-660. Imirzalioglu, C. et al. 2008. Hidden pathogens uncovered: metagenomic analysis of urinary tract infections. Andrologia 40:66-71. Goff, D.A. 2011. Antimicrobial stewardship: bridging the gap between quality care and cost. Curr. Opin. Infect. Dis. 24(Suppl.1):S11-S20. Umscheid, C.A. et al. 2011. Estimating the proportion of healthcare-associated infections that are reasonably preventable and the related mortality and costs. Infect. Control Hosp. Epidemiol. 32:101-114. Brown, J. and J.A Paladino. 2010. Impact of rapid methicillin-resistant Staphylococcus aureus polymerase chain

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reaction testing on mortality and cost effectiveness in hospitalized patients with bacteraemia. Pharmacoeconomics 28:567-575. 27. Avolio, M. et al. 2010. Molecular identification of bloodstream pathogens in patients presenting to the emergency

department with suspected sepsis. Shock 34:27-30. 28. Obara, H. et al. 2011. The role of realtime PCR technology for rapid detection and identification of bacterial and fungal pathogens in whole-blood samples. J. Infect. Chemother. 17:327-333.

29. Sancho-Tello, S. et al. 2011. Performance of the LightCycler SeptiFast Test Mgrade in detecting microbial pathogens in purulent fluids. J. Clin. Microbiol. 49:2988-2991. 30. Tissari, P. et al. 2010. Accurate and rapid identification of bacterial species

Table 3. Integrated fluidic systems developed for infectious disease diagnostics Commercially available platform or test GeneXpert system and Xpert tests (Cepheid; www.cepheid.com; www.cepheidinternational.com)

Description, applications, and regulatory status • Microfluidic cartridge for sample preparation, nucleic acid extraction, and reverse transcriptionase (RT)-PCR • Platform is CLIAa moderate complexity. • Many Xpert tests for HAI and influenza are FDA 510(k)b and CE IVD.c • Xpert tests for Chlamydia trachomatis (CT) and C. trachomatis/Neisseria gonorrhoeae (CT/NG) are CE IVD. • GeneXpert Infinity-80 instrument is FDA cleared.

BD MAX (BD Diagnostics GeneOhm; www.bd.com/geneohm)

• Microfluidic cartridge for sample preparation, nucleic acid extraction, and RT-PCR for several infectious diseases • Platform is CLIA moderate complexity. • BD MAX GBS Assay for Group B Streptococcus is FDA 510(k). • Benchtop analyzer that combines automated nucleic acid extraction, purification, amplification (if required), and hybridization on functionalized gold nanoparticles in independent modules • Platform is CLIA moderate complexity. • Verigene Respiratory virus+ and S. aureus blood culture tests are FDA 510(k). • RUO tests include gram-positive and gram-negative bacterial panels from blood culture, enteric pathogen panel, and respiratory virus panel. • Microfluidic cartridge for sample preparation, nucleic acid extraction, and multiplex PCR for upper respiratory tract infections • FDA 510(k) • Applications in development for blood culture identification, gastrointestinal illness, sexually transmitted infections, and biothreat agents • Automatic and integrated sample purification, RT-PCR amplification, and real-time detection of influenza A/B virus • FDA 510(k) • Integrated cartridge and instrument for universal DNA extraction and detection of different microorganisms (gram-positive and -negative bacteria, viruses, fungi, mycobacteria, and other intracellular organisms) and antibiotic resistance genes • PCR amplification and detection using proprietary array technology • Unyvero diagnostic platform and P50 Pneumonia disposable cartridge-based test are CE IVD. • Integrated cartridge and instrument for nucleic acid extraction, PCR amplification, amplicon denaturation, microarray hybridization, and detection • Microbial panels in development include sexually transmitted infections and sepsis pathogens. • RUO • Molecular diagnostics platform for detection of viral or bacterial pathogens from blood, saliva, or urine using PCR and gel drop microarray platform • Nucleic acid extraction using TruTip technology • RUO • Automated sample-to-answer systems using APTIMA kits • Hybrid capture and detection by isothermal transcription-mediated amplification • APTIMA assays are FDA 510(k) and CE IVD. • Real-time PCR capability for Prodesse RT-PCR tests planned for 2012 • Integrated microfluidic cartridge for sample preparation, nucleic acid extraction, and multiplex PCR • PCR amplification coupled to molecular hybridization

Verigene system and tests (Nanosphere; www.nanosphere.us.)

FilmArray (Idaho Technology; www.idahotech.com)

Liat (Lab-in-a-tube; IQuum; www.iquum.com) Unyvero (Curetis AG; www.curetis.com)

Rheonix CARD (Rheonix; www.rheonix.com)

TruDiagnosis and TruArray (Akonni Biosystems; www.akonni.com)

PANTHER and TIGRIS DTS systems (Gen-Probe; www.genprobe.com)

Biocartis Molecular Diagnostics platform (Biocartis; www.biocartis.com) VereID Biosystem (Veredus Laboratories; www.vereduslabs.com) a

CLIA, Clinical Laboratory Improvement Amendments of 1988. FDA, cleared by U.S. Food and Drug Administration for in vitro diagnostics. CE IVD, Conformité Européenne marking for in vitro diagnostics. d RUO, research use only. b c

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31.

32.

33.

34.

35.

36.

37.

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from positive blood cultures with a DNAbased microarray platform: an observational study. Lancet 375:224-230. Jordan, B.R. 2010. Is there a niche for DNA microarrays in molecular diagnostics? Expert Rev. Mol. Diagn. 10:875882. Balada-Llasat, J.-M. et al. 2011. Evaluation of commercial ResPlex II v2.0, MultiCode®-PLx, and xTAG® respiratory viral panels for the diagnosis of viral infections in adults. J. Clin. Virol. 50:42-45. Dundas, N.E. et al. 2011. A lean laboratory – Operational simplicity and cost effectiveness of the Luminex xTAG® respiratory viral panel. J. Mol. Diagn. 13:175-179. Dumonceaux, T.J. et al. 2009. Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres. J. Clin. Microbiol. 47:4067-4077. Landlinger, C. et al. 2009. Speciesspecific identification of a wide range of clinically relevant fungal pathogens by use of Luminex xMAP technology. J. Clin. Microbiol. 47:1063-1073. Platts-Mills, J.A., D.J. Operario, and E.R. Houpt. 2012. Molecular diagnosis of diarrhea: current status and future potential. Curr. Infect. Dis. Rep. 14:4146. Emonet, S. et al. 2010. Application and use of various mass spectrometry

0196-4399/00 (see frontmatter)

38.

39.

40.

41.

42.

43.

44.

45.

46.

methods in clinical microbiology. Clin. Microbiol. Infect. 16:1604-1613. Ferroni, A. et al. 2010. Real-time identification of bacteria and Candida species in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight spectrometry. J. Clin. Microbiol. 48:1542-1548. Seng, P. et al. 2010. MALDI-TOF-mass spectrometry applications in clinical microbiology. Future Microbiol. 5:1733-1754. La Scola, B. 2011. Intact cell MALDITOF mass spectrometry-based approaches for the diagnosis of bloodstream infections. Expert Rev. Mol. Diagn. 11:287298. Kemp, M. et al. 2010. Routine ribosomal PCR and DNA sequencing for detection and identification of bacteria. Future Microbiol. 5:1101-1107. Jordan, J.A. et al. 2009. Utility of pyrosequencing in identifying bacteria directly from positive blood culture bottles. J. Clin. Microbiol. 47:368-372. Deng, Y.-M., N. Caldwell, and I.G. Barr. 2011. Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing. PLoS One 6:e23400. Petrosino, J.F. et al. 2009. Metagenomic pyrosequencing and microbial identification. Clin. Chem. 55:856-866. Metzker, M.L. 2010. Sequencing technologies – the next generation. Nat. Rev. Genet. 11:31-46. Barnard, R.T., R.A. Hall, and E.A. Gould.

© 2012 Elsevier

47.

48.

49.

50.

51.

52.

53.

54.

2011. Expecting the unexpected: nucleic acid-based diagnosis and discovery of emerging viruses. Expert Rev. Mol. Diagn. 11:409-423. Brzuszkiewicz, E. et al. 2010. Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: entero-aggregative-haemorrhagic Escherichia coli (EAHEC). Arch. Microbiol. 193:883-891. Bainbridge, M.N. et al. 2011. Wholegenome sequencing for optimized patient management. Sci. Transl. Med. 3:87re3. Natrajan, R. and J.S. Reis-Filho. 2011. Next-generation sequencing applied to molecular diagnostics. Expert Rev. Mol. Diagn. 11:425-444. Mirnezami, R., J. Nicholson, and A. Darzi. 2012. Preparing for precision medicine. N. Engl. J. Med. 366:489-491. Eicher, D. and C.A. Merten. 2011. Microfluidic devices for diagnostic applications. Expert Rev. Mol. Diagn. 11:505-519. Gibbs, J.N. 2011. Regulating molecular diagnostic assays: developing a new regulatory structure for a new technology. Expert Rev. Mol. Diagn. 11:367-381. Russek-Cohen, E. et al. 2011. FDA perspectives on diagnostic device clinical studies for respiratory infections. Clin. Infect. Dis. 52(S4):S305-S311. Mitka, M. 2010. Hospitalizations for extreme conditions mean extreme expenses, study verifies. JAMA 304:2579-2580.

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