key: cord-022053-idft1p6d
authors: Pecora, Nicole; Milner, Danny A.
title: New Technologies for the Diagnosis of Infection
date: 2017-07-21
journal: Diagnostic Pathology of Infectious Disease
DOI: 10.1016/b978-0-323-44585-6.00006-0
sha: 
doc_id: 22053
cord_uid: idft1p6d

nan

FilmArray (BioFire/BioMérieux) BD Max (BD Diagnostics) Luminex

Next-Generation Sequencing Conclusion T he clinical microbiology laboratory is constantly evolving and progressing. New technologies have revolutionized the characterization and diagnosis of pathogens. The purpose of this chapter is to review the features, benefits, and limitations of some of the key new methodologies in infectious disease diagnostics. The chapter will be organized by technology. New technologies have the most impact in the fields of bacteriology and virology, but there are also promising new developments in mycology and parasitology.

The mainstay of bacterial and mycologic diagnosis continues to be culture by traditional techniques, although increasingly these are automated ( Fig. 6.1 ). After an organism is isolated, however, a host of newer diagnostics have enhanced, and in some cases replaced, the biochemical test algorithm that has historically defined clinical bacteriology. The advent of matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry is one of the main forces behind this shift, allowing a cultured organism to be identified in minutes instead of hours to days or even weeks in the case of very slow-growing organisms, such as mycobacteria. Although still not in routine clinical use, the application of MALDI-TOF to direct patient samples, such as blood and urine, and applications such as antibiotic resistance are exciting developments. Beyond bacteria, MALDI-TOF is becoming established for the identification of yeasts and, with the development of more extensive and higher quality libraries, is expected to also do so for other fungi.

Nucleic acid-based technologies have also had a big impact. In particular, there are a growing number of test platforms that provide rapid diagnostics directly from primary specimens. Many of these are multiplexed to look at several pathogens at one time. The sequencing of the regions of ribosomal RNA (rRNA), such as the 16S rRNA gene in bacteria, already considered the gold standard for bacterial identification, is entering more and more into clinical diagnostics ( Fig. 6.2) . Similarly, the routine sequencing of the intergenic transcribed spacer (ITS) and 28S rRNA regions in fungi is a welcome addition to current laborious and morphology-heavy techniques for diagnosing fungal illnesses. The entry of next-generation sequencing (NGS) into clinical microbiology has also had a dramatic effect on both the profiling of individual specimens (i.e., members of a potential outbreak or highly drug resistant strains) as well as communities (i.e., microbiome analysis in health and disease). Finally, the emerging technology of polymerase chain reaction (PCR)-electrospray ionization (ESI) mass spectrometry holds great promise for all major phyla of pathogens. ITS   ITS1  ITS2   ITS5  ITS1  ITS2  ITS3  ITS4  NL1  NL4   ITS1  ITS2  D1/ testing. Immunohistochemistry (IHC) in anatomic pathology is based on the same principle as these rapid tests and has a wide range of uses for the detection and sometimes speciation of organisms in formalin-fixed, paraffin-embedded tissue (FFPE) sections. Organisms commonly identified this way include spirochetes, mycobacteria, DNA viruses, Aspergillus, Candida, and Toxoplasma, although special reference laboratories (e.g., The Infectious Disease Pathology Branch at the Centers for Disease Control and Prevention) have a wide range of antibodies for common to exotic pathogens for tissue confirmation. MALDI-TOF, a mass spectrometry-based method, allows for a rapid (minutes) assessment of the characteristic complement of ribosomal proteins in an isolate, providing a species-level identification in many cases. After a microbial colony forms, it can be spotted onto a MALDI plate in matrix compound and loaded onto the mass spectrophotometer. This topic has been reviewed in great detail. [3] [4] [5] Briefly, a laser is focused onto the sample/matrix spot, causing sublimation, ionization, migration through a time of flight tube, and detection by a mass spectrometer (MS).The readout is a spectrum of peaks representing mass to charge ratios (M/Z) of different analytes. The mass range (approximately 2 to 20 kDa) is highly enriched for ribosomal proteins, which make up a large share of the typical MALDI spectra on clinical instruments. Spectra are compared with curated databases and the result is returned along with a metric describing the strength of the identification.

This technology has been widely adopted in Europe and is gradually entering clinical microbiology labs in the United States. There are currently two US Food and Drug Administration (FDA)-approved systems: the Vitek MS (BioMérieux, Durham, North Carolina) and the MALDI Biotyper (Bruker Daltonics Inc., Billerica, Massachusetts). Although the technology behind these two systems is essentially equivalent, they differ in their curated libraries, software, identification algorithms, and scoring systems. 5 For example, the Vitek MS reports isolates with a confidence value of 1% to 100%, whereas the Biotyper returns a score that can range from 0 to 3.000. For most applications a score of 2.000 or greater is necessary for a species level identification. Because of this, it can be difficult to directly compare results between the two systems. There have been several studies using both platforms back to back. One study on a large collection of 642 strains of bacteria, yeasts, and molds demonstrated comparable performance, although noting that the library coverage of the test cohort was greater with the Biotyper (572 strains) than the Vitek MS (406) and that the Vitek was more likely to output a misidentification for strains not represented in the library. 6 For FDA-approved applications, both the Vitek MS and the Bruker may be used for a large library of aerobic gram-negative and gram-positive organisms, as well as several anaerobes and yeast. In general, performance has been reported to equal or exceed traditional biochemical systems, although it seems to be stronger for gram-negative than gram-positive organisms. 5, 7 Lackluster performance, in particular, has been noted for several grampositive rods. In a study with the Biotyper, the system was unable to identify several isolates of Nocardia, Tsukamurella, Kocuria, or Gordonia, although 89.7% of Listeria and 80% of Rhodococcus were correctly classified. 8 Members of Corynebacteria, Lactobacillus, and some Listeria also seem to present some difficulty, both in terms of difficulty discriminating between species and in requiring a lowered score cutoff (1.7) for species-level identification. 9 In similar studies using the Vitek platform, there were also reported problems in speciation of Listeria. 10 The misidentification of a Kocuria as a Corynebacterium has also been reported. 11 Among both gram-positive and gram-negative organisms, MALDI-TOF has several well-characterized difficulties discriminating certain species and complexes, including Streptococcus pneumoniae/ Streptococcus mitis, Escherichia coli/Shigella, and members of the Enterobacter cloacae complex. 3 Finally, it is important to note that the Biotyper requires a Security-Relevant Library for the identification of such organisms as Francisella tularensis, Burkholderia pseudomallei, and Brucella spp. 12 MALDI is rapidly becoming the method of choice for identifying anaerobic bacteria. 13 Several studies using large, diverse collections of anaerobes have demonstrated strong performances by both the BioTyper and the Vitek MS. 5 In one recent multicenter trial bringing together 651 anaerobic isolates for analysis by the Vitek MS 2.0, 91.2% of the isolates were correctly identified to species level. 14 The most notable difficulty was in identifying Fusobacterium nucleatum (42.9% identified correctly). The system also showed lower performance for certain species of Bacteroides, Actinomyces, and Prevotella. Similarly, in a study using the Biotyper platform to identify 197 anaerobes isolated from blood cultures, 86.8% of the strains were correctly identified to the species level (using a score of 2) and 94.9% to the genus level. 15 Fusobacterium spp. was again problematic, with only 23% being correctly identified to the species level. With formic acid/ethanol pretreatment, this number increased to 76.9%. In addition, 20.8% of the non-fragilis Bacteroides spp. was misidentified as other Bacteroides, with high confidence scores, and only 52.9% of the gram-positive anaerobic cocci were correctly identified to the species level. The latter was mostly a function of poor performance on several species of Peptoniphilus. 15 Interestingly, in a dedicated study looking at the performance of the Biotyper (2.0) on a collection of 277 isolates of Bacteroides spp., 97.5% were correctly identified to the species level. 16 The identification of yeasts is an FDA-approved application for both the Biotyper and Vitek MS. Both systems have demonstrated strong performance in multiple studies, reviewed by Cassagne et al. 17 One recent study with the Biotyper, for example, used a library of 303 clinical isolates representing a diverse array of yeasts and found an agreement with standard methods in 84.8%. 18 In an additional 26 isolates the Biotyper returned a result that was confirmed as true by sequencing in 21. The Biotyper failed to make identification in an additional 20 isolates. Bader et al. used an even larger cohort of clinical yeast isolates (1192) to compare the performance of traditional methods with the Bruker Biotyper and the Vitek SARAMIS (research use only database). They found an overall agreement between the methods of 95.1% and noted not only that both MALDI methods were able to discriminate species that traditional methods could not, but that the savings in time and money were substantial. 19 More recently, Chao et al. analyzed a collection of 200 clinical yeast isolates with both the Biotyper and the Vitek MS. 20 They found that the Biotyper slightly outperformed the Vitek MS (92.5% vs 79.5% correctly identified to the species or complex level). This compared with rates of 89% and 74% for two common phenotypic methods (Phoenix 100 YBC and the Vitek 2 Yeast ID, respectively). This trend was also observed by Mancini et al., who used a collection of 197 clinical yeast isolates, which included 157 Candida or related species and 40 non-Candida species. They found that the Biotyper identified 89.8% of isolates, whereas the Vitek MS identified 84.3% using the standard commercially available database. Importantly, the Vitek MS had a much higher rate of misidentification (12.1% vs 1%). 21 In general, it has been found that a formic acid/ethanol on the organism in question). 5, 7 Other benefits to MALDI in routine clinical microbiology include low costs of reagents and less production of waste materials (reduced consumables). There are a few shortcomings of this technology. For the most part, MALDI is limited to cultured isolates, so although it offers tremendous TAT advantages it is inherently limited by the time to growth of the organism. Sparse database coverage over certain phyla (e.g., filamentous fungi, some anaerobes) presents an issue in some cases, although commercially available libraries continue to be expanded. Another major caveat of MALDI is that it is not yet a practical method for assessing antibiotic resistance. Direct detection of resistance-conferring enzymes is not practical within the assay's limits of detection; however, some promising studies have been published looking at the ability of MALDI to detect breakdown products after incubation in the presence of antibiotic. 3 However, resistance testing is unlikely to be incorporated into a standard MALDI microbial identification platform in the near future.

Mass spectroscopy has been applied to human tissues, including FFPE, for a variety of purposes, but, to date, there is not a commercially available or routine method for using MS in the detection of organisms in FFPE. 44, 45 With the advent of rapid and inexpensive nucleic acid methods for FFPE, this field largely lies dormant, although incredible potential for detection and other data extraction from FFPE through MS may be possible in the future.

For more than 30 years, a wide spectrum of assays based on the detection of nucleic acids has been developed for the diagnostics of infectious disease. They range from simple probes to qualitative and quantitative target amplification to sequencing. For example, the first nucleic acid probe-based assay was launched in 1985 to test for legionnaires' disease (Gen-Probe). Real-time platforms, such as the Smartcycler and the Lightcycler, have an established place in both detection and quantification of pathogens, although the major impact has mostly been in virology, where the nucleic acid-based techniques were quickly adopted to overcome the difficulties inherent in viral culture. For example, hepatitis C virus (HCV) and HIV viral loads are routine applications of these technologies. There have been a number of newer nucleic acid-based technologies that have had a tremendous impact on microbial diagnostics, many of which are also geared towards bacterial pathogens. These include highly sensitive probes for use in direct specimens, to alternative amplification methods, rapid assays of single targets, and multiplexed systems that allow for the detection of many organisms in one assay. Sequencing assays are becoming more commonplace, with targets ranging from 16S rRNA to whole genomes and even metagenomic studies looking at the make-up of complex microbial ecosystems within the human host. In this section the technologies behind several of the most important new assays will be briefly introduced, followed by a discussion of their application to clinical syndromes, such as respiratory and gastrointestinal (GI) disease, bloodstream infections, meningitis, and reproductive health/sexually transmitted disease.

Probe hybridization assays were one of the earliest diagnostic techniques using nucleic acids as a target. The most commonly used probes are marketed by Hologic Gen-Probe Inc. (Marlborough, extraction method and lowered identity cutoffs (i.e., 1.7 on the Biotyper) are optimal for the analysis of yeasts. 22 Although not an FDA-approved application, MALDI has tremendous promise for the identification of mycobacteria and is already the preferred identification method of choice for these organisms in some clinical laboratories. 5 Caveats include the need for enhanced sample preparation methods for efficient cell breakage for biosafety reasons and to ensure high-quality spectra. 3, 23, 24 Several studies have shown promising results. Using the Biotyper equipped with the mycobacterial library version 1.0 and the Vitek MS, 84.7% to 93.8% of isolates were identified correctly to the species level. 24, 25 Bruker has subsequently released a new version of the mycobacterial library, version 3.0, containing 149 different species. It reportedly demonstrated high performance (>95% correct identification to species level) in a group of 1045 clinical samples. 26 Version 3.0 of the Biotyper mycobacterial library was also described by Rodriguez-Sanchez et al., who found 91.7% identification to species level on a group of 109 non-tuberculosis mycobacteria. 27 There is a great deal of interest in the identification of fungi other than yeast by MALDI, also not an FDA-approved application. Concerns with this group include not only efficient cell lysis but also the need for higher-quality reference databases. 17 Few studies have assessed the Biotyper or Vitek MS platforms on fungal isolates using only the commercially available databases. Iriart et al. analyzed a group of 236 clinical isolates with the Vitek MS (192 yeast and 44 Aspergillus) and found that 93.2% of the isolates were correctly identified, including 81.8% of the Aspergillus. 28 This compared with 94.1% and 88.6%, respectively, by routine laboratory methods. When the authors limited the study to only those species that were present in the database, 100% of the Aspergillus isolates were correctly identified. 28 In another study the Vitek MS was able to correctly discriminate closely related Aspergillus species, Aspergillus fumigatus and Aspergillus lentulus. 29 Schulthess et al. did a prospective study on 200 isolates using the Biotyper/Filamentous Fungi Library 1.0. They achieved a 79% species and 83.5% genus level identification. Particularly poor performance was noted for Mucor and Scopulariposis. 30 Chen et al. evaluated 50 clinical mold isolates with the Biotyper and found that several were not identified or identified with a low score (<1.7). 31 Twenty-eight isolates of Penicillium marneffei were not identified due to the lack of reference spectra in the database; however, the rate of identification went up to 85.7% after a single P. marneffei spectrum was added to the database. This result is consistent with a number of studies indicating high performance of MALDI on fungal isolates using custom databases. [32] [33] [34] [35] [36] [37] Although the use of MALDI-TOF in the microbiology lab has been confined, for the most part, to cultured organisms, there have been several studies exploring the feasibility of its use in primary samples. For example, the use of MALDI directly on positive blood cultures is attractive for both its rapidity and the breadth of detection possible with an open system. Sample processing directly from blood is not trivial and not yet FDA approved, but each major platform has a dedicated process and the results are promising with high clinical accuracy (80% to 90%). 3, 38, 39 Other promising applications include direct analysis of urine and cerebrospinal fluid (CSF), although, as with blood, additional screening and processing steps are required. 3, [40] [41] [42] [43] In summary, MALDI-TOF in the clinical microbiology lab has been developed to complement and in many cases replace the traditional biochemical diagnosis of bacteria and yeasts and can shorten turnaround time (TAT) by hours to days (depending

There are a variety of focused assays in the clinical microbiology lab and anatomic pathology samples that query one or a handful of genes by amplification of the target. The majority of these relies on the PCR and includes both qualitative and quantitative assays, such as those run on real-time PCR platforms (i.e., the Lightcycler [Roche Diagnostics, Indianapolis, Indiana] and the Smartcycler [Cepheid, Sunnyvale, California]). Target detection is typically by fluorescent probes, such as TaqMan. 60 Single-target PCR has been applied to fresh, frozen, and FFPE in anatomic pathology for a large range of microorganisms but largely through home-brew or in-house assays and/or performance by large specialty reference labs, due to the challenges of control tissue, validation, and complexity of interpretation.

Recent developments in this area are based on the same technology but are innovative in their convenience, detection method, and, in some instances, sensitivity. For example, using standard real-time PCR technology, FDA-approved assays, such as the Prodesse kits (Hologic Gen-Probe), provide convenient detection of small sets of respiratory viruses. The Xpert system (Cepheid) has had a large impact on the clinical lab due to its ease of use (sample-to-result platform), excellent performance, and rapid TAT (approximately 1 hour). Detection is by melt curve analysis rather than fluorescence. This platform offers a variety of cartridges (e.g., C. difficile, MRSA, MTB/RIF) focused on quick answers to major clinical decision points. [61] [62] [63] [64] Newer assays from Cepheid are in development for FFPE, which will include microbiologic applications.

For novel detection methods and increased sensitivity, the T2MR platform (T2 Biosystems, Lexington, Massachusetts) is one of the most innovative technologies. It is a complete sampleto-result platform that relies on detection of pathogen-specific amplicons by hybridization with tagged superparamagnetic particles. 65 The T2Candida Panel (T2 Biosystems) is the only FDA-approved system that directly analyzes blood (without culturing) and has demonstrated impressive results in clinical trials (91% sensitivity with a 4.2-hour TAT). 66 T2 Biosystems is currently developing a bacterial panel as well, which is reported to target Acinetobacter baumannii, E. faecium, K. pneumoniae, P. aeruginosa, and SA. 67 Although PCR remains the most common means of nucleic acid amplification, other technologies have been developed that address the presence of single targets. Transcription-mediated amplification (TMA) has been applied to mycobacteria, HCV, E. coli, and Listeria, where RNA is bound by a target oligo and then acted upon, in sequence, by reverse transcriptase (RT) (to produce cDNA), RNase H (to degrade RNA template), RT again to produce a double-stranded DNA promoter sequence, and then RNA polymerase to produce many copies of single-stranded RNA-then the process repeats itself. Another isothermal reaction occurs in loop-mediated isothermal amplification, which uses a group of four to six primers and Bst DNA polymerase, with the creation of complimentary sites to generate exponentially amplified loop structures. These assays have been developed for a wide range of targets and produce larger quantities of DNA than traditional PCR in a matter of minutes to hours. This technique has been applied to viruses, bacteria, protists, fungi, and mycobacteria in a point of care manner. [68] [69] [70] [71] [72] LAMP techniques are limited by the challenges of complex primer design, limited ability to multiplex, and common inhibitors of the enzymes in certain human samples, including blood. A last isothermal amplification is Massachusetts) and consist of single-stranded DNA probes tagged with acridinium esters that hybridize to rRNA. Tests are read in a luminometer. Because there is no amplification, probe-based assays generally suffer low sensitivity and are reserved for culture confirmation, where organisms are present in high numbers. AdvanDX (Woburn, Massachusetts) has produced a series of probe-based assays using peptide nucleic acid (PNA) probes tagged with fluorescent markers. 46 PNA probes have several advantages over traditional DNA probes, such as increased stability and ability to penetrate the bacterial cell wall. Fluorescent tags have the added advantage of allowing for multicolor detection and the creation of limited panels. These are rapid assays (less than 1 hour) and are mostly targeted towards rapid diagnostics of positive blood culture bottles. [51] [52] [53] [54] In situ hybridization (ISH) of Epstein Barr encoding region for confirmation of Epstein-Barr virus infection on FFPE is routinely used in cancer diagnostics, but few, if any, other common viral infections are confirmed this way, with IHC being preferred. Application of RNA-ISH to Aspergillus and Candida in FFPE showed less sensitivity than real-time PCR with sequencing (gold standard), although some FISH-positive, PCRnegative cases with obvious fungal elements were seen, suggesting refinements of this technique may be valuable for rapid identification of these common organisms, especially if mucormycosis is in the differential. 55 A unique probe-based approached to nucleic acid detection is the RNA hybridization and digital counting technology offered by NanoString (Seattle, Washington). This method uses a capture probe (usually a specific 50-mer) and a reporter probe (a second, adjacent, specific 50-mer attached to a unique molecular color barcode) to bind up individual molecules of RNA in a sample, capture them on a solid state matrix, and use digital imaging to count the barcodes in the sample. The current version can measure up to 500 independent capture:reporter pairs. The technology can be applied to any RNA target (human or microbial), and the great advantage in infectious disease is the extremely low input quantities that can be detected without interference from other RNA. Because of the counting algorithm, with proper probe selection in a given mixture, accurate quantification of RNA is possible without amplification. One pitfall to this technology is that the presence of any given target in a disproportionate ratio to other targets will flood the digital analysis (i.e., consider hemoglobin relative to cytokines in peripheral blood). Relative to sequencing, the technology remains expensive, but the benefit of quantification of extremely limited sample with minimal processing makes this an attractive target for future test development. For microorganisms, broad panels using signature genes and/or focused panels using genes in antibiotic metabolism pathways can be created. Studies in a range of diseases have shown that RNA from any source, including FFPE, is sufficient for NanoString analysis, [56] [57] [58] In one study, quantitative expression of Plasmodium falciparum schizonts from human tissue (both frozen and FFPE) were measured and, using imputation, the entire expression profile for the organisms determined. 59 pathogens include E. coli and Shigella, Salmonella, Campylobacter, and Clostridium perfringens. Typical viral pathogens included in these panels are adenovirus, norovirus, and rotavirus, representing the three most common symptomatic viral diarrheas. Diagnosis is moving more and more towards multiplex panels that cover major bacterial pathogens, as well as some parasitic and viral agents. [84] [85] [86] In addition to standard agents of infectious diarrhea, there are special considerations when testing for disease caused by C. difficile. These panels are, again, extremely valuable because the tools to diagnose these disease in anatomic pathology are often very limited and too invasive for most patient situations.

Parasitic enteric infections represent a major challenge in developing countries but remain rare in countries in which most new technologies are being developed. Despite this disparate situation, efforts have been made to approach parasitic infections, and thus the inherent challenges have become apparent. Extraction from stool for suspected parasite infections requires that protists (ciliates, ameoba, flagellates, coccidia), helminth larvae (nematodes), and helminth eggs (nematode, trematode, and cestode) be lysed sufficiently to release DNA efficiently. This challenge has led to eight or more different extraction protocols with a range of preparation requirements, including spin columns, automated magnetic bead assays, and "bead beating" methods. Molecular targets for subsequent detection assays as specific single targets across this range of organisms has included 18S rDNA, ITS, COX 1, pOV-46, 5.8S rDNA, Segment A, COWP, actin, HDP2, DL1, and 28S rDNA. 87 In commercially available, multiplex PCR assays, the majority of the platforms target only Cryptosporidium, Giardia, Entamoeba histolytica, and, rarely, Cyclospora. Because these first three represent 80% to 99% of parasites isolated by all methods from stool in developed nations, additions of other much less common pathogens is not common. Inclusion of nonpathogenic ameba is common in these assays but of questionable value.

Genitourinary samples (fluid from cervical samples, urethral swabs) are another sample that benefits from multiplex testing as public health screening of sexually transmitted diseases are extremely valuable for individual patient diagnosis, as well as epidemiologic survey, especially with the inclusion of HIV. Neisseria gonorrhea, Chlamydia trachomatis, and HPV in the Hologic Panther system can be performed on a single sample, and this system also can test for Trichomonas. The FilmArray from BioFire (Salt Lake City, Utah) detects Chlamydia, Neisseria, syphilis, Trichomonas, Mycoplasma/Ureaplasma, herpes simplex, and chancroid in a single sample. 88 Although relatively uncommon, meningitis and encephalitis (both nosocomial and community-acquired) carry high morbidity and mortality. [89] [90] [91] Time is of the essence when diagnosing a causative agent, particularly when trying to determine whether it is a bacterial pathogen that needs immediate antibiotic coverage along with herpes simplex, which needs immediate antivirals. Common bacterial pathogens include S. pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, Haemophilus influenzae, and Listeria monocytogenes, although there are also several other, less frequently isolated, pathogens. Viral pathogens in these panels include the primary actionable viruses (Cytomegalovirus, herpes simplex, varicella zoster virus), as well as other common viruses (Enterovirus, Parechovirus, and human herpesvirus (HHV)-6). Platforms also may include Cryptococcus species of fungi, which is very valuable in immunosuppressed populations. 92 Current diagnostics are heavily focused on CSF biochemical profile (glucose, protein, lactate), cell count, and Gram stain. The latter is the most specific, but the limit of detection is approximately 10 (4) accomplished by the introduction of helicase, which unwinds DNA, and single-stranded DNA binding proteins, which keep the templates separated. This is followed by primer hybridization and amplification to produce more single-stranded DNA and thus a continuous reaction. Assays to measure herpes simplex virus (HSV)-1 and HSV-2 using helicase have been developed with similar performance to traditional molecular methods and assays are available for Bordetella, group A Streptococcus, Trichomonas, and malaria. 73 Commonly used platforms that use these technologies include the Tigris and Panther systems by Gen-Probe, which are based on TMA, SmMIT-LAMP based on LAMP, and Ampli-Vue based on helicase. Use of these assays in FFPE tissue has shown some promise with improved performance over PCR for some organisms (such as HCV in TMA), equivalent performance to PCR (such as human papillomavirus [HPV] in head and neck cancers with LAMP), and detection of limited signatures (such as microcarcinomas in small biopsies with helicase assays). [74] [75] [76] 

The ability to detect multiple organisms in primary samples using PCR-based panels has both provided clinicians with tremendous diagnostic opportunities and presented new challenges. 77 Major platforms include FilmArray (BioMérieux), Verigene (Nanosphere), xTAG/xMAP (Luminex, Austin, Texas), with technologies that range from real-time PCR to solid and liquid phase arrays. For a comprehensive listing of platforms see www.captodayonline.com/ productguides. For an in-depth discussion of the major technologies see the articles included in the reference list. 78, 79 These systems are generally geared towards syndromic testing, with panels directed towards respiratory, GI, and sexually transmitted diseases, as well as meningitis and bloodstream infections. After discussing the specific challenges of each of the major syndromes, this section will focus on commonly used FDA-approved panels that are currently available from a number of manufacturers and that scan for a wide array of microbes, encompassing viral, bacterial, and fungal organisms.

Panels aimed at respiratory pathogens are focused most heavily on viruses but also incorporate several bacterial agents. 80, 81 Commonly covered organisms include Mycoplasma pneumoniae, Chlamydia pneumoniae, and Bordetella species. M. pneumoniae and C. pneumoniae, both very common causes of communityacquired pneumonia, are particularly well-suited to nucleic acid amplification tests because they are difficult to culture. 82 Although less common, Bordetella pertussis is considered to be the most common vaccine-preventable disease in the United States, is increasing in incidence, and is also difficult to culture. 83 The "bread and butter" of respiratory panels (RPs) focus on viruses with influenza A (and B), adenovirus, and respiratory syncytial virus (RSV) being always including, along with a mixture of other coronavirus, enterovirus, metapneumovirus, and the parainfluenza group. These panels are extremely powerful from the clinician's point of view but also provide a larger range of diagnostics for respiratory viruses than can be found in most anatomic pathology labs, which are largely limited to the DNA viruses. Although not all of these viral diagnoses are chemotherapeutically actionable, confirmation of a viral cause is valuable for prognosis and resource allocation.

Diarrheal diseases are a global public health concern. Although these diseases are a common cause of illness and hospitalization in the United States, they impose an enormous toll in the developing world, particularly on children. The most common bacterial 99.5% to 100%), but, as has been common with many panels and individual tests, laboratories have difficulty obtaining natural clinical cases of E. histolytica. 103 Currently, the only FDA-approved multiplex assay for agents of meningitis and encephalitis is the FilmArray meningitis panel. It covers 14 pathogens, including the following bacteria: E. coli K1, H. influenza, L. monocytogenes, N. meningitides, S. agalactiae, and S. pneumoniae. Although the panel was only recently approved by the FDA (October 2015) , there are a few reports of its performance. The pre-FDA evaluation was conducted both on archived samples and prospectively on a multicenter collection of 1560 samples of CSF. Among 235 archived samples (32 with bacteria), the percent positive and negative agreement was 100% for bacterial targets. Among the 1560 prospective samples, there were only eight with bacterial pathogens (none with L. monocytogenes or N. meningitides). Of those that were present, the FilmArray ME panel did not identify the only S. agalactiae. The archived arm of the evaluation included two S. agalactiae samples, both of which were correctly identified. 106 Since FDA approval, one US study has been published on the performance of the panel in several Texas medical centers. In that study of 48 patients with community-acquired meningitis and a negative Gram stain, the FilmArray detected two samples with bacterial pathogens, both S. pneumoniae. Culture detected only one of these, although the other one was positive for streptococcal urinary antigen. 107 Finally, the FilmArray blood culture identification (BCID) panel tests for an array of 19 bacterial targets, including: Enterococcus, L. monocytogenes, SA, Streptococcus (multiple), A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae. It also tests for five species of Candida and three bacterial resistance genes: mecA, vanA/B, and kpc. It has performed well on both monomicrobial and polymicrobial specimens in several clinical studies. 108, 109 

The BD Max is a sample-to-result real-time PCR-based platform. Amplicons are detected via fluorescent probes. A distinguishing feature is the capability to run laboratory-developed tests alongside FDA-approved panels. Current FDA-approved assays include GBS (for group B Streptococci), CT/GC/TV (for vaginitis), MRSA XT (for extended MRSA coverage), Cdiff (for C. difficile), and StaffSR (for Staphylococcus aureas and MRSA). The bacterial panel includes Salmonella spp., Campylobacter spp. (jejuni/coli), Shigellosis disease-causing agents (Shigella spp. and EIEC), as well as Shiga-toxin producing E. coli. It has demonstrated high sensitivity for the organisms in its library in several studies. [110] [111] [112] The parasite panel includes the three most common protist parasites in humans in developed countries, including Giardia lamblia, Cryptosporidium, and E. histolytica, but has the difficult challenge of very low population prevalence relative to the bacterial assays, making cost a challenge over other methods for uptake. 113

Verigene is another widely used multiplex PCR platform incorporating PCR of multiple viral and bacterial targets followed by hybridization to a solid-phase microarray. Detection occurs via sandwich hybridization with another analyte-specific probe bound to a gold nanoparticle. The signal is amplified via deposition of colloidal silver that is then detected by light diffraction. Verigene organisms per milliliter. 90 There is a great need for rapid molecular diagnostics in this area. In anatomic pathology, sample with culture and/or morphology with special stains and/or IHC can resolve many of these infections; however, optimal patient care and best outcomes occur in those patients who can be diagnosed early with these panels and not require an invasive biopsy.

Sepsis continues to be a tremendous cause of mortality, ranking as one of the top 10 causes of death in the United States. 93 Organisms commonly associated with bloodstream infections include SA, E. coli, Enterococcus spp., K. pneumoniae, CNS, P. aeruginosa, Candida albicans, E. cloacae, and Serratia marcescens. 94 The current gold standard for diagnosis is culture, with several automated systems (such as the BacT/Alert [BioMérieux Inc.]) available to clinical laboratories. 95 Although these systems are quite sensitive, approximately only one-third of patients with sepsis have positive cultures. 96 Some of this lack of sensitivity may be due to pretreatment with antibiotics, and some of it may be due to the low numbers of organisms present in many clinically significant bacteremias. 97 In addition to sensitivity, speed is also a major issue. The rapid diagnosis of bloodstream infections is critical for patient outcomes, with estimates of a 7.6% decrease in survival for every hour that appropriate antimicrobials are delayed after the onset of hypotension. 98 The need for sensitive, fast detection, ideally accompanied by some assessment of antibiotic resistance determinants, has inspired a number of new technologies geared towards improved speed and sensitivity of blood culture diagnostics. Several of these have been reviewed. [99] [100] [101] Platforms and Technologies FilmArray (BioFire/BioMérieux)

The FilmArray system consists of nested PCR followed by highresolution melt curve analysis. 102 All steps of the assay, from cell lysis to the final analysis, take place within a pouch containing freeze-dried reagents that can be stored at room temperature. FilmArray has a short TAT of approximately 1 hour. Disadvantages of the system include the relatively high price of the pouches and restriction of the platform to one test at a time. Laboratories must purchase multiple FilmArray platforms if they desire to run tests in parallel.

The FilmArray panel was the first FDA-approved RP to include bacterial pathogens, covering B. pertussis, C. pneumoniae, and M. pneumoniae, along with 18 common respiratory viruses. 102 For GI testing, the FilmArray is the most comprehensive of the current FDA-approved panels, covering an array of 22 bacteria, viral, and parasitic targets, including the common agents listed above, as well as Plesiomonas shigelloides, Yersinia enterocolitica, and several species of Vibrio. It can also differentiate between enteroaggregative, enteropathogenic, enterotoxigenic, Shiga toxin-producing, and enteroinvasive E. coli (EIEC). Studies have shown a sensitivity of 95.9% to 100% and a specificity of 96.6% to 100% for bacterial pathogens. 103, 104 In many cases the FilmArray detected pathogens in samples that were negative and was far more likely to diagnose mixed infections than standard techniques. 104, 105 For viral pathogens the FilmArray GI panel has shown value in the younger age groups (patients younger than 12 years) for most tested pathogens (sensitivity: 95.5% to 100%; specificity: 99.1% to 99.9%), whereas Norovirus appears to be valuable across all age groups (sensitivity: 94.5%; specificity: 98.8%). 103 Performance for parasitic pathogens in this panel is equally high for Cryptosporidium, Cyclospora, and Giardia (sensitivity: 100%; specificity:

Vibrio cholerae, and Y. enterocolitica. In one study comparing the xTAG with the FilmArray and conventional techniques, the two platforms behaved comparably, with the xTAG slightly trailing the FilmArray in the diagnosis of mixed infections (27% for Fil-mArray vs 14.1% for xTAG vs 8.3% for routine testing). 104 The performance of xTAG for viral pathogens when compared with realtime RT-PCR and multiplex realtime RT-PCR, showed a wide variation in sensitivity across viruses for xTAG (20% to 100%), with performance for RSV-A, RSV-B, H1N1, and influenza B being 13.3%, 47.3%, 54.2%, and 20%, respectively. 121 In headto-head comparisons of FilmArray, eSensor RVP, and xTAG, the three methods were essentially equivalent, with xTAG having the best performance for influenza A. 122 Some of the caveats of the xTAG system are addressed with newer Luminex platforms, such as the Aries, which is a sampleto-result platform for testing primary clinical specimens. The assay consists of real-time PCR using the Luminex MultiCode technology. This system uses primers that incorporate isobases, isoC and isoG, which, when incorporated, can pair only with each other. One isobase is tagged with a fluorescent reporter, and the other is tagged with a quencher. Upon the successful generation of product, both isobases are incorporated and fluorescence decreases. A subsequent melt curve analysis is performed, and fluorescence is restored. Unlike the xTAG technology, Multi-Code can be run on either commercially available real-time PCR systems or within the sample-to-result Aries platform from BD. There is only one FDA-approved test for this panel, an HSV-1 and HSV-2 panel for use in testing cutaneous and mucocutaneous lesions.

Although all of the nucleic acid-based systems described above demonstrate high levels of sensitivity and specificity, they are all inherently limited to a select group of organisms based on the probe/primer sets they contain. Open assays using broad-range primers that target nearly universal sequences are nearly all based on the 1977 discovery by Carl Woese that the 16S rRNA subunit could be used to determine the phylogenetic structure of the domains of life. 123 Although this idea represented a fundamental shift in bacteriology, it took many years for it to be widely accepted, and even longer for the use of 16S rRNA sequencing (16Sseq) to be used as a standard means for identifying bacteria in the clinical laboratory. 124 However, since 2000, 16Sseq has become the gold standard for determining bacterial species, and its analysis has led to a great deal of restructuring and renaming among phyla. 125 Briefly, the 16S gene encodes a portion of the small ribosomal (30S) subunit and is composed of nine variable regions alternating with nine conserved regions (Fig. 6.2) . The gene is sequenced using primers that bind to the highly conserved regions and then compared with a reference database to identify an isolate.

The choice of database is typically the most challenging part of the assay. There are a number of databases that can be used to identify the sequence, ranging from large, public, uncurated or partially curated databases, such as Genbank and the Ribosome Database Project (RDP, University of Michigan) to commercial, highly curated collections with a much more limited scope. Identification algorithms include Ripseq (Pathogenomix, Santa Cruz, California), the Integrated Database Network System has a TAT of approximately 2 hours and has the advantage of offering flexible pricing by which the laboratory is charged only for the tests they request, instead of the entire panel. Disadvantages include the need to select the appropriate cartridge based on Gram stain results for bloodstream diagnostics. Respiratory, GI, and bloodstream panels are available.

The Verigene platform can detect 13 respiratory viruses with high sensitivity (including multiple types of influenza, parainfluenza) and three Bordetella species (B. pertussis, Bordetella parapertussis/ Bordetella bronchiseptica, and Bordetella holmesii), 114, 115 although one advantage of this system is the flexibility to test limited panels based on a given population. The GI panel is somewhat more limited than the FilmArray, covering 22 targets, including 13 bacteria, 5 viruses, and 4 parasites. Bacteria include the Campylobacter group, Salmonella spp., Shigella spp., Vibrio group, and Y. enterocolitica, as well as Shiga toxin. In one study of 725 stool samples the Verigene demonstrated a 98.5% agreement rate with culture. 116 For the diagnosis of bloodstream infections, the Verigene system has separate cartridges for gram-positive and gram-negative organisms, necessitating an accurate Gram stain to pick the appropriate test and making polymicrobial testing a bit more complicated. Each panel assesses a few extra organisms than the FilmArray single panel. In addition, the gram-negative panel queries several more resistance targets than the FilmArray, including CTX-M, IMP, NDM, OXA, and VIM. 117 Verigene in RPs has excellent performance for the influenza viruses (A and B), including the ability to subtype viruses with perfect agreement with gold standard methods (molecular); however, mixed infections with non-influenza viruses dampen performance. 118 Headto-head comparisons of the limited Verigene panel (influenza A and B, RSV) with FilmArray RP showed equivalent detection of the three major pathogens at a slightly reduced cost for the Verigene; thus analysis of the clinical population and the desires of the local clinical team for reported virus and actionable diagnoses may help in choosing between these technologies. 114 xTAG The xTAG assay system from Luminex is a liquid array based on multiplex PCR followed by amplicon extension using a targetspecific primer. The extension step incorporates a hybridization tag onto the amplicon, along with a biotin label. Elongated, tagged amplicons are then incubated with polystyrene microbeads of various fluorescent profiles, each coated with a specific anti-tag sequence. Bead/amplicon pairs are then stained with another streptavidin-linked fluorophore. The beads are analyzed by a system of two lasers on the xMAP instrument. The first determines which beads are bound to amplicons by way of the streptavidinfluorophore. The second laser indicates which specific target was amplified by way of the unique fluorescence of that bound bead. 119 Although the Luminex platforms generally have the potential to detect the greatest number of targets, drawbacks include the need for a separate nucleic acid extraction step and longer TAT (4 hours or longer) then the other multiplex platforms.

There are two xTAG platforms focused on respiratory testing: the original xTAG platform, restricted to viral pathogens, and the next-generation system (NxTAG), which included several bacterial pathogens as well, including Legionella pneumophila. 120 The xTAG Gastrointestinal Pathogens Panel (GPP) tests for nine bacterial, three viral, and three parasitic targets. Among bacterial loci, the Luminex xTAG covers Campylobacter, C. difficile toxin A/B, E. coli O157, enterotoxigenic E. coli (ETEC) LT/ST, Shiga toxin-producing E. coli (STEC) stx1/stx2, Salmonella, Shigella, Sanger sequencing, which is generally unable to assess polymicrobial infections, although some analysis software packages have algorithms to deconvolute traces of 16Sseq from two or three different organisms. 148

NGS is a high-throughput method that generally refers to a massively parallel process that generates tremendous quantities of raw sequence reads. There are a number of commercially available platforms that vary in the quantity, quality, and length of the reads that they produce. The majority of these include a step for clonal amplification of the template and produce short (50 to 400 bp) reads. For excellent reviews see articles provided in the reference list. 149, 150 The advent of NGS was a groundbreaking event in clinical medicine and is a key component of initiatives for "precision medicine" for cancer. Beyond human genomic analysis, NGS has also had a tremendous influence on clinical microbiology that extends from infectious disease testing and epidemiology to analysis of the human microbiome in health, disease, and intervention. [151] [152] [153] Within the diagnostic clinical microbiology laboratory, NGS of microbial whole genomes has been applied as an alternative to traditional methods, such as pulsed-field gel electrophoresis, for clonality analysis in outbreak tracking. 154 The refinement made possible with total chromosomal single-nucleotide polymorphism analysis, for example, allowed one group to assess the spread of variants of a colonizing population of carbapenem-resistant K. pneumoniae from one patient to several others in multiple transmission events. These variants were differentiated from each other by fewer than a dozen nucleotides. 155 Other applications that are becoming more and more routine include the characterization of unusual pathogens, the tracking of antibiotic resistance genes, and even the characterization of the plasmids that carry them through horizontal gene transfer. [156] [157] [158] [159] Although not yet routine, NGS has been used to diagnose infections directly from primary samples in cases in which all conventional techniques have failed. 160 In one report, after 4 months of progressive neurologic symptoms and a thorough but unrevealing infectious disease workup, NGS was finally able to diagnose a case of neuroleptospirosis directly from the CSF of a 14-year-old boy. 161 In addition to the detection and characterization of pathogens, NGS has also proven to be an invaluable tool for the characterization of the human microbiome. The influence of the microbiome on human health and disease has become progressively more established. Examples are many and include the development of obesity, the metabolism of therapeutic drugs, and the development of inflammatory bowel disease. [162] [163] [164] The two most common ways of assessing the microbiome are by 16S rRNA or shotgun metagenomics.

NGS 16S analysis entails sequencing one portion of 16S rRNA from a mixed community (e.g., stool sample) with subsequent clustering of sequences either against a reference dataset or into operational taxonomic units (OTUs). It gives a picture of taxonomic diversity in a sample and allows for the analysis of changing taxonomic composition in the face of various perturbations and interventions, such as antibiotic administration. Shotgun metagenomics refers to the sequencing of total DNA from a mixed community. This technique allows for more refined organism identification and an analysis of biochemical pathways, resistance genes, etc. but requires a greater amount of raw sequence. 165, 166 (IDNS) (SmartGene, Raleigh, North Carolina), and MicroSEQ (Applied Biosystems, Thermo-Fisher, Waltham, Massachusetts). The first two products are solely bioinformatic reference solutions. The last includes both reagents and access to a curated database. None of these products are FDA approved. The Clinical and Laboratory Standards Institute (CLSI) has published guidelines for the identification of organisms by nucleic acid sequencing in which the recommended percentage identity to make a match and guidance for organisms requiring additional targets (e.g., rpoB, hsp65) for accurate speciation are laid forth. 126 The use of 16Sseq in the clinical microbiology laboratory has been shown to be particularly useful in such applications as the identification of unusual and/or slow-growing organisms and the identification of organisms from specimens in which culture is not possible or has failed, such as in specimens from antibiotictreated patients and fixed specimens. [127] [128] [129] [130] For example, the identification of mycobacterial species identified by acid-fast staining of FFPE is a commonly used application of 16S sequencing, although additional targets, such as IS6110 and hsp65, are often needed to differentiate species. 131, 132 Specimen types most successfully analyzed by direct 16Sseq include heart valves, CSF, and synovial fluid. [133] [134] [135] [136] [137] [138] Fungal pathogens are also identified using rRNA sequencing, although it is a less established practice than it is for bacterial targets. The fungal rRNA 18S corresponds to bacterial 16S, although it has fewer hypervariable regions and is generally not as useful for isolate discrimination. 139 The most common regions for fungal identification are the ITS regions 1 and 2, as well as the D1 and D2 hypervariable regions of the 28S rRNA large subunit component (Fig. 6.2) . [139] [140] [141] When using these targets to identify an isolate, criteria required for percentage identity and percentage difference between competing matches are not as clear for fungi as they are for bacteria. 126 The lack of high-quality databases has been a major difficulty in fungal sequencing, with estimates of up to 20% of the entries in GenBank being incorrectly identified down to species level. 142 To address this, several of the private databases that are available for bacterial sequencing also include curated or partially curated fungal databases (IDNS and RipSeq). 143 The MicroSEQ system also includes a fungal component, but it is limited to the D2 region of 28S. Recently, several public databases have become available with high-quality, curated content. Databases that are most focused on species of medical importance include the International Society for Human and Animal Mycology (ISHAM) ITS database contains 3200 sequences representing 524 human/ animal pathogenic fungal species and is a major resource for clinical laboratories. 144 It can be searched via a web-based platform (its.mycologylab.org). The National Center for Biotechnology Information RefSeq ITS database contains 3060 sequences representing 270 families from 39 classes. The smaller 28S RefSeq database contains approximately 500, representing greater than 100 families from 21 classes. 145 This technique is particularly promising for identifying fungi from fixed tissue. 55, 146, 147 Important caveats of rRNA sequencing include high cost and complexity, issues with uncurated databases (described above), and the potential for false positives. This is particularly the case when broad-range sequencing is applied to primary specimens to "fish" for an infectious agent in the absence of any corroborating culture or gram stain. Furthermore, rRNA should not be used to determine whether an infection has "cleared," due to the potential for lingering nucleic acid even in a successfully treated infection. Analysis should be restricted to normally sterile sites for tests using In FFPE in which an organism has been seen but not identified (such as bacteria, fungal elements, viral cytopathic or necrotic effect, or unidentified parasitic elements), NGS has enormous potential to rectify theses diagnoses but is currently limited by cost per sample and the enormous amounts of raw human data in comparison with the relatively small amount of the organism's sequence. As costs fall for sequencing and rapid bioinformatic methods for elucidating causative sequence emerge, NGS will become a primary tool for confirmation of microbial infections.

One last technology to consider is the hybrid system, PLEX-ID by Abbott, which combines a PCR step to amplify through a set of primers (PLEX-ID Viral IC, PLEX-ID BAC Spectrum BC, or PLEX-ID Flu) a broad range of organisms and then detects the PCR products through mass spectroscopy. This approach combines amplification of many sample types, including body fluids, tissue, FFPE, culture isolates, and blood, with a highly sensitive detection system to make subtle species determinations across organism types, including bacterial, fungal, viral, and some parasitic. [167] [168] [169] [170] As the database of spectra improves and the multiplex primers improve, this system will gain popularity, although it is still limited by initial organism burden and procedure time (up to 8 hours).

The microbiology laboratory remains the most important companion for anatomic pathologists who have a potentially infectious diagnosis under the microscope. Anecdotal and small studies have demonstrated that the yield from molecular tests that are performed blindly (without visualization) versus with pathologist's review is miniscule and extremely expensive. Therefore anatomic pathologists and their microbiologic colleagues must have a constant line of communication from the moment the specimen is collected until final reporting to ensure interpretation of all modalities is maximally beneficial to patient care. This dialogue should also include discussions of molecular modalities that are applied to clinical and anatomic pathology specimens and changes, advantages, pitfalls, and benefits of those modalities in an ever-evolving manner. As with any approach to medicine, evidence-based use of the most efficient and inexpensive method that produces the highest-impact result for the patient is the constant goal.

MS technology for microorganism identification in a public health reference laboratory

Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Nocardia, Rhodococcus, Kocuria, Gordonia, Tsukamurella, and Listeria species

Evaluation of the Bruker MALDI Biotyper for identification of Gram-positive rods: development of a diagnostic algorithm for the clinical laboratory

Multicenter evaluation of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Gram-positive aerobic bacteria

Kocuria rhizophila misidentified as Corynebacterium jeikeium and other errors caused by the Vitek MS system call for maintained microbiological competence in the era of matrix-assisted laser desorption ionization-time of flight mass spectrometry

Importance of using Bruker's securityrelevant library for Biotyper identification of Burkholderia pseudomallei, Brucella species, and Francisella tularensis

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new possibility for the identification and typing of anaerobic bacteria

Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK(R) MS system

Anaerobic blood culture isolates in a Norwegian university hospital: identification by MALDI-TOF MS vs 16S rRNA sequencing and antimicrobial susceptibility profiles

ESCMID Study Group on Antimicrobial Resistance in Anaerobic Bacteria. Species identification of clinical isolates of Bacteroides by matrixassisted laser-desorption/ionization time-of-flight mass spectrometry

Performance of MALDI-TOF MS platforms for fungal identification

MALDI-TOF mass spectrometry proteomic phenotyping of clinically relevant fungi

Improved clinical laboratory identification of human pathogenic yeasts by matrix-assisted laser desorption ionization time-offlight mass spectrometry

Comparison of the accuracy of two conventional phenotypic methods and two MALDI-TOF MS systems with that of DNA sequencing analysis for correctly identifying clinically encountered yeasts

Comparative evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry systems for identification of yeasts of medical importance

Interlaboratory comparison of sample preparation methods, database expansions, and cutoff values for identification of yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry using a yeast test panel

Clinical performance evaluation of the Sofia RSV FIA rapid antigen test for diagnosis of respiratory syncytial virus infection

Serological markers in early stages of human immunodeficiency virus infection in haemophiliacs

Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology

Matrix-assisted laser desorption ionization-time of flight mass spectrometry in clinical microbiology

MS for the diagnosis of infectious diseases

A side by side comparison of Bruker Biotyper and VITEK MS: utility of screening methods and matrix-assisted laser desorption ionizationtime of flight mass spectrometry

Direct identification of urinary tract pathogens from urine samples using the Vitek MS system based on matrix-assisted laser desorption ionization-time of flight mass spectrometry

Mass spectrometry: pneumococcal meningitis verified and Brucella species identified in less than half an hour

Direct application of MALDI-TOF mass spectrometry to cerebrospinal fluid for rapid pathogen identification in a patient with bacterial meningitis

Sample preparation for mass spectrometry analysis of formalin-fixed paraffin-embedded tissue: proteomic analysis of formalin-fixed tissue

Urinary pellet sample preparation for shotgun proteomic analysis of microbial infection and host-pathogen interactions

PNA for rapid microbiology

Peptide nucleic acid fluorescence in situ hybridization for rapid detection of Klebsiella pneumoniae from positive blood cultures

Direct identification of major blood culture pathogens, including Pseudomonas aeruginosa and Escherichia coli, by a panel of fluorescence in situ hybridization assays using peptide nucleic acid probes

Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes

Rapid detection of Enterococcus spp. direct from blood culture bottles using Enterococcus QuickFISH method: a multicenter investigation

Development of a peptide nucleic acid probe to Trichosporon species and identification of trichosporonosis by use of in situ hybridization in formalin-fixed and paraffin-embedded (FFPE) sections

Design and evaluation of peptide nucleic acid probes for specific identification of Candida albicans

Identification of Mycobacterium species from BACTEC MGIT positive cultures with Oligo-FISH and PNA-FISH methods

Development and evaluation of nucleic acid-based techniques for an auxiliary diagnosis of invasive fungal infections in formalin-fixed and paraffin-embedded (FFPE) tissues

Comparison of quantitative real time PCR with sequencing and ribosomal RNA-FISH for the identification of fungi in formalin fixed, paraffin-embedded tissue specimens

Development and verification of the PAM50-based Prosigna breast cancer gene signature assay

Evaluating robustness and sensitivity of the NanoString Technologies nCounter Platform to enable multiplexed gene expression analysis of clinical samples

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of mycobacteria in routine clinical practice

Comparison of sample preparation methods, instrumentation platforms, and contemporary commercial databases for identification of clinically relevant mycobacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Identification of mycobacteria from solid and liquid media by matrix-assisted laser desorption ionization-time of flight mass spectrometry in the clinical laboratory

Poster 1190: Mycobacteria Identification by MALDI Biotyper System: Evolution of Database Content and Evaluation Criteria

Evaluation of MALDI Biotyper Mycobacteria Library v3.0 for identification of nontuberculous mycobacteria

Routine identification of medical fungi by the new Vitek MS matrix-assisted laser desorption ionization-time of flight system with a new time-effective strategy

Discrimination of Aspergillus lentulus from Aspergillus fumigatus by Raman spectroscopy and MALDI-TOF MS

Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory

Evaluation of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Bruker Biotyper for identification of Penicillium marneffei, Paecilomyces species, Fusarium solani, Rhizopus species, and Pseudallescheria boydii

Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using the Vitek MS system for rapid and accurate identification of dermatophytes on solid cultures

Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

MALDI-TOF mass spectrometry identification of filamentous fungi in the clinical laboratory

Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: clinical evaluation of an extended reference spectra library

Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi

Detection of microorganisms in blood specimens using matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a review

Sepsis: a roadmap for future research

Direct identification of urinary tract pathogens from urine samples, combining urine of human papillomaviruses (HPV) in oropharyngeal squamous cell carcinoma (OPSCC)

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies

Point-counterpoint: large multiplex PCR panels should be first-line tests for detection of respiratory and intestinal pathogens

Emerging technologies for the clinical microbiology laboratory

Basic concepts of microarrays and potential applications in clinical microbiology

Molecular diagnosis of respiratory viruses

Molecular Pathology in Clinical Practice

Community-acquired pneumonia requiring hospitalization among U.S. adults

Pertussis: microbiology, disease, treatment, and prevention

Laboratory diagnosis of bacterial gastroenteritis

Molecular testing for viral and bacterial enteric pathogens: gold standard for viruses, but don't let culture go just yet?

Multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis

Advances in molecular diagnosis of parasitic enteropathogens

Multiplex PCR testing for nine different sexually transmitted infections

Nosocomial bacterial meningitis

State-of-the-art microbiologic testing for community-acquired meningitis and encephalitis

Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis

Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda

Two decades of mortality trends among patients with severe sepsis: a comparative meta-analysis

The clinical and prognostic importance of positive blood cultures in adults

Laboratory detection of bacteremia and fungemia

Predicting bacteremia in patients with sepsis syndrome. Academic Medical Center Consortium Sepsis Project Working Group

Quantitative aspects of septicemia

A cross comparison of technologies for the detection of microRNAs in clinical FFPE samples of hepatoblastoma patients

Plasmodium falciparum gene expression measured directly from tissue during human infection

Molecular microbiology

Comparison of the next-generation Xpert MRSA/SA BC assay and the GeneOhm StaphSR assay to routine culture for identification of Staphylococcus aureus and methicillin-resistant S. aureus in positive-blood-culture broths

Multicenter evaluation of the Xpert Norovirus assay for detection of Norovirus genogroups I and II in fecal specimens

Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares

Charnot-Katsikas A. Comparison of Cepheid Xpert(R) Flu/ RSV XC and BioFire FilmArray(R) for detection of influenza A, influenza B, and respiratory syncytial virus

T2 magnetic resonance enables nanoparticle-mediated rapid detection of candidemia in whole blood

T2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial

T2Bacteria: rapid and sensitive detection and identification of sepsis pathogens in whole blood specimens by T2MR (poster)

Evaluation of two commercial loop-mediated isothermal amplification assays for detection of avian influenza H5 and H7 hemagglutinin genes

A comparison of in-house real-time LAMP assays with a commercial assay for the detection of pathogenic bacteria

Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar

Up-date on diagnostic strategies of invasive aspergillosis

The Use of a Commercial Loop-Mediated Isothermal Amplification Assay (TB-LAMP) for the Detection of Tuberculosis

Clinical evaluation of a helicase-dependant amplification (HDA)-based commercial assay for the simultaneous detection of HSV-1 and HSV-2

Comparison of transcription mediated amplification (TMA) and reverse transcription polymerase chain reaction (RT-PCR) for detection of hepatitis C virus RNA in liver tissue

Comparative evaluation of the Nanosphere Verigene RV+ assay and the Simplexa Flu A/B & RSV kit for detection of influenza and respiratory syncytial viruses

Evaluation of the Verigene EP IUO test for the rapid detection of bacterial and toxin causes of gastrointestinal infection

Evaluation of FilmArray and Verigene systems for rapid identification of positive blood cultures

Evaluation and verification of the nanosphere Verigene RV+ assay for detection of influenza A/B and H1/H3 subtyping

Principles of the xTAG respiratory viral panel assay (RVP Assay)

Clinical evaluation of the Luminex NxTAG respiratory pathogen panel

Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG(R) RVP fast assay for the detection of respiratory viruses

Comparison of the Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP fast multiplex assays for detection of respiratory viruses

Phylogenetic structure of the prokaryotic domain: the primary kingdoms

Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories

Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology

Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline

Broad-range 16S rRNA gene polymerase chain reaction for diagnosis of culturenegative bacterial infections

Utility of 16S rDNA sequencing for identification of rare pathogenic bacteria

Identification of bacteria in formalin-fixed, paraffin-embedded heart valve tissue via 16S rRNA gene nucleotide sequencing

16S rRNA sequencing in routine bacterial identification: a 30-month experiment

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction

Rapid real-time PCR for detection of Mycobacterium tuberculosis complex DNA in formalin-fixed paraffin embedded tissues: 16% of histological 'sarcoid' may contain such DNA

Broad-range PCR and sequencing in routine diagnosis of infective endocarditis

Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock

Emerging technologies for rapid identification of bloodstream pathogens

ID learning unit-diagnostics update: current laboratory methods for rapid pathogen identification in patients with bloodstream infections

Nonculture techniques for the detection of bacteremia and fungemia

an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection

Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis

Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens

Spectrum of enteropathogens detected by the FilmArray GI panel in a multicentre study of community-acquired gastroenteritis

Multi-center clinical evaluation of a multiplex meningitis/encephalitis PCR panel for simultaneous detection of bacteria, yeasts and viruses in cerebrospinal fluid specimens

General Meeting

Enhancing pathogen identification in patients with meningitis and a negative Gram stain using the BioFire FilmArray((R)) meningitis/ encephalitis panel

Performance evaluation of the Verigene(R) (Nanosphere) and FilmArray(R) (BioFire(R)) molecular assays for identification of causative organisms in bacterial bloodstream infections

Clinical evaluation of the FilmArray blood culture identification panel in identification of bacteria and yeasts from positive blood culture bottles

Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes

Comparison of the BD MAX enteric bacterial panel to routine culture methods for detection of Campylobacter, enterohemorrhagic Escherichia coli (O157), Salmonella, and Shigella isolates in preserved stool specimens

Comparison of the BD MAX(R) enteric bacterial panel assay with conventional diagnostic procedures in diarrheal stool samples

Evaluation of the BD Max enteric parasite panel for clinical diagnostics

Comparison of three commercial RT-PCR systems for the detection of respiratory viruses

Next-generation sequencing for infectious disease diagnosis and management: a report of the Association for Molecular Pathology

Clinical detection and characterization of bacterial pathogens in the genomics era

Routine use of microbial whole genome sequencing in diagnostic and public health microbiology

Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing

Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemaseproducing Enterobacteriaceae

Klebsiella pneumoniae Carbapenemase (KPC)-producing K. pneumoniae at a single institution: insights into endemicity from whole-genome sequencing

Genomically informed surveillance for carbapenem-resistant enterobacteriaceae in a health care system

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences

A cloudcompatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples

Actionable diagnosis of neuroleptospirosis by next-generation sequencing

Mirror, mirror on the wall: which microbiomes will help heal them all

The microbiome revolution

The human microbiome: at the interface of health and disease

Sequencing the human microbiome in health and disease

Characterization of the gut microbiome using 16S or shotgun metagenomics

Broad-range direct detection and identification of fungi by use of the PLEX-ID PCR-electrospray ionization mass spectrometry (ESI-MS) system

Polymerase-chain reaction/electrospray ionization-mass spectrometry for the detection of bacteria and fungi in bronchoalveolar lavage fluids: a prospective observational study

Broad-range PCRelectrospray ionization mass spectrometry for detection and typing of adenovirus and other opportunistic viruses in stem cell transplant patients

Detection and identification of yeasts from formalin-fixed, paraffinembedded tissue by use of PCR-electrospray ionization mass spectrometry

Advantages and limitations of direct PCR amplification of bacterial 16S-rDNA from resected heart tissue or swabs followed by direct sequencing for diagnosing infective endocarditis: a retrospective analysis in the routine clinical setting

Using PCR-based detection and genotyping to trace Streptococcus salivarius meningitis outbreak strain to oral flora of radiology physician assistant

Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections

Whipple disease diagnosed with PCR using formalin-fixed paraffin-embedded specimens of the intestinal mucosa

Use of broad range16S rDNA PCR in clinical microbiology

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

DNA barcoding of fungi causing infections in humans and animals

Intraspecific ITS variability in the kingdom fungi as expressed in the international sequence databases and its implications for molecular species identification

Taxonomic reliability of DNA sequences in public sequence databases: a fungal perspective

Evaluation of nucleic acid sequencing of the D1/D2 region of the large subunit of the 28S rDNA and the internal transcribed spacer region using SmartGene IDNS [corrected] software for identification of filamentous fungi in a clinical laboratory

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database-the quality controlled standard tool for routine identification of human and animal pathogenic fungi

Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation

Utility of DNA sequencing for direct identification of invasive fungi from fresh and formalin-fixed specimens

Molecular methods to improve diagnosis and identification of mucormycosis

Direct sequencing and RipSeq interpretation as a tool for identification of polymicrobial infections

High-throughput sequencing technologies

A glimpse into past, present, and future DNA sequencing

Making the leap from research laboratory to clinic: challenges and opportunities for next-generation sequencing in infectious disease diagnostics