New Technologies for the Diagnosis of Infection

The 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.

Protein-Based Identification

Antigen-Based Identification uses rapid assessment for the presence of microbial antigens and provides a “point of care” solution for diseases for which immediate answers impact patient care decisions. The principle of all of these assays is that a microbial antigen is present at sufficient quantity for which an antibody has been generated for detection. Common platform types include lateral flow assays (antibodies bound to a matrix through which the patient sample containing antigen passes and results in a colorimetric readout; examples are BinaxNow for Malaria, Remel Xpect for Clostridium difficile assays, p24 antigen detection in human immunodeficiency virus [HIV]) and fluorescent immunoassays (similar to lateral flow concept but with automated detection and fluorescently labeled antibodies; examples are Sofia system for bacteria and viruses). These assays may be available commercially to consumers or used in microbiology laboratories as inexpensive tools to screen for common diseases (e.g., seasonal influenza, Streptococcal antigens, diarrhea in acutely ill patients). In many laboratories, these rapid tests are confirmed by a second test which may include additional protein testing or nucleic acid 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. 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. 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.

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. Lackluster performance, in particular, has been noted for several gram-positive 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 . 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. In similar studies using the Vitek platform, there were also reported problems in speciation of Listeria . The misidentification of a Kocuria as a Corynebacterium has also been reported. 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. 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.

MALDI is rapidly becoming the method of choice for identifying anaerobic bacteria. Several studies using large, diverse collections of anaerobes have demonstrated strong performances by both the BioTyper and the Vitek MS. 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. 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. 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 . 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.

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. 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%. 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. More recently, Chao et al. analyzed a collection of 200 clinical yeast isolates with both the Biotyper and the Vitek MS. They found that the Biotyper slightly out-performed 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%). In general, it has been found that a formic acid/ethanol extraction method and lowered identity cutoffs (i.e., 1.7 on the Biotyper) are optimal for the analysis of yeasts.

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. Caveats include the need for enhanced sample preparation methods for efficient cell breakage for biosafety reasons and to ensure high-quality spectra. 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. 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. 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.

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. 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 . 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. In another study the Vitek MS was able to correctly discriminate closely related Aspergillus species, Aspergillus fumigatus and Aspergillus lentulus . 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 . 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). 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.

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%). Other promising applications include direct analysis of urine and cerebrospinal fluid (CSF), although, as with blood, additional screening and processing steps are required.

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 on the organism in question). 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. 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. 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.

Nucleic Acid–Based Techniques

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-Based Assays

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, 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. 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. The AdvanDX systems (PNA fluorescence in situ hybridization [FISH] and QuickFISH) offer a variety of testing options, such as Staphylococcus ( Staphylococcus aureus [SA] vs coagulase-negative staphylococci [CNS]), Enterococcus ( Enterococcus faecalis vs Enterococcus faecium or other enterococci), and gram negatives ( E. coli vs Klebsiella pneumoniae , vs Pseudomonas aeruginosa ) that can be chosen based upon the Gram stain results. PNA probes have been used for rapid identification of organisms, such as fungi, bacteria, and mycobacteria, in human tissues sections for fresh, frozen, and FFPE. 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, PCR-negative 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.

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, 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.

Singleplex or Limited Target Assays

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. 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. 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 sample-to-result platform that relies on detection of pathogen-specific amplicons by hybridization with tagged superparamagnetic particles. 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). 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.

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. 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 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. 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 AmpliVue 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).

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here