Molecular microbiology

Abstract

Introduction and historical perspective

Since the first US FDA-approved/cleared molecular probe for Legionella in 1986 and the first nucleic acid (NA)-based amplification test for Chlamydia trachomatis in 1993, the landscape of molecular testing in microbiology has developed significantly. In 1996, the first endpoint HIV-1 viral load test was FDA-approved. During the following decade, there was an explosive increase in the use of molecular tools to diagnose infectious diseases primarily due to real-time polymerase chain reaction (rtPCR) in laboratory-developed tests. In the last 10 years, we have seen a substantial increase in the number of FDA-approved/cleared and European CE-IVD molecular tests for infectious diseases, which has facilitated the broad use of these tests in clinical laboratories. Simple, sample-in, answer-out molecular test systems are now widely available that can be deployed in various laboratory and clinical settings, including at the point of care. In recent years, technological advances in NA amplification techniques, automation, NA sequencing, and multiplex analysis have reinvigorated the field and created new growth opportunities.

Molecular microbiology remains the leading area in molecular pathology in terms of the number of tests performed and clinical relevance. NA-based tests have reduced the dependency of the clinical microbiology laboratory on more traditional antigen detection and culture methods and created new opportunities for the laboratory to impact patient care. This chapter reviews the technology currently available in clinical laboratories to diagnose infectious diseases and emerging technology that may impact the field soon. The application of these technologies to diagnose health care–associated infections (HAI); syndromes such as bloodstream, gastrointestinal, respiratory, and central nervous system infections; and infectious diseases at the point of care are reviewed while highlighting the unique challenges and opportunities these tests present for clinical laboratories. Readers are directed to Molecular Microbiology: Diagnostic Principles and Practice , 3rd edition, for a more comprehensive and in-depth examination of this dynamic and exciting discipline.

Overview of methodologies

The diagnosis of infectious diseases has witnessed multiple phases over the years which largely started with Gram stain and growth of organisms in culture in the late 1800s, and expanded to antigen detection methods, serology, and polymerase chain reaction (PCR). Different approaches for molecular detection in clinical microbiology laboratories have become available, extending from nonamplification techniques to PCR and rtPCR. rtPCR facilitated a transformation in the clinical microbiology laboratory and enabled the introduction of quantitative assays that revolutionized the virology laboratory. Multiple commercially available molecular platforms, technologies, and assays now exist; some of them are now also used as point of care or near-patient tests (see POC section below). Methods for single target detection, small panels, and large syndromic molecular panels are also available (see Syndromic section below). Broader nontargeted strategies are also expanding, such as metagenomics and the characterization of the host response to infectious diseases.

Probe hybridization

Identifying microorganisms by specifically targeting certain regions in their genome by DNA or RNA probes is a common method of microbial identification and can generate either qualitative or quantitative results. Probe hybridization methods have four main components: the target, the probe, the reporter or detection method, and the hybridization method. Common hybridization methods include liquid and solid phase and in situ hybridization. These methods were developed for detection from either direct clinical specimens (e.g. probe detection methods for vaginitis screen) or from pathogens after growth in cell culture (e.g. probe detection of mycobacteria and dimorphic fungi). Generally, these methods have lower sensitivity compared to amplification methods and require high loads of microorganisms. To increase sensitivity, many hybridization techniques include an amplification step either prior to or after hybridization.

Liquid-phase hybridization

The basic concept of liquid hybridization is that the reaction is carried out in solution, enhancing the chance of the probe binding to the target. A successful reaction depends on the probe’s ability to exist in a single-stranded form and not to self-hybridize. A common method for hybridization detection in solution is using an acridinium ester moiety attached to the probe, then treating the product with alkali after hybridization. Emission of light occurs with the probe-target hybridized product only (hybridization protection, HPA) ( Fig. 67.1 ). The Hologic (San Diego, CA) assays are the most commonly encountered liquid phase hybridization methods in the clinical microbiology laboratory. The AccuProbe Hologic assays for identification of mycobacteria, other select bacteria, and dimorphic fungi (from culture-grown organisms) include hybridization and HPA reactions. The Hologic Aptima (assays for detection of sexually transmitted infections [STI] and viral diagnosis/ viral load assays) combine target capture (capture probes), transcription-mediated amplification (TMA, specific complementary primers to the target are used for amplification, discussed below), and HPA.

Solid-phase hybridization

In solid-phase hybridization, the target is usually fixed to a membrane (nylon or nitrocellulose). The membrane is exposed to the probe in solution, and specific binding is detected by different methods that most commonly include fluorescence and luminescence. The BD Diagnostics (Franklin Lakes, NJ) vaginitis screen (BD Affirm) for the detection of Gardenerella vaginalis , Candida spp., and Trichomonas vaginalis is an example of an easily operated solid-phase hybridization method. Other applications include line probe assays (e.g., Innogenetics/ Fujirebio, Ghent, Belgium) that are used for genotyping (e.g., hepatitis C virus [HCV], human papillomavirus [HPV]), ^, and drug resistance mutations detection (e.g., HIV, hepatitis B virus [HBV] and Mycobacterium ). Line probe assays are a variation of solid-phase hybridization; as in this case, specific probes are fixed to the membrane, and the amplified targets are in the hybridization solution (reverse hybridization principle). Stringent hybridization conditions are required to only allow binding of sequences with perfect probe match to enable typing and single polymorphism detection ( Fig. 67.2 ). Mesa Biotech (San Diego, CA) has developed point of care testing assays on the Accula Dock or the Silaris Dock that use hybridization-based lateral flow capture of PCR amplicons by probes immobilized to a porous detection strip. The company has CLIA waived influenza A/B and RSV assays and received EUA for SARS-CoV-2 rapid assay.

The branched DNA assay (bDNA) relies on a different solid phase hybridization approach (sandwich hybridization). bDNA offers increased analytic sensitivity by amplifying the signal using a branched DNA molecule. In this assay, target NAs are captured to a solid surface (microwell) by target-specific probes. A second set of specific probes binds to both the target and the branched DNA molecule (each can bind to up to eight bDNAs), which subsequently binds to multiple enzyme-labeled probes. An appropriate substrate is added (dioxetane), and a positive reaction results in the emission of light that is detected by a luminometer; the intensity of the signal corresponds to the target concentration. ^, The use of isoC and isoG base pairs and their incorporation into both the preamplifier region of bDNA and the second target-specific probe reduces nonspecific binding and enhances the specificity of the reaction. ^, bDNA assays are available for the detection and quantification of HIV-1, HCV, and HBV (Siemens Healthcare Diagnostics, Deerfield, IL) ( Fig. 67.3 ).

In situ hybridization

In situ hybridization is solid-phase hybridization where NAs are targeted within fixed cells or tissues. Because the target can be visualized within intact tissues or cells, additional information can be collected that might include a change in the tissue morphology. A widely used approach for identifying microorganisms using this method is fluorescence in situ hybridization (FISH). ^, FISH allows for both identification of microbial targets and differentiation to species level using specific probes. Multiple probes used for FISH have been deposited in the “probeBase” database. ^, The use of peptide-nucleic-acid (PNA) probes as a substitution to traditional NA probes offers an advance to the assay. The PNA probe backbone is formed of glycine rather than deoxyribose and phosphate molecules ( Table 67.1 ). This difference enhances the penetration through the cell membrane due to its neutrally charged glycine backbone. ^, Advantages of FISH include the short time to result; the ability to identify microorganisms in primary clinical specimens, which is particularly advantageous for intracellular, fastidious and difficult to identify organisms; the ability to detect select antimicrobial resistance markers. The sensitivity of FISH remains lower than amplification methods, and the methodology requires skilled personnel. There are limited FISH assays available for microbiology applications. OpGen (AdvanDx, Wolburn, MA) offers multiple QuickFISH and PNA FISH products that include Candida PNA FISH for the direct and rapid detection of Candida spp. from positive blood culture bottles ( Fig. 67.4 ; see Syndromic section below). In addition, the Accelerate Pheno system (Tucson, AZ) is a fully automated FISH platform that offers automated sample preparation, target hybridization, microscopic examination, and analysis of data. This system is a recent addition to the multiplexed assays that offer rapid identification of pathogens of positive blood cultures and rapid antimicrobial susceptibility testing (see Syndromic section below). This methodology can also identify polymicrobial positive blood cultures to overcome that limitation when mass spectrometry (MS) or phenotypic biochemical methods are used.

TABLE 67.1

Examples of Probes and Dyes for Real-time Polymerase Chain Reaction

		Mechanism	Advantage/Disadvantage
Nonspecific Detection of dsDNA Products
SYBR Green		Binds to the minor grooves of dsDNA with subsequent fluorescence,	Less cost Melting curve analysis is required to confirm the PCR specificity Low level of quantification
Primer-Probe
Scorpions	Reprinted from Real-time PCR detection chemistry by E. Navarro, G. Serrano-Heras, M.J. Castano, J. Solera with permission from Elsevier	Fluorescence emission starts after the second denaturation step due to binding of complementary sequences of the probe to the newly synthesized DNA.	Combining the primer and probe reduces the cost Less background fluorescence No primer-dimer formation or nonspecific PCR products
Hydrolysis Probes
TaqMan	Reprinted from Real-time PCR detection chemistry by E. Navarro, G. Serrano-Heras, M.J. Castano, J. Solera with permission from Elsevier	Due to the proximity of the fluorophores (of which one serves as the reporter and the other serves as the quencher), the signal is quenched. During PCR extension, the 5′–3′ exonuclease activity of the DNA polymerase is associated with the emission of fluorescence due to the release of the reporter.	Probes are easy to design Primer-dimers can form if not appropriately designed
Hybridization Probes
FRET	Reprinted from Real-time PCR detection chemistry by E. Navarro, G. Serrano-Heras, M.J. Castano, J. Solera with permission from Elsevier	Annealing of the probes to complementary target sequences is designed to bring the quencher and reporter closer with an associated emission of fluorescence secondary to energy transfer by FRET.	Probes are easy to design Melting curve analysis could be performed
Molecular Beacons	Reprinted from Real-time PCR detection chemistry by E. Navarro, G. Serrano-Heras, M.J. Castano, J. Solera with permission from Elsevier	Upon annealing, fluorescence is released when the probe binds to the target sequence.	Melting curve analysis could be performed Highly specific, single nucleotide mismatch could be discriminated
Molecular Torches	The figure is courtesy of HOLOGIC, Inc. and affiliates	Hybridization separates the quencher from the reporter and is associated with the emission of fluorescence.	The linker facilitates the closed conformation in the absence of the target The linker enables the use of smaller target-binding region, which increases the sensitivity
Partially Double Stranded		Fluorescence is generated when the hybridization probe (blocked at the 3′ to prevent extension) with the fluorescent marker attached binds to target DNA.	Mismatch discrimination capability can be controlled High tolerance to mismatches allows the detection of genetically diverse targets
Nucleic Acid Analogues
PNAs	Reprinted from Real-time PCR detection chemistry by E. Navarro, G. Serrano-Heras, M.J. Castano, J. Solera with permission from Elsevier	Mechanism is dependent on the probe design (e.g., primer-probe) and is similar to oligonucleotide probes.	Resistant to proteases and nucleases DNA binding is possible at lower salt concentrations Due to their neutral charge, binding to DNA or RNA is of superior affinity than regular oligonucleotides

Hybridization arrays

DNA hybridization arrays, also called microarrays, macroarrays, or high-density oligonucleotide arrays, offer high throughput ways to look at multiple targets simultaneously, including the expression of genes. These methods rely on immobilizing an array of target-specific probes on a surface (membrane, a slide, or a chip); these sequences are then hybridized with NAs (amplified and/or labeled) from clinical specimens. Different array methods were developed that differ in the number of detectable targets, which varies from a few to thousands (low/moderate density versus high density microarrays) and the method of detection. Although quantitative PCR methods are generally more sensitive than hybridization arrays, array methods can overcome the challenges of developing target-specific standards, reaction kinetics requirements, and multiple channel detection, making microarray technology a practical multiplexing approach in the clinical laboratory. Clinical applications include microbial identification, host gene expression analysis, antimicrobial resistance detection, and strain typing.

The routine use of arrays in clinical microbiology laboratories is currently limited to low and moderate-density microarrays. Several commercially available platforms used for syndromic infectious disease testing employ microarrays as part of the detection system. For example, the eSensor technology (GenMark Dx, Carlsbad, CA) uses an electrochemical detection system with a gold-plated microarray of electrodes. The targets are initially amplified and converted by exonuclease to single strands, which hybridize to ferrocene-labeled probes. This oligonucleotide “sandwich” recruits ferrocene to the surface of the electrode and results in specific electric currents that can identify each target ( Fig. 67.5 ). ^, The company has also used its technology to develop an assay for the detection of SARS-CoV-2, for which they received an emergency use authorization (EUA). ^,

Another hybridization array system used in clinical microbiology laboratories is the Verigene system (Luminex, Austin, TX). The Verigene uses NanoGrid technology, which starts with the hybridization of amplified or fragmented NA to capture probes immobilized on a microarray followed by a secondary hybridization step of mediator oligonucleotides and gold nanoparticle probes. The signal is amplified using a silver staining step. Multiple syndromic panels are available that include the Verigene gram-negative and gram-positive blood culture and enteric panels ( Fig. 67.6 ; see Syndromic section below). ^, The same company also offers respiratory and enteric syndromic panels ^, using a different microarray technology, which is suspension bead microarray. In this assay, targets are amplified with biotinylated primers, denatured, and then mixed with color-coded beads to which capture probes are attached. Scanning for hybridization is measured using a streptavidin-bound fluorophore, and the bead suspension is analyzed by flow cytometry. As a response to the global SARS-CoV-2 outbreak, Luminex developed an extended panel (NxTAG CoV) for SARS-CoV-2 detection for which they received EUA.

BioFilmChip microarrays (Autogenomics, Vista, CA) used by the Infiniti system offer multiplexing assays, the majority of which are research use only (RUO) in the United States, but many are available as CE marked, including the Candida Vaginitis and Bacterial Vaginosis QUAD panels. The fluorescently labeled amplified targets are hybridized to specific probes on multi-layered chips that are scanned for automated analysis with a high-resolution confocal camera.

High-density microarrays offer a large multiplexing capacity that permits applications including microbial identification, antimicrobial resistance detection, epidemiologic surveillance, and viral discovery, among others. The general concept includes the use of hundreds to thousands of hybridization probes attached to a solid surface, to which labeled amplified targets hybridize. Signals are mapped to hybridization locations within the array. Affymetrix (Santa Clara, CA) offers GeneChips where oligonucleotides are synthesized in situ (on the surface of the chip, typically quartz). Probes are synthesized between 20 and 25 bp, and multiple probes per target are synthesized, which enhances the specificity. Customized high-density chips have been designed for viral typing and characterization, ^, diagnosis, and quantification of microbial populations. Due to the high cost associated with most of the high-density microarray methods, routine clinical laboratory applications, and commercially available assays are not yet available.

Target nucleic acid amplification strategies

NA amplification via PCR has markedly advanced molecular diagnostics and significantly improved analytical sensitivity. Different groups have developed novel strategies for NA amplification; however, they all share the requirement for a polymerase for amplification. In addition, all target-specific amplification-based assays require specific primers. Primer binding sites mark the region to be amplified by the polymerase in a reaction that results in the production of up to millions or billions of copies of the target. The basic PCR reaction requires a buffer that, besides the primers and a thermostable polymerase, contains deoxyribonucleotides triphosphate (dNTPs) and magnesium. Cycles of certain periods at specific temperatures (thermocycling) are required for double-stranded DNA denaturation, primer binding (annealing), and primer extension (polymerization). With each PCR cycle, the number of target NA amplicons doubles, which means for a PCR reaction with 30 cycles, 10 ⁹ -fold amplification occurs. This exponential amplification is limited by the efficiency of the reaction and could plateau with the depletion of the reaction components ( Fig. 67.7 ). Because this reaction starts with a DNA target, a reverse transcription reaction step is required before the PCR amplification of RNA targets (RT-PCR) that relies on enzymes required for the replication of RNA viruses (reverse transcriptase). ^, Reverse transcriptase requires primers for synthesizing the complementary DNA strand (cDNA); those primers are usually either random primers, target-specific, or in certain applications oligonucleotides bind to the poly A tail if mRNAs are targeted for amplification (Oligo [dT] primers).

Traditionally, the detection of amplicons relied on visualizing the PCR products after agarose or polyacrylamide gel electrophoresis. DNA staining was required for visualizing the PCR products and different DNA binding stains, the most widely used of which is ethidium bromide, were used. The correct product was identified based on the amplicon size. This methodology is still widely used for research purposes but is not practical for the clinical microbiology laboratory. Several methods were developed to provide PCR assays that fulfill the criteria needed for diagnostic assays, including detection with minimal manipulation of amplicons to reduce the risk of contamination and reduction in hands-on time.

Real-time polymerase chain reaction

rtPCR has transformed the diagnostic capabilities of clinical microbiology laboratories. rtPCR offers simultaneous target amplification and product detection in real-time. In addition, rtPCR assays add the significant advantage of not only target detection but also highly accurate and sensitive quantification. In general, the detection relies on fluorescence that is measured with every PCR cycle and normalized to the baseline. Fluorescence emission is associated with the use of fluorescent dyes that bind to double-stranded DNA products with every cycle (e.g., SYBR green) or fluorescently-labeled oligonucleotides designed to bind specifically to the target of interest with different mechanisms such that fluorescence is altered (i.e., quenched or emitted) with target amplification (see Table 67.1 ).

Because amplification and detection overlap, the thermocyclers used for performing rtPCR require special software for fluorescence analysis and generation of the rtPCR amplification plots. Typically, the amplification plot shows the cycle numbers on the X-axis and the intensity of fluorescence on the Y -axis (normalized). The initial cycles will not show a change in fluorescence signal, which defines the baseline. A positive rtPCR is defined by the reaction of which the fluorescence signal increases above the threshold of the assay. The cycle threshold (C _T ) value is the cycle number at which the fluorescence crosses the plot fixed threshold ( Fig. 67.8 ). Quantified standards (calibrators) can be used to convert the C _T to a target-quantified value (e.g., copies or units). Several companies currently offer rtPCR platforms, including Applied Biosystems (Foster City, CA), Bio-Rad (Hercules, California), and Roche Diagnostics (Indianapolis, IN). In addition, continuous systems and high-throughput closed platforms that offer qualitative and quantitative rtPCR assays for different targets are commercially available (e.g., GeneXpert from Cepheid, Roche cobas systems, and Abbott molecular platforms [Lake Forest, Illinois]).

The use of dsDNA dyes for amplification detection relies on the ability of these dyes to nonspecifically bind to the minor grove of dsDNA with a progressive increase in fluorescence during DNA amplification. The use of these dyes is associated with nonspecific fluorescence due to amplification of nonspecific products; hence a melting curve analysis is highly recommended to confirm the specificity of the reaction. Melting curve analysis relies on the fact that different PCR products have different melting (denaturation) temperatures and should give distinct peaks of loss of fluorescence (due to the dissociation of dye) when exposed to a certain range of temperature. In contrast, fluorophore-labeled oligonucleotides bind with high specificity to the PCR target of interest. The fluorophore-labeled oligonucleotides available for rtPCR belong to three main groups based on their structure, mechanism of signal quenching, and production and types of fluorescent particles: primer-probe (e.g., Scorpions), probes (hydrolysis, e.g., TaqMan probes, hybridization, e.g., FRET ^, and molecular beacons ), and NA analogs (e.g., PNA ) (see Table 67.1 ).

A different approach for rtPCR detection developed by Luminex involves the use of the nonstandardized base pairs isoG and isoC in a probe-free reaction and a loss of fluorescence rather than an increase in fluorescence approach. The MultiCode-RTx assays use a reporter-labeled primer that contains a single base of isoC at the 5′ end. The first cycle of PCR incorporates this labeled primer along with unlabeled reverse primer to the newly synthesized strands. An isoG with an attached quencher will then get incorporated, resulting in a decrease in fluorescence. The assays also include a final step of thermal melt, which restores fluorescence ( Fig. 67.9 ). ^,

Digital polymerase chain reaction

Digital PCR offers a more accurate way to quantify NA targets without the need for standards, an advantage that adds precision to quantification of reference materials and controls for clinical microbiology quantitative assays. Other applications include absolute quantification of viral load ^, and detection of mutations that confer drug resistance. As in rtPCR, fluorescent dyes are added to the PCR reaction; however, in digital PCR, the reaction is divided into thousands of partitions so that each partition likely ends up with none or one copy. Without a need for a standard curve, quantification is achieved by counting the number of positive and negative partitions and applying Poisson’s law to accurately calculate the concentration in the original sample. Partitioning can be attained by using chips, arrays, capillaries, or oil droplets (droplet digital PCR). Commercial digital PCR platforms are available, including QuantStudio instruments (Thermo Fisher, Waltham, MA), BioMark HD (Fluidigm, South San Francisco, CA), Clarity (JN Medsys, Singapore), RainDrop (RainDance, Billerica, MA), and QX100 and QX200 (Bio-Rad) ( Fig. 67.10 ). (For more details, please refer to Chapter 64 , Nucleic Acid Techniques.)

Non–polymerase chain reaction target and signal amplification approaches

Several amplification methods that are not PCR-based have been developed, including non-PCR target amplification and signal amplification. These approaches were developed to reduce the need for thermocycling, which is associated with a reduction in cost or to reduce the risk of amplicon contamination. Signal amplification techniques amplify the detection signal rather than the target and include bDNA (discussed previously), hybrid capture, and Invader assays. The non-PCR target amplification methods largely require an isothermal reaction and result in a very efficient target amplification. These methods include isothermal TMA, loop-mediated amplification (LAMP), and helicase-dependent amplification. ^,

Signal amplification techniques

Hybrid capture

The first commercially available clinical assay that used the hybrid capture approach was the HPV Hybrid Capture 1 (HC1) test by Digene (now QIAGEN). The hybrid capture methodology relies on using RNA probes for the detection of specific DNA target sequence, followed by the detection of RNA/DNA hybrids by specific antibodies. The methodology has progressed to offer the ability to detect RNA targets using DNA probes or the reverse with the introduction of Hybrid Capture 2 (HC2). The use of conjugated antibodies (e.g., alkaline phosphatase-conjugated) allows for signal amplification after adding a chemiluminescent substrate. HC2 assays were developed for the detection of C. trachomatis , Neisseria gonorrhoeae , HBV, cytomegalovirus (CMV), and herpes simplex viruses (HSV) 1 and 2 ( Fig. 67.11 ).

Invader technology

Third Wave Technologies (now Hologic) developed the Invader technology, an isothermal assay that uses highly specific DNA probes, an upstream invader oligonucleotide, and a downstream probe. Both probes hybridize to the target DNA to form a structure that has a single nucleotide overlap at the cleavage site. The 3′ end of the probe has a specific complementarity to the target DNA; however, the 5′ end makes a flap. When the invader oligo binds, the structure will be a substrate for the cleavase enzyme, and the probe will be cleaved to release the flap. Signal amplification is feasible due to the ability of the reaction to continuously proceed due to the temporary binding of the probes, as the temperature of the reaction is set very close to the melting temperature of the probe. As the oligos are present in the reaction in molar excess, cycles of binding of the signal and invader probes, formation of the cleavage substrate, and cleavage continue with the production of cleavage products (cleaved flaps). The signal stems from the binding of the flaps to FRET cassettes, which also, upon binding, form a cleavase substrate-specific structure. Cleavage of the FRET probe is associated with fluorescence. A robust linear signal amplification reaction can develop with this approach, with each flap being able to cleave 10 ³ to 10 ⁴ FRET probes per hour. ^, Invader technology has been used for qualitative and quantitative detection and identification of single base polymorphisms. The Cervista HPV test is FDA approved for the detection of high-risk HPV genotypes ( Fig. 67.12 ). ^,

Non–polymerase chain reaction target amplification

Transcription-based amplification

Isothermal transcription-based amplification includes TMA and nucleic acid sequence-based amplification (NASBA). The methodology is derived from the biology of replication of retroviruses. ^, The process is achieved by three enzymatic reactions: a primer-based reverse transcription step mediated by a reverse transcriptase to convert the RNA targets to cDNA, degradation of the RNA strand bound to the cDNA by RNAse H, and a second primer-based amplification of cDNA using the DNA-dependent DNA polymerase activity of the reverse transcriptase. In the first step, the primer has T7 polymerase promotor sequence incorporated, which allows the subsequent transcription of the DNA synthesized copies by the bacteriophage T7 RNA polymerase, which creates new RNA copies during subsequent cycles of amplification. This method can achieve 10 ⁹ fold amplification of target in less than 2 hours ( Fig. 67.13 A). RNA-based methods detect pathogens at low abundance due to the richness of rRNA in relation to a single copy of DNA per pathogen. Another advantage is the detection of the transcriptional activities of organisms, which rules out dead pathogens or latent viral infections.

The Aptima (Hologic) assays use TMA technology, and the detection method depends on HPA (as previously described). Commercially available clinical assays include detection of C. trachomatis , N. gonorrhoeae , T. vaginalis, and Mycoplasma genitalium . The impact of using an RNA-based assay for diagnosing a M. genitalium infection was highlighted in two European studies (NAME & FAME) where greater sensitivities of the Aptima assay were observed in contrast to the comparator DNA tests, largely attributed to the lower titers of M. genitalium compared to other STIs. ^, Real-time TMA assays are also available from Hologic, both quantitative (HIV, HCV, and HBV) and qualitative (HSV-1 and 2). These assays use real-time fluorescence detection of amplicons (see Fig. 67.13 B).

NASBA (bioMerieux, Durham, NC) is a similar technology to TMA; however, in addition to using a different RNA polymerase (avian myeloblastosis virus RT), the detection probes are labeled with tris ruthenium, and detection is via electrochemiluminescence. NASBA assays are marketed by bioMerieux as NucliSENS assays and incorporate the use of molecular beacon probes for real-time detection (NUCLISENS EASYQ). These products have been discontinued in the US.