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Molecular diagnostics of infectious diseases

Division of Clinical Microbiology, Department of Pathology and Laboratory Medicine, Hilton Bldg. 470, Mayo Clinic, 200 First St., SW, Rochester, MN 55905.
^a Authors for correspondence. Fax 507 284-4272; e-mail: tang.yi-wei{at}mayo.edu or persing.david{at}mayo.edu

	Abstract

Top Abstract Introduction Traditional Microbial Typing Nucleic Acid-Based Typing... Nucleic Acid Analysis Without... Polymerase Chain Reaction Other Nucleic Acid Amplification... Analysis of Amplification... Current Application of Molecular... Future Applications References

 
Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Microbial phenotypic characteristics, such as protein, bacteriophage, and chromatographic profiles, as well as biotyping and susceptibility testing, are used in most routine laboratories for identification and differentiation. Nucleic acid techniques, such as plasmid profiling, various methods for generating restriction fragment length polymorphisms, and the polymerase chain reaction (PCR), are making increasing inroads into clinical laboratories. PCR-based systems to detect the etiologic agents of disease directly from clinical samples, without the need for culture, have been useful in rapid detection of unculturable or fastidious microorganisms. Additionally, sequence analysis of amplified microbial DNA allows for identification and better characterization of the pathogen. Subspecies variation, identified by various techniques, has been shown to be important in the prognosis of certain diseases. Other important advances include the determination of viral load and the direct detection of genes or gene mutations responsible for drug resistance. Increased use of automation and user-friendly software makes these technologies more widely available. In all, the detection of infectious agents at the nucleic acid level represents a true synthesis of clinical chemistry and clinical microbiology techniques.

	Introduction

Top Abstract Introduction Traditional Microbial Typing Nucleic Acid-Based Typing... Nucleic Acid Analysis Without... Polymerase Chain Reaction Other Nucleic Acid Amplification... Analysis of Amplification... Current Application of Molecular... Future Applications References

 
Over the past century microbiologists have searched for more rapid and efficient means of microbial identification. The identification and differentiation of microorganisms has principally relied on microbial morphology and growth variables. Advances in molecular biology over the past 10 years have opened new avenues for microbial identification and characterization (1)(2)(3)(4)(5).

The traditional methods of microbial identification rely solely on the phenotypic characteristics of the organism. Bacterial fermentation, fungal conidiogenesis, parasitic morphology, and viral cytopathic effects are a few phenotypic characteristics commonly used. Some phenotypic characteristics are sensitive enough for strain characterization; these include isoenzyme profiles, antibiotic susceptibility profiles, and chromatographic analysis of cellular fatty acids (6)(7)(8)(9)(10)(11)(12)(13). However, most phenotypic variables commonly observed in the microbiology laboratory are not sensitive enough for strain differentiation. When methods for microbial genome analysis became available, a new frontier in microbial identification and characterization was opened.

Early DNA hybridization studies were used to demonstrate relatedness amongst bacteria. This understanding of nucleic acid hybridization chemistry made possible nucleic acid probe technology (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Advances in plasmid and bacteriophage recovery and analysis have made possible plasmid profiling and bacteriophage typing, respectively (26)(27)(28)(29)(30)(31). Both have proven to be powerful tools for the epidemiologist investigating the source and mode of transmission of infectious diseases (26)(28)(30)(32)(33)(34)(35)(36)(37)(38)(39)(40). These technologies, however, like the determinations of phenotypic variables, are limited by microbial recovery and growth.

Nucleic acid amplification technology has opened new avenues of microbial detection and characterization (1)(5)(41), such that growth is no longer required for microbial identification (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52). In this respect, molecular methods have surpassed traditional methods of detection for many fastidious organisms. The polymerase chain reaction (PCR) and other recently developed amplification techniques have simplified and accelerated the in vitro process of nucleic acid amplification. The amplified products, known as amplicons, may be characterized by various methods, including nucleic acid probe hybridization, analysis of fragments after restriction endonuclease digestion, or direct sequence analysis. Rapid techniques of nucleic acid amplification and characterization have significantly broadened the microbiologists' diagnostic arsenal.

	Traditional Microbial Typing

Top Abstract Introduction Traditional Microbial Typing Nucleic Acid-Based Typing... Nucleic Acid Analysis Without... Polymerase Chain Reaction Other Nucleic Acid Amplification... Analysis of Amplification... Current Application of Molecular... Future Applications References

 
biotyping
Traditional microbial identification methods typically rely on phenotypes, such as morphologic features, growth variables, and biochemical utilization of organic substrates. The biological profile of an organism is termed a biogram. The determination of relatedness of different organisms on the basis of their biograms is termed biotyping. Investigators must determine which profile variables have the greatest differentiating capabilities for a given organism (53)(54). For example, gram stain characteristics, indole positivity, and the ability to grow on MacConkey medium do not aid in the differentiation of nonenterohemorrhagic Escherichia coli from E. coli O157:H7. However, sorbitol fermentation has proven to be an extremely useful characteristic of the biochemical profile used to differentiate these strains.

Biograms that are identical have been used to infer relatedness between strains in epidemiological investigations (32)(55)(56). The biograms of organisms are not entirely stable, and several isotypes may exist from a single isolate (12). Biograms may be influenced by genetic regulation, technical manipulation, and the gain or loss of plasmids. In many instances, biotyping is used in conjunction with other methods to more accurately profile microorganisms (32).

antibiograms, resistograms, and bacteriocin typing
The susceptibility or resistance of an organism to a possibly toxic agent forms the basis of the following typing techniques. The antibiogram is the susceptibility profile of an organism to a variety of antimicrobial agents, whereas the resistogram is the susceptibility profile to dyes and heavy metals (26). Bacteriocin typing is the susceptibility of the isolate to various bacteriocins, i.e., toxins that are produced by a collected set of producer strains. These three techniques are limited by the number of agents tested per organism.

By far, the antibiogram is the most commonly used susceptibility/resistance typing technique, most probably because the data required for antibiogram analysis are available routinely from the antimicrobial susceptibility testing laboratory. Although antibiograms have been used successfully to demonstrate relatedness, this technology is limited (6)(10)(55). And although organisms with similar antibiograms may be related, such is not necessarily the case. The antibiogram of an organism is not always constant (57). Selective pressure from antimicrobial therapy may alter an organism's antimicrobial susceptibility profile (58), such that related organisms show different resistance profiles. These alterations may result from chromosomal point mutations or from the gain or loss of extrachromosomal DNA such as plasmids or transposons (26)(57)(59).

protein analysis
Commercially available antibodies are routinely used to specifically identify antigenic proteins from a wide variety of organisms. In some instances, the test may be used only to identify the genus and species of an organism. Examples of this include the cryptococcal antigen agglutination assay and the exoantigen assay for Histoplasma capsulatum. Other immunoassays are designed to subtype microbes (60). Monoclonal antibodies directed against the major subtypes of the influenza virus, as well as the various serotypes of Salmonella, are commonly used in speciation. Specific antigenic proteins may be detected by antibodies directed against these proteins in immunoblot methods (12)(61).

Electrophoretic typing techniques have been used to examine outer membrane proteins, whole-cell lysates, and particular enzymes (6)(55). Several electrophoretic methods are available to examine the protein profile of an organism. Generally, outer membrane proteins and proteins from cell lysates are examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. This technique denatures the proteins and separates them on the basis of molecular mass. The protein profile may be used to compare strains (8)(55)(62).

Nondenaturing conditions are used for the electrophoretic separation of active enzymes. Multilocus enzyme electrophoresis is the typing technique based on the electrophoretic pattern of several constitutive enzymes (63). Differences in electrophoretic migration of functionally similar enzymes (e.g., lactate dehydrogenase isoenzymes) represent different alleles. These differences or similarities, especially when numerous enzymes are examined, may be used to exclude or infer relatedness (6)(8)(10).

The results of these studies may be difficult to interpret, however. The absence of a particular protein may simply reflect downregulation of that particular gene product, rather than the loss of that particular gene. Additionally, the electrophoretic migration of proteins is dependent on molecular mass, net protein charge, or both. Mutations that do not alter these characteristics will not be detected.

phage analysis
Bacteriophages, viruses that infect and lyse bacteria, are often specific for strains within a species. A collection of bacteriophages, many of which often infect similar bacteria, is termed a panel. When a bacterial isolate is exposed to a panel of bacteriophages, a profile is generated—a listing of which bacteriophages are capable of infecting and lysing the bacteria. The bacteriophage profile may be used to type bacterial strains within a given species (31)(62). The more closely related the bacterial strains, the greater the similarity of the bacteriophage profiles. Bacteriophage profiles have been used successfully to type various organisms associated with epidemic outbreaks (64)(65). However, this typing method is labor-intensive and requires the maintenance of bacteriophage panels for a wide variety of bacteria. Additionally, bacteriophage profiles may fail to identify isolates, are often difficult to interpret, and may give poor reproducibility (62).

chromatographic analysis
Chromatographic analysis of short-chain fatty acid production is a routine method used to aid in the identification of anaerobic bacteria. Computer-aided gas–liquid chromatography is commercially available and is a means of microbial identification. This identification system utilizes the type and amount of cellular fatty acids present in the lysate of an organism. Many species have unique cellular fatty acid chromatographic profiles (9)(13). Relationships between strains of a particular species may be inferred from highly similar cellular fatty acid profiles (7).

Chromatographic analysis is reliable when organisms are grown under identical conditions and the cellular fatty acids are extracted without technical variation. These constraints, however, limit the accuracy of this technology with respect to strain and in some instances even species-level identification.

	Nucleic Acid-Based Typing Systems

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plasmid analysis
Plasmids are small, self-replicating circular DNA found in many bacteria. These often encode genes related to antibiotic resistance and certain virulence factors. In epidemiological studies, relatedness of isolated pathogenic bacterial strains can be determined from the number and size of plasmids the bacteria carry. Plasmid profile analysis was among the earliest nucleic acid-based techniques applied to the diagnosis of infectious diseases and has proven useful in numerous investigations (26)(27)(28)(29)(30)(60). This method has also been widely utilized for tracking antimicrobial resistance during nosocomial outbreaks (26)(66)(67). In studies of the epidemiology of plasmids, analysis of restriction fragments has proved valuable. This technique is widely used to monitor the spread of resistance-encoding plasmids between organisms and between hospitals, communities, or even countries (37)(38)(39)(40). The weakness of the analysis is inherent in the fact that plasmids are mobile, extrachromosomal elements, not part of the chromosomal genotype. Because plasmids can be spontaneously lost from or readily acquired by a host stain, epidemiologically related isolates can exhibit different plasmid profiles (68).

restriction enzyme pattern
Restriction endonucleases recognize specific nucleotide sequences in DNA and produce double-stranded cleavages that break the DNA into small fragments. The number and sizes of the restriction fragments, called restriction fragment length polymorphisms (RFLPs)¹ , generated by digesting microbial DNA are influenced by both the recognition sequence of the enzyme and the composition of the DNA. In conventional restriction endonuclease analysis, chromosomal or plasmid DNA is extracted from microbial specimens and then digested with endonucleases into small fragments. These fragments are then separated by size with use of agarose gel electrophoresis. The nucleic acid electrophoretic pattern can then be visualized by ethidium bromide staining and examination under UV light.

Restriction endonuclease analysis has the advantage of being highly reproducible, very accurate in determining the relatedness of microbial strains, and well within the technical capabilities of experienced laboratory technologists. However, the major limitation of this technique, especially for chromosomal DNA, is the difficulty of comparing the complex profiles generated, which consist of hundreds of fragments. To address this problem, pulse-field gel electrophoresis (PFGE) has been developed (69) to enable the separation of large DNA fragments. PFGE provides a chromosomal restriction profile typically composed of 5 to 20 distinct, well-resolved fragments ranging from ~10–800 kilobases (kb) (58). The relative simplicity of the RFLP profiles generated by PFGE facilitates application of the procedure in identification and epidemiological survey of bacterial pathogens (12)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80). Fingerprinting, which combines PFGE with Southern transfer and hybridization, has been widely used in studying the tuberculosis nosocomial outbreak in human immunodeficiency virus (HIV)-positive populations (81)(82)(83).

ribotyping
Restriction patterns can be obtained by hybridizing Southern-transferred DNA fragments with labeled bacterial ribosomal operon(s), which encode for 16S and (or) 23S rRNA. This method, called ribotyping, has been shown to have both taxonomic and epidemiological value (84)(85). All bacteria carry these operons, which are highly conserved and therefore typeable. Particular rRNA sequences that are species- or group-specific have been also exploited in construction of oligonucleotides that have been used as probes for in situ detection of bacteria.

Ribotyping assays have been used to differentiate bacterial strains in different serotypes and to determine the serotype(s) most frequently involved in outbreaks (12)(29)(73)(79)(86)(87)(88)(89). This technique is especially useful in epidemiological studies for organisms with multiple ribosomal operons, such as members of the family of Enterobacteriaceae. Ribotyping simplifies the microrestriction patterns by rendering visible only the DNA fragments containing part or all of the ribosomal genes. The technique is less helpful when the bacterial species under investigation contains only one or a few ribosomal operons. In these instances, ribotyping typically detects only one or two bands, which limits its utility for epidemiological studies (70). Most studies have indicated that PFGE is superior to ribotyping for analysis of common nosocomial pathogens.

random amplified polymorphic dna (rapd)
RAPD typing, originally developed by Welsh and McClelland in 1990, involves the use of a short (usually 10 to 15 mers), arbitrarily chosen primer to amplify nearly homologous sequences of the genomic DNA under low-stringency conditions (90). RAPD has been used to differentiate strains of various species, various serotypes within species, and various subtypes within a serotype (91)(92)(93)(94)(95). It is, therefore, useful for determining whether two isolates of same species are epidemiologically related. RAPD has been used to evaluate outbreaks of infection of drug-resistant bacteria (96)(97)(98). For potentially dangerous drug-resistant organisms such as the mycobacteria, RAPD may be a better choice than PFGE because the technique requires fewer open manipulations and the organisms are kept viable for a shorter period. RAPD is probably the simplest DNA-based subtyping method to date if a temperature-cycling instrument is available, although the usefulness for epidemiological investigations remains to be determined, particularly with regard to reproducibility concerns.

	Nucleic Acid Analysis Without Amplification

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nucleic acid probes
Nucleic acid probes are capable of identifying organisms at, above, and below the species level. The quantity of target detectable by the method depends on the size and homology of the probe chosen and the nature of the original specimen; identification of organisms in pure cultures or from isolated colonies is usually easier than detection of organism in a direct specimen. DNA probes facilitate the identification of infectious agents that do not grow rapidly. Additionally, this technique allows for the diagnosis of infections in which the organisms are not easily cultured or cannot be cultured at all. Detection of DNA with direct or culture-amplified gene probe technology has been applied to several organisms, including bacteria (14)(15)(16), viruses (17)(18)(19), mycobacteria (20)(21)(22), fungi (23)(24), and even certain parasites (25). The technique has been also used to monitor growth as an indicator of drug resistance (99)(100) or to directly detect genes associated with antibiotic resistance (101)(102).

Gen-Probe, MicroProbe, and Digene Diagnostics are currently manufacturing several direct detection and culture identification nucleic probes that have been cleared by the US Food and Drug Administration. The procedures for the use of DNA probes are now well standardized, and the advent of synthetic short oligonucleotide DNA probes has shortened the time required for probe assay. However, direct probe techniques appear to be of limited utility owing to poor sensitivity. Nucleic acid amplification methods, described in detail below, have been explored to address this problem.

branched dna signal amplification
Developed and manufactured by Chiron Corp., branched DNA (bDNA) probes are an example of signal amplification. Multiple probes as well as multiple reporter molecules are used to increase the signal in proportion to amount of target in the reaction (103)(104). In this process, multiple specific synthetic oligonucleotides hybridize to the target and capture the target onto a solid surface. Synthetic bDNA amplifier molecules, which are enzyme-conjugated, branched oligonucleotide probes, are added. Hybridization proceeds between the amplifiers and the immobilized hybrids. After addition of a chemiluminescent substrate, light emission is measured and may be quantified (103).

In bDNA assays, all hybridization reactions occur simultaneously and the observed signal is proportional to the amount of target DNA. DNA quantification can thus be determined from a calibration curve. Because the target molecules themselves are not amplified during the process, this procedure is less likely to have contamination problems, which may be encountered with nucleic acid amplification methods. bDNA is also highly reproducible, and thus represents an excellent technological platform for monitoring therapeutic response and quantifying nucleic acids (105)(106)(107)(108)(109). A separate section below deals with this particularly important issue. One of the disadvantages, however, is that the bDNA assay is generally less sensitive than enzymatic amplification techniques and usually can detect no fewer than 10³ to 10⁵ nucleic acid targets. As with many techniques, moreover, test specificities decline as greater sensitivity is sought.

	Polymerase Chain Reaction

Top Abstract Introduction Traditional Microbial Typing Nucleic Acid-Based Typing... Nucleic Acid Analysis Without... Polymerase Chain Reaction Other Nucleic Acid Amplification... Analysis of Amplification... Current Application of Molecular... Future Applications References

 
As mentioned above, for direct application to the diagnosis of infections, nucleic acid analysis without amplification often has the disadvantage of low sensitivity (high detection limits). Nucleic acid amplification techniques increase sensitivity dramatically while still retaining a high specificity. Invented by Cetus scientist Kary Mullis in 1983 (1)(2), PCR is the best-developed and most widely used method of nucleic acid amplification. An ingenious procedure, PCR is based on the ability of DNA polymerase to copy a strand of DNA by elongation of complementary strands initiated from a pair of closely spaced chemically synthesized oligonucleotide primers.

The basic technique of PCR includes repeated cycles of amplifying selected nucleic acid sequences (1)(2). Each cycle consists of three steps: (a) a DNA denaturation step, in which the double strands of the target DNA are separated; (b) a primer annealing step, performed at a lower temperature, in which primers anneal to their complementary target sequences; and (c) an extension reaction step, in which DNA polymerase extends the sequences between the primers. At the end of each cycle (each consisting of the above three steps), the quantities of PCR products are theoretically doubled. The whole procedure is carried out in a programable thermal cycler. Generally, performance of 30 to 50 thermal cycles results in an exponential increase in the total number of DNA copies synthesized (110)(111). Commercial systems for PCR detection of DNA targets of Chlamydia trachomatis and Mycobacterium tuberculosis are manufactured by Roche Molecular Systems (112).

reverse transcriptase (rt)-pcr
Numerous modifications of the standard PCR procedure have been developed since its inception (4)(5)(41). Some of these modifications effectively expand the diagnostic capabilities of PCR and have increased its utility in the clinical laboratory. RT-PCR was developed to amplify RNA targets. In this process, RNA targets are first converted to complementary DNA (cDNA) by RT, and then amplified by PCR. RT-PCR has played an important role in diagnosing RNA-containing virus infections, detecting viable Mycobacteria species, and monitoring the effectiveness of antimicrobial therapy (113)(114)(115). The conventional reverse transcription reactions are fastidious: The enzymes cannot tolerate higher temperatures, which limits wide application of the method in clinical diagnosis. The thermostable DNA polymerase (Tth pol) and its thermostable cousins derived from other organisms have efficient reverse transcription activity and therefore can be used in detection of RNA targets without the need for a separate RT step (116)(117). The higher reaction temperature increases stringency of primer hybridization and avoids the possible RNA secondary structure, so that the reaction is more specific and efficient than previous protocols that used avian myeloblastosis virus RT. Commercial kits for detection of HIV are now available that use this single enzyme technology.

nested pcr
Nested PCR, designed mainly to increase sensitivity (detect smaller quantities of target), uses two sets of amplification primers (4)(118). One set of primers is used for the first round of amplification, which consists of 15 to 30 cycles. The amplification products of the first reaction are then subjected to a second round of amplification with another set of primers that are specific for an internal sequence that was amplified by the first primer pair (118)(119)(120). Nested PCR has extremely high sensitivity because of the dual amplification process. The DNA product from the first round of amplification contains the hybridization sites for the second primer pair. The amplification by the second primer set, therefore, verifies the specificity of the first-round product. The major disadvantage of the nested-amplification protocol is the high probability of contamination during transfer of the first-round amplification products to a second reaction tube. This can be avoided either by physically separating the two amplification mixtures with a layer of wax or oil, or by designing the primer sets to utilize substantially different annealing temperatures (4).

multiplex pcr
Multiplex PCR is an amplification reaction in which two or more sets of primer pairs specific for different targets are introduced in the same tube. Thus, more than one unique target DNA sequence in a specimen can be amplified at the same time (121). Primers used in multiplex reactions must be carefully designed to have similar annealing temperatures, which often requires extensive empirical testing. This coamplification of multiple targets can be used for various purposes. For diagnostic uses, multiplex PCR can be set up to detect internal controls or to detect multiple pathogens from a single specimen (115)(120)(122)(123). Quantitative competitive PCR, a variation of multiplex PCR, can be used to quantify the amount of target DNA or RNA in a specimen (124)(125).

broad-range pcr
Another important technical modification is the development of broad-range PCR, in which conserved sequences within phylogenetically informative genetic targets are used to diagnose microbial infection. A broad-range PCR approach has identified several novel, fastidious, or uncultivated bacterial pathogens directly from infected human tissue or blood (126)(127)(128)(129)(130)(131). A universal primer set designed to target herpesvirus DNA polymerases might be widely useful for diagnosing herpesvirus infection (132). Broad-range rRNA PCR techniques offer the possibility of rapid bacterial identification through use of a single pair of primers targeting bacterial small-subunit (16S) rRNA or DNA (133)(134)(135)(136). The major obstacles to implementation of rapid, automated rDNA-based bacterial identification systems are background contamination and, needless to say, cost. Perkin-Elmer Applied Biosystems is developing a commercial system for broad-range bacterial amplification and sequencing.

	Other Nucleic Acid Amplification Techniques

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transcription-based amplification system (tas).
Described in 1989 by Kwoh et al., TAS includes synthesis of a DNA molecule complementary to the target nucleic acid (usually RNA) and in vitro transcription with the newly synthesized cDNA as a template (137). Variations on this process are referred to as self-sustaining sequence replication ("3SR"), nucleic acid sequence-based amplification ("NASBA"), or transcription-mediated amplification (TMA) (138)(139). Three enzymes, RT, RNase H, and T7 DNA-dependent RNA polymerase are used in the reaction. Amplification steps involve the formation of cDNAs from the target RNA by using primers containing a RNA polymerase-binding site. The RNase H then degrades the initial strand of target RNA in the RNA–DNA hybrid after it has served as the template for the first primer. The second primer binds to the newly formed cDNA and is extended, resulting in the formation of double-strand cDNAs in which one or both strands are capable of serving as transcription templates for RNA polymerase. Although technically less robust and less sensitive than PCR, TMA has various merits that make it an attractive option: It works at isothermal conditions in a single tube to help minimize contamination risks (138). Amplification of RNA not only makes it possible to detect RNA-containing viruses, but also lowers the detection limit for certain bacterial and fungal pathogens by using high-copy-number rRNA targets (139). A commercial system for detection of M. tuberculosis by TMA is now available from Gen-Probe.

ligase chain reaction (lcr)
Also called ligase amplification reaction, LCR is a probe amplification technique first described in 1989 by Wu and Wallace (141). Successful ligation relies on the contiguous positioning and correct base-pairing of the 3' and 5' ends of oligonucleotide probes on a target DNA molecule. In the process, oligonucleotide probes are annealed to template molecules in a head-to-tail fashion, with the 3' end of one probe abutting the 5' end of the second. DNA ligase then joins the adjacent 3' and 5' ends to form a duplicate of one strand of the target. A second primer set, complementary to the first, then uses this duplicated strand (as well as the original target) as a template for ligation. Repeating the process results in a logarithmic accumulation of ligation products, which can be detected by means of the functional groups attached to the oligonucleotides (142). The recently developed thermostable DNA ligase greatly simplifies this technique and has increased the specificity by helping avoid problems of blunt-end ligation at low annealing temperature (143). When used after a target amplification method, such as PCR, this technique can be sensitive and is useful for the detection of point mutations. Although convenient and readily automated, one potential drawback of LCR is the difficult inactivation of the postamplification products. The nature of the technique does not allow the most widely used contamination control methods to be applied. The inclusion of a detection system within the same reaction tube would greatly decrease the possibility of contamination, which is associated with the opening of reaction tubes. A combination LCR kit for detection of both Chlamydia trachomatis and Neisseria gonorrhea is now commercially available from Abbott Labs. (144).

strand displacement amplification (sda)
SDA is another non-PCR nucleic acid amplification technique, developed in 1991 (145)(146). In this system, DNA polymerase initiates DNA syntheses at a single-stranded nick and displaces the nicked strand during DNA synthesis. The displaced single-stranded molecule then serves as a substrate for additional simultaneous nicking and displacement reactions (145). This isothermal DNA amplification procedure uses specific primers, a DNA polymerase, and a restriction endonuclease to achieve exponential amplification of target. The key technology behind SDA is the generation of site-specific nicks by the restriction endonuclease. Although complicated, SDA has two important advantages. Except for the initial denaturation step, SDA is isothermal and requires no specialized thermocycler (146). In addition, SDA can be applied to either single- or double-stranded DNA.

qß replicase system
Initially described in 1988 (147), the Qß replicase system is based on the incorporation of a single-stranded oligonucleotide probe into an RNA molecule that can be exponentially amplified after target hybridization by the enzyme Qß replicase (148). The assay is technically straightforward. The enzyme specifically recognizes the secondary structure of the RNA from the Qß genome, which is hybridized to the specific target. After a given probe anneals to a target, the nonhybridized material can be removed by the enzyme RNase III and subsequent wash steps. The hybridized probe is then enzymatically replicated by Qß replicase to detectable quantities (147)(149). The potential advantages associated with the Qß replicase procedure include its remarkable speed (<30 min) and isothermal reaction conditions. The main drawback is that unbound reporter probes or nonspecifically bound reporter probes serve as templates for amplification, resulting in false-positive results. This formidable problem has been largely overcome by the use of target capture methods.

Practical information about current commercially available and Mayo Clinic-developed amplification techniques for detection of microbial pathogens are summarized in Table 1 .

	Analysis of Amplification Products

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After target amplification, the simple or conventional version of product detection is use of agarose gel electrophoresis after ethidium bromide staining. Several other techniques have been developed not only to "visualize" the products, but to enhance both the sensitivity and specificity of amplification techniques as well. A probe-based DNA detection system has the advantage of providing sequence specificity and decreased detection limits. After routine agarose gel electrophoresis, the DNA is transferred to a solid phase, e.g., nitrocellulose or nylon membrane, and probed by a specific probe. Radiolabeled probed membranes are directly exposed to x-ray film, whereas enzyme-labeled probed membrane may be visualized through either light or color production.

hybridization protection assay (hpa)
HPA is a homogeneous format. The probe and the product are incubated together in a single test tube, and the binding of probe to the target is measured without further manipulation (150). A probe labeled with an acridinium ester is added to a sample containing PCR products for identification. In a positive sample, the bound probe is protected from alkaline hydrolysis and, upon addition of peroxides, emits detectable light. The HPA does not require the binding of amplified DNA to a solid support by DNA capture or other means, can be performed in a few hours, and does not need to have excess unbound DNA probe removed (151)(152).

dna enzyme immunoassay (deia)
DEIA is another newly developed system for detecting nucleic acid previously amplified by means of PCR (153). An anti-dsDNA antibody exclusively recognizes the hybridization product resulting from the reaction between target DNA and a DNA probe. The final product is revealed by means of a colorimetric reaction (153). The DEIA increases the sensitivity of a previous PCR by including enzymatic reactions. The hybridization between specific probe and PCR-amplified target DNA, as well as the formation of target DNA/probe hybrids and anti-dsDNA antibody complex, also enhances the specificity. The system is now manufactured by Sorin Biomedica Diagnostics in Europe and Incstar in the US. Variations on DEIA capture techniques have been explored recently (112)(144).

automated dna sequencing technology
Direct sequencing offers direct, rapid, and accurate analysis of amplification products. As described earlier, broad-range PCR amplifies conserved regions of a wide range of organisms (128)(133). The amplicon sequence is first determined, then a DNA sequence-based phylogenetic analysis is performed and used to specifically identify the pathogen (154). Current sequencing technologies include one of two approaches: electrophoretic separation, based on polyacrylamide slab gels or glass capillaries, and solid-phase sequencing, determined by matrix hybridization (128)(133).

single-strand conformational polymorphisms (sscp)
SSCP was first described by Orita et al. (155). DNA is subjected to PCR with primers to a region of suspected polymorphism. The PCR products, which usually incorporate a detector marker, are examined after gel electrophoresis. Physical conformational changes in single-stranded DNA are based on the physiochemical properties of the nucleotide sequence. Variations in the physical conformation are reflected in differential gel migration. This technique is sensitive enough to detect single nucleotide substitutions. One area in which SSCP may prove to be of value is in the detection of mutations related to resistance mechanisms. SSCP, and variations on the technique, have been successfully used to examine the genes contributing to the multidrug resistance of M. tuberculosis [156, 157].

rflp analysis
In postamplification RFLP analysis, the amplified DNA fragments are cut by a restriction endonuclease, separated by gel electrophoresis, and then transferred to a nitrocellulose or nylon membrane. The fragment(s) containing specific sequences may then be detected by using a labeled homologous oligonucleotide as a probe. Variations in the number and sizes of the fragments detected are referred to as RFLPs and reflect variations in both the number of loci that are homologous to the probe and the location of restriction sites within or flanking those loci (158). An epidemiological application of RFLPs is discussed in more detail later.

	Current Application of Molecular Diagnostics

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clinical microbiology
Traditionally, the clinical medical microbiology laboratory has functioned to identify the etiologic agents of infectious diseases through the direct examination and culture of clinical specimens. Direct examination is limited by the number of organisms present and by the ability of the laboratorian to successfully recognize the pathogen. Similarly, the culture of the etiologic agent depends on the ability of the microbe to propagate on artificial media and the laboratorian's choice of appropriate media for the culture. When a sample of limited volume is submitted, it is often not possible to culture for all pathogens. In such instances, close clinical correlation is essential for the judicious use of the specimen available.

Some microorganisms are either unculturable at present, extremely fastidious, or hazardous to laboratory personnel. In these instances, the diagnosis often depends on the serologic detection of a humoral response or culture in an expensive biosafety level II–IV facility. In community medical microbiology laboratories, these facilities may not be available, or it may not be economically feasible to maintain the special media required for culture of all of the rarely encountered pathogens. Thus, cultures are often sent to referral laboratories. During transit, fragile microbes may lose viability or become overgrown by contaminating organisms or competing normal flora.

The addition of molecular detection methods to the microbiology laboratory has resolved many of these problems. The exquisite sensitivity and specificity of many molecular methods allow the accurate detection of very small numbers of organisms. The direct detection of M. tuberculosis nucleic acid from the sputa of smear-negative patients with tuberculosis clearly illustrates this point (159)(160)(161). The technology allows for the rapid and accurate identification of the etiologic agent in a time substantially shorter than traditional methods. This allows for earlier initiation of a focused antimicrobial regimen and decreases the likelihood of disease progression.

In selected situations, the limitations imposed by the ability of an organism to be cultured and the selection of appropriate media and culture conditions may be replaced by the use of molecular microbiology. Microbial DNA/RNA extracted from a clinical specimen may be analyzed for the presence of various organism-specific nucleic acid sequences regardless of the physiological requirements or viability of the organism (136)(162)(163)(164)(165). For example, the inability to culture and analyze the principal etiologic agent of non-A, non-B hepatitis limited medical advances in this area. Using various molecular methods, however, investigators have been able to isolate hepatitis C virus (HCV) nucleic acid (166). Analysis and cloning of the HCV genome has provided the viral antigens necessary for the development of specific serologic tests (167)(168)(169). Currently, RT-PCR allows for the identification, quantification, and sequence analysis of the HCV genome in infected individuals (117)(170)(171).

Another unculturable microbe that has been specifically detected by PCR and probe analysis is Tropheryma whippelii, the causative agent of Whipple disease (128)(172)(173). Because of the inability of this organism to grow on conventional media and the lack of a serologic test, diagnosis of Whipple disease is usually based on clinical and specific biopsy findings. Patients with Whipple disease often have gastrointestinal manifestations and undergo endoscopy. Small bowel biopsies reveal foamy histiocytes filling the lamina propria. The definitive diagnosis is made with the identification of non-acid-fast, periodic acid-shift-positive, diastase-resistant bacillary forms within the histiocytes. Extraintestinal Whipple disease, principally seen as arthritis and central nervous system involvement, may be missed entirely unless the clinician and pathologist have a high index of suspicion. Even so, the diagnosis in such instances may prove difficult. Advances in the molecular detection of T. whippelii have resolved this dilemma (128)(172)(173). On the basis of bacterial 16S rRNA gene sequence analysis, an emerging pathogen, Bordetella holmesii, has been successfully identified in the immunocompromised hosts (130)(131). Additionally, the DNA from a single clinical specimen, such as a knee fluid aspirate, may be tested for several etiologic agents in a differential diagnosis. In such instances, the specimen may also be analyzed for other fastidious and difficult-to-culture agents of infectious arthritis, such as N. gonorrhea or Borrelia burgdorferi [14, 15, 60, 103, 125, 174].

As alluded to earlier, molecular methods may also be useful in instances of limited specimen volume (175)(176). Even in low-volume specimens, enough DNA/RNA can often be extracted to allow performance of numerous molecular assays. However, though molecular methods are very sensitive, we emphasize that, like culture and direct examination, clinically relevant results are ultimately reliant on the submission of quality specimens (177)(178).

Some organisms, although not difficult to culture, are encountered infrequently and require special media for isolation. In these instances, culturing may not be cost-effective for smaller laboratories because the reagents may expire before usage; these samples may also be sent to reference laboratories for culturing, for the sake of economy. Again, fragile organisms may die in transit or become overgrown by contaminating bacteria, thereby making the subsequent culture useless. If molecular microbiology facilities are not available in community laboratories, nucleic acids extracted by the use of commercially available kits may be sent frozen to molecular reference facilities. Alternatively, if molecular facilities are available, PCR primers and probes for relatively rare microorganisms may be maintained frozen at -70 °C for extended periods and used when needed. This may eliminate the need for special culture media and circumvent problems related to specimen transit. As molecular techniques become more widely available, the spectrum of rapid and cost-effective clinical microbiology testing available to smaller laboratories can be extended.

Molecular methods of detection may also play a role in laboratory safety. Organisms such as Coxiella burnetti, M. tuberculosis, Coccidioides immitis, and several viruses causing severe hemorrhagic fevers are laboratory hazards (179)(180)(181)(182). These organisms are easily cultured, but may infect laboratory personnel and cause serious illness or death. The handling of these organisms requires specially trained personnel, special equipment, and expensive ventilated facilities—all of which increase laboratory costs. Molecular methods may be used to detect such organisms directly from clinical specimens, without exposing laboratory personnel to biologically amplified organisms. After the initial extraction procedure, only noninfectious materials are handled.

The molecular detection of microbes with a known susceptibility profile is an effective replacement of the traditional culture. An excellent example is the molecular detection of Bordetella pertussis [176]. This organism is a relatively slow grower, requires specially supplemented and more costly media, and has a known susceptibility profile. The molecular detection of Bordetella pertussis can save time, lower laboratory costs with regard to special media, and allow for the more rapid initiation of effective therapy (176). If variable antimicrobial susceptibility profiles exist, culture for susceptibility testing is still necessary. Molecular methods for the detection of antimicrobial-resistant strains are in development and in the future may replace traditional susceptibility testing (see below). Until then, molecular screening may be used to determine which patients should be cultured for subsequent susceptibility testing.

In recent years, the demand for quantification of nucleic acid targets has been growing (183)(184). By use of molecular methods, the microbial load of an infecting pathogen may be determined and its genotype may also be evaluated. Viral load data are used to monitor therapeutic responsiveness and may yield prognostic information regarding the progression of disease. Until recently, however, the task of quantitative nucleic acid amplification has been very difficult to accomplish. Because the amplification techniques yielded products in an exponential manner until a plateau was reached, any factor interfering with the exponential nature of the amplification process would therefore affect the result of the quantitative assay. In practice, many factors can affect the efficiency of the PCR reaction throughout the amplification procedures and result in the differences between theoretical and actual yields of the reaction. Now, however, kit-based technologies make it possible for many laboratories to carry out quantitative determinations.

Viral load determinations are currently used for evaluating HIV and HCV disease by the use of PCR and bDNA technology (185)(186)(187). When used with other surrogate markers such as CD4 cell count, determination of plasma HIV viral load is an early and accurate marker of disease progression (188)(189)(190)(191). This may result in better predictors of disease progression and outcome, as well as criteria for initiation and modification of antiviral therapy.

clinical epidemiology and infection control
The investigation and control of nosocomial infections is a complex issue that involves clinical, infection-control, and laboratory personnel. The efforts of both the microbiologist and the hospital epidemiologist are facilitated greatly by the availability of the newer molecular epidemiological typing techniques. Molecular diagnostic techniques have been successfully used in the investigation and control of classical and emerging nosocomial pathogens, such as the enterobacteriaceae, Pseudomonas aeruginosa, Staphylococcus aureus, coagulase-negative staphylococci, enterococci, Candida albicans, M. tuberculosis, and Chlamydia pneumoniae [79, 192–194]. Application of DNA probe-based assays allows the diagnosis of other nosocomial infections caused by respiratory syncytial virus (195), varicella-zoster virus, herpes simplex virus (196), and legionella (197) to be made in only a few hours. The molecular techniques have played an important role in the detection, identification, and antimicrobial susceptibility testing of many nosocomial pathogens (83)(96)(97)(198). A good example is the use of PCR-RFLP analysis in combination with Southern transfer and hybridization (fingerprinting) to study the multiple drug-resistant M. tuberculosis nosocomial outbreak in HIV-positive groups in Miami (81) and New York (82)(83).

The ability to rapidly and unambiguously characterize organisms suspected of causing a disease outbreak is critical to public health and hospital infection-control endeavors. Recent contributions to clinical and hospital epidemiology have depended on PCR. Several putative outbreaks of infections have been investigated by molecular techniques. Such examples include investigation of several temporally clustered cases of Streptococcus pyogenes invasive disease in Air Force recruits (199), a case cluster of lymphogranuloma venereum caused by Chlamydia trachomatis serovar L1 in homosexual men (200), and an outbreak of E. coli O157:H7 infection from contaminated deer jerky (80).

Significantly, a PCR analysis was recently successfully used to identify the hantavirus agent responsible for an outbreak of fatal infections in the US Southwest. In May 1993, a mysterious respiratory illness outbreak was reported in the Four Corners region, which includes New Mexico, Arizona, Colorado, and Utah. Patients were defined as having unexplained adult respiratory distress syndrome or acute bilateral pulmonary interstitial infiltrates. Mortality in confirmed patients was >75%. Preliminary serologic studies found antibodies in patients' sera in patterns suggesting cross-reactivity (but not identity) with previously known hantaviruses (180). By comparing genome sequences of available hantavirus strains, precise regions of sequence conservation within the G2 protein coding region of the M segment of the hantavirus genome were identified (201)(202). Deoxyoligonucleotide primers were designed for detection of small amounts of hantavirus genome by a nested RT-PCR assay. The genetic detection assay amplified hantavirus-specific DNA fragments from RNA extracted from the tissues of patients and deer mice caught at or near patients' residences, revealing the associated virus to be a new hantavirus and providing a direct genetic link between infection in patients and rodents (203).

Molecular techniques are being used increasingly in epidemiological and clinical investigations. Among viral infections, the human papillomavirus (HPV) is a common cause of dysplasia, intraepithelial neoplasia, and carcinoma in the female genital tract. Certain types, such as types 16 and 18, have been regarded as high-risk cancer-associated HPVs, whereas types 6 and 11 are regarded as low-risk HPVs (204)(205). Use of DNA hybridization assays in cervical swabs or fresh cervical biopsy specimens to determine HPV infection and viral types has provided helpful information for clinical assessment and treatment of patients (206)(207). In HCV infections, different genotypes have been reported to alter disease severity, change treatment response, and influence virus–host interactions (208). A specific primer set to the 5'-untranslated region has been designed to allow detection of HCV nucleic acids of different genotypes (209). By using PCR followed by automated direct sequencing, several studies have revealed that the most common genotypes of HCV in the US and Western Europe are 1a and 1b; other genotypes, including 2a, 2b, 3, 4, 5, and 6, have their own distinct global distributions (210)(211). A new PCR-based HCV genotyping system has been recently developed to identify HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a; it may be useful for a large-scale determination of HCV genotypes in clinical studies (212).

Molecular techniques have been used to directly detect resistance genes or mutations that result in resistance in organisms. The mecA gene that codes for resistance to methicillin in Staphylococci has been detected by PCR, multiplex PCR, and bDNA assays (123)(213)(214). Defining the mutations responsible for resistance to microbial agents has led to new methods for monitoring efficacy of antimicrobial therapy. Successful investigations have been carried out on both bacterial and viral resistance mechanisms. A PCR assay has been used to detect mutations in the rpoB locus associated with rifampin resistance in M. tuberculosis [157, 159, 215]. The previously discussed TMA technique has been described for detection of the point mutations resulting in zidovudine resistance in stains of HIV (140). Determination of the structural basis of resistance of HIV to viral polymerase inhibitors has been described in detail elsewhere (106)(216)(217). Another example is the finding that certain point mutations in the herpes simplex virus-encoded thymidine kinase gene are responsible for the occurrence of acyclovir resistance (218). Determining acyclovir resistance by detecting these point mutations is extremely important in patients undergoing long-term therapy and in patients with AIDS or other immunosuppressed states (156)(219)(220).

	Future Applications

Top Abstract Introduction Traditional Microbial Typing Nucleic Acid-Based Typing... Nucleic Acid Analysis Without... Polymerase Chain Reaction Other Nucleic Acid Amplification... Analysis of Amplification... Current Application of Molecular... Future Applications References

 
Molecular screening of particular at-risk populations for a group of possible pathogens is an exciting area of development in molecular microbiology. For example, numerous etiologic agents cause debilitating gastroenteritis in immunosuppressed patient populations, including mycobacteria (i.e., M. avium complex and M. genevense), parasites (i.e., Cryptosporidum, Microsporidum), viruses (i.e., rotovirus, Norwalk agent), and typical bacterial pathogens (E. coli variants, Salmonella, Shigella, and Campylobacter). Traditionally, different methods of detection are used for each group of intestinal pathogens. This requires special media, equipment, and expensive facilities for the culture of mycobacteria; expertise in the identification of parasites in ova and parasite stool preparations; virology facilities; and special media for the workup of bacterial enteric pathogens. Although these tests may be relatively inexpensive individually, an adequate workup for enteric pathogens can be quite costly.

Molecular techniques exist and are being developed that may be used to screen individuals within a particular patient population for the most probable etiologic agents of disease. Nucleic acids extracted from the stool of patients with gastroenteritis may be examined with organism- or group-specific nucleic acid primers and probes. In this manner, one single test may be used to single out the etiologic agent of disease among numerous possibilities.

The techniques being used for molecular screening include the newer nucleic acid "chip" technologies, multiplex PCR, and the use of broad-range PCR primers and subsequent nucleic acid sequence analysis. "DNA chips," developed and manufactured by several companies, are basically the product of bonding or direct synthesis of numerous specific DNA probes on a stationary, often silicon-based support (221)(222)(223)(224)(225). Within the particular well, hybridization reactions occur if the appropriate sequence or probe "feature" is present in the DNA or RNA analyte. Because numerous features are present on a single chip, several microbial pathogens or targets may be detected in one test. The chip may be tailored to particular disease processes. This technology is easily performed and readily automated.

Similarly, multiplex PCR utilizes numerous primers within a single reaction tube so as to amplify nucleic acid fragments from different targets. Specific nucleic acid amplification should occur if the appropriate target DNA is present in the sample tested (115)(120)(122)(123). Detection may then be accomplished by traditional Southern transfer and subsequent nucleic acid probe, by enzyme immunoassay methods, or by "gene-chip" analysis. This technology is limited by the number of primers that can be included in a single reaction, primer–primer interference, and nonspecific nucleic acid amplification.

Finally, several pathogens within taxonomically related groups may be screened with broad-range PCR primers and detected by nucleic acid sequence or probe analysis (126)(127)(128)(226). Primers are chosen on the basis of nucleic acid sequence comparisons to include pathogenic agents and, if possible, to exclude possible environmental contaminants. For example, the use of broad-range PCR primers and sequence analysis has successfully detected diseases caused by members of the Rickettsiaceae; in particular, the agents of ehrlichiosis have been identified and speciated (154). This technique is quite useful in instances in which the differential diagnosis can be limited to a particular group of organisms.

Future applications in the field of molecular microbiology include the rapid detection of microbial resistance and, we hope, with the development of more user-friendly systems, the expansion of these technologies to smaller institutions and hospitals. The use of these biochemical methods and reactions in the specific identification of infectious agents at the nucleic acid level truly represents a synthesis of the clinical chemistry and clinical microbiology laboratories.

	Footnotes

 
¹ Nonstandard abbreviations: RFLP, restriction fragment length polymorphism; PFGE, pulse-field gel electrophoresis; RAPD, random amplified polymorphic DNA; bDNA, branched DNA; RT, reverse transcriptase; TAS, transcription-based amplification system; TMA, transcription-mediated amplification; LCR, ligase chain reaction; SDA, strand displacement amplification; HPA, the hybridization protection assay; DEIA, DNA enzyme immunoassay; SSCP, single-strand conformational polymorphisms; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus.

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