Anal. Chem. 1999, 71, 366R-372R
Clinical Analyzers. Microbiology C. Lars Mouritsen* and David R. Hillyard*
Molecular Pathology Laboratory, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, Utah 84108 From October 1996 through September 1998 there were significant advances in the diagnostic technologies for microbiology and virology. During this time, a number of new automated and semiautomated analyzers were introduced into the clinical laboratory. These analyzers allow rapid, precise, and accurate detection of different types of organisms, including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycobacterium tuberculosis (Mtb), hepatitides, retroviruses, enteroviruses, parasites, and fungi. Standard diagnostic techniques for the identification of these organisms have also advanced during this time period. In addition, some major advances in the area of molecular diagnostics have begun to revolutionize the way that routine infectious disease testing is performed. This review will give some attention to advances in conventional methods, but will primarily focus on recent progress in molecular diagnostics for the diagnosis and identification of infectious diseases. NON-NUCLEIC ACID-BASED MICROBIAL IDENTIFICATION One system that has shown great promise for fully automated microbial identification and susceptibility testing is the Vitek AMS analyzer (bioMerieux Vitek, Inc., Hazelwood, MO). This system uses a sealed multiwell card platform to which culture isolates can be added. Identification is based on specific fluorescence detection within each well of the card. In a recent study of antimicrobial susceptibility tests, directly inoculated bioMerieux Vitek GNS cards were compared to those inoculated according to manufacturers’ recommendations and the NCCLS broth microdilution test. This study was performed using 50 Enterobacteriaceae isolates tested against 15 antimicrobial agents. The data indicated that if urinary infection-specific drugs and ampicillin/ sulbactam were removed from consideration, the error rates for both methods of susceptibility testing, utilizing the Vitek AMS analyzer, could be reduced to less than 4.5%. These results compared well to the standard broth microdilution method (L1). In a second evaluation of the bioMerieux Vitek AMS analyzer, four different study centers tested 1082 clinical isolates for susceptibility to 11 antimicrobial agents. Each participating laboratory also tested a challenge panel involving 200 organisms. In all, 11 902 clinical isolate comparisons were made yielding an error rate of 4.5% and 8800 challenge comparisons were made yielding a 5.9% error rate. The study showed very good interlaboratory reproducibility, however, false resistance rates increased if inoculum densities exceeded those recommended by the manufacturer (L2). Another study compared the Vitek GNI+ to the Becton Dickinson Microbiology Systems Crystal E/NF (L3). Both systems utilize non-nucleic acid techniques for the identification of Enterobacteriaceae and other glucose and non-glucose fermenting 366R Analytical Chemistry, Vol. 71, No. 12, June 15, 1999
Gram-negative rods. O’Hara et al. determined that there was no significant difference between either system with respect to accuracy and proper identification. Both analyzers showed improved accuracy when additional testing was performed as suggested by the system software. Differences between the GNI+ and the Crystal E/NF platforms were noted in identification times, 4.1-6.8 h for GNI+ and 18 h for Crystal E/NF. In a third study, Shetty et al. observed that the Vitek AMS system performed rapidly and accurately for the identification of common bacterial pathogens (L4). The authors also noted that certain manual laboratory techniques would still be required for identification and susceptibility testing of a small subset of organisms. Although this system may not be as technically advanced as some of the nucleic acid amplification methodologies, it still provides a valuable resource for the clinical microbiology laboratory. This is because the Vitek AMS instrument and other similar instruments are more cost-effective and generally require much less hands-on time than the nucleic acid amplification systems described below. These analyzers have also been standardized for analysis of a greater spectrum of organisms. HYBRID CAPTURE TECHNOLOGY There are primarily two hybrid capture systems that can be integrated into the clinical laboratory. The Gen-Probe PACE 2 (Gen-Probe, San Diego, CA) assay is based on magnetic particle separation and subsequent detection of chemiluminescently labeled single-stranded DNA probes that are specifically designed to bind to ribosomal RNA (rRNA). The second system, from Digene Diagnostics (Silver Spring, MD), utilizes universal substratebound capture antibodies that have specific affinity for RNA-DNA hybrids. Detection is made possible by a second anti-RNA-DNA hybrid antibody that is conjugated with alkaline phosphatase. Dioxetane is cleaved by the alkaline phosphatase and results in a manyfold chemiluminescent signal amplification. The Gen-Probe PACE 2 assay has been shown to be significantly better than throat culture for the identification gonococcal infections, but did not look to be as sensitive as culture when testing rectal swabs (L5). In a method comparison by Carroll et al., Gen-Probe PACE 2 demonstrated very good specificity but was lacking in sensitivity when compared to either the Abbott ligase chain reaction (LCR) test for CT or culture (L6). In the same study, the Gen-Probe assay also showed lower sensitivity for men than for women. In an earlier study for the detection of CT in the female urogenital tract, Binder et al. also found the sensitivity of the Gen-Probe PACE 2 system to be greatly lacking in comparison to an LCR amplification method (L7). The Digene Hybrid Capture system has been described as a simple method for the identification of human papillomavirus 10.1021/a19999123 CCC: $18.00
© 1999 American Chemical Society Published on Web 05/20/1999
(HPV) DNA in patients with borderline atypical squamous cells of undetermined significance (ASCUS) Pap smear results (L8). The hybrid capture method, however, has lower analytic sensitivity in comparison to a PCR-amplification method, but may still be helpful for patients with concurrent squamous intraepithelial lesions (SILs) (L9). In two different studies, hybrid capture HPV DNA testing did not significantly improve the cytologic testing algorithms that were previously associated with diagnosis of cervical intraepithelial neoplasia (CIN) (L10, L11). Despite these shortcomings, the Digene Hybrid Capture system is still the most widely used nucleic acid-based testing method for the detection of HPV DNA in relation to an ASCUS Pap smear. As a result, this method has become an important part of disease management of cervical cancer. According to the manufacturer, hybrid capture systems are also available for detection of CT, NG, and hepatitis B virus (HBV). SOLID-PHASE NUCLEIC ACID DETECTION SYSTEMS (MANUAL AND SEMIAUTOMATED) An influx of several different methods for the detection of nucleic acid has surfaced in the past few years. Some of these methods have been developed using standard enzyme immunoassay (EIA) chemistry. Although similar to the serologically based EIA, the newer nucleic acid detection technology has the capacity to be much more sensitive and specific when combined with an efficient nucleic acid extraction and subsequent amplification procedure. There have been many different modifications to this basic theme, but the essential idea is to bind synthetically made oligonucleotide capture probes to a substrate. The substrate may be silica, plastic, or a ferrous, metal-based bead, or it may be as simple as the well of a microtiter plate. Once the probe is bound to the substrate, denatured amplicon can be added and allowed to hybridize to the capture probe. Generally, the amplicon is created in the presence of biotin- or digoxigenin-labeled primers. Therefore, once the amplicon binds to the capture probe, it can be detected using horseradish peroxidase-labeled anti-biotin, streptavidin, or anti-digoxigenin. The final colorimetric reaction takes place by adding tetramethylbenzidene (TMB) or some related enzyme substrate. This method has been widely adopted during the past few years, and the result has been a flood of bead and microtiter plate (MTP) nucleic acid detection techniques. The methods described above have great application for the detection of infectious diseases. Nucleic acid analytes that were once detected by gel electrophoresis or Southern blot can now be detected using semiautomated bead or MTP detection systems. MTP technology allows the detection of nucleic acids with as good or better sensitivity and specificity than the older methods and with greater safety. One of the pioneers who has commercialized this technology for nucleic acid detection has been Roche Diagnostics. Roche has made this technology available for the identification of such organisms as Mtb, CT, hepatitis C (HCV), HIV-1, cytomegalovirus (CMV), and enterovirus in both qualitative and quantitative formats (L12-L21). Other examples of MTP detection platforms are effectively described by Tang et al. in a study comparing Southern blotting to four different commercial MTP immunoassay systems for the detection of herpes simplex virus in cerebrospinal fluid (L22). The MTP systems used in this study include PrimeCapture from ViroMed Laboratories, Inc.; Quanti-PATH from CPG, Inc.; GEN-
ETI-K from IncSTAR Corp./Sorin; and a PCR ELISA system by Boehringer Mannheim Corp. All of these systems exhibited nearly 100% specificity and sensitivity as compared to Southern blot analysis. One exception was the PrimeCapture system, which exhibited only 63% sensitivity. All of the systems in this study made use, in some form, of the detection chemistry outlined above. The IncSTAR/Sorin kit, however, utilizes nonlabeled primers that, once incorporated in the amplicon, bind to the capture probe and are detected using an HRP-labeled anti-double-stranded DNA antibody. DNA enzyme immunoassay (DEIA) technology has great potential for detection of amplicon generated from many different nucleic acid species. Using these methods, it is possible for the user to efficiently detect minute quantities (some have reported as few as 50 000 copies of amplicon per microplate well) of nucleic acid with great specificity and sensitivity. Additional benefits of MTP technology are its speed and automation potential (L23). Much of the same automation technology that is standard in the serology laboratory can be effectively applied to nucleic acid MTP detection. There are several points that may need to be assessed by the end-user prior to incorporating one of these products into the routine laboratory. Many of these products are relatively new to the market; therefore, careful attention must be paid during the validation process. The user must assess the level of QC utilized during the manufacturer’s evaluation of the system. Oftentimes there can be manufacturing problems with oligos and probes making the reliability of the system questionable. Lot-to-lot variations may not be noticed unless a rigid analytical sensitivity panel is run with each lot employed. As a result, low-titer specimens may not be detected if sensitivity is evaluated using only clinical specimens with high titers. Users must assess the company’s commitment to GMP/GLP and ISO9001. Users must also determine whether contamination control has been incorporated into the assay. Component or technology licensure issues may also be of concern for the clinical laboratory. Finally, one must be prepared to do a lot of fine-tuning. Many of the kits come with a suggested protocol, but few have protocols that are inclusive from initial sample preparation to final result acquisition. FULLY AUTOMATED NUCLEIC ACID AMPLIFICATION AND DETECTION SYSTEMS Recently, a number of semiautomated or fully automated nucleic acid analyzers have been introduced for use in the infectious disease clinical laboratory. Some of these platforms have great versatility in detecting different nucleic acid species. Logically, the development of these testing platforms and products has been based on marketability and volume. As a result, the most clinically relevant tests are the first to be developed and automated. There are several key organisms that fall into this primary wave of automated nucleic acid testing as follows: Mtb, CT, NG, human immunodeficiency virus (HIV-1), types B and C hepatitides, and CMV. As each analyzer is discussed, applications will be referenced for some of the listed organisms. Becton Dickinson (Sparks, MD) has developed strand displacement amplification (SDA), which is an isothermal technology that uses of both restriction enzyme and polymerase activities. As described in a study for the detection of CT, fluorescence polarization is used to monitor hybridization of a detector probe Analytical Chemistry, Vol. 71, No. 12, June 15, 1999
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to the amplified product while the product is created (L24). This system allows high-throughput processing in the presence of an internal amplification control (∼46 samples can be processed from nucleic acid isolation, producing a result in less than 6 h). Badak et al. initially demonstrated the clinical utility of this method for amplification and detection of Mtb from positive mycobacterial growth indicator tubes (L25). A follow-up clinical comparison between the BDProbetec MTB Strand Displacement Test from Becton Dickinson and conventional mycobacterial culture showed that the SDA method had good agreement and great clinical utility for the detection of Mtb in respiratory specimens. Additional studies have demonstrated the utility of the SDA technology for the detection and quantitation of HIV-1 RNA (L26, L27). Gen-Probe has developed a second nucleic acid analytical platform in this category. This method is referred to as transcription-mediated amplification (TMA) (L28). This technology has some similarities to nucleic acid sequence-based amplification (NASBA), as well as to the SDA method described above (L29, L30). TMA is based on isothermal amplification using two different enzymes. One enzyme allows reverse transcription and the other provides both RNase H activity and RNA polymerization activity. Amplified products are detected using a specific probe hybridization process known as the hybridization protection assay. TMA has been used for the effective detection of CT from urine specimens and has compared well to culture, LCR, and PCR for specific applications (L31-L34). The Gen-Probe amplification system has also been described for the detection of Mtb complex from urine specimens, spinal fluid, and clinical respiratory specimens (L35-L39). Gamboa et al. demonstrated the use of an enhanced version of the amplified Mtb direct test (AMTDT). The newer version (AMTDT2) showed better sensitivity and a faster turnaround time (L38). The application of TMA technology has been described not only for CT and Mtb but also for HIV-1 viral RNA detection, and for the simultaneous detection of HIV-1 and hepatitis C virus RNA in blood samples (L28). Another instrument that can be used for the automation of nucleic acid analysis is the Abbott LCx (Abbott Laboratories, Abbott Park, IL). LCx instrumentation is fully automated for amplification and detection and is based on ligase chain reaction technology. In its most basic form, this method relies on hybridization and ligation of specific oligonucleotide probes to a template nucleic acid sequence. Once ligation is complete, both the template strand and the ligated strands can be denatured to act as templates for the next amplification cycle. Each cycle of the reaction theoretically yields a doubling of the amplified product, much like PCR. Use of the LCx instrument has been described in many publications. However, recently the LCx has been used effectively for the combined detection of CT and NG (L40). Buimer et al. have shown that LCR for both CT and NG is far superior to conventional culture methods. Specifically, the sensitivity of LCR was 100% with male urethral swabs, 95.4% with female cervical swabs, 88.9% with male urine, and 50.0% with female urine. Buimer also indicated that use of LCR-based assays represented a major improvement in CT/NG diagnostics. It was further suggested that such methods could be useful for screening. In a second study, Dubuis et al. demonstrated that LCR was quite reliable with 100% sensitivity and specificity for both CT and NG (L41). Dubuis also 368R
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proved that 2-sucrose-phosphate was an acceptable transport medium for LCR. One drawback that was noted for LCR was the lack of amplification control material. The Abbott LCR method has also been described for the detection of Mtb (L42). The Roche COBAS (Roche Diagnostics, Branchburg, NJ) is older technology, yet its use as a nucleic acid testing platform is quite new. The primary tests that can be performed clinically on the COBAS are HCV qualitatively and quantitatively, Mtb, and CT/NG; however, none of these tests are FDA approved for use with this analyzer. The COBAS technology fully automates both the amplification and detection steps of PCR. Amplification is performed on samples that have been placed in small 200-µL microwells fixed in a 12well, ring (A-ring) configuration. The A-ring is loaded into one of two onboard Peltier thermocyclers and allowed to cycle through reverse transcription and amplification. The amplified product is then removed to a detection cup with the use of a robust liquidtransfer probe. The remaining detection steps are very similar to standard MTP enzyme immunoassay chemistry with the exception that the amplified product, from both wild type and internal control species, is captured by specific probes attached to magnetic particles (L43). COBAS has been used for qualitatively detecting amplicon from hepatitis C virus (L44-L46). Initial studies have indicated that the automated HCV COBAS AMPLICOR system gives the clinical microbiology laboratory a sensitive and specific PCR method for the rapid and reliable detection of HCV RNA. The COBAS has also proven useful for the detection of Mtb (L47L53). In addition, CT detection can be performed either individually or in conduction with NG (L54-L59). In either case, detection and amplification are performed in the presence of an internal control. COBAS has also shown some potential for multiplex testing (L57, L59). Currently, the COBAS analyzer provides a very advanced testing platform for the analysis of nucleic acid in the field of molecular infectious diseases. This analyzer provides great reproducibility and accuracy and allows the user to perform many tasks without direct “hands-on” intervention. CONTINUOUS FLUORESCENCE MONITORING At the time of this review, there are two instruments widely available for continuous fluorescence monitoring. They are the Roche LightCycler and the Roche TaqMan (also listed as the Perkin-Elmer ABI 7700 5′ nuclease). Both detection platforms are based on fluorescence resonance energy transfer and have the ability to monitor the amount of product during amplification. The continous-monitoring properties are possible through the use of an intercalator or minor groove binding dye and/or fluorogenic probes or primers. It should be noted that dyes work effectively but are much less specific than probes. One basic premise for this methodology is that the intensity of fluorescence is proportional to the amount of target DNA present. A second premise is that the specificity of amplified product can be measured by specific fluorescence patterns or by product-specific melting temperatures. Some features of this technology include very rapid amplification and detection compared to conventional technologies. An example of this method is described by Lay and Wittwer in their study on factor V Leiden mutation detection (L60). This study
indicates that detailed information about this genetic disease can be gathered in less than 1 h using the LightCycler as opposed to PCR coupled with restriction digest and gel electrophoresis, which can take as much as 6 h. Continuous fluorescence monitoring also provides self-contained amplification and detection, greatly reducing contamination risk. Results are generally unambiguous and easy to interpret. Kennedy and others described the use of TaqMan PCR for the identification of human herpes virus 8 (HHV8) in early cases of Kaposi’s sarcoma (L61). They reported that TaqMan PCR detected low copies of viral target in affected human tissues. Researchers indicated good correlation between tumor pathogenesis and HHV8 detection by TaqMan PCR. A total of 49% of cases from African tissues and 90% of cases from European specimens contained HHV8. In a later study, Kennedy et al. showed that HHV8 could be detected in 87% of cases of Kaposi’s sarcoma (L62). Few articles have been published highlighting the LightCycler technology for the detection of viruses. Some publications, however, have described the identification of Leptospira and Borrelia species. To begin with, Woo et al. effectively used the LightCycler to specifically identify Leptospira inadai apart from 16 other related strains (L63). DNA samples were amplified in the presence of SYBR green I dye, and final melting curve analysis was used to determine the specific product apart from primer artifact. Morrison et al. used the LightCycler in a manner similar to quantitate low levels of Borrelia burgdorferi transcripts (L64). NUCLEIC ACID SEQUENCE TECHNOLOGY Modern sequence technology has integrated a number of techniques, such as gel electrophoresis, fluorescent dye labeling, and PCR, to generate high-throughput and accurate sequencing platforms. Many sequence detection systems allow direct detection of PCR products with rapid cleanup methods to remove unwanted primers and oligos. Other methods require a restriction digestion step prior to detection. Nucleic acid sequence analysis is probably the most information rich, but sometimes the most problematic, area in nucleic acid technology. In many cases, this area of technology can provide very specific data about genetic variability, identity, drug resistance, and presence or absence of specific mutations. Generally, the processes that are used to manage, compare, and process data are complex and may challenge new users. Several biotechnology corporations have given careful thought and planning to the development of nucleic acid sequence-based technology. Sanger-modified dye primer or dye terminator chemistries (cycle sequencing), MTP hybridization, chip hybridization, restriction digestion, cleavage of the nucleic acid secondary structure, and dot or slot blot technologies are just some of the methods that have been described over the past several years. Not all of these approaches provide a complete nucleic acid sequence of the sample target. At one end of the spectrum are tests based on hybridization of a limited number of probes or digestion with a battery of restriction enzymes. Inferences can be made about the approximate sequence of nucleotides in a limited genomic domain but the analysis falls short of a complete sequence description. A complete sequence description depends on analysis by methods such as Sanger dideoxy sequencing or
use of a dense array of hybridization probes. Analysis of both strands of plasmids or PCR amplicons is required to ensure the most accurate sequence information. An example of the application of the spectrum of methods for clinical “sequencing” is the case of HCV genotyping. Cretel and others described a comparison of hybridization methods for hepatitis C virus typing (L65). They compared both the Murex HCV and the Chiron RIBA HCV serotyping assays to the molecular-based Innogenetics Inno-LIPA HCV and the Sorin GEN-ETI-K HCV genotyping assays. Both of the serological assays give typing results that are based on antibody response to a specific series of HCV antigens. The molecular-based assays, on the other hand, give genotypic information that is based on the PCR amplification of a specific region of the genome. The amplicon is then allowed to bind to a series of capture probes bound to a membrane strip, in the case of the Inno-LIPA (line probe assay), or to the walls of a microplate well, in the case of the Sorin GENETI-K assay. The results of this study indicate that all four tests agreed on the specimen serotype or genotype 44% of the time and that 92% of all samples could be typed by at least one test. All discrepant results were confirmed by actual sequence analysis of a continuous portion of the 5′-noncoding (5′NC) region of the HCV genome. These results suggest that cycle sequencing of the 5′NC region may be the best method for accurately obtaining the most complete and accurate genotype of the specimen. Affymetrix (Santa Clara, CA) has described another method for obtaining sequence information. Similar to the probe hybridization systems used by Inno-LIPA and Sorin, Affymetrix applies probes to specific locations on the surface of a silica-based microchip. In contrast to the other probe systems, Affymetrix has developed a method whereby as many as 400 000 probes can be affixed to the microchip surface. Probes are affixed in a designated area such that all four nucleotide bases for a certain position in the genome can be compared on the basis of levels of fluorescence or luminescence after the amplicon has hybridized (L66, L67). This technology allows the user to test a single specimen containing amplified product from a specific region of the genome. The outcome is that the amplified product anneals to the probes on the microchip surface and exhibits a specific fluorescence pattern that can be interpreted for each nucleotide location of the region being analyzed. This method of obtaining sequence data has been used clinically for resistant-strain typing for HIV-1 (L68, L69). Sequence analysis has been applied in many different ways using many different methods and instrumentation (L70). Although sequencing has been used to analyze many types of diseases and pathogens, the area of greatest interest and controversy seems to be that of genotypic analysis of HIV and HCV. In a study of genotypic variation of HCV, Le Pogam and others compared a DNA enzyme immunoassay and the Innogenetics Inno-LIPA test (L71). Together, these methods were capable of determining the genotype for 87% of 120 samples in the study. The remaining 13% of samples showed discrepancies between the two methods. Amplified products from the core and 5′-untranslated region (UTR), for the discrepant samples, were consequently resolved by cycle sequencing. In two more recent studies, Doglio et al. and Holland et al. separately described genotypic analysis of the 5′-noncoding region (NCR) of the hepatitis C genome Analytical Chemistry, Vol. 71, No. 12, June 15, 1999
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through the use of the Roche Amplicor method for RT-PCR (L72, L73). Amplification, in both studies, was followed by rapid, automated DNA cycle sequencing. Both studies reported good success using this method with few failures in detection. With respect to HIV, the area of resistance testing is complex and requires extremely comprehensive databases to aid in clinical decision making. For this purpose, cycle sequencing of a specific amplified region (or regions) of the HIV genome will likely be one of the better alternatives. In October of 1998, Eron et al. conducted a study of 11 men with HIV infection and detectable viral loads (L74). Eron utilized cycle sequencing of amplified product, from both the RT and protease RNA, to monitor the development of resistance to antiretroviral therapy. There are some side notes to sequence technology that deserve mentioning. Most commercial systems require specialized and expensive instrumentation and may not be feasible for the routine clinical laboratory. Therefore, careful cost accounting must be done to assess whether testing can be accomplished “in-house” or should be referred to a larger reference laboratory providing such services. On another note, nearly all sequencing methods are quite sensitive and very specific. It has been demonstrated, in some cases, that the techniques that provide full sequence data are far superior to those that may only provide data based on limited information. For example, it may be better to make clinical decisions based on sequence data from the entire RT gene of HIV-1 as opposed to hybridization or restriction digest data from a few select regions of the same gene. NUCLEIC ACID ISOLATION AND PURIFICATION Nucleic acid isolation and purification is an area that has, until recently, been lagging behind in the development of amplification and detection instrumentation. This area is one of the fundamental components of molecular diagnostics and can make or break the quality of testing that is performed. Several biotechnology organizations have devoted resources to nucleic acid purification methods in order to meet the growing needs of the research and clinical laboratories. Many methods for nucleic acid purification have been described and oftentimes the method used is dictated by the specimen source. Such methods have ranged from lengthy and toxic phenol/chloroform and ethanol precipitation to nucleic acid adsorption on silica beads or other matrixes. Further endeavors include automated (robotic) nucleic acid isolations in 96-well configurations. This section will include a brief summary of some of the cited methods and look at present and future involvement for automation in such procedures. Multiple procedures have been described resulting in initial degradation of nucleases, cell lysis, nucleic acid release, and nucleic acid capture onto a specific or nonspecific substrate. The capture substrate can be made of an array of materials but is generally made of silica, plastic, ferrous metal, or a combination of these materials. The substrate binds all nucleic acid species contained in the sample through covalent bonds or adsorption. In other systems, the substrate can be labeled with specific capture probes recognizing and binding other specific nucleic acid species. Substrate beads can then be separated from proteins and other unwanted cellular byproducts by centrifugation or magnetic current. The final purified nucleic acid can be eluted from the binding substrate or denatured from the capture probe attached 370R
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to the substrate. The result is a highly purified nucleic acid that can be added directly to the amplification reaction. Furthermore, once the substrate material has been formulated it can often be used directly in the amplification reaction without inhibition. The above processes are quite robust, and fairly sensitive and can be used for different sample types and nucleic acid species. Many different manufacturers have made these techniques widely available in either semiautomated or fully automated platforms. One method that is commonly used in the laboratory for nucleic acid isolation is the “Boom” extraction. This method, based on adsorption of nucleic acid to a silica-based substrate, has been utilized extensively since it was first introduced in 1990 (L75). Organon Teknika (OTC; Durham, NC) has provided this method in nonautomated and fully automated systems. Their system has been routinely used for manual extraction of HIV-1 in the NASBA/ NucliSens test and can provide high-quality RNA for other clinical testing purposes, such as sequence analysis. OTC has now refined its extraction system, providing a fully automated instrument with the capacity to extract 10 plasma samples/h with great reproducibility and virtually no contamination (L76). This nucleic acid extraction instrument provides complete walk-away processing and has a very low risk for sample-to-sample carry-over as a result of careful engineering. Another manufacturer, QIAgen (Valencia, CA), provides a silica-based adsorption product utilizing a silica gel membrane in a spin column format. Lysed sample is applied and centrifuged or vacuumed through the column. Degraded proteins are washed through the column while nucleic acids bind to the silica membrane. Further ethanol/salt washing is performed before the DNA or RNA is finally eluted in RNase-free water. This method was used by Inglis and Valadares Inglis to rapidly isolate doublestranded RNA from fungal specimens (L77). This same method has been applied in the forensics laboratory by Scherzinger et al. to extract DNA from blood specimens (L78). Scherzinger showed the method to be rapid and capable of high throughput, while at the same time giving high-quality DNA with 2-4-fold yields over standard proteinase K degradation and organic extraction. Hilbert and others used this method for high-throughput extraction of double-stranded plasmid DNA (L79). These extractions were performed using a robotic workstation and a 96-well spectrophotometer. The final DNA proved to be of high quality and was adequate for doing large-scale sequencing. This process has been used effectively for plasmid DNA isolation and further sequence analysis. Other potential applications of this technology could include isolation and purification of viral RNA, viral DNA, genomic DNA, or bacterial DNA. Other systems for the purification of nucleic acid have been developed; however, the two systems described above have clearly impacted the manner in which nucleic acid testing can be performed. Using this instrumentation, the clinical laboratory will be able to process as many as 96 samples in ∼2 h. This has the potential to significantly reduce the required labor component for each test, decrease turnaround time, reduce costs, and increase reproducibility. It must be mentioned that along with this highthroughput technology comes greater risk for sample-to-sample carry-over. Great caution must be taken by the user to assess the likelihood of such events prior to investing in this type of instrumentation. This instrumentation can also be very costly;
therefore, one must use the same rationale as listed above for sequence instrumentation. A review of recent literature highlights the rapid growth of automation within the infectious disease laboratory. This is especially true for the molecular laboratory. It is certain that clinical molecular tests will continue to be developed and will become more widely accepted as a means of doing routine microbiology and virology testing. When coupled with conventional methods of diagnostic infectious disease testing, molecular testing will surely add greater depth and a wealth of information that is just beginning to be understood. C. Lars Mouritsen received a B.S. in molecular biology from the University of Utah. He later became ASCP certified in immunology and more recently as a clinical laboratory specialist in molecular biology. He is currently the technical supervisor of the molecular pathology laboratory at ARUP Laboratories in Salt Lake City, UT. His research interests at ARUP have included the development and validation of numerous immunological tests as well as PCR-based extraction, amplification, and detection methods for the identification of human pathogens. David R. Hillyard is medical director of the Molecular Infectious Diseases section of the Molecular Pathology Laboratory at ARUP, Inc. He is an associate professor of pathology, with an adjunct appointment in biology, at the University of Utah. Dr. Hillyard received his M.D. degree from the Columbia University College of Physicians and Surgeons. His training was in anatomic and clinical pathology with fellowships in medical microbiology and microbial genetics.
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